2-methyl-2H-isothiazol-3-one

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 March - 13 April 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline (now OECD Guideline 225)

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):
No data

Analytical monitoring:

yes

Details on sampling:

The analytical method used for the analysis of 2-Methyl-4-Isothiazolin-3-One was developed by Wildlife International, Ltd. Samples were collected from analytical replicates of each test concentration on Days 0, 7 and 28 of the test. Samples of overlying water were sampled at mid-depth and transferred to a microcentrifuge tube, centrifuged at 14,000 rpm for approximately five minutes and placed in autosampler vials for direct HPLC analysis. The sediment portion of each sample was transferred to centrifuge tubes and centrihged at 2500 rpm for ~10 minutes. The pore water from each centrifuged sample was transferred into a graduated cylinder. An aliquot was removed, centrifuged again at 14,000 rprn for approximately five minutes, diluted as necessary with freshwater or 10% methanol:90% HPLC-grade bottled water, and placed in an autosampler vial for direct HPLC analysis.

Sediment samples (5.0 g) were weighed into French square bottles. To each sample, 50.0 mL of 10% methanol:90% HPLC-grade bottled water was added and samples were sonically disrupted for approximately five minutes. Following disruption, the samples were centrihged for approximately five minutes at 14,000 rpm. Samples were diluted where appropriate with 10% methanol:90% HPLC grade bottled water. An aliquot of each extract was transferred to an autosampler vial for immediate HPLC analysis.

Vehicle:

yes

Details on sediment and application:

Test Sediment
Formulated sediment based on the recommendations of OECD Guideline 2 18 (6) was used as the test sediment. The sediment was composed of approximately 5% peat, 20% kaolin clay, and 75% industrial quartz sand (Appendix 3). The dry constituents of the sediment were mixed in a mixer for approximately 20 minutes. The pH of the final mixture of sediment was found to be 7.1. The percent organic carbon of the sediment was found to be 1.9%. The sediment was stored under ambient conditions until used.

A 28-day ration of food (200 mg salmon starter) was dry mixed into the test chambers after the sediment was added, but immediately before adding the water to the test chambers, and 48 hours prior to adding the organisms. The percent organic carbon of the sediment is based solely on peat as the source of organic carbon. The food added to the test system during the test was not included in the measurement of organic carbon.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

The oligochaete, Lumbriculus variegatus, was selected as the test species for this study.

The organisms were held in water from the same source as the water used during the test. During the 14-day holding period immediately preceding the test, water temperature ranged from 20.1 to 23. 1°C, while the pH ranged from 8.3 to 8.7 and dissolved oxygen ranged from 6.9 to 8.4 mg/L.

During holding, the oligochaetes appeared normal. Sixteen days prior to test initiation, adult oligochaetes from the culture were synchronized and transferred to a new holding tank. Synchronization was achieved by artificially fragmenting the worms in the median body region using a scalpel. Thirteen days prior to test initiation, the fragments were separated into anterior and posterior sections. The posterior sections were retained for use in the study and the anterior sections were discarded. At test initiation, "synchronized" adult oligochaetes were collected from the culture and impartially added one and two at a time into transfer chambers until each chamber contained 10 organisms. Each group of ten organisms was impartially assigned and transferred to a test chamber. All transfers were made below the water surface using wide-bore pipettes. All replicate test chambers were maintained with organisms during the study except the Day 0 analytical replicates and the Day 0 water quality replicates, which did not contain organisms.

Oligochaetes were fed a mixture of yeast, cereal grass and trout chow (YCT) during the holding period and salmon starter (Ziegler Brothers, Inc., Gardners, Pennsylvania) during the test. A 200 mg aliquot of food (enough food for the entire study) was weighed for each test chamber that contained organisms during the test and was added to the test chambers after adding the sediment, and mixed with a glass stir rod into the sediment before the overlying water was added. No additional food supplementation was made during the study.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

No data

Hardness:

Day 0 ranged from 140 - 144 mg/L. Day 28 ranged from 160 - 164 mg/L.

Test temperature:

within the 20+/- 2°C range

pH:

8.2 to 8.8 during the test.

Dissolved oxygen:

>/= 67% (6.0 mg/L) of saturation

Salinity:

No data

Ammonia:

Day 0 ammonia levels were <0.17 mg/L, the limit of quantification. By day 7 values ranged from 3.55 - 4.52 mg/L. By day 14, values ranged from 7.37 - 9.85 mg/L. On day 21, values ranged from 6.81 - 8.01 mg/L and on day 28, values ranged from 3.01 - 6.66 mg/L.

Due to the increasing levels of ammonia in the test system throughout the test, the overlying water was partially renewed on Days 14 and 21. The partial renewal consisted of removing 300 mL of overlying water from each test chamber. Clean UV sterilized well water was then added to each chamber (over a baffle so as to minimize the disturbance to the sediment) until reaching the water level mark made at the beginning of the test.

Nominal and measured concentrations:

Nominal test concentrations (3.1, 6.3, 13, 25 and 50 mg a.i./Kg ) were prepared on a dry weight basis, (i.e., mg test substance/Kg dry sediment).

The measured concentrations of 2-Methyl-4-isothiazolin-3-one in the 0.31, 0.63, 1.3, 2.5 and 5.0 mg a.i./mL stock solutions yielded 1 13, 109, 107, 103, and 98.0% of nominal values, respectively. These values indicate that the system was properly dosed.

Details on test conditions:

Preparation of Test Concentrations
The test substance was administered to the test organism in sediment. This route of administration was selected because it represents the most likely route of exposure to sediment dwelling organisms. Nominal test concentrations (3.1, 6.3, 13, 25 and 50 mg a.i./Kg ) were prepared on a dry weight basis, (i.e., mg test substance/Kg dry sediment). A primary stock solution was prepared by dissolving the test substance (Identification No. 7832) in acetone at a nominal concentration of 5.00 mg a.i./mL. The primary stock was mixed by inversion and appeared clear and colorless. Secondary stocks were prepared by serial dilution in acetone at nominal concentrations of 2.50, 1.25, 0.63 and 0.3 1 mg a.i./mL. A 15-mL volume of the appropriate stock was added to 150 grams of sediment and mixed by hand with a glass stir rod, then placed in a fume hood for an adequate amount of time for the acetone to partially dissipate. The 150 gram premixes were then added to 600 grams of untreated sediment in plastic Nalgene bottles and mixed on a rotary mixer for an adequate amount of time. Next, 750 grams of untreated sediment were added to the premixes to achieve a final weight of 1500 grams. The batch sediments were then mixed on a rotary mixer overnight.

Test chambers (one quart glass jars) were prepared by adding the appropriate dosed sediment to a 2 cm pre-calibrated depth in the corresponding replicates. A 28-day ration of food (approximately 200 mg of Salmon Starter) was added and gently stirred into the dry sediment. Finally, approximately 600 mL of ultraviolet-sterilized well water was added to the chambers. After the water was added to the chambers, the chambers were arranged in a random order in an environmental chamber. Gentle aeration was added to each test chamber for the duration of the study. The overlying water was partially renewed (300mL) on Days 14 and 21, due to rising levels of ammonia in the overlying water.

Fourteen replicate test chambers were prepared for the negative and solvent control groups, seven replicate test chambers were prepared for the 3.1, 6.3, 13 and 25 mg a.i./Kg treatment groups and twelve replicate test chambers were prepared for the 50 mg a.i./Kg treatment group. Four replicates in the treatment groups and six replicates in the controls were used for the evaluation of survival and growth. An additional three replicates were used for analytical confirmation purposes in each treatment group and control on Days 0, 7 and 14, and five replicates were prepared as sacrificial replicates for water quality measurements in the controls and the highest treatment group (50 mg a.i./Kg). The sedimentlwater mixtures were allowed to acclimate for approximately 49 hours prior to the introduction of the test organisms. At test initiation the overlying water in all test chambers appeared clear and colorless, and at test termination, the overlying water in all test chambers appeared slightly cloudy and colorless.

Dilution Water
The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water.

The well water was passed through a sand filter to remove particles greater than approximately 25 um, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water again was filtered (0.45 pm) to remove microorganisms and particles and
passed through an ultraviolet sterilizer.

Test Apparatus
Test chambers were one-quart glass mason jars. Each test chamber contained approximately 2.0 cm of sediment (2.0 cm in a representative test chamber) and approximately 600 mL of overlying water (8.4 cm in a representative test chamber). The ratio of the depth of the sediment layer to the depth of the overlying water was maintained at approximately 1:4. Aeration was applied to each test chamber through a glass pipette that extended to a depth not closer than 2 cm from the surface of the sediment. Air was bubbled into the test chamber at a rate greater than one bubble per second, but not so great as to disturb the sediment. The jars were arranged in a randomized block in an environmental chamber. Test chambers were labeled with the project number, test concentration and replicate.

Environmental Conditions
Lighting used to illuminate the culture and test chambers during holding and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (colortone@ 50). A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A
30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Light intensity at test initiation was 394 lux at the surface of the water over one representative test chamber.

The target test temperature during the study was 20 + 2OC. Temperature was measured in the overlying water of one alternating replicate test chamber daily during the test using a hand-held thermometer. Temperature also was measured continuously in a beaker of water adjacent to the test chambers using a Fulscope ERJC Recorder. The continuous recorder was verified with a hand-held thermometer prior to test initiation.

Dissolved oxygen measurements were made on samples of overlying water collected from one alternating replicate test chamber of each treatment and control group daily during the test. Measurements of pH were made on samples of overlying water collected from one alternating replicate test chamber of each treatment and control group at test initiation, once each week during the test, and at test termination. Hardness, alkalinity, specific conductance and ammonia were measured in a sample of overlying water from one sacrificed replicate from the control group replicates and the highest concentration treatment group (50 mg a.i./Kg) replicates at the beginning and end of the test. Additionally, ammonia was measured in a sample of overlying water from one sacrificed replicate from the control group replicates and the highest treatment group replicates weekly during the test. Due to the levels of ammonia in the overlying water, the overlying water was partially renewed on days 14 and 21. The partial renewal consisted of removing 300 mL of overlying water from each test chamber. Clean UV sterilized well water was then added to each chamber (over a baffle so as to minimize the disturbance to the sediment) until reaching the water level mark made at the beginning of the test.

Light intensity was measured using a SPER Scientific Model 840006C light meter. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter, and easurements of pH were made using a Thermo Orion Model 525Aplus pH meter. Hardness and alkalinity measurements were made by titration (7). Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Ammonia was measured using a Thermo Orion 720Aplus pH/ISE meter.

Observations
In addition to the organisms placed in the test compartments at the beginning of the test, an additional 20 organisms were impartially selected at the beginning of the test and measured for dry weight. The test compartments were observed daily to make visual assessments of abnormal behavior (e.g. leaving the sediment, climbing the walls of the test compartment). On Day 28 of the test, oligochaetes were removed from the sediment, and the numbers of live or dead worms were enumerated. Since Lumbriculus variegatus generally reproduces over a 28-day period, an increase in the number or size of worms in each test compartment is not unexpected. However, it is not possible to differentiate between the organisms placed in the compartment at the start of the test and the offspring. Therefore, survival and reproduction were considered one endpoint, i.e. the total number of organisms present at test termination.

Dry Weight Measurements
At test termination, surviving worms were rinsed of excess sediment, placed in a pre-weighed labeled aluminum pan and dried for approximately 24 hours at approximately 60°C. The worms were weighed after allowing the aluminum pans to cool to room temperature in a desiccator for approximately 30 minutes.

Statistical Analyses
The results of the test were based on the nominal sediment concentrations. Since the percent reduction in the number of organisms present at test termination in comparison to the negative control group was less than 50% in all treatment groups, the 28-day EC50 value was estimated to be greater
than the highest sediment concentration tested.

The no-observed-effect-concentration (NOEC) and lowest-observed-effect-concentration (LOEC) were determined by visual interpretation of the dose-response pattern and statistical analyses of the survival/reproduction and dry weight data. Using the statistical program TOXSTAT Version 3.5 the survival/reproduction and dry weight (growth) data were evaluated for normality (Chi-Square Test) and homogeneity of variances (Bartlett's Test). After the data was deemed normal with homogeneous variance, the survival/reproduction and dry weight data were analyzed using Bonferroni t-test to identify those treatment levels that were statistically different Q.KO.05) from the pooled control group (Finney (1971) and West and Gulley (1996)).

References:
Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London.

West, Inc. and D.D. Gulley. 1996. TOXSTAT Version 3.5. Western Ecosystems Technology, Inc. Cheyenne, Wyoming.

Reference substance (positive control):

not specified

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: survival/reproduction and dry weight data

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

50 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: survival/reproduction and dry weight data

Details on results:

All replicates appeared normal during the test, with few observations of abnormal behavior in the treatment and control groups. At test termination, there was an increase in the numbers of oligochaetes present in some test compartments from the 10 worms originally placed in each replicate, indicating that reproduction had occurred. The mean number of worms in the negative and solvent control groups at test termination was 28 and 30 worms, respectively. The mean number of worms in the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups was 30, 27, 21, 28 and 18, respectively, at test termination. The mean number of worms in the 50 mg a.i./Kg treatment group was significantly different (p < 0.05) from the pooled control group. The LOEC for survival was determined to be 50 mg a.i./Kg dry sediment. The NOEC for survival was determined to be 25 mg a.i./Kg dry sediment. The EC50 for survival was determined to be >50 mg a.i./Kg dry sediment, the highest concentration tested.

At the beginning of the test, a group of 20 organisms from the culture was impartially selected and used for the determination of dry weight. It was determined that the average individual dry weight of those twenty organisms was 0.41 mg. The average dry weight per worm in the negative and solvent control groups was 0.921 and 0.872 mg, respectively. The average dry weight per worm in the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups was 0.862, 1.022, 0.918, 0.899 and 0.951 mg, respectively. The dry weights of the treatment groups were not significantly different (p > 0.05) from the pooled control weights, and were not concentration dependent.

Results with reference substance (positive control):

No data.

Reported statistics and error estimates:

The mean number of worms in the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups was 30, 27, 21, 28 and 18, respectively, at test termination. The mean number of worms in the 50 mg a.i./Kg treatment group was significantly different (p < 0.05) from the pooled control group. The LOEC for survival was determined to be 50 mg a.i./Kg dry sediment. The NOEC for survival was determined to be 25 mg a.i./Kg dry sediment. The EC50 for survival was determined to be >50 mg a.i./Kg dry sediment, the highest concentration tested.

The average dry weight per worm in the negative and solvent control groups was 0.921 and 0.872 mg, respectively. The average dry weight per worm in the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups was 0.862, 1.022, 0.918, 0.899 and 0.951 mg, respectively. The dry weights of the treatment groups were not significantly different (p > 0.05) from the pooled control weights, and were not concentration dependent.

Measured concentrations of 2-Methyl-4-isothiazolin-3-one in the sediment samples collected on Day 0 from the 3.1, 6.3, 13, 25

and 50 mg a.i./Kg treatment groups yielded <LOQ, <LOQ, <LOQ, 37.1 and 43.4% of nominal, respectively. Measured concentrations of 2-Methyl-4-isothiazolin-3-one in the sediment samples collected on Days 7 and 28 from the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups were all <LOQ (Table 1). Measured concentrations of the test substance in negative and solvent control sediment samples collected on Days 0, 7 and 28 were below the limit of quantitation (4.00 mg a.i./Kg dry sediment).

Measured concentrations of 2-Methyl-4-isothiazolin-3-one in the overlying water samples collected on Day 0 from the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups were <LOQ, <LOQ, 0.177, 0.916 and 2.83 mg a.i./L, respectively (Table 1). Measured concentrations of 2-Methyl-4-isothiazolin-3-one in the overlying water samples collected on Days 7 and 28 from the 3.1, 6.3, 13 and 25 mg a.i./Kg treatment groups were all <LOQ. The measured concentration in the 50 mg a.i./Kg treatment group on Day 7 was 0.200 mg a.i./L

and on Day 28 was <LOQ. Measured concentrations of the test substance in negative and solvent control overlying water samples collected on Days 0, 7 and 28 were below the limit of quantitation (<0.100 mg a.i./L).

Measured concentrations of 2-Methyl-4-isothiazolin-3-one in the pore water samples collected on Day 0 from the 3.1, 6.3, 13, 25

and 50 mg a.i./Kg treatment groups were <LOQ, <LOQ, 3.36, 19.8 and 53.7 mg a.i./L, respectively (Table 1). Measured concentrations of 2-Methyl-4-isothiazolin-3-one in the pore water samples collected on Days 7 and 28 from the 3.1, 6.3, 13 and 25 mg a.i./Kg treatment groups were all <LOQ. The measured concentration in the 50 mg a.i./Kg treatment group on Day 7 was 1.75 mg a.i./L and on Day 28 was <LOQ. Measured concentrations of the test substance in negative and solvent control pore water samples collected on Days 0, 7 and 28 were below the limit of quantitation (<0.100 mg a.i./L).

Mass Balance

The mass balance data indicate that 2-Methyl-4-isothiazolin-3-one disappeared quickly from the test system during the test (Table 1). A mass balance for 2-Methyl-4-isothiazolin-3-one was calculated on Days 0, 7 and 28 of the study in overlying water, pore water and sediment. Overlying water and pore water volumes were measured for each sample as well as the mass of dry sediment added to each test replicate.

On Day 0, the amount of 2-Methyl-4-isothiazolin-3-one in the nominal 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups was 0, 0, 9, 63 and 80% of nominal, respectively. After 7 days, the amount of 2-Methyl-4-isothiazolin-3-one in the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment group was 0, 0, 0, 0 and 1.9% of nominal. After 28 days, no test material was recovered in any treatment group in the sediment, overlying water or pore water (Table 1).

The percent of 2-Methyl-4-isothiazolin-3-one in the sediment samples collected on Day 0 from the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups yielded 0, 0, 0, 37 and 43% of the total test substance applied, respectively. The percent of 2-Methyl-4-isothiazolin-3-one in the sediment samples collected on Days 7 and 28 from the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups were all 0%. No test substance was detected in the negative and solvent control sediment samples collected on Days 0, 7 and 28.

The percent of 2-Methyl-4-isothiazolin-3-one in the overlying water samples collected on Day 0 from the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups were 0, 0, 5, 14 and 22% of the total test substance applied, respectively. The percent of 2-Methyl-4-isothiazolin-3-one in the overlying water samples collected on Days 7 and 28 from the 3.1, 6.3, 13 and 25 mg a.i./Kg treatment groups were all 0%. The percent of test material in the 50 mg a.i./Kg treatment group on Day 7 was 1.5% of the total test substance applied and on Day 28 was 0%. No test substance was detected in the negative and solvent control overlying water samples collected on Days 0,7 and 28.

The percent of 2-Methyl-4-isothiazolin-3-one in the pore water samples collected on Day 0 from the 3.1, 6.3, 13, 25 and 50 mg a.i./Kg treatment groups were 0, 0,4, 12 and 15% ofthe total test substance applied, respectively. The percent of 2-Methyl-4-isothiazolin-3-one in the pore water samples collected on Days 7 and 28 from the 3.1, 6.3, 13 and 25 mg a.i./Kg treatment groups were all 0%. The measured concentration in the 50 mg a.i./Kg treatment group on Day 7 was 0.43% of the total test substance applied and on Day 28 was 0%. No test substance was detected in the negative and solvent control pore water samples collected on Days 0,7 and 28.

On Day 0, the mean percent of 2-Methyl-4-isothiazolin-3-one in the sediment, overlying water, and pore water in all test systems was 16, 8.2 and 6% of the total test substance applied, respectively. On Day 7, the mean percent of 2-Methyl-4-isothiazolin-3-one in the sediment, overlying water, and pore water in all test systems was 0, 0.3 and 0.1% of the total test substance applied, respectively. On Day 28, the mean percent of 2-Methyl-4-isothiazolin-3-one in the sediment, overlying water, and pore water in all test systems was 0, 0 and 0% of the total test substance applied, respectively.

Observations and Measurements

Temperatures were within the 20 + 2°C range established for the test. Dissolved oxygen concentrations were > 67% (6.0 mgL) of saturation throughout the test. Measurements of pH ranged from 8.2 to 8.8 during the test. Due to the increasing levels of ammonia in

the test system throughout the test, the overlying water was partially renewed on Days 14 and 21 to prevent toxicity caused by the ammonia concentrations.

Table 1 Mass Balance Approximations For Days 0, 7 and 28

Study Day	Nominal Test Conc.(mg a.i./kg)	Nominal 2 -Methyl-4 -Isothiazolin-3 -One in Test System (mg)	Mass of Sediment in Test System (kg)	Volume of Overlying Water in Test System (L)	Pore Water Volume (L)	2 -Methyl-4 -Isothiazolin-3 -One in Sediment1 (mg)	2 -Methyl-4 -Isothiazolin-3 -One in Overlying Water2 (mg)	2 -Methyl-4 -Isothiazolin-3 -One in Pore Water 3 (mg)	Total-2-Methyl-4 -Isothiazolin-3 -One in Test System (mg)	Percent of Nominal 2 -Methyl-4 -Isothiazolin-3 -One in Test System
Day 0	3.1	0.468	0.151	0.6	0.0235	0.000	0.000	0.000	0.000	0
	6.3	1.008	0.160	0.6	0.0225	0.000	0.000	0.000	0.000	0
	13	2.093	0.161	0.6	0.0230	0.000	0.106	0.077	0.183	9
	25	3.925	0.157	0.6	0.0230	1.455	0.550	0.455	2.460	63
	50	7.700	0.154	0.6	0.0210	3.342	1.698	1.128	6.168	80
Day 7	3.1	0.477	0.154	0.6	0.0258	0.000	0.000	0.000	0.000	0
	6.3	0.989	0.157	0.6	0.0256	0.000	0.000	0.000	0.000	0
	13	2.028	0.156	0.6	0.0214	0.000	0.000	0.000	0.000	0
	25	3.975	0.159	0.6	0.0210	0.000	0.000	0.000	0.000	0
	50	8.000	0.160	0.6	0.0198	0.000	0.120	0.035	0.155	1.9
Day 28	3.1	0.465	0.150	0.6	0.0195	0.000	0.000	0.000	0.000	0
	6.3	1.033	0.164	0.6	0.0220	0.000	0.000	0.000	0.000	0
	13	1.963	0.151	0.6	0.0220	0.000	0.000	0.000	0.000	0
	25	3.750	0.150	0.6	0.0195	0.000	0.000	0.000	0.000	0
	50	7.550	0.151	0.6	0.0215	0.000	0.000	0.000	0.000	0

¹2 -Methyl-4 -Isothiazolin-3 -One in Sediment = concentration of 2 -Methyl-4 -Isothiazolin-3 -One in sediment multiplied by the weight of sediment in the test system.

² 2 -Methyl-4 -Isothiazolin-3 -One in overlying water = concentration of 2 -Methyl-4 -Isothiazolin-3 -One in overlying water multiplied by the volume of water in the test system (0.6 L).

³2 -Methyl-4 -Isothiazolin-3 -One in pore water = concentration of 2 -Methyl-4 -Isothiazolin-3 -One in pore water multiplied by the volume of pore water in the test system.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value after 28-Days was >50 mg a.i./Kg, the highest concentration tested. Based on survival, the no-observed-effect concentration (NOEC) was 25 mg a.i./Kg, and the lowest observed effect concentration (LOEC) was 50 mg a.i./Kg.

Executive summary:

The objective of this study was to determine the effects of sediment-incorporated 2-Methyl-4- isothiazolin-3-one (51.4% a.i., Lot No. 8001J123, TD No. 01-119) to the oligochaete, Lumbriculus variegatus, during a 28-day exposure period under static conditions. The study was conducted according to procedures outlined in the protocol, "2-Methyl-4-isothiazolin-3-one: A Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment", Wildlife International, Ltd. Protocol Number 129/011007LUM-SED 28b/SUB129; Rohm and Haas Company Protocol Number 06P-227.

Oligochaetes, Lumbriculus variegatus, were exposed to a geometric series of five test concentrations, a negative control (untreated sediment) and a solvent control (sediment spiked with acetone) under static conditions for 28 days. Six replicates were tested for each control and four replicates were tested for each treatment group. Ten oligochaetes were placed in each replicate at the beginning of the test. Nominal test concentrations used in the study were 3.1, 6.3, 13, 25 and 50 mg active ingredient (a.i.)/Kg. The overlying water appeared clear and colorless in all test chambers at test initiation and slightly cloudy and colorless at test termination. The concentration of 2-Methyl-4-isothiazolin-3-one was measured in overlying water, pore water and sediment samples collected at test initiation, on Day 7 and at test termination.

All water quality parameters were within acceptable limits during the test. Temperature measurements ranged between 19.8 and 20.8°C throughout the 28-day exposure period. Dissolved oxygen concentrations remained ≥6.0 mg/L (67% of saturation). Measurements of pH ranged from 8.2 to 8.8.

Aside from a few observations of organisms on the surface of the sediment or climbing the walls of the test chambers in all treatment groups and controls, the organisms generally appeared normal and healthy throughout the study. The EC₅₀ value after 28-Days was >50 mg a.i./Kg, the highest concentration tested. Based on survival, the no-observed-effect concentration (NOEC) was 25 mg a.i./Kg, and the lowest observed effect concentration (LOEC) was 50 mg a.i./Kg.

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January - 24 February 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

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):
No data.

Analytical monitoring:

yes

Details on sampling:

The concentration of 14C 2-methyl-4-isothiazolin-3-one equivalents was measured in the overlying water, pore water, and sediment on days 0 (initiation), 14, and 28 (termination). The concentration of the 2-methyl-4-isothiazolin-3-one in the overlying solution of the control and the highest treatment level was also analyzed by high performance liquid chromatography (HPLC) on days 0 (initiation), 14 and 28 (termination. For each of these sampling events, the contents of two replicate test chambers were sampled. A 10 ml volume of overlying water was collected for each treatment replicate and added directly into a 20 ml scintillation vial for liquid scintillation counting analysis (LSC) of the total radioactive residue (TRR). Another 10 ml volume of overlying solution was collected from each treatment replicate and added to a 15 ml culture tube for high performance liquid chromatography (HPLC) analysis of the parent compound. The remaining overlying solutions were poured into amber bottles and stored under refrigeration if needed for future analysis.

After the decanting of the overlying solutions, the sediment from each replicate was added to individual 250 ml Nalgene centrifuge bottles. The sediment was centrifuged at 4000 rpm for 20 minutes at 20C. The pore water (i.e., interstitial water) was separated from the sediments and added to marked centrifuge tubes. The pore water was then centrifuged at 19,000 rpm for 20 minutes at 20C. A 10 ml volume of each pore water sample was collected. The remaining sediments were composited stored until sub-sampled. Triplicate sub-samples of approximately 240 to 270 mg of each sediment sample were analyzed for total radioactive residue of 14C 2-methyl-4-isothiazolin-3-one equivalents.

Vehicle:

yes

Details on sediment and application:

The formulated sediment used for the definitive test was prepared on 20 October 2004 by mixing the following ratio of constituents: 76% fine industrial sand, 20% kaolinite clay, and 4% sphagnum peat. The sediment constitutes were mixed based on dry weight equivalents. The peat moss was sieved to a finely ground consistency and did not contain any visible plant remains. Calcium carbonate, CaCO3, was added to the artificial sediment to adjust the pH to 7.0 +/-0.5 units.

Test organisms (species):

Chironomus riparius

Details on test organisms:

Cultures of Chironomus riparius at ABC Laboratories were maintained in laboratory freshwater under environmental conditions similar to that maintained during the definitive test. First instar larvae were used to initiate the test. The larvae were 1 to 4 days old at the time of test initiation. Measurements of the head capsule widths of a representative sample of larvae collected at test initiation and preserved in alcohol were used to confirm that the larvae were in their first instar of development. A calibrated dissecting microscope was used for this procedure.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

No data.

Hardness:

The total hardness ranged from 198 to 254 mg CaCO3/l, respectively.

Test temperature:

The temperatures recorded within the test chambers ranged from 18.8 to 19.5C. The electronically recorded water temperatures ranged from 18.8 to 19.9C throughout the test with a periodic average temperature of 19.3 +/-0.2C.

pH:

The pH of the overlying water ranged from 7.8 to 8.6.

Dissolved oxygen:

The dissolved oxygen values ranged from 2.6 to 8.8 mg/l (29 to 99% saturation). The 2.6 mg/L value was recorded at test termination and was not considered to be indicative of the dissolved oxygen experienced by the test organisms during the exposure.

Salinity:

No data

Ammonia:

At test initiaion and termination the un-ionized ammonia concentrations in the overlying water ranged from 1.1 to 7.7 and 167 to 773 ug/l, respectively.

Nominal and measured concentrations:

Nominal concentrations were 0 (control), 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediment treatments

Details on test conditions:

Two range-finding tests were performed from August 20 to October 9, 2004 at nominal concentrations of 0 (control), 0.10, 1.0, 10, 100 and 1000 mg 2-methyl-4-isothiazolin-3-one/kg dry sediment. Due to poor overall and/or questionable survival of the control and treatments from these first two tests, a third range-finding test was performed from November 12-26, 2004. The nominal sediment concentrations were 0 (control), 10, 50, 100, 500 and 1000 mg a.i./kg dry sediment. Duplicate test chambers containing 20 larvae were set up for each control and 2-methyl-4-isothiazolin-3-one treatment. After 14 days of exposure, mortality was 0, 0, 0, 5, 100, and 100% for the control, 10, 50, 100, 500 and 1000 mg a.i./kg dry sediment treatments. These results were used to establish the definitive test concentrations.

The definitive test was performed at nominal sediment concentrations of 0 (control), 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediment. A primary application stock solution was prepared by mixing 1.7895 g of 2-methyl-4-isothiazolin-3-one (i.e., 917.15 mg active ingredient) with 4.42 mg of the radioabeled substance and brought to a volume of 1000 ml with ABC reagent water. The nominal concentration of this solution was 922 mg 2-methyl-4-isothiazolin-3-one/l. A 500 ml volume of this primary solution was mixed with an additional 2.6801 g 2-methyl-4-isothiazolin-3-one (i.e., 1374 mg active ingredient) then diluted to 1000 ml with ABC reagent water, which resulted in a working standard of 1830 mg a.i./l (i.e., 1.83 mg a.i./ml). Stock solution aliquots were measured with Class A glassware.

To prepare the dosed sediments, 8.75, 17.5, 35.0, 70.0, 140, and 280 ml volumes of the working standard solution was brought to a 560 ml volume with deionized water and added to 1615 g of air-dried sediment (oven dry equivalent of 1.6 kg) in a stainless steel pan and mixed to a uniform consistency. These procedures produced a series of dosed sediments with nominal 2-methyl-4-isothiazolin-3-one concentrations of 10, 20, 40, 80, 160 and 320 mg a.i./kg dry sediment, which were hydrated to approximately 35% of the sediment dry weight. The control sediment consisted of 1615 g of air-dried sediment (oven dry equivalent of 1.6 kg), which was hydrated with 560 ml of deionized water resulting in a percent moisture content of approximately 35% of the sediment dry weight.

The test chambers were 1-L glass jars that were approximately 17 x 9.5 cm. Approximately 200 g (approximately 2 cm sediment depth) of formulated sediment was added to each replicate test chamber. A 600 ml volume of dilution water or prepared test solution was carefully added to the test chambers. A plastic deflector was used to minimize the disturbance to the sediments. Four replicate test chambers were prepared for the biological parameters. A total of six additional replicate chambers were prepared for the various analyses of the overlying water, pore water, and sediment samples. The biological replicates were covered with an emergence trap that consisted of a polyethylene jar with a screen mesh lid. The analytical replicates were covered with a perforated plastic lid. The test chambers were prepared two days prior to test initiation (i.e., addition of the test organisms).

The test chambers were placed in a temperature-controlled water bath in a randomized block arrangement. Temperature was set at a target range of 20 +/-2C. A 16-hour light:8-hour dark photoperiod with 30-min dawn and dusk periods was maintained. Aeration was provided at an intial rate of 60-100 bubbles per minute to each test chamber through a glass pipette. The pipette was inserted such that its tip was 2-3 cm from the sediment surface. Aeration was discontinued during the addition of the larvae and was resumed approximately 24 hours later. During the course of the test, the aeration was adjusted as deemed necessary in an attempt to maintain the dissolved oxygen concentrations within each test chamber. After the test chambers were set-up, they were inoculated daily for two days prior to test initiation with 2.0 ml of a concentrated green algae (Pseudokirchneiiella subcapitata (formerly known as Selenastrum capriocornutum) solution, in order to provide an initial food source for the larvae.

Measurements of temperature, dissolved oxygen concentration, and pH of the overlying water were measured at test initiation and weekly in each replicate test chamber. The test solution dissolved oxygen concentrations and temperatures were measured with a WTW OXi 330 dissolved oxygen meter. Test solution pH wa measured with a WTW 330i pH meter. Daily water bath temperature was continuously recorded with an electronic data logger. The thermistor of the data logger was located within a centrally located surrogate test chamber containing water and sediment. On days 0 and 28, composite samples were taken from each chamber for measurement of total hardness and ammonia concentrations. Total hardness was measured using a colorimetric titration procedure adapted from Standard Methods (3). Pore ammonia samples were measured using an Accumet Model 25 pH meter equipped with an ammonium specific probe. Light intensity was measured with a LI-CO model LI-189 light meter with a photometric sensor.

At study initiation, a total of 20 chironomids (Lot number CR0105) were added to each vial in a set of labeled glass vials. Each glass vial was randomly assigned to a treatment replicate by a random number generator program. The individuals within a vial were released into each biological replicate and the day 14 and 28 analytical replicates (replicates G-J). Observations of the biological replicates were recorded daily. Any abnormal activity (i.e., sediment avoidance, inactivity, etc.) was noted if observed. During the exposure, the larvae were fed from 2 to 5 ml of a 2 g/l flake food suspension on a daily basis.

Daily emergence observations (i.e., adult flies retained within the midge traps) were recorded from the biological replicates (i.e., replicates A-D) starting with the recording of the first emergent adult chironomid (i.e., test day 17) and ending at test termination. Evidence of emergence was noted by the presence of exuviae as well as adults. Where possible, the adult flies observed in the midge traps were identified and enumerated by gender and also for total emergence. if an exuviae was present on the water's surface but ther was no adult fly present (i.e., escaped) or if there was a greater number of exuviae present than was accounted for by the number of emergent adults, then these missing adults were recorded to be of an unknown gender. At test termination, the sediments were sieved and surviving animals were retained by the mesh and were recorded. These animals were included with the total number of emergent adults to determine the 28-day survival values for each treatment level.

Reference substance (positive control):

not specified

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

27.5 other: mg/L pore water

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

12.4 other: mg/l pore water

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Details on results:

After 28 days of exposure, the mean adult emergence was 90, 96, 89, 98, 75, 1, and 0% in the control, 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg 14-2-methyl-4-isothiazolin-3-one equivalents/kg of dry sediment treatments, respectively. The control emergence occurred between 12 and 23 days after the addition of the animals and the percent emergence of 90% met the acceptability criterion for these parameters set by the protocol and the test guideline. A total of 21 adult midge were recovered from the D replicate of the control and 7.28 mg/kg treatment. It was apparent that these addional animals were inadvertently added into each of the replicate chambers on day 0 and therefore it was assumed that 100% of the animals within each of these replicate test chambers emerged as adults. The male to female gender ratios were 0.50, 0.47, 1.1, 1.1, 1.1, 0, and 0 males to females for the control, 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg 14C equivalents/kg dry sediment treatments, respectively. Total percent emergence rates of 90 and 96% for the control and 7.28 mg 14C equivalents/kg dry sediment treatment suggest that there is no evidence of preferential mortality of males within the control and 7.28 mg 14C equivalents/kg dry sediment treatment, therefore the variance in the gender ratios should not be considered as a concentration dependent affect of the test substance. Because it is not possible to evaluate larval phenotypic gender, it is subsequently not possible to discern whether emerged organism gender ratios are simply a function of random selection (most likely) or other mechanisms. The Study Director is not aware of data indicative of chemically induced alterations in gender ratios in exposed chironomids. Taken together with the lack of a dose response for this parameter, it is improbable that the variation in the observed gender ratios evident in the study is a function of exposure to 2-methyl-4-isothiazolin-3-one. Thefore statistical analysis of the emergence rates was based upon total adult emergence.

The mean development rates were 0.0517, 0.0512, 0.0524, 0.0447, 0.0418, 0.0091, and 0 for the control, 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg 14C-2-methyl-4-isothiazolone-3-one equivalents/kg of dry sediment treatments, respectively. The development rates for the emergent males ranged from 0.0364 to 0.0565 for the controls and all treatment levels except for the 121 mg 14C equivalents/kg dry sediment treatment, which did not have any emergent males. The female development rates ranged from 0.0380 to 0.0545 for the controls and all treatment levels except for the 76.0 and 121 mg 14C equivalents/kg dry sediment treatments, which did not have any emergent females. The exposure to 2-methyl-4-isothiazoline-3-one did not differentially inhibit the development of the midge based upon specific gender or based upon total development values. The total adult development rate data were analyzed to determine statistical differences between the control and the treatment data. After 28-days of exposure, larvae and pupae were recovered in the 42.9, 76.0, and 121 mg/kg treatment levels at test termination and these animals were included in the survival values. The 28-day survival rates were 90, 96, 89, 98, 81, 61, and 46% in the control, 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg of dry sediment treatments, respectively. The surviving larvae exposed to the 121 mg/kg of dry sediment treatment were noticeably smaller than what would be expected by this time point (i.e., 4th instar).

Based on mean pore water concentrations of 2-methyl-4-isothiazolin-3-one, the 28-day NOEC and LOEC values for adult emergence exposure were 12.4 and 27.5 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l, respectively. The calculated MATC value for adult emergence was 18.5 14C-2-methyl-4-isothiazolin-3-one equivalents/l. The 28-day EC50 for percent adult emergence was 35.0 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l with 95% confidence interval of 31.9 to 38.9 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l. The NOEC and LOEC values for the development rates after 28 days of exposure were 4.90 and 12.4 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l, respectively. The calculated MATC value for development rate was 7.79 14C-2-methyl-4-isothiazolin-3-one equivalents/l. The 28-day development rate EC50 was 41.7 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l (95% confidence interval: 29.1 to 54.4 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l). The NOEC and LOEC values for the percent survival after 28 days of exposure were 27.5 and 64.9 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l, respectively. The calculated MATC value for survival was 42.2 14C-2-methyl-4-isothiazolin-3-one equivalents/l. The 28-day LC50 was 132 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l (95% confidence interval: 80.6 to 216 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l).

Based on mean sediment concentrations of 2-methyl-4-isothiazolin-3-one, the 28-day NOEC and LOEC values for adult percent emergence were 23.6 and 42.9 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment, respectively. The calculated MATC value for adult emergence was 31.8 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment. The 28-day EC50 for percent adult emergence was 50.3 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment with 95% confidence interval of 47.2 to 54.0 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment. The NOEC and LOEC values for the development rates after 28 days of exposure were 13.0 and 23.6 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment, respectively. The calculated MATC value for development rate was 17.5 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment. The 28-day development rate EC50 was 56.7 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment (95% confidence interval: 45.2 to 68.2 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment). The NOEC and LOEC values for the percent survival after 28 days of exposure were 42.9 and 76.0 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment, respectively. The calculated MATC value for survival was 57.1 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment. The 28-day LC50 was 113 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment (95% confidence interval: 79.8 to 323 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment).

Results with reference substance (positive control):

No data.

Reported statistics and error estimates:

See results.

Radioactive Residue Analysis

Overlying Water

The day 0 overlying water ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalent concentrations for the control, 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediment treatments were <MQL (0.0267 mg/L), 0.615, 1.42, 2.95, 6.27, 13.2, and 26.6 mg 14C-2 -methyl-4 -isothiazolin-3 -one equivalents/l, respectively. the day 14 overlying water ¹⁴C-2 -methyl-4 -isothiazolin-3 -one concentrations for the control, 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediment treatments were <MQL(0.0271 mg/L), 0.327, 0.828, 2.27, 7.17, 21.6, and 50,2 mg 14C-2 -methyl-4 -isothiazolin-3 -one equivalents/l, respectively. The day 28 overlying water ¹⁴C-2 -methyl-4 -isothiazoline-e-one concentrations for the control, 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediment treatments were <MQL (0.0253 mg/l), 0.263, 0.620, 1.52, 4.14, 16.7, and 45.8 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/l, respectively. The control samples were used to determine the background cpm values for this matrix, therefore the results for the controls are reported as less than the MQL for these analyses. The control cpm values ranged from 36.2 to 47.8, which are typical of background levels at ABC Laboratories.

Pore Water

The day 0 pore water ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalent concentrations for the control, 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediment treatments were <MQL (0.0284 mg/l), 6.31, 13.2, 32.1, 68.4, 151, and 296 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/l, respectively. The day 14 pore water ¹⁴C-2 -methyl-4 -isothiazolin-3 -one concentrations for the control, 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediment treatments were <MQL (0.0284 mg/l), 0.349, 0.933, 3.52, 10.4, 29.8, and 63.3 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/l, respectively. The day 28 pore water ¹⁴C-2 -methyl-4 -isothiazolin-3 -one concentrations for the control, 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediement treatments were <MQL (0.0279 mg/l), 0.235, 0.556, 1.43, 3.57, 13.8, and 43.7 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/l, respectively. The mean measured pore water concentrations were <MQL (control), 2.30, 4.90, 12.4, 27.5, 64.9, and 134 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/l. The control samples were used to determine the background cpm values for this matrix, therefore the results for the controls are reported as less than the MQL for these analyses. The control cpm values ranged from 44.2 to 50.8, which are typical of background levels at ABC Laboratories.

Sediment

The mean sediment concentrations from the day 0 samples were <MQL (0.867 mg/kg) (control, 6.95, 13.7, 24.5, 46.9, 92.2, and 175 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents per kiligram of dry sediment, which represent a range of recoveries of 55 to 70 percent of the nominal concentrations. Day 14 mean measured concentrations in sediment were <MQL (<0.911 mg/kg) (control), 6.99, 13.2, 23.1, 39.1, 53.4, 85.5 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/kg dry sediment, which represent a range of recoveries of 27 to 70 percent of the nominal concentrations. Day 28 mean measured concentrations in sediment were <MQL (0.818 mg/kg) (control), 7.90, 12.2, 23.4, 42.7, 82.5, and 102 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/kg dry sediment, which represents a range of recoveries of 32 to 79 percent of the nominal concentrations. The overall mean measured sediment concentrations based on ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents were <MQL (control), 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/kg of dry sediment. The control samples were used to determine the background dpm values for this matrix, therefore the results for the controls are reported as less than the MQL for this analysis. The control cpm values ranged from 45.2 to 73.8, which are typical of background levels at ABC Laboratories.

HPLC Analysis - Overlying Water

The day 0, 14, and 28 mean overlying solution concentrations of 2 -methyl-4 -isothiazolin-3 -one were 26.5, 38.3, and 29.7 mg a.i./l. There were no residues identified in the control samples that were greater than the MQL, which was 0.220 mg a.i./l. The mean overlying solution concentration of 2 -methyl-4 -isothiazolin-3 -one was 31.5 mg a.i./l. The recoveries of the QC fortifications ranged from 86 to 99%.

Validity criteria fulfilled:

yes

Conclusions:

Based on mean pore water concentrations of 2-methyl-4-isothiazolin-3-one, the 28-day NOEC and LOEC values for adult emergence exposure were 12.4 and 27.5 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l, respectively.

Executive summary:

A 28 -day toxicity test was performed to estimate the chronic toxicity of 2 -methyl-4 -isothiazolin-3 -one (supplied as a 50% solution in water known as Kordek 573F Industrial Microbiocide) to larvae of the midge, Chironomus riparius. The protocol was designed to comply with OECD guideline 218.

The 28 -day definitive test was performed at nominal sediment concentrations of 0 (control), 10, 20, 40, 80, 160, and 320 mg a.i./kg dry sediment. The test chambers were 1l glass jars each containing approximately 200 g (2 cm sediment depth) of prepared sediment and 600 ml of overlying water (8cm depth). Four replicate test chambers were prepared for the biological parameters. A total of six replicate chambers were prepared for the various analyses of the overlying water, pore water, and sediment samples. Aeration was provided to each test chamber. At test initiation, a total of 20 first instar chironomid larvae were added to each replicate.

Adult emergence was recorded daily, beginning with the first day of emergence (test day 17). Surviving larvae or pupae recovered at test termination (if applicable) were included with the emergent adults for the calculation of the 28 -day percent survival. Total emergence, development rate, and overall survival data were statistically analyzed to determine the median effect or lethal (i.e., EC₅₀ or LC₅₀) concentrations, no-observed-effect concentration (NOEC), and lowest-observed-effect concentration (LOEC) for these parameters. The maximum acceptable toxicant concentrations (MATC) were calculated as the geometric mean of the NOEC and LOEC values for each biological parameter as appropriate.

The concentrations of total activity of the ¹⁴C 2 -methyl-4 -isothiazolin-3 -one equivalents (TRR) in the overlying and pore water as well as the treated sediments were determined within samples collected from the control and all treatment levels on test day 0 (initiation), 14, and 28 (termination). The mean overlying test solution concentrations were <0.0271 (control), 0.402, 0.956, 2.25, 5.86, 17.2 and 40.9 mg ¹⁴C 2 -methyl-4 -isothiazolin-3 -one equivalents/l. The mean pore water concentrations were <0.0284 (control), 2.30, 4.90, 12.4, 27.5, 64.9 and 134 mg ¹⁴C 2 -methyl-4 -isothiazolin-3 -one equivalents/l.

The day 0 sediment concentrations ranged from 6.43 to 177 mg ¹⁴C 2 -methyl-4 -isothiazolin-3 -one equivalents/kg dry sediment. The percent recoveries at test initiation ranged from 54 to 75% of the nominal sediment concentration. The low recoveries of the water soluble test substance within the sediment can be attributed to the partitioning of the test substance into the pore water and overlying solutions. The mean sediment concentrations were <0.911 (control), 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg ¹⁴C 2 -methyl-4 -isothiazolin-3 -one equivalents/kg dry sediment, which represents recoveries ranging from 38 to 73% of the nominal sediment concentrations.

High performance liquid chromatography (HPLC) with UV detection was used to measure concentrations of 2 -methyl-4 -isothiazolin-3 -one in the overlying test solutions of the control and the highest test substance treatment. The samples were collected at day 0 (initiation), 14, and 28 (termination). The concentrations of 2 -methyl-4 -isothiazolin-3 -one as determined by HPLC in the individual overlying water samples ranged from 26.4 to 38.9 mg a.i./l over the course of the study. The mean concentration of the overlying water was 31.5 mg 2 -methyl-4 -isothiazolin-3 -one/l with a range of daily mean values of 26.5 to 38.3 mg 2 -methyl-4 -isothiazolin-3 -one/l.

After 28 days of exposure, the mean adult percent emergence was 90, 96, 89, 98, 75, 1, and 0% in the control, 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/kg of dry sediment treatments, respectively. The male to female gender ratios were 0.50, 0.47, 1.1, 1.1, 1.1, 0, and 0 males to females for the control, 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg ¹⁴C equivalents/kg dry sediment treatments, respectively. Total percent emergence rates of 90 and 96% for the control and 7.28 mg ¹⁴C equivalents/kg dry sediment treatment suggest that there is no evidence of preferential mortality of males within the control and 7.28 mg ¹⁴C equivalents/kg dry sediment treatment, therefore the variance in the gender ratios should not be considered as a concentration dependent affect of the test substance. The mean development rates were 0.0517, 0.0512, 0.0524, 0.0447, 0.0418, 0.0091, and 0 for the control, 7.28, 13.0, 23.6, 41.9, 76.0, and 121 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/kg of dry sediment treatments, respectively. Larvae and pupae were recovered in the 42.9, 76.0, and 121 mg/kg treatment levels at test termination and these animals were included in the survival values. The 28 -day survival rates were 90, 96, 89, 98, 81, 61, and 46% in the control, 7.28, 13.0, 23.6, 42.9, 76.0, and 121 mg ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/kg of dry sediment treatmetns, respectively. The surviving larvae exposed to the 121 mg/kg of dry sediment treatment were noticeably smaller than what would be expected by this time point (i.e., 4th instar).

Based on mean pore water concentrations of 2-methyl-4-isothiazolin-3-one, the 28-day NOEC and LOEC values for adult emergence exposure were 12.4 and 27.5 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/l, respectively. The 28-day EC₅₀ for percent adult emergence was 35.0 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/l with 95% confidence interval of 31.9 to 38.9 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/l. The NOEC and LOEC values for the development rates after 28 days of exposure were 4.90 and 12.4 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/l, respectively. The 28-day development rate EC₅₀ was 41.7 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/l (95% confidence interval: 29.1 to 54.4 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/l). The NOEC and LOEC values for the percent survival after 28 days of exposure were 27.5 and 64.9 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/l, respectively. The 28-day LC₅₀ was 132 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/l (95% confidence interval: 80.6 to 216 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/l). The calculated MATC value for the most sensitive endpoint, developmetn rate, was 7.79 ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/l.

Based on mean sediment concentrations of 2-methyl-4-isothiazolin-3-one, the 28-day NOEC and LOEC values for adult percent emergence were 23.6 and 42.9 mg 14C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment, respectively. The 28-day EC₅₀ for percent adult emergence was 50.3 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment with 95% confidence interval of 47.2 to 54.0 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment. The NOEC and LOEC values for the development rates after 28 days of exposure were 13.0 and 23.6 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment, respectively. The 28-day development rate EC50 was 56.7 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment (95% confidence interval: 45.2 to 68.2 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment). The NOEC and LOEC values for the percent survival after 28 days of exposure were 42.9 and 76.0 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment, respectively. The 28-day LC₅₀ was 113 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment (95% confidence interval: 79.8 to 323 mg ¹⁴C-2-methyl-4-isothiazolin-3-one equivalents/kg dry sediment). The calculated MATC value for the most sensitive endpoint, development rate, was 17.5 ¹⁴C-2 -methyl-4 -isothiazolin-3 -one equivalents/kg dry sediment.

Reference 3

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 October - 6 November 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline Study

Qualifier:

according to guideline

Guideline:

other: ASTM E 1706-00 Guideline

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1735 (Whole Sediment Acute Toxicity of Invertebrates, freshwater)

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:

Samples of the stock solutions used to spike the sediment were collected and analyzed on the day of preparation. Overlying water, pore water and sediment samples were collected and analyzed from the control group and from each concentration treatment groups just after the introduction of the
organisms on Day 0, on Day 7 and at test termination on Day 28. Water samples were collected from mid-depth in the water column. After the overlying water samples were taken, the remainder of the overlying water was poured out of the test compartment. The remaining sediment was then collected, centrifuged and split into separate samples of pore water and sediment. The samples were analyzed on the day of collection.

Vehicle:

yes

Details on sediment and application:

Formulated sediment, as described in Kemble et.al. (1999) was used as the test sediment. This artificial sediment is similar to that described in OECD Guideline 218 (2004), but uses alpha-cellulose as its source of organic matter instead of peat moss. Alpha-cellulose was selected by Kemble et.al. (1999) as a more standardized source of organic matter than peat moss. The sediment was composed of approximately <1% humic acid and dolomite, 5% alpha-cellulose, 14% silt and clay (kaolin clay) and 80% industrial quartz sand. The dry constituents of the sediment were mixed in a PK Twinshell® mixer for approximately 20 minutes. The dry soil was stored under ambient conditions until used. The final pH of the sediment was 6.6. Analyses were performed to determine the concentrations of selected organic and inorganic constituents in a representative sample of sediment similar to that which was used in the study. A sample of the sediment used in the test was sent to Agvise Laboratories, Northwood, ND where the sediment characterization was conducted. The percent organic carbon of the sediment was found to be 2.1%.

References:
Kemble, N.E., F.J. Dwyer, C.G. Ingersoll, T.D. Dawson, and T.J. Norberg-King. 1999. Tolerance of Freshwater Test Organisms to Formulated Sediments for use as Control Materials in Whole Sediment Toxicity Tests. Environ. Toxicol. Chem. 18:222-230.

OECD Guideline 218. 2004. Sediment-Water Chironomid Toxicity Test Using Spiked Sediment. Organization for Economic Cooperation and Development. Adopted 13 April 2004.

Test organisms (species):

Hyalella azteca

Details on test organisms:

The amphipod, Hyalella azteca, was selected as the test species for this study. Amphipods are representative of an important group of aquatic invertebrates and were selected for use in the study based upon past history of use and ease of culturing in the laboratory. The organisms were obtained from Aquatic Research Organisms, Hampton, NH. The identity of the species was verified by the supplier.

The organisms were held in water from the same source as the water used during the test. During the holding period immediately preceding the test, water temperature ranged from 22.0 to 24.7°C, while the pH ranged from 8.0 to 8.3 and dissolved oxygen ranged from 6.6 to 8.6 mg/L.

During holding, the amphipods appeared normal. At test initiation, 7 to 14-day old amphipods were collected from the culture and impartially added one and two at a time into transfer chambers until each chamber contained 10 organisms. Each group of ten organisms was impartially assigned and transferred to a test compartment. All transfers were made below the water surface using wide-bore pipettes. All replicate test chambers were maintained with organisms during the study except the Day 0 analytical replicates, which did not contain organisms.

Amphipods were fed a mixture of yeast, cereal grass and trout chow (YCT) during the holding period and during the test. Organisms were fed 1.0 mL of YCT on days 0-11 of the test. The amount of food was reduced to 0.5 mL YCT on Day 12 through to the end of the test due to the presence of fungal growth in the test compartments.

Study type:

laboratory study

Test type:

flow-through

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

Not applicable

Hardness:

Day 0 Hardness was 124 (Negative Control) and 128 (100 mg a.i./kg) mg/L as CaCO3. Day 28 hardness was 116 (Negative Control) and 124 (100 mg a.i./kg) mg/L as CaCO3.

Test temperature:

ranged between 22.5 and 23.3ºC

pH:

pH ranged from 8.1 to 8.4

Dissolved oxygen:

≥5.8 mg/L (68% of saturation).

Salinity:

Not applicable

Ammonia:

less than 0.17 mg/L (the lowest calibration standard used) during the test

Nominal and measured concentrations:

Nominal test concentrations used in the study were 6.3, 13, 25, 50 and 100 mg active ingredient (a.i.)/Kg (dry weight).

Measured concentrations of the test material in the overlying water samples collected on Day 0 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were
Measured concentrations of the test material in the overlying water samples collected on Day 7 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were

Details on test conditions:

Preparation of Test Concentrations
The test substance was administered to the test organism in sediment. This route of administration was selected because it represents the most likely route of exposure to sediment dwelling organisms. Nominal test concentrations (6.3, 13, 25, 50 and 100 mg a.i./Kg ) were prepared on a dry weight basis, (i.e., mg test substance/Kg dry sediment). A primary stock solution was prepared by dissolving the test substance (Identification No. 7832) in acetone at a nominal concentration of 10.0 mg a.i./mL. The primary stock was mixed by inversion and appeared clear and colorless. Secondary stocks were prepared by serial dilution in acetone at nominal concentrations of 5.0, 2.5, 1.3 and 0.63 mg a.i./mL. A 15-mL volume of the appropriate stock was added to 150 grams of sediment and mixed by hand with a glass stir rod, then placed in a fume hood for an adequate amount of time for the acetone to partially dissipate. The 150 gram premixes were then added to 600 grams of untreated sediment in plastic Nalgene bottles and mixed on a rotary mixer for an adequate amount of time. Next, 750 grams of untreated sediment were added to the premixes to achieve a final weight of 1500 grams. The batch sediments were then mixed on a rotary mixer overnight. Test compartments (300-mL glass beakers with stainless steel mesh screen covered holes on opposite sides of the chamber) were prepared by adding 100-mL of the appropriate dosed sediment to the corresponding replicates.

The test compartments were then indiscriminately and slowly placed into test chambers (diluter tanks) and grouped by test concentration (one treatment group per diluter tank). The water height was maintained in the diluter tank using a standpipe so that each test compartment contained 150 – 175 mL of overlying water.

Eleven replicate test compartments were prepared for each test concentration and control. Eight replicates were used for the evaluation of survival and growth. An additional three replicates were used for analytical confirmation purposes in each treatment group and control on Days 0, 7 and 28. The sediment/water mixtures were allowed to acclimate for approximately 48 hours prior to the introduction of the test organisms. At test initiation and termination, the overlying water in all test chambers appeared clear and colorless.

Dilution Water
The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water.

The well water was passed through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water again was filtered (0.45 μm) to remove microorganisms and particles.

Test Apparatus
The test apparatus consisted of a Wildlife International, Ltd. diluter unit that is designed to hold up to 14 tanks in a temperature controlled water bath. Each diluter tank can receive renewal rates that can range from static to greater than 10 volume additions per day. Test compartments were 300 mL glass beakers with stainless steel mesh covered holes on opposite sides of the beaker. Each test compartment contained approximately 100 mL of sediment (3.3 cm in a representative test compartment) and approximately 150 - 175 mL of overlying water (6.2 cm in a representative test chamber). The water level in the beakers was maintained by the water levels in the diluter tanks. Each replicate beaker received approximately two volume additions of water per day. The dilution water was delivered directly into the test compartments, passively forcing out the water through the holes in the sides of the beakers and exchanging the water overlying the sediment. Dilution water delivery was verified prior to test initiation. Test compartments were labeled with the project number, test concentration and replicate.

Environmental Conditions
Lighting used to illuminate the culture and test chambers during holding and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone® 50). A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Light intensity at test initiation was 213 lux at the surface of the water over one representative test compartment.

The target test temperature during the study was 23 ± 1°C. Temperature was measured in the overlying water of one alternating replicate test compartments daily during the test using a hand-held thermometer. Temperature also was measured continuously in a beaker of water adjacent to the test compartments using a Fulscope ER/C Recorder. The continuous recorder was verified with a hand-held thermometer prior to test initiation.

Dissolved oxygen measurements were made on samples of overlying water collected from one alternating replicate test compartment of each treatment and control group daily during the test. Measurements of pH were made on samples of overlying water collected from one alternating replicate test compartment of each treatment and control group at test initiation, once each week during the test, and at test termination. Hardness, alkalinity, specific conductance and ammonia were measured in a composite sample of overlying water from the negative control group replicates and the highest concentration treatment group (100 mg a.i./Kg, dry weight) replicates at the beginning and end of the test.

Light intensity was measured using a SPER Scientific Model 840006C light meter. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion Model 525Aplus pH meter. Hardness and alkalinity measurements were made by titration. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Ammonia was measured using a Thermo Orion 720Aplus pH/ISE meter.

Observations
In addition to the organisms placed in the test compartments at the beginning of the test, an additional 20 organisms were impartially selected at the beginning of the test and measured for dry weight. The test compartments were observed daily to make visual assessments of abnormal behavior (e.g. leaving the sediment, climbing the walls of the test compartment). On Day 28 of the test, amphipods were removed from the sediment, and the numbers of live or dead organisms were enumerated. When the total number of individuals found in each replicate at test termination were fewer than the number initially placed in each replicate at the beginning of the test, then those missing were considered dead.

Dry Weight Measurements
At test termination, surviving organisms were rinsed of excess sediment, placed in a pre-weighed labeled crucible and dried for approximately 69.5 hours at approximately 60°C. The amphipods were weighed after allowing the crucibles to cool to room temperature in a desiccator for approximately 2 hours.

Statistical Analyses
The results of the test were based on the nominal sediment concentrations. The mortality data were analyzed using the computer program of C. E. Stephan (1978). The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation (Thompson (1947), Stephan (1977), and Finney (1971)). In this study, binomial probability was used to calculate the 28-Day LC50 value. The no-observed-effect-concentration (NOEC) and the lowest-observed-effect-concentration (LOEC) were determined by visual interpretation of the dose-response pattern and statistical analyses of the survival and dry weight data.

Using the statistical program TOXSTAT Version 3.5 (West and Gulley (1996)), the survival and dry weight (growth) data were evaluated for normality (Chi-Square Test) and homogeneity of variances (Levene’s Test). A student t-test was used to compare the negative and solvent control groups. Since there were no statistically significant differences (p>0.05) between the controls, they were pooled for comparisons with the treatment groups. After the data was deemed normal with homogeneous variance, the survival and growth data were analyzed using Bonferroni t-test to identify those treatment levels that were statistically different (p<0.05) from the pooled control group (Stephan (1977), and Finney (1971)).

References:
Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London.

Stephan, C.E. 1977. “Methods for Calculating and LC50,” Aquatic Toxicology and Hazard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84.

Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication.

Thompson, W.R. 1947. Bacteriological Reviews. Vol.II, No. 2. Pp. 115-145.

West, Inc. and D.D. Gulley. 1996. TOXSTAT Version 3.5. Western Ecosystems
Technology, Inc. Cheyenne, Wyoming.

Reference substance (positive control):

not specified

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

34.9 mg/kg sediment dw

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 25-50 mg a.i./Kg (dry sediment)

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/kg sediment dw

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

25 mg/kg sediment dw

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

13 mg/kg sediment dw

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Measurement of Test Concentrations
The measured concentrations of the test material in the 0.63, 1.3, 2.5, 5.0 and 10.0 mg a.i./mL stock solutions yielded 109, 103, 104, 102 and 99.0% of nominal values, respectively. These values indicate that the system was properly dosed.

Measured concentrations of the test material in the sediment samples collected on Day 0 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups yielded 98.0, 77.7, 79.1, 89.2 and 79.4% of nominal, respectively. Measured concentrations of the test material in the sediment samples collected on Day 7 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups yielded
Measured concentrations of the test material in the overlying water samples are based on the total amount of material applied to the test system and converted to mass based on the concentration and compartment volume. Measured concentrations of the test material in the overlying water samples collected on Day 0 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were
Measured concentrations of the test material in the pore water samples are based on the total amount of material applied to the test system and converted to mass based on the concentration and compartment volume. Measured concentrations of the test material in the pore water samples collected on Day 0 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were 20.7, 36.0, 71.3, 144 and 241 mg a.i./L, respectively. Measured concentrations of the test material in the pore water samples collected on Day 7 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were 1.97, 16.8, 37.7, 81.1 and 148 mg a.i./L, respectively. Measured concentrations of the test material in the pore water samples collected on Day 28 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were
Mass Balance
The mass balance data indicate that MIT disappeared from the test system during the test. A mass balance for the test material was calculated on Days 0, 7 and 28 of the study in overlying water, pore water and sediment (Tables 1 and 2). Pore water volumes were measured for each sample as well as the mass of dry sediment added to each test replicate. An estimate of 150 mL overlying water was used for mass balance calculations.

On Day 0, the amount of MIT in the nominal 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups was 129.1, 103.0, 100.9, 118.4 and 99.7% of nominal, respectively. After 7 days, the amount of the test material in the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment group was 2.7, 43.8, 48.7, 49.4 and 52.4% of nominal. After 28 days, no test material was recovered in any treatment group in the sediment or overlying water. No test material was recovered in the pore water after 28 days in any of the treatment groups except the 100 mg a.i./Kg treatment group, where 0.5% of the total test material applied was recovered.

The percent of test material in the sediment samples collected on Day 0 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups yielded 98, 78, 79, 89 and 79% of the total test substance applied, respectively. The percent of test material in the sediment samples collected on Day 7 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups yielded 0, 33, 37, 35 and 42% of the total test substance applied, respectively. The percent of test material in the sediment samples collected on Day 28 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were all 0% (Table 6). No test substance was detected in the negative and solvent control sediment samples collected on Days 0, 7 and 28.

The percent of test material in the overlying water samples collected on Day 0 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were 0, 0, 0, 0.4 and 0.8% of the total test substance applied, respectively. The percent of test material in the overlying water samples collected on Day 7 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were 0, 0, 0, 0 and 0.2% of the total test substance applied, respectively (Table 6). The percent of test material in the overlying water samples collected on Day 28 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were all 0% (Table 6). No test substance was detected in the negative and solvent control overlying water samples collected on Days 0, 7 and 28.

The percent of test material in the pore water samples collected on Day 0 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were 31, 25, 22, 29 and 20% of the total test substance applied, respectively. The percent of test material in the pore water samples collected on Day 7 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were 3, 11, 12, 14 and 10% of the total test substance applied, respectively. The percent of test material in the pore water samples collected on Day 28 from the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry weight) treatment groups were 0, 0, 0, 0 and 0.5% (Table 6). No test substance was detected in the negative and solvent control pore water samples collected on Days 0, 7 and 28.

On Day 0, the mean percent of MIT in the sediment, overlying water, and pore water in all test systems was 85, 0.2 and 25% of the total test substance applied, respectively. On Day 7, the mean percent of the test material in the sediment, overlying water, and pore water in all test systems was 29, 0 and 10% of the total test substance applied, respectively. On Day 28, the mean percent of test material in the sediment, overlying water, and pore water in all test systems was 0, 0 and 0.1% of the total test substance applied, respectively.

Observations and Measurements
Temperatures measured in the test compartments were within the 23 ± 1°C range established for the test. Dissolved oxygen concentrations were ≥ 68% (5.8 mg/L) of saturation throughout the test. Measurements of pH ranged from 8.1 to 8.4 during the test. Measurements of ammonia in the overlying water were <0.17 mg/L at the beginning and end of the test. Hardness, alkalinity and conductivity are typical for Wildlife International, Ltd. well water.

Most organisms appeared normal during the test, with few observations of abnormal behavior in the treatment and control groups. Percent mortality at test termination in the 6.3, 13, 25, 50 and 100 mg a.i./L treatment groups was 10, 12.5, 32.5, 68.8 and 56.2%, respectively. The NOEC for survival was 13 mg a.i./Kg dry sediment. The LOEC for survival was 25 mg a.i./Kg dry sediment. The 28-Day LC50 was determined to be 34.9 mg a.i./Kg (dry sediment) with 95% confidence limits of 25 and 50 mg a.i./Kg (dry weight).

At the beginning of the test, a group of 20 organisms from the culture was impartially selected and used for the determination of dry weight. It was determined that the average individual dry weight of those twenty organisms was 0.070 mg. The average dry weight per amphipod in the negative and solvent control groups was 0.40 and 0.44 mg, respectively. The average dry weight per amphipod in the 6.3, 13, 25, 50 and 100 mg a.i./Kg (dry sediment) treatment groups was 0.32, 0.32, 0.27, 0.33 and 0.35 mg, respectively. The NOEC for growth was 100 mg a.i./Kg dry sediment. The LOEC for growth was >100 mg a.i./Kg dry sediment. The 28-Day EC50 for growth was determined to be >100 mg a.i./Kg (dry sediment), the highest concentration tested.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

Not applicable

Table 1 Mass Balance Approximations For Days 0, 7 and 28

Study Day	Nominal Test Conc.(mg a.i./kg)	Nominal Test Material in Test System (mg)	Mass of Sediment in Test System (kg)	Volume of Overlying Water in Test System (L)	Pore Water Volume (L)	Test Material in Sediment^1 (mg)	Test Material in Overlying Water^2 (mg)	Test Material in Pore Water^3 (mg)	Total Test Material in Test System (mg)	Percent of Nominal Test Material in Test System
Day 0	6.3	0.697	0.111	0.150	0.0105	0.683	0.000	0.217	0.900	129.1
	13	1.424	0.110	0.150	0.0100	1.106	0.000	0.360	1.466	103.0
	25	2.793	0.112	0.150	0.0085	2.212	0.000	0.606	2.818	100.9
	50	5.490	0.110	0.150	0.0110	4.897	0.021	1.584	6.503	118.4
	100	11.140	0.111	0.150	0.0090	8.845	0.092	2.169	11.106	99.7
Day 7	6.3	0.691	0.110	0.150	0.0096	0.000	0.000	0.019	0.019	2.7
	13	1.490	0.115	0.150	0.0098	0.488	0.000	0.165	0.653	43.8
	25	2.803	0.112	0.150	0.0088	1.034	0.000	0.332	1.365	48.7
	50	5.520	0.110	0.150	0.0098	1.932	0.000	0.795	2.727	49.4
	100	10.890	0.109	0.150	0.0076	4.563	0.0019	1.125	5.707	52.4
Day 28	6.3	0.687	0.109	0.150	0.0115	0.000	0.000	0.000	0.000	0.0
	13	1.457	0.112	0.150	0.0140	0.000	0.000	0.000	0.000	0.0
	25	2.805	0.112	0.150	0.0105	0.000	0.000	0.000	0.000	0.0
	50	5.740	0.115	0.150	0.0120	0.000	0.000	0.000	0.000	0.0
	100	11.620	0.116	0.150	0.0120	0.000	0.000	0.063	0.063	0.5

¹ Test Material in Sediment = concentration of 2-methyl-4-isothiazolin-3-one in sediment multiplied by the weight of sediment in the test system.

² Test Material in Overlying Water = concentration of 2-methyl-4-isothiazolin-3-one in overlying water multiplied by the volume of water in the test system (0.15 L).

³ Test Material in Pore Water = concentration of 2-methyl-4-isothiazolin-3-one in pore water multiplied by the volume of pore water in the test system.

Table 2 Percentage of 2-methyl-4-isothiazolin-3-one in Sediment, Overlying Water and Pore Water based on Mass Balance Calculations

Nominal Test Concentration (mg a.i./Kg)	Study Day	% in Sediment	% in Overlying Water	% in Pore Water	Total %
6.3	0	97.9	0.0	31.2	129.1
	7	0.0	0.0	2.7	2.7
	28	0.0	0.0	0.0	0.0
13	0	77.7	0.0	25.3	103.0
	7	32.8	0.0	11.1	43.8
	28	0.0	0.0	0.0	0.0
25	0	79.2	0.0	21.7	100.9
	7	36.9	0.0	11.8	48.7
	28	0.0	0.0	0.0	0.0
50	0	89.2	0.4	28.9	118.4
	7	35.0	0.0	14.4	49.4
	28	0.0	0.0	0.0	0.0
100	0	79.4	0.8	19.5	99.7
	7	41.9	0.2	10.3	52.4
	28	0.0	0.0	0.5	0.5

Validity criteria fulfilled:

yes

Conclusions:

The 28-day LC50 value for amphipods (Hyalella azteca) exposed to 2-methyl-4-isothiazolin-3 -one in sediment was 34.9 mg a.i./Kg (dry sediment), with 95% confidence limits of 25 and 50 mg a.i./Kg (dry sediment), based on survival (the most sensitive endpoint). The 28-day EC50 value for amphipods (Hyalella azteca) exposed to 2-methyl-4-isothiazolin-3-one in sediment was >100 mg a.i./Kg (dry sediment), based on growth. Based on survival, the LOEC was 25 mg a.i./Kg dry weight of sediment and the NOEC was 13 mg a.i./Kg dry weight of sediment.

Executive summary:

The objective of this study was to determine the effects of sediment-incorporated test substance (2-methyl-4-isothiazolin-3-one; 51.4% a.i., Lot No. 8001J123, TD No. 01-119) to the amphipod, Hyalella azteca, during a 28-day exposure period under flow-through conditions.

Amphipods, Hyalella azteca, were exposed to a geometric series of five test concentrations, a negative control (untreated sediment) and a solvent control (sediment spiked with acetone) under flow-through conditions for 28 days. Eight replicates were tested for each control and treatment group. Ten amphipods were placed in each replicate at the beginning of the test. Nominal test concentrations used in the study were 6.3, 13, 25, 50 and 100 mg active ingredient (a.i.)/Kg (dry weight). The overlying water appeared clear and colorless in all test chambers at test initiation and test termination. The concentration of 2-methyl-4-isothiazolin-3-one (a.k.a. MIT) was measured in overlying water, pore water and sediment samples collected at test initiation, on Day 7 and at test

termination.

All water quality parameters were within acceptable limits during the test. Temperature measurements ranged between 22.5 and 23.3ºC throughout the 28-day exposure period. Dissolved oxygen concentrations remained ≥5.8 mg/L (68% of saturation). Measurements of pH ranged from 8.1 to 8.4. Ammonia measurements were less than 0.17 mg/L (the lowest calibration standard used) during the test.

Most organisms appeared normal during the test, with few observations of abnormal behavior in the treatment and control groups. The LC₅₀ value after 28-days was 34.9 mg a.i./Kg, dry weight, with 95% confidence limits of 25 and 50 mg a.i./Kg, dry weight. Based on survival, the no-observed-effect-concentration (NOEC) was 13 mg a.i./Kg, dry weight, and the lowest observed effect

concentration (LOEC) was 25 mg a.i./Kg, dry weight.

Description of key information

The objective of this study was to determine the effects of sediment-incorporated 2-Methyl-4 -isothiazolin-3-one (51.4% a.i.) to the oligochaete, Lumbriculus variegatus, during a 28-day exposure period under static conditions. The EC50 value after 28-Days was >50 mg a.i./Kg, the highest concentration tested. Based on survival, the no-observed-effect concentration (NOEC) was 25 mg a.i./Kg, and the lowest observed effect concentration (LOEC) was 50 mg a.i./Kg.

A 28 -day toxicity test was performed to estimate the chronic toxicity of 2 -methyl-4 -isothiazolin-3 -one (supplied as a 50% solution in water known as Kordek 573F Industrial Microbiocide) to larvae of the midge, Chironomus riparius. Based on mean pore water concentrations of 2-methyl-4-isothiazolin-3-one, the 28-day NOEC and LOEC values for adult emergence exposure were 12.4 and 27.5 mg 14C-2-methyl-4-isothiazolin-3 -one equivalents/l, respectively.

The objective of this study was to determine the effects of sediment-incorporated test substance (2 -methyl-4 -isothiazolin-3 -one; 51.4% a.i.) to the amphipod, Hyalella azteca, during a 28-day exposure period under flow-through conditions. The 28-day LC50 value for amphipods (Hyalella azteca) exposed to 2-methyl-4-isothiazolin-3 -one in sediment was 34.9 mg a.i./Kg (dry sediment), with 95% confidence limits of 25 and 50 mg a.i./Kg (dry sediment), based on survival (the most sensitive endpoint). The 28-day EC50 value for amphipods (Hyalella azteca) exposed to 2-methyl-4-isothiazolin-3-one in sediment was >100 mg a.i./Kg (dry sediment), based on growth. Based on survival, the LOEC was 25 mg a.i./Kg dry weight of sediment and the NOEC was 13 mg a.i./Kg dry weight of sediment.

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:

50 mg/kg sediment dw

EC10, LC10 or NOEC for freshwater sediment:

25 mg/kg sediment dw