Registration Dossier

Diss Factsheets

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:
2011-08-09 to 2011-08-12
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-(cyclohexylimino)bisethanol
EC Number:
224-809-5
EC Name:
2,2'-(cyclohexylimino)bisethanol
Cas Number:
4500-29-2
Molecular formula:
C10H21NO2
IUPAC Name:
2,2'-(cyclohexylimino)bisethanol

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:All samples were stored at room temperature until start of analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water and agitated.
- Eluate: Dilution water
- Differential loading: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared and agitated.
- Eluate: Dilution water
- Differential loading: 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L
- Controls: Six replicated without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 22.5 °C, Min: 22 °C, Max: 23 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 9.11 8.10
32.0 8.72 8.06
10.0 8.44 8.49
3.20 8.11 8.79
1.00 8.01 8.82
0.320 7.98 8.72
Control 7.91 8.63
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to Any other information on materials and methods inc. tables
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 4910 cells/mL
- Control end cells density: Mean 683511 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 hours)

Nominal
Test Item Concentration [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 92 100
10.0 8 32
1.00 2 11
0.100 - 1 - 4
100 (neutralized) 95 100









Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: (8,8-21)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
8.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: (5-19 mg/L)
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: Yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.560 (CI 0.531 - 0.593) mg/L
Yield Inhibition: 0.260 (CI 0.234 - 0.289) mg/L
Reported statistics and error estimates:
NOEC/LOEC were determined by calculation of statistical
significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.
Normality test and Equal Variance Test failed for growth rate calculation. The growth rate values were transformed using Graph Pad Prism function (Y = Y squared).
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions
> 10 % are significant.

Any other information on results incl. tables

NOEC, LOEC, EC - values and 95 % Confidence Intervals of

                   2,2’-(Cyclohexylimino)diethanol(0-72 hours)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

3.20

LOEC

10.0

ErC10

9.40 (5.37 – 22.4)

ErC20

10.6 (6.25 – 22.7)

ErC50

12.5 (10.2 – 24.1)

Inhibition of Yield

NOEC

0.320

LOEC

0.320

EyC10

0.320

EyC20

1.98 (1.21 – 3.10)

EyC50

9.89 (9.04 – 11.2)

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

100

1

4910

8900

6673

5691

 

2

4910

10793

7167

7360

 

3

4910

9361

5536

8795

 

Mean

4910

9685

6459

7282

 

  32.0

1

4910

4910

4910

4910

 

2

4910

4910

4910

4910

 

3

4910

4910

4910

4910

 

Mean

4910

4910

4910

4910

 

  10.0

1

4910

19089

91291

347803

 

2

4910

17295

78824

350985

 

3

4910

18795

81214

322687

 

Mean

4910

18393

83776

340492

 

    3.20

1

4910

19495

114150

509437

 

2

4910

19611

119959

559264

 

3

4910

21435

106260

512416

 

Mean

4910

20180

113456

527039

 

    1.00

1

4910

18791

123340

550531

 

2

4910

22143

128557

522909

 

3

4910

19336

118744

575817

 

Mean

4910

20090

123547

549752

 

    0.320

1

4910

17739

128797

605368

 

2

4910

18781

114448

565763

 

3

4910

19485

123127

607839

 

Mean

4910

18668

122124

592990

 

Control

1

4910

19208

122068

644702

 

2

4910

22288

122423

696255

 

3

4910

25142

141115

703533

 

4

4910

18778

126357

691685

 

5

4910

21506

120917

654653

 

6

4910

20768

121262

710235

 

Mean

4910

21282

125690

683511

 

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

0.049

  97.0

781

  99.9

2

0.135

  91.8

2450

  99.6

3

0.194

  88.2

3885

  99.4

Mean

(+)

0.126

  92.3

(+)

2372

  99.7

  32.0

1

0.000

100

0

100

2

0.000

100

0

100

3

0.000

100

0

100

Mean

(+)

0.000

100

(+)

0

100

  10.0

1

1.42

  13.7

342893

  49.5

2

1.42

  13.5

346075

  49.0

3

1.40

  15.2

317777

  53.2

Mean

(+)

1.41

  14.1

(+)

335582

  50.6

    3.20

1

1.55

    5.94

504527

  25.7

2

1.58

    4.05

554354

  18.3

3

1.55

    5.82

507506

  25.2

Mean

(+)*

1.56

    5.27

(+)

522129

  23.1

    1.00

1

1.57

    4.37

545621

  19.6

2

1.56

    5.41

517999

  23.7

3

1.59

    3.46

570907

  15.9

Mean

(+)*

1.57

    4.41

(+)

544842

  19.7

    0.320

1

1.61

    2.45

600458

  11.5

2

1.58

    3.82

560853

  17.4

3

1.61

    2.36

602929

  11.2

Mean

(+)*

1.60

    2.88

(+)

588080

  13.3

Control

1

1.63

639792

2

1.65

691345

3

1.66

698623

4

1.65

686775

5

1.63

649743

6

1.66

705325

Mean

1.65

678601

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.36

1.85

1.66

1.63

0.245

15.1

10.5

2

1.51

1.70

1.74

1.65

0.121

7.35

3

1.63

1.73

1.61

1.66

0.062

3.76

4

1.34

1.91

1.70

1.65

0.286

17.3

5

1.48

1.73

1.69

1.63

0.135

8.25

6

1.44

1.77

1.77

1.66

0.187

11.3

Mean

1.65

 

SD ±

0.01

 

CV [%]

0.81

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study 2,2’-(Cyclohexylimino)diethanol was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 3.20 mg/L and < 0.320 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 10.0 mg/L and 0.320 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 12.5 (10.2 – 24.1) and 9.89 (9.04 – 11.2) mg/L, respectively.
All effect levels are given based on nominal concentrations of 2,2’-(Cyclohexylimino)diethanol.
Executive summary:

The toxicity of 2,2’-(Cyclohexylimino)diethanol(batch number: ESD0011452) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from August 09 to 12, 2011. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5 x 103 cells/mL. Based on a preliminary test 6 concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of 2,2’-(Cyclohexylimino)diethanolwere analytically verified by LC-MS/MS at test start and test end of all test concentrations and the control.
The measured concentrations of 2,2’-(Cyclohexylimino)diethanolat test start were in the range of 101 to 105 % and after 72 hours in the range of 110 to 115% of the nominal values. Therefore, all effect values given are based on the nominal concentrations of the test item 2,2’-(Cyclohexylimino)diethanol.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of

                   2,2’-(Cyclohexylimino)diethanol(0-72 hours)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

3.20

LOEC

10.0

ErC10

9.40 (5.37 – 22.4)

ErC20

10.6 (6.25 – 22.7)

ErC50

12.5 (10.2 – 24.1)

Inhibition of Yield

NOEC

0.320

LOEC

0.320

EyC10

0.320

EyC20

1.98 (1.21 – 3.10)

EyC50

9.89 (9.04 – 11.2)