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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

2008-02-04 to 2008-02-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Complete report, test performed under GLP, 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:

OECD Guideline 201 (Alga, Growth Inhibition Test)

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: Each sample was diluted 1:2 with 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: 20 mg/L, freshly prepared with dilution water 30 min before application, sonificated for 30 min at 40 °C
- Differential loading: 10-32-100-320-1000 µg/L (factor: v10)
- Controls: six replicates without test item, tested under the same conditions as the test replicates
- 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):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE•m-2•s-1 for 24 h per day

ACCLIMATION
- Culturing media and conditions (same as test or not): Inoculum: Nutrient medium Z according to Luettge (1994), conditions same as for test; test replicates: natural river water, other conditions same as for test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h algae were transferred from nominal concentrations 320 – 1000 µg/L and control into fresh untreated medium (0.5 – 1 mL algae suspension from each replicate were filled up with dilution water to 10 mL) and allowed to grow for further 5 d to determine whether the effect of the item is reversible.

Hardness:

Not measured

Test temperature:

Mean: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
1000 7.76 7.70
320 7.77 7.66
100 7.73 7.87
32 7.76 8.04
10 7.86 7.76
Control 7.92 8.03

Dissolved oxygen:

Not measured

Salinity:

Not measured

Nominal and measured concentrations:

Nominal test item concentrations: 10-32-100-320-1000 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 4971 cells/mL
- Control end cells density: Mean 774306 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

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 50 % of the components of the dilution water acc. to the guideline were added to enable a sufficient growth of algae
- 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: Nutrient medium Z according to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: 24 h
- Light intensity and quality: 60-120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Microscopic
- Chlorophyll measurement: fluorimeter; Chlorophyll-a-fluorescence, excitation at 435 nm, emission at 685 nm; measurements at test start and every 24 h, each replicate was measured 6-fold, value (flash) no. 3 - 6 were used for calculations
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: v10
- Range finding study
- Test concentrations: 10-100-1000 µg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

507 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 468 - 543

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

188 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 167-206

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: none
- Colour differences: none
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: at test end of the concentration levels 320 - 1000 µg/L
- Other: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None

The study meets the validity criteria of the guideline:
- The cell growth increased 156-fold after 72 h in the control (required: 16-fold).
- The temperature during the test was in the range of 21-24 °C. The difference from the mean was within ± 2 °C: min.: 21 °C, max.: 22 °C mean value: 21.5 °C.
- The pH-value in the control replicates increased not higher than 1.5 units: from 7.92 to 8.03.
- The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was 27.71 % (required: < 35 %).
- Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1.94 % (required: < 7 %).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 1.01 (CI 0.90 - 1.13) mg/L
Yield inhibition: 0.42 (CI 0.37 - 0.48) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using maximum likelihood analyses.

Table 1: EC₁₀-_, EC₂₀- and EC₅₀- values (0-72 h) of Duomeen OV

                based on nominal test item concentrations [µg/L]

                            

	Inhibition of yield
EyC10 (95 % confidence interval)	113 (98 – 131)
EyC20 (95 % confidence interval)	137 (121 – 158)
EyC50 (95 % confidence interval)	214 (186 – 243)
	Rate-related inhibition
ErC10 (95 % confidence interval)	188 (167 – 206)
ErC20 (95 % confidence interval)	267 (244 - 291)
ErC50 (95 % confidence interval)	507 (468 – 543)

Table 2:     Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 h		24 h	48 h	72 h
1000		1		4971		10547	7050	12720
		2		4971		9255	8373	13602
		3		4971		11649	9948	9255
		Mean		4971		10484	8457	11859
320		1		4971		38393	108669	207390
		2		4971		33920	100605	159573
		3		4971		39653	142595	230732
		Mean		4971		37322	117290	199232
100		1		4971		42519	223266	667416
		2		4971		43275	210351	685056
		3		4971		41291	215076	808536
		Mean		4971		42362	216231	720336
32		1		4971		41984	239961	892326
		2		4971		41417	260751	868071
		3		4971		45858	219171	848856
		Mean		4971		43086	239961	869751
10		1		4971		39716	237126	789006
		2		4971		44945	288156	777981
		3		4971		42992	198066	896421
		Mean		4971		42551	241116	821136
Control		1		4971		40661	240276	730101
		2		4971		44756	225471	674346
		3		4971		42897	241536	798456
		4		4971		38298	218226	794361
		5		4971		41921	201216	748056
		6		4971		44409	252246	900516
		Mean		4971		42157	229829	774306

Table 3     Evaluation after 72 h

        

Nominal test item concentration	Replicate	Yield		Inhibition of yield	Growth rate		Rate-related inhibition
[µg/L]	No.			[%]	[d^-1]		[%]
1000	1		7749	98.99		0.313	81.37
	2		8631	98.88		0.336	80.04
	3		4284	99.44		0.207	87.68
	Mean		6888	99.10		0.285	83.03
320	1		202419	73.69		1.244	26.04
	2		154602	79.90		1.156	31.23
	3		225761	70.66		1.279	23.92
	Mean		194261	74.75		1.226	27.06
100	1		662445	13.89		1.633	2.86
	2		680085	11.60		1.642	2.35
	3		803565	-4.45		1.697	-0.94
	Mean		715365	7.01		1.657	1.42
32	1		887355	-15.34		1.730	-2.89
	2		863100	-12.19		1.721	-2.35
	3		843885	-9.69		1.713	-1.90
	Mean		864780	-12.41		1.721	-2.38
10	1		784035	-1.91		1.689	-0.45
	2		773010	-0.48		1.684	-0.17
	3		891450	-15.87		1.732	-2.98
	Mean		816165	-6.09		1.702	-1.20
Control	1		725130			1.663
	2		669375			1.637
	3		793485			1.693
	4		789390			1.691
	5		743085			1.671
	6		895545			1.733
	Mean		769335			1.681

Negative values = increase of growth

Table 4:     Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	VC %	Mean VC %
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	2.102	1.777	1.111	1.663	0.505	30.35	27.71
	2	2.198	1.617	1.096	1.637	0.551	33.68
	3	2.155	1.728	1.196	1.693	0.481	28.39
	4	2.042	1.740	1.292	1.691	0.377	22.31
	5	2.132	1.569	1.313	1.671	0.419	25.08
	6	2.190	1.737	1.273	1.733	0.459	26.46
				Mean	1.681
				SD ±	0.03
				VC %	1.94

SD = Standard deviation        VC %= percent coefficient of variation

Table 5:     Recovery of Alga Cells

Nominal test item concentration                   [µg/L]	Incubation time                    [d]	Cell density [cells/mL]		Growth rate after incubation time [d^-1]
		Initial^#	Final
1000	5	1186	72255	0.82
			76917	0.83
			54867	0.77
320		19923	242229	0.50
			203295	0.46
			217407	0.48
Control		38715	201909	0.33
			250041	0.37

^#) calculated from the cell density of the definitive test after 72 h

Validity criteria fulfilled:

yes

Remarks:

The study meets the validity criteria of the guideline

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.

Executive summary:

The toxicity of Duomeen OV (CAS No. 7173-62-8) (Batch no.: S000902)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from February 04 to 12, 2008. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

An individual test design was applied. Natural river water was used as dilution medium.

The study was conducted under static conditions with an initial cell density of nominal 2 to 5 x 10³cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of v10, nominal: 10 - 32 - 100 - 320 - 1000 µg/L. Three replicates were tested for the test item concentrations and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of C18 : 1 diamine, main component of Duomeen OV were analysed at all concentration levels at test start and test end via LC-MS/MS analysis. The measured concentrations at test start were in the range of 91 to 98 % of the nominal values. At the end of the test Duomeen OV gave recoveries below the limit of quantification up to 12 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, low DOC and suspended matter content and also because the river water was frozen before use to minimize the microbial activity. This is supported by the monitoring results from the chronic daphnia tests with this test item. In this test only a limited decrease of the test substance was observed over 48 hours. This decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. The larger decrease of the test item during the algae test is therefore most likely caused by sorption of the test substance to algae. These points support the conclusion that the test organisms were fully exposed to the test substance during the test. At the end of the test Duomeen OV gave recoveries ranging from LOQ (2.04 µg/L) to 12 % of the nominal values. Therefore all effect values are given based on nominal concentrations of the test item. In this study Duomeen OV was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal concentrations): The EC₁₀-values with 95 % confidence intervals for inhibition of yield (E_yC₁₀) and specific growth rate (E_rC₁₀) after 72 h were 113 (98 to 131) and 188 (167 to 206) µg/L, respectively. The EC₅₀-values with 95 % confidence intervals for inhibition of yield (E_yC₅₀) and specific growth rate (E_rC₅₀) after 72 h were 214 (186 to 243) and 507 (468 to 543) µg/L, respectively.

After 72 h algae were transferred from the nominal concentrations 320 to 1000 µg/L and control to fresh untreated medium and allowed to grow for further 5 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 1000 µg/L (highest test concentration)

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Study period:

2008-01-15 to 2008-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

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:

OECD Guideline 201 (Alga, Growth Inhibition Test)

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: 3.2 - 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: 20 mg/L, freshly prepared with natural river water 30 min before application, sonificated for 30 min at 40 °C
- Differential loading: 3.2 - 10 - 32 - 100 - 320 - 1000 µg/L (factor: v10)
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates
- 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):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE•m-2•s-1 for 24 h per day

ACCLIMATION
- Culturing media and conditions (same as test or not): Inoculum: Nutrient medium Z according to Luettge (1994), conditions same as for test; test replicates: natural river water, other conditions same as for test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h algae were transferred from nominal concentrations 320 – 1000 µg/L and control into fresh untreated medium (0.5 – 1 mL algae suspension from each replicate were filled up with dilution water to 10 mL) and allowed to grow for further 3 d to determine whether the effect of the item is reversible.

Hardness:

Not measured

Test temperature:

Mean: 22, Min: 21, Max: 23

pH:

Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
1000 7.96 7.71
320 7.96 7.89
100 7.93 7.95
32 7.90 8.00
10 7.90 8.09
3.2 7.93 7.96
Control 8.03 8.07

Dissolved oxygen:

Not measured

Salinity:

Not measured

Nominal and measured concentrations:

Nominal concentrations: 3.2-10-32-100-320-1000 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 4971 cells/mL
- Control end cells density: Mean 863661 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

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 50 % of the components of the dilution water acc. to the guideline were added to enable a sufficient growth of algae
- Particulate matter: 2.5 mg/L
- Conductivity: 149 µS (natural river water)
- Salinity: Not measured
- Culture medium different from test medium: Nutrient medium Z according to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: 24 h
- Light intensity and quality: 60-120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Microscopic
- Chlorophyll measurement: fluorimeter; Chlorophyll-a-fluorescence, excitation at 435 nm, emission at 685 nm; measurements at test start and every 24 h, each replicate was measured 6-fold, value (flash) no. 3 - 6 were used for calculations
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: v10
- Range finding study
- Test concentrations: 1-10-100-1000 µg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

596 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 520 - 667

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

193 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 158 - 235

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: at the concentration levels 320 and 1000 µg/L at test end
- Other:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate related inhibition: 1.01 (CI 0.90 to 1.13) mg/L
Yield inhibition: 0.42 (CI 0.37 to 0.48) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using maximum likelihood analyses.

Table 1: EC₁₀-_, EC₂₀- and EC₅₀- values (0-72 h) of N-(hydrogenated tallow alkyl)trimethylenediamines

                                based on nominal test item concentrations [µg/L]

                          

	Inhibition of yield
EyC10 (95 % confidence interval)	44.6 ( 3.20 – 83.1)
EyC20 (95 % confidence interval)	89.3 (56.6 – 139)
EyC50 (95 % confidence interval)	223 (161 – 307)
	Rate-related inhibition
ErC10 (95 % confidence interval)	193 (158 – 235)
ErC20 (95 % confidence interval)	299 (257 – 350)
ErC50 (95 % confidence interval)	596 (520 – 667)

Table 2:     Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 h		24 h	48 h	72 h
1000		1		4971		13035	9224	19083
		2		4971		17256	13161	24375
		3		4971		12279	5979	10515
		Mean		4971		14190	9455	17991
320		1		4971		32565	128136	295086
		2		4971		28974	99786	231141
		3		4971		34833	154281	301701
		Mean		4971		32124	127401	275976
100		1		4971		35904	255081	617331
		2		4971		35274	288471	793416
		3		4971		45701	388011	781761
		Mean		4971		38960	310521	730836
32		1		4971		45417	348636	755616
		2		4971		44598	325641	679071
		3		4971		39275	309261	702381
		Mean		4971		43097	327846	712356
10		1		4971		36534	284691	776721
		2		4971		42047	279021	774201
		3		4971		35904	252876	758451
		Mean		4971		38162	272196	769791
3.2		1		4971		37353	357456	798141
		2		4971		35684	313986	1134876
		3		4971		41543	299181	830271
		Mean		4971		38193	323541	921096
Control		1		4971		40598	282801	848541
		2		4971		36440	252561	833736
		3		4971		36219	301701	813576
		4		4971		36093	218226	992496
		5		4971		36944	255711	876891
		6		4971		40913	309261	816726
		Mean		4971		37868	270044	863661

Table 3:     Evaluation after 72 h

            

Nominal test item concentration	Replicate	Yield		Inhibition of yield	Growth rate		Rate-related inhibition
[µg/L]	No.			[%]	[d^-1]		[%]
1000	1		14112	98.36		0.448	73.91
	2		19404	97.74		0.530	69.16
	3		5544	99.35		0.250	85.47
	Mean		13020	98.48		0.409	76.18
320	1		290115	66.21		1.361	20.79
	2		226170	73.66		1.280	25.52
	3		296730	65.44		1.369	20.36
	Mean		271005	68.44		1.337	22.22
100	1		612360	28.69		1.607	6.47
	2		788445	8.18		1.691	1.60
	3		776790	9.54		1.686	1.89
	Mean		725865	15.47		1.661	3.32
32	1		750645	12.58		1.675	2.55
	2		674100	21.50		1.639	4.62
	3		697410	18.78		1.650	3.96
	Mean		707385	17.62		1.655	3.71
10	1		771750	10.12		1.684	2.01
	2		769230	10.42		1.683	2.07
	3		753480	12.25		1.676	2.47
	Mean		764820	10.93		1.681	2.18
3.2	1		793170	7.63		1.693	1.48
	2		1129905	-31.58		1.810	-5.34
	3		825300	3.89		1.706	0.72
	Mean		916125	-6.69		1.736	-1.05
Control	1		843570			1.713
	2		828765			1.707
	3		808605			1.699
	4		987525			1.766
	5		871920			1.724
	6		811755			1.701
	Mean		858690			1.718

Negative values = increase of growth

Table 4:     Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 to 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	VC %	Mean VC %
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	2.100	1.941	1.099	1.713	0.538	31.41	28.19
	2	1.992	1.936	1.194	1.707	0.445	26.08
	3	1.986	2.120	0.992	1.699	0.616	36.26
	4	1.982	1.799	1.515	1.766	0.236	13.35
	5	2.006	1.935	1.232	1.724	0.428	24.79
	6	2.108	2.023	0.971	1.701	0.633	37.23
				Mean	1.718
				SD ±	0.02
				VC %	1.45

SD = Standard deviation        VC %= percent coefficient of variation

Table 5:     Recovery of Alga Cells

Nominal test item concentration                   [µg/L]	Incubation time                    [d]	Cell density [cells/mL]		Growth rate after incubation time [d^-1]
		Initial^#	Final
1000	3	1799	38865	1.02
			21099	0.82
			18075	0.77
320		13799	103377	0.67
			236937	0.95
			191577	0.88
Control		43183	687261	0.92
			708681	0.93

^#) calculated from the cell density of the definitive test after 72 h

Validity criteria fulfilled:

yes

Conclusions:

In this study N-(hydrogenated tallow alkyl)trimethylenediamines was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal concentrations): The EC50-values with 95 % confidence intervals for inhibition of yield (EyC50) and specific growth rate (ErC50) after 72 h were 223 (161 – 307) and 596 (520 – 667) µg/L, respectively.
The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 3 days of growth under test conditions.
All effect levels are given based on nominal concentrations of N-(hydrogenated tallow alkyl)trimethylenediamines.

Executive summary:

The toxicity of N-(hydrogenated tallow alkyl)trimethylenediamines (CAS No. 68603-64-5) (Batch no.: S000905) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from January 15 to 21, 2008. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

An individual test design was applied. Natural river water was used as dilution medium.

The study was conducted under static conditions with an initial cell density of nominal 2–5 x 10³cells/mL. 6 concentrations were tested in a geometrical series with a dilution factor of v10, nominal:3.2 - 10 - 32 - 100 - 320 - 1000 µg/L. Three replicates were tested for the test item concentrations and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of N-(hydrogenated tallow alkyl)trimethylenediamines were analysed at all concentration levels at test start and test end via LC-MS/MS analysis. The measured concentrations at test start were in the range of 92 - 135 % of the nominal values. At the end of the test N-(hydrogenated tallow alkyl)trimethylenediamines gave recoveries of 24 - 28 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, low DOC and suspended matter content and also because the river water was frozen before use to minimize the microbial activity. This is supported by the monitoring results from the chronic daphnia tests with this test item. In this test only a limited decrease of the test substance was observed over 48 hours. This decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. The larger decrease of the test item during the algae test is therefore most likely caused by sorption of the test substance to algae. These points support the conclusion that the test organisms were fully exposed to the test substance during the test.

Therefore all effect values are given based on nominal concentrations of the test item.

N-(hydrogenated tallow alkyl)trimethylenediamines was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal concentrations): The EC₁₀-values with 95 % confidence intervals for inhibition of yield (E_yC₅₀) and specific growth rate (E_rC₅₀) after 72 h were 44.6 ( 3.20 – 83.1) and 193 (158 – 235) µg/L, respectively. The EC₅₀-values with 95 % confidence intervals for inhibition of yield (E_yC₅₀) and specific growth rate (E_rC₅₀) after 72 h were 223 (161 – 307) and 596 (520 – 667) µg/L, respectively.

After 72 h algae were transferred from the nominal concentrations 320 – 1000 µg/L and control to fresh untreated medium and allowed to grow for further 3 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 1000 µg/L (highest test concentration).

Description of key information

The 72-EC50 and EC10 of (Z)-N-9-octadecenylpropane-1,3-diamine to algae are 507 µg/L and 188 µg/L, respectively in an OECD TG 201 test under GLP. 
These data have been used as read-across for Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates. They are then converted to the corresponding value of the salt, resulting to an EC50 of about 690 µg/L and an EC10 of about 256 µg/L (MW of C18-unsaturated diamine = 316, MW of diamine diacetate = 430). 

Key value for chemical safety assessment

EC50 for freshwater algae:

690 µg/L

EC10 or NOEC for freshwater algae:

256 µg/L

Additional information

One toxicity test with algae has been performed on (Z)-N-9-octadecenylpropane-1,3-diamine.

The 72 h ErC10 is 188 µg/L and the 72 h ErC50 = 507 µg/L based on the test substance (92.3% diamine and 7.7% primary alkyl amine). The effects are expressed as nominal values because the tests were performed with river water as they are intended to be used in an evaluation of the environmental risks based on the Bulk approach.

Thus value is used as a read-across for the N-C16-18-alkyl-(evennumbered) C18 unsaturated) propane-1,3-diamine. 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 toxicity data were obtained with Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine: a 72h ErC10 of 193 µg/L and a 72h ErC50 of 596 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 toxicity data obtained for algae can be used as read-across for Amines, N-(C16 -18 and C18 -unsatd. alkyl)trimethylenedi-, diacetates (see the read-across justification document).