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Ecotoxicological information

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:
from 2018-05-21 to 2018-05-28 with the definitive exposure phase from 2018-05-22 to 2018-05-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Determination of the test item
All concentration levels and the control were analytically verified via LC-QTOF at the start (0 hours) and at the end of the exposure (72 hours) with algae. The samples were analysed with a LC-QTOF method, implemented under non-GLP and documented in the GLP raw data. The method was validated.
Vehicle:
no
Details on test solutions:
Water Accommodated Fraction (WAF)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure is expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction
Five water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 0.100 – 0.316 – 1.00 – 3.16 – 10.0 mg/L (spacing factor √10). The loading levels are based on the results of a preliminary range finding test (non-GLP, open system).
For the loading levels 1.00, 3.16 and 10.0 mg/L an appropriate amount of the test item was pipetted onto the surface of the dilution water. For this purpose, the density was taken into account. These dispersions were shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the aqueous phase or WAFs were removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fractions (WAF) were used in the test. The loading levels 0.100 and 0.316 mg/L were prepared by diluting the WAF with a loading of 1.00 mg/L.

Test loading
Per definition of the WAF, all terms related to concentration level are given as loading level. Since the analytical recoveries were inside the nominal range (±20%), these correspond to the actual test item concentrations.

Control
Six replicates (without test item) were exposed under the same conditions as the item replicates.


Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A three day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 h per day.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
Not specified
Test temperature:
min.: 22.0 max.: 24.0 mean value: 23.0
pH:
Nominal test item loading pH-value
[mg/L] Start; 0 hours End; 72 hours
10.0 7.70 8.03
3.16 7.75 8.23
1.00 7.75 8.63
0.316 7.87 8.88
0.100 7.96 8.87
Control 8.10 8.90
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
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 5 x 103 - 104 cells/mL. Actual: 5523 cells/mL

- Control end cells density: Mean 2161234 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 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2

This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE ∙ m-2 ∙ s-1, mean value: 6105 lux, range of the measured values: 5205 to 6744


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the concentration levels of 10.0 mg/L.


Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.66 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
29.7% A.I.
Basis for effect:
growth rate
Remarks on result:
other: CI (0.448 - 0.891)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.97 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.203 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
29.7% a.i.
Basis for effect:
other: Yield Inhibition
Remarks on result:
other: CI (0.133 - 0.292)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.316 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield Inhibition
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.51 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield Inhibition
Remarks on result:
other: CI (2.00 - 3.26)
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: All test solutions were visually clear throughout the exposure period (possible turbidity related to algae growth not taken into account).
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-04-09 to 2018-04-12, with headspace. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.574 0.752 ± 0.554
95 % confidence interval 0.549 – 0.596
Yield inhibition
EyC50 0.337 0.399 ± 0.294
95 % confidence interval 0.313 – 0.345
SD = Standard deviation
Reported statistics and error estimates:
Since the two main ingredients of the test item are considered to be soluble and analytical recoveries were inside the nominal range throughout the exposure phase, loading rate and actual test item concentration are considered to be interchangeable in this study. Therefore, all effect values are reported based on concentration of the test item.

EC-values and statistical analyses
EC10-, EC20- and EC50-value with confidence intervals of growth rate and yield inhibition after 72 hours was calculated sigmoidal dose response.

NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a 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. 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. The Normality Test failed for the calculation of growth rate. Therefore, data was transformed (Y square).

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Cell Densities

Nominal test item loading

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

10.0

1

5523

13909

48816

228786

2

5523

15566

48801

246657

3

5523

11153

31196

122397

Mean

5523

13543

42938

199280

 3.16

1

5523

22488

120542

992088

2

5523

31821

101196

915558

3

5523

26563

127151

824063

Mean

5523

26957

116296

910570

 1.00

1

5523

27857

141032

1459807

2

5523

31779

183310

1668331

3

5523

35330

226882

2054561

Mean

5523

31655

183741

1727566

 0.316

1

5523

36278

246791

2329429

2

5523

31492

322930

2112952

3

5523

27771

253547

2238749

Mean

5523

31847

274423

2227043

 0.100

1

5523

33497

325281

2111905

2

5523

37026

328393

2464352

3

5523

25990

300972

2326307

Mean

5523

32171

318215

2300855

Control

1

5523

31018

291298

2239764

2

5523

32263

297275

2356077

3

5523

33660

345361

2142218

4

5523

22972

234628

2034647

5

5523

28484

218843

2188250

6

5523

33935

275029

2006445

Mean

5523

30389

277072

2161234

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 loading

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

10.0

1

 

1.24

38

 

223263

90

2

 

1.27

36

 

241134

89

3

 

1.03

48

 

116874

95

Mean

(+)

1.18

41

(+)

193757

91

 3.16

1

 

1.73

13

 

986565

54

2

 

1.70

14

 

910035

58

3

 

1.67

16

 

818540

62

Mean

(-)

1.70

15

(+)

905047

58

 1.00

1

 

1.86

7

 

1454284

33

2

 

1.90

4

 

1662808

23

3

 

1.97

1

 

2049038

5

Mean

(-)

1.91

4

(+)

1722043

20

 0.316

1

 

2.02

-1

 

2323906

-8

2

 

1.98

0

 

2107429

2

3

 

2.00

-1

 

2233226

-4

Mean

(-)

2.00

-1

(-)

2221520

-3

 0.100

1

 

1.98

0

 

2106382

2

2

 

2.03

-2

 

2458829

-14

3

 

2.01

-1

 

2320784

-8

Mean

(-)

2.01

-1

(-)

2295332

-6

Control

1

 

2.00

 

 

2234241

 

2

 

2.02

 

 

2350554

 

3

 

1.99

 

 

2136695

 

4

 

1.97

 

 

2029124

 

5

 

1.99

 

 

2182727

 

6

 

1.97

 

 

2000922

 

Mean

 

1.99

 

 

2155711

 

 

 


Section-by-Section and Average Specific Growth Ratesof the Control Group

                     (0 – 72 hours)

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.73

2.24

2.04

2.00

0.259

12.9

14.58

2

1.77

2.22

2.07

2.02

0.232

11.5

3

1.81

2.33

1.83

1.99

0.296

14.9

4

1.43

2.32

2.16

1.97

0.479

24.3

5

1.64

2.04

2.30

1.99

0.333

16.7

6

1.82

2.09

1.99

1.97

0.140

7.11

 

 

 

Mean

2.06

 

 

 

 

 

SD ±

0.163

 

 

 

 

CV [%]

7.80

 

SD = Standard deviation CV = Coefficient of variation

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of Sodiumcocopropylenediamine propionate were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-QTOF.

The measured concentrations of the test item were between 94% and 114% of the nominal value at the start of the exposure (0 hours). At the end of the exposure (72 hours) the concentration of the test item were between 85% and 119%.

 

 Measured Concentrations and Percent of Nominal Concentration of theTest Item in Fresh Medium (0 hours) with Algae

Sampling date

0 hours

72 hours

Nominal

loading of the

Sodium cocopropylenediamine propionate

(C12 Tripropionate)

test item

[mg/L]

Active ingredient

[mg a.i./L]

Meas. conc.

[mg a.i./L]

[%]

Meas. conc.

[mg a.i./L]

[%]

10.0

1.90

1.88

99

1.83

96

3.16

0.601

0.593

99

0.514

86

1.00

0.190

0.188

99

0.172

91

0.316

0.0601

0.0607

101

0.0563

94

0.100

0.0190

0.0216

114

0.0213

112

Control

< LOQ

< LOQ

Meas. Conc.   = measured concentration of the active ingredient of the test item

                           (dilution and weighing factor taken into account)

a.i.                   = active ingredient (C12-Tripropionate)

%                     = percentage of the nominal concentration of the active ingredient

LOQ                         = limit of quantification of the analytical method (0.08 mg test item/L)

Measured Concentrations and Percent of Nominal Concentration of theTest Item in Fresh Medium (0 hours) with Algae

Sampling date

0 hours

72 hours

Nominal

loading of the

Sodium cocopropylenediamine propionate

(C14 Tripropionate)

test item

[mg/L]

Active ingredient

[mg a.i./L]

Meas. conc.

[mg a.i./L]

[%]

Meas. conc.

[mg a.i./L]

[%]

10.0

0.475

0.454

96

0.466

98

3.16

0.150

0.142

94

0.127

85

1.00

0.0475

0.0458

97

0.0440

93

0.316

0.0150

0.0142

94

0.0141

94

0.100

0.00475

0.00448

94

0.00566

119

Control

< LOQ

< LOQ

Meas. Conc.   = measured concentration of the active ingredient of the test item

                           (dilution and weighing factor taken into account)

a.i.                   = active ingredient (C14-Tripropionate)

%                     = percentage of the nominal concentration of the active ingredient

LOQ                         = limit of quantification of the analytical method (0.08 mg test item/L)

 

 

Since the measured concentrations of the two main constituents of the test item were inside the nominal range throughout the exposure period, loading rate and actual test item concentration are considered to be interchangeable

Validity criteria fulfilled:
yes
Conclusions:
In this study, Sodium cocopropylenediamine propionate CAS no. 97659-50-2 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item loadings*): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 10.0 mg/L (>2.97 mg a.i./L) and 2.51 (2.00 – 3.26) mg/L, respectively. The 72h EC10-value for growth is 2.22 mg/L (0.66 mg a.i./L)
All effect levels are based on nominal test item loadings*.
* Due to the high water solubility of the test item, loading rate and actual test item concentration are considered to be interchangeable.
Executive summary:

The toxicity of Sodium cocopropylenediamine propionate (Batch no.:48724) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2018-05-21 to 2018-05-28 with the definitive exposure phase from 2018-05-22 to 2018-05-25. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.

The test item Sodium cocopropylenediamine propionate is a yellow liquid, which is soluble in water.The study was conducted under static conditions with an initial cell density of 5523 cells/mL. Five water accommodated fractions (WAF) were prepared in a geometrical series with a factor of 10: 0.100 – 0.316 – 1.00 – 3.16 – 10.0 mg/L. For the loading levels 10.0 – 3.16 – 1.00 mg/L, an appropriate amount of the test item was pipetted onto the water surface (density of the test item taken into account). These dispersions were shaken at 20 rpm for 24 hours at room temperature. After a separation phase of 1 hour at room temperature, the WAFs were removed by siphoning and used in the test. The loading levels of 0.316 – 0.100 mg/L were prepared by dilution of the WAF with a nominal loading of 1.00 mg/L. The environmental conditions were within the acceptable limits. The test solutions were clear throughout the test period.

All loading levels and the control were analytically verified by LC-QTOF at the start and the end of the exposure.The concentration levels of the test item Sodium cocopropylenediamine propionate were between 94% and 114% of the nominal value at the start of the exposure (0 hours) and between 86% and 112% of the nominal value at the end of the exposure (72 hours).

All effect values given are based on the nominal loading* of the test item.

NOEC, LOEC, EC-values and 95% Confidence Intervals ofSodiumcocopropylenediaminepropionate (0 - 72 hours)

based on nominal loading of the test item* [mg/L]

 

Growth Rate Inhibition
Nominal test item loading* [mg/L]

NOEC

1.00

LOEC

3.16

ErC10

2.22 (1.51 – 3.00)

ErC20

4.36 (3.39 – 6.11)

ErC50

> 10.0

 

Yield Inhibition

Nominal test item loading* [mg/L]

NOEC

0.316

LOEC

1.00

EyC10

0.684 (0.449 – 0.986)

EyC20

1.03 (0.733 – 1.36)

EyC50

2.51 (2.00 – 3.26)

* Due to the high water solubility of the test item, loading rate and actual test item concentration are considered to be interchangeable

Description of key information

The results presented here are based on the draft algae test report and will be updated when the final report is available.

The test item has an active ingredient content of 29.7%. The ErC10 and ErC50 based on active ingredient are 0.66 and >2.97 mg/L respectively

Key value for chemical safety assessment

EC50 for freshwater algae:
2.97 mg/L
EC10 or NOEC for freshwater algae:
0.66 mg/L

Additional information

The toxicity of Sodium cocopropylenediamine propionateto the freshwater green algaPseudokirchneriella subcapitatawas determined according to the principles of OECD 201 andEC method C.3.The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.

 

Sodium cocopropylenediamine propionateis a light yellow solid substance, which is soluble in water.The study was conducted under static conditions with an initial cell density of 5523 cells/mL. Five water accommodated fractions (WAF) were prepared in a geometrical series with a factor of10: 0.100 – 0.316 – 1.00 – 3.16 – 10.0 mg/L. For the loading levels 10.0 – 3.16 – 1.00 mg/L, an appropriate amount of the test item was pipetted onto the water surface (density of the test item taken into account). These dispersions were shaken at 20 rpm for 24 hours at room temperature. After a separation phase of 1 hour at room temperature, the WAFs were removed by siphoning and used in the test. The loading levels of 0.316 – 0.100 mg/L were prepared by dilution of the WAF with a nominal loading of 1.00 mg/L. The environmental conditions were within the acceptable limits. The test solutions were clear throughout the test period.

 

All concentration levels and the control were analytically verified by LC-QTOF at the start and the end of the exposure. The concentration levels of the test item

Sodium cocopropylenediamine propionate were between 99% and 114% of the nominal value at the start of the exposure (0 hours).

 

All effect values given are based on the nominal loading of the test item.

 

NOEC, LOEC, EC-values and 95% Confidence Intervals ofSodiumcocopropylenediamine propionate (0 - 72 hours)

                  based on nominal loading of the test item [mg/L]

 

Growth Rate Inhibition
Nominal test item loading [mg/L]

NOEC

3.16

LOEC

10.0

ErC10

2.22 (1.51 – 3.00)

ErC20

4.36 (3.39 – 6.11)

ErC50

> 10.0

 

Yield Inhibition

Nominal test item loading [mg/L]

NOEC

0.316

LOEC

1.00

EyC10

0.684 (0.449 – 0.986)

EyC20

1.03 (0.733 – 1.36)

EyC50

2.51 (2.00 – 3.26)

The test item has an active ingredient content of 29.7%. The ErC10 and ErC50 based on active ingredient are 0.66 and >2.97 mg/L respectively