3-{[3-(C10-16-oxy)propyl]amino}propan-1-aminium acetate

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This is a non GLP study used only to compare toxicity between different susbtances, and thereby understand if read across was feasible. The study is reliable as long as only used for comparision between substances. The study is not reliable to use alone for derviing key values for the CSA. See furhter explanation in this RSS.

Justification for type of information:

More information on the justification for the analogue approach can be found in Section 13.2.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 225 Sedimentwater Lumbriculus toxicity test using spiked sediment

Deviations:

yes

Remarks:

See text box "Any other information on material and methods including tables"

GLP compliance:

no

Remarks:

The test was performed on a GLP ceritifed Lab, however, the particular test was not GLP.

Analytical monitoring:

yes

Details on sampling:

Samples of sediment were taken for chemical analysis at the start and at the end of the test for chemical analysis. Enough sediment was sampled to allow analysis, dry weight determination and any required repeats of results. To ensure the consistency of the experimental replicates, samples at the end of the test were taken in at least 2 replicates in the sampled concentrations. Water samples were taken at the start and end of the test. Sediment samples were extracted using an accelerated solvent extraction in order to quantify the concentration of the test substance on the sediment.

Vehicle:

no

Details on sediment and application:

Sediment was prepared by adding the dry ingredients together. The peat used in the formulated sediment had been previously conditioned and pH adjusted by slow addition of calcium carbonate over an extended period of 2 – 3 weeks.

The composition of the formulated sediment used according to OECD Guideline 225 and is described in a table below. A detailed description is found under "Any othter information on material and methods including tables"

The desired test solutions were prepared separately per concentration. The appropriate amount of the test chemical needed to achieve the desired concentration in the sediment was weighed out in a glass beaker on an analytical balance. Then 80 – 100 ml of deionised water was added and the substance was sonicated until a homogeneous solution without precipitate was formed. Care was taken not to excessively overheat the stock solution. After emptying of the initial stock solution (described below) the procedure was repeated with DSW only until no visible trace of the test substance remained in the beaker. This was repeated in the same manner for all test concentrations / substances as required. For controls only deionised water was used to wet the sediment.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

The test animals were taken from the environmental chemistry laboratories Lumbriculus Variegatus stock originating from Wageningen University. The test animals were cultured, subcultured and synchronised in conformity with laboratory Standard Operation Procedures. In preparation for testing 2 weeks prior to the planned start of the test the animals were sub cultured to allow more optimal growth and increase of body mass. 2 weeks prior to testing the sub cultured organisms were synchronized and the tail ends of the worms were allowed to recover for a maximum of 14 days before being used in the test. Test animals of a similar size were chosen from this synchronized batch for use in the test. All selected test animals were therefore considered to be of a similar physiological state. A representative sample of this batch was sampled and the dry weight was determined to allow an increase in dry weight endpoint to be calculated at the end of the test if required. The maintained Lumbriculus V stock was reference tested twice a year to check the condition of the culture as indicated in the test guideline.

The number of animals used per concentration was 30. Animals were equally divided over 3 replicates of 10 animals and exposed to the test concentrations. The control contained 6 replicates of 10 animals. Before the addition of the worms the spiked sediment was left for 2 days to equilibrate.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

The water hardness was checked periodically in the dilution water and was not specifically checked for this study.

Test temperature:

The toxicity test was carried out in a temperature-controlled room. The test temperature was set at 20 °C and the actual temperature difference between vessels should not exceed 1°C.

pH:

Dissolved oxygen content, pH, and ammonium concentration in the test replicates were largely not checked during the study as historical data has demonstrated that there is no cause for concern providing aeration remains constant. Aeration was therefore was therefore checked daily

Dissolved oxygen:

Dissolved oxygen content, pH, and ammonium concentration in the test replicates were largely not checked during the study as historical data has demonstrated that there is no cause for concern providing aeration remains constant. Aeration was therefore was therefore checked daily

Ammonia:

The total ammonia concentration was checked once only during the study at the middle concentration for each of the test substances and in a single control replicate.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

134 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

ETHERDIAMINE

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

68 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

DIAMINE HT

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

134 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

ETHERDIAMINE

Basis for effect:

other: biomass

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

68 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

DIAMINE HT

Basis for effect:

other: biomass

This study shows that the target for the read across (etherdiamine), is not more toxic to sediment organisms then the source (diamine HT). This is based on the reported NOECs of the two substance (68 mg/kg dw for the source, diamine HT and 134 mg/L dw for the target, etherdiamine).

ECx were also calculated but limitation of the data regarding its use for ECx determination should be understood. 3 widely spaced concentrations (based on previous Duomeen HT data) in triplicate were selected for a toxicity comparison not for determination of definitive ECx values. For definitive ECx values a narrower range with 4 replicates is ideally required. This data should therefore only be used for relative comparison between the tested substance.

Read across to the definitive GLP Duomeen HT sediment endpoints for registration purposes is considered justified and sufficiently conservative (worst case) when considering a comparison of chronic toxicity of the tested substances to Lumbriculus variegatus.

In conclusion calculation of the required endpoints to allow a toxicity comparison between the three test substances was achieved. The analytical quality criteria were met. The control performance was lower than desired. 180% reproduction was not achieved in all cases. This is likely due to the condition of the starter culture. For definitive studies the cultures are usually allowed a greater length of time to reach optimum condition. For this study preconditioning of 2-3 weeks was used. However due to this study not being used for definitive endpoint determination but as a toxicity comparison between the tested substances used the data is still considered valid for this purpose.

Validity criteria fulfilled:

yes

Executive summary:

The study was conducted to support read across for the sediment toxicity usint the REACH registered Diamine HT ( Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine, EC number 696-364-9) as a source with Etherdiamine (1,3-Propanediamine, N-[3-((C11-14, C13-rich)oxy)propyl]- branched acetate, EC number 931-295-2) as a target.

The performed study showed that the source for the read across (diamine HT), is more toxic to sediment organisms then the target, etherdiamine. Hence, using sediment data from diamine HT for risk assessment of ehterdiamine is by the registrans considered as a worst case. This is based on the reported NOECs of the two substance (68 mg/kg dw for the source, diamine HT and 134 mg/L dw for the target, etherdiamine).

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

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

Adequacy of study:

supporting study

Study period:

21-01-2009 to 25-02-2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

All data is presented in the report, study is full GLP compliant and is complete but there is a significant scientific uncertainty due to the large difference between the solvent control and normal control reproduction, therefore the study is considered to have reliability 2. In addition no NOEC could be derived for reproduction.

Justification for type of information:

More information on the justification for the analogue approach can be found in Section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD 225 (2007)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of 1 to 10 mL were taken from stock solution (=C5) and from the test solution C3, and the control (solvent without test item), immediately after spiking of the sediment using a glass beaker, and filled into glass bottles. These samples were stored as a reserve. Samples were taken at start of the exposure phase (7 d after spiking the sediment and setting up the sediment-water systems) and at the end of the test (35 d after spiking the sediment and setting up the sediment-water systems) as shown in Table 3. Two different types of samples were taken:
Overlying water and Sediment.

Vehicle:

yes

Details on sediment and application:

The test solutions for application of the test item to the sediment were obtained by diluting the stock solution or application solutions with. From these test solutions an appropriate volume was mixed with an appropriate amount of quartz sand for each treatment (10 g of quartz sand per replicate). The methanol was evaporated to dryness before the sediment of each concentration level was prepared. The amount of sand provided by the test-item-and-sand mixture was taken into account when preparing the formulated sediment. To ensure that the test item added to sediment was evenly distributed within the sediment, the bulk formulated sediments were thoroughly mixed. From these bulk concentration levels, the sediment was distributed to the individual replicates of each concentration level.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

The test system used in this study was the endobenthic oligochaete Lumbriculus variegatus (Müller). This species is tolerant to a wide range of sediment types, and is widely used for sediment toxicity and bioaccumulation testing. The species has been cultured at ECT Oekotoxikologie GmbH since Jan. 1998. The animals were originally obtained from Fischfutter Etzbach (D-53894 Mechernich-Bergheim, Germany). The species identity of the cultured organisms was confirmed according to Brinkhurst (1971).

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

18.0-26.2 dH

Test temperature:

19.6-20.6

pH:

7.9-8.6

Dissolved oxygen:

6.9-8.5 mg/L

Ammonia:

0.1-4.4 mg/L

Nominal and measured concentrations:

te doen

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

< 68 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

No NOEC due to large difference in controls

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

68 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

68 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

204 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Details on results:

The test vessels were inspected one day after addition of the worms, and it was noted that in the controls and up to 1833 mg/kg sediment d.w. mostly no worms were visible on the sediment surface, indicating no sediment avoidance. At the highest concentration level (5500 mg/kg sediment d.w.) worms were not buried. During the regular inspections (at least three times per week), concentration-dependent production of fecal pellets could be observed in the three highest concentration level (611 to 5500 mg/kg sediment d.w.), which indicated different feeding behaviour throughout the treatments. The water phase in the three highest concentrations showed less or no turbidity; additionally the growth of bacteria was reduced in comparison to the control level.

Reported statistics and error estimates:

The total number of worms per replicate, and the total dry weight of the worms per replicate were assessed. In order to estimate mortalities, the numbers of worms that did not react to a gentle stimulus or showed signs of decomposition were considered dead. In cases where the number of worms found on day 28 of the exposure period was less than the number of worms introduced to the vessel, the number of missing worms was added to the number of worms observed dead. In these cases, survival is defined as the difference between the number of worms introduced and the sum of dead and missing worms.

To determine significant differences between the controls (control and solvent control) the replicates of each control were tested for normal-distribution (Range-to-standard-deviation-ratio Test) and for homogeneity of variances (F test); thereafter a pairwise comparison test (Student-t Test) was used.

Normal-distribution of data was tested with the Kolmogorov-Smirnov-test modified after Lilliefors 1967 (two-sided, p ≤ 0.05). Cochran´s test (two-sided, p ≤ 0.05) was used to test variance homogeneity. Subsequent multiple-comparison tests (Dunnett`s t-test; one-sided, smaller) were used to calculate whether there were significant differences (p ≤ 0.05) between the controls and the test item concentrations.

ECx-values were calculated using Probit analyses.

The statistical software package ToxRat Professional 2.09 was used for these calculations.

After 28 days of exposure, mortality was observed at the highest concentration level (5500 mg/kg sediment dry weight (d.w.)). In all other replicates, 10 or more worms were found.

 

The total number of worms found at the end of the test was evaluated as parameter of reproduction.

 

Table10: Total number of worms per replicate (mean values and SD per treatment; n = 4; n = 6 for the solvent control) after four weeks of exposure.

treatment	nominal conc. [mg/kg sed. d.w.]	mean	Std. dev.
control	0	21.3	5.1
solvent control	0	30.2	4.0
C1	68	21.3	3.8
C2	204	16.0	3.5
C3	611	10.3	0.5
C4	1833	10.0	0.0
C5	5500	0.0	0.0

Table11: Total biomass of the animals per replicate (mean values and SD per treatment; n = 4; n = 6 for the solvent control) in mg dry weight.

treatment	nominal conc. [mg/kg sed. d.w.]	mean	Std. dev.
control	0	24.43	3.28
solvent control	0	33.40	3.20
C1	68	30.60	0.88
C2	204	20.23	3.31
C3	611	6.35	1.03
C4	1833	3.25	0.59
C5	5500	0.00	0.00

Validity criteria fulfilled:

yes

Conclusions:

With respect to reproduction and biomass the test showed a clear concentration-response relationship. The test item was acutely toxic with respect to survival, since mortality was observed at the highest concentration level. The study meets all criteria and is valid. the report is considered complete and thus the study is reliable. The large difference between the solvent control and normal control is however an indication that spiking without solvent could remove this uncertainty. In addition would this allow for a more realistic distribution of the substance over the sediment. Unfortunately no NOEC could be determined in this study due to the large difference in the controls.

Executive summary:

In accordance with GLP regulations the chronic toxicity of N-(Hydrogenated tallow)-1,3-diaminopropane t

o the sediment dwelling oligochaeteLumbriculus variegatuswas investigated in a 28-day static sediment-water study.

 

Report:	Daniel Gilberg: N-(Hydrogenated tallow)-1,3-diaminopropane : A study on the chronic toxicity to the sediment dwellerLumbriculus variegatus
	Source: ECT Oekotoxikologie GmbH, unpublished report No.: 09BQ1LA
	June ##, 2009
Guideline:	OECD (2007) Guideline for the Testing of Chemicals No. 225: "Sediment-waterLumbriculus toxicity test using spiked sediment".Organisation for Economic Co-Operation and Development,.
Deviations from the Guideline:	None
GLP:	Yes (certified laboratory)

-	Chemical name:			N-(Hydrogenated tallow)-1,3-diaminopropane
-	CAS number:			68603-64-5
-	Batch no. (test item):			S000905
-	Content/purity:			-           active content diamine: 90.4% (w/w) -           primary fatty amine: 9.6% (w/w)
		-	C-chain distribution of active content		-       C14 part: 2% (w/w) -        C16 part: 31% (w/w) -        C18 part: 67% (w/w)

Test organism:	Lumbriculus variegatus(Müller),synchronised adult worms of similar size
Test medium:	spiked artificial sediment according to OECD guideline No. 225 (OECD 2007); peat content 5% of sediment dry weight; addition ofUrtica-powder (0.25% on dry sediment) and cellulose-powder (0.25% on dry sediment) before application of test item; no additional feeding
Overlying water:	reconstituted water according to OECD guideline No. 203 (OECD 1992); sediment-water volume ratio approx. 1 : 4
Control media:	uncontaminated artificial sediment and reconstituted water (sediment-water volume ratio approx. 1 : 4)
Endpoints:	reproduction and biomass (dry weight) (ECxand NOEC/LOEC)
Biological parameters:	total number of worms, dry weight of worms in each replicate
Test duration (exposure):	28 d
Test units:	glass vessels, 250 mL for biological and analytical samples measurements, covered by plastic lids
Aeration of test chambers:	gentle aeration during acclimation and exposure
Water renewal:	static; periodic compensation of evaporated water
Feeding during exposure:	feed in sediment (Urticapowder and cellulose)
Sediment conditioning:	7 d prior to application of test item
Sediment spiking:	test item suspended in methanol; test item coated on quartz sand, then mixed with wet sediment
Equilibration:	7 d prior to addition of organisms
Temperature:	20 ± 2°C; (min / max: 19.6 / 20.6 °C; manual measurements)
Light regime:	16 h light : 8 h dark
Number of organisms per test chamber:	10 in replicates for biological measurements (25 in replicates for analytical measurements)
Number of test concentrations:	5 plus a solvent control and a control
Test concentrations:	5500, 1833, 611, 204, and 68 mg/kgsediment dry weight plus a solvent control and a control
Number of replicates per treatment:	6 in the solvent controls; 4 in each other treatment; additional vessels for chemical analysis
Sampling for chemical analysis of test concentrations:	sediment and overlying water, at start and at end of exposure
Determination of organic carbon content in sediment:	4 control samples before equilibration
Data evaluation:	ANOVA and multiple comparison tests (Dunnett`s t-test); Probit analyses
Chemical analysis of test concentrations:	samples from control, the highest and one intermediate treatment (sediment and overlying water) were analysed at start and at end of exposure; not part of this study report.

 

Findings:

With respect to reproduction and biomass, concentration-dependent effects were found. Therefore, EC_x-values were calculated. Mortalities were observed in the highest concentration level.

 

Summary of results based on statistical evaluation of biological results and nominal concentrations in mg kg^-1sediment dry weight.

Endpoint	NOEC [mg kg^-1]	LOEC [mg kg^-1]	EC10  [mg kg^-1]	EC50   [mg kg^-1]
reproduction	< 68	68	11	261
biomass	68	204	70	268

 

 

The biological part of the study was conducted at ECT Oekotoxikologie GmbH, Flörsheim am,. Samples of the overlying water and sediment were analysed under the responsibility of the analytical laboratory at Dr. U. Noack-Laboratorien, Sarstedt, Germany. The analyses were not part of the study report.

 

 

Validity of the test:

 

number of worms in the control vessels at the end of exposure:
(required:at least 80% higher than at the start of exposure)	start level + 113%
pH of the overlying water:
(required:between 6 and 9 at the start and at the end of the test)	at start:7.9 – 8.4 at end:8.1 – 8.4
Dissolved oxygen concentration measured in at least one replicate per concentration level and control:
(required:above 60% of the air saturation value (ASV)	≥ 77% (ASV)

 

Reference 3

Endpoint:

sediment toxicity: long-term

Type of information:

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

Adequacy of study:

supporting study

Study period:

2010-08-26 to 2010-09-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

More information on the justification for the analogue approach can be found in Section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Draft ISO/DIS 10872 (2008)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates:
Modified artificial sediment:
- 70 % quartz sand (with > 50 % particles sized between 50 and 200 µm)
- 20 % Al2O3
- 4.5 % Fe2O3
- 4 % peat, air dried and finely ground
- 0.3 % CaCO3
- 0.5 % dolomite (clay)
The pH-value and the moisture of the artificial sediment were determined. One day prior to introduction of the nematodes the sediment was moistened with M9 medium to a moisture of 47.4 %.
- Details of spiking:
The test item was pounded in a mortar and the respective test amount for the limit concentration was weighed out and thoroughly mixed with quartz sand (20 % of the entire sediment amount). Subsequently, the treated quartz sand was given to the sediment and thoroughly mixed to ensure a homogenous distribution of the test item. M9 medium was added to the sediment to obtain a water content of 47.4 %. Subsequently, 0.5 g sediment wet weight was filled into each test well.
- Equilibration time: 1 day
- Equilibration conditions: 6 +/- 2 °C, dark
- Controls: Moistened test sediment was tested under the same conditions as the test replicates.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): quartz sand
- Concentration of vehicle in test medium (stock solution and final test solution): 20 % of the entire sediment amount
- Evaporation of vehicle before use: No

Test organisms (species):

Caenorhabditis elegans

Details on test organisms:

TEST ORGANISM
- Common name: Nematode
- Strain/clone: Wilde type strain N2 var Bristol
- Source: Caenorhabditis Genetics Center, Minneapolis
- Breeding conditions:Breeding is performed at the test facility at 20 +/- 2 °C in the dark on agar plates containing NGM (nematode growth medium) agar inoculated with Escherichia coli OP 50.
- Age of animals at beginning of exposure: First stage juveniles were obtained by filtering nematode solutions from culture plates through nylon nets (10 µm and subsequently 5 µm mesh size). The size of 30 representative juvenile nematodes, which were not used in the test, was determined prior to nematode insertion.
- Feeding during test
LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. After inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.



ACCLIMATION
- Acclimation period: None, test conditions = breeding conditions

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

yes

Duration:

96 h

Exposure phase:

total exposure duration

Nominal and measured concentrations:

Nominal: 1000 mg test item/kg sediment dry weight

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Nunc Polystyrol Multiwells (12 well microtiter plates)
- Sediment volume: 0.5 g sediment wet weight per replicate
- Overlying water volume: 0.5 mL food medium (see above)
- Aeration: no
- Replacement of evaporated test water, if any: No


EXPOSURE REGIME
- No. of organisms per container (treatment): 60
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 6
- Type and preparation of food: LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 17 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.


CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- Composition (if artificial substrate):
Modified artificial sediment:
- 70 % quartz sand (with > 50 % particles sized between 50 and 200 µm)
- 20 % Al2O3
- 4.5 % Fe2O3
- 4 % peat, air dried and finely ground
- 0.3 % CaCO3
- 0.5 % dolomite (clay)
The pH-value and the moisture of the artificial sediment were determined. One day prior to introduction of the nematodes the sediment was moistened with M9 medium to a moisture of 47.4 %.
- Maturation of artificial substrate (if any): no

OTHER TEST CONDITIONS
- Continuous dark
The initial pH of the artificial sediment at test start was 7.25. The initial sediment moisture was
1 %. The room temperature ranged from 20.5 to 21.5 °C.



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The recovery / mortality of the inserted nematodes was analysed by counting the adult nematodes under a microscope. The body length of each female adult nematode was determined with a microscale.
The reproduction was determined by counting the juvenile nematodes in a subsample of the supernatant.


VEHICLE CONTROL PERFORMED: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: None limit test
- Range finding study
Range Finding Test (96 hours, non-GLP): Recovery, Fertility and Reproduction
Test Item Concentration
Recovery of adult nematodes Fertility of adult females Reproduction
[mg test item/kg soil dry weight] [%] [%] [Offspring per replicate]
Control 86.7 100 98.7
10 90.0 100 80.9
100 86.7 100 82.2
1000 83.3 100 86.8

Reference substance (positive control):

yes

Remarks:

Benzyldimethylhexadecylammonium chloride

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality, Fertility, Reproduction, Growth

Details on results:

- Mortality of test animals at end of exposure period:
In the artificial sediment a recovery of 90 % was observed in the control and at 1000 mg test item/kg sediment dry weight after 4 days of exposure.
No evident mortality was observed at the reference item concentration of 5 mg/L. However, at 10, 15 and 20 mg/L the reference item induced mortality of 100 % of adult nematodes.
- No. of offspring produced: The reproduction in artificial sediment was not statistically significantly reduced compared to the control at the limit test item concentration of 1000 mg test item/kg sediment dry weight
- Morphological abnormalities: No
- Behavioural abnormalities: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:


Effects Reference Item Benzyldimethylhexadecylammonium chloride
[mg/L]
Mortality 10
Fertility 15
Reproduction 10
Growth 5
LOEC
(Mortality, Fertility, Reproduction, Growth) 5
NOEC
(Mortality, Fertility, Reproduction, Growth) < 5
EC50
(Mortality) 15.0 (14.2 – 15.8)
EC50
(Reproduction) 10.2 (9.75 – 10.9)
EC50
(Size) 15.9 (11.5 – 23.4)

Reported statistics and error estimates:

For growth and reproduction data of the test item test t-tests were used for LOEC and NOEC calculations. Prior to running the t-tests a Normality and an Equal Variance Test were done. P-values for both Normality and Equal Variance Test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.

For growth data of the reference item test a t-test was performed. Prior to running the t-test a Normality and an Equal Variance Test were done. For reproduction data of the reference item One Way Analysis of Variance (ANOVA) were used. A Normality Test and an Equal Variance test were conducted prior to ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The -value for ANOVA (acceptable probability of incorrectly concluding that there is no difference) is =0.05. Due to a failed Equal Variance Test with original data (p = 0.002) the ANOVA was repeated with log transformed data.

Recovery / Mortality after 4 Days of Exposure to N-(Hydrogenated tallow)-1,3-
                  diaminopropane (CAS no. 68603-64-5)

Nominal test item concentration [mg test item/kg sediment dry weight]	[%] Recovered test organisms								Mortality [%]
	Replicate						MV	SD
	1	2	3	4	5	6
Liquid Control	100	100	110	100	–	–	103	5.00	0.00
Control	80.0	90.0	90.0	100	90.0	90.0	90.0	6.32	10.0
1000	90.0	90.0	90.0	100	80.0	90	90.0	6.32	10.0

Nematode Reproduction after 4 Days of Exposure to N-(Hydrogenated tallow)-1,3-
                    diaminopropane (CAS no. 68603-64-5)     

Nominal test item concentration [mg test item/kg sediment dry weight]	Number of offspring per inserted female nematode
	Replicate						MV	SD	Inhibition [%]
	1	2	3	4	5	6
Control	105	126	94.6	98.6	95.2	110	105	11.9	–
1000	109	99.7	85.2	110	80.6	107	98.6	12.8	6.01

Nematode Growth after 4 Days of Exposure to N-(Hydrogenated tallow)-1,3-
                         diaminopropane (CAS no. 68603-64-5)     

Nominal test item concentration [mg test item/kg sediment dry weight]	Mean Growth [µm]
	Replicate						MV	SD	Inhibition [%]
	1	2	3	4	5	6
Control	793	800	790	811	790	769	792	13.9	–
1000	805	784	799	768	757	704	770	36.9	3.00

Validity criteria fulfilled:

yes

Conclusions:

In this study N-(Hydrogenated tallow)-1,3-diaminopropane (CAS no. 68603-64-5) did not induce significant mortality of C. elegans after an exposure to the limit test item concentration of 1000 mg test item/kg sediment dry weight for 96 hours. Also, adult nematode fertility as well as nematode reproduction was not affected at 1000 mg test item/kg sediment dry weight. No reduction of growth was observed at the limit test item concentration of 1000 mg test item/kg sediment dry weight after four days of exposure to the test item compared to the control.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set at > 1000 mg test item/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentrations of N-(Hydrogenated tallow)-1,3-diaminopropane
(CAS no. 68603-64-5).

Executive summary:

The effects of the test item N-(Hydrogenated tallow)-1,3-diaminopropane (CAS no. 68603-64-5) (batch no.S000905) on the bacterivorous nematode Caenorhabditis elegans in a sediment system were determined at Dr.U.Noack-Laboratorien, Sarstedt, Germany, from August 26^th to September 13^th, 2010 with the definitive exposure phase from September 8^th to September 13^th, 2010. The study was carried out according to the Draft guideline ISO/DIS 10872 (2008). Test duration was 96 hours after insertion of the test organisms. The study was performed by spiking the test item into the sediment with the limit test item concentration of 1000 mg test item/kg sediment dry weight. Six replicates per control and limit test item concentration were set up.

After four days of exposure to the limit test item concentration of 1000 mg test item/kg sediment dry weight, N-(Hydrogenated tallow)-1,3-diaminopropane (CAS no. 68603-64-5) did not induce biologically significant mortality of C. elegans. Also, adult nematode fertility as well as nematode reproduction was not affected at 1000 mg test item/kg sediment dry weight. Accordingly, no statistically significant reduction of adult nematode growth was observed at the limit test item concentration of 1000 mg test item/kg sediment dry weight after an exposure of 96 hours to the test item.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set at > 1000 mg test item/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentration of N-(Hydrogenated tallow)-1,3-diaminopropane.

Summary of all Effects and resulting LOEC, NOEC based on nominal concentrations

Effects	N-(Hydrogenated tallow)-1,3-diaminopropane (CAS no. 68603-64-5)
	[mg test item/kg sediment dry weight]
Mortality	> 1000
Fertility	> 1000
Reproduction	> 1000
Growth	> 1000
LOEC (Mortality, Fertility, Reproduction, Growth)	> 1000
NOEC (Mortality, Fertility, Reproduction, Growth)	1000

Reference 4

Endpoint:

sediment toxicity: long-term

Type of information:

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

Adequacy of study:

supporting study

Study period:

10.01.2011-30.03.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

More information on the justification for the analogue approach can be found in Section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD 225

Deviations:

no

Principles of method if other than guideline:

No deviation but

•A solvent was not used to spike the test chemical to the sediment. The sediment was spiked as a complete sediment with a refined and extended
spiking procedure to allow complete distribution and equilibration of the test chemical in all components of the sediment without the use of a solvent.

•The test concentrations were not generated by subsequent mixing of the spiked sediment with un- spiked sediment components. This has led to an uneven distribution and recovery of the test chemical. Observed both biologically by worm behaviour and analytically by extraction and subsequent
chemical analysis in preliminary testing. Each concentration was spiked separately and agitated for an extended period at elevated temperature with
the appropriate amount of the test chemical. This ensured an accurate exposure concentration and even distribution of the test chemical through all of the sediment components.

•Due to the sacrificial nature of the sediment sampling for chemical analysis 100 g of formulated sediment was used per replicate to ensure enough
sediment remains to allow adequate growth of the test organisms.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of sediment were taken for chemical analysis prior to equilibration, after equilibration and at the end of the test for chemical analysis.
Enough sediment was sampled to allow analysis, dry weight determination and any required repeats of results. To ensure the consistency of the
experimental replicates, samples at the end of the test were taken in at least 2 replicates in the sampled concentrations. Water samples were taken
at the start and end of the test. Sediment samples were extracted using an accelerated solvent extraction and a manual extraction method. See
analytical methods below. Chemical analysis was conducted after spiking, after equilibration and at the end of the test in order to quantify the concentration of the test substance on the sediment. The analytical method used was an LC/ MS/MS method. Details of the method are shown below.

Vehicle:

no

Details on sediment and application:

OECD formulated sediment

Sediment according to OECD 225 was used. The table is presented in the other information section below.


Preparation of the stock solution
The desired test solutions were prepared separately per concentration. The appropriate amount of the test chemical needed to achieve the desired concentration in the sediment was weighed out in a glass beaker on an analytical balance. Then 80 – 100 mL of DSW was added and the substance was sonicated until a homogeneous milky white solution without precipitate was formed. Care was taken not to excessively overheat the stock
solution. After emptying of the initial stock solution (described below) the procedure was repeated with DSW only until no visible trace of the test
substance remained in the beaker. This was repeated in the same manner for all test concentrations required.

Preparation of the spiked sediment
The stock solutions were added to 400 g of wetted sediment and agitated for 24 hours at approximately 40ºC. After spiking the resulting sediment
was checked for pH and adjusted with calcium carbonate if required. The resulting spiked sediment was then sampled for analysis and then
transferred evenly into the test vessels and left to equilibrate for a 6 day period under gentle aeration. Identical procedures without the test chemical were followed for control sediment. The following test concentrations were prepared: 45, 90, 180, 360 and 720 mg/kg dw.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

Test animals
The test animals were taken from the environmental chemistry laboratories Lumbriculus Variegatus stock originating from Wageningen University.
The test animals were cultured, sub-cultured and synchronised in conformity with laboratory Standard Operation Procedures. In preparation for
testing 4 weeks prior to the planned start of the test the animals were sub cultured to allow optimal growth and increase of body mass. 2 weeks prior
to testing the sub cultured organisms were synchronized and the tail ends of the worms were allowed to recover for a maximum of 14 days before
being used in the test. Test animals of a similar size were chosen from this synchronized batch for use in the test. All selected test animals were
therefore considered to be of a similar physiological state. A representative sample of this batch was sampled and the dry weight was determined to allow an increase in dry weight endpoint to be calculated at the end of the test if required. The test animals were reference tested twice a year to check
the condition of the culture as indicated in the test guideline.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

Post exposure worms were counted and left to purge for 48 Hours in fresh water to allow more accurate dry weight determination. Worms were
observed for any visible abnormalities. No abnormalities were obseved except for the highest concentration being slightly smaller than in the other
concentrations and the control.

Hardness:

Hardness was measured ( as calcium carbonate) but calcium carbonate was used to stabilize sediment pH. The water hardness was therefore
influenced and was higher at the end of the study than at the beginning. There was however little variation between replicates and between the
control and the highest concentration. The hardness measurements demonstrated concistancy of conditions between replicates and the control
but did not indicate acurate hardness of the dilution water. The composition of water used for dilution in indicated below in other information.

Test temperature:

Min 19.0ºC Max 20.9ºC

pH:

pH Water Min 8.0 Max 8.5
pH Sediment Min 6.4 Max 6.5

Dissolved oxygen:

Min-7.0 mg/L Max- 9.1mg/L

Salinity:

Not Measured

Ammonia:

Min- 0.08mg/L Max 2.72 mg/L

Nominal and measured concentrations:

The results of the measured concentrations in the sediment is presented below in any other information results. The nominal test concentrations
were as follows:
45, 90, 180, 360 and 720 mg/kg dw.

Details on test conditions:

Method

The test was performed as a 28 day static test. The number of animals used per concentration was 40. Animals were equally divided over 4 replicates of 10 animals and exposed to the test concentrations. The control contained 6 replicates of 10 animals. Before the addition of the worms the spiked sediment was left for 6 days to equilibrate. Equilibration took place under the same conditions as the final test with gentle aeration to allow stabilization of the microbial component and distribution of the test chemical between the overlying water and sediment.

Synchronized worms were randomly placed in the test fluids and the test vessels were placed in a random manner on the laboratory work surface. The test vessels were clearly labelled and gently aerated for the full test duration. During the test, the animals were not additionally fed as the food components were included in the formulated sediment. The test was inspected at least 6 times a week, biological observations were recorded and relevant physical chemical parameters were measured according to the study plan. At the end of the test, surviving worms were gently sieved from the sediment with a 250µm sieve and counted for use in endpoint calculations.

Worms were considered dead if no active movement occured after stimulation. Due to the nature of the a sediment test any dead worms are likely to have been decomposed during the test period and very difficult or impossible to find in the sediment. Dead worms were recorded in the raw data if
observed. After counting the worms, living worms were transferred to clean continually aerated DSW for 48 hours to purge the worms of ingested
sediment. Then they were then transferred to appropriate vessels for dry weight determination. Dry weight determination was carried out by weighing of the oven dishes before use and then placing the entire worm population per replicate in the dish and oven drying for 24 hours at approximately 100ºC to drive of all moisture. Reweighing after cooling allowed the dry biomass to be determined for use in endpoint calculations..

Reference substance (positive control):

yes

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

86 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Analytical Recovery >80% of nominal. (Nominal concentrations used)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

180 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Analytical recovery >80% of nominal (Nominal concentrations used)

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

237 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Dry Weight

Remarks on result:

other: Analytical recovery >80% ( based on Nominal Concentrations)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

360 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Dry Weight

Remarks on result:

other: Analytical recovery >80% ( based on Nominal Concentrations)

Details on results:

Extraction of the sediment at the end of the test gave analytical recoveries of around or greater than 80% of the nominal concentrations. Nominal
concentrations were therefore used for calculating of the endpoints. Due to the inaccuracies involved with reproduction counts not taking into
account the size of the individual the dry weight enpoint is considered the most accurate as this is dertermined per replicate as a whole and is not
influenced by the presence of small or large worms.

Biological observations
During the test period no abnormal behaviour (e.g. sediment avoidance) in the test concentrations was observed. Worms burrowed into all
concentrations and were visibly feeding. Production of faecal pellets was observed at all concentrations in all replicates. In replicate 2 of 360
mg/kg no biological activity was observed after 31/1/2011. In this replicate an aeration malfunction occurred subsequently influencing this
replicate. This effect was not concentration (test substance) related and was therefore not used in data calculations. All worms were active at the
time of counting and no malformations were visible. Two dead worms were found at 180 mg/kg in replicates 2 and 4. Worms appeared slightly smaller and thinner at higher concentrations. Grouping of the worms at the highest concentration was also observed. At all other concentrations the distribution of the animals was even throughout the sediment.

Results with reference substance (positive control):

The laboratory culture was reference tested as indicated in :
"German Federal Environment Agency (2005) Validation of a Sediment Toxicity Test with Endobenthic Aquatic Oligochaete Lumbriculus Variegatus by an International Ring Test. FKZ 20267 429" as part of the GLP maintainance. The results obtained fell inbetween the minimum and maximum
values obtained in the ring test. The culture was therefore considered suitible for testing.

Reported statistics and error estimates:

Lumbriculus worms reproduce via a-sexual fragmentation. The number of worms observed in a test therefore depends on the timing of
fragmentation and the moment of counting the worms. It is unlikely despite of the synchronisation that this fragmentation will occur exactly at the
same time. The dry-weight of the total number of living worms is independent of the timing of fragmentation. It is therefore considered to be a more
reliable endpoint than the reproduction based on the number of viable worms. For guideline compliance however both reproduction and dry weight
endpoints were calculated using the TOXCALC version 5.023. The Dunnett`s t-test and Probit analyses were conducted to determine the NOEC/LOEC and the ECx values respectively.

Measured analytical recovery in the lowest middle and highest concentrations

Nominal Test Concentration mg/kg dw	T=28 Manual Acidified Extraction % of Nominal
Control	0
45	84.0
180	79.0
720	89.6

 

Summary of results

 

Endpoint	NOEC [mg/kg dw]	LOEC [mg/kg dw]	EC10  [mg/kg dw]	EC50   [mg/kg dw]
Reproduction	180	360	86	593
Biomass	360	720	237	638

Validity criteria fulfilled:

yes

Conclusions:

The study can be considered a reliable representation of the toxicity of the test substance to the test organism without significant restrictions.

Executive summary:

Study was conducted to GLP and to the appropriate guideline. Quantification was conducted with a validated analytical and suitible extraction methods. Sufficient test substance identification (Analytical certificate present) was also provided. Critical guideline criteria were met and minor data discrepancies, deviations and amendments were reported, discussed and excluded from valid data points where appropriate. Spiking method was adapted from the guideline without using solvent. Chemical analysis indicates spiking method was acceptable.

Description of key information

No sediment toxicity data are currently available for the registered substance. 
Long-term sediment toxicity studies are not required for Annex VIII substances under the REACH Regulation. However, due to the high sediment adsorption potential of the substance, it is appropriate to assess the potential significance of this compartment as part of the chemicals safety assessment. Data have been read across from structural analogues.  

One non-GLP OECD 225 sediment study has been performed on the highly similar source substance ehterdiamine C11-13. This test was done to compare sediment toxicity with diamine HT. The results  indicate that the etherdiamine C11-13 is less toxic to sediment compared to diamine HT. The read across for the target substance etherdiamine C12-14 ace. is therefore supported and the most reliable Lumbriculus study was used as a key value for the chemical risk assessment.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

86 mg/kg sediment dw

Additional information

One study was conducted to support read across for the sediment toxicity using the REACH registered Diamine HT ( Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine, EC number 696-364-9) as one of the source substances and with Etherdiamine C11-13 (1,3-Propanediamine, N-[3-((C11-14, C13-rich)oxy)propyl]- branched acetate, EC number 931-295-2) as another source substance.

 

The performed non-GLP study showed that the source substance diamine HT is more toxic to sediment organisms then the source substance etherdiamine C11-13. Using sediment data from diamine HT for risk assessment of etherdiamine C12-14 ace. is by the registrant considered as a worst case, based on the reported NOECs of the related substances (68 mg/kg dw for diamine HT and 134 mg/L dw for the etherdiamine C11-13).

 

For the group of diamines, four test results are available regarding the sediment toxicity. However, for this read across, only the three studies performed on Diamine HT were used. The PNEC for the etherdiamine C12-14 ace., is derived exactly as the PNEC for the supporting substance 2 (diamine HT).

 

The long term studies were performed with the hydrogenated diamine C16 -18 and were performed with Lumbriculus variegatus and and Caenorhabditis elegans.The long-term study with Caenorhabditis elegans showed no effects up to 1000 mg/kg dw. Two long-term tests with Lumbriculus variegatus were performed applying two different spiking approaches. In the first test a solvent was used to spike the test substance on the sand fraction and in the second test the test substance was spiked onto the whole sediment in the water phase at a slightly elevated temperature. In the first test with solvent spiking, a significant difference in reproduction between the normal control and solvent control was observed. It was not clear why this difference was observed but considering to unrealism in using a solvent to spike the test substance it was decided to repeat the test using an environmentally more realistic solvent free spiking procedure of the whole sediment. For this second long-term Lumbriculus variegatus a NOEC and EC10 for reproduction was observed of resp. 180 mg/kg dw and 86 mg/kg dw. The NOEC and EC10 based on dry weight of resp 360 and 237 mg/kg dw are higher probably because the moment of splitting of the worms is slightly influenced by the test substance.

 

The spiking procedure using a solvent to spike the sand fraction is unrealistic for cationic surfactants. Cationic surfactants which may enter surface water are normally sorbed to dissolved organic matter or suspended matter and may redistribute slowly to thermodynamically more favourable sites when available. Quartz sand has a very low cation exchange capacity and no organic matter. The use of natural sediment spiked without using solvent is far more realistic and could allow a more evenly distribution of the test substance over the sediment. In addition it would allow the ingestion of the test substance more realistically. In addition the solvent apparently had a positive influence on the reproduction which limits the reliability of the result.

 

Overall, the EC10 from the most reliable Lumbriculs study was used as key value for the chemical safety assessment.