reaction mass of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2H -isothiazol-3-one

Bioaccumulation: aquatic / sediment

Administrative data

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 165-4 (Laboratory Studies of Pesticide Accumulation in Fish)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas Company, Batch No.: 902.0002 (14C-CMIT) and JK-30/70 (12C-CMIT)

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 99.7% (14C-CMIT)
- Specific activity: 41.00 mCi/g
- Locations of the label: 4 and 5 positions

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: All solutions were stored in the freezer until needed.
- Stability under test conditions: 14C-CMIT was stable in the treated aquaria. In all the aquaria samples analyzed the percent 14C-activity present as parent compound ranged from 97.9% to 100%.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: Dosing solutions were initially prepared by dissolving the 14C–CMIT (175.9 mg) into 25 ml of acetone. A stock solution for the proportional diluter associated with the aquarium dosed at 0.010 mg/L was prepared by combining 7.5 ml of 14C CMIT and 162 mg of 12C-CMIT and making up to 25 m with acetone. For the aquarium dosed at 0.12 mg/L, 17.5 ml of 14C-CMIT plus 2430 mg of 12C-CMIT were combined and made up to 50 ml with acetone. 18.30 ml of the 0.010 mg/L stock solution and 35.16 ml of the 0.12 mg/L stock solution were diluted with acetone to a volume of 1500 ml for use in the proportional diluters.

OTHER SPECIFICS: Purity of non-labeled test material (Batch No. JK-30/70) was 99.86%

Radiolabelling:

yes

Sampling and analysis

Details on sampling:

- Sampling intervals/frequency for test organisms: 0, 4 h, and 1, 3, 7, 14, 21, and 28 d
- Sampling intervals/frequency for test medium samples: 0, 4 h, and 1, 3, 7, 14, 21, and 28 d
- Sample storage conditions before analysis: Fish were separately pooled and frozen
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): Frozen fish were homogenized in a blender with dry ice and after the dry ice had sublimed, aliquots were taken and combusted in a sample oxidizer. The trapped 14C was quantitated by liquid scintillation spectrometry. Water samples were radioassayed by liquid scintillation spectrometry.

Test solutions

Vehicle:

yes

Remarks:

acetone

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Proportional diluter
- Controls: Vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): ca. 0.01% v/v
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not described

Test organisms

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Source: Osage Catfisheries, Inc. (Osage Beach, Missouri)
- Age at study initiation (mean and range, SD): < 1 year
- Length at study initiation (lenght definition, mean, range and SD): 51± 3.0 mm
- Weight at study initiation (mean and range, SD): 4.5 ± 0.92 g
- Weight at termination (mean and range, SD): 5.1 ± 1.8 g
- Health status: Test fish were in good health at study initiation
- Feeding during test
- Food type: Salmon Starter
- Amount: ca. 6.3 grams per aquarium per day
- Frequency: Daily

Study design

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural sediment: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Test conditions

Hardness:

260-282 (as CaCO3)

Test temperature:

20-21°C

pH:

7.5-8.4

Dissolved oxygen:

6.9-8.7 mg/L

TOC:

< 1.0 mg/L

Salinity:

Not applicable

Conductivity:

440-520 µMhos/cm

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Material, size, headspace, fill volume: 100L size, filled with 70 mL of well water
- Aeration: Yes
- Type of flow-through (e.g. peristaltic or proportional diluter): Proportional diluter
- Renewal rate of test solution (frequency/flow rate): 6.5-7 volume additions per day
- No. of organisms per vessel: 70
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well water
- Holding medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light : 8 hour dark : 30 minutes transition

Nominal and measured concentrations:

Nominal: 0.12 mg/L and 0.010 mg/L
Measured: 0.12 ± 0.004 and 0.010 ± 0.0005 mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
- Estimation software: BIOFAC
- Result based on measured log Pow of: 0.401

Results and discussion

Bioaccumulation factor

Bioaccumulation factor 1

Key result

Conc. / dose:

0.01 mg/L

Temp.:

ca. 20 °C

pH:

8

Type:

BCF

Value:

ca. 54 dimensionless

Basis:

whole body w.w.

Time of plateau:

5.2 d

Calculation basis:

steady state

Bioaccumulation factor 2

Key result

Conc. / dose:

0.12 mg/L

Temp.:

ca. 20 °C

pH:

8

Type:

BCF

Value:

ca. 41 dimensionless

Basis:

whole body w.w.

Time of plateau:

2.1 d

Calculation basis:

steady state

Depuration

Depuration 1

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.64 d

Remarks on result:

other: 0.12 mg/L treatment

Depuration 2

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

1.6 d

Remarks on result:

other: 0.010 mg/L treatment

Rate constants

Rate constants 1

Key result

Rate constant:

other: uptake rate constant

Remarks:

mg/kg fish/mg/L water/day

Value:

45

Remarks on result:

other: 0.12 mg/L treatment

Rate constants 2

Key result

Rate constant:

other: uptake rate constant

Remarks:

mg/kg fish/mg/L water/day

Value:

24

Remarks on result:

other: 0.010 mg/L treatment

Rate constants 3

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

1.1

Remarks on result:

other: 0.12 mg/L treatment

Rate constants 4

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

0.44

Remarks on result:

other: 0.010 mg/L treatment

Details on kinetic parameters:

- Indication of bi- or multiphasic kinetics: No
- Computation / data analysis: The steady-state bioconcentration factor for whole fish was determined using the BIOFAC computer program

Metabolites:

Metabolites were not isolated or identified. Given the low BCF and the resulting low concentration of metabolites in the tissue this was neither practical nor applicable.

Results with reference substance (positive control):

n/a

Details on results:

- Mortality of test organisms: The only mortality that occured is in the 0.12 mg/l treated aquarium on days 1, 2 and 5 of the exposure phase; mortality affects 5 fish in total, or 7.1% of the 70 fish added to that aquarium.
- Behavioural abnormalities: Not examined
- Observations on feeding behavior: Not examined
- Observations on body length and weight:
- Reproduction during test period: Not examined
- Other biological observations: No
- Organ specific bioaccumulation: Not examined
- Bound residues forming a plateau: Not examined
- Mortality and/or behavioural abnormalities of control: No
- Loss of test substance during test period: No
- Non-eliminated residues (NER) at the end of elimination phase: 12% and 26% for the 0.12 and 0.010 mg/L treatments, respectively
- Results with vehicle control: No effects

Reported statistics:

In evaluating the data obtained from the bioconcentration study, a steady-state approach was used. This consists of a two compartment model (water and fish) which is used to describe the movement of the test substance into and out of the test fish. This approach is used to determine the steady-state bioconcentration factor, the uptake rate constant and the depuration rate constant.
Bioconcentration factors for the exposure phase were determined by dividing the test substance tissue concentration for each label by the appropriate test substance concentration in water for that day. The uptake rate constant and depuration rate constant were determined by the Dow BIOFAC computer program. This is a non-linear kinetic modeling program which provides optimal parameter estimates of rate constants by utilizing the actual (observed) bioconcentration study data. The bioconcentration factor at steady-state, the time to reach 90 % of steady-state and the time to reach 50% clearance (depuration) of the test substance were also calculated from the estimated rate constants. A measure of the variability of the estimated parameters was provided by the standard deviation of each estimate. All calculations are based on the values for whole fish analysis.

Any other information on results incl. tables

Uptake Phase Concentration in Water and Whole Fish

Uptake Day	Concentration based on^14C-CMIT Equivalents^1
	Low Dose—0.010 mg/L		High Dose—0.12 mg/L
	Water (mg/L)	Fish (mg/kg)	Water (mg/L)	Fish (mg/kg)
0	0.011		0.12
0.17	0.011	0.14	0.12	1.3
1	0.010	0.37	0.12	4.3
3	0.010	0.37	0.12	4.4
7	0.0099	0.37	0.12	5.0
14	0.010	0.51	0.12	5.3
21	0.010	0.46	0.12	4.0
28	0.010	0.46	0.13	5.0

¹ Total¹⁴C-residues (parent and metabolites)

 

Depuration Phase Concentration in Water, Whole Fish and Percent Depuration.

Depuration Day	Concentration Based on^14C-CMIT Equivalents^1
	Low Dose—0.010 mg/L			High Dose—0.12 mg/L
	Water (mg/L)	Fish (mg/kg)	Depuration (%)	Water (mg/L)	Fish (mg/kg)	Depuration (%)
1	0.0002	0.28	39	0.0005	2.5	50
3	<MLQ^2	0.19	59	<MLQ^2	1.4	72
7	<MLQ	0.15	67	<MLQ	0.82	84
10	<MLQ	0.11	76	<MLQ	0.65	87
14	<MLQ	0.12	74	<MLQ	0.61	88

¹ Total¹⁴C-residues (parent and metabolites)

² MLQ = minimum limit of detection

Characterization of Test Substance in Aquaria, Diluter Stock Solution, and Spiking of Control Aquarium Water.

Uptake Day	Sample	Percent^14C-CMIT (by HPLC)
		Low Dose (0.010mg/L	High Dose (0.12 mg/L
0	Aquaria Diluter Stock Spike^a	100 100 100	100 100 100
14	Aquaria Diluter Stock Spike^a	97.9 100 100	100 100 100
28	Aquaria Diluter Stock Spike^a	100   100	98.6   100

^a Water from the Control Aquarium was spiked with ¹⁴C-CMIT prior to concentration and HPLC analysis

Water Quality Measurements

Study Day	Control (0 mg CMIT/L)			Low Dose (0.010 mg CMIT/L)			High Dose (0.12 mg CMIT/L)
	Temp. (°C)	D.O. (mg/L)^1	pH	Temp. (°C)	D.O. (mg/L)^1	pH	Temp. (°C)	D.O. (mg/L)^1	pH
0	20	8.7	8.3	20	8.7	8.3	20	8.7	8.2
0.17	20	7.8	8.2	20	8.0	8.2	20	7.7	8.1
1	20	7.7	8.1	20	7.8	8.1	20	7.5	8.0
3	21	7.4	8.1	21	7.8	8.2	21	7.8	8.1
7	21	7.8	8.1	21	8.1	8.1	21	7.6	8.0
14	21	7.9	8.3	21	8.1	8.4	21	7.9	8.3
21	21	8.0	7.5	21	8.0	7.7	21	6.9	7.6
28	21	7.9	8.0	21	8.2	8.1	21	7.9	8.0
29	20	8.0	8.1	20	8.3	8.2	20	8.1	8.2
31	21	8.0	8.1	21	8.3	8.1	21	8.1	8.1
35	20	8.4	8.0	20	8.5	8.3	20	8.3	8.2
38	20	8.4	8.3	20	8.5	8.2	20	8.3	8.1
42	20	8.4	8.1	20	8.3	8.1	20	8.2	8.0

 

¹ Dissolved oxygen saturation at 20°C and 21°C are 8.7 mg/L and 8.5 mg/L, respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study provided fulfills the requirement for CMIT fish bioaccumulation. The Bioconcentration Factor (BCF) of total 14C residues, ≤ 54, is very low; well below regulatory thresholds. Additionally 14C-residues depurate very rapidly. These results were expected given the low log Pow value and the high water solubility of CMIT. At environmentally relevant concentrations, the bioaccumulation of parent compound will be significantly less than the toxicity.

Executive summary:

U.S. EPA 40 CFR § 158, Pesticide Assessment Guidelines Subdivision N § 165-4 and OECD 305 were followed. Initially Bluegill sunfish were dosed with14C-CMIT at 0.010 mg/L and 0.12 mg/L and water and fish samples taken on Days 0, 0.17, 3, 7, 14, 21, and 28 (uptake phase). On day 28 fish were transferred to water free of the active substance and water and tissue samples taken on Days 29, 32, 35, 38, and 42 (depuration phase). Aliquots of water were quantitated (total14C-residues) by radioassaying. Tissue was pooled, homogenized and aliquots radioassayed for total14C-residues.  The study provided fulfills the requirement for CMIT fish bioaccumulation. The Bioconcentration Factor (BCF) of total 14C residues, ≤ 54, is very low; well below regulatory thresholds. Additionally 14C-residues depurate very rapidly. These results were expected given the low log Pow value and the high water solubility of CMIT. At environmentally relevant concentrations, the bioaccumulation of parent compound will be significantly less than the toxicity.