Amines, di-C16-18-alkyl

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:

2010-07-26 to 2010-09-09

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 loadings all loading levels and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates of the second lowest and the second highest test loading (nominal: 0.32 and 3.2 mg/L) were prepared without algae for analysis after 72 h. Analysis after 72 h of these loadings were carried out. Sorption to the walls of the glass container was checked out of one test replicate (prepared with algae) of a loading level close to the EC50-value (the second highest test loading of 3.2 mg/L was analysed).
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions (3.2 - 10.0 mg/L were be weighed out) were prepared with test medium and shaken with 20 rpm for 24 hours (WSF) at room temperature. After a separation phase of 24 hours the WSF was taken from the homogeneous liquid phase.
- Eluate: Natural river water
- Differential loading: 0.1 - 0.32 - 1.0 - 3.2 and 10 mg/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): 5 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, 0.1 - 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from initial measured test item concentrations of 10.0 – 29.0 µg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 – 6 days under test conditions. The test item effect was observed to be reversible at these loadings. Therefore, there is potential for recovery following exposure up to 29.0 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: 23 °C

pH:

Initial measured
test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
29.0 8.01 8.36
10.0 8.00 9.28
3.00 7.98 9.30
(0.949) 7.99 9.31
(0.300) 7.99 9.58
Control 7.98 9.42

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: 9846 cells/mL
- Control end cells density: Mean 2636591 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 Leine was used. This river is located near D-31171 Nordstemmen, Marienbergstraße, Germany (52° 10’ 14.98‘’, 9° 46’ 7.64’’).
River Leine
Location D-31171 Nordstemmen, Marienbergstrasse
Sampling Date 2009-12-15
Weather on Day of Sampling cloudy, ca. - 1 °C
Colour Yellowish, clear
pH 7.97
Conductivity [µS/cm] 386
DOC [mg C/L] 3.9
TOC [mg C/L] 3.9
Ammonium-N [mg N/L] 0.042
Nitrate-N [mg N/L] 2.62
o-Phosphate-P [mg P/L] 0.062
Total Phosphate [mg P/L] 0.053
Suspended Matter [mg/L] 16.2
Total Hardness [mg CO3/L] 154


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)
The water accommodated fractions was prepared by shaking with 20 rpm for 24 hours at room temperature. After a separation phase of 1 hour the WAF was taken from the homogeneous liquid phase and used for the range finding test .

Nominal Loading of the Water Accommodated Fraction (WAF)
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
10.0 97 100
1.00 3 17
0.10 0 - 1
0.01 0 3

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

8.79 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (8.57-9.02)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

17.7 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (17.4-18.1)

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: 0.74 (CI 0.71 - 0.78) mg/L
Yield Inhibition: 0.36 (CI < 0.35 - 0.36) mg/L

Reported statistics and error estimates:

EL10-, EL20- and EL50-values after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals of EL10-, EL20- and EL50-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

EL₁₀-_, EL₂₀-and EL₅₀- values and 95 % Confidence Intervals of
Amines, bis (hydrogenated tallow alkyl)(CAS No. 61789-79-5) (0-72 h)

                                based on initial measured test item concentrations [µg/L]

                               

	Rate-related inhibition
ErL10	8.79 (8.57 – 9.02)
ErL20	11.6 (11.3 – 11.9)
ErL50	17.7 (17.4 – 18.1)
	Inhibition of yield
EyL10	4.38 (3.17 – 6.14)
EyL20	5.89 (4.47 – 7.18)
EyL50	9.44 (8.65 – 10.0)

Cell Densities

	Initial measured test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 h		24 h	48 h	72 h
29.0		1		9846		1410	d.l.	3040
		2		9846		1964	d.l.	3800
		3		9846		7054	1950	4560
		Mean		9846		3476	n.a.	3800
10.0		1		9846		48842	218418	1210141
		2		9846		45237	192338	1168828
		3		9846		51770	244075	1242806
		Mean		9846		48616	218277	1207258
3.00		1		9846		52019	538370	2397126
		2		9846		63177	592561	2625546
		3		9846		72314	518442	2544894
		Mean		9846		62503	549791	2522522
(0.949)		1		9846		69301	577662	2405586
		2		9846		93873	671568	2718982
		3		9846		68629	626307	2662018
		Mean		9846		77268	625179	2595529
(0.300)		1		9846		74222	585182	2545176
		2		9846		91513	722328	2701592
		3		9846		83222	748037	2558430
		Mean		9846		82986	685182	2601733
Control		1		9846		72361	669124	2675836
		2		9846		57960	598577	2567454
		3		9846		54205	592561	2087490
		4		9846		73023	724631	2919578
		5		9846		59093	649713	2798506
		6		9846		84820	701930	2770682
		Mean		9846		66910	656089	2636591

Evaluation after 72 h

             

Initial measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
29.0	1		-0.392	100		-6806	100
	2		-0.317	100		-6046	100
	3		-0.257	100		-5286	100
	Mean		-0.322	100		-6046	100
10.0	1		1.604	13.8		1200295	54.3
	2		1.592	14.5		1158982	55.9
	3		1.613	13.4		1232960	53.1
	Mean		1.603	13.9		1197412	54.4
3.00	1		1.832	1.60		2387280	9.12
	2		1.862	-0.03		2615700	0.42
	3		1.852	0.53		2535048	3.49
	Mean		1.848	0.70		2512676	4.34
(0.949)	1		1.833	1.54		2395740	8.79
	2		1.874	-0.65		2709136	-3.14
	3		1.867	-0.27		2652172	-0.97
	Mean		1.858	0.21		2585683	1.56
(0.300)	1		1.852	0.53		2535330	3.48
	2		1.872	-0.54		2691746	-2.47
	3		1.853	0.44		2548584	2.98
	Mean		1.859	0.14		2591887	1.33
Control	1		1.868			2665990
	2		1.855			2557608
	3		1.786			2077644
	4		1.897			2909732
	5		1.883			2788660
	6		1.880			2760836
	Mean		1.861			2626745

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	1.995	2.224	1.386	1.868	0.4332	23.2	25.3
	2	1.773	2.335	1.456	1.855	0.4450	24.0
	3	1.706	2.392	1.259	1.786	0.5704	31.9
	4	2.004	2.295	1.394	1.897	0.4600	24.2
	5	1.792	2.397	1.460	1.883	0.4752	25.2
	6	2.153	2.113	1.373	1.880	0.4395	23.4
				Mean	1.861
				SD ±	0.04
				VC %	2.14

Validity criteria fulfilled:

yes

Conclusions:

Amines, bis (hydrogenated tallow alkyl) (CAS No. 61789-79-5) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (initial measured test item concentrations): The EL10-values with 95 % confidence intervals for inhibition of specific growth rate (ErL10) and yield (EyL10) after 72 h were 8.79 (8.57 – 9.02) and 4.38 (3.17 – 6.14) µg/L, respectively. The EL50-values with 95 % confidence intervals for inhibition of specific growth rate (ErL50) and yield (EyL50) after 72 h were 17.7 (17.4 – 18.1) and 9.44 (8.65 – 10.0) µg/L, respectively.
The test item effect was observed to be reversible at 10.0 – 29.0 µg/L. Therefore, there is potential for recovery following exposure up to 29.0 µg/L (highest test item concentration).
All effect levels are given based on initial measured concentrations of Amines, bis (hydrogenated tallow alkyl) (CAS No. 61789-79-5).

Executive summary:

The toxicity of Amines, bis (hydrogenated tallow alkyl)(CAS No. 61789-79-5) (Batch no.: S001020)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 July 26 to September 09, 2010 with the definitive exposure phase from July 28 to 31, 2010. The aim of the study was the determination of EL₁₀- , EL₂₀ - and EL₅₀ - values of growth rate and yield over a period of 72 h.

Amines, bis (hydrogenated tallow alkyl)(CAS No. 61789-79-5) is poorly soluble in water and also has a strong tendency to adsorb to surfaces such as suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many sorbing substances in general but long chain dialkyl amines in particular rank among the most difficult items 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 bioavailability and exposure assessment for substances like the dialkyl amines 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 these substances.

The aquatic ecotoxicity tests with dialkyl amines 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 a standard laboratory toxicity study valid, it is of particular importance that – besides information on test item, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test item 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. Prior to the preparation of the WSF solutions the river water was filtered using a 0.45 µm filter.

The study was conducted under static conditions with an initial cell density of approximately 1 x     10⁴ cells/mL. Two water soluble fractions (WSF) and three further dilution levels, with nominal loadings of 0.10 - 0.32 - 1.0 - 3.2 and 10 mg/L were tested in a geometrical series with a dilution factor of √10, corresponding to initial measured concentrations of 3.00 – 10.0 – 29.0 µg/L. The two lowest concentrations were extrapolated from the third lowest concentration level considering a dilution factor of √10 to be 0.300 and 0.949 µg/L. The range of measured and extrapolated test concentrations, respectively was 0.300 – 0.949 – 3.00 – 10.0 – 29 µg/L. Three replicates were tested for the test item loadings and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Amines, bis (hydrogenated tallow alkyl)(CAS No. 61789-79-5) were analysed at all loading levels (prepared without algae) and the control at test start via LC-MS analysis. At the end of the test Amines, bis (hydrogenated tallow alkyl)was analysed from the control and the loading levels 0.32 and 3.20 mg/L. At the level 0.32 mg/L the measured concentration was below LOQ (as being at the test start) whereas the measured concentration at the nominal level 3.2 mg/L was 14 µg/L and indicated stability throughout exposure. All effect values are given based on the measured initial test item concentrations.

Amines, bis (hydrogenated tallow alkyl) (CAS No. 61789-79-5) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (initial measured concentrations): The EL₁₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rL₁₀) and yield (E_yL₁₀) after 72 h were 8.79 (8.57 – 9.02) and 4.38 (3.17 – 6.14) µg/L, respectively. The EL₅₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rL₅₀) and yield (E_yL₅₀) after 72 h were 17.7 (17.4 – 18.1) and 9.44 (8.65 – 10.0) µg/L, respectively.

After 72 h algae were transferred from the initial measured concentrations of 10.0 – 29.0 µg/L and the control to fresh untreated medium and allowed to grow for further 3 – 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 29.0 µg/L (highest test concentration).

Description of key information

The test substance is poorly soluble in water and the test solutions were prepared by dilution of a water soluble fraction. The toxicity to aquatic algae resulted in a 72 hour EL10 and EL50 based on growth of resp. 8.79 and 17.7 µg a.i./L.

Key value for chemical safety assessment

EC50 for freshwater algae:

17.7 µg/L

EC10 or NOEC for freshwater algae:

8.79 µg/L

Additional information

The test substance is poorly soluble in water and the test solutions were prepared by dilution of a water accommodated fraction (WAF). The toxicity to aquatic algae resulted in a 72 hour EL10 and EL50 based on growth of resp. 8.79 and 17.7 µg a.i./L. The test is performed in river water but for the preparation of the WAF the river water was filtered using a 0.45 µg/L filter to remove any undissolved particles.