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

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:

2009-12-14 to 2009-12-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 (32 and 320 µg/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 were carried out. 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 highest test concentration of 320 µg/L was analysed).
- Sample storage conditions before analysis: All samples were stored at 6 ± 2°C until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared with test medium. Dispersion treatment was 15 min ultrasound at room temperature, equilibration phase for 1 h before application.
- Eluate: Natural river water
- Differential loading: 10 - 32 - 100 - 320 - 1000 µg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 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, 0.2 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from test concentrations 320 – 1000 µg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 4 – 6 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 1000 µg/L (highest test concentration).
For details please refer to the table under information on materials and methods

Hardness:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 21.5 °C

pH:

Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
1000 7.92 7.97
320 7.93 8.02
100 7.94 8.09
32 7.95 8.12
10 7.95 8.15
Control 7.96 8.12

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- 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: 9787 cells/mL
- Control end cells density: Mean 3464189 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 Hüpeder Bach
Location D-30982 Pattensen, Am Pfingstanger
Sampling Date 2009-10-07
Weather on Day of Sampling dry, few clouds, ca. 22 °C
Colour Yellowish, clear
pH 7.79
Conductivity [µS/cm] 531
DOC [mg C/L] 3.8
TOC [mg C/L] 3.7
Ammonium-N [mg N/L] < 0.015
Nitrate-N [mg N/L] 3.06
o-Phosphate-P [mg P/L] 0.116
Total Phosphate [mg P/L] 0.140
Suspended Matter [mg/L] 17.6
Total Hardness [mg CO3/L] 170

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
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition[%] Yield Inhibition [%]
10.0 100 100
1.00 100 100
0.10 0 - 1
0.01 - 3 - 16
0.001 - 6 - 32
0.0001 - 6 - 37

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

184

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

97.8% Active ingredient

Basis for effect:

growth rate

Remarks on result:

other: (137-229)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

420 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

97.8% active ingredient

Basis for effect:

growth rate

Remarks on result:

other: (376 - 510)

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: 0.82 (CI 0.78 - 0.85) mg/L
Yield Inhibition: 0.37 (CI 0.36 - 0.40) 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 of EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

EC₁₀-_, EC₂₀-and EC₅₀- values and 95 % Confidence Intervals of
N-(3-(Tridecyloxy)propyl)-1,3-propane diamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine

                   (CAS No. 102047-27-8 and 68479-04-9) (0-72 h)

                                based on nominal test item concentrations [µg/L]

                               

	Rate-related inhibition
ErC10	184 (137 – 229)
ErC20	252 (210 – 277)
ErC50	420 (376 – 510)
	Inhibition of yield
EyC10	83.9 ( 10 – 122)
EyC20	118 (92.7 – 153)
EyC50	188 (153 – 225)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 h		24 h	48 h	72 h
1000		1		9787		d.l.	d.l.	3420
		2		9787		d.l.	d.l.	3040
		3		9787		d.l.	d.l.	3800
		Mean		9787		d.l.	d.l.	3420
320		1		9787		18177	79328	406049
		2		9787		29344	74525	572480
		3		9787		25203	107190	646289
		Mean		9787		24241	87014	541606
100		1		9787		48064	451361	2500557
		2		9787		51283	537747	3114151
		3		9787		60185	612830	3326403
		Mean		9787		53177	533979	2980370
32		1		9787		53976	717936	3214543
		2		9787		41531	574675	3004763
		3		9787		61995	792187	3531558
		Mean		9787		52501	694933	3250288
10		1		9787		62201	718505	3443175
		2		9787		58987	741789	3441464
		3		9787		64650	727355	3297023
		Mean		9787		61946	729216	3393887
Control		1		9787		60655	708409	3457402
		2		9787		59635	675354	3535544
		3		9787		55081	598692	3467601
		4		9787		59438	678489	3431495
		5		9787		60326	644635	3361221
		6		9787		74238	729869	3531868
		Mean		9787		61562	672575	3464189

Evaluation after 72 h

             

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1000	1		-0.350	100		-6367	100
	2		-0.390	100		-6747	100
	3		-0.315	100		-5987	100
	Mean		-0.352	100		-6367	100
320	1		1.24	36.5		396262	88.5
	2		1.36	30.7		562693	83.7
	3		1.40	28.6		636502	81.6
	Mean		1.33	31.9		531819	84.6
100	1		1.85	5.55		2490770	27.9
	2		1.92	1.81		3104364	10.1
	3		1.94	0.690		3316616	3.99
	Mean		1.90	2.68		2970583	14.0
32	1		1.93	1.27		3204756	7.23
	2		1.91	2.42		2994976	13.3
	3		1.96	-0.330		3521771	-1.95
	Mean		1.93	1.12		3240501	6.19
10	1		1.95	0.100		3433388	0.610
	2		1.95	0.110		3431677	0.660
	3		1.94	0.840		3287236	4.84
	Mean		1.95	0.350		3384100	2.04
Control	1		1.96			3447615
	2		1.96			3525757
	3		1.96			3457814
	4		1.95			3421708
	5		1.95			3351434
	6		1.96			3522081
	Mean		1.96			3454402

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	1.82	2.46	1.59	1.96	0.451	23.1	20.4
	2	1.81	2.43	1.66	1.96	0.409	20.8
	3	1.73	2.39	1.76	1.96	0.372	19.0
	4	1.80	2.44	1.62	1.95	0.427	21.9
	5	1.82	2.37	1.65	1.95	0.375	19.3
	6	2.03	2.29	1.58	1.96	0.359	18.3
				Mean	1.96
				SD ±	0.01
				VC %	0.32

Validity criteria fulfilled:

yes

Conclusions:

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

Executive summary:

The toxicity of N-(3-(Tridecyloxy)propyl)-1,3-propane diamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine (CAS No. 102047-27-8 and 68479-04-9) (Batch no.: S001086) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from December 14 to 23, 2009 with the definitive exposure phase from December 14 to 17, 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.

N-(3-(Tridecyloxy)propyl)-1,3-propane diamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine(CAS no. 102047-27-8 and 68479-04-9) have a low solubility in water and also 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 etherdiamines 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 etherdiamines as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavaibility that occurs in the environment with cationic surface-active substances.

The aquatic ecotoxicity tests with etherdiamines 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 1 x     10⁴ cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of √10, 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 N-(3-(Tridecyloxy)propyl)-1,3-propane diamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine were analysed at the concentration levels 32 and 320 µg/L (prepared without algae) and the control at test start and test end via UPLC-MS/MS analysis. The measured concentrations at test start were in the range of 86 – 92 % of the nominal values. At the end of the test N-(3-(Tridecyloxy)propyl)-1,3-propane diamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine gave recoveries of LOQ –

12 % 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 is attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. 8.93 µg/L test item could be recovered from the glassware (9 %). 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.

N-(3-(Tridecyloxy)propyl)-1,3-propane diamine, monoacetate and N-(3-(Tridecyloxy)propyl)-1,3-propane diamine (CAS No. 102047-27-8 and 68479-04-9) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata 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 184 (132 – 229) and 83.9 ( 10 – 122) µg/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 420 (376 – 510) and 188 (153 – 225) µg/L, respectively.

After 72 h algae were transferred from the nominal concentrations of 320 – 1000 µg/L and the control to fresh untreated medium and allowed to grow for further 4 – 6 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

One algae test has been performed, the EC50 for inhibition of specific growth rate was determined to be 420 µg/L and the EC10 for inhibition of specific growt rate was determined to 184 µg/L. The effects are expressed as nominal values because the tests were performed with river water as they were intended to be used in an evaluation of the environmental risks based on the Bulk approach.

Key value for chemical safety assessment

EC50 for freshwater algae:

420 µg/L

EC10 or NOEC for freshwater algae:

184 µg/L