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Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1993-05-05 to 1993-05-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: ASTM (1991) E1415-91 Standard guide for conducting static toxicity tests with Lemna gibba G3.
Principles of method if other than guideline:
no data
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:
yes
Details on sampling:
- Concentrations: The following nominal concentrations were tested : control, 19, 38, 75, 150, 300, 600, 1200 and 2400 mg/L
- Sampling method: Samples of each test solution were taken at the start of the test, using the excess remaining after filling the test vessels, to determine the concentration of the test substance by radiochemical analysis (liquid scintillation counting). At the end of the est, each replicate solution was analysed. The dry tissues from each replicate vessel were analysed for 14C residues, by liquid scintillation counting following sample oxidation. The fresh weight and dry weight of approximately 390 fronds (excess remaining after inoculation of test vessels) were determined to provide a fresh weight/dry weight ratio.
- Sample storage conditions before analysis: not specified.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A primary stock solution was prepared containing 4.8 g of sodium trifluoroacetate and 0.0003 g of trifluoro[2-14C]acetic acid in 25 mL of deionised water (192,000 mg/L). The specific activity of this mixture was 1.0 GBq/µg. A volume (20mL) of the primary stock solution was filter (0.2 µm) sterilised and added to sterile culture medium to give a total volume of 1600 mL at a concentration of 2400 mg/L, which was the highest nominal concentration tested. The remaining test concentrations were prepared by the addition of aliquots of the nominal 2400 mg/L solution to sterile culture medium. Using aseptic techniques, 160 mL volumes of the appropriate test solution were dispensed to each of the triplicate test vessels and the remaining test solutions used for physical and chemical analysis. Each replicate test vessel was inoculated with 3 plants, each consisting of 4 fronds (total of 12 fronds)
- Eluate: no data
- Differential loading: no data
- Controls: The control consisted of culture medium only.
- 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):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: the duckweed Lemna gibba
- Strain: G3
- Source (laboratory, culture collection): University of Waterloo, Canada.
- Age of inoculum (at test initiation): no data
- Method of cultivation: axenic conditions

ACCLIMATION
- Acclimation period: The cultures were incubated at 25+/-1°C under continuous illumination using "warm-white" lights.
- Culturing media and conditions (same as test or not): same as test, and known as M-Hoagland's medium.
- Any deformed or abnormal cells observed: no data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
168 h
Remarks on exposure duration:
no remarks
Post exposure observation period:
none
Hardness:
no data
Test temperature:
The daily temperature measurements recorded, by thermometer, in the incubator ranged from 24.7 to 25.1°C.
pH:
The pH values was determined by a Corning Model 240 pH meter, at the start and finish of the test ranged from 4.6 to 4.7 at the start, and from 5.0 to 5.6 at the end of the study. See table 1 in "Any other information on materials and methods incl. tables" field.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
See table 2 in "Any other information on results incl. tables" field. The measured concentrations at the start of the test ranged from 102 to 113 % of the nominal values. At each nominal concentration the mean measured values at the end were the same as those at the start of the test.
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: yes, the cultures were incubated at 25+/-1°C under continuous illumination using "warm-white" lights
- Test vessel: The test vessels were borosilicate glass crystallising
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: glass, dishes of 400 mL nominal capacity with loose-fitting lids.
- Type of cover: no data
- Aeration: no data
- Agitation: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Control end cells density: 100 fronds in average
- No. of colonies per vessel: not applicable
- No. of fronds per vessel: 3 plants, each consisting of 4 fronds (total of 12 fronds per test vessel).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes, M-Hoagland's medium
- Detailed composition if non-standard medium was used: not applicable

SEDIMENT USED (for rooted aquatic vascular plants)
- Origin: not applicable
- Textural classification (% sand, %silt and clay): not applicable
- organic carbon (%): not applicable
- Geographic location: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: no data
- 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: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 9220 lux on test day 1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: On days 2, 5 and 7 the number of plants and the number of fronds were counted and recorded for each test vessel. All fronds which visibly projected beyond the edge of the parent frond were counted. See table 3 in "Any other information on results incl. tables" field and figure 1 in "illustration" field.
- Determination of biomass: At the end of the test (7days) the duckweed from each vessel was rinced briefly in distilled water and dried to constant weight at 60°C. The dry weight of the tissue was determined. See table 4 in "Any other information on results incl. tables" field.
- Determination of frond area: no specified
- Other: Any other symptoms of toxicity were recorded. The bioconcentration factor (BCF) after 7 days is also determined (See table 5 in "Any other information on results incl. tables" field) for each exposure concentration and was calculated as follows : Mean tissue concentration (mg/kg fresh weight) / Mean measured water concentration (mg/L).

RANGE-FINDING STUDY
- Test concentrations: no
- Results used to determine the conditions for the definitive study: no
Reference substance (positive control):
no
Duration:
168 h
Dose descriptor:
EC50
Effect conc.:
1 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NaTFA
Basis for effect:
frond number
Remarks on result:
other: 95% CL = 960 to 1200 mg/L
Duration:
168 h
Dose descriptor:
EC50
Effect conc.:
915 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
TFA
Basis for effect:
frond number
Duration:
168 h
Dose descriptor:
NOEC
Effect conc.:
300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NaTFA
Basis for effect:
frond number
Duration:
168 h
Dose descriptor:
NOEC
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
TFA
Basis for effect:
frond number
Duration:
168 h
Dose descriptor:
EC50
Effect conc.:
1 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NaTFA
Basis for effect:
biomass
Remarks on result:
other: 95% CL = 780 to 1900 mg/L
Duration:
168 h
Dose descriptor:
EC50
Effect conc.:
999 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
TFA
Basis for effect:
biomass
Duration:
168 h
Dose descriptor:
NOEC
Effect conc.:
300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NaTFA
Basis for effect:
biomass
Duration:
168 h
Dose descriptor:
NOEC
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
TFA
Basis for effect:
biomass
Details on results:
- Any visual signs of phytotoxicity (abnormalities): no data
- Decrease in frond size: no data
- Necrosis / chlorosis: no data
- Sinking of fronds: no data
- Other: From day 5 onwards, plants in the nominal 600, 1200 and 2400 mg/L exhibited pale, misshapen fronds with decreased root growth, compared with the control. There were no observed symptoms at or below a nominal concentration of 300 mg/L compared with the control.
- Any stimulation of growth found in any treatment: At nominal concentrations of 75 and 150 mg/L, the increase in number of fronds was significantly greater (p=0.05) than the control. This apparent stimulation should be interpreted with caution.
- 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
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
no data

Table 2 : Radiochemical analysis of test solutions

Nominal concentration (mg/L)

Measured concentration (mg/L)

Mean as % nominal

Day 0

Day 7

Mean

(days 0-7)

Reps

Mean

19

20

20

20

20

20

20

105

38

39

39

39

39

39

39

103

75

79

81

79

78

79

79

105

150

170

170

170

170

170

170

113

300

320

320

320

320

320

320

107

600

610

620

610

610

610

610

102

1200

1300

1300

1300

1300

1300

1300

108

2400

2500

2600

2400

2500

2500

2500

104

Table 3 : Increase in number of fronds

Nominal concentration (mg/L)

Increase in number of fronds (days 0 -7) #

% Inhibition

Rep A

Rep B

Rep C

Mean

Control

90

84

90

88

-

19

81

106

97

95

-

38

94

104

97

98

-

75

153

122

121

132

-

150

120

145

139

135

-

300

112

104

111

109

-

600

52

48

68

56*

36

1200

32

38

36

35*

60

2400

27

26

34

29*

67

# Increase = (No. of fronds at day 7 - No. of fronds (12) at day 0).

* Significant decrease (p=0.05, one-sided) from the control.

Table 4 : Dry weights

Nominal concentration (mg/L)

Tissue dry weight (day 7) (mg)

Mean increase # (days 0-7) (mg)

% Inhibition

Rep A

Rep B

Rep C

Mean

Control

15.4

14.2

19.7

16.4

14.4

-

19

14.7

20.6

19.0

18.1

16.1

-

38

15.2

19.6

17.5

17.4

15.4

-

75

27.8

18.5

19.4

21.9

19.9

-

150

16.8

21.1

21.2

19.7

17.7

-

300

14.9

14.1

13.5

14.2

12.2

15

600

11.6

10.5

11.1

11.1

9.1*

37

1200

8.5

9.3

9.3

9.0

7.0*

51

2400

7.6

7.5

8.3

7.8

5.8*

60

# Increase = (Dry weight at day 7 - estimated day 0 dry weight).

Dry weight at day 0 estimated from control dry weight to be 2.0 mg/12 fronds.

(Calculated from day 7 determination, (300) fronds weighing 49.3 mg).

* Significant decrease (p=0.05, one sided) from the control.

Table 5 : Tissue concentrations (based on 14C residues) and bioconcentration factors

Mean measured water concentration (mg/L)

Rep

Tissue concentration (mg/kg dry wt)

Mean

(mg/kg dry wt)

Mean

(mg/kg fresh wt)*

BCF**

20

A

B

C

600

570

560

580

31

1.6

39

A

B

C

1,100

1,100

980

1,100

58

1.5

79

A

B

C

1,700

1,900

2,300

2,000

110

1.4

170

A

B

C

5,200

3,600

4,200

4,300

230

1.4

320

A

B

C

10,000

9,300

7,900

9,100

480

1.5

610

A

B

C

14,000

15,000

16,000

15,000

790

1.3

1300

A

B

C

24,000

29,000

25,000

26,000

1,400

1.1

2500

A

B

C

47,000

53,000

48,000

49,000

2,600

1.0

* Calculated from fresh/dry weight ratio = 19

** BCF = Mean tissue concentration (mg/kg fresh wt) / Mean measured water concentration (mg/L)

Validity criteria fulfilled:
yes
Remarks:
the doubling time of frond number in the control is less than seven-fold increase in seven days
Conclusions:
The duckweed, Lemna gibba G3, was cultured in a range concentrations of sodium trifluoroacetate under static test conditions at 25°C for 7 days. The procedures employed were based on the relevant ASTM standard. The EC50 (frond increase) and EC50 (weight increase) were 915 and 999 mg/L of TFA, respectively.
= 1200 mg/L (95% Confidence Limits = 780 - 1900 mg/L)
Executive summary:

The duckweed, Lemna gibba G3, was cultured in a range concentrations of sodium trifluoroacetate under static test conditions at 25°C for 7 days. The following nominal concentrations tested were 19, 38, 75, 150, 300, 600, 1200 and 2400 mg/L. A control was performed with culture inoculum only. Triplicate cultures of the control and of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 3 plants, each consisting of 4 fronds (total of 12 fronds). The pH values determined at the start and finish of the test ranged from 4.6 to 4.7 at the start, and from 5.0 to 5.6 at the end of the study. The daily temperature measurements in the incubator ranged from 24.7 to 25.1°C. The light intensity was 9220 Lux. The test substance was mixed with 14C-labelled trifluoroacetic acid to enable radiochemical analysis of the test solutions at the start and finish, and analysis of the plant tissue at the end of the exposure. Moreover, the bioconcentration factor was determined for each measured concentrations. The measured concentrations at start of the test ranged from 102 to 113% of the nominal values. At each nominal concentration the mean measured values at the finish were the same as those at the start of the test. The number of fronds and the dry weights of tissue were determined during the test. Furthermore, other symptoms of toxicity were determined : from day 5 onwards, plants in the nominal 600,1200 and 2400 mg/L exhibited pale, misshapen fronds with decreased root growth, compared with the control. There were no observed symptoms at or below a nominal concentration of 300 mg/L compared with the control.

The EC50 for increase in frond number and increase in frond dry weight were as follows:

EC50 (frond increase) = 915 mg/L of TFA

EC50 (weight increase) = 999 mg/L of TFA

These values were based on nominal concentrations which were confirmed by radiochemical analysis. There were no significant (p=0.05) inhibitory effects on frond or weight increase, at a nominal concentration of 300 mg NaTFA/L (250 mg TFA/L). The tissues showed only slight bioconcentration of the test substance after 7 days, with bioconcentration factors ranging from 1.0 to 1.6, based on radiochemical analysis.

Description of key information

This endpoint is covered by two studies: a key study on the duckweed Lemna gibba, showing an EC50 frond number/weight of 915 mg TFA/L and a NOEC of 250 mg/l after 7 days of exposure. A supporting study in microcosms show no effect on somatic endpoints of TFA at a concentration up to 10 mg/l during 49 days of exposure.

Key value for chemical safety assessment

EC50 for freshwater plants:
915 mg/L
EC10 or NOEC for freshwater plants:
250 mg/L

Additional information

This endpoint is covered by two studies: a key study reliable without restriction on the duckweed Lemna gibba and a supporting study published on the macrophytes Myriophyllum spicatum and Myriophyllum sibiricum.

The key study, performed according to standard guideline and GLP, shows that the EC50 after 7 days of exposure for frond number and frond dry weight were 915 mg/L and 999 mg/L of TFA, respectively. There were no significant inhibitory effects at a concentration of 250 mg/L of TFA. The key value EC50 for chemical safety assessment on Lemna gibba was 915 mg/L of TFA.

Moreover, a non standard but reliable supporting study in microcosms on the aquatic macrophytes Myriophyllum spicatum and Myriophyllum sibiricum shows that no effect on somatic endpoints were observed after 49 days of exposure to TFA concentrations up to 10 mg/l . There were statistically significant effects of the Trichloroacetic acid (TCA) and TFA mixture on certain pigment concentrations immediately after the start of exposure (2 -7 days) but the plants showed no signs of stress thereafter.