trisodium 3-[{3-[bis(2-carboxylatoethyl)amino]propyl}(C12-18-(even numbered) and C18-(unsaturated) alkyl)amino]propanoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

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

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)

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):

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): 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

Nominal and measured concentrations:

Nominal test item concentrations: 0.1, 0.316, 1, 3.16 and 10 mg/L
See any other information on results

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 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 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 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