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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:
2013-03-19 to 2013-03-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study, no deficiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

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:
sodium/triethanolamine 4-((2-hydroxyethyl)amino)-3-pentaproenyl-4-oxobutanoate
EC Number:
800-765-8
Cas Number:
1424149-03-0
Molecular formula:
C21H40NO4.1/2Na.1/2C6H15NO3
IUPAC Name:
sodium/triethanolamine 4-((2-hydroxyethyl)amino)-3-pentaproenyl-4-oxobutanoate
Details on test material:
- Name of test material (as cited in study report): Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts
- Physical state: Brown, viscous liquid
- Analytical purity: 100 % (w/w)
- Purity test date: 2011-11-15
- Lot/batch No.: ESD0010887
- Expiration date of the lot/batch: 2013-12-13
- Stability under test conditions: Not applicable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.00 - 3.16 - 10.0 - 31.6 - 100 - 316 mg/L
- Sampling method: Each test item concentration and the control were analyticallyverified at the beginning and end of the test. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and the dilution water compound MES monohydrate, due to difficulties at the determination of the test item. These replicates were incubated under test conditions.
- Sample storage conditions before analysis:
All original samples were stored at room temperature until sample preparation. Prepared samples were stored at room temperature until 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 500 mg/L was freshly prepared with dilution water
- Eluate: Dilution water
- Differential loading: 1.00 - 3.16 - 10.0 - 31.6 - 100 - 316 mg/L
- Controls: Six replicates (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 every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

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:
0.24 mmol Ca+Mg/l
Test temperature:
min.: 22.0 max.: 23.0, mean value: 22.5 °C
pH:
Environmental Conditions

Nominal
test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
316 8.27 8.44
100 8.16 8.73
31.6 8.13 9.24
10.0 8.12 9.41
3.16 8.12 9.43
1.00 8.14 9.46
Control 8.17 9.34
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
See window "Any other information on materials and methods incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- 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: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4332 cells/mL
- Control end cells density: Mean 357349 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH 8.1  0.2




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, mean value: 101



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 17 59
10.0 - 2 -10
1.00 1 8
0.1 -2 -11
















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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
159 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 100-316 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 10.0 - 100 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: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.631 0.682 ± 0.452
95 %
confidence
interval 0.583 - 0.687
Yield Inhibition
EyC50 0.256 0.314 ± 0.144
95 %
confidence
interval 0.247 - 0.265
SD = Standard deviation


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 a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05. Normality test failed for calculation of yield values. No transformation was found to pass Normality test.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values of growth rate was estimated directly from the concentration levels. Calculation of the confidence intervals EC10-, EC20- and EC50-values of yield was carried out using standard procedures.

Any other information on results incl. tables

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

316

1

4332

8087

6206

5586

 

2

4332

7936

5237

4655

 

3

4332

7392

5222

4504

 

Mean

4332

7805

5555

4915

 

100

1

4332

11662

33030

94955

 

2

4332

12043

29765

94722

 

3

4332

14392

32850

101201

 

Mean

4332

12699

31882

96959

 

  31.6

1

4332

14597

51805

160811

 

2

4332

13210

48676

163943

 

3

4332

13540

54585

167548

 

Mean

4332

13782

51689

164101

 

  10.0

1

4332

19921

126175

416685

 

2

4332

20871

134251

403885

 

3

4332

18830

113362

457664

 

Mean

4332

19874

124596

426078

 

    3.16

1

4332

19531

107286

361931

 

2

4332

20752

100524

366585

 

3

4332

17056

117552

358597

 

Mean

4332

19113

108454

362371

 

    1.00

1

4332

20082

100814

457413

 

2

4332

19805

102814

458230

 

3

4332

22447

114658

449550

 

Mean

4332

20778

106095

455064

 

Control

1

4332

26620

106022

374919

 

2

4332

17987

122043

323184

 

3

4332

22337

91178

331267

 

4

4332

17044

109582

349413

 

5

4332

18883

98247

414075

 

6

4332

21053

96172

351238

 

Mean

4332

20654

103874

357349

 

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

Growth rate inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

316

1

0.085

94

1254

100

2

0.024

98

323

100

3

0.013

99

172

100

Mean

(+)

0.041

97

(+)

583

100

100

1

1.03

30

90623

74

2

1.03

30

90390

74

3

1.05

29

96869

73

Mean

(+)

1.04

30

(+)

92627

74

  31.6

1

1.21

18

156479

56

2

1.21

18

159611

55

3

1.22

17

163216

54

Mean

(+)

1.21

18

(+)

159769

55

  10.0

1

1.52

-4

412353

-17

2

1.51

-3

399553

-13

3

1.55

-6

453332

-28

Mean

(+)*

1.53

-4

(+)*

421746

-19

    3.16

1

1.48

0

357599

-1

2

1.48

-1

362253

-3

3

1.47

0

354265

0

Mean

(-)

1.48

0

(-)

358039

-1

    1.00

1

1.55

-6

453081

-28

2

1.55

-6

453898

-29

3

1.55

-5

445218

-26

Mean

(+)*

1.55

-6

(+)*

450732

-28

Control

1

1.49

370587

2

1.44

318852

3

1.45

326935

4

1.46

345081

5

1.52

409743

6

1.47

346906

Mean

1.47

353017

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

1.82

1.38

1.26

1.49

0.291

19.6

17.8

2

1.42

1.91

0.974

1.44

0.471

32.7

3

1.64

1.41

1.29

1.45

0.178

12.3

4

1.37

1.86

1.16

1.46

0.360

24.6

5

1.47

1.65

1.44

1.52

0.113

  7.44

6

1.58

1.52

1.30

1.47

0.150

10.3

Mean

1.47

 

SD ±

0.03

 

CV [%]

2.04

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for inhibition of growth rate and inhibition of yield after 72 hours was 10.0 mg/L mg/L, respectively. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 159 (100 – 316) and 35.9 (26.9 – 49.7) mg/L, respectively.
All effect levels are given based on nominal concentrations of Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts.

Executive summary:

The toxicity of Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts (batch number: ESD0010887)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2006) at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2013-03-19 to 2013-03-22. 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 4332 cells/mL. With regard to the volatility of the test item glass flasks without headspace were used to reduce losses of test item. Based on a preliminary test 6 concentration levels were tested in a geometrical series with a nominal dilution factor of : 1.00 - 3.16 - 10.0 - 31.6 - 100 - 316 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 the test item Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts were analytically verified by LC-MS/MS at the start and the end of the exposure of all test concentrations and the control.
The measured concentrations of the
test item at the start of the exposure were in the range of 97 to 108 % and at the end of the exposure (72 h) in the range of 97 to 105 % of the nominal values. Therefore, all effect values given are based on the nominal test item concentrations.

NOEC, LOEC, EC-values and 95 % Confidence Intervals of Pentapropylensuccinicanhydride, reaction products with ethanolamine, sodium and triethanolamine salts (0-72 hours)

                   based on nominal test item concentrations [mg/L]

Inhibition of Growth Rate

Nominal test item concentration [mg/L]

NOEC

10.0

LOEC

31.6

ErC10

31.6 (10.0 – 100)

ErC20

62.9 (31.6 – 100)

ErC50

159 (100 – 316)

Inhibition of Yield

Nominal test item concentration [mg/L]

NOEC

10.0

LOEC

31.6

EyC10

15.5 (10.2 – 22.0)

EyC20

19.6 (13.4 – 26.5)

EyC50

35.9 (26.9 – 49.7)