Butanoic acid, 4-[(2-hydroxyethyl)amino]-4-oxo-2-(pentapropenyl)-, sodium salt, compd. with 2,2',2''-nitrilotris[ethanol] (1:?:?)

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

Test material information

Constituent 1

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 concentrations

Effect concentrations 1

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

Effect concentrations 2

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)