N-{3-[C12-14-(even numbered)-alkyloxy]propyl}propane-1,3-diamine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2022-02-15 to 2022-03-10, with the definitive exposure phase from 2022-02-17 to 2022-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

via LC-MS/MS

Details on sampling:

Determination of the test item
All loading rates and the control were analyzed via LC-MS/MS at least 3 times during the test (e.g. once within a period of 7 days) in the fresh media at the start of an renewal interval (0 hours, without algae) as well as in the old media at the end of an renewal interval (24 hours, with daphnia and algae). Additional, samples with test media, but without daphnids and algae, were prepared for the sampling of the old media at least one time during the test.
The samples were analyzed under GLP with a LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data. The analytical method was validated according to Annex I.

Sampling for the analytical monitoring
At the start of the renewal intervals, samples of the fresh media were taken after preparation of all loading rates and analyzed.
At the end of the renewal intervals (24 hours), samples of the old media were taken from the test vessels containing daphnia and algae. Additional replicates with test media, but without daphnids and algae, were prepared for the sampling of the old media. These replicates were incubated under test conditions until sampling.

Peak distribution (Finger print)
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the highest test item concentration, once during the test under non-GLP and documented finally in the GLP raw data. The signal distribution was compared with an analytical standard prepared in solvent.

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction
Water accommodated fractions (WAF) were prepared because the test item is an UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2019).
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.
 
Preparation of the water accommodated fractions
Six water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.253 to 80.0 µg/L set up in a geometric series with a factor of 3.16:
0.253 – 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L.
The test item was melted at 80 °C before preparation of the WAFs. For each loading rate an appropriate amount of stock solution containing the test item with methanol was placed on a curved glass slide. The methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour (at room temperature), the WAF was removed by siphoning (from the approximate middle of the glass flask). The first 25 mL were discarded. The WAFs were checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. The resulting water accommodated fractions (WAF) were used in the test.

Test loading
The loading rates are based on the results of a preliminary range finding test (non-GLP, open system with headspace).

Test organisms (species):

Daphnia magna

Details on test organisms:

The test species was Daphnia magna STRAUS, obtained from continuous laboratory cultures.

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.

The Daphnia magna culture has shown to meet the validity criteria for the reproduction test of a mean of ≥ 60 living juveniles per survived parent animal when cultured under conditions described.

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. 1500 lx.

Culture medium
Elendt M4, according to OECD 211, Annex 2 is used.

Feeding of the culture stocks
The daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Culture Medium Elendt M4 according to OECD 211, Annex 2

Limit test:

no

Total exposure duration:

21 d

Hardness:

The total hardness was above 140 mg/L as CaCO3.
For details see section "Any other information on results incl. tables" below.

Test temperature:

The temperature was within the range of 20 ± 2 °C.
For details see section "Any other information on results incl. tables" below.

pH:

The pH-values should be in the range of 6 – 9. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.
For details see section "Any other information on results incl. tables" below.

Dissolved oxygen:

The dissolved oxygen concentration was above 3 mg/L.
For details see section "Any other information on results incl. tables" below.

Nominal and measured concentrations:

Six water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.253 to 80.0 µg/L set up in a geometric series with a factor of 3.16:
0.253 – 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L.

The measured concentrations of the compounds of the test item in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14 without algae) were < LOQ to 118% (C12 ether diamine) and < LOQ to 86% (C14 ether diamine) of the nominal values.
The measured concentrations of the test item in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8 and 15 with daphnia and algae) were < LOQ to 64% (C12 ether diamine) and < LOQ to 42% (C14 ether diamine) of the nominal values.
The additional replicates with test media, but without daphnids and algae were investigated on day 8. Amounts of < LOQ to 48% (C12 ether diamine) and < LOQ to 20% (C14 ether diamine) of the nominal loading rates were measured.

Details on test conditions:

Control
Dilution water without test item incubated under the same conditions as the test groups. The control solution was prepared without test item following the same method as specified for the test item.

Test method
The study was performed under semi-static conditions with a daily renewal of the test solutions.
 
Test duration 21 days

Test vessels
Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses were used.

Test volume 50 mL

Test medium Same composition as the culture medium (Table 2)

Number of daphnids and replicates
10 daphnids and 10 replicates were used for all loading rates and the control with one daphnid per replicate, which will be held individually.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this, 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 this 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 composition of the dilution water is equivalent to the culture medium.

Application
50 g test solution per replicate were weighed into the test vessels. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.

Renewal of the test solutions
The test solutions and the control were renewed daily. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by pipette (see ‘Application’).

Feeding
Daily feeding with unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, max. 0.2 mg C / daphnia and day

Test temperature (target)
The temperature should be in the range of 18 – 22 °C and should not vary by more than 2 °C.

Light intensity (target) Max. 1500 lx

Photoperiod 16/8 hours light/dark cycle

Aeration Test vessels were not aerated during the test.
 
Pre-treatment
A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure and each renewal, the test container were emptied and refilled with freshly prepared test solutions.

Biological Parameters

Mortality of parental Daphnids
The number of parental daphnids, which were immobilized or dying during the test and the dates of death were observed and recorded once a day at least at the same time as the number of offspring was counted. Dead specimens were removed as soon as they were detected. Accidental mortality did not occur.

Offspring
Trials were checked daily for first appearance of juveniles. The number of neonates (alive and dead progeny) was counted and the dates of release were recorded once a day from the first day of production of neonates until the end of the exposure. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and the dates of observation were recorded. At the end of the test (21 days), the total number of living offspring was assessed.

Additional observations /Condition of parental daphnids
Abnormalities (e.g. swimming behavior, number of males andwinter eggs) were observed and recorded on each day of observation.

Total body length / Mean dry weight of parental daphnids
At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of all WAFs and the control were determined. As stated in the OECD protocol, this may be useful additional information but should not be used for determination of a NOEL.

Additional endpoints
The time to first brood and the number and size of first brood per animal were reported, but not used for endpoint calculations.
 
Water Quality Parameters

Measurements
The water quality parameters (i.e. pH-values, dissolved oxygen concentration, total hardness and water temperature) were measured once within 7 days, in the fresh media at the start of an exposure-renewal interval (0 hours) as well as in the old media at the end of an exposure-renewal interval (24 hours), in one replicate of the control and the highest loading rate of the test item with a living parent animal.
The water quality parameters in the fresh media were measured in an additional replicate without daphnids of the highest loading rate of the test item and the control. At the end of the exposure-renewal interval, the water quality parameters of the old media were measured in a test vessel of the highest loading rate of the test item and the control, which contains daphnids and food algae.
The incubator temperature (measurement in air with a thermo-hygrograph) was recorded throughout the period of the test.
Acid capacity and acidity of the dilution water are determined at least quarterly and were reported.

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LL50

Effect conc.:

56.89 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

86.33 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

59.08 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

7.59 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

19.86 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.01 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

10.4 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

25.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

33.4 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LL50

Effect conc.:

57 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

5.99 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.01 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

10.4 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

25.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

59.2 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

86.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

7.61 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

19.9 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Biological Data

Reproductive Output

The number of juveniles in all replicates of the control and the test groups was counted and recorded every day.

The average number of living juveniles at the end of the test after 21 days per introduced parental female daphnid was 131 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.

Accidental and inadvertent mortality were not observed. Since the Cochran-Armitage trend test revealed a significant increasing trend (p ≤ 0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced (female) parent.

A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the nominal loading rates.

Adult Mortality

Accidental and inadvertent mortality as defined in the OECD test guideline were not observed.
The Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality. A statistically significant dose-response was detected.
Intrinsic Rates of Natural Increase (IR)

The intrinsic rate of population increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations it will be zero. For growing populations it will be positive and for shrinking populations it will be negative.

First Appearance of living Juveniles

The first appearance of living juveniles was observed between days 7 and 8 at the introduced parental daphnids of the control and the nominal loading rates of 0.253 to 80.0 µg/L of the test item.

Analytical Results

The concentrations of the two compounds C12 ether diamine and C14 ether diamine of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 7 and 14 without algae) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 8 and 15 with daphnia and algae) in all WAFs and in the control. Additional, samples with test media, but without daphnids and algae, were prepared for the sampling of the old media at least one time during the test (24 hours).

The stability of the test item under test conditions was monitored after 24 hours by quantifying the concentration of parallel samples which were prepared without daphnids and algae.

The measured concentrations of the compounds of the test item in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14 without algae) were < LOQ to 118% (C12 ether diamine) and < LOQ to 86% (C14 ether diamine) of the nominal values.
The measured concentrations of the test item in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8 and 15 with daphnia and algae) were < LOQ to 64% (C12 ether diamine) and < LOQ to 42% (C14 ether diamine) of the nominal values.
The additional replicates with test media, but without daphnids and algae were investigated on day 8. Amounts of < LOQ to 48% (C12 ether diamine) and < LOQ to 20% (C14 ether diamine) of the nominal loading rates were measured.
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the limit loading of the test item after 0 hours. The signal distribution was compared with an analytical standard prepared in solvent.

Water Quality Parameters

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits. The incubator temperature was in the range of 19 to 21°C.

Reported statistics and error estimates:

All calculations are based on the control and the nominal loading rates 0.253 – 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L, corresponding to the time weighted mean measured test item concentrations 0.124 – 0.294 – 1.01 – 3.01 – 10.4 – 53.4 µg/L.

Statistical evaluation The NOEL/NOEC and LOEL/LOEC for the reproduction and adult mortality were determined with the software ToxRat Professional as specified below. Significant differences were determined in comparison to the control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test), a Williams Multiple t-test for reproduction and a step-down Cochran-Armitage test for adult mortality. P values of the normality and the variance homogeneity test were 0.01.
For the effects on reproduction, the juveniles per introduced parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were counted. Since no accidental or inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output. Details of the statistical analyses are presented in section 13.
The coefficients of variation around the mean number of living offspring produced per introduced parent and per survived parent in the control and the test groups were evaluated.

Determination of the The EL/ECx – and LL/LCx- values for the reproductive output and
EL/ECx- / LL/LCx-values adult mortality were calculated by non linear regression.

Calculation of the The intrinsic rate of natural increase (IR) was calculated for the
intrinsic rate survived parental daphnids with the following EULER-LOTKA-equation with the software ToxRat Professional.
 (e -A  IR) * Sx * Jx = 1
A = Age
IR = Intrinsic rate of natural increase
Sx = Survival rate on day x
Jx = Reproduction rate on day x

The intrinsic rate of natural increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations, it will be zero. For growing populations, it will be positive and for shrinking populations, it will be negative. Clearly, the latter is not sustainable and ultimately will lead to extinction.
 
Software All data are 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:
- ToxRat Professional, TOXRAT SOLUTIONS GMBH
- Excel, MICROSOFT CORPORATION
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

 

Preparation of the WAFs in the Definitive Test

	Stock solution 1		Stock solution 2*
Stock solution	1000		100
[mg/L in MeOH]
Weighing out [mg]	10		1 mL of Stock solution 1
Volume of the stock solution [mL]	10		10
Volume of the stock solution placed on a curved glass slide [µL/L]	80.0	25.3	80.0	25.3	8.00	2.53
Final test item	80.0	25.3	8.00	2.53	0.800	0.253
Loading rate [µg/L]

* = This stock solution will be prepared out of stock solution 1

 

Effects on Reproduction for all Introduced Parents
(based on the nominal loadings of the water accommodated fractions of the test item [µg/L])

Nominal loading rate	Mean number of offspring
of the test item	per introduced parental daphnid
[µg/L]	Mean	SD	CV
80.0	61.0	29.9	49.0
25.3	97.2	39.0	40.2
8.00	117	37.2	31.9
2.53	113	37.0	32.9
0.800	102	29.3	28.6
0.253	111	37.4	33.7
Control	131	46.5	35.5

 

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure
(n = 10)

Nominal loading rate	Adult Mortality [%]
of the test item [µg/L]	7 days	14 days	21 days
80.0	10	20	60
25.3	10	10	30
8.00	0	0	10
2.53	0	0	10
0.800	0	0	10
0.253	0	10	10
Control	0	0	0

 

Intrinsic Rates of Natural Increase

Nominal loading rate	Mean IR	CV
of the test item [µg/L]	MV ± SD	[%]
80.0	0.342 ± 0.0286	8.40
25.3	0.389 ± 0.0363	9.30
8.00	0.419 ± 0.0281	6.70
2.53	0.426 ± 0.0353	8.30
0.800	0.397 ± 0.0423	10.7
0.253	0.396 ± 0.0155	3.90
Control	0.402 ± 0.0521	13.0

 

First Appearance of Living Juveniles in the Individual Groups

Nominal loading rate	Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.										First
of the test item											appearance
[µg/L]	1	2	3	4	5	6	7	8	9	10	(mean day)
80.0	8	8	8	8	--	8	8	7	8	7	7.8
25.3	--	8	8	7	8	7	8	8	8	7	7.7
8.00	8	8	8	7	7	7	7	7	8	7	7.4
2.53	7	7	8	7	7	7	8	7	7	7	7.2
0.800	8	7	7	8	7	7	8	8	7	7	7.4
0.253	7	8	8	7	8	7	7	7	7	8	7.4
Control	7	8	8	7	8	8	7	7	8	8	7.6

= not applicable, since no living juveniles occurred

Analytical Results

The concentrations of the two compounds C12 ether diamine and C14 ether diamine of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 7 and 14 without algae) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 8 and 15 with daphnia and algae) in all WAFs and in the control. Additional, samples with test media, but without daphnids and algae, were prepared for the sampling of the old media at least one time during the test (24 hours).

The stability of the test item under test conditions was monitored after 24 hours by quantifying the concentration of parallel samples which were prepared without daphnids and algae.

The measured concentrations of the compounds of the test item in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14 without algae) were < LOQ to 118% (C12 ether diamine) and < LOQ to 86% (C14 ether diamine) of the nominal values.
The measured concentrations of the test item in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8 and 15 with daphnia and algae) were < LOQ to 64% (C12 ether diamine) and < LOQ to 42% (C14 ether diamine) of the nominal values.
The additional replicates with test media, but without daphnids and algae were investigated on day 8. Amounts of < LOQ to 48% (C12 ether diamine) and < LOQ to 20% (C14 ether diamine) of the nominal loading rates were measured.
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the limit loading of the test item after 0 hours. The signal distribution was compared with an analytical standard prepared in solvent.

Measured Concentrations of the Test Item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) during the Definitive Test with daphnids (C12 ether diamine)
(Days 0, 1, 7, 8, 14 and 15)

Sampling date		Day 0		Day 1		Day 7		Day 8
		Fresh medium,		Old medium,		Fresh medium,		Old medium,
		0 hours		24 hours		0 hours		24 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C12 ether diamine
test item	a.s.	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
80.0	50.4	44.3	88	17.6	35	50.7	101	25.4	50
25.3	44819	13.6	85	3.61	23	17.8	112	1.95	12
8.00	44656	3.14	62	0.909	18	5.05	100	0.894	18
2.53	21551	0.940	59	0.304	19	1.55	97	0.412	26
0.800	0.504	0.257	51	< LOQ		0.450	89	< LOQ
0.253	0.159	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling date		Day 14		Day 15
		Fresh medium,		Old medium,
		0 hours		24 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C12 ether diamine
test item	a.s.	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.
		[µg a.s./L]		[µg a.s./L]
80.0	50.4	59.6	118	32.0	64
25.3	15.9	17.0	107	2.09	13
8.00	5.04	4.47	89	1.06	21
2.53	1.59	1.47	92	0.485	30
0.800	0.504	0.517	103	< LOQ
0.253	0.159	< LOQ		< LOQ
Control		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
a.s. = active substance
% = percent of the nominal measured test item concentration
LOQ = limit of quantification (0.250 µg/L of the test item, corresponding to 0.158 µg a.s./L)

 

Measured Concentrations of the Test Item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) during the Definitive Test without daphnids and algae (C12 ether diamine)
(Day 8)

Sampling date		Day 8
		Old medium,
		24 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C12 ether diamine
test item
[µg/L]
test item	a.s.	Meas.	%
[µg/L]	[µg a.s./L]	conc.
		[µg a.s./L]
80.0	50.4	24.4	48
25.3	15.9	3.54	22
8.00	5.04	1.26	25
2.53	1.59	0.182	11
0.800	0.504	< LOQ
0.253	0.159	< LOQ
Control		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
a.s. = active substance
% = percent of the nominal measured test item concentration
LOQ = limit of quantification (0.250 µg/L of the test item, corresponding to 0.158 µg a.s./L)

 

Measured Concentrations of the Test Item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) during the Definitive Test with daphnids (C14 ether diamine)
(Days 0, 1, 7, 8, 14 and 15)

Sampling date		Day 0		Day 1		Day 7		Day 8
		Fresh medium,		Old medium,		Fresh medium,		Old medium,
		0 hours		24 hours		0 hours		24 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C14 ether diamine
test item	a.s.	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
80.0	16.0	13.7	86	3.69	23	10.1	63	3.16	20
25.3	5.06	2.84	56	0.617	12	3.59	71	0.590	12
8.00	1.60	0.396	25	0.105	7	0.699	44	0.125	8
2.53	0.506	0.113	22	< LOQ		0.141	28	0.0575	11
0.800	0.160	< LOQ		< LOQ		0.0912	57	< LOQ
0.253	0.0506	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling date		Day 14		Day 15
		Fresh medium,		Old medium,
		0 hours		24 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C14 ether diamine
test item	a.s.	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.
		[µg a.s./L]		[µg a.s./L]
80.0	16.0	11.9	74	6.74	42
25.3	5.06	2.54	50	0.234	5
8.00	1.60	0.673	42	0.154	10
2.53	0.506	0.126	25	< LOQ
0.800	0.160	0.0807	50	< LOQ
0.253	0.0506	< LOQ		< LOQ
Control		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
a.s. = active substance% = percent of the nominal measured test item concentration
LOQ = limit of quantification (0.250 µg/L of the test item, corresponding to 0.0500 µg a.s./L)

 

Measured Concentrations of the Test Item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) during the Definitive Test without daphnids and algae (C14 ether diamine)
(Day 8)

Sampling date		Day 8
		Old medium,
		24 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C14 ether diamine
test item
[µg/L]
test item	a.s.	Meas.	%
[µg/L]	[µg a.s./L]	conc.
		[µg a.s./L]
80.0	16.0	3.16	20
25.3	5.06	0.653	13
8.00	1.60	0.253	16
2.53	0.506	< LOQ
0.800	0.160	< LOQ
0.253	0.0506	< LOQ
Control		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
a.s. = active substance
% = percent of the nominal measured test item concentration
LOQ = limit of quantification (0.250 µg/L of the test item, corresponding to 0.0500 µg a.s./L)

Time weighted Mean Measured Concentrations of the Test Item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)

Nominal loading	Time Weighted Mean Measured Test Item Concentration ^1)
rate of the test item
test item	[µg/L]
[µg/L]
80.0	53.4
25.3	10.4
8.00	3.01
2.53	1.01
0.800	0.294
0.253	0.124
Control	< LOQ

1) 1/2 LOQ was used for calculations when the measured value was < LOQ

 

Stillborn Juveniles and Aborted Eggs produced by the Survived Parental Daphnids after 21 Days

Nominal	Number of		Total no.
loading rate
of the test item			å
[µg/L]	stillborn	aborted
	juveniles	eggs
80.0	22	14	36
25.3	7	17	24
8.00	1	16	17
2.53	3	15	18
0.800	3	14	17
0.253	0	7	7
Control	0	0	0

 

Total Body Length and Dry Weight of the Survived Parental Daphnids

Nominal	Total length of the survived parent animals [mm]										MV	N	Dry weight
loading rate													[mg]
of the test item	Replicate no.
[µg/L]	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
80.0	-	-	-	-	-	4.0	-	3.5	4.0	4.0	3.88	4	1.20	0.30
25.3	-	4.0	-	4.0	4.5	4.0	4.0	-	4.5	4.5	4.21	7	3.30	0.47
8.00	-	5.0	4.5	3.0	4.0	4.0	4.5	4.0	4.5	4.5	4.22	9	9.20	1.02
2.53	4.5	4.5	4.0	4.0	4.5	4.5	-	4.5	4.5	4.5	4.39	9	6.80	0.76
0.800	4.5	4.5	4.5	-	4.5	4.0	5.0	4.5	4.5	4.0	4.44	9	5.60	0.62
0.253	4.5	5.0	5.0	-	4.5	4.0	4.5	4.5	4.5	4.5	4.56	9	5.40	0.60
Control	5.0	4.0	4.5	5.0	4.5	4.5	5.0	4.5	4.5	4.5	4.60	10	4.50	0.45

N = number of the survived parental female daphnids
- = not applicable, due to the mortality of the parental daphnid(s)

 

Observations in the Control

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	24	-	-	41	-	-	46	-	-	51	-	49	-	-	50	261
2	-	-	-	-	-	-	-	12	-	-	20	-	-	35	-	-	33	-	-	4	24	128
3	-	-	-	-	-	-	-	14	-	-	11	-	-	34	-	-	23	-	-	-	31	113
4	-	-	-	-	-	-	14	-	-	15	-	-	33	-	-	1	15	-	-	29	-	107
5	-	-	-	-	-	-	-	19	-	-	22	-	-	36	-	-	26	-	-	2	28	133
6	-	-	-	-	-	-	-	13	-	-	12	-	-	30	-	-	32	-	-	20	6	113
7	-	-	-	-	-	-	16	-	-	17	1	-	24	-	-	25	-	-	-	27	-	110
8	-	-	-	-	-	-	17	-	-	17	-	-	27	-	-	29	-	-	-	19	-	109
9	-	-	-	-	-	-	-	14	1	-	19	-	-	27	-	-	26	-	-	-	31	118
10	-	-	-	-	-	-	-	9	-	-	21	-	-	30	-	-	-	4	32	-	21	117
Based on all parental daphnids inserted at the start of the exposure:																						1309
Based on parental daphnids survived until the end of the test:																						1309
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

- = no juveniles observed

 

Observations in the Nominal Loading Rate of 0.253 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	13	-	-	22	-	-	24	-	-	34	-	44	-	-	29	166
2	-	-	-	-	-	-	-	17	-	-	20	-	-	25	-	49	-	-	47	-	-	158
3	-	-	-	-	-	-	-	15	-	-	11	-	-	27	-	-	21	-	-	-	22	96
4	-	-	-	-	-	-	14	-	-	17M												31
5	-	-	-	-	-	-	-	16	-	-	18	-	-	31	-	-	17	-	-	-	34	116
6	-	-	-	-	-	-	15	-	-	12	-	-	19	-	-	-	-	52	-	-	30	128
7	-	-	-	-	-	-	17	-	-	13	-	-	-	30	-	-	9	-	-	26	-	95
8	-	-	-	-	-	-	13	-	-	14	-	-	27	-	-	-	20	-	-	24	-	98
9	-	-	-	-	-	-	11	-	-	18	-	-	33	-	-	-	22	-	-	26	-	110
10	-	-	-	-	-	-	-	14	-	-	25	-	-	28	-	-	18	-	-	-	29	114
Based on all parental daphnids inserted at the start of the exposure:																						1112
Based on parental daphnids survived until the end of the test:																						1081
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	1	1	1	1	1	1	1
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	10	10	10	10	10	10	10	10	10	10	10	10	10

- = no juveniles observed
M = dead parental daphnid

 

Observations in the Nominal Loading Rate of 0.800 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	-	13	-	-	24	-	-	21	-	-	17	-	-	26	-	101
2	-	-	-	-	-	-	15	-	-	15	-	-	16	-	-	-	17	-	-	21	-	84
3	-	-	-	-	-	-	10	-	-	18	-	-	24	-	-	-	15	-	-	20	-	87
4	-	-	-	-	-	-	-	16	-	-	29	-	-	16	-	M						61
5	-	-	-	-	-	-	12	-	-	21	-	-	31	-	-	26	-	-	-	28	-	118
6	-	-	-	-	-	-	19	-	-	46	-	-	40	-	-	28	-	-	40	-	-	173
7	-	-	-	-	-	-	-	10	-	-	11	-	-	28	-	-	29	-	-	-	22	100
8	-	-	-	-	-	-	-	11	-	-	18	-	-	29	-	-	2	28	-	-	20	108
9	-	-	-	-	-	-	15	-	-	11	-	-	24	1	-	-	18	-	-	20	-	89
10	-	-	-	-	-	-	9	-	-	18	-	-	25	-	-	-	26	-	-	25	-	103
Based on all parental daphnids inserted at the start of the exposure:																						1024
Based on parental daphnids survived until the end of the test:																						963
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	1
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	10	10	10	10	10	10	10

- = no juveniles observed
M = dead parental daphnid

 

Observations in the Nominal Loading Rate of 2.53 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	11	-	-	13	-	-	24	-	-	-	11	-	-	14	-	73
2	-	-	-	-	-	-	17	-	-	18	-	-	26	-	-	19	-	-	-	20	-	100
3	-	-	-	-	-	-	-	14	-	49	-	-	46	-	-	20	-	-	30	-	-	159
4	-	-	-	-	-	-	13	-	-	18	-	-	26	-	-	-	21	-	-	20	-	98
5	-	-	-	-	-	-	16	-	-	44	-	-	30	-	-	32	-	-	41	-	-	163
6	-	-	-	-	-	-	17	-	-	46	-	-	36	-	-	30	-	-	-	16	-	145
7	-	-	-	-	-	-	-	9	-	-	16	-	-	25	-	M						50
8	-	-	-	-	-	-	20	-	-	41	-	-	29	-	-	24	-	-	2	18	-	134
9	-	-	-	-	-	-	16	-	-	18	-	-	28	-	-	30	-	-	-	17	-	109
10	-	-	-	-	-	-	14	-	-	21	-	-	26	-	-	-	15	-	-	20	-	96
Based on all parental daphnids inserted at the start of the exposure:																						1127
Based on parental daphnids survived until the end of the test:																						1077
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	1
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	10	10	10	10	10	10	10

- = no juveniles observed
M = dead parental daphnid

 

Observations in the Nominal Loading Rate of 8.00 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	-	12	-	-	15	-	-	201	M							47
2	-	-	-	-	-	-	-	9	-	39	-	-	31	-	-	29	-	-	36	-	-	144
3	-	-	-	-	-	-	-	13	-	45	-	-	30	-	-	30	-	-	31	-	-	149
4	-	-	-	-	-	-	15	-	-	17	-	-	30	-	-	18	-	-	-	20	-	100
5	-	-	-	-	-	-	14	-	-	21	-	-	24	-	-	1	35	-	-	14	-	109
6	-	-	-	-	-	-	8	-	-	23	-	-	28	-	-	28	-	-	21	-	-	108
7	-	-	-	-	-	-	12	-	-	21	-	-	24	-1	-1	-1	-1	-1	-1	101	-1	67
8	-	-	-	-	-	-	19	-	-	47	1	-	20	-	-	32	-	-	30	-	-	149
9	-	-	-	-	-	-	-	16	-	32	2	-	30	-	-	29	-	-	29	-	-	138
10	-	-	-	-	-	-	18	-	-	46	-	-	23	-	-	36	-	-	31	-	-	154
Based on all parental daphnids inserted at the start of the exposure:																						1165
Based on parental daphnids survived until the end of the test:																						1118
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	1	1
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	10	10	10	10	10	10	10	10

- = no juveniles observed
1 = pale parental daphnid
M = dead parental daphnid

 

Observations in the Nominal Loading Rate of 25.3 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	M																					0
2	-	-	-	-	-	-	-	14	-	42	-	-	23	-	-	28	-	-	21	-	-	128
3	-	-	-	-	-	-	-	13	-	43	-	-	30	-	-	31	M					117
4	-	-	-	-	-	-	10	-	-	24	-	-	27	-	-	28	-	-	24	-	-	113
5	-	-	-	-	-	-	-	16	-	-	22	-	-	21	-	-	22	-	-	-	-	81
6	-	-	-	-	-	-	11	-	-	18	-	9	17	-	-	14	-	16	-	-1	111	96
7	-	-	-	-	-	-	-	11	-	-	17	-	5	15	-	26	-	-	-	14	-	88
8	-	-	-	-	-	-	-	10	-	45	-	-	28	-	-	35	-	-	25	-1	M	143
9	-	-	-	-	-	-	-	12	-	-	17	-	-	19	-	-	27	-	-	1	18	94
10	-	-	-	-	-	-	11	-	-	28	-	-	29	-	-	20	-	-	24	-	-	112
Based on all parental daphnids inserted at the start of the exposure:																						972
Based on parental daphnids survived until the end of the test:																						712
Mortality of Parental (N)	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	2	2	2	2	3	3
Mortality of Parental [%]	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	20	20	20	20	30	30

- = no juveniles observed
1 = pale parental daphnid
M = dead parental daphnid

 

Observations in the Nominal Loading Rate of 80.0 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	-	11	-	-	20	-	M									31
2	-	-	-	-	-	-	-	7	-	13	-	-	18	-	-	20	-	M				58
3	-	-	-	-	-	-	-	7	-	-	9	-	-	17	-	M						33
4	-	-	-	-	-	-	-	12	-	44	-	-	28	-1	M							84
5	-	-	-	-	-	M																0
6	-	-	-	-	-	-	-	6	-	-	11	-	1	24	-	-	16	-	-	14	-	72
7	-	-	-	-	-	-	-	4	-	-	9	-	27	-	-	21	6	-	-	16M		83
8	-	-	-	-	-	-	7	-	-	18	-	-	24	-	-	17	-	-	21	-	-	87
9	-	-	-	-	-	-	-	11	-	-	13	-	-	17	-	-	27	-	-	12	-	80
10	-	-	-	-	-	-	6	-	-	17	-	-	20	-	-	25	-	-	-	14	-	82
Based on all parental daphnids inserted at the start of the exposure:																						610
Based on parental daphnids survived until the end of the test:																						321
Mortality of Parental (N)	0	0	0	0	0	1	1	1	1	1	1	1	2	2	3	4	4	5	5	6	6	6
Mortality of Parental [%]	0	0	0	0	0	10	10	10	10	10	10	10	20	20	30	40	40	50	50	60	60	60

- = no juveniles observed
1 = pale parental daphnid
M = dead parental daphnid

 

Water Quality Parameters

Comparison of water quality parameters in fresh and old medium once within 7 days.

pH-Values

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	7.75	8.22	7.69	7.48	7.86	7.47
Control	7.86	7.35	7.74	7.46	7.83	7.77

The pH-values should be in the range of 6 – 9. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

Dissolved Oxygen Concentration [mg/L]

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	8.49	10.44	7.73	7.21	8.53	8.08
Control	8.24	4.77	8.37	8.43	8.67	7.98

The dissolved oxygen concentration was above 3 mg/L.

Total Hardness [mg/L] as CaCO3

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	235	236	253	258	252	250
Control	240	234	250	250	252	246

The total hardness was above 140 mg/L as CaCO3.

Temperature [°C]

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	21.6	21.4	21.1	21.6	20.6	20.6
Control	21.6	21.6	21.0	21.7	20.6	20.6

The temperature was within the range of 20 ± 2 °C.

 

Gradient Table

Time [min]	A [%]	B [%]
0.00	85	15
0.50	85	15
1.50	5	95
2.50	5	95
2.60	85	15
3.00	85	15

 

Dilution Steps (fresh media)

Nominal	Dilution	Sample	Final
test item concentration	Factor*	volume	volume
[mg/L]		[mL]	[mL]
80.0	40	0.05	1.0
25.3	10	0.2	1.0

* including factor 2 of sampling

 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Calibration	≥ 5 standard concentrations,	0.075 to 5.0 µg test item/L (n = 8),	ü
	r ≥ 0.99 (R^2≥ 0.9801 for 2^nd order regression)	R^2≥ 0.9801
	Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation
Matrix effects	Matrix effects are considered significant if they exceed ±20%.	Matrix effects: 1 to 4%	ü
		(matrix-matched calibration‑standards were used)
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace	S/N for 0.075 µg standard/L	ü
	S/N ≥ 3 for qualifier ion trace	C12 Diamine
		4901 (Quantifier), 1430 (Qualifier)
		C14 Diamine
		1839 (Quantifier), 923 (Qualifier)
Limit of Quantification (LOQ)	Should be at or below lowest nominal test concentration	0.250 µg test item/L (1 x LOQ)	ü
		100 µg test item/L (400 x LOQ)
Accuracy (Fortified samples)	Mean recovery rate of 70-120%	C12 Diamine	ü
	per fortification level (2 levels)	1 x LOQ: 112% (n = 4)
		400 x LOQ: 117% (n = 4)
		C14 Diamine
		1 x LOQ: 113% (n = 5)
		400 x LOQ: 117% (n = 4)
Precision	Relative standard deviation ≤ 20% per fortification level	C12 Diamine	ü
		1 x LOQ: 10%
		400 x LOQ: 4.8%
		C14 Diamine
		1 x LOQ: 17%
		400 x LOQ: 8.0%

Stability	Samples: Stable if 70 – 120% of nominal concentration	See section 17.1	ü
	Calibration standards: Stable if ±10% of freshly prepared standard
Specificity: LC-MS/(MS)	Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity).	C12 Diamine	ü
		quantifier [m/z]: 301.35 > 98.11
		qualifier [m/z]: 301.35 > 70.16
		C14 Diamine
		quantifier [m/z]: 329.38 > 98.17
		qualifier [m/z]: 329.38 > 312.45
	Blank values < 30% of the LOQ	Blank values < 30% of LOQ	ü
Procedural recovery	Procedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value.	See section 17.2	ü

 

 = criterion fulfilled

 

Preparation of Fortified Samples of the Test Item

LOQ Level	Control	1	400
Stock solution	-	1000 mg test item/L in acetonitrile
Spiking solution	-	25.0	10000
[µg test item/L]		(dilution medium)	(acetonitrile)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	0.250	100
[µg test item/L]
Medium for preparation	Daphnia dilution water
Volume of spiking solution [mL]	-	0.05	0.05
Volume of medium [mL]	10^a)	9.95^a)	4.95
Dilution factor	2^a)	2^a)	200
Dilution medium	Dilution medium		Acetonitrile containing 2% formic acid ^1)
			Dilution medium^2)
Sample volume [mL]	10^1)a)	10^1)a)	5.0^1)
			0.1^2)
Finale volume [mL]	10^1)a)	10^1)a)	10^1)
			10^2)
Final expected concentrations for analyses	-	0.0788 µg C12 ether diamine/L,	0.315 µg C12 ether diamine/L,
		0.0250 µg C14 ether diamine/L  and	0.100 µg C14 ether diamine/L

Dilution medium: daphnia dilution water : acetonitrile (50 : 50) containing 1% formic acid
a) Spiking solution was given directly into dilution medium including dilution factor 2
1) First dilution step
2) Second dilution step

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
Fortified concentrations*: 0.250 µg test item/L (1 x LOQ) and 100 µg test item/L (400 x LOQ), corresponding to 0.163 µg C12 ether diamine/L and 0.0517 µg C14 ether diamine/L (1 x LOQ) and 65.1 µg C12 ether diamine/L and 20.7 µg C14 ether diamine/L (400 x LOQ)

Replicate	C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
	C12 ether diamine
	1 x LOQ		400 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
1	0.155	95	71.1	109
2	0.188	115	75.4	116
3	0.310^1)	190^1)	78.2	120
4	0.195	120	79.3	122
5	0.194	119	207^1)	318^1)
Mean	0.18	112	76	117
SD ±	0.02		4
CV [%]	10		4.8
Replicate	C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
	C14 ether diamine
	1 x LOQ		400 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
1	0.0492	95	23.0	111
2	0.0472	91	22.5	109
3	0.0620	120	24.4	118
4	0.0695	134	26.8	130
5	0.0633	122	1611)	778^1)
Mean	0.06	113	24	117
SD ±	0.01		2
CV [%]	17		8.0

Meas. conc. = measured concentration of the test item, dilution factor taken into account
a.s. = active substance
% = percent concentration of the fortified sample
* = weighing factor taken into account
SD = standard deviation
CV = coefficient of variation
1) = outlier determined by Grubb’s Test for outliers

 

Stability

The stability of the standard stock solution (1000 mg test item/L in acetonitrile) was checked after 28 days and was 107 to 110% of the nominal concentration and was found to be stable over this period.

The responses of a 2 and 6 days old calibration standard (1 µg test item/L) was compared with the responses of a freshly prepared calibration standard with the same concentration. The deviation was < 10% after 2 days and > 10% after 6 days for both components.

The stability of the fortified samples at the 1xLOQ was checked after 6 days. Samples were found to be stable over this period.

Measured Concentrations and Percent of Nominal Concentration of the stored fortified samples

	6 days
Component	Meas. conc.	%
	[µg a.s./L]
C12 Diamine	0.151	92
C14 Diamine	0.0526	102

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
a.s. = active substance

 

Procedural Recovery

A procedural recovery (Quality Control) on 1 x LOQ Level was freshly prepared on each day of analysis. It was treated in parallel to the test samples.

Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test

Sampling	Day 0		Day 1		Day 7		Day 8
date
Component	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
C12 Diamine	0.127	78	0.188	115	0.184	113	0.154	95
					0.159	98
C14 Diamine	0.0587	114	0.0469	91	0.0484	94	0.0527	102
					0.0455	88
Sampling	Day 14		Day 15
date
Component	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
C12 Diamine	0.144	88	0.145	89
C14 Diamine	0.0472	91	0.0416	80

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
a.s. = active substance
Quality Control = 0.250 µg test item/L, weighing factor taken into account

 

Preliminary Tests (non-GLP)

Solubility Test

One WAF was prepared with a nominal loading of 10.0 mg/L. For the loading rate, an appropriate amount of the test item was pipetted on the surface of the dilution water. A slow stirring procedure was applied at room temperature. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate bottom of the glass flask). The WAF was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect for 10.0 mg/L was positive. After 24, 48 and 72 hours samples were taken as shown below and analyzed via LC-MS.

Measured Concentrations of C12 – C14 alkyletherdiamine during the Solubility Test (non-GLP)
Analytical system: LC-MS

Nominal test item loading rate	C12 – C14 alkyletherdiamine
[mg/L]	Components	Measured concentration
		[mg a.s./L]
		24 hours	48 hours	72 hours
10.0	C12	9.52	11.3	9.20
	C14	3.05	3.11	2.63

LCL = lowest calibration standard = 0.5 µg test item/L, corresponding to 0.315 µg C12/L and 0.1 µg C14/L

 

Preliminary Range Finding Test

A non-GLP preliminary range finding test was conducted at the test facility as a shortened reproduction test (at least until observation of the first brood in all test groups) under semi-static conditions with renewal of the loading rates every day over a period of 14 days. A control and three water accommodated fractions of the test item of 0.50, 5.00 and 50.0 µg/L were tested in the range finding test. The Tyndall effect was negative during the exposure phase in the WAFs.
The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). Five daphnids, one daphnia per replicate, were exposed to each loading rate and the control.

Immobilization and Offspring in the non-GLP Preliminary Range Finding Test after 14 Days
(n = 5, divided into 5 replicates with 1 daphnid each)

Nominal test item loading rates	Mortality [%] after 14 days						Number of offspring after 14 days per inserted parent						First appearance of living juveniles
[µg/L]	Replicates						Replicates
	1	2	3	4	5	MV	1	2	3	4	5	MV	[d]
50.0	100^x	0	0	100^x	0	40	7	37	34	22	42	28	7 - 8
5.00	0	0	100	0	0	20	41	49	12	42	47	38	7 - 8
0.50	0	0	0	0	0	0	50	40	43	40	38	42	8
Control	0	0	0	0	0	0	44	55	54	55	39	49	8

x = animal was pale

 

Stillborn Juveniles and Aborted Eggs produced by the Survived Parental Daphnids after 14 Days

Nominal	Number of		Total no.
loading rate
of the test item			å
[µg/L]	stillborn	aborted
	juveniles	eggs
50.0	30	6	36
5.00	27	0	27
0.50	0	0	0
Control	0	0	0

 

Measured Concentrations of the Test Item during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS

Sampling		Fresh media,		Old media,		Glass Adsorption
		0 hours		24 hours		24° hours
Nominal test item loading rates		C12 – C14 alkyletherdiamine
		(C12-Diamine)
[µg/L]	[µg a.s./L]	Meas. conc.	%	Meas. conc.	%	Meas. conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
50.0	31.5	49.1	156	21.2	67	28.4
5.00	3.15	3.52	112	0.968	31	1.75
0.500	0.315	0.289	92	0.0912	29	0.253
Control		< LCL		< LCL		< LCL
Sampling		Fresh media,		Old media,		Glass Adsorption
		0 hours		24 hours		24° hours
Nominal test item loading rates		C12 – C14 alkyletherdiamine
		(C14-Diamine)
[µg/L]	[µg a.s./L]	Meas. conc.	%	Meas. conc.	%	Meas. conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
50.0	10.0	11.4	114	5.09	51	7.85
5.00	1.00	0.513	51	0.182	18	0.402
0.500	0.100	0.0360	36	< LCL		0.0459
Control		< LCL		< LCL		< LCL

% = Percent of the nominal test item concentration
LCL = lowest calibration standard = 0.1 µg test item/L, corresponding to 0.063 µg C12/L and 0.020 µg C14/L
a.s. = active substance
Meas. Conc. = measured concentration of the test item, dilution factors taken into account

 

Fingerprint – MS spectrum

The peak distribution of the test item was analyzed in fresh prepared medium in the highest test item concentration 80.0 µg/ L (WAF approach) from the main study and the control. An analytical standard of the test item was prepared in acetonitrile and diluted to 40.0 µg/L with acetonitrile. The highest test item concentration and the control was diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The standard dilution and the test item dilution were analytical verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared and were quite similar. The mass of C12 ether diamine (301.35 Da ± 0.5 Da) and the mass of C14 ether diamine (329.38 Da± 0.5 Da) in the measured concentrations were too low for significant signals.
For analyses the C12 and C14 ether diamine were chosen, because both were found in a higher concentrated analytical standard. During analysis of the test item solutions of the study, another detection mode (MRM) was chosen, because this is more specific and sensitive for concentration control analysis. This modus was suitable to detect both analytes.

Validity criteria fulfilled:

yes

Conclusions:

The effects on reproduction were evaluated based on the reproduction per female parent animal inserted at the start of the exposure. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effect values for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor.
The overall effect threshold for effects of the test item under the test conditions was 8.00 µg/L (NOEL) and 25.3 µg/L (LOEL) based on the nominal loading rates of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) and 3.01 µg/L (NOEC) and 10.4 µg/L (LOEC) based on the time weighted mean measured test item concentrations.

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (batch number: S20210420) was conducted according to OECD 211 (2012) from 2022-02-15 to 2022-03-10, with the definitive exposure phase from 2022-02-17 to 2022-03-10 at Noack Laboratorien.
Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per WAF loading rate and control. At the start of the exposure the daphnids were less than 24 hours old. The study was carried out under semi-static conditions with a daily renewal of the test solutions. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, the number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.
The test item is a UVCB substance with compounds of different water solubility. The aqueous media were prepared following OECD guidance document No. 23 (2019), which is specifically developed for aquatic toxicity testing of UVCBs, 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 WAF was separated by a siphon. This procedure was followed for each renewal of the test solutions. Six WAFs were prepared and tested at nominal loading rates 0.253 – 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L (separation factor 3.16), corresponding to the time weighted mean measured test item concentrations 0.124 – 0.294 – 1.01 – 3.01 – 10.4 – 53.4 µg/L.
The WAFs were checked for any undissolved or emulsified material by Tyndall effect, which was negative.
The concentrations of the two compounds C12 ether diamine and C14 ether diamine of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 7 and 14 without algae) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 8 and 15 with daphnia and algae) in all WAFs and in the control. Additional, samples with test media, but without daphnids and algae, were prepared for the sampling of the old media at least one time during the test (24 hours).

The stability of the test item under test conditions was monitored after 24 hours by quantifying the concentration of parallel samples which were prepared without daphnids and algae.

The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.
 
The effects on reproduction were evaluated based on the reproduction per female parent animal introduced at the start of the exposure. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effect values for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor. All reasonable efforts were taken to produce a solution of all soluble components of the test item in test media. Since the test item is a multicomponent mixture (UVCB), the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD guidance document No. 23 (2019), for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or loading rate (for mixtures). All evaluations are based on the nominal loading rates of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) and the time weighted mean measured test item concentrations.
A summary of all endpoints based on the nominal loading rates of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) and the time weighted mean measured test item concentrations is given in the table below.

Endpoints for Reproduction and Mortality
(based on the nominal loading rates of the test item and the time weighted mean measured test item concentrations)

Effect values	C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
	based on	based on
	the nominal loading rates	the time weighted mean measured concentrations
	[µg/L]	[µg/L]
EL/EC10 Reproduction (introduced)	19.9 (5.06 – 77.8)	7.61 (1.18 – 49.3)
EL/EC50 Reproduction (introduced)	86.5 (11.5 – 583#)	59.2 (3.88 – 781#)
LOEL/LOEC Reproduction (introduced)	25.3	10.4
NOEL/NOEC Reproduction (introduced)	8.00	3.01
LL/LC20 Adult mortality after 21 days	15.9 (10.0 – 24.1)	5.99 (2.84 – 12.0)
LL/LC50 Adult mortality after 21 days	57.0 (46.0 – 72.1)	33.4 (22.4 – 52.6)
LL/LC100 Adult mortality after 21 days	> 80.0	> 53.4
LOEL/LOEC Adult mortality after 21 days	25.3	10.4
NOEL/NOEC Adult mortality after 21 days	8.00	3.01

CI = 95% confidence limits
# value extrapolated

 

 

Description of key information

The long-term dahpnia test has been performed by using the WAF approach. The EL₁₀ Reproduction was 19.86 based on loading rate and 99.8% active. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Remarks:

WAF approach: The EL10 (repro) = 19.86 µg a.i./L based on loading rate and 99.8% active ingredient.

Effect concentration:

19.86 µg/L

Additional information

One long-term daphnia study for etherdiamine C12-14 is available. The study was performed semi-statically (daily water renewal) according to OECD TG 211 with specific chemical analysis (LC-MS/MS) under GLP conditions.

 

Etherdiamine C12-14 is a multicomponent mixture (UVCB) of cationic surface-active constituents with different water solubilities. The fate of cationic surfactants in general deviates from standard chemicals. These substances are therefore considered as difficult substances for which the results of standard guideline studies are very difficult to interpret when considering them in a standard way. The reasons are intrinsic properties like the relatively low water solubility and strong sorption to equipment and organisms. Classical ecotoxicity testing with this type of substance using reconstituted water often leads to test results which are poorly reproducible and are associated with high uncertainty. Because of the complex sorption mechanisms (van der Waals and ionic mechanisms) the actual dissolved exposure concentration cannot reliably be estimated.

 

The long-term daphnia test was performed according to the Water Accommodated Fraction (WAF) approach as described in “OECD guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (No. 23 Feb. 2019) with a daily refreshment of the test solutions. The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration.

 

The test solutions for the WAF approach were prepared by gentle mixing the test item with test medium for a prolonged period (24 hours) sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period (1 hour), the WAF was separated by siphoning. This procedure was followed for each renewal of the test solution (daily water renewal). Six WAFs were prepared and tested at nominal loading rates 0.253 - 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L (separation factor 3.16), corresponding to the time weighted mean measured test item concentrations were 0.124 – 0.294 – 1.01 – 3.01 – 10.4 – 53.4 µg/L. No undissolved or emulsified material was observed in the WAF solutions based on the Tyndall effect check. Adsorptive losses to glass vessels was kept as low as possible by pre-conditioning the test vessels already with appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure and each renewal, the test containers were emptied and refilled with freshly prepared test solutions.

The results are presented based on nominal test loadings and on time weighted average (TWA) measured concentrations. The EL10/EC10 for reproduction after 21 days is 19.9/7.61 µg/L. The EL50/EC50 for adult mortality after 21 days is 57.0/33.4 µg/L. The TWA results are given despite the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates (mass-to-volume ratio of the substance to the medium) because partly dissolved compounds and mixtures cannot be related to concentrations. Analytical verifications of selected components can be helpful and deliver supporting information, but they do not represent the whole test substance and therefore, toxicity results will be evaluated based on WAF loading rate (Wheeler, Lyon et al. 2020). Several guidance documents suggest to use the WAF loading rate for the environmental hazard classification of chemical substances e.g. the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (OECD 2002, OECD 2019) as well as OECD guidance documents on the classification of chemicals which are Hazardous for the Aquatic Environment

 

 

 

 

 

 

References:

OECD (2002). Guidance Document on the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for the Aquatic Environment.

 

OECD (2019). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.

 

Wheeler, J. R., D. Lyon, C. Di Paolo, A. Grosso and M. Crane (2020). "Challenges in the regulatory use of water-accommodated fractions for assessing complex substances." Environmental Sciences Europe 32(1): 1-10.