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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rohm and Haas, Batch No. 0004436406
- Expiration date of the lot/batch: April 5, 2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions
- Stability under test conditions: Stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Step dilution in saltwater algal medium
- Final dilution of a dissolved solid, stock liquid or gel: 0.75, 1.5, 3.0, 6.0 and 12 μg a.i./L

OTHER SPECIFICS: Purity of TS was 14.27% Total AI (3.45% MIT and 10.82% CMIT)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.75, 1.5, 3.0, 6.0 and 12 μg a.i./L
- Sampling method: Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to addition of the algae. Samples at 24, 48, and 72 hours were collected from individual analytical replicates. At exposure termination, samples were collected from the pooled biological replicates from each treatment and control group. Abiotic replicates from each experimental group were sampled at 24, 48, 72 and 96 hours.
- Sample storage conditions before analysis: All samples were collected in glass vessels and processed immediately for analysis

Vehicle:

yes

Remarks:

Water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dilutiuon in water
- Controls: Negative control, abiotic control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Marine diatom
- Strain: Skeletonema costatum
- Source (laboratory, culture collection): Provasoli-Guillard National Center for Culture of Marine Phytoplankton
- Age of inoculum (at test initiation): Cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. The culture was last transferred to fresh medium three days prior to test initiation.
- Method of cultivation: Batch culture

ACCLIMATION
- Acclimation period: 2 weeks
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

Observations at 0, 24, 48, 72 and 96h

Hardness:

Not reported

Test temperature:

20 ± 2°C

pH:

8.0

Dissolved oxygen:

Not reported

Salinity:

30 parts per thousand

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 0.75, 1.5, 3.0, 6.0 and 12 μg a.i./L
Samples of test medium collected on
Day 0 had recoveries that ranged from 109 to 113% of nominal concentrations. Analyses of samples collected at approximately 24, 48, 72 and 96 hours after initiation showed that levels of the test substance declined to

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml Erlenmeyer flasks
- Material, size, headspace, fill volume: 100 mL of test or control medium
- Aeration: No
- Initial cells density: 10,000 cells/mL
- Control end cells density: 3,263,987 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: 0.72 mg/L FeCl3•6H2O, 2.16 mg/L MnCl2•4H2O, 0.675 mg/L ZnSO4•7H2O, 2.36 μg/L CuSO4•5H2O, 6.06 μg/L CoCl2•6H2O, 17.1 mg/L H3BO3, 15.0 mg/L Na2EDTA•2H2O, 3.0 mg/L K3PO4, 50.0 mg/L NaNO3, 20.0 mg/LNa2SiO3•9H2O, 0.25 mg/L Thiamine Hydrochloride, 0.05 μg/L Biotin, 0.5 μg/L B12.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Wildlife International, Ltd. Well Water
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Pesticides: Not detected
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: 10% HCl
- Photoperiod: 16 hours of light and 8 hours darkness
- Light intensity and quality: 4310 ± 650 lux, cool-white fluorescent lighting
- Salinity (for marine algae): 30 parts per thousand

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter, 24, 48, 72 and 96 hours
- Chlorophyll measurement: No
- Other: Cells in the replicate test chambers were assessed for adherence of the cells to the test chamber. Subsamples of one replicate of each treatment and control group were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in the subsamples also were assessed for aggregations or flocculations of cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 0.75, 1.5, 3.0, 6.0 and 12 μg a.i./L

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.49 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19.9 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

37.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Adherence to test vessels: No
- Aggregation of algal cells: There were aggregations and flocculations in the control and each exposure concentration, but these were consistent between the control and treatment groups and were not considered to be treatment related.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: No

Reported statistics and error estimates:

Growth rate was calculated for each replicate of the control and treatment groups using the following formula:
μ = (ln Nn - ln N0) / (tn -t0)
where: μ = Average specific growth rate
N0= Nominal cell density (cells/mL) at t0
Nn = Measured cell density (cells/mL) at tn
t0 = Time of beginning of test (hours)
tn = Time after beginning of test (hours)

Yield was calculated for each treatment and control replicate, and was defined as the cell density at the end of the exposure period minus the cell density at the start of the exposure period.

Inhibition values were calculated for each treatment group as the percent reduction in cell density, yield, and growth rate relative to the negative control replicates.

EC50 values in this study represent the theoretical test concentration that would produce a 50% reduction in an effect relative to the control. Non-linear regression was used to calculate EC50 values and their corresponding 95% confidence intervals for cell density (EC50), yield (EyC50), and growth rate (ErC50) for each 24-hour exposure period, when possible. The data were evaluated for normality and homogeneity of variance (p=0.05) using the Shapiro-Wilk’s and Levene’s tests, respectively. The treatment groups were compared to the negative control using Dunnett’s test (p=0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC for cell density, yield, and growth rate at 24, 48, 72 and 96 hours.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour ErC50 value for growth rate was 19.9 μg a.i./L

Executive summary:

The marine diatom, Skeletonema costatum, was exposed to six concentrations of a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one in a ratio of 3:1, and evaluated for effects on cell density, yield, and growth rate. The results of the study are based on the Day 0 measured concentrations, ranging from 0.82 to 27 μg a.i./L. The 24, 48, 72 and 96-hour ErC50 values for growth rate were >27, 17.6, 19.9 and 19.3 μg a.i./L, respectively. The 24, 48, 72 and 96-hour NOECs for growth rate were 6.6, 1.6, 6.6, and 6.6 μg a.i./L, respectively.

Description of key information

In accordance with the assessment of CMIT/MIT as an active substance under the BPR.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.037 mg/L

EC10 or NOEC for marine water algae:

0.004 mg/L