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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
Study period:
19 October 2010 - 22 October 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP. However, concentration of test substance decreased to below the LOD within 24 hours of exposure.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Temperature ranged between 21.2 and 24.1 deg C, thus slightly greater than a 2 deg C difference. Algal growth was not considered affected by this as confirmed by the growth in the control.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Series on Testing and Assessment No. 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report: MTDID 21721
- Physical state: liquid
- Lot/batch no.: TFEE5-276/12/09-1
- Expiration date of the lot/batch: 31 December 2012
- Storage condition of test material: Room temperature in the dark under nitrogen

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Sampling occured at 0, 24 and 72-hours. Three mL singular samples were taken from the center of the test vessels of all test concentrations and the control. Samples were taken in a 10 mL GC vial containing 3 mL of tertiary butyl methyl ether (TBME) and then shaken for 30 seconds prior to storage. At the end of the exposure period, the replicates were pooled at each concentration before sampling. Only the results from the blank control, the highest test concentration with algae, and the highest test concentration without algae were reported
- Sample storage conditions before analysis: Frozen after addition of TMBE.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Magnetic stirring for 25 hours was applied to a water solution prepared at a loading rate of 100 mg/L. Final solution was clear and colorless with a precipitate in the form of droplets. The solution was settled for 2 hours after which the water soluble fraction (WSF) was collected by siphoning from the middle. Three lower test concentration solutions were prepared by subsequent dilution of the WSF in test medium.

- Controls: Negative control (test medium without test substance or other additives)

- Evidence of undissolved material (e.g. precipitate, surface film, etc): None noted in final test solutions.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 4 days prior to test initiation, cells from algal stock culture were inoculated in culture medium at a cell density of 10,000 cells/mL.
- Method of cultivation: In M1 growth medium, continuously aerated and exposed to 60- 120 uE/m2/s in the wavelength range of 400 to 700 nm at 21 - 24 deg. C.

ACCLIMATION
- Acclimation period: 4 days in M2 pre-culture medium
- Culturing media and conditions (same as test or not): Same during acclimation period
- Any deformed or abnormal cells observed: None noted

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
Combined limit and range-finding test of a water soluble fraction
Post exposure observation period:
None

Test conditions

Hardness:
Not reported
Test temperature:
21.2 - 24.1 deg. C
pH:
8.1
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: Control, 0.10, 1.0, 10, 100% of a water soluble fraction (WSF). Only highest concentration (100% WSF) was analyzed for test substance concentration. At time 0, 100% WSF = 0.396 mg/L; at 24- and 72-hours 100% WSF = < LOD (<0.14 mg/L). Time Weighted Average exposure concentration over 72-hours was 0.10 mg/L in the 100% WSF solution.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass
- Type (delete if not applicable): open / closed: Not reported
- Material, size, headspace, fill volume: 50 mL
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Static
- Initial cells density: 10,000 cells/mL
- Control end cells density: 651,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes/no: Yes - M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by Milli-RO reverse osmosis and susequently passed over activated carbon and ion-exchange cartridges (Milli-Q water).
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no: Not reported
- Adjustment of pH: None
- Photoperiod: 24-hours light
- Light intensity and quality: Cool-white 30 watt TLD bulbs at 5184 - 5940 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Counting chamber at day 0, spectophotometer thereafter
- Other: Appearance of cells in highest test concentration evaluated by microscopic observation at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: Combined limit/range-finding test.
- Range finding study: combined with limit test
Reference substance (positive control):
yes

Results and discussion

Effect concentrations
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 72-hour ErC50 and EyC50 both exceeded a time-weighted average concentration of 0.10 mg/L, as measured in the WSF prepared at a loading rate of 100 mg/L.
Details on results:
- Exponential growth in the control (for algal test): yes/no: yes
- Observation of abnormalities (for algal test): Exposed cells were normal and healthy as compared to the control cells at the end of the test in the highest concentration tested.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test substance could not be detected in test solutions after 24 hours. The lowest detection limit was 0.14 mg/L. An exposure concentration of 0.10 mg/L in the 100% water soluble fraction was derived as the Time Weighted Average over 72-hours.
- Effect concentrations exceeding solubility of substance in test medium: No significant effects were seen at an initial loading of 100 mg/L. Slight effects observed in the 1.0 and 10% of WSF were not considered treatment related.
72-hr NOEC (growth and yield) = 100 mg/L (nominal loading).
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 1.4 (0.97 - 2.0) mg/L
- Other: Reference substance study conducted with potassium dichromate 21 September 2010 - 24 September 2010.
Reported statistics and error estimates:
Statistical analysis of the data not needed as the effects recorded were not significant (< 10%).

Any other information on results incl. tables

Table 1:  Cell Density, Yield Inhibition and Growth Rate Reductions

Initial Dilution of WSF*, %

Vessel Number

Individual Cell Densities, 104 cells/mL

Yield

Yield Inhibition (%)

Growth Rate (µ)

Growth Rate (µ) Reduction (%)

Exposure Period (hours)

0

24

48

72

0 - 72 h

0 - 72 h

0 - 72 h

0 - 72 h

Control

1

1.00

7.42

21.12

64.00

63.00

 

0.05776

 

2

1.00

7.25

19.53

65.18

64.18

 

0.05802

 

3

1.00

7.55

18.88

55.80

54.80

 

0.05586

 

4

1.00

7.75

19.60

59.90

58.90

 

0.05684

 

5

1.00

7.78

24.03

80.22

79.22

 

0.06090

 

6

1.00

7.32

13.71

65.55

65.55

 

0.05809

 

Mean

 

 

 

 

65.11

64.11

 

0.05791

 

CV (%)

 

 

 

 

 

 

 

3

 

0.1

1

1.00

7.11

18.44

56.03

55.03

14

0.05592

3

2

1.00

7.61

20.20

63.84

62.84

2

0.05773

0

3

1.00

8.03

23.55

75.32

74.32

-16

0.06002

-4

Mean

 

 

 

 

65.06

64.06

0.1

0.05789

0.0

1.0

1

1.00

7.68

20.12

61.36

60.36

6

0.05718

1

2

1.00

7.55

16.82

48.22

47.22

26

0.05383

7

3

1.00

7.79

20.69

65.68

64.68

-1

0.05812

0

Mean

 

 

 

 

58.42

57.42

10.4

0.05638

2.7

10

1

1.00

7.59

18.65

50.93

49.93

22

0.05459

6

2

1.00

7.67

17.78

50.92

49.92

22

0.05459

6

3

1.00

8.09

21.86

68.01

67.01

-5

0.05861

-1

Mean

 

 

 

 

56.62

55.62

13.2

0.05593

3.4

100**

1

1.00

7.89

19.18

57.11

56.11

12

0.05618

3

2

1.00

7.60

16.70

56.04

55.04

14

0.05592

3

3

1.00

8.04

22.34

69.17

68.17

-6

0.05884

-2

4

1.00

7.76

21.27

72.69

71.69

-12

0.05953

-3

5

1.00

7.59

18.96

61.37

60.37

6

0.05718

1

6

1.00

7.45

19.56

67.65

66.65

-4

0.05853

-1

Mean

 

 

 

 

64.01

63.01

1.7

0.05770

0.4

*WSF prepared at an initial loading rate of 100 mg/L.  **TWA concentration = 0.10 mg/L

Table 2: Section-by-section Growth Rates

Initial Dilution of WSF*, %

Vessel Number

Growth Rate (µ)

Growth Rate Reduction (%)

Exposure Period (hours)

0 - 24h

24-48h

48-72h

0 - 24h

24-48h

48-72h

Control

1

0.08351

0.04359

0.04619

 

 

 

2

0.08254

0.04129

0.05022

 

 

 

3

0.08423

0.03819

0.04515

 

 

 

4

0.08532

0.03866

0.04655

 

 

 

5

0.08548

0.04699

0.05023

 

 

 

6

0.08294

0.02615

0.06520

 

 

 

Mean

0.08400

0.03914

0.05059

 

 

 

CV (%)

1

18

15.00

 

 

 

The mean CV for section-by-section specific growth rate was:  35%

0.1

1

0.08173

0.03971

0.04631

3

-1

8

2

0.08456

0.04068

0.04795

-1

-4

5

3

0.08680

0.04483

0.04844

-3

-15

4

Mean

0.08436

0.04174

0.04757

-0.4

-6.6

6.0

1.0

1

0.08494

0.04013

0.04646

-1

-3

8

2

0.08423

0.03338

0.04388

0

15

13

3

0.08554

0.04070

0.04813

-2

-4

5

Mean

0.0849

0.03807

0.04616

-1.1

2.7

8.8

10

1

0.08445

0.03746

0.04186

-1

4

17

2

0.08489

0.03503

0.04384

-1

11

13

3

0.08711

0.04142

0.04729

-4

-6

7

Mean

0.08548

0.04115

0.04433

-1.8

-5.1

12.4

100**

1

0.08607

0.03701

0.04546

-2

5

10

2

0.08451

0.03280

0.05044

-1

16

0

3

0.08685

0.04258

0.04709

-3

-9

7

4

0.08537

0.04201

0.05120

-2

-7

-1

5

0.08445

0.03815

0.04894

-1

3

3

6

0.08368

0.04022

0.05170

0

-3

-2

Mean

0.08515

0.0388

0.04914

-1.4

0.9

2.9

*WSF prepared at an initial loading rate of 100 mg/L.  **TWA concentration = 0.10 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by an average factor of > 16 within two days. The mean COV of control sec.-by-sec. spec. growth rates was not > 35%. The COV of av. spec. growth rates in replicate control vessels during the entire test period was not > 7%.
Conclusions:
The 72-hour ErC50 of TFEE-5 is > 100 mg/L (nominal concentration of water soluble fraction)
Executive summary:

The toxicity of TFEE-5 to Pseudokirchneriella subcapitata was evaluated according to OECD TG201. Due to the low water solubility of this substance, toxicity was determined in a combination range-finder/ limit test using water soluble fractions. Test concentrations were 0.1, 1.0, 10, and 100% of a water soluble fraction prepared at 100 mg/L. No statistically significant reduction in yield or growth rate was observed after 72 hours of exposure to 100% of the water soluble fraction. Therefore, the 72-hour EC50 is > 100 mg/L. TFEE-5 is not toxic to Pseudokirchneriella subcapitata according to GHS criteria.

The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice standards. However, test substance concentration was not maintained throughout the 72-hour exposure period. Therefore, this study is reliable with restrictions.

Results Synopsis

Test Type: static

EC50: > 100 mg/L       95% C.I.: Not applicable

NOEC: 100 mg/L       Probit slope: not applicable