Quaternary ammonium compounds, C12-14-alkyltrimethyl, Me sulfates

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:

From March 12, 2018 to March 19, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

Deviations are minimal and which doesn’t have any impact on the outcome of the study.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

Deviations are minimal and which doesn’t have any impact on the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start and at the end of the test, the content of the test substance in the test solutions was determined.

Vehicle:

yes

Remarks:

emineralised water enriched with minerals but without algae

Details on test solutions:

A stock solution containing 100.0 mg/L in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution of this stock solution with algal medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Specification:
Unicellular freshwater green alga.
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae

Source and Culture
Source: The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
Culture: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent cul-ture was prepared. From an aliquot of the permanent culture, the pre-culture was pre-pared.

Test type:

not specified

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

19.2 – 22.4°C

pH:

7.6 - 8.1

Salinity:

Fresh wter

Nominal and measured concentrations:

Nominal concentration: 0.32, 1, 3.2, 10 and 32 µg/L
Measured concentrations: < LOQ, < LOQ, < LOQ, 1.89, 4.98 and 19.30 µg/L respectively to nominal concentrations

Details on test conditions:

Treatments tested: 0.32 / 1 / 3.2 / 10 / 32 µg/L nominal concentration. The concentrations to be tested based on non GLP pre-tests
Number of replicates:
6 replicates for the blank control
3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 h
Lighting:: 5000 lux
Temperature: 19.2 – 22.4 °C
Replicates (Blank control): 45 ±1 mL algal medium and alga
Replicates (Treatments): 45 ±1 mlL test solution and alga

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.89 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and yeild

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.3 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.08 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.68 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.73 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Results with reference substance (positive control):

The results of the last study with the positive control K2Cr2O7 are presented below.

Biological Results of K2Cr2O7
Parameter Value 95% confidence interval
72h ErC50 0.90 mg/L 0.77 – 1.06 mg/L
72h EyC50 0.42 mg/L 0.35 - 0.51 mg/L

Results:

Cell uumbers: The cell numbers were determined with an electronic substance counter. The measns and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table:

 

 Cell Number/mL

Nominal Concentration in µg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2000	7383	45433	304340
Blank control	SD	0	759	2225	12011
0.32*	Mean	2000	7147	34640	229193
0.32*	SD	0	843	2344	35138
1	Mean	2000	8393	50933	299167
1	SD	0	947	1781	11725
3.2	Mean	2000	8360	49093	302160
3.2	SD	0	625	2833	3120
10	Mean	2000	7360	24500	81793
10	SD	0	1129	3296	5307
32	Mean	2000	5600	4187	8480
32	SD	0	212	1157	503

SD = Standard Deviation

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation.

Light intensity: In the following table,the light intensity during the test is stated:

Light Intensity

	0 h	24 h	48 h	72 h
Light intensity [Lux]	5000	5000	5000	5000

pH values:

In the following table,the pH values measured at the start and the end of the test are stated:

pH values

Nominal Concentrationin µg/L	0 h	72 h
Blank control	7.6	8.1
0.32*	7.6	8.1
1	7.6	8.2
3.2	7.6	8.3

10	7.6	7.8
32	7.6	7.6

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation

Microscopical observation:

In the following table, the appearance of the algae at the end of the test is stated:

 Microscopical Observations

Nominal Concentrationin µg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
0.32*	Yes
1	Yes
3.2	Yes
10	Yes
32	No cells visible

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation

 

Analytical determination:

The test substance was only detectable at the beginning of the test and in the 3 highest concentrations. The measured concentrations lay between 50 % and 60 % of the nominal concentrations at the beginning of the test.

During validation the test substance was stable under test conditions. In this study it might be possible that the measurable amount of test substance was biased by the test organism (inges-tion or adsorption of test substance onto the algae cells). Nevertheless a clear dose-response relationship could be observed. Therefore, the biological results were based on the meas-ured concentration at the beginning of the test in the 3 highest concentrated treatments. This procedure is in agreement with the OECD guideline see point 40 on page 6.

The details are given in the following tables:

 Measured Concentrations and % of Nominal

Nominal Concentration Test Substance	Measured concentrations  t = 0 h	Measured concentrations  t = 72 h	% of Nominal  t = 0 h	% of Nominal  t = 72 h
µg/L	µg/L	µg/L
Blank control	< LOQ	< LOQ	--	--
0.32	< LOQ	< LOQ	--	--
1	< LOQ	< LOQ	--	--
3.2	1.89	< LOQ	58.93	--
10	4.98	< LOQ	49.82	--
32	19.30	< LOQ	60.32	--

LOQ (Limit of quantification) = 1 µg/L

Growth Rate, Yield

From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Growth Rate µ, Yield

Nominal Concentration in µg/L	Parameter	Growth Rate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.67	302340
	SD	0.01	12011
0.32*	Mean	1.58	227193
	SD	0.05	35138
1	Mean	1.67	297167
	SD	0.01	11725
3.2	Mean	1.67	300160
	SD	0.00	3120
10	Mean	1.24	79793
	SD	0.02	5307
32	Mean	0.48	6480
	SD	0.02	503

SD = Standard Deviation

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation

 

Inhibition:

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

 Inhibition Values

Nominal concentration in µg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
0.32*	5.79	24.86
1	0.34	1.71
3.2	0.13	0.72
10	26.17	73.61
32	71.27	97.86

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation

 

 Biological Results

The following values were determined:

Biological Results of the Test Substance

Parameter	Value	95% confidence interval
NOEC (Growth Rate) 72 h	1.89 µg/L	--
NOEC (Yield) 72 h	1.89 µg/L	--
LOEC (Growth Rate) 72 h	4.89 µg/L	--
LOEC (Yield) 72 h	4.98 µg/L	--
72h ErC10	2.68 µg/L	2.52 – 2.85 µg/L
72h EyC10	2.73 µg/L	1.76 – 4.22 µg/L
72h ErC50	10.30 µg/L	9.58 – 11.04 µg/L
72h EyC50	4.08 µg/L	2.60 – 6.53 µg/L

 

Cell Numbers measured

Nominal Concentration in µg/L	Cell Number/mL
	0 h	24 h	48 h	72 h
Blank control	100	367	2423	15490
	100	425	2255	15177
	100	401	2349	16089
	100	360	2291	15083
	100	337	2209	15220
	100	325	2103	14243
0.32*	100	406	1856	13035
	100	332	1717	11779
	100	334	1623	9565
1	100	398	2477	14362
	100	387	2516	14979
	100	474	2647	15534
3.2	100	438	2514	14935
	100	434	2557	15151
	100	382	2293	15238
10	100	385	1055	3864
	100	414	1236	4023
	100	305	1384	4382
32	100	276	195	395
	100	292	273	437
	100	272	160	440

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation

Cell Numbers calculated

Nominal Conc. in µg/L	Replicate	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	1	2000	7340	48460	309800
	2	2000	8500	45100	303540
	3	2000	8020	46980	321780
	4	2000	7200	45820	301660
	5	2000	6740	44180	304400
	6	2000	6500	42060	284860
	Mean	2000	7383	45433	304340
	SD	0	759	2225	12011
0.32*	1	2000	8120	37120	260700
	2	2000	6640	34340	235580
	3	2000	6680	32460	191300
	Mean	2000	7147	34640	229193
	SD	0	843	2344	35138
1	1	2000	7960	49540	287240
	2	2000	7740	50320	299580
	3	2000	9480	52940	310680
	Mean	2000	8393	50933	299167
	SD	0	947	1781	11725
3.2	1	2000	8760	50280	298700
	2	2000	8680	51140	303020
	3	2000	7640	45860	304760
	Mean	2000	8360	49093	302160
	SD	0	625	2833	3120
10	1	2000	7700	21100	77280
	2	2000	8280	24720	80460
	3	2000	6100	27680	87640
	Mean	2000	7360	24500	81793
	SD	0	1129	3296	5307
32	1	2000	5520	3900	7900
	2	2000	5840	5460	8740
	3	2000	5440	3200	8800
	Mean	2000	5600	4187	8480
	SD	0	212	1157	503

SD = Standard Deviation

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation

Growth Rate µ

Nominal Conc. in µg/L	Replicate	Growth Rate µ [day^-1]
		24 h	48 h	72 h
Blank control	1	1.30	1.59	1.68
	2	1.45	1.56	1.67
	3	1.39	1.58	1.69
	4	1.28	1.57	1.67
	5	1.21	1.55	1.68
	6	1.18	1.52	1.65
	Mean	1.30	1.56	1.67
	SD	0.10	0.02	0.01
0.32*	1	1.40	1.46	1.62
	2	1.20	1.42	1.59
	3	1.21	1.39	1.52
	Mean	1.27	1.43	1.58
	SD	0.11	0.03	0.05
1	1	1.38	1.60	1.66
	2	1.35	1.61	1.67
	3	1.56	1.64	1.68
	Mean	1.43	1.62	1.67
	SD	0.11	0.02	0.01
3.2	1	1.48	1.61	1.67
	2	1.47	1.62	1.67
	3	1.34	1.57	1.68
	Mean	1.43	1.60	1.67
	SD	0.08	0.03	0.00
10	1	1.35	1.18	1.22
	2	1.42	1.26	1.23
	3	1.12	1.31	1.26
	Mean	1.29	1.25	1.24
	SD	0.16	0.07	0.02
32	1	1.02	0.33	0.46
	2	1.07	0.50	0.49
	3	1.00	0.24	0.49
	Mean	1.03	0.36	0.48
	SD	0.04	0.14	0.02

SD = Standard Deviation

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation

Yield

Nominal Conc. in µg/L	Replicate	Yield [Cell Concentration/mL]
		72 h
Blank control	1	307800
	2	301540
	3	319780
	4	299660
	5	302400
	6	282860
	Mean	302340
	SD	12011
0.32*	1	258700
	2	233580
	3	189300
	Mean	227193
	SD	35138
1	1	285240
	2	297580
	3	308680
	Mean	297167
	SD	11725
3.2	1	296700
	2	301020
	3	302760
	Mean	300160
	SD	3120
10	1	75280
	2	78460
	3	85640
	Mean	79793
	SD	5307
32	1	5900
	2	6740
	3	6800
	Mean	6480
	SD	503

SD = Standard Deviation

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for statistical evaluation

 

Inhibition Values at the end of the test (72 hours)

Nominal Conc. in µg/L	Replicate	Inhibition Growth Rate µ 72 hours	Inhibition Yield 72 hours
Blank control	1	-0.37	-1.81
	2	0.04	0.26
	3	-1.12	-5.77
	4	0.16	0.89
	5	-0.02	-0.02
	6	1.30	6.44
	Mean	0!	0!
	SD	0.79	3.97
0.32*	1	3.07	14.43
	2	5.08	22.74
	3	9.23	37.39
	Mean	5.79	24.86
	SD	3.14	11.62
1	1	1.14	5.66
	2	0.30	1.57
	3	-0.42	-2.10
	Mean	0.34	1.71
	SD	0.78	3.88
3.2	1	0.36	1.87
	2	0.07	0.44
	3	-0.04	-0.14
	Mean	0.13	0.72
	SD	0.21	1.03
10	1	27.27	75.10
	2	26.47	74.05
	3	24.76	71.67
	Mean	26.17	73.61
	SD	1.28	1.76
32	1	72.66	98.05
	2	70.65	97.77
	3	70.51	97.75
	Mean	71.27	97.86
	SD	1.20	0.17

SD = Standard Deviation

*Contamination is suspected. Excluded from evaluation Treatments 0.32 µg/L and 1 µg/L not used for al evaluation

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h EC10 were determined to be at 2.68 µg/L (growth rate) and 2.73 µg/L (yield) respectively. The 72 h EC50 were determined to be 10.30 (growth rate) and 4.08 µg/L (yield) respectively

Executive summary:

A study was conducted to determine the short term toxicity of the test substance to the algae Desmodesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Algae were exposed to the test substance at nominal concentrations of 0, 0.32, 1, 3.2, 10 and 32 µg/L. Algal cell concentrations were determined spectrophotometrically. All the validity criteria were fulfilled. Analytical assessment was performed for the test substance in aqueous samples, at the beginning, mid and at the end of the test. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (HPLC-MS/MS).The test substance was only detectable at the beginning of the test and in the top three concentrations. The measured concentrations lay between 50% and 60% of the nominal concentrations at the beginning of the test. During validation the test substance was stable under test conditions. In this study it might be possible that the measurable amount of test substance was biased by the test organism (ingestion or adsorption of test substance onto the algae cells). Nevertheless a clear dose-response relationship could be observed. Therefore, the biological results were based on the measured concentration at the beginning of the test. This is in agreement with the OECD Guideline. Significant inhibition of algal growth was observed at the top two concentrations. Inhibition of algal growth was also observed at the lowest tested concentration. This was caused probably due to contaminated test vessels because no inhibition occurred at the next higher concentration. This concentration was therefore not used for evaluation. Because significant inhibition of algal growth was observed only at the top two concentration, therefore, only the top three concentrations were used for statistical evaluation. Under the study conditions, the 72 h EC10 were determined to be at 2.68 µg/L (growth rate) and 2.73 µg/L (yield) respectively. The 72 h EC50 were determined to be 10.30 (growth rate) and 4.08 µg/L (yield) respectively (Muckle, 2018).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.01 mg/L

EC10 or NOEC for freshwater algae:

0.003 mg/L

Additional information

A study was conducted to determine the short term toxicity of the test substance to the algae Desmodesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Algae were exposed to the test substance at nominal concentrations of 0, 0.32, 1, 3.2, 10 and 32 µg/L. Algal cell concentrations were determined spectrophotometrically. All the validity criteria were fulfilled. Analytical assessment was performed for the test substance in aqueous samples, at the beginning, mid and at the end of the test. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (HPLC-MS/MS).The test substance was only detectable at the beginning of the test and in the top three concentrations. The measured concentrations lay between 50% and 60% of the nominal concentrations at the beginning of the test. During validation the test substance was stable under test conditions. In this study it might be possible that the measurable amount of test substance was biased by the test organism (ingestion or adsorption of test substance onto the algae cells). Nevertheless a clear dose-response relationship could be observed. Therefore, the biological results were based on the measured concentration at the beginning of the test. This is in agreement with the OECD Guideline. Significant inhibition of algal growth was observed at the top two concentrations. Inhibition of algal growth was also observed at the lowest tested concentration. This was caused probably due to contaminated test vessels because no inhibition occurred at the next higher concentration. This concentration was therefore not used for evaluation. Because significant inhibition of algal growth was observed only at the top two concentration, therefore, only the top three concentrations were used for statistical evaluation. Under the study conditions, the 72 h EC10 were determined to be at 2.68 µg/L (growth rate) and 2.73 µg/L (yield) respectively. The 72 h EC50 were determined to be 10.30 (growth rate) and 4.08 µg/L (yield) respectively (Muckle, 2018).