C10-13-alkyl(branched, unsaturated)-succinic acid-4-(3-methyl-butyl)-ester

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-2-[2-hydroxy-2-(3-methylbutoxy)ethyl]-3-methyltridec-4-enoic acid; (4E)-3-methyl-2-[2-(3-methylbutoxy)-2-oxoethyl]dodec-4-enoic acid

Molecular formula:

C18H32O4 - C22H40O4

IUPAC Name:

(4E)-2-[2-hydroxy-2-(3-methylbutoxy)ethyl]-3-methyltridec-4-enoic acid; (4E)-3-methyl-2-[2-(3-methylbutoxy)-2-oxoethyl]dodec-4-enoic acid

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 concentrations

Effect concentrations 1

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

Effect concentrations 2

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

Effect concentrations 3

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

Effect concentrations 4

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

Effect concentrations 5

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

Effect concentrations 6

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

Effect concentrations 7

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

Effect concentrations 8

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:

Effect concentrations 9

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:

Effect concentrations 10

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