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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 April 2017 - 07 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted 23 March 2006; Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Inspection dates: 7 - 11, 14 and 16 September 2015. Date of the certificate: 03 November 2015.
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Appearence: clear colourless liquid
- Test item storage: in refrigerator (2-8°C)
Analytical monitoring:
yes
Details on sampling:
- Nominal test concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L. Exact volumes of the test item (84 µL per litre) were pipetted just below the surface and then a 10-minute period of low intensity stirring was applied to ensure homogenisation of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
- Controls: Test medium without test item or other additives
- Chemical name of vehicle: No vehicle
- Concentration of vehicle in test medium: No vehicle
- Evidence of undissolved material: Completely soluble in test medium at the concentrations tested
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Not specified (initial algal cell density: 10E4 cells/mL)
- Method of cultivation: Fresh water culture

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/mL.
- Culturing media and conditions (same as test or not): The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Light intensity: 60 to 120 µE/m2 /s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
21-24°C
pH:
7.1
Nominal and measured concentrations:
Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Measured concentrations (t= 0h): 0.252, 0.758, 2.52, 2.49, 7.47, 23.5 and 69.6 mg/L
Measured concentrations (t= 72h): 0.271, 0.793, 2.43, 2.42, 7.73, 21.8 and 69.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL, purge & trap vials
- Type: closed
- Material, size, headspace, fill volume: containing 40 mL of test solution. Closed, no headspace.
- Aeration: contenuously aerated
- Initial cells density: 1 x 10E4 cells/mL
- Control end cells density: Yes, test medium without test item or other additives
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes, standard OECD medium (only the stock culture medium changed)
- Detailed composition is provided in section "Attached background material"

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, standard OECD medium
- Source/preparation of dilution water: Tap water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA)
- Culture medium different from test medium: yes
- Intervals of water quality measurement: Not specified

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes
- Photoperiod: Continuously illuminated using TLD-lamps
- Light intensity and quality: range of 88 to 92 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm. Algal medium was used as blank.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the control and the test concentration closest to the to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 3 (nominal concentrations)

COMBINED/RANGE-FINDING STUDY
- Limit test concentration: 100 mg/L
- Test concentrations: 0.10, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: Effects on algal growth increased with increasing concentration from 1.0 mg/L upwards. Based on these results samples taken from nominally 1.0 and 100 mg/L were analysed. The initial measured concentrations were 1.0 and 76 mg/L for 1.0 and 100 mg/L, respectively. Both concentrations remained stable during the test period (92-101% of initial). Furthermore the expected EC50 for both growth rate and yield inhibition was between 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.26 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%-CI: 18-20 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95%-CI: 4.5-6.7
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%-CI: 60-64 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95%-CI: 18-22 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 23 mg/L (effects > 10%) when compared to the control.
Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ErC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.2 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EyC50) was 0.43 mg/L with a 95% confidence interval ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller

Growth rate and percentage inhibition for the total test period

 1,1,1 -trifluoroacetone concentration average (mg/L)  Mean  Std. Dev.  n  %Inhibition

Control

0.26

0.78

2.5

7.6

23

69

1.760

1.755

1.736

1.734

1.692

1.527

0.796 

0.0192

0.0273

0.0148

0.0173

0.0205

0.0207

0.0185 

6

3

3

3

3

3

0.3

1.4

1.5

3.8*#

13.2*

54.8* 

*: effect was statistically significant

#: effect biologically not relevant (< 10%)

Summary of the effect parameters

  Parameter (mg/L)

NOEC1 

NOEC2  EC10  EC20  EC50 

Growth rate

Value

lower 95%-CI

upper 95%-CI

2.5 

7.6 

19

18

20 

29

27

30 

62

60

64 

Yield 

Value

lower 95%-CI

upper 95%-CI 

0.26 

2.5 

5.6

4.5

6.7 

8.7

7.4

10 

20

18

22 

1 NOEC based on statistical significance

2 NOEC based on biological relevance

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, 1,1,1-trifluoroacetone reduced growth rate of this fresh water algae species statistically significantly at 7.6 mg/L and higher. The EC50 for growth rate inhibition (72h-ErC50) was 62 mg/L and the EC10 for growth rate inhibition (72h-ErC10) was 19 mg/L.
Executive summary:

The ability to produce toxic effects of 1,1,1-trifluoroacetone in Pseudokirchneriella subcapitata during an exposure period of 72 hours was evaluated using the Fresh water algal growth inhibition test according to the OECD guideline No. 201 and under GLP compliance.

A final EC50 test was performed based on the results obtained in a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal test item concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal concentrations). The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the exposure period. Analysis of the samples taken at the start and at the end of the final test allowed calculating the average exposure concentrations of 0.26, 0.78, 2.5, 7.6, 23 and 69 mg/L.

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, 1,1,1-trifluoroacetone reduced growth rate of this fresh water algae species statistically significantly at 7.6 mg/L and higher.

Based on the growth rate inhibition, the effect parameters obtained were:

- 72h-ErC10 = 19 mg/L with a 95% confidence interval ranging from 18 to 20 mg/L

- 72h-ErC50 = 62 mg/L with a 95% confidence interval ranging from 60 to 64 mg/L

- 72h-NOEC = 2.5 mg/L

Based on the yield inhibition, the effect parameters obtained were:

- 72h-EyC10 = 5.6 mg/L with a 95% confidence interval ranging from 4.5 to 6.7 mg/L

- 72h-EyC50 = 20 mg/L with a 95% confidence interval ranging from 18 to 22 mg/L

- 72h-NOEC = 0.26 mg/L

Results are based on average exposure concentrations and statistical analysis.

Description of key information

Harmful to aquatic plant (OECD 201, GLP compliant, Key Rel. 1)

Key value for chemical safety assessment

EC50 for freshwater algae:
62 mg/L
EC10 or NOEC for freshwater algae:
19 mg/L

Additional information

One key study was identified for acute toxicity to aquatic plant in freshwater .

The ability to produce toxic effects of 1,1,1-trifluoroacetone in Pseudokirchneriella subcapitata during an exposure period of 72 hours was evaluated using the Fresh water algal growth inhibition test according to the OECD guideline No. 201 and under GLP compliance.

A final EC50 test was performed based on the results obtained in a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal test item concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal concentrations). The initial algal cell density was 104cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the exposure period. Analysis of the samples taken at the start and at the end of the final test allowed calculating the average exposure concentrations of 0.26, 0.78, 2.5, 7.6, 23 and 69 mg/L.

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, 1,1,1-trifluoroacetone reduced growth rate of this fresh water algae species statistically significantly at 7.6 mg/L and higher.

Based on the growth rate inhibition, the effect parameters obtained were:

- 72h-ErC10 = 19 mg/L with a 95% confidence interval ranging from 18 to 20 mg/L

- 72h-ErC50 = 62 mg/L with a 95% confidence interval ranging from 60 to 64 mg/L

- 72h-NOEC = 2.5 mg/L

Based on the yield inhibition, the effect parameters obtained were:

- 72h-EyC10 = 5.6 mg/L with a 95% confidence interval ranging from 4.5 to 6.7 mg/L

- 72h-EyC50 = 20 mg/L with a 95% confidence interval ranging from 18 to 22 mg/L

- 72h-NOEC = 0.26 mg/L

Results are based on average exposure concentrations and statistical analysis.