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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 June 2012 to 23 January 2013
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:
yes
Remarks:
The increase of pH was 1.6 unit instead 1.5, without effect on study
GLP compliance:
yes (incl. QA statement)
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 trifluoromethanesulfonic acid concentration. 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 control.

At 72 hours of exposure, a sample was removed from the replicate flask (D) of the 6.4 mg/L test concentration, which did not contain algae and analyzed for trifluoromethanesulfonic acid concentration. The result of this analysis was compared with that obtained for the 72-hour analysis of the 6.4 mg/L solution containing algae to assess the impact that algae had on the test substance concentration.

In addition, three quality control (QC) samples, fortified with trifluoromethanesulfonic acid, were prepared at each sampling interval and remained with the set of exposure solution samples throughout the analytical process. These QC samples were prepared in dilution water at concentrations of trifluoromethanesulfonic acid similar to the treatment level range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.
Vehicle:
no
Details on test solutions:
A 100 mg/L primary stock solution was prepared prior to test initiation by placing 0.2001 g of trifluoromethanesulfonic acid in a 2.0 L volumetric flask and bringing it to volume with AAP medium. The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance following mixing on a magnetic stir plate with a Teflon®-coated stir bar. The pH of the stock solution was 3.65 and outside the acceptable range for testing (≥6.0). At the sponsor’s request, the pH of the primary stock solution was adjusted to pH 7.43 using dilute sodium hydroxide. This pH-adjusted primary stock solution was then used to prepare the nominal test solutions 1, 2.6, 6.4, 16, 40 and 100 mg/L.

After addition of the pH-adjusted primary stock solution, the test solutions were observed to be clear and colorless with no visible undissolved test substance following multiple shakes and inversions. In addition, a control only containing untreated AAP medium was established and maintained under the same conditions as the test solutions.

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 6.4 mg/L nominal treatment level was prepared. This flask, which was not inoculated with algae, was analyzed after 72 hours of exposure for trifluoromethanesulfonic acid concentrations. The results of these analyses were compared with the 72-hour results for the 6.4 mg/L solution containing algae.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: strain 1648, Class Chlorophyceae
- Source (laboratory, culture collection): . The alga was obtained from University of Texas, Austin, Texas and was maintained in stock culture at Smithers Viscient culture facility.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 1 ºC and continuous illumination at the surface of the medium with an intensity range of 4400 to 5900 lux (420 to 550 footcandles). Lighting was supplied by Premira VitaLux® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber.

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not): same as test.
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 ± 1ºC
pH:
7.1 to 7.5 at test initiation. At test termination, the pH of the test and control solutions ranged from 7.5 to 9.0 .
Nominal and measured concentrations:
Nominal: 1.0, 2.6, 6.4, 16, 40 and 100 mg/L.
Measured concentrations: 0.90, 2.3, 5.7, 14, 35 and 89 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks
- Material, size, headspace, fill volume: One hundred milliliters of the appropriate exposure solution was then placed in each replicate flask. All test vessels were fitted with stainless steel caps which permit gas exchange.
- Aeration:
- Initial cells density: 1.0 x 10E4 cells/mL
- Control end cells density: a 1.440-mL inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 69.58 x 10E4 cells/mL, was aseptically introduced into each flask.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes, AAP medium. The initial pH of this medium was adjusted, if necessary, to 7.5 ± 0.1 prior to use


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The AAP medium used to prepare the exposure solutions was formulated in the same manner as the culture medium.
- Total organic carbon:
- Conductivity: 100 to 160 µS/cm


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, at test initiation.
- Photoperiod: none
- Light intensity and quality: Light intensity of the test area ranged from 440 to 510 footcandles (4700 to 5500 lux) throughout the exposure period.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : yield and growth rate.
- Determination of cell concentrations: hemacytometer (Neubauer Improved) and compound microscope.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study
- Test concentrations: 0.10, 1.0, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study:
A preliminary exposure was conducted at nominal trifluoromethanesulfonic acid concentrations of 0.10, 1.0, 10, 100 and 1000 mg/L, and a control. Prior to dilution to the remaining treatment levels, the primary stock solution was pH adjusted from 2.40 to 7.54 therefore, the pH of all test solutions at test initiation ranged from 7.4 to 7.6. All exposure solutions were observed to be clear and colorless with no visible undissolved test substance following preparation. Two exposure vessels were established for each concentration and the control. At test termination, cells exposed to the 0.10, 1.0 and 10 mg/L treatment levels tested and the control were observed to be normal. Cells exposed to the 100 and 1000 mg/L treatment levels were observed to be bloated. Cell density in the control averaged 73.25 x 10E4 cells/mL. Cell density in the 0.10, 1.0, 10, 100 and 1000 mg/L treatment levels averaged 81.88, 86.88, 35.38, 5.50 and 1.0 x 10E4 cells/mL, respectively.

Based on the results of the preliminary exposure and in consultation with the Study Sponsor, treatment levels of 1.0, 2.6, 6.4, 16, 40 and 100 mg/L and a control were selected for the definitive exposure.
Reference substance (positive control):
yes
Remarks:
zinc chloride
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.5 - 10
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 31 - 67
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: ND - 4.1
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 2.7 - 19
Details on results:
The results of the analysis of the exposure solutions for trifluoromethanesulfonic acid concentration showed that the measured concentrations closely approximated the desired nominal concentrations at test initiation and termination. The mean measured concentrations ranged from 89 to 90% of nominal concentrations and were defined as 0.90, 2.3, 5.7, 14, 35 and 89 mg/L.

The analytical result of the 72-hour trifluoromethanesulfonic acid sample from the 6.4 mg/L treatment level, with algae present, was 5.8 mg/L. The equivalent test solution without algae present resulted in a measured concentration of 5.6 mg/L, and demonstrated that the presence of algae had a negligible impact on the concentration of trifluoromethanesulfonic acid in the test solution.

Analysis of the quality control (QC) samples associated with the trifluoromethanesulfonic acid test samples resulted in measured concentrations that were consistent with the predetermined recovery range and ranged from 95.1 to 108% (N = 6) of the nominal fortified levels (0.515, 20.6 and 103 mg/L). Based on these results, it was demonstrated that satisfactory precision and quality control were maintained during the analysis of the exposure solutions.

A significant reduction in growth rate was detected in the 14, 35 and 89 mg/L treatment levels tested compared to the control data. Therefore, the 72 hour NOEC for growth rate was determined to be 5.7 mg/L. The 72-hour ErC50 value was determined to be 48 mg/L with lower and upper 95% confidence limits of 31 and 67 mg/L, respectively.

The 0- to 72-hour yield in the control averaged 91.38 x 10E4 cells/mL. The 0- to 72-hour yield in the 0.90, 2.3, 5.7, 14, 35 and 89 mg/L treatment levels averaged 78.33, 89.50, 57.75, 35.33, 15.00 and 2.92 x 10E4 cells/mL, respectively. Based on the results of Shapiro-Wilks' and Bartlett's Tests, this data set passed the requirement for normality and homogeneity; therefore, Bonferroni’s Adjusted t-Test was used to determine treatment-related effects. A significant reduction in yield was detected in the 5.7, 14, 35 and 89 mg/L treatment levels tested compared to the control data. Therefore, the 72 hour NOEC for yield was determined to be 2.3 mg/L. The 72-hour EyC50 value was determined to be 9.3 mg/L with lower and upper 95% confidence limits of 2.7 and 19 mg/L, respectively.
Results with reference substance (positive control):
- Results with reference substance valid? YES
Based on the results of this test, the 96-hour EC50 value based on cell density was determined to be 0.14 mg Zn/L, with 95% confidence intervals of 0.13 to 0.15 mg Zn/L. Reference testing conducted at Smithers Viscient determined a range of EC50 values for Pseudokirchneriella subcapitata and zinc chloride to be 0.068 to 0.14 mg Zn/L with a mean EC50 value of 0.088 mg Zn/L (± 0.022 mg Zn/L standard deviation). These values closely approximate the mean EC50 value (95% confidence limits) of 0.064 mg Zn/L (0.042 to 0.085 mg Zn/L), indicating the culture used for this study was of appropriate sensitivity for use in toxicity tests.
Reported statistics and error estimates:
Bonferroni’s Adjusted t-Test was used to determine treatment-related effects.

Calculated growth rates ofPseudokirchneriella subcapitataafter 24, 48 and 72 hours of exposure to trifluoromethanesulfonic acid.

Mean Measured

Concentration

(mg/L)

Growth Rate (days-1)

Observation Interval (Hours)

 

0 - 24

0 - 48

0 - 72

% Reductionab

Control

A

1.66

1.39

1.43

 

 

B

1.61

1.46

1.53

 

 

C

2.11

1.71

1.57

 

 

D

1.43

1.50

1.54

 

 

E

1.61

1.57

1.42

 

 

F

1.55

1.76

1.60

 

 

Mean (SD)b

1.66(0.23)

1.57(0.14)

1.52(0.08)

NAc

0.90

A

1.29

1.62

1.37

 

 

B

1.55

1.72

1.56

 

 

C

1.71

1.52

1.45

 

 

Mean (SD)

1.52(0.21)

1.62(0.10)

1.46(0.09)

4

2.3

A

1.49

1.67

1.40

 

 

B

2.08

1.91

1.52

 

 

C

1.36

1.67

1.60

 

 

Mean (SD)

1.64(0.38)

1.75(0.14)

1.51(0.10)

1

5.7

A

1.43

1.30

1.35

 

 

B

1.43

1.39

1.32

 

 

C

1.93

1.19

1.42

 

 

Mean (SD)

1.60(0.29)

1.29(0.10)

1.37(0.05)

10

14

A

1.43

1.31

1.31

 

 

B

1.36

1.02

1.14

 

 

C

1.76

1.25

1.15

 

 

Mean (SD)

1.52(0.21)

1.19(0.15)

1.20(0.10)d

21

35

A

1.04

0.91

0.89

 

 

B

1.04

1.18

1.05

 

 

C

1.13

1.00

0.81

 

 

Mean (SD)

1.07(0.05)

1.03(0.14)

0.92(0.13)d

40

89

A

0.42

0.81

0.51

 

 

B

1.21

0.73

0.56

 

 

C

0.71

0.41

0.23

 

 

Mean (SD)

0.78(0.40)

0.65(0.21)

0.43(0.17)d

71

a      Percent reduction relative to the control.

b         Mean, standard deviation (SD) and percent reduction are calculated from original raw data, not from the rounded values presented in this table.

c      NA = Not Applicable.

d         Significantly reduced compared to the control, based on Bonferroni’s Adjusted t-Test.

Validity criteria fulfilled:
yes
Remarks:
The 72-hour cell density in the control was 92.38 x 10E4 cells/mL (Exceed 16 x 10E4 cells/mL). The mean daily CV for growth rates was 18% (< 35%) and the CV for 0- to 72-hour growth rate was 5.3% (< 7%).
Conclusions:
Trifluoromethanesulfonic acid is considered harmful to algae.
Executive summary:

The objective of the study was to determine the effect of trifluoromethanesulfonic acid on the growth of the freshwater green alga,Pseudokirchneriella subcapitata. Procedures used in this study followed the OECD Guideline for Testing of Chemicals #201 and the GLP.

Although the test item concentration was found to be stable during the study, the results are based on mean measured concentrations of trifluoromethanesulfonic acid.

The 72-hour NOEC for growth rate was determined to be 5.7 mg/L. The 72-hour ErC50value was determined to be 48 mg/L with lower and upper 95% confidence limits of 31 and 67 mg/L, respectively.

The OECD validation criteria were fulfilled.

In these test conditions, trifluoromethanesulfonic acid is harmful to algae.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The read-across justification is provided in the attached document.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.8 mg/L
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
48 mg/L
Basis for effect:
growth rate
Conclusions:
By analogy with its degradation product in water, the 72-hour ErC50 and ErC10 values of trifluoromethanesulfonic anhydride are considered to be 48 and 5.8 mg/L respectively.

Description of key information

Trifluoromethanesulfonic anhydride being rapidly and completely hydrolysed in water, the effect concentration obtained with its degradation product, i.e. trifluoromethanesulfonic acid, is considered as relevant for the assessment of its aquatic toxicity.

Key value for chemical safety assessment

EC50 for freshwater algae:
48 mg/L
EC10 or NOEC for freshwater algae:
5.8 mg/L

Additional information

The read-across justification with trifluoromethanesulfonic acid is provided in the attached document in the endpoint study record.

The determination the effect of trifluoromethanesulfonic acid on the growth of the freshwater green alga,Pseudokirchneriella subcapitata was performed in accordance with the OECD 201 and the GLP.

Although the test item concentration was found to be stable during the study, the results are based on mean measured concentrations of trifluoromethanesulfonic acid.

The 72-hour NOEC for growth rate was determined to be 5.7 mg/L and the 72-hour ErC10 5.8 mg/L. The 72-hour ErC50 value was determined to be 48 mg/L with lower and upper 95% confidence limits of 31 and 67 mg/L, respectively.

The OECD validation criteria were fulfilled.

In these test conditions, trifluoromethanesulfonic acid is harmful to algae.