Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 201 compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
August 2013

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqueous solution

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
At test initiation (0 hour) and test termination (72 hours), a single sample was removed from each test concentration and the control and analyzed for bistrifluoromethanesulphonimide (TFSIH). Samples analyzed at 0 hour were removed from the test and control solutions prior to division into the replicate test vessels. Samples analyzed at 72 hours were removed from individually composited replicate solutions of the treatment levels and the control.
At 72 hours of exposure, a sample was removed from the replicate flask (D) of the 13 mg/L test concentration, which did not contain algae. The result of this analysis was compared with that obtained for the 72-hour analysis of the 13 mg/L solution containing algae to assess the impact that algae had on the test substance concentration.

Test solutions

Vehicle:
no
Details on test solutions:
Prior to exposure initiation, a 100 mg/L primary stock solution was prepared by placing 0.2397 g (0.1999 g as active ingredient) of the test substance in a 2000-mL volumetric flask and bringing it to volume with AAP medium. Following mixing using a magnetic stir bar and Teflon®-coated stir bar, the 100 mg/L primary stock solution was observed to be clear and colorless with no visible undissolved test substance. The initial pH of the 100 mg/L primary stock solution was 3.81 and was adjusted to a final pH of 7.57 by the addition of 0.70 mL of 1.0 N sodium hydroxide and 0.30 mL of 0.10 N hydrochloric acid, as recommended by the OECD 201 guideline.
The resulting exposure solutions were observed to be clear and colorless with no visible undissolved test substance following preparation. Additional untreated AAP medium was used for the control. Control vessels were maintained under the same conditions as the treatment levels but contained no bistrifluoromethanesulphonimide (TFSIH).
In order to estimate the impact that the presence of algal biomass had on the test substance concentration, an additional replicate flask (D) of the 13 mg/L nominal treatment level was prepared. This flask, which was not inoculated with algae, was analyzed after 72 hours of exposure for bistrifluoromethanesulphonimide (TFSIH) concentration. The result of this analysis was compared with the 72-hour result for the 13 mg/L solution containing algae.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 1648, class Chlorophyceae
- Source (laboratory, culture collection): algae from The University of Texas, Austin, Texas and was maintained in stock culture at Smithers Viscient.
- Age of inoculum (at test initiation): The inoculum used to initiate the toxicity test with bistrifluoromethanesulphonimide was taken from a stock culture that had been transferred to fresh medium four days before testing.
- Method of cultivation: The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water. Representative samples of the dilution water used in the preparation of the culture medium were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations that are considered toxic in any of the water samples analyzed in agreement with ASTM guidelines (ASTM, 2002). In addition, a representative sample of AAP medium was analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the sample collected in May 2014 was 0.41 mg/L.
If necessary, the pH of the culture medium was adjusted to pH 7.5 ± 0.1 with dilute hydrochloric acid or sodium hydroxide. Stock cultures were grown in 250-mL glass flasks each containing 100 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.
The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 2 ºC and continuous illumination at the surface of the medium with an intensity range of 2900 to 3900 lux. The photosynthetically-active radiation (PAR) of the test area was maintained with a range of 51 to 69 µE/m2/S.
Lighting was supplied by Premira VitaLux® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker table. Temperature was controlled using an environmental chamber.

ACCLIMATION
- Culturing media and conditions (same as test or not): yes

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
23 to 25°C
pH:
adjusted to 7.5
Nominal and measured concentrations:
Nominal: 6.3, 13, 25, 50 and 100 mg/L
Measured: 5.7, 13, 24, 46 and 94 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250-mL Erlenmeyer flasks. One hundred milliliters of the appropriate exposure solution was then placed
in each replicate flask.
- Aeration: no
- Initial cells density: 1 x 10E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: AAP medium, the same as culture medium.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 62 to 75 µE/m2/S (4400 to 5900 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

- Range finding study
- Test concentrations: 0.010, 0.10, 1.0, 10 and 100 mg/L
A preliminary range-finding exposure was conducted at Smithers Viscient at nominal bistrifluoromethanesulphonimide (TFSIH) concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/L, and a control. Two exposure vessels were established for each concentration and the control.
Test solutions were prepared in the same manner as in the definitive study (including pH adjustment of the high stock solution). At test termination, cells exposed to all treatment levels tested and the controls were observed to be normal. Following 72 hours of exposure, cell densities in the 0.010, 0.10, 1.0, 10 and 100 mg/L treatment levels averaged 102, 104, 104, 98 and 40 x 10E4 cells/mL, respectively. Cell density in the control averaged 101 x 10E4 cells/mL.
Based on these results, nominal concentrations of 6.3, 13, 25, 50 and 100 mg/L were selected for the definitive exposure.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 29 - 37
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The pH of the test solutions ranged from 7.3 to 7.4 at test initiation. At test termination, the test solution pH ranged from 7.8 to 8.5. The increase in pH during the exposure is common in static algal cultures and is due to photosynthesis by the algae.
Conductivity measured at test initiation in the treatment and control solutions ranged from 96 to 130 µS/cm, and increased in conductivity as test concentrations increased.
Cells exposed to all treatment levels tested and the control were observed to be normal throughout the exposure period with the exception of cell fragments, which were observed in the 50 and 100 mg/L treatment levels at the 72-hour observation interval.
The 72-hour cell density in the control averaged 128.21 × 10E4 cells/mL.
The 0 to 72-hour yield in the control averaged 127.21 × 10E4 cells/mL. The 0 to 72-hour yield in the 6.3, 13, 25, 50 and 100 mg/L treatment levels averaged 140.06, 124.75, 122.00, 48.42 and 46.83 × 10E4 cells/mL, respectively. Based on the results of Shapiro-Wilks’ and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance; therefore, Williams’ Multiple Comparison Test was used to determine treatment-related effects.
A significant reduction in yield was detected in the 50 and 100 mg/L treatment levels compared to the control data.
Therefore, the 72-hour NOEC and LOEC for yield were determined to be 25 and 50 mg/L, respectively. The 72-hour EyC50 value for yield was determined by linear interpolation to be 42 mg/L, with a corresponding 95% confidence interval of 37 to 46 mg/L.

The 0 to72-hour average specific growth rate in the control averaged 1.65 days-1.
The 0 to 72-hour average specific growth rate in the 6.3, 13, 25, 50 and 100 mg/L treatment levels averaged 1.68, 1.64, 1.63, 1.33 and 1.31 days-1, respectively. Based on the results of Shapiro-Wilks’ and Bartlett’s Tests, this data set passed the requirement for normality and homogeneity of variance; therefore, Williams’ Multiple Comparison Test was used to determine treatment-related effects.
A significant reduction in growth rate was detected in the 50 and 100 mg/L treatment levels compared to the control data. Therefore, the 72-hour NOEC and LOEC for growth rate were determined to be 25 and 50 mg/L, respectively. Since no concentration tested resulted in ≥ 50% inhibition of growth rate, the 72-hour ErC50 value for average specific growth rate was empirically estimated to be > 100 mg/L, the highest nominal concentration tested.

Any other information on results incl. tables

Validity criteria fulfilled:

The following acceptance criteria were required by the protocol: the cell growth in the control must increase from the initial density (1.0 × 10E4 cells/mL) by more than 16 times after 72 hours of growth. Additionally, the mean coefficient of variation (CV) for section-by-section specific growth rates (days 0 to 1, 1 to 2 and 2 to 3) in the control replicates should not exceed 35%.

The CV for the average growth rate of the control for the entire test period (0- to 72-hour) should not exceed 7%.

During the study, the 72-hour cell density in the control averaged 128.21 × 10E4 cells/mL, which meets the above criterion. The mean daily CV for growth rate was 14% and the CV for 0- to 72−hour growth rate was 2.1%, which meet the above criteria.

Table 1: Calculated growth rates of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to bis trifluoromethanesulphonimide (TFSIH).

Nominal

Concentration

(mg/L)

Replicate

Growth Rate (days-1)

72 Hour Percent Inhibitionab

Observation Interval (Hours)

0-24

0-48

0-72

 

 

 

 

 

 

Control

A

1.36

1.61

1.67

 

 

B

1.66

1.57

1.63

 

 

C

1.55

1.67

1.61

 

 

D

2.11

1.58

1.71

 

 

E

1.55

1.57

1.65

 

 

F

1.43

1.54

1.62

 

 

Mean (SDb)

1.61 (0.27)

1.59 (0.04)

1.65 (0.03)

NAc

6.3

A

1.89

1.54

1.69

 

 

B

1.61

1.69

1.71

 

 

C

1.36

1.61

1.63

 

 

Mean (SD)

1.62 (0.26)

1.62 (0.07)

1.68 (0.04)

-2

13

A

1.80

1.76

1.68

 

 

B

1.85

1.66

1.62

 

 

C

1.55

1.55

1.62

 

 

Mean (SD)

1.73 (0.16)

1.66 (0.11)

1.64 (0.04)

0

25

A

2.11

1.74

1.60

 

 

B

1.76

1.63

1.69

 

 

C

1.36

1.61

1.61

 

 

Mean (SD)

1.74 (0.37)

1.66 (0.07)

1.63 (0.05)

1

50

A

1.66

1.51

1.31

 

 

B

1.36

1.46

1.34

 

 

C

1.55

1.49

1.33

 

 

Mean (SD)

1.53 (0.15)

1.49 (0.03)

1.33d(0.02)

20

100

A

1.61

1.46

1.30

 

 

B

1.49

1.33

1.33

 

 

C

1.43

1.42

1.31

 

 

Mean (SD)

1.51 (0.09)

1.40 (0.06)

1.31d(0.02)

20

 

 

 

 

 

 

Table 2: ErC10, ErC20, ErC50and No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) values for bis trifluoromethanesulphonimide (TFSIH) from results (growth rate) after 72 hours of exposure with Pseudokirchneriella subcapitata.

Biological Parameter

Based on Nominal Concentrations (mg/L)

ErC10

(95% CLa)

ErC20

(95% CL)

ErC50

(95% CL)

NOEC

LOEC

 

 

 

 

72-Hour Average Specific Growth Rate

34

(29 - 37)

49

(44 - NDb)

> 100

(NAc)

25

50

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Bis trifluoromethanesulphonimide is not harmful to algae.
Executive summary:

The purpose of the study was to determine the effect of bis trifluoromethanesulphonimide (TFSIH) on the growth of the freshwater green alga, Pseudokirchneriella subcapitata, according to the OECD 201 guideline and under GLP conditions.

Nominal concentrations tested were 6.3, 13, 25, 50 and 100 mg/L.

In this study the 72 -hour EC50 (growth rate) was higher than 100 mg/L.

The 72-hour EC10 (growth rate) was 34 mg/L.

The 72-hour NOEC (growth rate) was 25 mg/L.

In these conditions, bis trifluoromethanesulfonimide is not considered as harmful to algae.