Bis(hydroxyethyl)methyloleylammonium chloride

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

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:

Read-across data is presented from quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808-53-2, EC Number 276-038-9. This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

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:

- Name of test material (as cited in study report): Cocobis(2-hydroxyethyl) methylammonium chloride (CAS No. 70750-47-9)
- Physical state: Paste-like, yellow-brownish
- Active ingredient content: 99.4%
97.9 % Cocobis(2-hydroxyethyl) methylammonium chloride
1.5 % Cocobis(2-hydroxyethyl) amine
- Purity test date: 2009-06-02
- Lot/batch No.: S001700
- Expiration date of the lot/batch: 2019-04-28
- Storage condition of test material: Room temperature, protected from moisture and light, under nitrogen gas.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: From the series of concentrations the second lowest and the second highest test concentration (2.00 and 200 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 200 µg/L was analysed).
- 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: A stock solution of 10 mg/L was freshly prepared. Dispersion treatment was 5 min ultrasound at room temperature and an equilibration phase for 1 h before application.
- Eluate: Natural river water
- Differential loading: 0.640 - 2.00 - 6.40 - 20.0 - 64.0 - 200 - 640 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:

semi-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 200 – 640 µg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 4 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 640 µg/L (highest test concentration).

Hardness:

Not measured

Test temperature:

Mean: 23.5 °C, Min: 23 °C, Max: 24 °C

pH:

Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
640 8.04 8.13
200 8.05 8.41
64.0 8.04 8.92
20.0 8.05 8.96
6.40 8.03 8.94
2.00 8.02 8.94
0.640 7.96 8.93
Control 7.96 8.86

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): 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 3370253 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 79 99
0.10 9 39
0.01 3 15
0.001 3 16
0.0001 3 13

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

121 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: (100 - 140)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

413.5 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: (361 - 467)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

122 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

99.4% purity

Basis for effect:

growth rate

Remarks on result:

other: (101-141)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

416 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

99.4% purity

Basis for effect:

growth rate

Remarks on result:

other: (363 - 470)

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.

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 h		24 h	48 h	72 h
640		1		9787		26923	42819	43195
		2		9787		27887	39327	35552
		3		9787		25847	40149	37893
		Mean		9787		26886	40765	38880
200		1		9787		30763	194187	1034288
		2		9787		38894	218711	1128274
		3		9787		31924	196302	976008
		Mean		9787		33860	203067	1046190
64.0		1		9787		37263	341118	2409034
		2		9787		45582	493224	3090534
		3		9787		39839	409108	2489357
		Mean		9787		40895	414483	2662975
20.0		1		9787		40920	554611	3216320
		2		9787		52242	557755	3357959
		3		9787		42551	549718	3329021
		Mean		9787		45238	554028	3301100
6.40		1		9787		39914	516884	3090252
		2		9787		43162	514548	3255095
		3		9787		53436	582741	3548718
		Mean		9787		45504	538058	3298022
2.00		1		9787		46936	561595	3444655
		2		9787		49135	593311	3671411
		3		9787		43712	499240	3377948
		Mean		9787		46594	551382	3498005
0.640		1		9787		49479	583337	2604361
		2		9787		45187	569566	3559392
		3		9787		47801	536544	3306339
		Mean		9787		47489	563149	3156697
Control		1		9787		45606	531901	3339225
		2		9787		41291	518045	3208875
		3		9787		48844	587596	3425573
		4		9787		44741	591765	3268349
		5		9787		42034	674315	3396776
		6		9787		49742	575103	3582722
		Mean		9787		45376	579788	3370253

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]		[%]
640	1		0.495	74.6		33408	99.0
	2		0.430	77.9		25765	99.2
	3		0.451	76.8		28106	99.2
	Mean		0.459	76.4		29093	99.1
200	1		1.55	20.2		1024501	69.5
	2		1.58	18.7		1118487	66.7
	3		1.53	21.2		966221	71.3
	Mean		1.56	20.1		1036403	69.2
64.0	1		1.84	5.74		2399247	28.6
	2		1.92	1.47		3080747	8.32
	3		1.85	5.18		2479570	26.2
	Mean		1.87	4.13		2653188	21.0
20.0	1		1.93	0.790		3206533	4.58
	2		1.95	0.050		3348172	0.370
	3		1.94	0.200		3319234	1.23
	Mean		1.94	0.350		3291313	2.06
6.40	1		1.92	1.47		3080465	8.33
	2		1.94	0.580		3245308	3.43
	3		1.96	-0.890		3538931	-5.31
	Mean		1.94	0.390		3288235	2.15
2.00	1		1.96	-0.380		3434868	-2.21
	2		1.98	-1.48		3661624	-8.96
	3		1.95	-0.050		3368161	-0.230
	Mean		1.96	-0.640		3488218	-3.80
0.640	1		1.86	4.40		2594574	22.79
	2		1.97	-0.950		3549605	-5.63
	3		1.94	0.320		3296552	1.90
	Mean		1.92	1.26		3146910	6.35
Control	1		1.94			3329438
	2		1.93			3199088
	3		1.95			3415786
	4		1.94			3258562
	5		1.95			3386989
	6		1.97			3572935
	Mean		1.95			3360466

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	1.54	2.46	1.84	1.94	0.468	24.1	27.4
	2	1.44	2.53	1.82	1.93	0.553	28.6
	3	1.61	2.49	1.76	1.95	0.470	24.0
	4	1.52	2.58	1.71	1.94	0.567	29.3
	5	1.46	2.78	1.62	1.95	0.719	36.9
	6	1.63	2.45	1.83	1.97	0.428	21.8
				Mean	1.95
				SD ±	0.01
				VC %	0.66

Validity criteria fulfilled:

yes

Conclusions:

Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750-47-9, EC Number 274-846-6 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 122 (101 – 141) and 39.7 (24.5 – 61.7) µg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 416 (363 – 470) and 132 (108 – 161) µg/L, respectively.
The test item effect was observed to be reversible at 200 – 640 µg/L. Therefore, there is potential for recovery following exposure up to 640 µg/L (highest test concentration).

Executive summary:

The toxicity to aquatic algae and cyanobacteria of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl chlorides, CAS Number 70750 -47 -9, EC Number 274-846-6, [Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9], was investigated in a study conducted according to OECD guideline 201 (Alga, Growth Inhibition Test).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750-47-9, EC Number 274-846-6 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 122 (101 – 141) and 39.7 (24.5 – 61.7) 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 416 (363 – 470) and 132 (108 – 161) µg/L, respectively.

Description of key information

...........................

In a supporting study the toxicity to aquatic algae and cyanobacteria of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl chlorides, CAS Number 70750 -47 -9, EC Number 274-846-6, [Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9], was investigated in a study conducted according to OECD guideline 201 (Alga, Growth Inhibition Test).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750-47-9, EC Number 274-846-6was 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 122 (101 – 141) and 39.7 (24.5 – 61.7) 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 416 (363 – 470) and 132 (108 – 161) µg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.414 mg/L