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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:
from 2016-09-26 to 2016-09-30, with the definitive exposure phase from 2016-09-27 to 2016-09-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
C10-13-alkyl(branched, unsaturated)-succinic acid-4-(3-methyl-butyl)-ester
Molecular formula:
C18H32O4 - C22H40O4
IUPAC Name:
C10-13-alkyl(branched, unsaturated)-succinic acid-4-(3-methyl-butyl)-ester
Test material form:
liquid: viscous

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the Saturated solution
A saturated solution with a nominal loading of 193 mg test item/L was prepared once with dilution water 24 ± 1 hours prior to the start of the exposure. The test item was placed on to the surface of the dilution water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 24 ± 1 hours. After a separation phase of 1 hour at room temperature the saturated solution was removed from the approximate bottom of the glass flask. Due to the density of the test item, any undissolved material was expected to float on the surface. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). No Tyndall effect was observed.

Test concentrations
The saturated solution of the test item with a nominal loading of 193 mg/L and two dilution levels of 25 and 50 % of the saturated solution were tested in an extended limit test. The concentrations are based on the results of a preliminary range finding test (non-GLP).

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

Test organisms

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): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ 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:
Not specified
Test temperature:
min.: 21.0 max.: 22.0 mean value: 21.5
pH:
Geometric mean measured active ingredient concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
15.9 7.90 8.76
8.37 7.93 8.78
4.46 7.97 8.86
Control 8.00 8.76
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Nominal: 25.0 - 50.0 - 100% of the saturated solution
geometric mean measured concentration levels: 4.46 - 8.37 - 15.9 mg/L.
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 25 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 10^3 cells/mL, Actual: mean 6839 cells/mL
- Control end cells density: Mean 1619091 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



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: 5213 lux,


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 range finding test at the saturated solution with a nominal loading of 193 mg/L.

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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 8.37 - < 15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other:
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other:
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 15.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
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: The test item concentrations were clear during the exposure. The algae in the saturated solution were agglutinated after 72 hours. No agglutination of the algae was observed in the control replicates.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA, 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 2016-04-26 to 2016-04-29 (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 0.460 0.780 ± 0.473
95 % confidence interval 0.443 – 0.481
Yield inhibition
EyC50 0.317 0.411 ± 0.276
95 % confidence interval 0.208 – 0.347
SD = Standard deviation
Reported statistics and error estimates:
EC-values and statistical analyses
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of only three treatment levels (Limit test design).

NOEC, LOEC and statistical analyses
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test was 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

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully 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.

Any other information on results incl. tables

Cell Densities

Geometric Mean Measured Test Item Concentration

Replicate

Cell Density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

15.9

1

6305

29708

214545

1373481

2

6305

25158

212425

1468988

3

6305

21020

184426

1032156

Mean

6305

25295

203799

1291542

 8.37

1

6305

28795

291571

1587894

2

6305

23994

202107

1433187

3

6305

23817

262727

1666600

Mean

6305

25535

252135

1562560

 4.46

1

6305

36279

345639

1786859

2

6305

39330

282708

1846847

3

6305

27323

326538

1627642

Mean

6305

34311

318295

1753783

Control

1

6305

40996

307532

1830553

2

6305

33982

288628

1907174

3

6305

31436

310171

1559140

4

6305

26895

240784

1533149

5

6305

26678

207029

1505071

6

6305

21584

233088

1379457

Mean

6305

30262

264539

1619091

 


 

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Geometric Mean Measured Test Item Concentration

Replicate

Growth Rate

Inhibition of Growth Rate

Yield

Inhibition of Yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

15.9

1

 

1.80

3

 

1367176

15

2

 

1.82

2

 

1462683

9

3

 

1.70

8

 

1025851

36

Mean

(-)

1.77

4

(-)

1285237

20

 8.37

1

 

1.84

0

 

1581589

2

2

 

1.81

2

 

1426882

12

3

 

1.86

-1

 

1660295

-3

Mean

(-)

1.84

1

(-)

1556255

4

 4.46

1

 

1.88

-2

 

1780554

-10

2

 

1.89

-2

 

1840542

-14

3

 

1.85

0

 

1621337

-1

Mean

(-)

1.88

-2

(-)

1747478

-8

Control

1

 

1.89

 

 

1824248

 

2

 

1.90

 

 

1900869

 

3

 

1.84

 

 

1552835

 

4

 

1.83

 

 

1526844

 

5

 

1.83

 

 

1498766

 

6

 

1.80

 

 

1373152

 

Mean

 

1.85

 

 

1612786

 

 

negative inhibition = increase of growth


 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

1.87

2.02

1.78

1.89

0.117

 6.17

18.3

2

1.68

2.14

1.89

1.90

0.228

12.0

3

1.61

2.29

1.62

1.84

0.392

21.3

4

1.45

2.19

1.85

1.83

0.371

20.3

5

1.44

2.05

1.98

1.83

0.333

18.2

6

1.23

2.38

1.78

1.80

0.575

32.0

 

 

 

Mean

1.85

 

 

 

 

 

SD ±

0.04

 

 

 

 

CV [%]

2.24

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study no inhibition of growth on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item Tetrapropylene succinic acid monoisobutylester. The nominal EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were >15.9 mg/L, respectively. The nominal NOEC-values for inhibition of growth rate and yield after 72 hours were 15.9 mg/L, respectively.
All effect values given are based on geometric mean measured concentrations of the test item Tetrapropylene succinic acid monoisobutylester.
Executive summary:

The toxicity of Tetrapropylene succinic acid monoisobutylester to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3 from 2016-09-26 to 2016-09-30, with the definitive exposure phase from 2016-09-27 to 2016-09-30 at the test facility. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

 

A saturated solution of 193 mg test item/L was prepared with dilution water one day prior to the start of the exposure. The test item was placed by pipetting onto the surface of the demineralized water. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer for 24 hours at room temperature. After one hour of separation the test item phase was separated by siphon from the bottom and was used for testing including further dilution.The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion), it showed no Tyndall effect.

 

The study was conducted under static conditions with an initial cell density of 6305 cells/ mL. Based on preliminary range finding tests, the saturated solution and twodilution levels of the saturated solution were tested in a geometrical series with a dilution factor of 2:25.0 - 50.0 - 100% of the saturated solutionas an extended limit test. These dilution levels are corresponding to geometric mean measured concentration levels of 4.46 - 8.37 - 15.9 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The test media were visually clear throughout the test period. The concentrations of the test item Tetrapropylene succinic acid monoisobutylester in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of exposure.
The measured concentrations of Tetrapropylene succinic acid monoisobutylesterin the fresh media (0 h) were in the range of 4.69 to 16.4 mg/L at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 90 to 94% of the initial measured test item concentrations.

Therefore, all effect values given are based on the geometric mean measured test item concentrations of Tetrapropylene succinic acid monoisobutylester.

 


 

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

                  based on geometric mean measured test item concentrations [mg/L]

 

Inhibition of Growth Rate
geometric mean measured test item concentrations [mg/L]

NOEC

15.9

LOEC

> 15.9

ErC10

> 15.9

ErC20

> 15.9

ErC50

> 15.9

 

Inhibition of Yield
geometric mean measured test item concentrations [mg/L]

NOEC

15.9

LOEC

> 15.9

EyC10

8.37 < EyC10< 15.9

EyC20

15.9

EyC50

> 15.9

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