Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-05-30 to 2017-06-01, with the definitive exposure phase from 2017-05-30 to 2017-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All WAFs and the control were analytically verified via LC-MS/MS in fresh media at the start of exposure and at water renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure (24 and 48 hours). The constituents (300 Da, 325 Da and 327 Da represent together 87.8% of the amine fraction) analyzed are considered representative for the exposure to the total amine (Diamines + monoamines) fraction (52.2% of the test item) of the product. Quantification was focused on the three main constituents of the diamine fraction (C18:1, C16 and C18) as it is considered more ecotoxic when compared to the other components of the test item. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000). Details of the analytical method are presented

Sampling for the analytical monitoring
At the start of the exposure and at renewal (0 and 24 hours), samples of the fresh media were taken after preparation of the WAFs and analyzed. At renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.

Criteria for the analytical monitoring
Recoveries of the test item should be within ± 20% of the initially measured concentrations.

Determination of adsorption of the test item
Adsorption to glass walls of the test vessels was analytically verified on glass from all loading levels at the end of the exposure intervals. After sampling for the test medium analysis (aqueous phase), the glassware was emptied and an appropriate amount of a suitable solvent was added. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration. Before this procedure, the test vessels were carefully rinsed with demineralized water to remove the remaining test media.

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. At the completion of mixing and following a settlement period, the test item phase was separated by siphoning and the test organisms 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 nominal loadings of the test item of: 0.0625 – 0.125 – 0.250 – 0.500 – 1.00 mg/L (spacing factor 2).
The loading levels were selected based on the results of two preliminary range finding tests. For the loading level 1.00 mg/L, an appropriate amount of the test item was weighed on a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After completion of shaking and following a separation phase of at least 1 hour of standing, the aqueous phase or WAF was
removed from the approximate center of the water body by siphoning.
The resulting water accommodated fractions (WAF) were used as the highest loading level in the test and as a stock solution for preparation of the loading levels 0.0625 to 0.500 mg/L by dilution of the WAF 1.00 mg/L with dilution water. The WAF 1.00 mg/L was prepared one day before the start of the exposure (at -24 hours) and one day before the renewal of the test solutions (at 0 hours).

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 of the test substance.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5).

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2°C, in an incubator, 16 hours illumination, light intensity of max. 20 µEm-2  s-1

Culture medium
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used. The composition of the culture medium is presented in Table 2.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany


Composition of the Culture Medium according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2 ± 0.8

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 160
24 hours:Total hardness [mg CaCO3/L]: 166

Test temperature:

18 - 22°C, constant within ± 1°C
20.8 °C

pH:

Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in all replicates containing daphnids)
Nominal loading pH-values Dissolved O2
of the test item concentration
[mg/L] [mg/L]

Replicates Replicates
1 2 3 4 1 2 3 4
1.00 - - - - - - - -
0.500 7.82 7.84 7.87 7.90 8.95 8.74 8.94 8.92
0.250 7.80 7.84 7.82 7.87 8.84 8.85 8.93 8.90
0.125 7.80 7.79 7.81 7.79 8.99 8.94 9.00 8.98
0.0625 7.72 7.79 7.76 7.79 8.92 8.90 8.99 8.99
Control 7.75 7.75 7.69 7.75 8.53 8.65 8.82 8.77

Water Quality Parameters of the Dilution Water at the Start of the Exposure and at Renewal (0 and 24 hours)

Dilution water pH-Value Dissolved O2 Temperature Conductivity Total hardness
dated: concentration
[mg/L] [°C] [µS/cm] [mg CaCO3/L]
0 hours: 2017-05-30 8.06 9.56 20.8 430 160
24 hours: 2017-05-31 7.75 9.12 20.8 430 166

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 430 [µS/cm]
Dilution water day 1: 430 [µS/cm]

Nominal and measured concentrations:

Nominal test substance concentrations: 0, 0.0625, 0.125, 0.250, 0.5 and 1 mg/L
Nominal amine fraction concentrations: 0, 0.0326, 0.06525, 0.1305, 0.261 and 0.522 mg/L (=52.2% of test substance).
Nominal main constituent concentrations: 0, 0.0286, 0.0573, 0.1146, 0.2292 and 0.458 mg/L (main constituents C18:1 diamine, C16 diamine and C18 diamine represent 87.8% of amine fraction in the test item)
Measured main constituent concentrations: 0, 0.0120, 0.0179, 0.0244, 0.0768 and 0.148 mg/L (Geometric mean measured concentrations of the main constituents)
Calculated concentration of the amine fraction in the test item: 0, 0.0137, 0.0204, 0.0278, 0.0875 and 0.1686 mg/L (calculated from geometric mean by multiplying by 1/0.878)
Calculated concentration of the test item: 0, 0.0262, 0.0391, 0.0532, 0.168 and 0.323 mg/L (calculated from geometric mean of the amine fraction and multiplying by 1/0.522)

Details on test conditions:

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids were used for each loading level and the control.

Age of the daphnidsat the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization was not necessary, because the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by pipette.

Test temperature (target)
18 - 22°C, constant within ± 1°C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Exposure concentration back calculated from three main constituents

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.025 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Amine fraction in the test item

Remarks:

52.2% of the test item is the amine fraction

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.022 other: mg m.c./L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Main constituents of the amine fraction

Remarks:

represting 87.8% of the amine fraction

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

0.49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The CMC of the amine fraction (Diamine T: N-C16-18-alkyl-(evennumbered, C18 unsaturated) propane-1,3-diamine) in the test item is 36 mg/L at pH7 and it is therefore assumed that the amine fraction is completely soluble.

Results with reference substance (positive control):

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in ISO test water (acc. to OECD 202 (2004), Annex 3) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 7.4.

Reference item Potassium dichromate p.a. (SIGMA)
Purity 99.0%
Batch number MKBV0900V
Expiry date 2021-11-25
Test concentrations 1.00 – 2.00 – 4.00 mg/L
Ranges of validity EC50 (24 hours): 0.6 mg/L - 2.4 mg/L, according to AQS P 9/2 (clone 5),
EC50 (24 hours): 0.6 mg/L - 2.1 mg/L, according to OECD 202 (clone A)

Exposure phase 2017-05-04 to 2017-05-05
The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017-05-04 to 2017-05-05. For results of the most recent of the monthly performed reference tests, see Table 15.

Table 15: EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range EC50 1.60 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5 95% confidence limits 1.43 - 1.79 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Reported statistics and error estimates:

The EC/EL100-values (after 24 and 48 hours) were estimated empirically from the observed immobilization rates.
All effect levels (EL10 / 50 / 100) are given based on the nominal loading rates of the test item. Per definition of the WAF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Exposures were expressed in terms of the original concentrations of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentrations of the test item in the WAFs.
The three analysed constituents are considered representative for the exposure to the diamine fraction of the product.
Therefore, and since the measured concentrations of the components of the test item Redicote 814 M were not within ± 20% of the nominal concentrations, the EC10 / 50 / 100-values based on the geometric mean measured concentrations summed up of all components were calculated and are reported additionally to the nominal loading levels.
EC/ELx-values and The EC/EL10- and the EC/EL50-values after 24 and 48 hours of statistical analyses exposure were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism. The concentration-effect relationship for immobilization after 48 hours is shown graphically.
The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

Software
All data were computer-processed and rounded for presentation.
Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently.
Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from
2017-05-04 to 2017-05-05. For results of the most recent of the monthly performed reference tests, see Table 15.

Table 15: EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)

Current Study	Valid Range
EC50	1.60 mg/L	0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits	1.43 - 1.79 mg/L

Measured Concentrations of theTest Itemat the Start of Exposure and at Renewal (0 and 24 hours

Sampling date		0 hours Fresh media
Nominal test item loading   [mg/L]	Nominal concentration ofthe main constituents^1) [mg m.c./L]	C16 diamine	C18:1 diamine	C18:0 diamine	Total amount of the main constituents^1)
		Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	∑ [mg m.c./L]	%
1.00	0.458	0.0666	0.0941	0.0838	0.245	53
0.500	0.229	0.0348	0.0479	0.0419	0.125	54
0.250	0.115	0.0161	0.0230	0.0205	0.0596	52
0.125	0.0573	0.00918	0.0128	0.0109	0.0328	57
0.0625	0.0286	0.00398	0.00540	0.00509	0.0145	51
Control		< LOQ	< LOQ	< LOQ	< LOQ
Sampling date		24 hours Old media
Nominal test item loading   [mg/L]	Nominal concentration ofthe main constituents^1) [mg m.c./L]	C16 diamine	C18:1 diamine	C18:0 diamine	Total amount of the main constituents^1)
		Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	∑ [mg m.c./L]	%
1.00	0.458	0.0216	0.0256	0.0321	0.0793	17
0.500	0.229	0.0108	0.0128	0.0146	0.0382	17
0.250	0.115	0.00284	< LOQ	0.00382	0.00878^2)
0.125	0.0573	< LOQ	< LOQ	< LOQ	0.00878^2)
0.0625	0.0286	< LOQ	< LOQ	< LOQ	0.00878^2)
Control		< LOQ	< LOQ	< LOQ	< LOQ

Meas. conc. = measured concentration of the main constituent, mean value of all injections, dilution factors taken into
account
LOQ = limit of quantification of the analytical method (0.0200 mg test item/L)
% = Percentage of the nominal m.c. concentrations (sum of the three analyzed constituents)
1) = C16 diamine, C18:1 diamine and C18:0 diamine content taken into account

2) = ½ x LOQ of the main constituents in the test item is used for evaluation for the geometric mean measured concentration in Table 13

Measured Concentrations of theTest Itemat Renewal and at the End of Exposure (24 and 48 hours)

Sampling date		24 hours Fresh media
Nominal test item loading   [mg/L]	Nominal concentration ofthe main constituents^1) [mg m.c./L]	C16 diamine	C18:1 diamine	C18:0 diamine	Total amount of the main constituents^1)
		Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	∑ [mg m.c./L]	%
1.00	0.458	0.0438	0.0708	0.0525	0.167	36
0.500	0.229	0.0433	0.0712	0.0522	0.167	73
0.250	0.115	0.0197	0.0331	0.0250	0.0777	68
0.125	0.0573	0.0107	0.0172	0.0132	0.0410	72
0.0625	0.0286	0.00470	0.00798	0.00611	0.0188	66
Control		< LOQ	< LOQ	< LOQ	< LOQ
Sampling date		48 hours Old media
Nominal test item loading   [mg/L]	Nominal concentration ofthe main constituents^1) [mg m.c./L]	C16 diamine	C18:1 diamine	C18:0 diamine	Total amount of the main constituents^1)
		Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	∑ [mg m.c./L]	%
1.00	0.458	-	-	-	-
0.500	0.229	0.0119	0.0162	0.0157	0.0438	19
0.250	0.115	0.00334	0.00536	0.00787	0.00878^2)
0.125	0.0573	< LOQ	< LOQ	0.00322	0.00878^2)
0.0625	0.0286	< LOQ	< LOQ	< LOQ	0.00878^2)
Control		< LOQ	< LOQ	< LOQ	< LOQ

Meas. conc. = measured concentration of the main constituent, mean value of all injections, dilution factors taken into
account
LOQ = limit of quantification of the analytical method (0.0200 mg test item/L)
% = Percentage of the nominal m.c. concentrations (sum of the three analyzed components)
1) = C16 diamine, C18:1 diamine and C18:0 diamine content taken into account
2) = ½ x LOQ of the main constituents in the test item is used for evaluation for the geometric mean measured
concentration in Table 13

Nominal test item loading	Nominal concentration ofthe active substances^1)	Geometric mean measured concentrations^2)	Geometric mean measured concentrations^3) Amine fraction
[mg/L]	[mg m.c./L]	[mg m.c./L]	[mg a.s./L]
1.00	0.458	0.148	0.1686
0.500	0.229	0.0768	0.0875
0.250	0.115	0.0244	0.0278
0.125	0.0573	0.0179	0.0204
0.0625	0.0286	0.0120	0.0137

1) = C16 diamine, C18:1 diamine and C18:0 diamine content taken into account
2) = total amount of the main constituents of Table 11 and Table 12 were taken for evaluation

3) = total amount of the amine fraction calculated from geometric mean by multiplying with 1/0.878

Determination of Adsorption to Glass Walls of the Test Vessels of all Components of theTest Itemat Renewal and at the End of Exposure (24 and 48 hours)

Sampling date		24 hours Old media
Nominal test item loading   [mg/L]	Nominal concentration ofthe main constituents^1) [mg m.c./L]	C16 diamine	C18:1 diamine	C18:0 diamine	Total amount of the main constituents^1)
		Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	∑ [mg m.c./L]	%
1.00	0.458	0.0239	0.00350	0.00254	< LOQ
0.500	0.229	0.00445	0.00701	0.00521	< LOQ
0.250	0.115	0.00543	0.00849	0.00641	0.0203	18
0.125	0.0573	0.00559	0.00894	0.00664	0.0212	37
0.0625	0.0286	0.00274	0.00412	0.00313	< LOQ
Control		< LOQ	< LOQ	< LOQ	< LOQ
Sampling date		48 hours Old media
Nominal test item loading   [mg/L]	Nominal concentration ofthe main constituents [mg m.c./L]	C16 diamine	C18:1 diamine	C18:0 diamine	Total amount of the main constituents^1)
		Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	Meas. conc. [mg m.c./L]	∑ [mg m.c./L]	%
1.00	0.458	0.00367	0.00113	0.00720	< LOQ
0.500	0.229	0.00247	0.00124	0.00307	< LOQ
0.250	0.115	0.00824	< LOQ	0.00194	< LOQ
0.125	0.0573	< LOQ	< LOQ	0.000601	< LOQ
0.0625	0.0286	< LOQ	< LOQ	< LOQ	< LOQ
Control		< LOQ	< LOQ	< LOQ	< LOQ

Meas. conc. = measured concentration of the main constituent, mean value of all injections, dilution factors taken into
account
LOQ = limit of quantification of the analytical method (0.0200 mg test item/L)
% = Percentage of the nominal m.c. concentrations (sum of the three analyzed constituents)

1) = C16 diamine, C18:1 diamine and C18:0 diamine content taken into account

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading of the test item, the 48 hours-EL50 for Daphnia magna was 0.200 mg/L (95% confidence limits: 0.156 – 0.228 mg/L).
Based on the geometric mean measured concentrations of the main constituents of the test item, the 48 hours-EC50 for Daphnia magna was 0.0221 mg m.c./L (95% confidence limits: 0.0197 – 0.0234 mg m.c./L).
Based on the geometric mean measured concentrations of the test item calculated from the geometric mean measured concentrations of the main constituents, the 48 hours-EC50 for Daphnia magna was 0.0482 mg/L (95% confidence limits: 0.0430 – 0.0511 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004) from 2017-05-29 to 2017-06-01, with the definitive exposure phase from 2017-05-30 to 2017-06-01. The study was conducted under semi-static conditions over a period of 48 hours with a WAF (1.00 mg/L) of the test item and four dilution levels (nominal: 0.0625 to 0.500 mg/L) prepared out of the WAF in a geometric series with a separation factor of 2.

The test item is a salt of diamine T (N-C16-18-alkyl-(evennumbered, C18 unsaturated) propane-1,3-diamine, CAS no 1219010 -04 -4) and a mixture of tallic, 2 -ethylhexoic and acetic acid.

Due to the complex structure of the test item, the WAF approach was used. As the tested concentrations were too low for exact direct weighing, the lower concentrations were diluted from the WAF. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. The concentrations of the main constituents (diamine C18:1, diamine C16 and diamine C18) of the test item were analytically verified via LC-MS/MS in fresh media at the start of the exposure and at renewal (0 and 24 hours) and in old media at renewal and at the end of the test (24 and 48 hours) in all concentration levels and the control. Additionally, the adsorption to glass walls of the test vessels was analytically verified from all loading levels at the end of the exposure intervals.

The chemical analysis was focused on the amine fraction in the test solutions because this fraction is considered to be the most toxic fraction in the test item.

The summed measured concentrations of the main constituents C16 diamine, C18:1 diamine and C18:0 diamine in the test item in fresh media were in the range of 51 to 57% at the start of the exposure (0 hours) and <LOQ to 73% at renewal (24 hours) of the nominal concentrations. The summed measured concentrations of the main constituents C16 diamine, C18:1 diamine and C18:0 diamine of the test item in old media were in the range of <LOQ to 17% at renewal (24 hours) and <LOQ to 19% of the nominal concentration at the end of the test (48 hours). The fraction of the main constituents of

the test item sorbed to glassware at the end of the exposure intervals was between <LOQ and 37% at renewal (24 hours) and <LOQ at the end of the test (48 hours). The analytical results are presented in Table 11 to Table 14. Per definition of the WAF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Therefore, the EL10/50/100-values given in Table 1 were based on the nominal loading levels. The three analyzed constituents are considered representative for the exposure to the diamine fraction of the product. Therefore, and since the measured concentrations of the components of the test item were not within ± 20% of the nominal concentrations, the geometric mean measured concentrations of the summed up constituents were calculated and are reported additionally to the nominal loading levels. The evaluation of the EC10/50/100-values based on the geometric mean measured concentrations of the main constituents is reported in Table 2. The evaluation of the EC10/50/100 -values based on the geometric mean measured concentrations of the amine fraction in the test item is reported in Table 3. Table 4 finally presents the effect values for the test item which were calculated from the geometric mean measured concentrations of the main constituents.

The validity criteria of the test guideline were fulfilled EL10-, EL50- (with 95% Confidence Limits) and EL100-Values (based on the nominal loading levels of the test item).

Redicote814 M
Effect values	Test duration [hours]	Nominal test item loadings [mg/L]
EL10 (with 95% confidence limits)	24	0.458		(CI: 0.247 – 0.907)
	48	0.141	(Cl: < 0.0625 – 0.195)
EL50 (with 95% confidence limits)	24	0.490	(Cl: 0.260 – 0.924)
	48	0.200	(Cl: 0.156 – 0.228)
EL100	24	1.00
	48	0.500

Table 2: EC10-, EC50- (with 95% Confidence Limits) and EC100-Values
(based on the geometric mean measured concentrations (total amount of the main constituents))

Redicote814 M
Effect concentrations	Test duration [hours]	Geometric mean measured concentrations (total amount of the main constituents^1) [mg m.c./L]
EC10 (with 95% confidence limits)	24	0.0664	(CI: 0.0239 – 0.148)
	48	0.0189	(Cl: < 0.0120 – 0.0218)
EC50 (with 95% confidence limits)	24	0.0744	(Cl: 0.0260 – 0.148)
	48	0.0221	(Cl: 0.0197 – 0.0234)
EC100	24	0.148
	48	0.0768

Table 3: EC10-, EC50- (with 95% Confidence Limits) and EC100-Values

(based on the geometric mean measured concentrations (total amount of the amine fraction in the test item))

Redicote814 M
Effect concentrations	Test duration [hours]	Geometric mean measured concentrations (total amount of the amine fraction^1) [mg m.c./L]
EC10 (with 95% confidence limits)	24	0.0756	(CI: 0.0272 – 0.1696)
	48	0.0215	(Cl: < 0.0136 – 0.0248)
EC50 (with 95% confidence limits)	24	0.0874	(Cl: 0.0296 – 0.1696)
	48	0.0252	(Cl: 0.0224 – 0.0267)
EC100	24	0.1696
	48	0.0875

¹⁾                             = Main constituents C16 diamine, C18:1 diamine and C18:0 diamine represent 87.8% of the amine fraction in the test item

Table 4: EC10-, EC50- (with 95% Confidence Limits) and EC100-Values

( Based on the geometric mean measured concentrations of the test item calculated from the geometric mean measured concentrations of the main constituents)

Redicote814 M
Effect concentrations	Test duration [hours]	Geometric mean measured concentrations (calculated for the test item)1 [mg/L]
EC10 (with 95% confidence limits)	24	0.1449	(CI: 0.0521 – 0.323)
	48	0.0412	(Cl: < 0.0262 – 0.0476)
EC50 (with 95% confidence limits)	24	0.0162	(Cl: 0.0567 – 0.323)
	48	0.0482	(Cl: 0.0430 – 0.0511)
EC100	24	0.323
	48	0.168

¹⁾                             = calculated effect concentrations for the test item based on the geometric mean measured concentrations of the main constituents C16 diamine, C18:1 diamine and C18:0 diamine

Description of key information

One short term toxicity test with daphnia according to OECD 202 has been performed with "Reaction mass of Amines, N-tallow alkyltrimethylenedi-, (2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3 -diaminopropane ditallate" (R814M). This substance is the salt of tallow-1,3 -diaminopropane (CAS no 1219010 -04 -4) and a mixture of talloil fatty acids, 2 -ethylhexoic and acetic acid.

Due to the complex structure of the test item, the WAF approach was used for the preparation of the stock solution. The concentrations of the main constituents (diamine C18:1, diamine C16 and diamine C18) of the test item were analytically verified via LC-MS/MS in fresh media at the start of the exposure and at renewal (0 and 24 hours) and in old media at renewal and at the end of the test (24 and 48 hours) in all concentration levels and the control. The chemical analysis was focused on the amine fraction in the test solutions because this fraction is considered to be the most toxic fraction in the test item.

The measured concentrations of the three main constituents of the amine fraction of the test item did not remain within ± 20% of the nominal concentrations and therefore the geometric mean measured concentrations of the sum of the three main constituents was calculated and are reported in addition to the nominal loading levels. The 48h EC₅₀ (semi-static) is 0.0221 mg m.c./L (95% CL: 0.0197 – 0.0234).

Because the three main constituents are considered to be representative for the amine fraction and completely soluble (CMC = 36 mg/L) the effect data are also presented for amine fraction. The 48h EC₅₀ (semi-static) is 0.0252 mg amine fraction/L (95% CL: 0.0224 – 0.0267).

Finally these results were recalculated to the effect data for R814M resulting in an 48h EC₅₀ (semi-static) is 0.0482 mg test item/L (95% CL: 0.0430 – 0.0511).

These results of R814M are in good agreement with the results observed by CECA (1999) for oleyl-1,3 -diaminopropane (CAS no 7173 -62 -8) where an EC50 of between 0.025 and 0.048 mg/L (recalculated to R814M) was observed.

The long term daphnia toxicity test with oleyl-1,3 -diaminopropane (OECD 211, river water test, reliability 1) resulted in a NOEC for reproduction is 1000 µg/L based on the test substance (92.3% diamine and 7.7% primary alkyl amine; R814M also contains this fraction of primary alkyl amine). The NOEC and EC50 for parental mortality are resp. 100  and 290 µg/L. The effects are expressed as nominal values because the tests were performed with river water as they are intended to be used in an evaluation of the environmental risks based on the Bulk approach.

Recalculating these results to R814M using the tallow-1,3 -diaminopropane content of 52.2% in R814M gives a NOEC for reproduction of 1.92 mg/L and a NOEC and EC50 for parental mortality of 0.192 and 0.56 mg/L. This NOEC for parental mortality of 0.192 mg/L will as a worst-case be used as Key value.

For classification and labelling the value as observed in the actual daphnia study with R814M of 0.0482 mg/L will be used. The environmental risk assessment will however be based on the long term endpoints of the 72h algae and 21d daphnia study of oleyl-1,3 -diaminopropane in natural river water (recalculated to R814M).

For intermittent release also the river water test data from the alkyl-1,3 -diaminopropanes is used. In the absence of an acute EC50 value for daphnia the long term daphnia 21d EC50 for parental mortality of 290 µg/L as observed for oleyl-1,3 -diaminopropane is used. This value is like the other alkyl-1,3 -diaminopropane data recalculated to R814M using the tallow-1,3 -diaminopropane content of 52.2% in R814M. EC50 acute daphnia is then 0.556 µg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.556 mg/L

Additional information

One short term toxicity test with daphnia has been performed. The effects of the test item were determined according to OECD 202 (2004). The study was conducted under semi-static conditions over a period of 48 hours with a WAF (1.00 mg/L) of the test item and four dilution levels (nominal: 0.0625 to 0.500 mg/L) prepared out of the WAF in a geometric series with a separation factor of 2.

The test item is a salt of tallow-1,3 -diaminopropane (N-C16-18-alkyl-(evennumbered, C18 unsaturated) propane-1,3-diamine, CAS no 1219010 -04 -4) and a mixture of tallic, 2 -ethylhexoic and acetic acid.

Due to the complex structure of the test item, the WAF approach was used. As the tested concentrations were too low for exact direct weighing, the lower concentrations were diluted from the WAF.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. The concentrations of the main constituents (diamine C18:1, diamine C16 and diamine C18) of the test item were analytically verified via LC-MS/MS in fresh media at the start of the exposure and at renewal (0 and 24 hours) and in old media at renewal and at the end of the test (24 and 48 hours) in all concentration levels and the control. Additionally, the adsorption to glass walls of the test vessels was analytically verified from all loading levels at the end of the exposure intervals.

The chemical analysis was focused on the amine fraction in the test solutions because this fraction is considered to be the most toxic fraction in the test item.

The summed measured concentrations of the main constituents C16 diamine, C18:1 diamine and C18:0 diamine in the test item in fresh media were in the range of 51 to 57% at the start of the exposure (0 hours) and <LOQ to 73% at renewal (24 hours) of the nominal concentrations. The summed measured concentrations of the main constituents C16 diamine, C18:1 diamine and C18:0 diamine of the test item in old media were in the range of <LOQ to 17% at renewal (24 hours) and <LOQ to 19% of the nominal concentration at the end of the test (48 hours). The fraction of the main constituents sorbed to glassware at the end of the exposure intervals was between <LOQ and 37% at renewal (24 hours) and <LOQ at the end of the test (48 hours).

Per definition of the WAF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Therefore, dose effect data were derived using the nominal loading concentrations.The 48h EL₅₀ (semi-static) is 0.2 mg/L (95% CL: 0.156 – 0.228).

The three analysed main constituents are considered representative for the exposure to the amine fraction of the product. Therefore, and since the measured concentrations of the components of the test item were not within ± 20% of the nominal concentrations, the geometric mean measured concentrations of the summed up main constituents were calculated and are reported additionally to the nominal loading levels. The 48h EC₅₀ (semi-static) is 0.0221 mg/L (95% CL: 0.0197 – 0.0234).

Because the three main constituents are considered to be representative for the amine fraction (CMC of tallow-1,3 -diaminopropane is 36 mg/L at pH 7) in the test item the effect data are also presented for amine fraction. The 48h EC₅₀ (semi-static) is 0.0252 mg/L (95% CL: 0.0224 – 0.0267).

Finally these results were recalculated to the effect data for R814M resulting in an 48h EC₅₀ (semi-static) is 0.0482 mg test item/L (95% CL: 0.0430 – 0.0511).

Under environmental conditions a large extend of the salt is expected to be dissociated and ecotoxicity observed will originate mainly from tallow-1,3 -diaminopropane. No effects (NOEL>=100 mg/L) were observed for the WAF of tall oil fatty acid to algae and daphnids and for 2 -ethyl hexanoic acid the lowest NOEC (21d daphnia) was 25 mg/L (ECHA) . Therefore available ecotoxicity data on alkyl-1,3 -diamines were included in the dataset. The ecotoxicity of tallow-1,3 -diaminopropane is expected to be reduced when forming a salt with talloil fatty acids or 2 -ethylhexoic acid. The available data on alkyl-1,3 -diaminopropanes was included for comparison reasons.

For the alkyl-1,3 -diaminopropanes the 21d EC50 parental daphnia magna mortality as determined in riverwater of 290 µg/L (= 0.56 mg R814M/L) is used as key value for the acute toxicity to aquatic invertebrates in the chemical safety assessment because the two acute daphnia test results available on oleyl-1,3-diaminopropane (CAS no 7173 -62-8) were performed in a period when no reliable specific method of analyses was available. The concentrations were therefore not analytically verified. The substance is known for its strong tendency to adsorb onto the walls of test vessels. Both studies are therefore of lower reliability. The study from CECA (1999) which did not use presoaked glassware as in the study from Sewel (1992) resulted in a 48h EC50 ranging from 0.013 to 0.025 mg/L which corresponds to 0.025 – 0.048 mg R814M/L which is in good agreement with the results observed for R814M.

As Key study for classification and labelling the value as observed in the actual daphnia study with R814M of 0.0482 mg/L will be used because the risk assessment will be based on the long term endpoints of the 72h algae and 21d daphnia study (recalculated to R814M).