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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:
5 June - 8 June 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test substance is reactive with water, but neither the parent material nor the potential degradation products other than trifluoroacetic acid were detected in the test solutions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
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
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report: MTDID 20422
- Substance type: Pure active substance
- Physical state: Volatile liquid (VP 87.5 kPa at 20 °C)
- Analytical purity: >99%
- Expiration date of the lot/batch: 16 Feb 2013
- Stability under test conditions: Reactive to water
- Storage condition of test material: Sealed container

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: 2 or 4 replicates of each group were taken for sampling (volume: 3 mL) at times 0h, 24h, 48h, and 72h. Samples at t = 0h were taken from excess prepared solution. Samples at 24h and 48h were taken from additional test vessels set aside for sample collection. Samples from 72h were taken from the test solutions. Duplicate samples were taken and analyzed at 24 hours.
- Sample storage conditions before analysis: Not applicable, samples were transported on dry ice to the test site and analysed on the day of sampling. Samples were analysed starting at 1.5 hours after arrival at the laboratory.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A volume of 64 µL CAS# 756-12-7 corresponding to 100 mg/L was injected into 1 L of test medium and magnetically stirred for 17 min, after which no test substance was visible in the medium. The flask was closed during stirring, but a small amount of headspace was present. Lower test concentrations were prepared by susbsequent dilutions of the highest concentration in test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None, final test solutions were all clear and colorless.

Test organisms

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): Three or four days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E+04 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
ACCLIMATION
- Acclimation period: None
- Culturing media and conditions (same as test or not): Pre-culture was maintained under the same conditions as used in the test.
- Any deformed or abnormal cells observed: not reported

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22.2 - 23.2 °C
pH:
7.2 - 10
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32, and 100 mg/L (CAS# 756-12-7)
Measured: 1, 2, 4, 10, and 31 mg/L (Degradation product Trifluoro acetic acid)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, air-tight closed, containing 75 ml of test solution (headspace reduced to minimum)
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking.
- Control end cells density: 64.9E+04 cells/ml
- No. of colonies per vessel: Initial cell density was 1E+04 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
1 replicate of the highest concentrations without algae
2 or 4 replicated of each group for sampling purposes

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used to make growth medium was purified by reverse osmosis.
- Culture medium different from test medium: yes, propagation in M1, test in adjusted M2 (NaHCO3 increased to 100 mg/L to compensate for sealed bottle test).
- Intervals of water quality measurement: pH at beginning, 24, 48, and 72 hours, temperature continuously

OTHER TEST CONDITIONS
- Adjustment of pH: no, the pH in a number of groups exceeded 9.0 at the end of the test due to accumulation of CO2 produced during exposure. Exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met. Therefore, it was assumed that this observation had no influence on the results of this study.
- Photoperiod: continuous illumination using 30 Watt 'Cool-white' TLD-Iamps. Light intensity within the range of 99 to 106 µE/(m²∙s)
-Other: Algae were introduced within 66 minutes after preparation of the initial stock solution

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Other: Cell counts at 0h, 24h, 48h, and 72h were determined by spectrophotometric measurement at 720 nm using a Shimadzu Spectrophotometer UV-1800 with immersion probe (UVProbe 2.33 software: Algal cell density) (pathlength =20 mm). Algal medium was used as blank.
- Cells were observed microscopically after the end of the 72-h exposure period in the two highest concentrations to check for any abnormal appearance of the algae.

RANGE-FINDING STUDY
- Test concentrations: Control and a range of 0.1 to 100 mg/l (nominal concentrations of CAS# 756-12-7) increasing by a factor of 10
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
98 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % C.I. 67.7 - 140 mg/L. Concentration is stoicheometric equivalent of measured TFA concentration.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.3 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Concentration is stoicheometric equivalent of measured TFA concentration.
Details on results:
- Exponential growth in the control (for algal test): yes/no: Yes (Figure 1)
- Growth in controls (validity criteria):
- cell density increase to hour 48: factor of >16
- % Coefficient of variance in average specific growth rate at hour 72: 2% (Table 1)
- % Coefficient of variance in section-specific growth rates to hour 72: 22% (Table 2)

- Any visual signs of phytotoxicity (abnormalities): Observation was done at the end of the test and no abnormalities were found.
- Any stimulation of growth found in any treatment: No Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: CAS#756-12-7 is reactive with water. The concentrations of CAS# 756-12-7, as well as the potential degradation products were measured. The concentrations of CAS# 756-12-7 and all degradation products except trifluoroacetic acid were below the limit of detection of the analytical method, i.e. 1 mg/L. The measured concentrations of the degradation product, trifluoroacetic acid remained stable during the entire test period (75-100% of initial) (Table 3). The effect parameters were calculated in terms of initially measured concentrations of trifluoro acetic acid. Effects concentrations were then expressed in terms of stoicheometric equivalent concentration of CASVAF#
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.98 mg/L, 95% C.I. 0.74 - 1.3 mg/L. The historical ranges for growth rate reduction for Pseudokirchneriella subcapitata lie between 0.82 and 2.3 mg/l. Hence, the growth rate reduction EC50: 72h for the Pseudokirchneriella subcapitata tested corresponds with this range.
Reported statistics and error estimates:
ANOVA, Bonferroni t-test and TOXSTAT Release 3.5 were utilized to evaluate statistically significant differences from the controls. Calculation of the EC50 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the initially measured concentrations of trifluoro acetic acid.

Any other information on results incl. tables

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

Measured Concentration, Trifluoroacetic acid, mg/L

(nominal concentration, CAS# 756-12-7, mg/L)

Vessel Number

Individual Cell Densities, 10^4 cells/mL

Yield

Yield Inhibition (%)

Growth Rate (µ)

Growth Rate Reduction (%)

Exposure Period (hours)

0

24

48

72

0 - 72 h

0 - 72 h

0 - 72 h

0 - 72 h

Control

1

1.00

4.95

23.13

61.84

60.84

0.05728

2

1.00

3.95

21.22

54.43

53.43

0.05551

3

1.00

4.21

24.79

74.00

73.00

0.05978

4

1.00

4.06

24.65

65.36

64.36

0.05805

5

1.00

3.86

23.15

65.18

64.18

0.05802

6

1.00

3.67

21.97

68.49

67.49

0.05870

Mean

63.88

0.05789

CV, 2%

1 (1)

1

1.00

3.68

20.32

58.96

57.96

9

0.05662

2

2

1.00

3.38

17.75

54.55

53.55

16

0.05554

4

3

1.00

2.60

21.07

56.94

55.94

12

0.05614

3

Mean

55.81

12.6

0.05610

3.1

2 (3.2)

1

1.00

1.93

14.76

44.94

43.94

31

0.05285

9

2

1.00

3.25

18.99

57.32

56.32

12

0.05623

3

3

1.00

3.20

16.62

52.46

51.46

19

0.05500

5

Mean

50.57

20.8

0.05469

5.5

4 (10)

1

1.00

2.51

12.27

34.90

33.90

47

0.04934

15

2

1.00

2.53

11.59

29.34

28.34

56

0.04693

19

3

1.00

2.60

10.98

33.79

32.79

49

0.04889

16

Mean

31.68

50.4

0.04839

16.4

10 (32)

1

1.00

2.07

8.56

18.10

17.10

73

0.04022

31

2

1.00

2.10

8.49

16.85

15.85

75

0.03922

32

3

1.00

1.80

9.28

19.12

18.12

72

0.04098

29

Mean

17.02

73.4

0.04014

30.7

31 (100)

1

1.00

2.67

7.05

9.80

8.80

86

0.03170

45

2

1.00

2.14

6.69

9.44

8.44

87

0.03118

46

3

1.00

2.33

7.36

10.51

9.51

85

0.03267

44

Mean

8.92

86.0

0.03185

45.0

Table 2: Section-by-section Growth Rates

Measured concentration, Trifluoroacetic acid, mg/L

(nominal concentration, CAS# 756-13-8, mg/L)

Vessel Number

Growth Rate (µ)

Growth Rate Reduction (%)

Exposure Period (hours)

Exposure Period (hours)

0 - 24h

24-48h

48-72h

0 - 24h

24-48h

48-72h

Control

1

0.06664

0.06425

0.04097

2

0.05720

0.07010

0.03925

3

0.05986

0.07391

0.04556

4

0.05837

0.07516

0.04063

5

0.05625

0.07466

0.04314

6

0.05414

0.07460

0.04738

Mean

0.05874

0.07211

0.04282

CV (%)

7

6

7

The mean CV for section-by-section specific growth rate was: 22%

1 (1)

1

0.05427

0.07122

0.04438

8

1

-4

2

0.05076

0.06909

0.04678

14

4

-9

3

0.03986

0.08712

0.04143

32

-21

3

Mean

0.04829

0.07581

0.04419

17.8

-5.1

-3.2

2 (3.2)

1

0.02742

0.08475

0.04639

53

-18

-8

2

0.04905

0.07361

0.04604

17

-2

-8

3

0.04850

0.06861

0.04789

17

5

-12

Mean

0.04166

0.07565

0.04677

29.1

-4.9

-9.2

4 (10)

1

0.03841

0.06606

0.04355

35

8

-2

2

0.03871

0.06339

0.03868

34

12

10

3

0.03976

0.06006

0.04685

32

17

-9

Mean

0.03896

0.06317

0.04303

33.7

12.4

-0.5

10 (32)

1

0.03039

0.05908

0.03117

48

18

27

2

0.03099

0.05814

0.02854

47

19

33

3

0.02451

0.06830

0.03013

58

5

30

Mean

0.02863

0.06184

0.02995

51.3

14.2

30.1

31 (100)

1

0.04098

0.04042

0.01370

30

44

68

2

0.03168

0.04752

0.01433

46

34

67

3

0.03523

0.04793

0.01487

40

34

65

Mean

0.03596

0.04529

0.01430

38.8

37.2

66.6

 

Table 3. Measured concentrations in the final test

Time of sampling (hours)

Nominal concentration, CAS# 756-12-7 (mg/L)

Analysed concentration, Trifluoroacetic acid (mg/l)

Relative to initial (%)

0

control

< LOD ¹

1

1

3.2

2

10

4

32

10

100

31

100 WA ¹

31

24

Control

< LOD

 

Control

< LOD

1.0

1

 

1.0

1 (1) ²

100

3.2

2

 

3.2

2 (2)

100

10

3

 

10

3 (3)

75

32

10

 

32

10 (10)

100

100

31

 

100

29 (30)

97

100 WA

31

 

100 WA

32 (32)

102

48

Control

< LOD

1.0

1

100

3.2

2

100

10

3

75

32

11

110

100

31

100

100 WA

30

97

72

Control

< LOD

1.0

1

100

3.2

2

100

10

4

100

32

10

100

100

31

100

100 WA

31

100

1, LOD – limit of detection (LOD=1 mg/l); WA – without algae

2, () – average concentration calculated on the basis of two measurements.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour EC50 of CAS# 756-12-7 to Pseudokirchneriella subcapitata is 98 mg/L. The 72-hour NOEC of CAS# 756-12-7 to Pseudokirchneriella subcapitata is 2.3 mg/L.
Executive summary:

The toxicity of CAS# 756-12-7 to the green algae, Pseudokirchneriella subcapitata, was assessed in a 72-hour toxicity test conducted according to the OECD 201 (2011) method. Test containers were sealed to avoid volatilization losses of test material, and growth medium was amended with 100 mg/L NaHCO3 to provide sufficient carbon dioxide and buffering to maintain exponential growth. An exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met.

CAS# 756-12-7 is reactive with water. The concentrations of parent, as well as the potential degradation products were measured. The concentrations of CAS# 756-12-7 and all degradation products except trifluoroacetic acid were below the limit of detection of the analytical method, i.e. 1 mg/L. The measured concentrations of the degradation product, trifluoroacetic acid (TFA), remained fairly stable during the entire test period (75-100% of initial). Based on the obtained results, the effect parameters were expressed in terms of initially measured concentrations of TFA. The measured concentrations for the degradation product, TFA were: 1, 2, 4, 10, and 31 mg/L. Effect concentrations for the parent were calculated from TFA using the molecular weight ratio on a stoicheometric basis.

The 72-hour ErC50 of CAS# 756-12-7 to Pseudokirchneriella subcapitata was 98 mg/L based on growth rate. Neither the parent material nor other degradation products were detected in the test solutions.

The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. However, neither the parent material nor degradation products, other than TFA, were detected in the test solutions. Therefore, this study is reliable with restrictions and the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis.

Results Synopsis

Test Type: static (based on data obtained using OECD201 methodology)

EC50: 98 mg/L 95% C.I.: 67.7 to 140 mg/L

NOEC: 2.3 mg/L