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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 9th 2002- September 22nd 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth 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

Test material

Constituent 1
Chemical structure
Reference substance name:
1-BUTANESULFONAMIDE, 1,1,2,2,3,3,4,4,4-NONAFLUORO-N-METHYL-
EC Number:
614-396-3
Cas Number:
68298-12-4
Molecular formula:
C5H4F9NO2S
IUPAC Name:
1-BUTANESULFONAMIDE, 1,1,2,2,3,3,4,4,4-NONAFLUORO-N-METHYL-
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M SMMD EHSR, Lot #3
- Purity: 97.25%
- Physical state: off white crystalline powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.1, 2.3, 4.5, 9.0, 18, 36 mg/L
- Sampling method: Samples of exposure solution were collected at test initiation and exposure termination to measure dissolved concentrations of MeFBSA. Test initiation samples were taken from batch test solutions of each treatment concentration and control before distribution into test chambers. Exposure termination samples were collected from pooled replicates of each respective concentration and control group. All samples were placed in 20 mL plastic scintillation vials.
- Sample storage conditions before analysis: Samples were analyzed immediately

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 1L stock solution was prepared in algal medium at a concentration of 36 mg/L. The primary stock was mixed for 24 hours on a magnetic stir plate using a Teflon coated stir bar. After mixing, the primary stock solution was proportionally diluted with algal medium to prepare 500 mL stocks of the other test conditions. Test concentrations were not corrected for percent active ingredient of the test material. All secondary stocks were mixed via inversion. After mixing, 100 mL aliquots of each test solution were placed in each respective test vessel and placed in the environmental chamber for 24 hours for conditioning. The volumetric flasks used to prepare the exposure solutions were conditioned with the remaining solution. The procedure for preparing the primary stock and test solutions were repeated to prepare the exposure solutions using the volumetric flasks that were allowed to condition for a 24-hour period.
- Controls: Negative control (dilution water), test medium without substance or other additives.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None, solution appeared clear and colorless
- Other relevant information: None

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): Wildlife International Ltd. Cultures
- Method of propagation: Algal cells were grown in culture medium for two weeks prior to test initiation. Prior to test, the chambers were inoculated with algal cells and shaken continuously at 100 rpm.
- Growth medium: MgCl2.6H2O 12.164 mg/L, CaCl2.2H20 4.410 mg/L, H3BO3 0.1855 mg/L, MnCl2.4H2O 0.4154 mg/L, ZnCl2 3.27 ug/L, FeCl3.6H20 0.1598 mg/L, CoCl2.6H2O 1.428 ug/L, Na2MoO4.2H2O 7.26 ug/L, CuCl2.6H2O 0.012 ug/L, Na2EDTA.2H2O 0.300 mg/L, NaNO3 25.5 mg/L, MgSO4.7H2O 14.70 mg/L, K2HPO4 1.044 mg/L, NaHCO3 15.00 mg/L prepared with reverse osmosis purified water, sterilized via filtration (0.2 um) prior to use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
At exposure termination, the 15 and 31 mg/L treatment groups exhibited no visual evidence of algal growth. A 0.5 mL aliquot of test solutions was taken from each replicate test chamber and pooled by treatment. The solution was diluted in 100 mL of culture medium to a concentration of test substance that was assumed would not hinder growth. The control flask was prepared for comparison by diluting 0.5 mL of solution from a single negative control replicate to 100 mL with culture medium. Growth of these subcultures was monitored for 9 days to determine if inhibition of growth observed at the test concentrations were reversible. Samples were collected for cell counts at recovery phase initiation, Days 3 and 6, and at termination of the recovery phase. The 15 mg/L treatment was terminated on Day 6 when algal growth was sufficient to indicate that the algal cells has recovered from effects of the test substance. The 31 mg/L treatment level was terminated on Day 9 after algal cells has recovered.

Test conditions

Test temperature:
23.1-23.8
pH:
7.2-8.8
Nominal and measured concentrations:
Nominal: 1.1, 2.3, 4.5, 9.0, 18, 36 mg/L
Mean measured: 0.92, 1.9, 3.8, 7.8, 15, 31 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL polycarbonate Erlenmeyer flasks
- Type: Closed with foam stoppers
- Fill volume: 100 mL
- Aeration: shaking at 100 RPM
- Renewal rate of test solution: None
- Initial cells density: 10,000 cells/mL (nominal)
- Control end cells density: 2,310,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Yes, AAP medium per ASTM1218-E90

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 4300 ± 10 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were performed using a hemacytometer with 10 grids and a microscope. Samples were diluted as needed using an electrolyte solution (Isoton) to maintain counting accuracy.

RANGE FINDING STUDY
- Results used to determine the conditions for the definitive study: A range finding study was conducted and used to establish definitive test concentrations. However, details were not provided.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% CI: 2.1-4.7 mg/L
Key result
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
6.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% CI: 5.7-8.2 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% CI: 10-14 mg/L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% CI: 12-14 mg/L
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities: The morphology of the cells of all treatment groups appeared normal when compared to the negative control. There was no evidence of aggregation, flocculation of cells, nor evidence of algal cells adhering to the test chambers.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: No
- Recovery Test Results: Based on the growth observed in the recovery phase, the effect on algal growth was found to be algistatic at 15 to 31 mg/L treatrments. See Table 3.

Results with reference substance (positive control):
No reference substance test run
Reported statistics and error estimates:
Statistics run: Levene's test for homogeneity, Shapiro-Wilk's Test for normality, Dunnett's Multuple Comparison Test for comparing the treatment groups to the negative control. EC10 and EC50 growth rates were calculated using non-linear regression or linear interpolation using TOXSTAT v3.5 software.

Any other information on results incl. tables

Table 1. Measured concentrations of MeFBSA in Freshwater

Concentration (mg/L)

Sampling Time (Day)

Measured concentration MeFBSA (mg/L)

% of Nominal

Mean Measured concentration (mg/L)

Mean Measured % of Nominal

Negative control

0

<LOQ

-

<LOQ

-

 

4

<LOQ

-

 

 

1.1

0

1.3

114

0.92

84

 

4

0.59

54

 

 

2.3

0

2.4

103

1.9

83

 

4

1.3

58

 

 

4.5

0

4.8

106

3.8

84

 

4

2.7

61

 

 

9

0

9.8

109

7.8

87

 

4

5.8

65

 

 

18

0

19

104

15

83

 

4

11

64

 

 

36

0

38

107

31

86

 

4

23

64

 

LOQ was 0.200 mg/L, calculated as the product of the lowest calibration standard (0.1 mg/L) and the dilution factor of the matric blanks (2.00)

Table 2. Mean Growth Rates

Mean Measured Test Concentration (mg/L)

72 hour Mean Growth Rate

72 hour % Inhibition

96 hour Mean Growth Rate

96 hour % Inhibition

Negative control

0.0568

 --

0.0567

 --

0.92

0.0553

2.7

0.0553

2.4

1.9

0.0547

3.7

0.0563

0.75

3.8

0.0507*

11

0.0520*

8.3

7.8

0.0380*

33

0.0441*

22

15

0.0214*

62

0.0227*

60

31

0.0019*

97

0.0020*

97

There were statistically significant differences (p<0.05) at 72 and 96 hours when compared to the negative control replicates using Dunnett's test.

Table 3. Cell Densities During the Nine-Day Recovery Phase

Mean Measured Test Concentration (mg/L)*

Cell Densities (Cells/ml) Day 0

Cell Densities (Cells/ml) Day 3

Cell Densities (Cells/ml) Day 6

Cell Densities (Cells/ml) Day 9

Negative control

15000

870000

5280000

6480000

15

2000

121000

5280000

 --

31

0

3000

79000

4395000

* The treatment group was diluted to a concentration of the test substance that theoretically would not inhibit growth. Due to the method used to prepare recovery phase test solutions, initial cell densities were not equivalent between the groups.

-- The treatment group was fully recorded and terminated on Day 3

Table 4. Cell Densities By Replicate Over the 96-hour Exposure Period

Mean Measured Test Concentration (mg/L)*

Replicate

Cell Densities (Cells/ml) 24 hours

Cell Densities (Cells/ml) 48 hours

Cell Densities (Cells/ml) 72 hours

Cell Densities (Cells/ml) 96 hours

Negative control

A

32000

101000

610000

2360000

 

B

34000

121000

595000

2440000

 

C

19000

123000

585000

2130000

0.92

A

22000

100000

510000

2030000

 

B

31000

101000

565000

1950000

 

C

30000

98000

530000

2080000

1.9

A

30000

87000

470000

1950000

 

B

28000

142000

580000

2390000

 

C

28000

118000

495000

2330000

3.8

A

25000

111000

410000

1740000

 

B

26000

55000

320000

1220000

 

C

21000

86000

435000

1490000

7.8

A

8000

63000

147000

660000

 

B

10000

55000

185000

650000

 

C

18000

64000

135000

770000

15

A

18000

22000

46000

77000

 

B

10000

19000

38000

80000

 

C

9000

25000

58000

111000

31

A

6000

7000

7000

16000

 

B

10000

12000

15000

11000

 

C

9000

11000

8000

7000

Prior to the test initiation, the cell density of the stock culture was determined and an inoculum volume was administered to each test chamber to yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours).

Table 5. Specific Growth Rates

 

24 HRS

48 HRS

72 HRS

(0-72 H)

CV (%)

 

(0-24H)

(24-48H)

(48-72H)

 

 

Rep A

1.16

1.15

1.80

1.37

27.06

Rep B

1.22

1.27

1.59

1.36

14.77

Rep C

0.64

1.87

1.56

1.36

47.01

 

 

 

 

 

 

Mean

1.01

1.43

1.65

1.36

29.61

Std Dev

0.32

0.38

0.13

0.01

 

CV (%)

31.69

26.93

7.84

0.51

 


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
control biomass increase >16x (61x), CV for section-by-section growth rates <35% (30.7%), CV for average specific growth rates <7% (0.51%)
Conclusions:
72 hour EC50 of 12 mg/L (OECD 201 and EPA 850.5400) in Pseudokirchneriella subcapitata.
72 hour EC10 of 3.6 mg/L (OECD 201 and EPA 850.5400) in Pseudokirchneriella subcapitata.
Executive summary:

The 72 hour and 96 hour EC50, EC10 and NOEC, of MeFBSA to Pseudokirchneriella subcapitata was determined according to OECD 201 and EPA 850.5400 guidelines. Nominal concentrations of 1.1, 2.3, 4.5, 9.0, 18, 36 mg/L and a blank control, were run with three replicates for each treatment level and the negative control. A 72 hour EC50 of 12 mg/L and a 72 hour EC10 of 3.6 mg/L (measured concentrations) were determined. Percent of nominal concentrations of the test material ranged from 104-114% on Day 0 to 54-65% on Day 4. A 9 day recovery test was run at 15 and 31 mg/L and demonstrated algistatic effects on the population.

The study was well-documented, followed a international standard methods and was GLP compliant. The study is considered reliable without restrictions. The results from this study are considered suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.