1,3-Propanediamine, N-[3-((C11-14, C13-rich)oxy)propyl]- branched acetate

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:

2009-10-14 to 2009-12-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Two concentration levels 30.0 and 270 µg/L and the control were analysed at least once within 7 days in the fresh (0 h) and old media (48 h).
- Sampling method: For the longest exposure interval of 72 h samples were taken from the fresh (0 h) and old media (72 h) once within the test period, respectively. For the analyses of the old media additional replicates without algae and test organisms were prepared and stored under test conditions.
The sorption of the test item to the glassware was quantified once during the test period after the first sampling interval. Therefore, one test vessel of the test concentration 30.0 µg/L, containing daphnia and algae, was emptied after 72 hours of exposure and rinsed twice with water. Thereafter an extraction of the test vessel was carried out.
The analytical method was validated.
- Sample storage conditions before analysis:All samples were stored at 6 ± 2°C until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared with natural water and treated with ultrasound for 15 minutes at room temperature.
- Eluate: NAtural river water
- Differential loading: 10 - 30 - 90 - 270 - 810 µg/L
- Controls: 10 replicates of dilution water without test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
Oct. 14th Oct. 16th Oct. 23rd Oct. 26th Oct. 28th Oct. 30th
810 156 137 n.a. n.a. n.a. n.a.
270 -- -- 158 148 157 160
Control 159 151 158 153 164 155

Test temperature:

Temperature [°C]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
Oct. 14th Oct. 16th Oct. 23rd Oct. 26th Oct. 28th Oct. 30th
810 20.4 22.0 n.a. n.a. n.a. n.a.
270 -- -- 21.8 21.7 21.4 22.0
Control 19.9 21.9 21.7 21.7 19.3 21.9

pH:

pH-Values
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
Oct. 14th Oct. 16th Oct. 23rd Oct. 26th Oct. 28th Oct. 30th
810 7.74 8.81 n.a. n.a. n.a. n.a.
270 -- -- 7.84 8.07 7.85 7.85
Control 7.82 7.65 7.85 7.94 7.72 7.85

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
Oct. 14th Oct. 16th Oct. 23rd Oct. 26th Oct. 28th Oct. 30th
810 8.51 6.72 n.a. n.a. n.a. n.a.
270 -- -- 10.17 8.29 9.72 7.24
Control 9.52 6.47 10.32 8.02 10.41 7.70

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information in materials and methods incl. tables

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water of the river Hüpeder Bach was used. This river is located near D-31171 Nordstemmen, Germany. Specifications of the natural river water are given in the table below.
River: Hüppeder Bach
Location D-30982 Pattensen
Sampling Date 2009-10-07
Weather on Day of Sampling Dry, few clouds, ca. 22 °C
Colour yellowish, clear
pH 7.79
Conductivity [µS/cm] 531
Dissolved Oxygen [mg/L] 8.12
DOC [mg C/L] 3.8
TOC [mg C/L] 3.7
Ammonium-N [mg N/L] < 0.015
Nitrate-N [mg N/L] 3.06
Total Nitrogen [mg N/L] 3.71
o-Phosphate-p [mg P/L] 0.116
Total Phosphate [mg P/L] 0.140
Suspended Matter [mg/L] 17.6
Total Hardness [mg CO3/L] 170
Storage conditions 6 ± 2 °C





OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
- Test concentrations: 10 - 1 - 0.1 mg/L
Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
10 100 100 100 100 100 100
1 100 100 100 100 100 100
0.1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

90 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

156 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

adult

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality of 100 % at the concentration levels 270 and 810 µg/L after 21 days. Adult mortality > 20 % is seen to be biologically significant. At the other concentration levels and the control all parental daphnids survived till the end of the study. The estimation of an EC10-value for the adult mortality was not applicable to the data obtained. The EC50 for adult mortality after 21 days was calculated to be 156 µg/L (CI: 90.0 - 270 µg/L).

- No. of offspring produced per day per female:
Five broods were released by all surviving animals of the control and the test groups.
The mean number of offspring alive produced per parent animal surviving at the end of the test was 140 juveniles at the control group. At the concentration level 10.0 µg/L the reproductive output was comparable. The reproductive output was statistically significantly increased at the concentration levels 30.0 µg/L (+14 %) and 90.0 µg/L (+23 %) when compared to the control (One Way Analysis of Variance, DUNNETT’S METHOD, p = 0.05). Due to mortality of all parental daphnids at the concentration levels 270 and 810 µg/L no reproduction was observed.

- Body length and weight of parent animals:
The mean dry body weight at the concentration levels 10.0 to 90.0 µg/L ranged from 0.88 to 0.98 mg per daphnid. The mean dry body weight in the control was 0.95 mg per daphnid.
The mean body lengths at the tested concentrations were in the range of 5.85 to 5.93 mm per daphnid and 5.75 mm per daphnid at the control group.

- Type and number of morphological abnormalities:
Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was ≤ 2 %, which is not biologically significant. At the control no dead juveniles or aborted eggs were observed.
- Number of males and females (parental): No males were observed in either the control or the test groups during the test.
The first day of appearance of juveniles at all test item and control groups producing juveniles was day 8

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.58 (CI 1.41 - 1.77)

Reported statistics and error estimates:

The NOEC value for the adult mortality as the most sensitive effect was determined directly from the observation data. Significant deviations were evaluated in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance.
Statistical evaluation of the reproduction rates, the intrinsic rates of natural increase and the body length was carried out using One Way Analysis of Variance, p = 0.05. The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.
The EC50-value of the reference test was calculated by
EC-VALUES sigmoidal dose-response regression. The confidence interval for the EC50-value was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.
The EC50 for adult mortality was calculated as the geometric mean of the lowest concentration causing 100 % adult mortality (1000 µg/L) and the highest concentration causing 0 % adult mortality (320 µg/L), because only one partial effect was observed. These concentrations were also used as confidence limits.
The estimation of an EC10- or EC50-value for the reduction or increase of the reproductive output could not be performed due to the absence of effects on reproduction.

Number of Juveniles alive in the Control and Test Groups after 21 Days

Nominal conc. [µg/L]												No.	Mean No. of Juveniles alive			Comparison
	Number of Juveniles alive in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Inc.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
810	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
270	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
90.0	169	168	156	162	183	167	173	187	175	176	1716	10	172 ±	9	5	23	yes
30.0	167	164	171	159	141	142	163	167	163	157	1594	10	159 ±	10	6	14	yes
10.0	162	147	138	130	163	161	153	147	141	166	1508	10	151 ±	12	8	8	no
Control	136	146	133	130	138	157	143	133	141	142	1399	10	140 ±	8	6	—	—

First Appearance of Living Juveniles in the Individual Groups

Nominal concentration	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
[µg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
810	--	--	--	--	--	--	--	--	--	--	--
270	--	--	--	--	--	--	--	--	--	--	--
90.0	8	8	8	8	8	8	8	8	8	8	8.0
30.0	8	8	8	8	8	8	8	8	8	8	8.0
10.0	8	8	8	8	8	8	8	8	8	8	8.0
Control	8	8	8	8	8	8	8	8	8	8	8.0

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

                 (n = 10)

Nominal concentration	Adult Mortality [%]
[µg/L]	7 days	14 days	21 days
810	100*	100*	100*
270	40*	60*	100*
90.0	0	0	0
30.0	0	0	0
10.0	0	0	0
Control	0	0	0

Total Body Length and Dry Weight of the Parent Animals

Nominal concentration [µg/L]	Total Length of the Parent Animals [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
810	--	--	--	--	--	--	--	--	--	--	--	0	--	--
270	--	--	--	--	--	--	--	--	--	--	--	0	--	--
90.0	5.75	6.00	6.00	6.00	6.00	5.75	6.00	5.75	5.75	6.2505	5.93	10	9.3	0.93
30.0	6.00	6.00	5.50	5.50	6.00	5.50	6.00	6.00	6.00	6.00	5.85	10	8.8	0.88
10.0	6.00	5.75	6.00	5.50	5.75	6.00	6.00	6.00	5.75	6.255.75	5.90	10	9.8	0.98
Control	6.00	5.75	6.00	5.75	5.75	5.75	5.50	5.50	5.75	5.75	5.75	10	9.5	0.95

Validity criteria fulfilled:

yes

Conclusions:

The test is considered valid as all validity criteria have been fulfilled; Based on the test setup (GLP, Guideline study) and the extensive effort which has been paid to ascertain a realistic exposure of the test organisms and to quantify the extent of this exposure the results are considered to be reliable. Finally, the test result is considered adequate for the evalution of the environmental risks in the EU applying the bulk approach considering the realistic worst case (low) concentration of suspended matter (17.6 mg/L) and DOC (3.8 mg C/L) in the river water used.

Executive summary:

The Daphnia magna Reproduction Test with Natural River Water (Semi-Static, 21 d) of the test item N-(3-(Tridecyloxy)propyl)-1,3-propanediamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine(CAS numbers 102047-27-8 and 68479-04-9, batch number S001086) was conducted according to OECD 211 (2008) from 2009-10-14 to 2009-12-02 , with the definitive exposure phase between 2009-10-14 to 2009-11-04, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.

Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration level and control. At the beginning of the test they were 2 to 24 hours old. The test method was semi-static. The test solutions were renewed 3 times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects on parameters such as adult mortality, intrinsic rate of natural increase, time of production of first brood, occurrence of aborted eggs and stillborn juveniles, dry body weight and length of the parent animals.

The test item N-(3-(Tridecyloxy)propyl)-1,3-propanediamine, monoacetate and N-(3-(Tridecyloxy) propyl)-1,3-propane diamine (CAS No. 102047-27-8 and 68479-04-9) has a low water solubility and also has a strong tendency to adsorb to negatively charged surfaces such as suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many cationic substances in general but long chain ether diamines in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavailability and exposure assessment for cationic surface-active substances like the ether diamines as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavailability that occurs in the environment with these substances.

The aquatic ecotoxicity tests with ether diamines were therefore performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulkapproach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing ‘total aquatic concentrations’. To characterize the risk to the aquatic compartment the PEC_aquatic,bulk is compared with the PNEC_aquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2001).

In order to class standard laboratory toxicity study valid, it is of particular importance that - besides information on test substance, test method / conditions and test organism used - suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

For ecotoxicity tests performed using the bulkapproach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier to interpret, more environmental realistic, and if compared to PEC_bulk clearly provide a more appropriate assessment of risks for the environment.

Nominal concentrations of the test item N-(3-(Tridecyloxy)propyl)-1,3-propanediamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine were selected after a preliminary acute immobilization test (48 h, static) as follows: 10.0 - 30.0 - 90.0 - 270 - 810 µg/L. The test item N-(3-(Tridecyloxy)propyl)-1,3-propanediamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine was analytically verified in the concentration levels 30.0 and 320 µg/L and in the control via UPLC-MS/MS of samples taken on days 0, 9, 14 (fresh media) and on days 2, 16 (old media, 48 hours) and 12 (old media, 72 hours).
The recoveries in the fresh media were in the range of 111 to 130 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 38 to 83 %. Biodegradation as possible reason for this is very unlikely considering the short time frame between the refreshments of the test solutions. No adsorption ( LOQ; day 12) of the test item to the glassware was observed. The adsorbed fraction of the test item concentration was determined to be 30 % of the nominal concentration at the concentration level 30.0 µg/L. The limited concentration decrease between fresh and old media is most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. The results of the chemical analyses show that the test organisms were fully exposed to the test substance during the test. Therefore, all effect values given are based on the nominal test item concentrations.

·   The average number of juveniles per parent in the control group was 140 after 21 days. At the concentration level 10.0 µg/L the reproductive output was comparable. The reproductive outputwas statistically significantly increased at the concentration levels 30.0 µg/L (+14 %) and 90.0 µg/L (+23 %) when compared to the control (One Way Analysis of Variance, Dunnett’s method, p = 0.05). Due to mortality of all parental daphnids at the concentration levels 270 and 810 µg/L no reproduction was observed. The EC₁₀-value for the reproductive output was calculated by sigmoidal dose-response regression to be 16.1 µg/L. An EC₅₀-value for the reproductive output could not be calculated because no effects ≥ 50 % (reduction or increase of the reproductive output) occurred within the tested concentration range. As adverse effects the NOEC was assessed to be 90.0 µg/L (the highest test concentration with no reduction of the reproductive output).

·   The coefficient of variation of the mean number of living offspring produced per parent alive in the control group was 6 %.

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance (p = 0.05). There was no statistical significant difference determined at any of the concentration levels.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles (sum of stillborn juvenile and aborted eggs) was ≤ 2 %, which is not biologically significant. At the control no dead juveniles or aborted eggs were observed.

·   The the first brood was released at day 8 by all surviving daphnids of the test and control groups. Five broods were released by all surviving animals of the control and the test groups.

The statistically significant increase of the reproductive output at the concentration levels 30.0 and 90.0 µg/L was the most sensitive effect in this study. A summary of all test item related effects on reproduction is given in Table 1.

Table 1:        Test Item Related Effects on Reproduction (NOEC and EC₅₀)

Effects	Nominal Concentration [µg/L]
	Control	10.0	30.0	90.0	270	810
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	140 ± 8	151 ± 12	159 ± 10^1)	172 ± 9^1)	--	--
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	6	8	6	5	--	--
Number of Dead Juveniles and Aborted Eggs	0	11	12	37	--	--
Mean Intrinsic Rates of Natural Increase	0.51	0.51	0.50	0.51	--	--
Appearance of First Brood [Mean Day]	8.0	8.0	8.0	8.0	--	--
Number of broods	5	5	5	5	--	--
EC50, Reproduction with Confidence Interval (CI) p = 95 %	Not determinable (CI: Not applicable)
NOEC (adverse effect)	90.0 µg/L^2)

SD    = Standard deviation

¹⁾       = Statistically significant increase of the reproductive output (One Way Analysis of Variance, Dunnett’s

            method, p = 0.05)

²⁾       = Adverse effect, hormesis not taken into account

--       = Not applicable due to the mortality of all parental animals

A summary of test item related effects concerning the adult mortality and growth (weight, length) is given in Table 2.

·   The test item induced significant adult mortality of 100 % at the concentration levels 270 and 810 µg/L after 21 days. At the other concentration levels and the control all parental daphnids survived till the end of the study. The estimation of an EC₁₀-value for the adult mortality was not applicable to the data obtained. The EC₅₀ for adult mortality after 21 days was calculated to be 156 µg/L (CI: 90.0 - 270 µg/L).

·   The mean dry body weight and mean total body length of the surviving parental daphnids at the tested concentration levels were determined at the end of the study at the concentration levels 10.0 to 90.0 µg/L and the control group.
The mean dry body weight at the concentration levels 10.0 to 90.0 µg/L ranged from 0.88 to 0.98 mg per daphnid. The mean dry body weight in the control was 0.95 mg per daphnid.
The mean body lengths at the tested concentrations were in the range of 5.85 to 5.93 mm per daphnid and 5.75 mm per daphnid at the control group.  

Table 2:    Test Item Related Effects on Adult Mortality (EC₁₀ and EC₅₀) and Growth (weight, length)

Effects	Nominal Concentration [µg/L]
	Control	10.0	30.0	90.0	270	810
Adult Mortality after 21 Days [%]	0	0	0	0	100^1)	100^1)
Parent Animals: Mean Dry Weight [mg]	0.95	0.98	0.88	0.93	--	--
Parent Animals: Mean Body Length [mm]	5.75	5.90	5.85	5.93	--	--
EC10, Adult Mortality	Not applicable
EC50, Adult Mortality with Confidence Interval (CI) p = 95 %	156 µg/L (CI: 90.0 - 270 µg/L)

¹⁾       = Mortality > 20 % biologically significant

--       = Not applicable due to 100 % mortality of all parental animals

·       No males and ephippia (winter eggs) were observed at the control or test groups.

 

·   Water quality parameters as pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits.

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromateas reference item once per month.
The EC₅₀ of the reference item at 1.58 mg/L after 24 hours was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC₅₀-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Description of key information

One long term test according to OECD 211 have been performed with Daphnia magna, the NOEC was determined to be 90 µg/L. The effects are expressed as nominal values because the tests were performed with river water as they were intended to be used in an evaluation of the environmental risks based on the Bulk approach.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

90 µg/L