Amines, N-(C18-unsatd. alkyl)trimethylenedi-, ethoxylated

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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:

2009-11-03 to 2009-11-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, all validity criteria fulfilled, identification complete, Including chemical analyses

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The short term toxicity test result as observed for the tallow based product is read across to the Oleyl based product. The products differ only in their alkylchain distribution. Both tallow and Oleyl are natural fats for which the composition slightly changes over time due to changes in the environmental considitions.

The tallow alkyl fat which is used for the production of CAS no 1268344-02-0 are for 99 % straight chain alkyl fats

Commercial tallow alkyl fats have the following distribution:

C12 about 2 %
C14 about 4 %
C16 about 31 %
C18 about 65 %,
where 40 –50 % are unsaturated.

The oleyl fate which is used for the production of CAS no 1290049-56-7 are also straight chain fates

Commercial Oleylfats have the following distribution:

C12 1 %
C14 2 - 4 %
C16 and C16’ 12 - 14 %
C18, C18’ and C18’’ > 80 % (C18: 8 %, C18’cis: 46 %, C18’trans: 21 %, C18’’: 5 %)
C20 1 %
(a single prime indicates one double bond, a double prime indicates two double bonds):

When the water solubility of cationic surfactants based on tallow are compared to oleyl based cationic surfactants it is consistently the tallow product which has a sligthly lower water solubility and due to that also a slightly lower bioavailability. This lower bioavailability regularly results is slight differences in biodegradation rate and ecotoxicity. The substances are however difficult to test substances and the variation in the test results is especially in ecotoxicity testing frequently larger than the difference in bioavailability. It is therefore considered justified to use the tallow product test results for read across for the oleyl based product.

Reason / purpose for cross-reference:

read-across source

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 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 highest test concentration of 320 µg/L was analysed).
- Sample storage conditions before analysis:All samples were stored at room temperature 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. After agitation, an equilibration phase of 1 h before application was carried out.
- 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): 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, 0.2 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from test concentrations 320 – 1000 µg/L and the control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 – 12 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: 23 °C, Min: 22 °C, Max: 24 °C

pH:

Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
1000 8.20 7.99
320 8.18 7.99
100 8.18 8.02
32 8.17 8.02
10 8.15 8.01
Control 8.16 8.02

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: 9815 cells/mL
- Control end cells density: Mean 1155765 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 6 28
0.01 0 -3

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

256 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (238 - 272)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

86.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (73.0 - 102)

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:

EEC10-, 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
Tris (2-hydroxyethyl) oleyl diaminopropane (CAS No. 90367-27-4) (0-72 h)

                                based on nominal test item concentrations [µg/L]

                               

	Rate-related inhibition
ErC10	86.3 (73.0 – 102)
ErC20	131 (116 – 147)
ErC50	256 (238 – 272)
	Inhibition of yield
EyC10	32.4 (17.5 – 45.6)
EyC20	53.3 (42.9 – 64.5)
EyC50	105 (94.5 – 118)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 h		24 h	48 h	72 h
1000		1		9815		2911	0	3420
		2		9815		2121	0	4180
		3		9815		3038	0	3420
		Mean		9815		2690	0	3673
320		1		9815		11460	18712	47603
		2		9815		10553	24254	58597
		3		9815		11587	24714	77570
		Mean		9815		11200	22560	61257
100		1		9815		18529	93550	630859
		2		9815		16964	94505	611913
		3		9815		22698	155243	641354
		Mean		9815		19397	114433	628042
32		1		9815		19121	133867	994710
		2		9815		20024	116679	978673
		3		9815		29626	164441	1057826
		Mean		9815		22924	138329	1010403
10		1		9815		22642	159929	1212545
		2		9815		25570	162969	1046222
		3		9815		25880	180383	1123231
		Mean		9815		24697	167760	1127333
Control		1		9815		24761	192819	1177638
		2		9815		22519	156305	1085645
		3		9815		32295	155666	1131470
		4		9815		24559	216183	1390572
		5		9815		26101	165409	1144372
		6		9815		27102	159745	1004890
		Mean		9815		26223	174355	1155765

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		-0.351	100		-6395	100
	2		-0.285	100		-5635	100
	3		-0.351	100		-6395	100
	Mean		-0.329	100		-6142	100
320	1		0.526	66.9		37788	96.7
	2		0.596	62.5		48782	95.7
	3		0.689	56.6		67755	94.1
	Mean		0.604	62.0		51442	95.5
100	1		1.39	12.6		621044	45.8
	2		1.38	13.2		602098	47.5
	3		1.39	12.3		631539	44.9
	Mean		1.39	12.7		618227	46.1
32	1		1.54	3.05		984895	14.1
	2		1.53	3.39		968858	15.5
	3		1.56	1.75		1048011	8.55
	Mean		1.55	2.73		1000588	12.7
10	1		1.61	-1.11		1202730	-4.95
	2		1.56	1.99		1036407	9.56
	3		1.58	0.49		1113416	2.84
	Mean		1.58	0.46		1117518	2.48
Control	1		1.60			1167823
	2		1.57			1075830
	3		1.58			1121655
	4		1.65			1380757
	5		1.59			1134557
	6		1.54			995075
	Mean		1.59			1145950

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	0.925	2.05	1.81	1.60	0.593	37.2	34.3
	2	0.830	1.94	1.94	1.57	0.639	40.8
	3	1.19	1.57	1.98	1.58	0.396	25.0
	4	0.917	2.18	1.86	1.65	0.655	39.7
	5	0.978	1.85	1.93	1.59	0.529	33.3
	6	1.02	1.77	1.84	1.54	0.458	29.7
				Mean	1.59
				SD ±	0.04
				VC %	2.27

Validity criteria fulfilled:

yes

Conclusions:

Tris (2-hydroxyethyl) oleyl diaminopropane (CAS No. 90367-27-4) 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 (ErC10) and yield (EyC10) after 72 h were 86.3 (73.0 – 102) and 32.4 (17.5 – 45.6) µg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 256 (238 – 272) and 105 (94.5 – 118) µg/L, respectively.
The test item effect was observed to be reversible at 320 – 1000 µg/L. Therefore, there is potential for recovery following exposure up to 1000 µg/L (highest test concentration).
All effect levels are given based on nominal concentrations of Tris (2-hydroxyethyl) oleyl diaminopropane (CAS No. 90367-27-4).

Executive summary:

The toxicity of Tris (2-hydroxyethyl) oleyldiaminopropane (CAS No. 90367-27-4) (Batch no.: S001023)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 November 03 to 18, 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.

Tris (2-hydroxyethyl) oleyl diaminopropane (CAS no. 90367-27-4) 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 ethoxylated diamines in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavaibility and exposure assessment for cationic surface-active substances like the ethoxylated diamines 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 ethoxylated diamines 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 Tris (2-hydroxyethyl) oleyldiaminopropane were analysed at the concentration levels 32 and 320 µg/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 78 – 83 % of the nominal values. At the end of the test Tris (2-hydroxyethyl) oleyldiaminopropane was analysed at concentration levels 32 and 320 µg/L (prepared without algae) and gave recoveries of LOQ – 8 % 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. Negligible adsorption (4 % of nominal) 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.

Tris(2-hydroxyethyl) oleyl diaminopropane (CAS No. 90367-27-4) 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 86.3 (73.0 – 102) and 32.4 (17.5 – 45.6) 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 256   (238 – 272) and 105 (94.5 – 118) µg/L, respectively.

After 72 h algae were transferred from the nominal concentration of 320 – 1000 µg/L and the control to fresh untreated medium and allowed to grow for further 3 – 12 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 toxicity test with algae has been performed. The 72 h ErC10  was determined to 86.3 µg/L and the 72 h ErC50 to 256 µg/L. 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.

Key value for chemical safety assessment

EC50 for freshwater algae:

256 µg/L

EC10 or NOEC for freshwater algae:

86.3 µg/L