Dodecyltrimethylammonium bromide

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:

2018-04-23 to 2018-05-04

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:

2011-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009-08-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The samples were taken from the biological phase of the study. One sample from the freshly prepared stock solution and duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment. All samples were diluted by a factor of two with acetonitrile. Additional samples of the control and of the dilution solvent acetonitrile were taken at each sampling without any sample treatment.
- Sample storage conditions before analysis: All samples were stored in a freezer (< - 20 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Volumes of approximately 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass dishes and incubated in a water bath.
- Controls:
Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above.
- Evidence of undissolved material: No

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81 SAG
- Source: Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut fur Pflanzenwissenschaften, Universitat Gottingen", 37073 Gottingen, Germany.
- Age of inoculum: Exponentially growing

ACCLIMATION
- Culturing media and conditions: same as test
- 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:

None

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22.0 to 23.7 °C

pH:

At test start 7.8 to 7.9, at the end of the test 8.0 to 9.9

Dissolved oxygen:

Not available

Salinity:

Not applicable

Conductivity:

Not available

Nominal and measured concentrations:

Nominal: 0.10, 0.032, 0.010, 0.0032 and 0.0010 mg test item/L
Geometric mean measured: 94.6, 28.6, 6.51, 1.58 and 0.620 µg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size: Glass, 50 mL, closed with glass dishes
- Initial cells density: Approx. 5000 algal cells per mL test medium
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Reconstituted water (OECD Medium) prepared with deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity: Mean light intensity: 6820 lux (6230 to 7250 lux).


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Spectrophotometer
- Observation intervals: 24, 48 and 72 hours

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

59.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval: 54.5 - 65.8 µgL

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.12 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval: 4.05 - 6.42 µg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

11.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval: 10.1 - 14.0 µg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.58 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.51 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.53 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. interval: 6.00 - 9.45 µg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.64 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. interval: 1.93 - 3.64 µg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.53 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. interval: 1.00 - 2.30

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.58 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.51 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Any stimulation of growth found in any treatment: No
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by non-linear regression using the 3-param. normal CDF. For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat® Solutions GmbH.

Table 1: Growth rate during the Test Period of 72 Hours

Geometric mean measured concentration [µg test item/L]	Flask No.	Growth rates [1/day] after
		0 - 24 hours	0 - 48 hours	0- 72 hours
Control	1	2.06	1.886	1.685
	2	2.001	1.841	1.631
	3	2.169	1.882	1.642
	4	2.06	1.841	1.66
	5	2.169	1.857	1.671
	6	1.938	1.803	1.639
	m	2.066	1.852	1.655
	s	0.092	0.031	0.021
0.62	1	2.169	1.836	1.632
	2	1.938	1.846	1.647
	3	2.06	1.846	1.672
	m	2.055	1.843	1.65
	s	0.116	0.006	0.02
1.58	1	1.87	1.797	1.628
	2	1.938	1.82	1.644
	3	2.06	1.836	1.668
	m	1.956	1.818	1.647
	s	0.096	0.019	0.02
6.51	1	2.001	1.659	1.473
	2	2.06	1.586	1.374
	3	2.06	1.62	1.423
	m	2.04	1.622	1.423
	s	0.034	0.037	0.05
28.6	1	1.442	1.283	1.074
	2	1.544	1.299	1.093
	3	1.72	1.329	1.135
	m	1.569	1.304	1.101
	s	0.141	0.023	0.032
94.6	1	1.202	0.923	0.69
	2	1.056	0.923	0.663
	3	0.885	0.923	0.649
	m	1.048	0.923	0.667
	s	0.159	0	0.021

m: mean value; s: standard deviation

Table 2: Yield y and Percentage Inhibition of y during the Test Period

Geometric mean measured concentration [ug test item/L]	Yield y [10000 cells/mL] and % inhibition of y
	0 - 24 hours		0 - 48 hours		0 - 72 hours
	y	%	y	%	y	%
Control	3.46	-	19.822	-	71.254	-
0.62	3.423	1.1	19.44	1.9 *	70.208	1.5
1.58	3.046	12	18.465	6.8 *	69.503	2.5
6.51	3.347	3.3	12.341	37.7 *	35.522	50.1 *
28.6	1.917	44.6	* 6.286	68.3 *	13.122	81.6 *
94.6	0.938	72.9	* 2.667	86.5 *	3.204	95.5 *

* mean value significantly different from the control (tested with Williams t-test,a= 0.05, one-sided)

Table 3: Growth Rates µ and Percentage Inhibition of µ during theTest Period

Geometric mean measured concentration [µg test item/L]	Growth rate µ [1/day] and % inhibition of µ
	µ	%	µ	%	µ	%
Control	2.066	-	1.852	-	1.655	-
0.62	2.055	0.5	1.843	0.5	1.65	0.3
1.58	1.956	5.3	1.818	1.8 *	1.647	0.5
6.51	2.04	1.3	1.622	12.4 *	1.423	14
28.6	1.569	24.1	* 1.304	29.6 *	1.101	33.5
94.6	1.048	49.3	* 0.923	50.2 *	0.667	59.7

* mean value significantly different from the control (tested with Williams t-test,a= 0.05,

Table 4: Results for the Determination of the Test Item in the Test Samples

Sample description			Concentration found	D.F.	Concentration calculated nominal		% of nominal1	Geometric mean measured concentration
	sample age
[mg test item/L]	day	[h]	[ug test item/L]^1		[µg test item/L]^1	[µg test item/L]^1		[µg test item/L]
Control (untreated)	0	0	<LOD	1	n.a.	0	n.a.
Control (untreated)	3	72	<LOD	1	n.a.	0	n.a.
Dilution Solvent	0	-	<LOD	1	n.a.	0	n.a.
Dilution Solvent	3	-	<LOD	1	n.a.	0	n.a.
Control	0	0	<LOQ	2	n.a.	0	n.a.
Control	0	0	<LOQ	2	n.a.	0	n.a.
Control	3	72	<LOQ	2	n.a.	0	n.a.
Control	3	72	<LOQ	2	n.a.	0	n.a.
0.001	0	0	0.574	2	1.147	1	115
0.001	0	0	0.523	2	1.047	1	105
0.001	3	72	0.12	2	0.24	1	24	1/2 LOQ used
0.001	3	72	0.17	2	0.341	1	34	1/2 LOQ used
						initial mean value (n=2): RSD (n=2):	110 6
					mean value of aged test media (n=2):		29
						RSD (n=2):	24
					geometric mean value (n=2):		62	0.62
0.0032	0	0	1.519	2	3.037	3.2	95
0.0032	0	0	1.677	2	3.355	3.2	105
0.0032	3	72	0.394	2	0.787	3.2	25
0.0032	3	72	0.384	2	0.768	3.2	24
						initial mean value (n=2):	100
						RSD (n=2):	7
					mean value of aged test media (n=2):		24
						RSD (n=2):	2
					geometric mean value (n=2):		49	1.58
0.01	0	0	5.321	2	10.642	10	106
0.01	0	0	4.968	2	9.936	10	99
0.01	3	72	2.171	2	4.343	10	43
0.01	3	72	1.942	2	3.884	10	39
						initial mean value (n=2):	103 5
						RSD (n=2):
					mean value of aged test media (n=2):		41
						RSD (n=2):	8
					geometric mean value (n=2):		65	6.51
0.032	0	0	16.958	2	33.916	32	106
0.032	0	0	16.958	2	33.916	32	106
0.032	3	72	12.106	2	24.211	32	76
0.032	3	72	11.929	2	23.858	32	75
						initial mean value (n=2):	106 0
						RSD (n=2):
					mean value of aged test media (n=2):		75
						RSD (n=2):	1
					geometric mean value (n=2):		89	28.6
0.1	0	0	48.946	2	97.892	100	98
0.1	0	0	57.288	2	114.576	100	115
0.1	3	72	37.891	2	75.781	100	76
0.1	3	72	46.434	2	92.867	100	93
						initial mean value (n=2):	106 11
						RSD (n=2):
					mean value of aged test media (n=2):		84
						RSD (n=2):	14
					geometric mean value (n=2):		95	94.6
					overall mean value (n=18):		83
						RSD (n=18):	37

¹ The tabulated results represent rounded results calculated on the exact raw data

² Calculated using mean of duplicates and according to OECD Guidance Document No. 23, Annex 2

LOD: Limit of Detection = 0.06 µg test item/L

LOQ: Limit of Quantification = 0.7 ug test item/L corresponding to 0.35 µg test item/L after dilution by factor 2 n.a.: not applicable; RSD: Relative Standard Deviation; D.F.: Dilution factor

italic: Values below the LOQ. Results reported for information only and excluded from evaluation. For values <LOQ, half the LOQ was used for calculating measured values according to OECD Guidance Document No. 23.

Validity criteria fulfilled:

yes

Conclusions:

In a valid Growth Inhibition Test according to OECD TG 201, EU Method C.3 with Desmodesmus subspicatus the 72-hour EyC50 of the test item was calculated to be 7.53 µg/L and the ErC50 59.9 µg/L. The 72-hour EyC10 was calculated to be 1.53 µg test item/L and the ErC10 5.12 µg test item/L.

Executive summary:

A Growth Inhibition Test according to OECD TG 201, EU Method C.3 and GLP principles was carried out in order to determine the inhibitory effect of the test item on the growth of the freshwater green algae Desmodesmus subspicatus (strain No.86.81 SAG). For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to 5 concentrations of the test item (0.10, 0.032, 0.010, 0.0032 and 0.0010 mg test item/L in nominal) with three replicates per test concentration and six replicates for the blank control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours. The initial concentrations and the maintenance of the exposure concentrations during the test were verified by LC-MS/MS analysis to be 94.6, 28.6, 6.51, 1.58 and 0.620 µg/L (geometric mean). Thus, all reported results refer to geometric mean measured values since the concentrations of the test item were not within ± 20% of the measured initial concentrations during the test. The72-hour EyC50 was calculated to be 7.53 µg test item/L and the ErC50 59.9 µg test item/L. The 72-hour EyC10 was calculated to be 1.53 µg test item/L and the ErC10 5.12 µg test item/L. The 72-hour NOEyC was determined to be 1.58 µg test item/L and the associated 72-hour LOEyC of 6.51 µg test item/L. The 72 -hour NOErC was determined to be 1.58 µg test item/L and the associated 72-hour LOErC is 6.51 µg test item/L. All validity criteria of the guidelines were fulfilled.

Description of key information

In a Growth Inhibition Test according to OECD TG 201, EU Method C.3 with Desmodesmus subspicatus the 72-hour EyC50 of the substance was calculated to be 7.53 µg/L and the ErC50 59.9 µg/L. The 72-hour EyC10 was calculated to be 1.53 µg test item/L and the ErC10 5.12 µg test item/L (reference 6.1.5 -1).

Key value for chemical safety assessment

EC50 for freshwater algae:

59.9 µg/L

EC10 or NOEC for freshwater algae:

5.12 µg/L

Additional information

A valid 72-hour Growth Inhibition Test according to OECD TG 201, EU Method C.3 and GLP principles was carried out in order to determine the inhibitory effect of the test item on the growth of the freshwater green algae Desmodesmus subspicatus (strain No.86.81 SAG). For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to 5 concentrations of the test item (0.10, 0.032, 0.010, 0.0032 and 0.0010 mg test item/L in nominal) with three replicates per test concentration and six replicates for the blank control.). The test item concentration in the test media was verfied by LC-MS/MS analysis to be 94.6, 28.6, 6.51, 1.58 and 0.620 µg/L (geometric mean, ± 20% of the measured initial concentrations) and hence, results are based on geometric mean measured concentrations. The72-hour EyC50 was calculated to be 7.53 µg test item/L and the ErC50 59.9 µg test item/L. The 72-hour EyC10 was calculated to be 1.53 µg test item/L and the ErC10 5.12 µg test item/L. The 72-hour NOEyC was determined to be 1.58 µg test item/L and the associated 72-hour LOEyC of 6.51 µg test item/L. The 72 -hour NOErC was determined to be 1.58 µg test item/L and the associated 72-hour LOErC is 6.51 µg test item/L.