Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

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:

From March 29, 2010 to April 15, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 as RA study

Justification for type of information:

Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose:

read-across source

Qualifier:

according to

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 (125 and 500 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 500 µg/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: A stock solution of 10 mg/L was freshly prepared. Dispersion treatment was 5 min ultrasound at room temperature, equilibration phase for 1 h before application was carried out.
- Eluate: NAtural river water
- Differential loading: 62.5 - 125 - 250 - 500 - 1000 µg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 500 – 1000 µg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 5 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: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
1000 8.02 8.01
500 8.03 8.01
250 8.06 8.37
125 8.06 8.92
62.5 8.07 9.07
Control 8.06 9.14

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 2742164 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’’). 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
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)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
10.0 100 100
1.00 89 99
0.10 1 3
0.01 - 6 - 32
0.001 - 7 - 37

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

139.8 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

Corrected for a.i. and 45% sorption

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

269 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (200 - 351)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

200 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

103.95 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

Corrected for a.i. and 45% sorption

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

453 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (388-514)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

235.4 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

corrected for a.i. and 45% sorption

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

277 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

143.9 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

corrected for a.i. and 45% sorption

Basis for effect:

other: Yield

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:

EC10-, EC20- and EC50-values of the growth rate and yield
analyses inhibition after 72 h were calculated by sigmoidal dose-response regression and 0 – 100 variable slope equation. 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,N,N',N',N’’-Pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride (CAS No. 68607-29-4) (0-72 h)

                                based on nominal test item concentrations [µg/L]

                               

	Rate-related inhibition
ErC10	269 (200 – 351)
ErC20	322 (254 – 388)
ErC50	453 (388 – 514)
	Inhibition of yield
EyC10	200 (152 – 236)
EyC20	225 (178 – 248)
EyC50	277 (254 – 375)

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		23896	15906	17373
		2		9787		21109	19972	10638
		3		9787		19013	18628	15013
		Mean		9787		21339	18169	14341
500		1		9787		26919	36676	45220
		2		9787		27172	32615	52557
		3		9787		44520	74567	363923
		Mean		9787		32870	47953	153900
250		1		9787		35303	276296	1560834
		2		9787		39524	310150	1805422
		3		9787		42678	353094	2091520
		Mean		9787		39168	313180	1819259
125		1		9787		59410	586397	2864605
		2		9787		68838	659571	3158989
		3		9787		73857	749999	3237056
		Mean		9787		67368	665322	3086883
62.5		1		9787		64274	745257	3107670
		2		9787		69585	646980	2771469
		3		9787		78642	722533	3017782
		Mean		9787		70834	704923	2965640
Control		1		9787		65675	645222	2583972
		2		9787		60190	554785	2438766
		3		9787		74703	806164	2544032
		4		9787		66206	809642	3065234
		5		9787		65402	695714	2922748
		6		9787		71376	715299	2898233
		Mean		9787		67259	704471	2742164

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		0.191	89.8		7586	99.7
	2		0.028	98.5		851	100
	3		0.143	92.4		5226	99.8
	Mean		0.121	93.6		4554	99.8
500	1		0.510	72.8		35433	98.7
	2		0.560	70.2		42770	98.4
	3		1.21	35.8		354136	87.0
	Mean		0.76	59.6		144113	94.7
250	1		1.69	9.94		1551047	43.2
	2		1.74	7.36		1795635	34.3
	3		1.79	4.75		2081733	23.8
	Mean		1.74	7.35		1809472	33.8
125	1		1.89	-0.84		2854818	-4.48
	2		1.93	-2.58		3149202	-15.3
	3		1.93	-3.01		3227269	-18.1
	Mean		1.92	-2.14		3077096	-12.6
62.5	1		1.92	-2.28		3097883	-13.4
	2		1.88	-0.25		2761682	-1.07
	3		1.91	-1.76		3007995	-10.1
	Mean		1.90	-1.43		2955853	-8.18
Control	1		1.86			2574185
	2		1.84			2428979
	3		1.85			2534245
	4		1.92			3055447
	5		1.90			2912961
	6		1.90			2888446
	Mean		1.88			2732377

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	1.90	2.29	1.39	1.86	0.450	24.2	26.3
	2	1.82	2.22	1.48	1.84	0.371	20.2
	3	2.03	2.38	1.15	1.85	0.634	34.2
	4	1.91	2.50	1.33	1.92	0.586	30.6
	5	1.90	2.36	1.44	1.90	0.465	24.5
	6	1.99	2.31	1.40	1.90	0.459	24.2
				Mean	1.88
				SD ±	0.03
				VC %	1.63

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the EyC50 and ErC50 (0-72 h) were 277 µg/L(equivalent to 143.9µg a.i./L) and 453µg/L (i.e., equivalent to 235.4µg a.i./L), respectively. The ErC10, EyC10, values were determined at 269 µg/L (i.e., equivalent to 139.8 µg a.i./L), and 200 µg/L (i.e., equivalent to 103.95µg a.i./L) (nominal) respectively

Executive summary:

A study was conducted to determine the toxicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride to aquatic algae, according to OECD Guideline 201, in compliance with GLP.Pseudokirchneriella subcapitatawere exposed for 72 h to nominal concentrations of62.5, 125, 250, 500 and 1000 µg/Lof the test substance.Three replicates were tested for each of the test substance concentrations and six replicates for the control. Analytical dose verification was performed. Chemical analysis of duplicate samples at125 and 500 µg/Ltaken at the beginning (64 - 67 %recovery) and at the end (25 - 31%recovery) of the test indicated 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 adsorbed fraction. Test substance recovered from the glassware (227 µg/L at concentration level 500 µg/L corresponding to 45 % of the nominal concentration). All effect values are given based on nominal concentrations of the test substance.Growth rate and biomass were recorded throughout the experiment. The EC10 for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 269 and 200 µg/L, respectively. The EC50 for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 453 and 277 µg/L, respectively.Under the study conditions, the EyC50 and ErC50 (0 -72 h) were277 µg/L(equivalent to 143.9µg a.i./L) and453µg/L (equivalent to 235.4µg a.i./L), respectively and the ErC10 and EyC10 were269µg/L (equivalent to 139.8µg a.i./L) and 200µg/L(equivalent to 103.95µg a.i./L) (nominal) respectively (Scheerbaum, 2010).

Key value for chemical safety assessment

EC50 for freshwater algae:

235.4 µg/L

EC10 or NOEC for freshwater algae:

139.8 µg/L

Additional information

A study was conducted to determine the toxicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride to aquatic algae, according to OECD Guideline 201, in compliance with GLP. Pseudokirchneriella subcapitata were exposed for 72 h to nominal concentrations of 62.5, 125, 250, 500 and 1000 µg/L of the test substance. Three replicates were tested for each of the test substance concentrations and six replicates for the control. Analytical dose verification was performed. Chemical analysis of duplicate samples at 125 and 500 µg/L taken at the beginning (64 - 67 % recovery) and at the end (25 - 31% recovery) of the test indicated 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 adsorbed fraction. Test substance recovered from the glassware (227 µg/L at concentration level 500 µg/L corresponding to 45 % of the nominal concentration). All effect values are given based on nominal concentrations of the test substance. Growth rate and biomass were recorded throughout the experiment. The EC10 for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 269 and 200 µg/L, respectively. The EC50 for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 453 and 277 µg/L, respectively. Under the study conditions, the EyC50 and ErC50 (0 -72 h) were 277 µg/L (equivalent to 143.9µg a.i./L) and 453 µg/L (equivalent to 235.4 µg a.i./L), respectively and the ErC10 and EyC10 were 269 µg/L (equivalent to 139.8 µg a.i./L) and 200 µg/L(equivalent to 103.95 µg a.i./L) (nominal) respectively (Scheerbaum, 2010).