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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 May 17th 2017 to September 27th 2017.
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
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate No 2017/12
Analytical monitoring:
yes
Details on sampling:
Test concentrations were analyzed:
- at T0h, before algae addition
- at T72h with and without algae.
In order to remove algae at T72h, 50 mL of test concentration were sampled and centrifugated at 4000G during 10 minutes. Analyses were performed on the supernatant.
For test concentrations higher than 100 mg/L (maximum value of the working range) an appropriate dilution (factor 2 to factor 20) was applied.
All samples were analyzed immediately after sampling.
Vehicle:
no
Details on test solutions:
Preliminary experiments showed that pH of test concentrations higher than 60 mg/L were not appropriate to a suitable algal growth (pH < 6).

Therefore, the following steps were performed to prepare the range of test concentrations:
- preparation of a stock solution at 1 460 mg/L in growth medium,
- preparation of an intermediate solution at 58.4 mg/L by diluting stock solution in growth medium. This solution was used to prepare the lowest test concentrations (0.9 to 54.7 mg/L),
- adjustment of the stock solution pH with concentrated NaOH solution (10 M), to reach a pH consistent with suitable algal growth (≥ 6.5). This adjusted stock solution was used to prepare the highest test concentrations (153.1 to 1200.0 mg/L).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Species: Pseudokirchneriella subcapitata, green alga belonging to the order of Chlorococcales; strain CCAP 278/4
- Origin: Liquid culture obtained from the Culture Center for Algae and Protozoa (CCAP) Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria, U.K.
- Sensitivity: controlled in May 2017 (See chapter IV - Reference item)
- Pre-culture : 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium.The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.7 – 22.2°C (continuous measurement during the study)
pH:
8.2 ± 0.1 (after equilibriation with air)
Nominal and measured concentrations:
Initial nominal concentrations: 0.9, 2.5, 7.0, 19.5, 54.7, 153.1, 428.6, 1 200.0 mg/L, corresponding to the following mean measured concentrations (geometric mean of measured concentration at T0h and at T72h): 0.9, 2.7, 7.2, 20.2, 55.6, 157.6, 438.0, 1207.0 mg/L
Details on test conditions:
- Test medium: OECD Growth medium as described in OECD guideline 201
- Vessels: 250 mL conical flasks with air permeable stoppers filled with 100 mL of test solutions
- Initial cell concentration: 10E4 cells/mL taken from an exponentially growing pre-culture
- Test concentrations: (initial nominal) (mg/L) 0.9 – 2.5 – 7.0 – 19.5 – 54.7– 153.1 – 428.6 – 1 200.0 This range of concentrations was selected, in agreement with the sponsor representative, after preliminary tests, to determine accurate EC50, EC10and NOEC values.
- Number of replicates: 6 for control conditions + 1 without algae (analysis) 3 per concentration with algae 1 per concentration, without algae (blank control and analysis)
- Incubation temperature: 21.7 – 22.2°C (continuous measurement during the study)
- Incubation illumination: 79 – 118 µE m-2s-1(obtained with plant growth light) corresponding to the range 2570 – 3790 lux.
- After equilibration with air, the pH of growth medium was 8.2 ± 0.1.
- Agitation: Orbital shaking 125 rpm
- Duration: 72h
- Renewal: None
- Determination of algal cells: Every 24 hours with a particle counter (Coulter counter Z2, BECKMAN COULTER Life Sciences)
- Algal cells microscopic observation: At the beginning of the test to verify the normal and healthy appearance of the inoculum culture. After 72h to observe any abnormal appearance of the algae (as may be caused by the exposure to the test item).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: logarithmic increase of biomass (average specific growth rate) during the exposure period
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: logarithmic increase of biomass (average specific growth rate) during the exposure period
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
237.07 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: logarithmic increase of biomass (average specific growth rate) during the exposure period
Details on results:
- NOEC: 2.7 mg/L (measured concentration) corresponding to 2.5 mg/L in nominal concentration
- ErC10 (72h): 5.80 mg/L (3.36 – 10.06), corresponding to 5.59 mg/L (3.00 – 9.69) in nominal concentration.
- ErC50 (72h): 241.95 mg/L (195.45 – 292.66), corresponding to 237.07 mg/ (192.21 – 289.23) in nominal concentration.

Cell density measurements, performed with a particle counter at 0h, 24h, 48h and 72h are summarized in Table 1. Measurement done on blank controls, performed for all concentrations, demonstrated the lack of interference for measurements with particle counter. Microscopic observations were performed at T0h (inoculum) and at T72h for each test concentration. Some aggregations of algal cells were noticed at the highest test concentrations (mainly in the range between 153.1 and 1200 mg/L). On the other hand, no changes in color and cell shape was observed for any test concentration.
Averages specific growth rates calculated for tests concentrations and for control are summarized in Table 2. Effects on algal growth increased with increasing concentration of trifluoroacetic acid.

Analyses were performed on:
- the stock solution at 1460 mg/L and the intermediate solution 58.4 mg/L (dilution of the stock solution) at T0h (see Table 3).
- the test concentrations (including control) at T0h, before algae addition and at T72h with and without algae (See Table 4).
These results showed that:
- the concentrations of trifluoracetic acid were stable during the exposure period,
- results are comparable in test concentrations with and without algae demonstrating a lack of interaction between TFA and algae,
- the test item has been satisfactorily maintained within ± 20 % of the nominal concentration in all test conditions.
For each test concentration, an exposure concentration can be calculated by performing a geometric mean of measured values at T0h and T72h. Results are presented in Table 5.
According to these results, either nominal or measured concentrations can be used for toxicity values determination.
Results with reference substance (positive control):
Sensitivity of the strain of Pseudokirchneriella subcapitata was checked using potassium dichromate.
The test gave the following result: ErC50 (72h): 1.05 mg/L (0.99 – 1.14 mg/L)
This value was calculated using a log-logistic regression analysis with bootstrap estimation of the 95% confidence limits.
This value is included in the warning limits of the laboratory (mean ErC50±2 SD) on this species corresponding to two standard deviations from the historic geometric mean of themedian inhibitory concentration (0.75 – 1.19 mg/L).
This result confirmed the appropriate sensitivity of the algal strain.
Reported statistics and error estimates:
Statistics have been performed using Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming.
Normality and homogeneity of the data have been checked and the significance of the effect on growth was determined, by performing a two tailed ANOVA (α-value = 0.05) followed by the Bonferroni post-hoc test (α-value = 0.05). ErC10 and ErC50 (72 h) values have been calculated using log-logistic regression with bootstrap estimation of 95% confidence limits.

Table 1. Cell density measurements, performed with a particle counter at 0h, 24h, 48h and 72h

Test concentrations (initial nominal concentrations) in mg/L

Cell Density x 104cell/mL (particle counter)

pH

T0

Mean

T24h

Mean

T48h

Mean

T72h

Mean

T0

T72h

Control

1.00

1.00

1.00

1.00

1.00

1.00

1.00

3.13

3.91

4.76

3.82

3.59

3.26

3.75

17.76

22.66

18.91

19.08

19.11

19.36

19.48

104.88

112.44

119.58

120.77

116.72

98.89

112.21

8.3

8.8

0.9

1.00

1.00

1.00

1.00

4.57

4.76

4.71

4.68

17.56

19.20

24.99

20.58

97.40

113.65

108.18

106.41

8.1

8.6

2.5

1.00

1.00

1.00

1.00

4.05

4.25

4.90

4.40

16.58

19.84

21.19

19.20

97.60

86.61

91.27

91.83

8.0

8.6

7.0

1.00

1.00

1.00

1.00

3.66

4.00

4.19

3.95

16.86

18.45

20.06

18.46

85.73

91.30

79.40

85.48

7.6

8.5

19.5

1.00

1.00

1.00

1.00

4.09

3.10

4.15

3.78

15.42

13.88

13.13

14.14

70.04

68.48

47.39

61.97

7.0

8.1

54.7

1.00

1.00

1.00

1.00

2.91

2.71

3.37

3.00

7.33

7.14

7.75

7.41

17.41

18.86

19.65

18.64

6.2

7.4

153.1

1.00

1.00

1.00

1.00

3.29

3.06

3.29

3.21

7.24

6.38

7.00

6.87

14.97

15.15

17.64

15.92

7.9

7.9

428.6

1.00

1.00

1.00

1.00

2.54

2.45

2.70

2.56

5.14

4.31

4.97

4.81

7.90

7.53

7.12

7.52

7.6

7.8

1200.0

1.00

1.00

1.00

1.00

2.81

2.60

2.28

2.56

3.37

3.13

3.35

3.28

4.08

4.19

4.20

4.16

7.1

7.7

Table 2. Average specific growth rates and growth rates inhibitions.

Test concentrations (nominal concentrations) in mg/L

Average specific growth rate (days–1) - 0-72h

Mean

CV (%)

Growth rate inhibition (%)

Control

1.551

1.574

1.595

1.598

1.587

1.531

1.573

1.7

/

0.9

1.526

1.578

1.561

1.555

1.7

1.1

2.5

1.527

1.487

1.505

1.506

1.3

4.3

7.0

1.484

1.505

1.458

1.482

1.6

5.8

19.5

1.416

1.409

1.286

1.370

5.3

12.9

54.7

0.952

0.979

0.993

0.975

2.2

38.0

153.1

0.902

0.906

0.957

0.922

3.4

41.4

428.6

0.689

0.673

0.654

0.672

2.7

57.3

1200.0

0.469

0.478

0.478

0.475

1.1

69.8

Table 3. Stock solution and intermediate solution analysis

 

Nominal concentration
(mg/L)

Measured concentration (mg/L)

Stock solution

1 460

1 448.0

Intermediate solution

58.4

59.8

Table 4. Concentrations of TFA measured during the test

Nominal concentration (mg/L)

T0h

T72h
with algae

T72h
without algae

Measured concentration
(µg/L)

% Nominal concentration

Measured concentration
(µg/L)

% Nominal concentration

Measured concentration
(µg/L)

% Nominal concentration

Control

ND

/

ND

/

ND

/

0.9

1.0

113

0.9

99

0.9

103

2.5

2.7

108

2.7

106

2.7

108

7.0

7.3

105

7.2

103

7.2

103

19.5

20.2

103

20.1

103

19.8

101

54.7

55.7

102

55.5

101

55.0

101

153.1

157.1

103

158.0

103

158.1

103

428.6

436.7

102

439.3

103

438.9

102

1200.0

1208.7

101

1205.4

100

1208.4

101

Table 5. Mean measured concentration of trifluoroacetic acid in test concentrations

Nominal concentration (mg/L)

Mean measured concentration (mg/L)

% of nominal concentration

0.9

0.94

106

2.5

2.66

107

7.0

7.23

104

19.5

20.15

103

54.7

55.60

102

153.1

157.57

103

428.6

437.98

102

1200.0

1207.04

101

Validity criteria fulfilled:
yes
Remarks:
biomass of controls increased by a factor > 16, mean CV for section by section specific growth rates for control < 35% (mean CV: 14.7%), CV of average specific growth rates during the whole test period in replicate control cultures < 7% (CV: 1.7%)
Conclusions:
In a GLP-compliant and OECD guideline 201 toxicity study on the growth of the unicellular alga Pseudokirchneriella subcapitata, the following toxicity values have been determined for the test item trifluoroacetic acid based on nominal concentrations:
- NOEC: 2.5 mg/L
- ErC10 (72h): 5.59 mg/L
- ErC50 (72h): 237.07 mg/L
Executive summary:

In a GLP-compliant study conducted according to the procedure described in the OECD Guideline 201 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test", the effects of the test item trifluoroacetic acid on the growth of the unicellular green algal species Pseudokirchneriella subcapitata was investigated.

Green algae were exposed during 72h to the test item in static conditions. The initial nominal concentrations were 0.9 – 2.5 – 7.0 – 19.5 – 54.7 – 153.1 – 428.6 – 1200.0 mg/L (corresponding to the following geometric mean of measured concentration at T0h and at T72h: 0.9 – 2.7 – 7.2 – 20.2 – 55.6 – 157.6 – 438.0 – 1207.0 mg/L). The test concentrations were analytically monitored by ion-exchange chromatography with gradient elution and conductimetric detection. The test item has been satisfactorily maintained within ± 20 % of the nominal concentration in all test conditions. The endpoint was inhibition of growth, expressed as logarithmic increase of biomass (average specific growth rate) during the exposure period.

All validity criteria were fulfilled. In conclusion, the following toxicity values have been determined for the test item trifluoroacetic acid based on nominal concentrations:

- NOEC (72h): 2.5 mg/L

- ErC10 (72h): 5.59 mg/L

- ErC50 (72h): 237.07 mg/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 1996-05-31 to 1996-06-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD 201 guideline with GLP statement. Some deviation is reported but is not expected to impair the validity of the results. The concentrations were not measured during this study but the test substance was shown to be very stable in the marine algae medium during a previous study (Smith et al, 1994).
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
No chemical analyses were performed because the test substance was found to be stable in previous studies.
GLP compliance:
yes
Specific details on test material used for the study:
Aqueous solutions of trifluoroacetic acid have naturally low pH and for testing on organisms either the sodium salt or pH adjustment were required.
Sodium trifluoroacetate (TFANa, CAS RN: 2923-18-4) is appropriate as test material in replacement of trifluoroacetic acid because of the following elements :
Both substances are very soluble in water (water solubility at 25°C of 625 g/L for TFANa and 1520 g/L for TFA) and have a low potential of bioaccumulation (QSAR estimated LogKow values of -3.31 for TFANa and 0.79 for TFA). TFANa is a crystalline solid with a low vapour pressure (Vapour pressure at 25°C of 10E-5 Pa for TFANa estimated by QSAR) whereas TFA is a liquid with a medium vapour pressure (12.4 kPa at 20°C). Despite this different volatility, in the aquatic environment, both substances are ionized into the trifluoroacetate anion. This is justified by the fact that trifluoroacetic acid is a strong organic acid with a pKa of 0.23 meaning that it is under dissociated form in all environmental compartments.
Therefore, in the aquatic environment the bioavailability of the trifluoroacetate moiety should be the same for both substances. Additionally, as the toxicity of the cation Na is of low concern, the toxicity of the trifluoroacetate moiety is taken into account to assess the toxicity of TFA and TFANa.
Moreover, it should be noted that pH adjustment would be required when testing TFA in water due to pH effect on the organisms. Using the sodium salt of the acid is therefore a relevant approach.
The results obtained on TFANa are recalculated to be expressed in mg of TFA moiety solely.
Analytical monitoring:
no
Details on sampling:
- Concentrations: 0, and 117 mg/L
- Sampling method: Before the test initiation, the absorption (at 750 nm) of the preculture was determined by using a spectrophotometer. A sample of 10 mL of the preculture was taken and the absorption appeared to be 0.821. At test initiation, a quantity of 2.44 mL of the preculture was added to all erlenmeyers. This resulted in an initial absorption of 0.020 in the test solutions. Due to inoculation with the preculture the total test solution volume was 102.4 mL and therefore the nominal sodium trifluoroacetate concentration on the test solutions was 117 mg/L. At day 1, 2, 3, the absorption at 750 nm was measured in all erlenmeyers, using a spectrophotometer and a cuvette with a pathlength of 5 cm. The volume, used for measurement of the absportion (10 mL), was not returned to the erlenmeyers.
- Sample storage conditions before analysis: no data
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: three different stock solution with a concentration of 12 g/l were prepared:
1) 171 mg NaTFA was dissolved in 14.25 ml algal medium.
2) 146 mg NaTFA was dissolved in 12.17 ml algal medium.
3) 126 mg NaTFA was dissolved in 10.50 ml algal medium.
Test solutions were placed in 250-ml erlenmeyer flasks. Six control erlenmeyers (no. 1-6) were prepared which contained 100 ml algal medium. Hereafter, six erlenmeyers were each filled with 99ml algal medium. A volume of 1.0 mL stock solution 1 was added to erlenmeyers 7 and 8, 1.0 mL stock solution 2 was added to erlenmeyers 9 and 10. Finally, 1.0 mL stock solution 3 was added to erlenmeyers 11 and 12.
Before inoculation of test solutions with algae ,the erlenmeyers were placed in the thermostated shaking incubator for 1 hour.
- Eluate: no data
- Differential loading: no data
- Controls: 6 (no. 1-6)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc):no data
Test organisms (species):
Phaeodactylum tricornutum
Details on test organisms:
TEST ORGANISM
- Common name: marine alga
- Strain: CCAP 1052/1A
- Source (laboratory, culture collection): Dunstaffnage Marine Laboratory, P.O. Box 3, Oban, Argyll, PA34 4AD, Scotland.
- Age of inoculum (at test initiation): The preculture used for this study was transferred into fresh algal medium 3 days prior to test initiation.
- Method of cultivation: The algae were cultured in a 100-ml erlenmeyer with 50ml algal medium. Each week, an inoculum of this culture was transferred into fresh algal medium. The culture chamber was illuminated 24 hours a day with fluorescent lamps.

ACCLIMATION
- Acclimation period: continuously during the test
- Culturing media and conditions (same as test or not): same as test. The erlenmeyers were placed in a shaking incubator for 1 hour. The shaking speed in the incubator was 80-120 rotations per minute. These erlenmeyers were illuminated continuously during the experiment with fluorescent lamps.
- Any deformed or abnormal cells observed: no data
Test type:
static
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
Limit test at 117 mg/l
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
21-22°C
pH:
at the sart of the test: 7.8
at the end of the test: 8.9-9.1
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Nominal concentrations : 0 and 117mg/l
Measured concentrations : none. No chemical analyses were performed because the test substance is stable and very water soluble.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyers
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: 100 mL
- Aeration: no data
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): no data
- Initial cells density: no data
- Control end cells density: no data
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: water prepared by reversed osmosis, followed by anion, cation and mixed bed chromatography and filtered through a 0.22mm cellulose filter; the water is prepared according to Local Release Specification 1322 830 78115.
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: the conductivity of the water is less than 4 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: none
- Photoperiod: continuously during the experiment
- Light intensity and quality: At day 0, the light intensity was measured = 80-120 µE/(m2.s)
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At 24, 48, and 72 hours after test initiation, 3 the absorption at 750 nm was measured in all erlenmeyers, using a spectrophotometer (Hitachi U-2000) and a cuvette with a pathlength of 5 cm. The volume, used for measurement of the absorption (10 ml), was not returned to the erlenmeyers.
- Chlorophyll measurement: no data

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: no data
- Range finding study: none
- Test concentrations: 0 and 117 mg/l
- Results used to determine the conditions for the definitive study: Previous tests showed that NaTFA is generally not toxic to algae and for this reason a limit test was performed at a concentration of 117 mg/l.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
117 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NaTFA
Basis for effect:
growth rate
Remarks:
and biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
97 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
TFA
Basis for effect:
growth rate
Remarks:
and biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 117 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NaTFA
Basis for effect:
growth rate
Remarks:
and biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 97 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
TFA
Basis for effect:
growth rate
Remarks:
and biomass
Details on results:
- Exponential growth in the control (for algal test): yes, see table 2 in "any other information on results incl. tables" field.
- Observation of abnormalities (for algal test): no data
- Unusual cell shape: no data
- Colour differences: no data
- Flocculation:no data
- Adherence to test vessels: no data
- Aggregation of algal cells: no data
- Any stimulation of growth found in any treatment: no data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: no data
- Other : none.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The biomass integral data at test termination were analysed with Student's t-test (two-sided). No statistical significant difference in biomass integral (p = 0.15) was found and therefore a nominal concentration of 117 mg/L can be considered a NOEC.

Table: Biomass integral and growth rate during the test period.

Nominal concentration (mg/L) Replicates Biomass integral Growth rate (day-1)
Day 0-1 Day 0-2 Day 0-3 Day 0-3
0 1 0.045 0.318 1.097 1.34
2 0.047 0.325 1.105 1.34
3 0.041 0.299 1.076 1.35
4 0.045 0.325 1.122 1.35
5 0.044 0.319 1.161 1.38
6 0.041 0.293 1.062 1.35
Mean 0.044 0.313 1.104 1.35
117 1 0.048 0.33 1.095 1.33
2 0.044 0.314 1.089 1.34
3 0.045 0.315 1.102 1.35
4 0.041 0.306 1.088 1.35
5 0.041 0.311 1.119 1.36
6 0.046 0.302 1.037 1.33
Mean 0.044 0.313 1.088 1.34
Validity criteria fulfilled:
yes
Remarks:
criteria of the version of 1984 of the OECD TG
Conclusions:
The toxicity of sodium trifluoroacetate to algae (Phaeodactylum tricornutum) was tested according to OECD guideline 201 (1984) and OECD GLP guidelines (1981). The EbC50 (72h) and ErC50 (72h) were > 97 mg/L of TFA.
Executive summary:

The toxicity of sodium trifluoroacetate to algae (Phaeodactylum tricornutum) was tested according to OECD guideline 201 (1984) and according to OECD GLP guidelines (1981). Previous tests showed that sodium trifluoroacetate is generally not toxic to algae and for this reason a limit test was performed at a concentration of 117 mg/L. Algae were exposed for three days using erlenmeyers containing 102 mL test solution. No chemical analyses were performed because the test substance is stable and very water soluble. Degradation and/or adsorption are not likely to occur. To determine the growth of the algae, the absorption of the test solutions at 750 nm was measured daily in each erlenmeyer during the test. The light absorption at 750 nm is considered to be a parameter for the algal cell density of the test solutions. The results of the test showed that the absorption at 750 nm increased 56 -62 times in the control solutions which meets the validity criterion of a minimal 16 times increase. The absorption data of each erlenmeyer were used to calculate the biomass integral and the growth rate for each erlenmeyer. The biomass integral data at test termination were analysed with Student's t-test (two sided). No statistical significant difference in biomass integral between the control and the exposed algae was found (p=0.42) and therefore a nominal concentration of 117 mg sodium trifluoroacetate per liter can be considered a No Observed Effect Concentration (NOEC) for the algae.

Description of key information

Data of various reliability are available for seven freshwater species and three marine water species. Selenastrum capricornutum was the only species showing adverse effects.

For marine algae no ErC50 can be derived and the lowest NOEC value of 97 mg/l was defined for Phaedactylum tricornutum.

Key value for chemical safety assessment

EC50 for freshwater algae:
237 mg/L
EC10 or NOEC for freshwater algae:
5.6 mg/L
EC10 or NOEC for marine water algae:
97 mg/L

Additional information

Algal toxicity data of various reliability, produced from international guidelines under GLP or not, are available for seven freshwater species and three marine water species (see Table below). In aqueous solution, the pH of the substance is naturally low (pH<4) and for testing on organisms either the sodium salt (NaTFA) or pH adjustment were required.

Not all studies were performed with analytical monitoring. But the studies including chemical analysis confirmed the stability of the test substance during freshwater and marine algae tests and therefore high stability during other algae tests was postulated.

Among all the species tested, a toxic effect (growth inhibition) was found only for Pseudokirchneriella subcapitata, (formerly known as Selenastrum capricornutum). These effects have been demonstrated in several studies giving ErC50 in a range of 6.4 – 237 mg/L. Due to the high variability of the toxicity results, the available data have been closely assessed in line with the validity criteria of the OECD TG 201 (OECD Testing Guideline 201, last version adopted on the 23 March 2006) and using the CRED (Criteria for Reporting and Evaluating Ecotoxicity Data) evaluation method (Moermond et al., 2016).

The study of Groeneveld A.H.C., de Kok H.A.M. and van den Berg G. (1992) was performed in 1992 with a GLP statement and according to the OECD TG 201 adopted in 1984. The experiment was scheduled for a duration of 4 days, but due to unexpectedly high growth of the algae in the control, the pH had increased from 7.3 at the start of the test to 9.4 after 3 days and therefore the study was not continued after 3 days. This high increase of pH observed in the control medium has also been observed in the lowest tested concentration (i.e. from 7.3 to 9.2). On one hand, a high pH could limit the growth of algae and therefore can mask the potential effect of the substance. On the other hand, a high increase of pH can be the result of an insufficient aeration demonstrating the absence of optimal growth conditions and therefore can enhance the stress generated by the test substance. Even if this criterion is not reported in the performance criteria of the guideline, it is however indicated that “The pH of the control medium should not increase by more than 1.5 units during the test”. Therefore, there are uncertainties on the reliability of this study which showed an EC 50 of 133 mg/L based on growth rate inhibition.

The study of Berends A.G. and Molenaar J.A. (1993) was performed in 1992 with a GLP statement and according to the OECD TG 201 adopted in 1984. The objective of this study was to determine the effects of TFA at low concentrations in order to clarify the results of the previous study performed in the same laboratory (i.e. Groeneveld et al., 1992, discussed above) which showed 11% of the inhibition of the biomass at the lowest tested concentration of 0.3 mg/L. This additional study was performed at concentrations ranging from 0.03 to 1 mg/L in order to determine the NOEC based on biomass inhibition. The highest concentration tested resulted in an inhibition of only 6% of the growth rate which doesn't allow the derivation of robust NOEC, EC10 or EC50 values for growth rate. It is noted that no NOEC and EC10 based on the growth rate inhibition were reported in the study. This study can be considered as not relevant for the derivation of PNECwater because no results on the growth rate can be derived.

The study of Groeneveld A.H.C. and Berends A.G. (1995) was performed in 1995 with a GLP statement and according to the OECD TG 201 adopted in 1984. The objective of this study was to compare the toxicity of sodium trifluoroacetate, difluoroacetic acid, sodium monofluoroacetate and sodium fluoride to the alga Pseudokirchneriella subcapitata by comparison of rough estimation of EC50. Therefore, even if this study complies with the validity criteria of the OECD TG 201 published in 1984, its objective was not to determine NOEC or EC10 value, and the study design may not be adequate for this determination. This is supported by the fact that only 4 concentrations were tested and separated by a large ratio of 10, although the OECD TG 201 indicates at least 5 concentrations arranged in a geometric series with a factor not exceeding 3.2. Additionally, only 2 replicates were performed per test concentration while the OECD TG 201 requests 3 replicates. These elements show that the study design is not appropriate to obtain statistically reliable results, what is also indicated in the study report itself as follows: “Due to the large ratio between the test concentrations (10) and the low number of replicates (only 2 erlenmeyers per concentration) the EC50 values can only be considered as rough estimates”. It is also noted that the reliability criterion of the current version of the OECD TG 201 on low variability of the growth rate for section-by-section in the control is not fulfilled. This indicates that there are uncertainties on the reliability of this study which is not appropriate for the derivation of PNECwater because no robust EC10 or NOEC on the growth rate can be derived.

The study of Thiebaud H. (1996) was performed in 1996 according to the OECD TG 201 adopted in 1984. However, 6 concentrations were tested with a non constant separating factor ranging from 2 to 10 and only 3 replicates were performed for the control. These elements show that the study design may not be appropriate to obtain statistically reliable results. Similarly to the previous study, the reliability criterion of the current version of the OECD TG 201 on low variability of the growth rate for section-by-section in the control is not fulfilled. Therefore, there are uncertainties on the reliability of this study which showed an EC50 and an EC10 based on the growth rate inhibition of 6.4 mg/l an 1 mg/L respectively. 

 The study of Barthel Y. (2008) was performed in 2008 according to the norm NF EN ISO 8692 2005. Although some information is lacking to enable a full reliability assessment (i.e. source of the inoculum) and the number of replicates in the control is 5 instead of 6 recommended in the OECD TG 201, it can still be assumed with reasonable certainty that the results are reliable. The EC50 and EC10 based on the growth rate inhibition were determined to be 145 mg/l and 15.4 mg/L respectively. It is recognized that these results being more than 10 times higher than the results of the study of Thiebaud H. (1996), they need to be confirmed, what was done in the study of INERIS (2017).

A new GLP study has been performed at INERIS in 2017 (Chabot L., 2017), in compliance with the most recent version of the OECD TG 201 (OECD Testing Guideline 201, last version adopted on the 23 March 2006). In pre-tests, the following parameters of the test conditions have been varied to check if they can explain the difference of results observed between the available studies :

• the method of pH adjustment to reach relevant pH for algae growth,

• the composition of the test medium by comparing two different growth media,

• the method applied for algae cells counting: fluorimetry or particle counter.

None of the above parameters influenced significantly the effects observed and the preliminary results were as follows :72h-ErC50 = 604.8 mg/L; 72h-ErC10 = 22.7 mg/L; 72h-NOEC = 10 mg/L

 

Based on these preliminary results, the final study was performed with 8 test concentrations ranging from 0.9 to 1200 mg/L. The pH of the test solutions from 0.9 to 54.7 mg/L was above 6 and therefore was not adjusted. The test solutions of concentrations from 153.1 to 1200 mg/L were prepared using a pH adjusted stock solution of 1460 mg/L. Analytical verification showed a good correlation between the nominal and the measured concentrations. Therefore, the results were based on nominal concentrations.

The following results were determined based on the inhibition of the growth rate of the algal cells: 72h-ErC50 = 237 mg/L; 72h-ErC10 = 5.6 mg/L; 72h-NOEC = 2.5 mg/L

All validity criteria were met, therefore the test is considered to be reliable without restrictions. This study confirms that Pseudokirchneriella subcapitata is the most sensitive species tested to TFA.

This new study being assessed as reliable without restrictions is considered as the key study. The studies of Thiebaud H. (1996) and Barthel Y. (2008) provide ErC10 values of 1 mg/L and 15.4 mg/L respectively. Although of lower reliability, the mean value of these results is 8.2 mg/L which is consistent with the ErC10 value of the key study. Therefore it can be concluded that the results of the key study are relevant for the determination of the aquatic PNEC.

Concerning the remaining studies, although they are supporting the conclusion that TFA has toxic effects on the growth of Pseudokirchneriella subcapitata, their design do not enable the determination of ErC10 values. They are therefore not considered for the derivation of the PNEC.

No toxicity (at concentration up to 2 g/l) was found for the three marine algae tested. Then no ErC50 can be derived and the lowest NOEC value corresponds to the lowest tested concentration of 97 mg/l on the diatom Phaedactylum tricornutum.

Several supporting studies investigate the mode of action of TFA on Selenastrum capricornutum. An effect on the chlorophyll content was found but it is unknown if this is a primary or secondary effect. Another study showed that the exposure to high concentrations (up to 999 mg/l) of TFA does not result in mortality of the algae because a recovery of the growth was observed when the substance was removed from the test solutions. Further, it is hypothesised that the activated products of TFA could result in inhibition of the citric acid cycle according to two possible routes: the defluorination to monofluoroacetate (MFA) which is highly toxic to the algae and/or a direct effect on acetyl CoA which would result in the formation of difluorocitrate, that could inhibit aconitase. If activated products of TFA are inhibitors of the citric acid cycle, the enzymatic differences could explain the variation in toxicity of TFA between species. But there is only some evidence that the mode of action of TFA is inhibition aconitase. Moreover, possible accumulation of TFA in the algae cells from the media and conversion of TFA to DFA or MFA are not clearly demonstrated.

Freshwater species

Species

NOEC growth rate

EC10 growth rate

EC50 growth rate

Method

Reference

ESR name

Pseudokirchneriella subcapitata

2.5 mg/L

5.6 mg/L

237 mg/L

OECD TG 201 (2006)

Chabot L. 2017

P.suba.V1 2017INERIS

Pseudokirchneriella subcapitata

6 mg/L

15.4 mg/L

145 mg/L

NF-EN-ISO 8692 (2005)

Barthel Y. 2008

P.suba.V2 2008IPL

Pseudokirchneriella subcapitata

n.d.

1 mg/L

6.4 mg/L

OECD TG 201 (1984)

Thiebaud H. 1996

P.suba.V2 1996ElfAto

Pseudokirchneriella subcapitata

n.d.

n.d.

11.4 mg/L

OECD TG 201 (1984)

Groeneveld A.H.C., Berends A.G. 1995

P.suba.V2 1995Solvay

Pseudokirchneriella subcapitata

n.d.

n.d.

n.d.

OECD TG 201 (1984)

Berends A.G. and Molenaar J.A. 1993

P.suba.V2 1993Solvay

Pseudokirchneriella subcapitata

n.d.

n.d.

133 mg/L

OECD TG 201 (1984)

Groeneveld AHC et al. 1992

P.suba.V2 1992Solvay

Chlorella vulgaris

≥ 999 mg/L

n.d.

> 999 mg/L

OECD TG 201 (1984)

Berends A.G. 1993

C.vulga. V2 1993Solvay

Desmodesmus subspicatus

n.d.

n.d.

> 99.9 mg/L

OECD TG 201 (1984)

Berends A.G. et al. 1995

D.subspi. V2 1995Solvay

Clamydomonas reinhardtii

≥ 99.9 mg/L

n.d.

> 99.9 mg/L

OECD TG 201 (1984)

Berends A.G. 1995 and Groeneveld A.H.C., 1996

C.reinhard. V1 1996Solvay

Microcystis aeruginosa

≥ 97 mg/L

n.d.

> 97 mg/L

OECD TG 201 (1984)

Berends A.G. 1995 and Groeneveld A.H.C., 1996

M.aerugi. V2 1996Solvay

Navicula pelliculosa

499 mg/L

n.d.

1997 mg/L

OECD TG 201 (1984)

Smyth D.V. et al., 1994

N.pellic. V2 1994Zeneca

Anabaena flos-aquae

499 mg/L

n.d.

> 1997 mg/L

OECD TG 201 (1984)

Smyth D.V. et al., 1994

A.flos-a. V1 1994Zeneca

Marine species

Species

NOEC growth rate

EC10 growth rate

EC50 growth rate

Method

Reference

ESR name

Dunaliella tertiolecta

< 103 mg/L

n.d.

> 103 mg/L

OECD TG 201 (1984)

Berends A.G. 1996

D.tertiolecta. V1 1996Solvay

Phaeodactylum tricornutum

≥ 97 mg/L

n.d.

> 97 mg/L

OECD TG 201 (1984)

Berends A.G. 1996

Ph.tricor. V1 1996Solvay

Skeletonema costatum

≥ 1997 mg/L

n.d.

> 1997 mg/L

OECD TG 201 (1984)

Smyth D.V. et al., 1994

S.costa. V1 1994Zeneca

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Berends A.G. and J. A. Molenaar (1993). The toxicity of sodium trifluoroacetate to the alga selenastrum capricornutum at low concentrations. Testing laboratory: SOLVAY DUPHAR B. V., Environmental Research Department, Noordereinde 56, 1243 JJ 's-Graveland, The Netherlands. Report no.: 56635/61/92. Owner company: SOLVAY DUPHAR B. V. Study number: C. SOL.51.045. Report date: 1993-04-15.

Chabot L. (2017). ALGA, GROWTH INHIBITION TEST Effect of the trifluoroacetic acid on the growth of the unicellular alga Pseudokirchneriella subcapitata, according to OECD guideline 201.Testing Laboratory : INERIS, Parc Technologique ALATA, BP 2, 60550 Verneuil-En-Halatte, France.Owner Company : RHODIA OPERATIONS – SOLVAY. Study number: 17-005-167094. Report date : 2017-09-27.

Groeneveld A. H. C., Berends A. G. (1995). A comparison of the toxicity of sodium trifluoroacetate, difluoroacetic acid, sodium monofluoroacetate and sodium fluoride to the alga selenastrum capricornutum. Testing laboratory: Solvay Duphar B. V. Report no.: 56835/44/94. Owner company: Solvay. Study number: C. SOL.51.062. Report date: 1995-01-31.

Groeneveld AHC, de Kok HAM and G van den Berg (1992). The toxicity of sodium trifluoroacetate to the alga Selenastrum capricornutum. Testing laboratory: SOLVAY DUPHAR B. V. Environmental Research Department. Noordereinde 56. 1243 JJ 's-Graveland. The Netherlands. Report no.: 56635/52/92. Owner company: SOLVAY. Study number: C. SOL.51.038. Report date: 1992-10-15.

Moermond, C.T., Kase, R., Korkaric, M., Ågerstrand, M. (2016) CRED: Criteria for reporting and evaluating ecotoxicity data. Environmental Toxicology and Chemistry, 35, 1297–1309.

THIEBAUD H. (1996). Toxicity of sodium trifluoroacetate to the alga Raphidocelis subcapitata.Testing laboratory: ELF ATOCHEM, Centre d'application de Levallois, Service analyse environnement, 95 rue Danton, 92300 LEVALLOIS-PERRET.Report no.: 96/SAE6/1398/HT. Report date: 1996-09-05.