Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2008-01-25 - 2008-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance, including chemical analyses

Justification for type of information:

The category and read-across approach are justified in both documents, please find this further information in the attached documentation part in the dedicated ESR for aquatic toxicity "Supporting Read-Across document".

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 211

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 9-20 °C
- Boiling point: > 250 °C
- Vapour pressure: < 1 hPa (20 °C)
- Water solubility (under test conditions): Insoluble
- Henry's law constant: Not applicable
- log Pow: Not applicable
- pKa: Not applicable
- Stability in water: Not applicable
- Stability in light: Not applicable
- pH dependance on stability: Not applicable

OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: Not applicable
- Other:
- Density: 840 kg/m3 (25 °C)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10.0 - 32.0 - 100 - 320 - 1000 µg/L
- Sampling method: 5 mL of each sample were diluted with 5 mL 2-propanol containing 4 % formic acid directly after sampling for stabilisation
- Sample storage conditions before analysis: Room temperature

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For weighing out a part of the test item was filled in a glass flask with screw top and gently heated up to 40 °C to get it homogeneous liquid. Stock solutions of 10 mg/L test item were freshly prepared with natural river water for each water renewal and sonificated for 30 min at 40 °C.
- Differential loading: 10.0 - 32.0 - 100 - 320 - 1000 µg/L, prepared by dilution of the stock solution with dilution water (natural river water containing 80 % of the components of the culture medium Elendt M4)
- Controls: Dilution water without test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not observed

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: recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 d
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris
- Amount: 2 mg C/daphnia per day
- Frequency: daily


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

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

none

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
Concentration
[µg/L] I F I F I F I F
Day 0 Day 3 Day 5 Day 7 Day 10 Day 12 Day 17 Day 19
Jan. 25. Jan. 28. Jan. 30. Feb. 01. Feb. 04. Feb. 06. Feb. 11. Feb. 13.
1000 148 156 -- -- -- -- -- --
320 -- -- 169 157 168 166 180 166
Control 167 152 170 154 175 159 174 170
The total hardness should be more than 140 mg/L as CaCO3.

Test temperature:

Temperature [°C]
Nominal
Concentration
[µg/L] I F I F I F I F
Day 0 Day 3 Day 5 Day 7 Day 10 Day 12 Day 17 Day 19
Jan. 25. Jan. 28. Jan. 30. Feb. 01. Feb. 04. Feb. 06. Feb. 11. Feb. 13.
1000 20.2 20.1 -- -- -- -- -- --
320 -- -- 20.8 21.1 20.4 21.6 20.7 20.7
Control 20.2 20.4 20.8 21.1 19.8 21.4 20.1 20.7
The temperature should be within the range 18-22 °C.

pH:

pH-Values
Nominal
Concentration
[µg/L] I F I F I F I F
Day 0 Day 3 Day 5 Day 7 Day 10 Day 12 Day 17 Day 19
Jan. 25. Jan. 28. Jan. 30. Feb. 01. Feb. 04. Feb. 06. Feb. 11. Feb. 13.
1000 7.91 7.99 -- -- -- -- -- --
320 -- -- 7.81 8.12 7.89 7.52 7.80 7.82
Control 7.94 8.04 7.87 7.61 8.01 7.37 8.06 7.60
The pH-values should be in the range of 6 – 9.
Deviations < 1.5 units between the initial and final pH-values are regarded to be not significant.

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Concentration
[µg/L] I F I F I F I F
Day 0 Day 3 Day 5 Day 7 Day 10 Day 12 Day 17 Day 19
Jan. 25. Jan. 28. Jan. 30. Feb. 01. Feb. 04. Feb. 06. Feb. 11. Feb. 13.
1000 8.30 9.16 -- -- -- -- -- --
320 -- -- 8.26 8.11 8.19 8.28 8.21 8.68
Control 8.29 9.23 8.31 8.27 8.24 8.27 8.23 8.52
The dissolved oxygen concentration should be above 3 mg/L.

Salinity:

Not measured

Nominal and measured concentrations:

Nominal test item concentrations: 10.0 - 32.0 - 100 - 320- 1000 µg/L (factor v10)
Mean measured test item concentrations: 7.98 - 25.3 - 77.8 - 243 - 771 µg/L

Details on test conditions:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water, river grane, located in the low mountain range "Harz", D-38685 Langelsheim, Herzog-Julius-Hütte, Im Granetal; additionally 80 % of the components of the culture medium acc. to Elendt (1990) were added to enable a sufficient total water hardness of more than 140 mg CaCO3/L and a sufficient growth and reproduction of the daphnids.
- Total organic carbon: > 140 mg CaCO3/L
- Particulate matter: 2.5 mg/L
- DOC: 1.5 mg C/L
- Conductivity: 149 µS (natural river water)
- Salinity: Not measured
- Culture medium different from test medium: Elendt M4 acc. to Elendt (1990)
- Intervals of water quality measurement: once per week in fresh and old medium

OTHER TEST CONDITIONS
- Adjustment of pH: Not necessary
- Photoperiod: 16/8 h light/dark cycle
- 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 weigth): test end

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 0.1 - 1.0 - 10 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

290 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL: 288 - 291

Details on results:

- Mortality of parent animals: The nominal test item concentrations 320 and 1000 µg/L had a biologically significant effect on adult mortality (> 20 %) after 21 d of exposure. The EC50 (with 95 % Confidence Interval) for the adult mortality was calculated by sigmoidal dose-response regession to be 290 µg/L (CI: 288 - 291 µg/L).

- No. of offspring produced per day per female: The mean number of offspring alive produced per parent animal surviving at the end of the test was 98 juveniles in the control group. The mean number of juveniles of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance, BONFERRONI T-TEST, p = 0.05. There was no statistically significant difference at any of the tested concentration levels when compared to the control.

- Body length and weight of parent animals: The body weight and total length of the parental daphnids in the tested concentration levels were comparable to the control group. The mean dry body weight in the concentration levels 10.0 - 100 µ/L ranged from 1.11 to 1.26 mg per daphnid. The mean dry body weight in the control was 1.14 mg per daphnid. The mean value of the total body length in the tested concentrations was in the range of 5.18 to 5.35 mm per daphnid and 5.33 mm per daphnid in the control group.

- Type and number of morphological abnormalities: None
- Type and number of behavioural abnormalities: None
- Number of males and females (parental): No males occurred

- Time to first brood release or time to hatch: The mean day of release of the first brood was day 8.7 in the control group. In the tested concentration levels 10.0 to 1000 µg/L the mean day of release of the first brood was in the range of 8.3 to 9.0. The first brood was released until day 9 by all daphnids of the control group and the tested concentration levels.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 (24 h) = 1.60 (CI 1.59 - 1.61) mg/L

Reported statistics and error estimates:

The NOEC and LOEC for the adult mortality were determined directly from the results. Significant deviations were determined in comparison to the control group using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance. For the determination of significant deviations for the reproduction rate One Way Analysis of Variance, BONFERRONI t-test, p = 0.05 was used. One Way Analysis of Variance, p = 0.05 was used for the determination of significant deviations for the body length. Statistical evaluation of age of first reproduction, occurrence of stillborn juveniles and aborted eggs could not be carried out because Normality Test failed. The coefficient of variation around the mean number of living offspring produced per parent in the control and test groups was evaluated.

Reproductive output:

Table 1:   Number of Juveniles in the Control and Test Groups after 21 Days

Nominal conc. [µg/L]												No.	Mean No. of Juveniles			Comparison
	Number of Juveniles in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV±SD		[%]	[%]
1000	--	--	--	--	--	--	--	--	--	--	--	--	--		--	--	--
320	--	--	--	--	--	--	117	--	--	--	117	1	117±	n.a.	n.a.	-19*	no
100	122	105	109	113	108	108	109	102	93	101	1070	10	107±	8	7	-9*	no
32.0	98	102	77	108	90	91	87	105	89	81	928	10	93±	10	11	5	no
10.0	95	87	80	80	94	80	98	78	96	112	900	10	90±	11	12	8	no
Control	104	88	105	99	95	96	99	92	109	88	975	10	98±	7	7	—	—

Nominal conc.   = Nominal concentration

N                  = Number of parents producing juveniles alive

MV ± SD         = Mean value ± Standard deviation

CV                 = Coefficient of variation

Red.              = Reduction of reproduction

Stat.               = Statistical significance (Bonferroni t-test, p = 0.05)

no                 = Statistically not significant

n.a.                 = Not applicable,because juveniles of only one surviving daphnid were counted

--                   = Not applicable, due to mortality of the parent animals

*)                  = Increase of reproductive output in comparison to the control group

For the mean number of living offspring produced per parent in the control group the coefficient of variation amounts to 7 %. At the test concentrations 10.0 to 1000 µg/L the coefficient of variation ranged from 7 to 12 %.

The mean number of juveniles of the surviving daphnids of the treatment groups were comparedtothe control byOne Way Analysis of Variance,Bonferroni t-test, p= 0.05. There was no statistically significant difference at any of the tested concentration levels when compared to the control.

Intrinsic rate of natural increase:

The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and offspring numbers were used for calculation of population growth and maintenance. For the concentration level 1000 µg/L no intrinsic rates could be calculated due to mortality of all parent animals.

Table 2:   Intrinsic Rates of Natural Increase

Nominal concentration	Intrinsic Rate of Natural Increase in Replicate No.										Mean IR		CV
[µg/L]	1	2	3	4	5	6	7	8	9	10	MV±SD		[%]
1000	--	--	--	--	--	--	--	--	--	--	--	--	--
320	--	--	--	--	--	--	0.46	--	--	--	0.46±	n.a.	n.a.
100	0.51	0.49	0.47	0.46	0.47	0.50	0.49	0.45	0.45	0.44	0.47±	0.03	5
32.0	0.45	0.48	0.45	0.51	0.48	0.46	0.49	0.47	0.47	0.44	0.47±	0.02	5
10.0	0.44	0.43	0.41	0.43	0.42	0.40	0.47	0.42	0.42	0.44	0.43±	0.02	4
Control	0.46	0.45	0.46	0.48	0.49	0.46	0.46	0.45	0.47	0.44	0.46±	0.01	3

IR               = Intrinsic rate of natural increase

MV ± SD    = Mean value ± Standard deviation

CV             = Coefficient of variation

n.a.            = Not applicable, due to only one surviving daphnid

--             = Not applicable, due to mortality of the parent animal

Stillborn juveniles and aborted eggs:

The percentage number of dead juveniles (sum of stillborn juveniles and aborted eggs) was related to the total number of produced juveniles (alive, stillborn juveniles and aborted eggs) of the surviving daphnids. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles came to a maximum of 3 % in the concentration levels of 10.0 to 1000 µg/L. In the control no stillborn juveniles or aborted eggs occurred.

Table 3:   Stillborn Juvenilesand Aborted Eggs after 21 Days

Nominal concentration	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
[µg/L]	Juveniles	Eggs	å	N	MV±SD
1000	--	--	--	0	--
320	1	0	1	1	1.00±n.a.
100	11	15	26	9	2.60±1.78
32.0	5	27	32	10	3.20±1.99
10.0	0	10	10	10	1.00±1.33
Control	0	0	0	10	0±0

  MV ± SD    = Mean value ± Standard deviation

   --               = Not applicable, due to mortality of the parent animal

   N               = Number of parents producing juveniles alive

   n.a.            = Not applicable, due to only one surviving daphnid

Table 4:   Relative Number of Dead (Stillborn + Aborted Eggs)to Total Number of Juveniles

Nominal concentration	Number of Juveniles			Percentage of Dead Juveniles * [%]
[µg/L]	Dead	Alive	Total
1000	--	--	--	--
320	1	117	118	1
100	26	1070	1096	2
32.0	32	928	960	3
10.0	10	900	910	1
Control	0	975	975	0

  Dead   = Aborted eggs + stillborn juveniles

   Total = Dead + alive juveniles

   *)       = Related to the total number of juveniles

   --        = Not applicable, due to mortality of the parent animal

Appearance of first brood:

The first day of appearance of juveniles at all test item and control groups producing juveniles was between day 8 and day 9.

Table 5:   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
1000	--	--	--	--	--	--	--	--	--	--	--
320	--	--	--	--	--	--	9	--	--	--	9.0
100	8	8	9	9	8	8	8	9	8	9	8.4
32.0	9	8	8	8	8	9	8	9	8	8	8.3
10.0	9	9	9	8	9	9	8	9	9	9	8.8
Control	9	9	9	8	8	9	9	8	9	9	8.7

 --  = Not applicable, due to mortality of the parent animal

Adult mortality:

The test concentrations 320 and 1000 µg/L had a biologically significant effect on adult mortality (> 20 %) after 21 d ofexposure.The EC₅₀(with 95 % Confidence Interval) for the adult mortality was calculated by sigmoidal dose-response regression to be 290 µg/L (CI: 288 - 291 µg/L).

Table 6:      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
1000	100*	100*	100*
320	80*	90*	90*
100	0	0	0
32.0	0	0	0
10.0	0	0	0
Control	0	0	0

 *)  = Biologically significant effect, mortality > 20 %

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 (2.5 mg/L) and DOC (1.5 mg C/L) in the river water used.


NOECadult mortality : 100 µg/L
LOECadult mortality : 320 µg/L
EC50 (adult mortality) : 290 µg/L (CI: 288 - 291 µg/L)

Executive summary:

The Daphnia magna Reproduction Test with natural river water (Semi-Static, 21 d) of Duomeen OV (batch No. S000902) was conducted according to OECD 211 (1998) from2008-01-25 to 2008-02-15atDr.U.Noack-Laboratorienin Sarstedt, Germany .Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration and control. At test start they were 2 to 24 hours old.The test methodwas 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 occurrence of aborted eggs and stillborn juveniles, time of production of first brood, adult survival, intrinsic rate of natural increase, body weight and length of the parents.Duomeen OV(CAS No.7173-62-8) is poorly soluble in water 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 alkyl 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 EU Technical Guidance and RIP Documents do not provide sufficient guidance concerning bioavailability and exposure assessment for cationic surface-active substances like the 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 diamines were therefore performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulkapproach, 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,bulkis compared with the PNEC_aquatic,bulkderived from river water ecotoxicity studies (ECETOC, 2001).In order to class standardlaboratory 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 thebulk approach, 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 minimal. The results of these bulk approach tests are therefore much easier to interpret, more environmental realistic, and if compared to PEC_bulkclearly provide a more appropriate assessment of risks for the environment.

Nominal concentrations of Duomeen OV were selected after a preliminary acute immobilization test (48 h, static) as follows: 10.0 - 32.0 - 100 - 320 - 1000 µg/Lcorresponding to the mean measured concentrations 7.98 - 25.3 - 77.8 - 243 - 771 µg/L. The C18 diamine, main component of Duomeen OV, of all concentrations and control were analytically verified via LC-MS/MS of samples on days 5, 10, 17 (fresh media, 0 h) and on days 7, 12, 19 (old media, 48 h). The average recovery rate in the fresh media (0 h) was 92 % (78 to 108). The discrepancy observed between the nominal and monitored concentrations in fresh media is most likely caused by sorption of the test item to suspended matter and DOC in the river water during the preparation of the test solutions. The average recovery rate in the old media (after 48 h) was 62 % (50 to 76 %). 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 are given based on the nominal test item concentrations.

·  Theadult mortality after 21 days amounted to 100 % at the test concentration 1000µg/L and to 90 % at the test concentration 320 µg/L. The test item did not induce significant mortality (£20 %) of parent animals at any of the other concentrations and in the control. In the control as well as at the test concentrations 10.0 to 100 µg/L all daphnids survived until the end of the study. The EC₅₀for adult mortality after 21 dayswas calculated by sigmoidal dose-response regressionto be 290 µg/L.

·  The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC)after 21 days based on adult mortality as the most sensitive effect are summarized inTable 1. Effects on reproduction capacity did not occur.

·  The average number of juveniles per parent in the control group was 98 after 21 days. The reproductive output was not statistically significant reduced in any of the tested concentrations when compared to the control (One Way Analysis of Variance,Bonferronit-test, p = 0.05.

·  The coefficient of variation of the mean number of living offspringproduced per parent in the control group was 7 %.

·  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 andmaintenance.

· No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was=3 % at all tested concentration levels.

· The mean day ofrelease of the first brood was day 8.7 in the control group. In the tested concentration levels 10.0 to 1000 µg/L the mean day of release of the first brood was in the range of 8.3 to 9.0. The first brood was released until day 9 by all daphnids of the control group and the tested concentration levels.

·  The body weight and total length of the parental daphnids in the tested concentration levels were comparable to the control group. The mean dry body weight at the concentration levels 10.0 to 100 µg/L ranged from 1.11 to 1.26 mg per daphnid. The mean dry body weight in the control was 1.14 mg per daphnid. The dry body weight at the concentration level 320 µg/L could not be determined due to the low weight of only one surviving daphnid. The mean value of the total body length in the tested concentrations was in the range of 5.18 to 5.35 mm per daphnid and 5.33 mm per daphnid in the controlgroup.

Table1:       Test Item Related Effects, NOEC, LOEC and EC₅₀

Effects	Nominal Concentration [µg/L]
	Control	10.0	32.0	100	320	1000
Adult mortality after 21 days [%]	0	0	0	10	90^1)	100^1)
Mean number of juveniles per producing parent (Reproduction Rate±SD)	98±7	90±11	93±10	107±8	117^2)	---
Intrinsic rates of natural increase	0.46	0.43	0.47	0.47	0.46	---
Appearance of first brood [day]	8.7	8.8	8.3	8.4	9.0	---
Parent animals: dry weight of [mg]	1.14	1.14	1.11	1.26	n.d.	---
Parent animals: total length [mm]	5.33	5.35	5.18	5.25	5.25	---
NOECadult mortality	100 µg/L
LOECadult mortality	320 µg/L
EC50 (adult mortality) with Confidence Interval (CI) p = 95 %	290 µg/L (CI: 288 - 291 µg/L)

SD    = Standard deviation

¹⁾      = Biological significant effect, mortality > 20 %

²⁾      = Standard deviation was not calculable, because Juveniles of only one surviving daphnid were counted.

n.d.  = Not determined, because weight of only one surviving daphnid was to low.

---    = Not applicable due to 100 % mortality of the parent animals before test end

·      Nomales and ephippia(winter eggs) occurred in 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 EC₅₀of the reference item at 1.60 mg/L after 24 hours was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according toAQS 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.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2008-01-18 - 2008-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Complete report, test performed under GLP conditions, all validity criteria fulfilled

Justification for type of information:

The category and read-across approach are justified in both documents, please find this further information in the attached documentation part in the dedicated ESR for aquatic toxicity "Supporting Read-Across document".

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 211

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 45-50 °C
- Boiling point: > 250 °C
- Vapour pressure: < 1 hPa (20 °C)
- Water solubility (under test conditions): Insoluble
- Density: 840 kg/m3 (60 °C)

Analytical monitoring:

yes

Details on sampling:

Concentrations: 10.0 - 32.0 - 100 - 320 - 1000 µg/L
- Sampling method: 5 mL of all test item concentrations and the control were diluted with 5 mL 2-propanol containing 4 % formic acid directly after sampling
- Sample storage conditions before analysis: Room temperature

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions of 10 mg/L test item were freshly prepared with natural river water for each water renewal and sonificated for 30 min at 40 °C.
- Differential loading: Nominal 10.0 - 32.0 - 100 - 320 - 1000 µg/L, prepared by dilution of the stock solution with dilution water (natural river water containing 80 % of the components of the culture medium Elendt M4)
- Controls: Dilution water without test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not observed

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:

None

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal Concentration
[µg/L] Day 0 Day 3 Day 5 Day 7 Day 12 Day 14 Day 17 Day 19
Jan. 18. Jan. 21. Jan. 23. Jan. 25. Jan. 30. Feb. 01. Feb. 04. Feb. 06.
1000 180 145 164 138 -- -- -- --
320 -- -- -- -- 180 150 174 155
Control 168 124 180 160 170 152 175 167
The total hardness should be more than 140 mg/L as CaCO3.

Test temperature:

Temperature [°C]
Nominal Concentration
[µg/L] Day 0 Day 3 Day 5 Day 7 Day 12 Day 14 Day 17 Day 19
Jan. 18. Jan. 21. Jan. 23. Jan. 25. Jan. 30. Feb. 01. Feb. 04. Feb. 06.
1000 21.6 21.1 21.8 20.4 -- -- -- --
320 -- -- -- -- 20.8 20.4 20.3 21.3
Control 21.7 21.3 21.4 20.4 20.8 20.4 19.8 21.3
The temperature should be within the range 18-22 °C.

pH:

pH-Values
Nominal Concentration
[µg/L] Day 0 Day 3 Day 5 Day 7 Day 12 Day 14 Day 17 Day 19
Jan. 18. Jan. 21. Jan. 23. Jan. 25. Jan. 30. Feb. 01. Feb. 04. Feb. 06.
1000 7.92 8.39 7.88 8.52 -- -- -- --
320 -- -- -- -- 7.85 7.71 7.86 7.54
Control 7.95 8.53 8.05 7.96 7.87 7.63 8.01 7.45
The pH-values should be in the range of 6 – 9.
Deviations < 1.5 units between the initial and final pH-values are regarded to be not significant.

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal Concentration
[µg/L] Day 0 Day 3 Day 5 Day 7 Day 12 Day 14 Day 17 Day 19
Jan. 18. Jan. 21. Jan. 23. Jan. 25. Jan. 30. Feb. 01. Feb. 04. Feb. 06.
1000 8.53 8.68 8.15 9.14 -- -- -- --
320 -- -- -- -- 8.21 8.16 8.36 8.22
Control 8.31 8.03 8.04 8.96 8.31 8.19 8.24 8.20
The dissolved oxygen concentration should be above 3 mg/L.

Salinity:

Not measured

Nominal and measured concentrations:

Nominal test item concentrations: 10.0 - 32.0 - 100 - 320- 1000 µg/L (factor v10)
Mean measured test item concentrations: 7.52 - 22.4 - 73.1 - 229 - 713 µg/L

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, 5 (ID) x 8 (H) cm, 50 mL headspace, 50 mL fill volume
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): None

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water, river Grane, located in the low mountain range “Harz”, D-38685 Langelsheim, Herzog-Julius-Hütte, Im Granetal; additionally 80 % of the components of the culture medium acc. to ELENDT (1990) were added to the natural river water to enable a sufficient total water hardness of more than 140 mg CaCO3/L and a sufficient growth and reproduction of the daphnids.
- Total organic carbon: > 140 mg CaCO3/L
- Particulate matter: 2.5 mg/L
- Conductivity: 149 µS (natural river water)
- Salinity: Not measured
- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: once per week in fresh and old medium

OTHER TEST CONDITIONS
- Adjustment of pH: Not necessary
- Photoperiod: 16/8 h light/dark cycle
- 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 weigth): test end

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 0.1 - 1.0 - 10 mg/L
- Results used to determine the conditions for the definitive study: Yes

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

566 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Mortality of parent animals: The nominal test item concentration 1000 µg/L had a biologically significant effect on adult mortality (> 20 %) after 21 d of exposure. The EC50 (with 95 % Confidence Interval) for the adult mortality was calculated as geometric mean of the highest concentration causing no mortality and the lowest concentration causing 100 % mortality to be 566 µg/L (CI: 320 - 1000 µg/L).

- No. of offspring produced per day per female: The mean number of offspring alive produced per parent animal surviving at the end of the test was 83 juveniles in the control group. The mean number of juveniles of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance, BONFERRONI T-TEST, p = 0.05. There was no statistically significant difference at any of the tested concentration levels when compared to the control.

- Body length and weight of parent animals: The body weight and total length of the parental daphnids in the tested concentration levels were comparable to the control group. The mean dry body weight in the concentration levels ranged from 0.85 to 1.30 mg per daphnid. The mean dry body weight in the control was 1.04 mg per daphnid. The mean value of the total body length in the tested concentrations was in the range of 5.14 to 5.25 mm per daphnid and 5.10 mm per daphnid in the control group.

- Type and number of morphological abnormalities: None
- Type and number of behavioural abnormalities: None
- Number of males and females (parental): No males occurred

- Time to first brood release or time to hatch: The mean day of release of the first brood was day 8.2 in the control group. In the tested concentration levels 10.0 to 1000 µg/L the mean day of release of the first brood was in the range of 8.1 to 8.3. The first brood was released until day 9 by all daphnids of the control group and the tested concentration levels. Four broods were released by all surviving animals of the test and control groups.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 (24 h) = 1.60 (CI 1.59 - 1.61) mg/L

Reported statistics and error estimates:

The NOEC and LOEC for the adult mortality were determineddirectly from the results. Significant deviations were determined in comparison to the control group using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance. For the determination of significant deviations for the reproduction rate One Way Analysis of Variance, BONFERRONI t-test, p = 0.05 was used. One Way Analysis of Variance, p = 0.05 was used for the determination of significant deviations for the body length. Statistical evaluation of age of first reproduction, occurrence of stillborn juveniles and aborted eggs could not be carried out because Normality Test failed. The coefficient of variation around the mean number of living offspring produced per parent in the control group was evaluated.

Reproductive output:

Table 1:   Number of Juveniles in the Control and Test Groups after 21 Days

Nominal conc. [µg/L]												No.	Mean No. of Juveniles			Comparison
	Number of Juveniles in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV±SD		[%]	[%]
1000	--	--	--	--	--	--	--	--	--	--	--	--	--		--	--	--
320	98	87	108	101	116	99	81	74	83	108	955	10	96±	14	14	-16	no
100	80	--	83	104	83	79	91	92	109	91	812	9	90±	11	12	-9	no
32.0	84	81	78	98	89	88	57	77	72	84	808	10	81±	11	14	2	no
10.0	90	87	95	84	115	114	90	88	88	108	959	10	96±	12	12	-16	no
Control	74	91	70	69	97	72	91	86	73	102	825	10	83±	12	15	—	—

Nominal conc.             = Nominal concentration

N                           = Number of parents producing juveniles alive

MV ± SD                  = Mean value ± Standard deviation

CV                         = Coefficient of variation

Red.                      = Reduction of reproduction

Stat.                       = Statistical significance(Bonferroni t-test, p = 0.05)

no                          = Statistically not significant

--                            = Not applicable, due to mortality of the parent animals

For the mean number of living offspring produced per parent in the control group the coefficient of variation amounts to 15 %. At the test concentrations 10.0 to 1000 µg/L the coefficient of variation ranged from 12 to 14 %.

Intrinsic rate of natural increase:

The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and offspring numbers were used for calculation of population growth and maintenance. For the concentration level 1000 µg/L no intrinsic rates could be calculated due to mortality of all parent animals.

Table 2:   Intrinsic Rates of Natural Increase

Nominal concentration	Intrinsic Rate of Natural Increase in Replicate No.										Mean IR		CV
[µg/L]	1	2	3	4	5	6	7	8	9	10	MV±SD		[%]
1000	--	--	--	--	--	--	--	--	--	--	--	--	--
320	0.50	0.45	0.54	0.51	0.50	0.47	0.50	0.50	0.48	0.52	0.50±	0.02	5
100	0.49	--	0.50	0.54	0.49	0.53	0.51	0.52	0.49	0.47	0.50±	0.02	4
32.0	0.49	0.45	0.46	0.51	0.50	0.48	0.40	0.46	0.44	0.49	0.47±	0.03	7
10.0	0.50	0.50	0.51	0.49	0.51	0.53	0.51	0.50	0.46	0.52	0.50±	0.02	4
Control	0.43	0.50	0.47	0.40	0.50	0.44	0.49	0.49	0.46	0.49	0.47±	0.03	7

IR           = Intrinsic rate of natural increase

MV ± SD    = Mean value ± Standard deviation

CV          = Coefficient of variation

--            = Not applicable, due to mortality of the parent animal

Stillborn juveniles and aborted eggs:

The number of stillborn juveniles and aborted eggs producing no juveniles deriving from the surviving parents of the test and control groups is shown inTable 3.The percentage number of dead juveniles (sum of stillborn juveniles and aborted eggs) was related to the total number of produced juveniles (alive, stillborn juveniles and aborted eggs) of the surviving daphnids. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was=4 % in the concentration levels of 10.0 to 1000 µg/L. In the control no stillborn juveniles or aborted eggs occurred.

Table 3:   Stillborn Juvenilesand Aborted Eggs after 21 Days

Nominal concentration	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
[µg/L]	Juveniles	Eggs	å	N	MV±SD
1000	--	--	--	0	--
320	6	17	23	10	2.30±1.77
100	7	29	36	9	4.00±2.12
32.0	4	33	37	10	3.70±2.67
10.0	2	19	21	10	2.10±1.79
Control	0	0	0	10	0±0

  MV ± SD    = Mean value ± Standard deviation

  --             = Not applicable, due to mortality of the parent animal

  N             = Number of parents producing juveniles alive

Table 4:   Relative Number of Dead (Stillborn + Aborted Eggs)to Total Number of Juveniles

Nominal concentration	Number of Juveniles			Percentage of Dead Juveniles * [%]
[µg/L]	Dead	Alive	Total
1000	--	--	--	--
320	23	955	978	2
100	36	812	848	4
32.0	37	808	845	4
10.0	21	959	980	2
Control	0	825	825	0

  Dead   = Aborted eggs + stillborn juveniles

  Total   = Dead + alive juveniles

  *)         = Related to the total number of juveniles

  --         = Not applicable, due to mortality of the parent animal

Appearance of first brood:

The first day of appearance of juveniles at all test item and control groups producing juveniles was between day 8 and day 9.

Table 5:   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
1000	--	--	--	--	--	--	--	--	--	--	--
320	8	9	8	8	8	8	8	8	8	8	8.1
100	8	--	8	8	8	8	8	8	8	9	8.1
32.0	8	9	8	8	8	8	9	8	9	8	8.3
10.0	8	8	8	8	8	8	8	8	9	8	8.1
Control	9	8	8	8	8	9	8	8	8	8	8.2

-- = Not applicable, due to mortality of the parent animal

Adult mortality:

The nominal test item concentration 1000 µg/L had a biologically significant effect on adult mortality (> 20 %) after 21 d ofexposure.The EC₅₀(with 95 % Confidence Interval) for the adult mortality was calculated as geometric mean of the highest concentration causing no mortality and the lowest concentration causing 100 % mortality to be566 µg/L (CI: 320 - 1000 µg/L).

Table 6:      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
1000	80*	100*	100*
320	0	0	0
100	10	10	10
32.0	0	0	0
10.0	0	0	0
Control	0	0	0

 *)   = Biologically significant effect, mortality > 20 %

Validity criteria fulfilled:

yes

Conclusions:

The Lowest Observed Effect Concentrations (LOEC) and the No Observed Effect Concentrations (NOEC) for the adult mortality as most sensitive effect were determined directly from the results. Mortality of > 20 % is seen to be significant. All effect values are given based on the nominal concentrations.
NOEC : 320 µg/L
LOEC : 1000 µg/L
EC50 : 566 µg/L (CI: 320 - 1000 µg/L)

Executive summary:

The Daphnia magna Reproduction Test with natural river water (Semi-Static, 21 d) of N-(hydrogenated tallow alkyl)trimethylenediamines(batch No. S000905) was conducted according to OECD 211 (1998) from2008-01-18 to 2008-02-08atDr.U.Noack-LaboratorieninSarstedt, Germany.

Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration and control. At test start they were 2 to 24 hours old.The test methodwas 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 occurrence of aborted eggs and stillborn juveniles, time of production of first brood, adult survival, intrinsic rate of natural increase, body weight and length of the parents.

N-(hydrogenated tallow alkyl)trimethylenediamines (CAS No.68603-64-5) is poorly soluble in water 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 alkyl 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 EU Technical Guidance and RIP Documents do not provide sufficient guidance concerning bioavailability and exposure assessment for cationic surface-active substances like the 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 diamines were therefore performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulkapproach, 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,bulkis compared with the PNEC_aquatic,bulkderived from river water ecotoxicity studies (ECETOC, 2001).

In order to class standardlaboratory 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 bulk approach, 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 minimal. The results of these bulk approach tests are therefore much easier to interpret, more environmental realistic, and if compared to PEC_bulkclearly provide a more appropriate assessment of risks for the environment.

Nominal concentrations of N-(hydrogenated tallow alkyl)trimethylenediamines were selected after a preliminary acute immobilization test (48 h, static) as follows: 10.0 - 32.0 - 100 - 320 - 1000 µg/Lcorresponding to the mean measured concentrations 7.52 - 22.4 - 73.1 - 229 - 713 µg /L. The C16 and C18 diamines, main components of Duomeen HT, of all concentrations and control were analytically verified at four sampling dates via LC-MS/MS of samples on days 10, 12, 17, 19 (fresh media, 0 h) and on days 12, 14, 19, 21 (old media, 48 h). The average recovery rate of 83 (71 - 94 %) in the fresh media (0 h) decreased to 66 % (24 to 74 %) in the old media (after 48 h).The recoveryrates of all samples were used for calculating of the mean measured concentrations. The mean recovery rate of new and old media was 72 %. The discrepancy between the nominal and observed concentrations in the fresh media is most likely caused by sorption of the test item to suspended matter and DOC in the river water during the preparation of the test solutions. 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 are given based on the nominal test item concentrations.

At the nominal test concentration 1000µg/L theadult mortality amounted to 100 % after 21 days. The test item did not induce significant mortality (>20 %) of parent animals at any of the other concentrations and in the control. In the control as well as in the test concentrations 10.0, 32.0 and 320 µg/L all daphnids survived until the end of the study. At the concentration level 100 µg/L the adult mortality amounted to 10 %. The EC₅₀for adult mortality after 21 dayswas calculatedto be 566 µg/L.

·  The average number of juveniles per parent in the control group was 83 after 21 days. The reproductive output was not statistically significant reduced in any of the tested concentrations when compared to the control (One Way Analysis of Variance,Bonferronit-test, p = 0.05).

·  The coefficient of variation of the mean number of living offspringproduced per parent in the control group was 15 %.

·  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 andmaintenance.

·  No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was=4 % at all tested concentration levels.

·  The mean day ofrelease of the first brood was day 8.2 in the control group. In the tested concentration levels 10.0 to 1000 µg/L the mean day of release of the first brood was in the range of 8.1 to 8.3. The first brood was released until day 9 by all daphnids of the control group and the tested concentration levels.Four broods were released by all surviving animals of the test and control groups.

·  The body weight and total length of the parental daphnids in the tested concentration levels were comparable to the control group. The mean dry body weight in the concentration levels ranged from 0.85 to 1.30 mg per daphnid. The mean dry body weight in the control was 1.04 mg per daphnid. The mean value of the total body length in the tested concentrations was in the range of 5.14 to 5.25 mm per daphnid and 5.10 mm per daphnid in the controlgroup.

A summary of all test item related effects is given inTable 1. The effect levels (NOEC, LOEC and EC₅₀) are given based on the adult mortality as the most sensitive effect.

Table1:       Test Item Related Effects, NOEC, LOEC and EC₅₀

Effects	Nominal Concentration [µg/L]
	Control	10.0	32.0	100	320	1000
Adult mortality after 21 days [%]	0	0	0	10	0	100^1)
Mean number of juveniles per producing parent (Reproduction rate±SD)	83±12	96±12	81±11	90±11	96±14	---
Intrinsic rates of natural increase	0.47	0.50	0.47	0.50	0.50	---
Appearance of first brood [day]	8.2	8.1	8.3	8.1	8.1	---
Parent animals: dry weight of [mg]	1.04	1.30	0.990	0.967	0.850	---
Parent animals: total length [mm]	5.10	5.25	5.20	5.14	5.25	---
NOEC	320 µg/L
LOEC	1000 µg/L
EC50, with Confidence Interval (CI) p = 95 %	566 µg/L (CI: 320 - 1000 µg/L)

SD    = Standard deviation

¹⁾      = Biological significant effect, mortality > 20 %

---    = Not applicable due to 100 % mortality of the parent animals before test end

·      Nomales and ephippia(winter eggs) occurred in 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 dichromate as reference item once per month. The EC₅₀of the reference item at 1.60 mg/L after 24 hours was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according toAQS 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.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

October 2020

Justification for type of information:

REPORTING FORMAT FOR THE READ-ACROSS APPROACH
The category and read-across approach are justified in both documents, please find this further information in the attached documentation part in this dedicated ESR for aquatic toxicity.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

REACH TGD, Chapter R.6: QSARs and grouping of chemicals, May 2008

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

REACH Practical guide 6: How to report read-across and categories, December 2012

Deviations:

not specified

GLP compliance:

no

Remarks:

Not applicable

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

290 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

See read-across attached document for more details

Description of key information

The 21d-EC50 of (Z)-N-9-octadecenylpropane-1,3-diamine to daphnia is 290 µg/L in an OECD TG 211 test under GLP. This data has been used as read-across for Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates. This is then converted to the corresponding value of the salt, resulting to an EC50 of about 395 µg/L (MW of C18-unsaturated diamine = 316, MW of diamine diacetate = 430). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

395 µg/L

Additional information

A higher tier chronic invertebrate study with analytical monitoring of the test concentrations is available for (Z)-N-9-octadecenylpropane-1,3-diamine (CAS 7173-62-8). These higher tier tests were performed in river water to allow a PECaquatic,bulk/PNECaquatic,bulk approach for the environmental risk assessment. The long term 21d EC50 for parental mortality determined in the OECD211 test with river water (reliability 1) was used as the key value for acute toxicity for daphnia in the chemical safety assessment.

This value is used as a read-across for the N-C16-18-alkyl-(evennumbered) C18 unsaturated) propane-1,3-diamine (CAS 133779 -11 -0). The substances have the same hydrophilic group and nearly the same hydrophobic group. The only difference concerns the unsaturation level which is higher for (Z)-N-9-octadecenylpropane-1,3-diamine. Similar toxicty data were obtained with Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine: EC50 for adult mortality of 560 µg/L was obtained using same conditions of test than used for (Z)-N-9-octadecenylpropane-1,3-diamine. It confirms a similar toxicity profile between these diamines.

As confirmed by the physico-chemical properties of diamines acetates and fish toxicity result, the read-acoss from diamines is justified since the presence of acetates has no effect on the ecotoxicity profile of diamines acetates. Thus these long-term toxicity data obtained for daphnia can be used as read-across for Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates (for more details, please see the read-across justification document).