N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

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:

2020-12-10 to 2020-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Preparation of the test item solutions:

Out of the stock solution 5 concentrations were tested in a geometric series with a dilution factor of 2: 0.0313 – 0.0625 – 0.125 – 0.250 – 0.500 mg/L, corresponding to the geometric mean measured test item concentrations of 0.0209 – 0.0399 – 0.0905 – 0.176 – 0.375 mg/L. The concentrations are based on the results of a preliminary range finding test (non-GLP, open system with headspace).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4 (axenic)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567–5130 lux for 24 h per day
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

river water total hardness [mg CO3/l]: 347

Test temperature:

Nominal range: 21 - 24°C, controlled at ± 2°C

pH:

start: 8.18 - 8.20 (test concentrations 0.0209 - 0.375 mg/l), 8.18 (control); end: 8.22 - 8.39 (test concentrations 0.0209 - 0.375 mg/l), 8.32 (control)

Dissolved oxygen:

8.99 mg O2/L (river water)

Conductivity:

1001 µS/m (river water)

Nominal and measured concentrations:

0.0209, 0.0399, 0.0905, 0.176, 0.375 mg/L geometric mean measured test item concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, provided with cotton wool plugs. The test container were pretreated with the appropriate test solutions for 72 hours under test conditions. Before the start of the exposure phase, the test container were emptied and refilled with freshly prepared test solutions.
- Fill volume: 100 mL
- Initial cells density: nominal: approximately 5 x 10 E03 - E04 cells/mL; actual: 5416 cells/mL
- Control end cells density: 753084 (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no, natural river water, for details see section "any other information on materials and methods"

TEST MEDIUM / WATER PARAMETERS
river water as specified in section "any other information on materials and methods"

OTHER TEST CONDITIONS
- Sterile test conditions: The natural river water was stored at 6 ± 2 °C and at - 18± 2 °C for at least 24 hours. Freezing was found to be suitable to minimize the content of vital natural algae cells of the water as well as to reduce microbial (bacterial) activity
- Adjustment of pH: no
- Light regime: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: delution factor of 2
Range finding study
- Test concentrations: 0.0055 - 0.05 - 0.5 mg/L nominal
- Results used to determine the conditions for the definitive study:
Growth Rate Inhibition: 0.5: 100%, 0.05: 1%, 0.005: -4% each concentration mg/l nominal
Yield Inhibition: 0.5: 100%, 0.05: 4%, 0.005: -20% each concentration mg/l nominal

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.044 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.021 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.099 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.

Results with reference substance (positive control):

- Results with reference substance valid?
- ErC50: 0.940 (95 % c.l. 0.732 - 1.33 mg/L); valid range: 0.758 ± 0.535
- EyC50: 0.660 (95 % c.l. 0.393 - 0.70), valid range: 0.414 ± 0.335
- Other: tested from 2019-03-05 to 2019-03-08

Reported statistics and error estimates:

Statistical Significance (NOEL/LOEL) – Growth Rate

t-test

Normality Test (Shapiro-Wilk): Passed (P = 0.222)
Equal Variance Test (Brown-Forsythe): Passed (P = 0.860)

The result of the equal variance test indicates the likelihood that the two groups are sampled from populations with equal variances. but does not guarantee the equality or inequality of the two variances.

Group Name N Missing Mean Std Dev SEM
Control 6 0 1.815 0.0237 0.00969
100 mg/L 6 0 1.816 0.0188 0.00767

Difference of means -0.001000
Use the results of Welch's test. where equal variances are not assumed. if the equality of the population variances of the two groups is in doubt.

Equal Variances Assumed (Student's t-test):

t = -0.0809 with 10 degrees of freedom.

95 percent two-tailed confidence interval for difference of means: -0.0285 to 0.0265
Two-tailed P-value = 0.937

The difference in the mean values of the two groups is not great enough to reject the possibility that the difference is due to random sampling variability. There is not a statistically significant difference between the input groups (P = 0.937).

Equal Variances Not Assumed (Welch's t-test):

t = -0.0809 with 9.501 degrees of freedom.

95 percent two-tailed confidence interval for difference of means: -0.0287 to 0.0267
Two-tailed P-value = 0.937

The difference in the mean values of the two groups is not great enough to reject the possibility that the difference is due to random sampling variability. There is not a statistically significant difference between the input groups (P = 0.937).

The program states no significant difference for the limit loading rate compared to the control.  

Table 4:    Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
0.375	1	5416	<LOQcell density	<LOQcell density	<LOQcell density
	2	5416	<LOQcell density	<LOQcell density	<LOQcell density
	3	5416	<LOQcell density	<LOQcell density	<LOQcell density
	Mean	5416	n.a.	n.a.	n.a.
0.176	1	5416	2632	3208	6743
	2	5416	2812	8232	15488
	3	5416	2924	5238	14957
	Mean	5416	2789	5559	12396
0.0905	1	5416	8884	31158	84983
	2	5416	8237	22897	90543
	3	5416	8682	28147	89915
	Mean	5416	8601	27401	88480
0.0399	1	5416	14422	87583	443049
	2	5416	11559	78640	530990
	3	5416	20083	99217	529797
	Mean	5416	15355	88480	501279
0.0209	1	5416	21617	151821	718578
	2	5416	20952	160233	762463
	3	5416	19248	153087	695747
	Mean	5416	20606	155047	725596
Control	1	5416	21390	168606	750731
	2	5416	22291	162102	724468
	3	5416	17294	144850	753676
	4	5416	23939	149017	739194
	5	5416	24309	177748	749879
	6	5416	23951	199255	800555
	Mean	5416	22196	166930	753084

LOQ_{cell density}            = Limit of quantification of the cell density (2280 cells/mL).      n.a.     = not applicable

 

Table 5: Evaluation after 72 hours

           Statistically significant differences of growth rates and yield compared to
             control values are marked (+), not significant differences are marked (-).

 

Geometric mean measured test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
0.375	1		n.a.	100		n.a.	100
	2		n.a.	100		n.a.	100
	3		n.a.	100		n.a.	100
	Mean	(+)	n.a.	100	(+)	n.a.	100
0.176	1		0.073	96		1327	100
	2		0.350	79		10072	99
	3		0.339	79		9541	99
	Mean	(+)	0.254	85	(+)	6980	99
0.0905	1		0.918	44		79567	89
	2		0.939	43		85127	89
	3		0.937	43		84499	89
	Mean	(+)	0.931	43	(+)	83064	89
0.0399	1		1.47	11		437633	41
	2		1.53	7		525574	30
	3		1.53	7		524381	30
	Mean	(+)	1.51	8	(+)	495863	34
0.0209	1		1.63	1		713162	5
	2		1.65	0		757047	-1
	3		1.62	2		690331	8
	Mean	(-)	1.63	1	(-)	720180	4
Control	1		1.64			745315
	2		1.63			719052
	3		1.65			748260
	4		1.64			733778
	5		1.64			744463
	6		1.67			795139
	Mean		1.65			747668

n.a.     = not applicable

Table 6: Section-by-Section and Average Specific Growth Rates of the Control Group
(0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.37	2.07	1.49	1.64	0.369	22.5	20.6
	2	1.42	1.98	1.50	1.63	0.308	18.8
	3	1.16	2.13	1.65	1.65	0.482	29.3
	4	1.49	1.83	1.60	1.64	0.174	10.6
	5	1.50	1.99	1.44	1.64	0.301	18.3
	6	1.49	2.12	1.39	1.67	0.396	23.7
				Mean	1.65
				SD ±	0.01
				CV [%]	0.68

 

SD = Standard deviationCV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

72 h NOEL of Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates to the green alga Pseudokirchneriella subcapitata based on growth rate and yield inhibition are 0.0209 mg/l based on geometric mean measured test item concentration.
ErC10 was 0.0445 mg/L, ErC50 was 0.099 mg/L, EyC10 was 0.0264 mg/L, EyC50 was 0.0487 mg/L based on geometric mean measured test item concentration.

Executive summary:

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4 (axenic) were exposed to Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates at concentrations of 0.0313 – 0.0625 – 0.125 – 0.250 – 0.500 mg/L in natural river water under static conditions.

72 h NOEL of Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates to the green alga Pseudokirchneriella subcapitata based on growth rate and yield inhibition are 0.0209 mg/l based on geometric mean measured test item concentration.

ErC10 was 0.0445 mg/L, ErC50 was 0.099 mg/L, EyC10 was 0.0264 mg/L, EyC50 was 0.0487 mg/L based on geometric mean measured test item concentration.

Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for the algal growth inhibition test in Pseudokirchneriella subcapitata.

 

Key value for chemical safety assessment

EC50 for freshwater algae:

99 µg/L

EC10 or NOEC for freshwater algae:

44.5 µg/L

Additional information

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of Pseudokirchneriella subcapitata HINDÁK,CCAP 278/4 (axenic) were exposed to Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates at concentrations of 0.0313 – 0.0625 – 0.125 – 0.250 – 0.500 mg/L in natural river water under static conditions.

72 h NOEL of Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates to the green alga Pseudokirchneriella subcapitata based on growth rate and yield inhibition are 0.0209 mg/l based on geometric mean measured test item concentration.

ErC10 was 0.0445 mg/L, ErC50 was 0.099 mg/L, EyC10 was 0.0264 mg/L, EyC50 was 0.0487 mg/L based on geometric mean measured test item concentration.

Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.