Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-02-09 to 2015-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented guideline study under GLP without deviations.
Cross-reference
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
other information
Justification for type of information:
Justification for the read-across from the introduced read-across supporting substance to the registration substance:
The introduced read-across supporting substance (Reaction products resulting from the esterification of C18 unsaturated fatty acid with glycerol oligomers) is structurally related to the registration substance. Both substances are fatty acid esters of polyglycerols, the only difference being that the introduced read-across source substance is synthesized by using a C18-unsaturated fatty acid while the registration substance by using a C16-18 fatty acid.
Both substances are expected to undergo enzymatic hydrolysis resulting into the release of polyglyerols and the corresponding fatty acids. Both fatty acids are well-investigated substances and occurs also endogenously. No significant difference in aquatic toxicity can be derived.
Reason / purpose for cross-reference:
reference to other study
Conclusions:
In this read-across study, Reaction mass of C18 (unsaturated) esters with glycerine oligomers was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEL-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L. Consequently, the LOEL-values for both parameters after 72 hours were 100 mg/L. The EL50-value for inhibition of growth rate (ErC50) after 72 hours was determined to exceed 100 mg/L. The corresponding EC20- and EC10-values for inhibition of growth rate (ErC20, ErC10) with 95 % confidence intervals after 72 hours were determined to be 60.9 (46.7 – 81.2) and 47.3 (39.1 – 63.8) mg/L, respectively.
The EC50-value for inhibition of yield (EyC50) with 95 % confidence intervals after 72 hours was 69.7 (35.5 – 98.5) mg/L. The corresponding EC20- and EC10-values for inhibition of yield (EyC20, EyC10) with 95 % confidence intervals after 72 hours were determined to be 48.4 (33.1 – 95.7) and 42.2 (31.6 – 94.8) mg/L, respectively.
All effect levels are given based on nominal test item loading levels of Reaction mass of C18 (unsaturated) esters with glycerine oligomers.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

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
Reference substance name:
Reaction products resulting from the esterification of C18 unsaturated fatty acid with glycerol oligomers
EC Number:
943-350-8
Molecular formula:
not applicable (UVCB)
IUPAC Name:
Reaction products resulting from the esterification of C18 unsaturated fatty acid with glycerol oligomers
Test material form:
other: UVCB substance

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.
- Sampling method: All loading levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) measured as NPOC (Non Purgeable Organic Carbon), at the start and at the end of the test. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and incubated under test conditions.
- Sample storage conditions before analysis: None

Test solutions

Vehicle:
no
Details on test solutions:
Water accommodated fraction
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. At the completion of mixing and following a 1-hour settlement period, the test item phase was separated by siphoning 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).
Exposures are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading level) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fractions
Five water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range of 1.00 to 100 mg/L set up in a geometric series with a factor of 3.16: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.
For each loading level an appropriate amount of the test item was weighed. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a brown glass flask with an appropriate amount of dilution water. 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.

Test loadings
Per definition of the WAF, all terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The loadings are based on the results of a non-GLP preliminary range finding test. For the preliminary test a stock solution was used with a test item loading level of 25 mg/L (24 hours magnetic stirring at 1100 rpm). Further test loading levels were diluted out of this stock solution.

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

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test organism
Desmodesmus subspicatus CHODAT SAG 86.81 (formerly Scenedesmus subspicatus CHODAT)

Reason for the selection of the test system
According to the guideline, Desmodesmus subspicatus CHODAT SAG 86.81 is suitable for this type of study.

Origin
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility
Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE•m-2•s-1 for 24 h per day.

Culture medium
Nutrient medium Z according to LÜTTGE et al. (1994)


ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): yes
- 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:
The medium had a nominal hardness of 0.24 mmol Ca+Mg/L.
Test temperature:
min.: 20.0
max.: 24.0
mean: 22.0
pH:
Nominal Test Item Loading pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.66 7.50
31.6 7.68 7.71
10.0 7.71 7.98
3.16 7.79 8.04
1.00 7.91 8.09
Control 8.11 8.31
Dissolved oxygen:
not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 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 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 4998 cells/mL
- Control end cells density: Mean 470043 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: 71.3



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the definitive test at the loading level 100 mg/L.



The results of the preliminary range finding test are presented below:

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Loading
[mg/L] Growth Rate Inhibition
[%] Yield Inhibition
[%]
25.0 13 48
2.50 - 3 - 18
0.250 - 12 - 80
negative inhibition = increase of growth
TOC Content of the Test Media at the Start and the End of the Exposure in the Preliminary Test (non-GLP)

Nominal Test Item Loading
[mg/L] TOC mg/L as NPOC
Test start (without algae), 0 hours Test end (without algae), 72 hours
25.0 16.5 12.3
2.50 3.10 2.58
0.250 < 2.00 < 2.00
Control < 2.00 < 2.00








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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EL10
Effect conc.:
47.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 39.1 - 63.8
Duration:
72 h
Dose descriptor:
other: EL20
Effect conc.:
60.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 46.7 - 81.2
Duration:
72 h
Dose descriptor:
other: EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
other: EL10
Effect conc.:
42.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 31.6 - 94.8
Duration:
72 h
Dose descriptor:
other: EL20
Effect conc.:
48.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 33.1 - 95.7
Duration:
72 h
Dose descriptor:
other: EL50
Effect conc.:
69.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 35.5 - 98.5
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: The test solutions were concentration related turbid
- Any stimulation of growth found in any treatment: 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):
The toxicity of potassium dichromate (SIGMA, batch number MKBH9127V, purity 99.9 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2014-10-14 to 2014-10-17. 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 0.586 0.678 ± 0.414
95 % confidence interval 0.522 - 0.818
Yield Inhibition
EyC50 0.401 0.319 ± 0.141
95 % confidence interval 0.285 - 0.469
SD = Standard deviation
Reported statistics and error estimates:
EL-values and statistical analyses
EL10-, EL20- and EL50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response. Calculation of the confidence intervals EL10-, EL20- and EL50-values were carried out using standard procedures.

NOEL, LOEL and statistical analyses
The values for NOEL/LOEL were determined by calculation of statistical significance of inhibition of growth rate and yield. As standard methods, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL 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.

Any other information on results incl. tables

Cell Densities

 

Nominal Test Item Loading

Replicate

Cell Density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

4998

17646

117093

107343

2

4998

19756

87150

126436

3

4998

16529

103943

150615

Mean

4998

17977

102729

128131

 31.6

1

4998

16745

112277

506066

2

4998

20934

75125

410817

3

4998

22563

121949

570562

Mean

4998

20081

103117

495815

 10.0

1

4998

24815

114460

635665

2

4998

16005

108560

476894

3

4998

25374

139487

600710

Mean

4998

22065

120836

571090

  3.16

1

4998

14874

96952

615610

2

4998

16745

102895

552976

3

4998

21744

110981

520577

Mean

4998

17788

103609

563054

  1.00

1

4998

16416

99360

610542

2

4998

18456

105987

537946

3

4998

26452

128689

579139

Mean

4998

20441

111345

575876

Control

1

4998

19933

106529

480510

2

4998

18040

74922

431521

3

4998

14229

74207

409907

4

4998

20180

83827

464441

5

4998

17794

90602

483542

6

4998

21008

127277

550334

Mean

4998

18531

92894

470043

 

 


 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 [mg/L]

Replicate

Growth Rate

Growth Rate Inhibition

Yield

Inhibition of Yield

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.02

32

 

102345

78

2

                                                                                        

1.08

29

 

121438

74

3

 

1.14

25

 

145617

69

Mean

(+)

1.08

29

(+)

123133

74

 31.6

1

 

1.54

-2

 

501068

-8

2

 

1.47

3

 

405819

13

3

 

1.58

-4

 

565564

-22

Mean

(-)

1.53

-1

(-)

490817

-6

 10.0

1

 

1.62

-7

 

630667

-36

2

 

1.52

0

 

471896

-1

3

 

1.60

-6

 

595712

-28

Mean

(-)

1.58

-4

(-)

566092

-22

  3.16

1

 

1.60

-6

 

610612

-31

2

 

1.57

-4

 

547978

-18

3

 

1.55

-2

 

515579

-11

Mean

(-)

1.57

-4

(-)

558056

-20

  1.00

1

 

1.60

-6

 

605544

-30

2

 

1.56

-3

 

532948

-15

3

 

1.58

-5

 

574141

-23

Mean

(-)

1.58

-5

(-)

570878

-23

Control

1

 

1.52

 

 

475512

 

2

 

1.49

 

 

426523

 

3

 

1.47

 

 

404909

 

4

 

1.51

 

 

459443

 

5

 

1.52

 

 

478544

 

6

 

1.57

 

 

545336

 

Mean

 

1.51

 

 

465045

 

negative inhibitions = 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

1.38

1.68

1.51

1.52

0.147

9.66

15.0

2

1.28

1.42

1.75

1.49

0.240

16.1

3

1.05

1.65

1.71

1.47

0.367

25.0

4

1.40

1.42

1.71

1.51

0.175

11.6

5

1.27

1.63

1.67

1.52

0.221

14.5

6

1.44

1.80

1.46

1.57

0.203

13.0

 

 

 

Mean

1.51

 

 

 

 

 

SD ±

0.03

 

 

 

 

CV [%]

2.25

 

CV= Coefficient of variation

SD= Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Reaction mass of C18 (unsaturated) esters with glycerine oligomers was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEL-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L. Consequently, the LOEL-values for both parameters after 72 hours were 100 mg/L. The EL50-value for inhibition of growth rate (ErC50) after 72 hours was determined to exceed 100 mg/L. The corresponding EC20- and EC10-values for inhibition of growth rate (ErC20, ErC10) with 95 % confidence intervals after 72 hours were determined to be 60.9 (46.7 – 81.2) and 47.3 (39.1 – 63.8) mg/L, respectively.
The EC50-value for inhibition of yield (EyC50) with 95 % confidence intervals after 72 hours was 69.7 (35.5 – 98.5) mg/L. The corresponding EC20- and EC10-values for inhibition of yield (EyC20, EyC10) with 95 % confidence intervals after 72 hours were determined to be 48.4 (33.1 – 95.7) and 42.2 (31.6 – 94.8) mg/L, respectively.
All effect levels are given based on nominal test item loading levels of Reaction mass of C18 (unsaturated) esters with glycerine oligomers.
Executive summary:

The toxicity of Reaction mass of C18 (unsaturated) esters with glycerine oligomers (batch number: ESD0017954) to the unicellular freshwater green alga Desmodesmus subspicatuswas determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No. 440/2008/ Method C.3 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany, from 2015-02-09 to 2015-02-13 with the definitive exposure phase from 2015-02-10 to 2015-02-13. The aim of the study was to assess the effects of the test item on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4998 cells/mL. Based on a preliminary test, 5 water accommodated fractions (WAF) with nominal loadings of the test item with a dilution factor of 3.16 were tested: 1.00 – 3.16 – 10.0 – 31.6 – 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.

 

All test loading levels of Reaction mass of C18 (unsaturated) esters with glycerine oligomers and the control were analytically verified by analysis of total organic carbon (TOC), measured as NPOC (Non-Purgeable Organic Carbon) at the start (0 hours) and the end of exposure (72 hours).
All effect values given are based on the nominal test item loading levels of
Reaction mass of C18 (unsaturated) esters with glycerine oligomers.

 

NOEL, LOEL, EL-values and Confidence Intervals ofReactionmass of C18 (unsaturated) esters with glycerine oligomers (0-72 hours)    

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

 

 

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

NOEL

31.6

LOEL

100

ErL10

47.3 (39.1 – 63.8)

ErL20

60.9 (46.7 – 81.2)

ErL50

> 100

 

Inhibition of Yield

Nominal test item loading [mg/L]

NOEL

31.6

LOEL

100

EyL10

42.2 (31.6 – 94.8)

EyL20

48.4 (33.1 – 95.7)

EyL50

69.7 (35.5 – 98.5)