Amines, N-(3- aminopropyl)-N’-[3-(C18 and C18-unsatd. alkyl amino)propyl]trimethylenedi and amines, N-(3-aminopropyl)-N’-(C18 and C18-unsatd. alkyl)trimethylenedi-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-05-26 to 2009-07-03

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: From the series of concentrations the second lowest and the second highest test concentration (0.32 and 3.20 mg/L) and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates for the test item analysis after 0 and 72 h were prepared without algae. Analysis after 72 h of these concentrations was carried out to demonstrate stability of the test item. Sorption to the walls of the glass container was checked out of one test replicate (prepared with algae) of a concentration level close to the EC50-value (the second lowest test concentration of 0.32 mg/L).
- Sample storage conditions before analysis: All samples were stored at room temperature, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (10 mg/L) was treated with ultrasound for 15 min at 40 °C. An equilibration phase for 1 h before application was carried out.
- Eluate: Natrual river water
- Differential loading: 0.10 - 0.32 - 1.00 - 3.20 - 10-0 mg/L
- Controls: Six replicates wihtout test item were tested under the same test conditions as the test replicates.

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, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- 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 of exposure, 0.5 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from test concentrations 1.0 – 10.0 mg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 – 7 days 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 10.0 mg/L.
Recovery of Alga Cells

Nominal test item concentration [mg/L] Incubation time [d] Cell density [cells/mL] Growth rate after incubation time [d-1]
Initial# Final
10.0 7 3 1881 0.91
3952 1.01
8781 1.13
3.20 18 40558 1.10
4795 0.79
12226 0.93
1.00 3 10956 415223 1.21
299421 1.10
308339 1.11
Control 51801 1599371 1.14
1576467 1.14

Hardness:

Not measured

Test temperature:

Mean: 23 °C, Min: 22 °C, Max: 24 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
10.0 8.05 8.10
3.20 8.04 8.01
1.00 8.02 8.04
0.32 8.03 8.10
0.10 8.02 8.09
Control 7.91 7.87

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- 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 appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 4996 cells/mL
- Control end cells density: Mean 1036016 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



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water of the river Innerste was used. This river is located in
Sarstedt / Ruthe. Water parameters are given in Table 1. Additionally 50 % of the components concentrations of the dilution water (total application volume 6.5 mL/L) acc. to the guideline were added to enable a sufficient growth of algae
Characterisation Data of Natural River Water
River Innerste
Location D-31157 Sarstedt / Ruthe, Ruther Straße, Innerste
Sampling Date 2009-02-12
Weather on Day of Sampling dry, sunny, ca. 1 °C
Colour Slightly, turbid, yellowish
pH 8.08
Conductivity [µS/cm] 887
DOC [mg C/L] 2.8
TOC [mg C/L] 2.9
Ammonium-N [mg N/L] 0.163
Nitrate-N [mg N/L] 6.10
o-Phosphate-P [mg P/L] 0.103
Total Phosphate [mg P/L] 0.152
Suspended Matter [mg/L] 24
Total Hardness [mg CO3/L] 338

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: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.

TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 h)

Nominal Rate Yield
Test Item related
Concentration Inhibition Inhibition
[mg/L] [%] [%]
10.0 100 100
1.00 15 59
0.10 0 -1
0.01 -1 -3

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.518 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.348 - 0.594

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1.02 - 1.14

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.356 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: (0.314-0.398)

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: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.320 (CI 0.294 - 0.358) 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 standard procedures.

EC₁₀-_, EC₂₀-and EC₅₀- values and 95 % Confidence Intervals of
Oleyl dipropylene triamine (CAS No. 28872-01-7) (0-72 h)

                                based on nominal test item concentrations [mg/L]

                               

	Rate-related inhibition
ErC10	0.518 (0.348 – 0.594)
ErC20	0.728 (0.674 – 0.776)
ErC50	1.09 (1.02 – 1.14)
	Inhibition of yield
EyC10	0.118 ( 0.10 – 0.155)
EyC20	0.181 (0.148 – 0.217)
EyC50	0.356 (0.314 – 0.398)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
10		1		4996		0	0	0
		2		4996		37	0	0
		3		4996		365	0	99
		Mean		4996		134	0	33
3.20		1		4996		5306	0	0
		2		4996		6011	1011	0
		3		4996		7026	1170	553
		Mean		4996		6114	727	184
1.00		1		4996		20525	69569	117019
		2		4996		17899	65256	96756
		3		4996		22581	69439	114898
		Mean		4996		20335	68088	109558
0.32		1		4996		28617	159856	563210
		2		4996		31168	143130	544471
		3		4996		30859	155446	621879
		Mean		4996		30215	152811	576520
0.10		1		4996		27758	231311	834453
		2		4996		24487	196410	1004975
		3		4996		30835	201006	1030083
		Mean		4996		27693	209576	956504
0 (Control)		1		4996		26614	210558	1054429
		2		4996		28257	211573	982343
		3		4996		31891	208980	1017811
		4		4996		30401	222007	1086460
		5		4996		28593	213080	1028917
		6		4996		23782	184790	1046137
		Mean		4996		28256	208498	1036016

Evaluation after 72 h

             

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
10	1		n.a.	100.00		-4996	100.00
	2		n.a.	100.00		-4996	100.00
	3		-1.307	100.00		-4897	100.00
	Mean		n.a.	100.00		-4963	100.00
3.20	1		n.a.	100.00		-4996	100.00
	2		n.a.	100.00		-4996	100.00
	3		-0.734	100.00		-4443	100.00
	Mean		n.a.	100.00		-4812	100.00
1.00	1		1.051	40.88		112023	89.13
	2		0.988	44.44		91760	91.10
	3		1.045	41.22		109902	89.34
	Mean		1.028	42.18		104562	89.86
0.32	1		1.575	11.42		558214	45.86
	2		1.564	12.05		539475	47.68
	3		1.608	9.56		616883	40.17
	Mean		1.582	11.01		571524	44.57
0.10	1		1.706	4.05		829457	19.55
	2		1.768	0.56		999979	3.01
	3		1.776	0.10		1025087	0.58
	Mean		1.750	1.57		951508	7.71
0 (Control)	1		1.784			1049433
	2		1.760			977347
	3		1.772			1012815
	4		1.794			1081464
	5		1.776			1023921
	6		1.781			1041141
	Mean		1.778			1031020

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	1.673	2.068	1.611	1.784	0.2481	13.9	12.4
	2	1.733	2.013	1.535	1.760	0.2401	13.6
	3	1.854	1.880	1.583	1.772	0.1643	9.27
	4	1.806	1.988	1.588	1.794	0.2004	11.2
	5	1.745	2.009	1.575	1.776	0.2186	12.3
	6	1.560	2.050	1.734	1.781	0.2485	13.9
				Mean	1.778
				SD ±	0.01
				VC %	0.64

Validity criteria fulfilled:

yes

Conclusions:

Oleyl dipropylene triamine (CAS No. 28872-01-7) 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 specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.518 (0.348 – 0.594) and 0.118 (< 0.10 – 0.155) mg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 1.09 (1.02 – 1.14) and 0.356 (0.314 – 0.398) mg/L, respectively.
The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 3 – 7 days of growth under test conditions.
The concentrations of Oleyl dipropylene triamine were analysed at the concentration levels 0.32 and 3.20 mg/L (prepared without algae) and the control at test start via LC-MS/MS analysis. The measured concentrations at test start were in the range of 49 – 78 % of the nominal values. At the end of the test Oleyl dipropylene triamine was analysed at concentration levels 0.32 and 3.20 mg/L (prepared without algae) and 3.2 mg/L (prepared with algae). The measured concentrations were in the range of LOQ to 6% of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No adsorption ( LOQ) of the test item to glassware was observed. Therefore all effect values are given based on nominal concentrations of Oleyl dipropylene triamine (CAS No. 28872-01-7).

Executive summary:

The toxicity of Oleyl dipropylene triamine (CAS No. 28872-01-7) (Batch no.: S001256)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from May 26 to July 03, 2009with the definitive exposure phase being conducted between June 23 to 26, 2009. The aim of the study was the determination of EC₁₀ - , EC₂₀- and EC₅₀ - values of growth rate and yield over a period of 72 h.

Oleyl dipropylene triamine(CAS no. 28872-01-7) is practically insoluble 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 polyamines in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavaibility and exposure assessment for cationic surface-active substances like the polyamines 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 cationic surface-active substances.

The aquatic ecotoxicity tests with polyamines were therefore performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulkapproach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing 'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PEC_aquatic,bulkis compared with the PNEC_aquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

         

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

         

For ecotoxicity tests performed using the 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 negligible. The results of these bulk approach tests are therefore much easier and more realistic, and if compared to PEC_bulk clearly provide a more appropriate assessment of risks for the environment.

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 approximately 5 x  10³cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of √10, nominal: 0.10 - 0.32 - 1.00 - 3.20 - 10.0 mg/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 Oleyl dipropylene triamine were analysed at the concentration levels 0.32 and 3.20 mg/L (prepared without algae) and the control at test start via LC-MS/MS analysis. The measured concentrations at test start were in the range of 49 – 78 % of the nominal values. At the end of the test Oleyl dipropylene triamine was analysed at concentration levels 0.32 and 3.20 mg/L (prepared without algae) and 3.2 mg/L (prepared with algae). The measured concentrations were in the range of LOQ to 6% of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No adsorption ( LOQ) of the test item to glassware was observed. Therefore all effect values are given based on nominal concentrations of the test item. For details of the analytical results please refer to part 9.

Oleyldipropylene triamine (CAS No. 28872-01-7) 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 specific growth rate (E_rC₁₀) and yield (E_yC₁₀) after 72 h were 0.518 (0.348 – 0.594) and 0.118 ( 0.10 – 0.155) mg/L, respectively. The EC₅₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rC₅₀) and yield (E_yC₅₀) after 72 h were 1.09 (1.02 – 1.14) and 0.356 (0.314 – 0.398) mg/L, respectively.

After 72 h algae were transferred from the nominal concentration of 1.00 – 10.0 mg/L and the control to fresh untreated medium and allowed to grow for further 3 – 7 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 10.0 mg/L (highest test concentration)

Description of key information

A valid 72 hour algae test according to OECD 201 has been performed using natural river water as test water. An EC50 for growth of 1090 µg/L and an EC10 for growth of 518 µg/L were observed.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.09 mg/L

EC10 or NOEC for freshwater algae:

518 µg/L

Additional information

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 evaluation of the environmental risks in the EU applying the bulk approach considering the realistic worst case (low) concentration of suspended matter and DOC in the river water used.