Amines, N-(3-aminopropyl)-N'-(C16-18 and C18-unsatd. alkyl)trimethylenedi-

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 2022-07-02 to 2022-11-10, with the definitive exposure phase from 2022-07-06 to 2022-07-27

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)

Specific details on test material used for the study:

Test item
N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (CAS No.: 1219458-14-6)

Common name Tallow dipropylenetriamine

Batch number 1929319

CAS No. 1219458-14-6

Purity (certified) 99.6 FID-area-%

Water content (certified) 0.41%

Appearance White solid

Water Solubility Slightly soluble

Stability under test
conditions Not specified

Expiry date 2023-01-29

Recommended storage
Store container tightly closed in a dry, well-ventilated place. Avoid elevated temperatures.

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 3 times during the test (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 one times during the test.
Additionally, all old media samples from one interval were centrifuged to separate the algae from the water phase. Then algae and water samples were analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae.
The samples were analyzed under GLP with a LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data.

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. On each sampling point the following constituents of the test item C16:0 propylenediamine, C18:0 propylenediamine, C18:1 propylenediamine, C16:0 dipropylenetriamine, C18:0 dipropylenetriamine, C18:1 dipropylenetriamine were analytically determined to distinguish how much test item is truly dissolved.

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. The signal distribution was compared with an analytical standard prepared in solvent and a control.

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction
Water accommodated fractions (WAFs) were prepared because the test item is a 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
Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.800 to 80.0 µg/L set up in a geometric series with a factor of 3.16: 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L.
For each loading rate an appropriate amount of stock solutions containing the test item in 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 72 ± 1 hour at room temperature (20-25 °C) with a magnetic stirrer bar placed at the bottom of the glass flask. The stirring speed was as low as technically feasible (Approx. 60 rpm). In a preliminary test, the optimal duration of stirring phase was determined to be 72 hours. After a separation phase of 1 hour at room temperature, the aqueous phase of 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 (formation of an emulsion). The Tyndall effect was negative and no presence of undissolved test item during the test was observed. The resulting water accommodated fractions (WAF) were used in the test. For the preparation of the water accommodated fractions a stirring phase of 72 hours was found to be suitable based on a solubility test

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
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. The composition of the culture medium is presented in Table 2.

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 CaCO3.
see section "Any other information on results incl. tables" below

Test temperature:

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

Dissolved oxygen:

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

Nominal and measured concentrations:

Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.800 to 80.0 µg/L set up in a geometric series with a factor of 3.16: 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L corresponding to the time weighted mean measured test item concentrations 0.175 – 0.738 – 2.51 – 6.26 – 21.1 µg/L.

Details on test conditions:

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

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 WAFs and the control. 1 daphnid was held individually per replicate.

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 containers were emptied and refilled with freshly prepared test solutions.

Type and Frequency of Measurements

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 and winter 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 NOEC/NOEL.

Additional endpoints
The time to first brood, the intrinsic rate of the population increase and the size of the 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.

Quality criteria for the water quality parameters
• The oxygen concentration should be > 3 mg/L at the start and during the test period.
• The total hardness should be above 140 mg/L CaCO3.
• The pH-value should be in the range of 6 - 9.
• Deviations between the initial and the final pH-values should be < 1.5 units.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.738 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

2.53 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.51 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LC100

Effect conc.:

> 21.1 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LL100

Effect conc.:

> 80 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

10.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LL50

Effect conc.:

40.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

2.73 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LL20

Effect conc.:

8.92 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

< 0.175 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

< 0.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

<= 0.175 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

<= 0.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

6.96 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

24.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.179 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.808 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 113 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 all nominal loading rates. Therefore, the LOEL was determined to be ≤ 80.0 µg/L and the NOEL ˂ 80.0 µg/L for the cumulative offspring per introduced parent. Based on the time weighted mean measured test item concentration, the LOEC was determined to be ≤ 0.175 µg/L and the NOEC ˂ 0.175 µg/L for the cumulative offspring per introduced parent.

The EL/EC10-, and the EL/EC50-values were calculated by sigmoidal dose-response regression from the best-fit values with the software GraphPad Prism. The EL10 for the test item based on the nominal loading rate was 0.808 µg/L and the EL50 24.3 µg/L per introduced parent. The EC10 for the test item based on the time weighted mean measured concentration was 0.179 µg/L and the EC50 6.96 µg/L per introduced parent.


Adult Mortality

Accidental and inadvertent mortality as defined in the OECD test guideline were not observed. In the control no mortality of the introduced adults was observed.
The Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality. A statistically significant dose-response was detected. The LL50 was 40.3 µg/L. The LOELAdult mortality was 8.00 µg/L. The NOELAdult mortality was 2.53 µg/L. The LC50 based on the time weighted mean measured test item concentration was 10.6 µg/L. The LOECAdult mortality was 2.51 µg/L. The NOECAdult mortality was 0.738 µg/L.

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 0.800 to 80.0 µg/L of the test item.


Analytical Results

Five WAFs were prepared and tested at nominal loading rates 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L (separation factor 3.16). The test item is an UVCB substance.
The test item concentrations of N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (CAS No.: 1219458-14-6) 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 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8 and 15) 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 the end of an exposure-renewal interval (day 1).
Additionally, all old media samples from one interval (day 8) were centrifuged to separate the algae from the water phase. Then algae and water samples were analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae.

The measured concentration of the test item in the fresh media (0 hours, on test days 0, 7, 14 without algae) was < LOQ to 89% of the nominal values and < LOQ to 54% in the aged media after 24 hours (on test days 1, 8, 15 with daphnids and algae). The additional replicates with test media, but without daphnids and algae were investigated on day 1. Amounts between < LOQ and 52% of nominal loading rates were measured. Centrifuged samples and algae adsorption were investigated on day 8. Amounts between < LOQ to 33% (centrifuged samples) and < LOQ to 53% (algae adsorption) of nominal loading rates were measured.
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium at the highest loading rate of the test item after 0 hours. The signal distribution was compared with an analytical standard prepared in solvent and a control.

Additional Analytical Results

Additional analytical measurements were carried out to investigate the behavior of the test item in water and algae.
Freshly prepared WAFs (water phase) and 24 hours old WAFs (water phase and algae) were sampled and analyzed. The algae were separated via centrifugation. Water and algae were analyzed separately.

Findings:

- The tendency for adsorption to the wall of the centrifuge tubes increases with the chain length of the analytes.
- In the lower nominal loading rates the results of the truly dissolved fraction partly fell below the limit of quantification.
- A mass balance, calculated as the sum of the results of the truly dissolved fraction and the adsorbed fraction from the centrifuge tubes, shows that the desorption of the analytes from the centrifuge tubes is not complete.
The determination of the truly dissolved fraction is not possible with N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine (CAS No.: 1219458-14-6).

Fingerprint of the Test Item (Non-GLP)

The peak distribution of the highest test item concentration (80 µg test item/L) and the control was analyzed. A solution of the analytical standard of the test item was prepared and diluted to 35 µg/L with methanol. The sample and the control were diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The detected signals of the analytical standards and the sample solution were compared. However, the concentrations and solubility of the test item is too low for significant MS spectra and no signals related to the test item could be observed.

Reported statistics and error estimates:

All calculations are based on the control and the nominal loading rates 0.800 – 2.53 – 8.00 – 25.3 – 80.0 µg/L, corresponding to the time weighted mean measured test item concentrations 0.175 – 0.738 – 2.51 – 6.26 – 21.1 µg/L.

Time weighted mean measured concentrations
The time weighted mean measured concentrations were determined by the sumproduct function of excel, referring to the recoveries and purities of the six lead components of the samples. This procedure takes into account the mean measured concentration of main constituent as well as their content in the product.

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 Jonckheere-Terpstra 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. 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 EL/ECx- / LL/LCx-values
The EL/ECx– and the LL/LCx- values for the reproductive output were calculated by sigmoidal dose-response regression from the best-fit values with the software GraphPad Prism. The respective 95% confidence limits were derived from the observation data based on the cumulative offspring per introduced parent.

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, Version 3.3.0, TOXRAT SOLUTIONS GMBH
- Excel, MICROSOFT CORPORATION
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

 

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  of the test item  [µg/L]	Time weighted mean measured test item concentration [µg/L]	Mean number of offspring  per introduced parental daphnid
		Mean	SD	CV %	Reduction % (compared to control)
80.0	21.1	42.8	17.6	41.1	62.1
25.3	6.26	55.6	15.6	28.0	50.8
8.00	2.51	71.3	11.1	15.6	36.8
2.53	0.738	77.5	26.1	33.7	31.4
0.800	0.175	102	9.55	9.30	9.50
Control		113	14.1	12.5	-

 

 Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                 (n = 10)

Nominal loading rate  of the test item  [µg/L]	Time weighted mean measured test item concentration [µg/L]	Adult Mortality [%]
		7 days	14 days	21 days
80.0	21.1	0	30	70
25.3	6.26	0	0	30
8.00	2.51	0	0	20
2.53	0.738	0	10	10
0.800	0.175	0	0	0
Control		0	0	0

 

Intrinsic Rates of Natural Increase

Nominal loading rate  of the test item [µg/L]	Mean IR	CV
	MV ± SD	[%]
80.0	0.326 ± 0.0052	1.60
25.3	0.329 ± 0.0160	4.90
8.00	0.353 ± 0.0178	5.10
2.53	0.350 ± 0.0329	9.40
0.800	0.362 ± 0.0170	4.70
Control	0.365 ± 0.0253	6.90

 

 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 0.800 to 80.0 µg/L of the test item.

 

First Appearance of Living Juveniles in the Individual Groups

 

Nominal loading rate  of the test item [µg/L]	Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.										First appearance (mean day)
	1	2	3	4	5	6	7	8	9	10
80.0	8	8	8	8	8	8	8	8	8	8	8.00
25.3	8	8	8	8	8	8	8	8	8	8	8.00
8.00	7	8	8	7	8	8	8	7	8	7	7.60
2.53	8	7	8	8	8	8	8	7	8	7	7.70
0.800	7	7	7	8	7	8	8	8	8	8	7.60
Control	8	8	7	8	8	7	7	8	8	8	7.70

 

 Measured Concentrations of the compound C16:0 propylenediamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test with daphnia and algae

 (Days 0, 1, 7, 8, 14 and 15)

Sampling		Fresh media, Day 0		Old media, Day 1		Fresh media, Day 7		Old media, Day 8
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C16:0 propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	4.56	1.14	25	0.772	17	4.06	89	0.708	16
25.3	1.44	0.101	7	0.126	9	0.878	61	0.444	31
8.00	0.456	0.0324	7	0.0256	6	0.205	45	0.149	33
2.53	0.144	< LOQ		< LOQ		0.0216	15	< LOQ
0.800	0.0456	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling		Fresh media, Day 14				Old media, Day 15
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C16:0 propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%		Meas. conc. [µg a.s./L]		%
80.0	4.56	0.964		21		0.401		9
25.3	1.44	0.414		29		0.0819		6
8.00	0.456	0.0758		17		0.0309		7
2.53	0.144	0.0285		20		0.0204		14
0.800	0.0456	< LOQ				< LOQ
Control		< LOQ				< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0200 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C16:0 propylenediamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test without daphnia and algae, centrifuged samples and algae adsorption

Sampling date		Old media, Day 1
Nominal test item loading rates		without daphnia and algae
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%
80.0	4.56	0.456	10
25.3	1.44	0.0541	4
8.00	0.456	< LOQ
2.53	0.144	< LOQ
0.800	0.0456	< LOQ
Control		< LOQ
Sampling		Old Media Day 8			Old media, Day 8
Nominal test item loading rates		Centrifuged samples			Algae adsorption
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
80.0	4.56	0.526		12	0.901	20
25.3	1.44	0.386		27	0.249	17
8.00	0.456	0.0748		16	0.0983	22
2.53	0.144	< LOQ			< LOQ
0.800	0.0456	< LOQ			< LOQ
Control		< LOQ			< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0200 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C18:0 propylenediamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test with daphnia and algae

                      (Days 0, 1, 7, 8, 14 and 15)

Sampling		Fresh media, Day 0		Old media, Day 1		Fresh media, Day 7		Old media, Day 8
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:0 propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	5.36	2.83	53	1.41	26	1.73	32	0.429	8
25.3	1.70	0.707	42	0.510	30	0.491	29	0.314	19
8.00	0.536	0.250	47	0.187	35	0.152	28	0.120	22
2.53	0.170	0.0925	55	0.0693	41	0.0340	20	< LOQ
0.800	0.0536	0.0372	69	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling		Fresh media, Day 14				Old media, Day 15
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:0 propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%		Meas. conc. [µg a.s./L]		%
80.0	5.36	0.500		9		0.322		6
25.3	1.70	0.160		9		0.0739		4
8.00	0.536	0.0543		10		0.0274		5
2.53	0.170	< LOQ				< LOQ
0.800	0.0536	< LOQ				< LOQ
Control		< LOQ				< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0235 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C18:0 propylenediamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test without daphnia and algae, centrifuged samples and algae adsorption

Sampling date		Old media, Day 1
Nominal test item loading rates		without daphnia and algae
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%
80.0	5.36	1.88	35
25.3	1.70	0.522	31
8.00	0.536	0.144	27
2.53	0.170	0.0618	36
0.800	0.0536	0.0279	52
Control		< LOQ
Sampling		Old Media Day 8		Old media, Day 8
Nominal test item loading rates		Centrifuged samples		Algae adsorption
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	5.36	0.299	6	0.464	9
25.3	1.70	0.244	14	0.169	10
8.00	0.536	0.0728	14	0.0670	13
2.53	0.170	< LOQ		< LOQ
0.800	0.0536	< LOQ		< LOQ
Control		< LOQ		< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0235 µg a.s./L)

a.s.                   = active substance

 

 

Measured Concentrations of the compound C18:1 propylenediamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test with daphnia and algae

                      (Days 0, 1, 7, 8, 14 and 15)

Sampling		Fresh media, Day 0		Old media, Day 1		Fresh media, Day 7		Old media, Day 8
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:1 propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	6.72	1.37	20	0.598	9	4.96	74	0.524	8
25.3	2.13	0.0782	4	0.0574	3	0.973	46	0.330	16
8.00	0.672	< LOQ		< LOQ		0.234	35	0.127	19
2.53	0.213	< LOQ		< LOQ		< LOQ		< LOQ
0.800	0.0672	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling		Fresh media, Day 14				Old media, Day 15
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:1 propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%		Meas. conc. [µg a.s./L]		%
80.0	6.72	1.06		16		0.385		6
25.3	2.13	0.425		20		0.0851		4
8.00	0.672	0.0794		12		< LOQ
2.53	0.213	< LOQ				< LOQ
0.800	0.0672	< LOQ				< LOQ
Control		< LOQ				< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0294 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C18:1 propylenediamine of the Test Item N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test without daphnia and algae, centrifuged samples and algae adsorption

Sampling date		Old media, Day 1
Nominal test item loading rates		without daphnia and algae
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%
80.0	6.72	0.564	8
25.3	2.13	0.0431	2
8.00	0.672	< LOQ
2.53	0.213	< LOQ
0.800	0.0672	< LOQ
Control		< LOQ
Sampling		Old Media Day 8			Old media, Day 8
Nominal test item loading rates		Centrifuged samples			Algae adsorption
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
80.0	6.72	0.412		6	0.642	10
25.3	2.13	0.300		14	0.171	8
8.00	0.672	0.0636		9	0.0805	12
2.53	0.213	< LOQ			< LOQ
0.800	0.0672	< LOQ			< LOQ
Control		< LOQ			< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0294 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C16:0 dipropylenetriamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test with daphnia and algae

                      (Days 0, 1, 7, 8, 14 and 15)

Sampling		Fresh media, Day 0		Old media, Day 1		Fresh media, Day 7		Old media, Day 8
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C16:0 dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	17.0	5.37	32	2.40	14	10.5	62	1.80	11
25.3	5.39	3.30	61	2.22	41	2.47	46	1.30	24
8.00	1.70	0.979	57	0.700	41	1.06	62	0.878	52
2.53	0.539	0.344	64	0.292	54	0.257	48	0.218	40
0.800	0.170	0.131	77	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling		Fresh media, Day 14				Old media, Day 15
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C16:0 dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%		Meas. conc. [µg a.s./L]		%
80.0	17.0	2.53		15		1.32		8
25.3	5.39	0.813		15		0.396		7
8.00	1.70	0.319		19		0.139		8
2.53	0.539	0.102		19		< LOQ
0.800	0.170	< LOQ				< LOQ
Control		< LOQ				< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0746 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C16:0 dipropylenetriamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test without daphnia and algae, centrifuged samples and algae adsorption

Sampling date		Old media, Day 1
Nominal test item loading rates		without daphnia and algae
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%
80.0	17.0	2.73	16
25.3	5.39	2.71	50
8.00	1.70	0.577	34
2.53	0.539	0.248	46
0.800	0.170	0.0809	47
Control		< LOQ
Sampling		Old Media Day 8		Old media, Day 8
Nominal test item loading rates		Centrifuged samples		Algae adsorption
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%
80.0	17.0	1.25	7	2.35		14
25.3	5.39	0.984	18	0.746		14
8.00	1.70	0.436	26	0.643		38
2.53	0.539	0.179	33	0.285	53
0.800	0.170	< LOQ		< LOQ
Control		< LOQ		< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0746 µg a.s./L)

a.s.                   = active substance

 

 

Measured Concentrations of the compound C18:0 dipropylenetriamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test with daphnia and algae

                      (Days 0, 1, 7, 8, 14 and 15)

Sampling		Fresh media, Day 0		Old media, Day 1		Fresh media, Day 7		Old media, Day 8
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:0 dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	19.4	11.4	59	4.71	24	3.95	20	1.16	6
25.3	6.15	3.70	60	2.59	42	1.24	20	0.705	11
8.00	1.94	1.37	71	0.867	45	0.671	35	0.573	29
2.53	0.615	0.347	57	0.234	38	0.256	42	0.201	33
0.800	0.194	0.126	65	< LOQ		0.117	60	< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling		Fresh media, Day 14				Old media, Day 15
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (C18:0 dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%		Meas. conc. [µg a.s./L]		%
80.0	19.4	1.67		9		1.14		6
25.3	6.15	0.493		8		0.281		5
8.00	1.94	0.203		10		0.0979		5
2.53	0.615	< LOQ				< LOQ
0.800	0.194	< LOQ				< LOQ
Control		< LOQ				< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0851 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C18:0 dipropylenetriamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test without daphnia and algae, centrifuged samples and algae adsorption

Sampling date		Old media, Day 1
Nominal test item loading rates		without daphnia and algae
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%
80.0	19.4	5.30	27
25.3	6.15	2.96	48
8.00	1.94	0.790	41
2.53	0.615	0.271	44
0.800	0.194	< LOQ
Control		< LOQ
Sampling		Old Media Day 8			Old media, Day 8
Nominal test item loading rates		Centrifuged samples			Algae adsorption
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]		%
80.0	19.4	0.994		5	1.52		8
25.3	6.15	0.600		10	0.597		10
8.00	1.94	0.362		19	0.518		27
2.53	0.615	0.196		32	0.166	27
0.800	0.194	< LOQ			< LOQ
Control		< LOQ			< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0851 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C18:1 dipropylenetriamine of the Test Item

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test with daphnia and algae

                      (Days 0, 1, 7, 8, 14 and 15)

Sampling		Fresh media, Day 0		Old media, Day 1		Fresh media, Day 7		Old media, Day 8
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:1 dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	16.2	4.55	28	1.90	12	7.64	47	0.784	5
25.3	5.11	2.89	57	1.92	37	1.47	29	0.637	12
8.00	1.62	1.00	62	0.743	46	0.848	52	0.637	39
2.53	0.511	0.311	61	0.262	51	0.218	43	0.190	37
0.800	0.162	0.114	71	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling		Fresh media, Day 14				Old media, Day 15
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:1 dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]		%		Meas. conc. [µg a.s./L]		%
80.0	16.2	1.67		10		0.924		6
25.3	5.11	0.515		10		0.256		5
8.00	1.62	0.231		14		0.105		7
2.53	0.511	< LOQ				< LOQ
0.800	0.162	< LOQ				< LOQ
Control		< LOQ				< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0707 µg a.s./L)

a.s.                   = active substance

 

Measured Concentrations of the compound C18:1 dipropylenetriamine of the Test Item N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

(CAS No.: 1219458-14-6) during the Definitive Test without daphnia and algae, centrifuged samples and algae adsorption

Sampling date		Old media, Day 1
Nominal test item loading rates		without daphnia and algae
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%
80.0	16.2	2.54	16
25.3	5.11	2.09	41
8.00	1.62	0.542	34
2.53	0.511	0.225	44
0.800	0.162	0.0710	44
Control		< LOQ
Sampling		Old Media Day 8		Old media, Day 8
Nominal test item loading rates		Centrifuged samples		Algae adsorption
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%
80.0	16.2	0.662	4	1.21		8
25.3	5.11	0.614	12	0.393		8
8.00	1.62	0.409	25	0.405		25
2.53	0.511	0.166	33	0.227	44
0.800	0.162	< LOQ		< LOQ
Control		< LOQ		< LOQ

Meas. conc.     = measured concentration of the test item, dilution factors taken into account

%                      = percent of the nominal test item loading rate

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0707 µg a.s./L)

a.s.                   = active substance

 

Overview of Mean Measured Concentrations of the Test item and Calculated Time Weighted Mean Measured Test Item Concentrations (Study Day 0 to 15)

Sampling day	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Nominal concentration of the test item [µg/L]	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (CAS No.: 1219458-14-6)						Time weighted mean measured concentrations [µg/L]
	Calculated mean measured concentration per study day [µg/L]
0.800	0.501	0.175	0.261	0.175	0.175	0.175	0.175
2.53	1.30	1.02	0.932	0.744	0.272	0.187	0.738
8.00	4.22	2.94	3.66	2.86	1.10	0.485	2.51
25.3	12.5	8.53	8.71	4.26	3.24	1.36	6.26
80.0	30.9	13.6	37.9	6.40	9.74	5.35	21.1

 

Measured Concentrations of the compound C16:0 propylenediamine of the Test Item during the additional test

Sampling date		Day 0		Day 1		Day 1
		-		test media with algae		test media without algae
Nominal loading rate of the		Samples with organic solvent
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	4.56	1.40	31	0.553	12	0.762	17
25.3	1.44	0.515	36	0.133	9	0.220	15
8.00	0.456	< LOQ		< LOQ		< LOQ
2.53	0.144	< LOQ		< LOQ		< LOQ
0.800	0.0456	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Truly dissolved test item (centrifuged samples)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	4.56	0.131	3	< LOQ		< LOQ
25.3	1.44	0.0261	2	< LOQ		< LOQ
8.00	0.456	< LOQ		< LOQ		< LOQ
2.53	0.144	< LOQ		< LOQ		< LOQ
0.800	0.0456	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Adsorption to the centrifuge tubes
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	4.56	0.477	10	0.566	12	0.290	6
25.3	1.44	0.268	19	0.120	8	0.136	9
8.00	0.456	< LOQ		< LOQ		< LOQ
2.53	0.144	< LOQ		< LOQ		< LOQ
0.800	0.0456	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ

Meas. conc.     = measured concentration of the compound C16:0 propylenediamine, dilution factors taken into account

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0200 µg a.s./L)

a.s.                   = active substance

%                      = percent of the nominal loading rate of the active substance

 

Measured Concentrations of the compound C18:0 propylenediamine of the Test Item during the additional test

Sampling date		Day 0		Day 1		Day 1
		-		test media with algae		test media without algae
Nominal loading rate of the		Samples with organic solvent
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	5.36	0.783	15	0.270	5	0.568	11
25.3	1.70	0.274	16	0.147	9	0.284	17
8.00	0.536	0.0353	7	0.0287	5	0.0398	7
2.53	0.170	0.0238	14	< LOQ		< LOQ
0.800	0.0536	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Truly dissolved test item (centrifuged samples)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	5.36	0.0312	1	< LOQ		< LOQ
25.3	1.70	< LOQ		< LOQ		< LOQ
8.00	0.536	< LOQ		< LOQ		< LOQ
2.53	0.170	< LOQ		< LOQ		< LOQ
0.800	0.0536	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Adsorption to the centrifuge tubes
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	5.36	0.367	7	0.349	7	0.283	5
25.3	1.70	0.185	11	0.143	8	0.21	12
8.00	0.536	0.0284	5	0.0244	5	0.0289	5
2.53	0.170	< LOQ		< LOQ		< LOQ
0.800	0.0536	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ

Meas. conc.     = measured concentration of the compound C18:0 propylenediamine, dilution factors taken into account

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0235 µg a.s./L)

a.s.                   = active substance

%                      = percent of the nominal loading rate of the active substance

 

Measured Concentrations of the compound C18:1 propylenediamine of the Test Item during the additional test

Sampling date		Day 0		Day 1		Day 1
		-		test media with algae		test media without algae
Nominal loading rate of the		Samples with organic solvent
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	6.72	1.53	23	0.490	7	0.754	11
25.3	2.13	0.526	25	0.113	5	0.183	9
8.00	0.672	< LOQ		< LOQ		< LOQ
2.53	0.213	< LOQ		< LOQ		< LOQ
0.800	0.0672	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Truly dissolved test item (centrifuged samples)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	6.72	0.103	2	< LOQ		< LOQ
25.3	2.13	< LOQ		< LOQ		< LOQ
8.00	0.672	< LOQ		< LOQ		< LOQ
2.53	0.213	< LOQ		< LOQ		< LOQ
0.800	0.0672	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Adsorption to the centrifuge tubes
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	6.72	0.491	7	0.587	9	0.258	4
25.3	2.13	0.266	13	0.104	5	0.104	5
8.00	0.672	< LOQ		< LOQ		< LOQ
2.53	0.213	< LOQ		< LOQ		< LOQ
0.800	0.0672	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ

Meas. conc.     = measured concentration of the compound C18:1 propylenediamine, dilution factors taken into account

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0294 µg a.s./L)

a.s.                   = active substance

%                      = percent of the nominal loading rate of the active substance

 

Measured Concentrations of the compound C16:0 dipropylenetriamine of the Test Item during the additional test

Sampling date		Day 0		Day 1		Day 1
		-		test media with algae		test media without algae
Nominal loading rate of the		Samples with organic solvent
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	17.0	4.02	24	1.42	8	2.69	16
25.3	5.39	1.29	24	0.585	11	1.48	27
8.00	1.70	0.297	17	0.349	20	0.478	28
2.53	0.539	0.198	37	0.211	39	0.287	53
0.800	0.170	< LOQ		< LOQ		0.0748	44
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Truly dissolved test item (centrifuged samples)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	17.0	0.404	2	< LOQ		0.0849	0
25.3	5.39	< LOQ		< LOQ		< LOQ
8.00	1.70	< LOQ		< LOQ		< LOQ
2.53	0.539	< LOQ		< LOQ		< LOQ
0.800	0.170	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Adsorption to the centrifuge tubes
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	17.0	2.25	13	1.64	10	1.44	8
25.3	5.39	1.03	19	0.522	10	1.22	23
8.00	1.70	0.251	15	0.300	18	0.427	25
2.53	0.539	0.184	34	0.171	32	0.237	44
0.800	0.170	< LOQ		< LOQ		0.0774	45
Control		< LOQ		< LOQ		< LOQ

Meas. conc.     = measured concentration of the compound C16:0 dipropylenetriamine, dilution factors taken into account

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0746 µg a.s./L)

a.s.                   = active substance

%                      = percent of the nominal loading rate of the active substance

 

Measured Concentrations of the compound C18:0 dipropylenetriamine of the Test Item during the additional test

Sampling date		Day 0		Day 1		Day 1
		-		test media with algae		test media without algae
Nominal loading rate of the		Samples with organic solvent
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	19.4	1.79	9	0.863	4	2.04	10
25.3	6.15	0.801	13	0.678	11	1.17	19
8.00	1.94	0.195	10	0.257	13	0.330	17
2.53	0.615	0.200	33	0.210	34	0.286	47
0.800	0.194	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Truly dissolved test item (centrifuged samples)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	19.4	0.125	1	< LOQ		0.149	1
25.3	6.15	< LOQ		< LOQ		0.114	2
8.00	1.94	< LOQ		< LOQ		< LOQ
2.53	0.615	< LOQ		< LOQ		< LOQ
0.800	0.194	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Adsorption to the centrifuge tubes
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	19.4	1.33	7	1.21	6	1.36	7
25.3	6.15	0.553	9	0.541	9	0.939	15
8.00	1.94	0.162	8	0.225	12	0.274	14
2.53	0.615	0.154	25	0.176	29	0.230	37
0.800	0.194	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ

Meas. conc.     = measured concentration of the compound C18:0 dipropylenetriamine, dilution factors taken into account

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0851µg a.s./L)

a.s.                   = active substance

%                      = percent of the nominal loading rate of the active substance

 

Measured Concentrations of the compound C18:1 dipropylenetriamine of the Test Item during the additional test

Sampling date		Day 0		Day 1		Day 1
		-		test media with algae		test media without algae
Nominal loading rate of the		Samples with organic solvent
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	16.2	2.65	16	0.814	5	1.68	10
25.3	5.11	0.797	16	0.321	6	0.916	18
8.00	1.62	0.202	12	0.236	15	0.325	20
2.53	0.511	0.145	28	0.150	29	0.209	41
0.800	0.162	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Truly dissolved test item (centrifuged samples)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	16.2	0.187	1	< LOQ		< LOQ
25.3	5.11	< LOQ		< LOQ		0.0738	1
8.00	1.62	< LOQ		< LOQ		< LOQ
2.53	0.511	< LOQ		< LOQ		< LOQ
0.800	0.162	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Nominal loading rate of the		Adsorption to the centrifuge tubes
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
80.0	16.2	1.66	10	1.06	7	1.03	6
25.3	5.11	0.612	12	0.314	6	0.750	15
8.00	1.62	0.180	11	0.208	13	0.318	20
2.53	0.511	0.124	24	0.127	25	0.172	34
0.800	0.162	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ

Meas. conc.     = measured concentration of the compound C18:1 dipropylenetriamine, dilution factors taken into account

LOQ                 = limit of quantification (0.350 µg/L of the test item, corresponding to 0.0707 µg a.s./L)

a.s.                   = active substance

%                      = percent of the nominal loading rate of the active substance

 

Observations at Test and Control Groups

Stillborn Juveniles and Aborted Eggs

 

Aborted eggs were observed in the loading rates of 8.00 to 80.0 µg/L during the exposure period of 21 days. In the loading rates of 0.800 to 80.0 µg/L stillborn juveniles were observed.

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

 

Nominal  loading rate  of the test item  [µg/L]	Number of		Total no.   å
	stillborn juveniles	aborted eggs
80.0	12	2	14
25.3	19	3	22
8.00	9	2	11
2.53	2	0	2
0.800	2	0	2
Control	0	0	0

 

Growth (Total Body Length and Mean Dry Weight) of the Survived Parental Daphnids

 

At the end of the test the mean values of the body length (excluding the anal spine) of the survived parental female daphnids in the tested loading rates of 0.800 to 80.0 µg/L were in the range of 4.17 to 4.60 mm per daphnid and 4.65 mm per female daphnid in the control group.

On day 21 the mean dry weight of the survived parental daphnids was in the range of 0.07 to 0.48 mg per daphnid in the WAF loading rates of 0.800 to 80.0 µg/L and 0.68 mg per daphnid in the control.

 

Total Body Length and Dry Weight of the Survived Parental Daphnids

 Nominal 
loading rate 
of the test item 
[µg/L]

	Total length of the survived parent animals [mm]										MV	N	Dry weight [mg]
	Replicate no.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
80.0	4.50	-	-	-	-	-	4.00	4.00	-	-	4.17	3	0.20	0.07
25.3	-	-	4.50	4.00	-	4.00	4.50	4.50	4.50	4.00	4.29	7	3.50	0.50
8.00	4.00	4.50	4.50	4.50	4.50	-	.	4.00	4.00	5.00	4.38	8	4.40	0.55
2.53	4.50	4.50	4.50	4.50	4.50	4.50	5.00	4.50	-	4.00	4.50	9	3.70	0.41
0.800	4.50	4.50	5.00	4.50	4.50	5.00	4.50	4.50	4.50	4.50	4.60	10	4.80	0.48
Control	4.50	5.00	5.00	4.50	4.50	4.50	5.00	4.50	4.50	4.50	4.65	10	6.80	0.68

N    = number of the survived parental female daphnids

-     = not applicable, due to the mortality of the parental daphnid(s)

 Presence of Males

 

No male daphnids were observed in the control or in the test groups during the test.

 

 Occurrence of Ephippia (Winter Eggs)

 

No ephippia were observed in the control or in the test groups during the test.

 

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	-	-	-	-	-	-	-	12	-	16	-	-	-	31	-	-	29	-	-	28	-	116
2	-	-	-	-	-	-	-	14	-	-	19	-	-	34	-	35	-	-	34	-	-	136
3	-	-	-	-	-	-	3	10	-	18	-	-	26	-	-	-	30	-	-	26	-	113
4	-	-	-	-	-	-	-	7	-	-	3	-	-	34	-	-	31	-	-	29	-	104
5	-	-	-	-	-	-	-	14	-	-	15	-	-	36	-	-	29	-	-	-	28	122
6	-	-	-	-	-	-	8	-	-	18	-	-	28	-	-	-	35	-	-	30	-	119
7	-	-	-	-	-	-	9	1	-	14	-	-	24	-	-	-	27	-	-	29	-	104
8	-	-	-	-	-	-	-	10	-	-	14	-	-	32	-	-	31	9	-	-	30	126
9	-	-	-	-	-	-	-	10	-	-	13	-	-	35	-	-	29	-	-	-	-	87
10	-	-	-	-	-	-	-	8	-	-	3	-	-	30	-	-	34	-	-	5	22	102
Based on all parental daphnids inserted at the start of the exposure:																						1129
Based on parental daphnids survived until the end of the test:																						1129
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 0.800 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	-	-	-	-	-	-	2	9	-	10	-	-	23	-	-	-	29	-	-	26	-	99
2	-	-	-	-	-	-	9	-	-	13	-	-	24	-	-	20	-	-	-	30	-	96
3	-	-	-	-	-	-	6	-	-	7	-	-	28	-	-	-	24	-	-	27	-	92
4	-	-	-	-	-	-	-	10	-	13	1	-	-	29	-	-	28	-	-	-	26	107
5	-	-	-	-	-	-	9	-	-	12	-	-	26	-	-	16	-	-	-	24	-	87
6	-	-	-	-	-	-	-	8	-	-	5	-	-	31	-	-	29	-	-	28	-	101
7	-	-	-	-	-	-	-	15	-	-	22	-	-	27	-	-	28	-	-	26	-	118
8	-	-	-	-	-	-	-	10	-	12	-	-	-	30	-	-	31	6	-	-	25	114
9	-	-	-	-	-	-	-	13	-	-	17	-	-	26	-	-	21	-	-	24	-	101
10	-	-	-	-	-	-	-	12	-	-	17	-	-	29	-	-	-	23	-	-	26	107
Based on all parental daphnids inserted at the start of the exposure:																						1022
Based on parental daphnids survived until the end of the test:																						1022
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 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	-	-	-	-	-	-	-	10	-	-	8	-	-	21	-	-	20	-	-	-	16	75
2	-	-	-	-	-	-	8	-	-	20	-	-	24	-	-	-	23	-	-	20	-	95
3	-	-	-	-	-	-	-	8	-	-	7	-	-	27	-	-	18	-	-	19	-	79
4	-	-	-	-	-	-	-	12	-	-	16	-	-	25	-	-	-	7	-	-	23	83
5	-	-	-	-	-	-	-	14	-	-	16	-	-	29	-	-	6	-	-	24	-	89
6	-	-	-	-	-	-	-	5	-	4	-	-	-	27	-	-	19	-	-	20	-	75
7	-	-	-	-	-	-	-	11	-	-	26	-	-	21	-	-	17	-	-	-	21	96
8	-	-	-	-	-	-	7	-	-	19	-	-	22	-	-	-	17	-	-	15	-	80
9	-	-	-	-	-	-	-	7	-	-	M											7
10	-	-	-	-	-	-	10	-	-	15	-	-	-	25	-	-	28	-	-	-	18	96
Based on all parental daphnids inserted at the start of the exposure:																						775
Based on parental daphnids survived until the end of the test:																						768
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	1	1	1	1	1	1
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	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 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	-	-	-	-	-	-	7	-	-	17	-	-	20	-	-	11	-	-	-	16	-	71
2	-	-	-	-	-	-	-	9	-	-	11	-	-	21	-	-	7	-	-	-	19	67
3	-	-	-	-	-	-	-	7	-	-	7	-	-	24	-	-	15	-	-	20	-	73
4	-	-	-	-	-	-	9	-	-	16	-	-	24	-	-	-	19	-	-	18	-	86
5	-	-	-	-	-	-	-	13	-	-	15	-	-	19	-	-	20	-	-	-	16	83
6	-	-	-	-	-	-	-	13	-	-	17	-	23	-	-^1	M						53
7	-	-	-	-	-	-	-	10	-	-	24	-	-	21	M							55
8	-	-	-	-	-	-	7	-	-	9	-	-	19	-	-	-	13	-	-	19	-	67
9	-	-	-	-	-	-	-	9	-	-	12	-	-	22	-	-	23	-	-	-	13	79
10	-	-	-	-	-	-	8	-	-	12	-	-	20	-	-	-	18	-	-	21	-	79
Based on all parental daphnids inserted at the start of the exposure:																						713
Based on parental daphnids survived until the end of the test:																						605
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	2	2	2	2	2	2	2
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	10	20	20	20	20	20	20	20

 

-        = no juveniles observed

M      = dead parental daphnid

¹            = pale 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	-	-		-		-	-	-	-	6	-	-	6	-	-	20	M								32
2	-	-		-		-	-	-	-	10	-	-	20	-	-	19	-^1	-	M						49
3	-	-		-		-	-	-	-	8	-	-	18	-	-	21	-	-	12	-	-	-	10		69
4	-	-		-		-	-	-	-	9	-	-	12	-	-	23	-	-	-	9	-	-	-		53
5	-	-		-		-	-	-	-	6	-	-	6	-	-^1	17^1	M								29
6	-	-		-		-	-	-	-	9	-	-	12	-	-	23	-	-	11	-	-	9	2		66
7	-	-		-		-	-	-	-	7	-	-	17	-	-	18	-	-	16	-	-	-	10		68
8	-	-		-		-	-	-	-	8	-	-	14	-	-	18	-	-	12	-	-	10	-		62
9	-	-		-		-	-	-	-	9	-	-	11	-	-	15	-	-	10	-	-	-	8		53
10	-	-		-		-	-	-	-	10	-	-	17	-	-	21	-	-	-	16	-	-	11		75
Based on all parental daphnids inserted at the start of the exposure:																									556
Based on parental daphnids survived until the end of the test:																									446
Mortality of Parental (N)	0	0	0		0		0	0	0	0	0	0	0	0	0	0	2	2	3	3	3	3		3	3
Mortality of Parental [%]	0	0	0		0		0	0	0	0	0	0	0	0	0	0	20	20	30	30	30	30		30	30

 

-        = no juveniles observed

M      = dead parental daphnid

¹            = pale 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	-	-	-	-	-	-	-	9	-	-	12	-	-	14	-	-	12	-	-	-	6	53
2	-	-	-	-	-	-	-	9	-	-	23	-^1	-^1	M								32
3	-	-	-	-	-	-	-	8	-	-	12	-^1	M									20
4	-	-	-	-	-	-	-	6	-	-	20	-	-	13	-	-	10	M				49
5	-	-	-	-	-	-	-	10	-	-	26	-	-	12^1	M							48
6	-	-	-	-	-	-	-	7	-	15	1	-	18	-	-	-	15	M				56
7	-	-	-	-	-	-	-	6	-	-	14	-	-	15	-	-	19	-	-	9	-	63
8	-	-	-	-	-	-	-	8	-	10	-	-	10	-	-	-	16	-	-	8	-	52
9	-	-	-	-	-	-	-	9	-	-	21	-	-	18	-	M						48
10	-	-	-	-	-	-	-	7M														7
Based on all parental daphnids inserted at the start of the exposure:																						428
Based on parental daphnids survived until the end of the test:																						168
Mortality of Parental (N)	0	0	0	0	0	0	0	1	1	1	1	1	2	3	4	5	5	7	7	7	7	7
Mortality of Parental [%]	0	0	0	0	0	0	0	10	10	10	10	10	20	30	40	50	50	70	70	70	70	70

 

-        = no juveniles observed

M      = dead parental daphnid

¹            = pale 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	7.57	7.68	7.61	7.37	8.02	7.27
Control	7.54	7.61	7.62	7.43	8.06	7.38

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	8.42	8.50	7.73	7.59	7.61	6.94
Control	8.21	8.29	7.76	6.23	8.13	6.06

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	253	249	265	267	273	275
Control	259	266	272	260	271	263

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	20.2	20.4	20.9	20.6	21.0	20.9
Control	20.3	20.4	20.8	20.7	21.0	20.8

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

Percentage of the Relevant Amine

	[%]
C16:0 propylenediamine	5.7
C18:0 propylenediamine	6.7
C18:1 propylenediamine	8.4
C16:0 dipropylenetriamine	21.3
C18:0 dipropylenetriamine	24.3
C18:1 dipropylenetriamine	20.2

 

Gradient Table

Time [min]	A [%]	B [%]
0.00	80	20
0.30	80	20
1.30	10	90
2.50	10	90
2.60	80	20
3.00	80	20

 

   Dilution Steps

Nominal loading rates of the test item [µg/L]	Dilution Factor*	Sample volume [mL]	Final volume [mL]
80.0	20	0.1	1.0
	10	0.2	1.0
25.3	10	0.2	1.0
8.00	4	0.5	1.0

                                                            * including dilution factor 2

                                                           

Dilution Steps (centrifuged samples)

Nominal loading rates of the test item [µg/L]	Dilution Factor*	Sample volume [mL]	Final volume [mL]
80.0	10	0.2	1.0
25.3	10	0.2	1.0

                                                            * including dilution factor 2

 

 

Dilution Steps (algae adsorption)

Nominal loading rates of the test item [µg/L]	Dilution Factor*	Sample volume [mL]	Final volume [mL]	Total Factor
80.0	20^b)	0.05	1.0	DF 5
25.3	20^b)	0.05	1.0	DF 5
8.00	20^b)	0.05	1.0	DF 5
2.53	2^a)	0.7	1.4	EF 2
0.800	2^a)	0.7	1.4	EF 2
Control	2^a)	0.7	1.4	EF 2

       * including enrichment factor 4                                            DF: total dilution factor

1. a) dilution step with ultra-pure water                                    EF: total enrichment factor


2. b) dilution step with dilution medium

 

 

 

Dilution steps (amended samples)

Nominal test item concentration [µg/L]	Dilution Factor*	Sample volume [mL]	Final volume [mL]
80.0	20	0.1	1.0
	10	0.2	1.0
25.3	10	0.2	1.0
8.00	4	0.5	1.0

*including factor 2

 

Dilution steps

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

*including enrichment factor 1

 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Linearity	≥ 5 standard concentrations,  r ≥ 0.99 (R^2≥ 0.9801 for 2^nd order regression) Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation	0.05 to 5 µg test item/L (n = 8), R^2≥ 0.9801	ü
Matrix effects	Should be ≤ 20%	C16:0 propylenediamine: 28% C18:0 propylenediamine: 26% C18:1 propylenediamine: 21% C16:0 dipropylenetriamine: 30% C18:0 dipropylenetriamine: 26% C18:1 dipropylenetriamine: 27% Matrix-matched calibration standards were used	ü
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace S/N ≥ 3 for qualifier ion trace	S/N for 0.05 µg test item/L C16:0 propylenediamine 85 (quantifier), 22 (qualifier) C18:0 propylenediamine 101 (quantifier), 12 (qualifier) C18:1 propylenediamine 167 (quantifier), 45 (qualifier) C16:0 dipropylenetriamine 210 (quantifier), 38 (qualifier) C18:0 dipropylenetriamine 308 (quantifier), 105 (qualifier) C18:1 dipropylenetriamine 266 (quantifier), 160 (qualifier)	ü
Limit of Quantification (LOQ)	Should be at or below lowest test concentration	0.350 µg test item/L (1 x LOQ) 100 µg test item/L (286 x LOQ)	ü

ü             = criterion fulfilled

 

Table continued

Accuracy^1) (Fortified samples)	Mean recovery rate of 70-120% per fortification level (2 levels)	C16:0 propylenediamine 1 x LOQ: 117% (n = 5) 286 x LOQ: 102% (n = 5) C18:0 propylenediamine 1 x LOQ: 117% (n = 4) 286 x LOQ: 111% (n = 5) C18:1 propylenediamine 1 x LOQ: 116% (n = 5) 286 x LOQ: 104% (n = 5) C16:0 dipropylenetriamine 1 x LOQ: 118% (n = 5) 286 x LOQ: 103% (n = 5) C18:0 dipropylenetriamine 1 x LOQ: 120% (n = 5) 286 x LOQ: 109% (n = 5) C18:1 dipropylenetriamine 1 x LOQ: 115% (n = 5) 286 x LOQ: 110% (n = 5)	ü
Precision^1)	Relative standard deviation ≤ 20% per fortification level	C16:0 propylenediamine 1 x LOQ: 12% 286 x LOQ: 1.7% C18:0 propylenediamine 1 x LOQ: 4.2% 286 x LOQ: 2.1% C18:1 propylenediamine 1 x LOQ: 15% 286 x LOQ: 2.3% C16:0 dipropylenetriamine 1 x LOQ: 8.8% 286 x LOQ: 1.6% C18:0 dipropylenetriamine 1 x LOQ: 19% 286 x LOQ: 3.4% C18:1 dipropylenetriamine 1 x LOQ: 14% 286 x LOQ: 4.5%	ü

ü             = criterion fulfilled

¹⁾              = for results see Table 48 to Table 52

 

Table continued

Stability	Samples: Stable if 70 – 120% of nominal concentration Calibration standards: Stable if ±10% of freshly prepared standard	See section 17.1	ü
Specificity^1): 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).	C16:0 propylenediamine quantifier [m/z]: 299.30 > 282.29 qualifier [m/z]: 299.30 > 57.98 C18:0 propylenediamine quantifier [m/z]: 327.40 > 310.33 qualifier [m/z]: 327.40 > 57.98 C18:1 propylenediamine quantifier [m/z]: 325.32 > 308.32 qualifier [m/z]: 325.32 > 57.98 C16:0 dipropylenetriamine quantifier [m/z]: 356.43 > 282.33 qualifier [m/z]: 356.43 > 98.05 C18:0 dipropylenetriamine quantifier [m/z]: 384.46 > 310.31 qualifier [m/z]: 384.46 > 98.05 C18:1 dipropylenetriamine quantifier [m/z]: 382.44 > 308.36 qualifier [m/z]: 382.44 > 98.05	ü
	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

ü             = criterion fulfilled

¹⁾              = for results see Table 48 to Table 52

 

 

Preparation of the fortified samples    

    Preparation of Fortified Samples of the Test Item

LOQ Level	Control	1	286
Stock solution	-	1000 mg test item/L in methanol
Spiking solution [µg test item/L] (Medium)	-	35 (methanol)	10000 (methanol)
Replicates	2	5	5
Concentration of the LOQ  [µg test item/L]	-	0.350	100
Medium for preparation	Dilution medium*
Volume of spiking solution [mL]	-	0.05	0.05
Volume of medium [mL]	10	9.95	9.95
Dilution factor	1*	1*	20
Dilution medium	Dilution medium
Sample volume [mL]	10	10	0.05
Finale volume [mL]	10	10	1.0
Test item concentration in the final solution [µg/L]	-	0.175	2.5

Dilution medium = acetonitrile : daphnia dilution medium (50 : 50 v/v) containing 1% formic acid

*directly diluted in dilution medium due to low solubility in daphnia dilution medium

  (including dilution factor 2)

 

 

Nominal Concentrations of the Fortified Samples of the components of the test item

                      Fortified concentrations*: 0.352 µg test item/L (1 x LOQ) and 101 µg test item/L (286 x LOQ).

Component	1 x LOQ [µg a.s./L]	286 x LOQ [µg a.s./L]	Results see
C16:0 propylenediamine	0.0201	5.73	Table 49
C18:0 propylenediamine	0.0236	6.73	Table 50
C18:1 propylenediamine	0.0296	8.44	Table 50
C16:0 dipropylenetriamine	0.0750	21.4	Table 51
C18:0 dipropylenetriamine	0.0856	24.4	Table 51
C18:1 dipropylenetriamine	0.0711	20.3	Table 52

*           = weighing factor taken into account

a.s.       = active substance

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of the test item

Replicate	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine
	C16:0 propylenediamine
	1 x LOQ		286 x LOQ
	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
1	0.0203	101	5.79	101
2	0.0226	113	5.99	105
3	0.0231	115	5.82	102
4	0.0228	114	5.73	100
5	0.0282	141	5.83	102
Mean	0.023	117	5.8	102
SD ±	0.003		0.1
CV [%]	12		1.7

Meas. conc.     = measured concentration of the test item, dilution factor taken into account

a.s.                   = active substance

%                      = percent concentration of the fortified sample

SD                    = standard deviation

CV                    = coefficient of variation

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of the test item

Replicate	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine
	C18:0 propylenediamine
	1 x LOQ		286 x LOQ
	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
1	0.0262	111	7.36	109
2	0.0271	115	7.68	114
3	0.0288	122	7.30	108
4	0.0279	118	7.36	109
5	0.0357 ^1)	151 ^1)	7.54	112
Mean	0.028	117	7.5	111
SD ±	0.001		0.2
CV [%]	4.2		2.1
Replicate	C18:1 propylenediamine
	1 x LOQ		286 x LOQ
	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
1	0.0324	110	8.46	100
2	0.0314	106	8.82	104
3	0.0313	106	8.95	106
4	0.0330	111	8.92	106
5	0.0436	147	8.92	106
Mean	0.034	116	8.8	104
SD ±	0.005		0.2
CV [%]	15		2.3

Meas. conc.     = measured concentration of the test item, dilution factor taken into account

a.s.                   = active substance

%                      = percent concentration of the fortified sample

SD                    = standard deviation

CV                    = coefficient of variation

¹⁾                                   = outlier determined by Grubb’s Test for outliers.

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of the test item

Replicate	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine
	C16:0 dipropylenetriamine
	1 x LOQ		286 x LOQ
	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
1	0.0840	112	21.6	101
2	0.0891	119	22.4	105
3	0.0977	130	22.3	104
4	0.0784	105	22.1	103
5	0.0947	126	21.8	102
Mean	0.089	118	22.0	103
SD ±	0.008		0.3
CV [%]	8.8		1.6
Replicate	C18:0 dipropylenetriamine
	1 x LOQ		286 x LOQ
	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
1	0.0939	110	26.0	106
2	0.0807	94	27.1	111
3	0.105	123	25.8	106
4	0.101	118	26.4	108
5	0.133	156	28.1	115
Mean	0.10	120	26.7	109
SD ±	0.02		0.9
CV [%]	19		3.4

Meas. conc.     = measured concentration of the test item, dilution factor taken into account

a.s.                   = active substance

%                      = percent concentration of the fortified sample

SD                    = standard deviation

CV                    = coefficient of variation

 

 Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of the test item

Replicate	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine
	C18:1 dipropylenetriamine
	1 x LOQ		286 x LOQ
	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
1	0.0805	113	21.3	105
2	0.0732	103	23.0	114
3	0.0780	110	22.5	111
4	0.0764	107	21.3	105
5	0.102	144	23.4	115
Mean	0.08	115	22	110
SD ±	0.01		1
CV [%]	14		4.5

Meas. conc.     = measured concentration of the test item, dilution factor taken into account

a.s.                   = active substance

%                      = percent concentration of the fortified sample

SD                    = standard deviation

CV                    = coefficient of variation

 

 

Stability

 

The stability of the standard stock solution (1000 mg test item/L in methanol) was checked after 7 days and was between 92 and 99% of the nominal concentration for all components and was found to be stable over this period.

 

The responses of an 1 day old calibration standard (0.8 µg test item/L) was compared with the responses of a freshly prepared calibration standard with the same concentration. The deviation was > 10% and was not found to be stable over this period. Therefore the calibration standards were freshly prepared on each day of analysis.

 

Fortified Samples at 2 x LOQ Level (0.700 µg/L) were prepared and analyzed. The diluted fortified samples (factor 2 with acetonitrile containing 2% formic acid) were stored at room temperature. After 2 days these samples were measured once again to check the stability.

Results of the stored Fortified Samples of the test item after 2 days

Component	Fort. Conc. [µg a.s./L]	Meas. conc. [µg a.s./L]	%^1	%^2
C16:0 propylenediamine	0.0401	0.0411	103	107
C18:0 propylenediamine	0.0471	0.0511	108	103
C18:1 propylenediamine	0.0590	0.0590	100	103
C16:0 dipropylenetriamine	0.150	0.158	106	118
C18:0 dipropylenetriamine	0.171	0.198	116	122
C18:1 dipropylenetriamine	0.142	0.150	106	117

Fort. conc.       = fortified concentration, weighing factor taken into account

Meas. conc.     = measured concentration of the a.s, dilution factor taken into account

a.s.                   = active substance

%¹                     = percent concentration of the nominal concentration

%²                     = percent of the initially measured concentration

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 date	Day 0		Day 1		Day 7		Day 8
Quality Control*	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
Component
C16:0 propylenediamine	0.0236	118	0.0231	116	0.0175	86	0.0242	120
			0.0233	117
C18:0 propylenediamine	0.0256	109	0.0250	106	0.0207	87	0.0279	117
			0.0259	110
C18:1 propylenediamine	0.0343	116	0.0341	116	0.0266	89	0.0358	120
			0.0348	118
C16:0 dipropylenetriamine	0.0896	120	0.0875	117	0.0715	95	0.0896	119
			0.0817	109
C18:0 dipropylenetriamine	0.0998	117	0.0886	104	0.0929	108	0.0972	113
			0.0886	104
C18:1 dipropylenetriamine	0.0848	120	0.0812	115	0.0817	114	0.0850	119
			0.0826	117
Sampling date	Day 8 (reanalysis)		Day 14		Day 14 (reanalysis)		Day 15
Quality Control*	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
Component
C16:0 propylenediamine	0.0207	102	0.0195	97	0.0192	95	0.0194	96
C18:0 propylenediamine	0.0209	88	0.0212	89	0.0214	90	0.0212	89
C18:1 propylenediamine	0.0280	94	0.0272	91	0.0284	95	0.0272	91
C16:0 dipropylenetriamine	0.0884	117	0.0795	105	0.0795	105	0.0804	107
C18:0 dipropylenetriamine	0.0994	115	0.0930	108	0.100	117	0.102	119
C18:1 dipropylenetriamine	0.0831	116	0.0765	107	0.0781	109	0.0807	113

*                           = weighing factor taken into account

Meas. conc.                    = measured concentration of the a.s., dilution factors taken into account

a.s.                                   = active substance

%                             = percent of the fortified concentration

 

Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test (Amended samples)

Sampling date	Day 0		Day 1
Quality Control*	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
Component
C16:0 propylenediamine	0.0213	106	0.0196	98
			0.0189	94
C18:0 propylenediamine	0.0256	109	0.0250	106
			0.0232	98
C18:1 propylenediamine	0.0310	105	0.0281	95
			0.0271	92
C16:0 dipropylenetriamine	0.0889	119	0.0850	113
			0.0893	119
C18:0 dipropylenetriamine	0.101	118	0.0989	116
			0.0982	115
C18:1 dipropylenetriamine	0.0824	116	0.0781	110
			0.0778	109

*                           = weighing factor taken into account

Meas. conc.                    = measured concentration of the a.s., dilution factors taken into account

a.s.                                   = active substance

%                             = percent of the fortified concentration

 

Preliminary Tests (non-GLP)

Solubility Test

 

One Water Accommodated Fraction (WAF) was prepared with a nominal loading rate of 10.0 mg/L. 0.1000 g of the test item were weighed out and transferred with methanol into a measuring flask to achieve a stock solution in methanol with a nominal concentration of 10.0 g/L. 1 mL of this stock solution was placed by pipette onto two curved glass slides in appropriate steps (e.g. 100 µL). The methanol was evaporated after every step. The glass slide with the test item was inserted in a glass flask with 2000 mL of the dilution water (Elendt M4).

A slow stirring procedure was applied for 24 to 72 hours at 20 ± 2 °C. 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, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask) after 24, 48 and 72 hours of stirring. The first 25 mL were discarded. The WAF was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative and no undissolved particles were visually observed in all samples (after 24, 48 and 72 hours of stirring). The resulting water accommodated fractions (WAF) were analyzed.

Samples after 24, 48 and 72 hours of slow stirring were taken and analyzed via LC-MS/MS as presented in the tables below. Six components were measured.

For the definitive reproduction test a slow stirring phase of 72 hours was found to be optimal.

Measured Concentrations of N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine during the Solubility Test (non-GLP)

                 Analytical system: LC-MS

		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine
Stirring time		Components	24 hours		48 hours		72 hours
Nominal test item loading rate			Meas. conc. [mg a.s./L]	%	Meas. conc. [mg a.s./L]	%	Meas. conc. [mg a.s./L]	%
[mg/L]	[mg a.s./L]
10.0	0.570	C16:0 propylenediamine	0.274	48	0.347	61	0.456	80
	0.670	C18:0 propylenediamine	0.0776	12	0.0747	11	0.121	18
	0.840	C18:1 propylenediamine	0.783	93	0.798	95	0.994	118
	2.13	C16:0 dipropylenetriamine	1.81	85	2.42	114	2.69	126
	2.43	C18:0 dipropylenetriamine	0.741	30	0.859	35	1.32	54
	2.02	C18:1 dipropylenetriamine	1.66	82	2.40	119	3.03	150

%                      = percent of the nominal test item loading rate

 

Preliminary Range Finding Test

 

A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions with renewal of the test solutions three times per week over a period of 12 days. A control and three water accommodated fractions of the test item of 1.00, 10.0 and 100 µg/L were tested in the range finding test. The Tyndall effect was negative and no undissolved particles were visually observed in the freshly prepared WAF’s.

Five daphnids, one daphnia per replicate, were exposed to each nominal loading level and the control.

 

Immobilization and Offspring in the non-GLP Preliminary Range Finding Test after 12 Days

(n = 5, divided into 5 replicates with 1 daphnid each)

Nominal loading of the test item in the WAF [µg/L]	Mortality [%] after 12 days						Number of offspring after 12 days per inserted parent						First appearance of living juveniles
	Replicates						Replicates
	1	2	3	4	5	MV	1	2	3	4	5	MV	[d]
100	100^1	100^1	100^1	100^1	100^1	100	-	-	-	-	-	-	-
10.0	0	0	0	0	0	0	27	32	33	23	30	29	7 - 8
1.00	0	0	0	0	0	0	26	27	30	39	34	31	7 - 8
Control	0	0	0	0	0	0	27	34	27	31	35	31	7 - 8

¹ = dead on day 1

 

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, 0 hours		Old media, 48 hours
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C16:0 Propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
10.0	0.570	0.153	27	0.0802	14
1.00	0.0570	< LCL		0.152	27
Control		< LCL		< LCL
Sampling		Fresh media, 0 hours		Old media, 48 hours
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:0 Propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
10.0	0.670	0.669	100	0.437	65
1.00	0.0670	0.0542	81	0.0178	27
Control		< LCL		< LCL
Sampling		Fresh media, 0 hours		Old media, 48 hours
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:1 Propylenediamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
10.0	0.840	0.269	32	0.0645	8
1.00	0.0840	< LCL		< LCL
Control		< LCL		< LCL

%                   = Percent of the nominal test item loading rate

LCL               = lowest calibration standard = 0.1 µg test item/L

a.s.                = active substance

Meas. Conc. = measured concentration of the test item, dilution factors taken into account

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, 0 hours		Old media, 48 hours
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C16:0 Dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
10.0	2.13	2.01	94	0.999	47
1.00	0.213	< LCL		0.110	52
Control		< LCL		< LCL
Sampling		Fresh media, 0 hours		Old media, 48 hours
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:0 Dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
10.0	2.43	3.56	146	2.86	118
1.00	0.243	0.178	73	0.160	66
Control		< LCL		< LCL
Sampling		Fresh media, 0 hours		Old media, 48 hours
Nominal test item loading rates		N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine  (C18:1 Dipropylenetriamine)
[µg/L]	[µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
10.0	2.02	2.32	115	0.758	38
1.00	0.202	0.0751	37	0.0747	37
Control		< LCL		< LCL

%                   = Percent of the nominal test item loading rate

LCL               = lowest calibration standard = 0.1 µg test item/L

a.s.                = active substance

Meas. Conc. = measured concentration of the test item, dilution factors taken into account

 

 

 

 

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.
A summary of all endpoints based on the nominal loading rates of N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (CAS No.: 1219458-14-6) and the time weighted mean measured test item concentrations is given.
The overall effect threshold for effects of the test item under the test conditions was 0.808 µg/L (EL10 Reproduction) based on the nominal loading rates of the test item N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine and 0.179 µg/L (EC10 Reproduction) 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 N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (batch number: 1929319) was conducted according to OECD 211 (2012).

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 the 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 using a siphon. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 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 0.175 – 0.738 – 2.51 – 6.26 – 21.1 µg/L. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions. This procedure was followed for each renewal of the test solution.

The WAFs were checked for any undissolved or emulsified material by Tyndall effect, which was negative.

The test item concentrations of N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine 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 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8 and 15) in all WAFs and in the control.

On each sampling point the following constituents of the test item C16:0 propylenediamine, C18:0 propylenediamine, C18:1 propylenediamine, C16:0 dipropylenetriamine, C18:0 dipropylenetriamine, C18:1 dipropylenetriamine were analytically determined.

All evaluations are based on the nominal loading rates and the time weighted mean measured concentration of N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine. The test item is an UVCB substance.

The environmental conditions during the test 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 N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (CAS 1219458-14-6) and the time weighted mean measured test item concentrations.

A summary of all endpoints based on the nominal loading rates of N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (CAS 1219458-14-6) 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	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine (CAS 1219458-14-6)
	based on  the nominal loading rates [µg/L]	based on  the time weighted mean measured concentrations [µg/L]
EL/EC10 Reproduction (introduced)	0.808 (Cl: ˂ 0.800 – 47.1)	0.179 (Cl: ˂ 0.175 – > 21.1)
EL/EC50 Reproduction (introduced)	24.3 (Cl: 0.977 – > 25.3)	6.96 (Cl: 0.204 – > 21.1)
LOEL/LOEC Reproduction (introduced)	≤ 0.800	≤ 0.175
NOEL/NOEC Reproduction (introduced)	˂ 0.800	˂ 0.175
LL/LC20 Adult mortality after 21 days	8.92 (Cl: 2.04 – 19.4)	2.73 (Cl: 1.13 – 6.60)
LL/LC50 Adult mortality after 21 days	40.3 (Cl: 18.5 – > 80)	10.6 (Cl: 4.63 – > 21.1)
LL/LC100 Adult mortality after 21 days	> 80.0	> 21.1
LOEL/LOEC Adult mortality after 21 days	8.00	2.51
NOEL/NOEC Adult mortality after 21 days	2.53	0.738

CI   = 95% confidence limits

Description of key information

One long-term daphnia study with N-(3-aminopropyl)-N'-C16-18 (evennumbered), C18 unsaturated alkyl -propane-1,3-diamine (CAS 1219458-14-6) is available.

Abbreviation: Triamine T

Triamine T 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 the intrinsic properties like the relatively low water solubility and strong sorption to equipment and organisms. Classical ecotoxicity testing with these substances using reconstituted water often leads to test results which are poorly reproducible and are associated with high uncertainty. In addition, because of the complex sorption mechanisms (van der Waals and Ionic mechanisms) the actual dissolved exposure concentration cannot reliably be quantified.

To determine the intrinsic toxicity of Triamine T (for C&L purposes) a long-term daphnia test (OECD TG 211) is 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.

 For the preparation of the test solutions according to the WAF approach, all reasonable efforts were taken to produce a solution of all soluble components of the test item in test media. The test solutions were prepared daily, by gentle mixing the test item with test medium 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 siphoning. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 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 0.175 – 0.738 – 2.51 – 6.26 – 21.1 µg/L.
No undissolved or emulsified material was observed in the WAF solutions based on the Tyndall effect check. Adsorptive losses to the glass test vessels were 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. 

It should be noted that the test substance sorbs strongly to the food algae (van Wijk, 2009) which leads to an apparent reduction of the freely dissolve concentration (when algae are separated prior to analysis) but are still providing a secondary exposure route via ingestion.

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 0.808/0.179 µg/L. The EL50/EC50 for adult mortality after 21 days is 40.3/10.6 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. The test item concentrations of Triamine T were analytically verified via LC-MS/MS 3 times during the test in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8 and 15) in all WAFs and in the control.
The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.

A fingerprint was performed with the highest loading rate (80.0 µg/L) and compared with the analytical standard with the same concentration of the test item prepared in methanol. Both were verified via MS and evaluated by the software. The solutions were analytical verified via high resolution MS and evaluated by the software. The detected signals of the analytical standards and the sample solution were compared. However, the concentrations and solubility of the test item is too low for significant MS spectra and no signals related to the test item could be observed. 

 

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


• 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.


• OECD (2019): Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals. OECD series on testing and assessment no. 23 (second edition), ENV/JM/MONO(2000)6/REV1

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.808 µg/L