1-Propanaminium, 3-amino-N-ethyl-N,N-dimethyl-, N-rape-oil acyl derivs., Et 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 October 30, 2017 to February 02, 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)

Version / remarks:

Adopted 23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

liquid chromatography/mass spectrometry method

Details on sampling:

Samples for analysis at 0 h were taken from the excess test solutions and at 72 h from a single test replicate solution of the control and each test concentration (three samples taken from the nominal 0.0610 mg/L concentration, one from each replicate).

Vehicle:

yes

Remarks:

AAP media (test media)

Details on test solutions:

Preparation of test solutions: This study was run with a culture medium control and nominal exposure concentrations of 0.0191, 0.0610, 0.195, 0.625 and 2.0 mg/L. A primary stock concentrate of test substance, with a nominal concentration of 10 mg/L, was prepared by weighing a nominal 10 mg of test substance (actual weight: 9.999 mg) and making up to 1000 mL volume with the culture medium AAP in a volumetric flask. The stock was then stirred for 11 minutes. The resultant stock was observed to be a clear and colourless solution with bubbles on the surface and was used to prepare the test solutions. This was achieved by adding the relevant volumes of the primary stock to AAP media and making up to 1000 mL volume in a volumetric flask. The control consisted of culture medium only. The test solutions were all observed to be clear and colourless after 10 minutes stirring.
 
Nominal Concentration(mg/L) Volume Prepared (mL) Volume Stock Added(mL) Stock Concentration(mg/L)
Control 1000 N/A N/A
0.0191 1000 1.91 10
0.0610 1000 6.1 10
0.195 1000 19.5 10
0.625 1000 62.5 10
2.0 1000 200 10
The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.

Test organisms (species):

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

Details on test organisms:

Species: unicellular green alga Pseudokirchneriella subcapitata
Source: Scymaris laboratory culture
Strain: CCAP 278/4
Conditions: : from laboratory cultures maintained under axenic conditions.
Age: A 3-day old culture of the alga in the exponential growth phase was used as inoculum for the test.
Nominal test temperature: 22 ± 2°C

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22 ± 2°C

pH:

7.32 to 7.57

Nominal and measured concentrations:

Control and nominal concentrations of 0.0191, 0.0610, 0.195, 0.625 and 2.0 mg/L.
Control and mean measured concentrations of 0.0085, 0.023, 0.066, 0.18 and 0.58 mg/L.

Details on test conditions:

The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 22±2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. The position of each test replicate vessel in the incubator was randomised daily. One blank vessel (without algal inoculum) was incubated concurrently for each control and test concentration sampling occasion. The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 µm respectively. Each replicate test vessel was inoculated with 0.346 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.471 × 104 cells/mL. This value was used for growth calculations. After 24, 48, and 72 h, samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density. At the end of the test microscopic observations were made on samples taken from a single replicate of the control and each test substance concentration.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.057 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.023 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

in terms of cell particles per unit volume

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

in terms of cell particles per unit volume

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.066 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass

Growth rate:

The growth rate (0 to 72 h) was calculated for each replicate culture, according to the formula:

Groth rate = Logn (N2/N1)/t

where N₁       =         Cell density at start of test

           N₂       =         Celldensity at end of test

           t          =         Time interval (days)

The control was compared to the treatments by one-way analysis of variance and Equal Variance Two-Sample t test to identify significant differences (p<0.05). EC_Xwas calculated using the linear interpolation (ICPIN) method.

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows: 

	Test substance (mg/L)	95% Confidence Limits
NOEC	0.023	N/A
LOEC	0.066	N/A
ErC50	0.057	0.052-0.064
ErC20	0.037	0.033-0.039
ErC10	0.030	0.027-0.031

Yield:

This response is defined as the biomass at the end of the test minus the starting biomass For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. The control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). EC_Xwas calculated using the linear interpolation (ICPIN) method. The EC₅₀, EC₂₀and EC₁₀values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method. The yield mean values are shown in below table, together with the yields expressed as percentages of the control. The results obtained from these statistical analyses, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)	95% Confidence Limits
NOEC	0.023	N/A
LOEC	0.066	N/A
EyC50	0.046	0.040-0.047
EyC20	0.032	0.023-0.033
EyC10	0.027	0.015-0.028

Additional biological data:

The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the nominal 0.0191 and 0.0610 mg/L test concentrations appeared normal. Algal cells from the nominal 0.195 mg/L test concentration appeared small, deformed and some clumping was observed. No cells were observed in samples from the nominal 0.625 and 2.0 mg/L test concentrations.

Analytical results:

Nominal concentration of Test substance (mg/L)	Measured concentration of Test substance				Mean measured concentration*    (mg/L)	Mean measured concentration (% of nominal)
	0 hour		72 hour
	(mg/L)	% of nominal	(mg/L)	% of nominal
Culture medium control	<LOQ	-	<LOQ	-	-	-
0.0191	0.017	89	<LOQ	-	0.0085	44
0.0610	0.046^a	75	<LOQ^b	-	0.023	38
0.195	0.13	68	<LOQ	-	0.066	34
0.625	0.36	57	<LOQ	-	0.18	29
2.0	1.1	56	0.034	2	0.58	29

All measurements are quoted to 2 significant figures.

^a      Mean of triplicate analyses: 0.047, 0.046 and 0.044 mg/L

^b      Mean of triplicate analyses: <LOQ, <LOQ and <LOQ mg/L

* Arithmetic mean calculated for the control and all concentrations.

At 0 hours, the limit of quantification (LOQ) varied due to the dilution factor used for each concentration. The instrument LOQ was 0.002 mg/L. The culture medium control and nominal 0.0191 and 0.0610 mg/L concentrations were diluted x2, the nominal 0.195 mg/L concentration was diluted x4, the nominal 0.625 mg/L concentration was diluted x8 and the nominal 2.0 mg/L concentration was diluted x25. Therefore, the sample LOQs increased to 0.004, 0.008, 0.016 and 0.050 mg/L respectively.

At 72 hours, the instrument LOQ was 0.002 mg/L. The culture medium control and all test concentrations were diluted x2, giving a sample LOQ of 0.004 mg/L.

Where measured concentrations were less than the LOQ, values of zero were used to calculate the mean.

Algal cell particle density:

Nominal concentration of Test substance   (mg/L)	Mean measured concentration of Test substance (mg/L)	Replicate	Algal cell particle density (10^4cells/mL)
			24 hours	48 hours	72 hours
Culture medium control	0	A	2.00	7.46	35.7
		B	1.31	6.42	28.4
		C	2.15	6.93	32.6
		D	2.30	8.30	31.1
		E	2.04	8.38	35.1
		F	1.85	6.55	30.3
		Mean	1.94	7.34	32.2
0.0191	0.0085	A	2.40	9.04	37.4
		B	2.42	8.59	33.8
		C	2.60	7.70	34.2
		Mean	2.47	8.44	35.1
0.0610	0.023	A	2.46	7.47	36.2
		B	2.08	6.95	29.8
		C	2.29	7.78	34.1
		Mean	2.28	7.40	33.4
0.195	0.066	A	1.44	0.930	2.82
		B	1.92	1.08	1.71
		C	1.30	1.29	1.64
		Mean	1.55	1.10	2.06
0.625	0.18	A	0.556	0.720	2.12
		B	0.478	0.890	2.25
		C	0.952	1.60	4.28
		Mean	0.662	1.07	2.88
2.0	0.58	A	0 [-0.10]	0 [-0.65]	1.07
		B	0.246	0 [-0.54]	0 [-0.01]
		C	0 [-0.23]	0.200	0.302
		Mean	0.0820	0.0667	0.457

Inoculum (Day 0) cell density = 0.471 x 10⁴cells/mL.

Positive cell particle density values are quoted to 3 significant figures.

Where negative cell particle densities (shown in square parentheses) were calculated due to the subtracted blank particle density being higher than the test vessel particle density, these have been taken as zero (0) for calculation of the mean and statistical analysis.

Algae growth rate:

Nominal concentration of Test substance (mg/L)	Mean measured concentration of Test substance (mg/L)	Replicate	0-72 hours Growth rate (day^-1)
Culture medium control	0	A	1.442
		B	1.366
		C	1.412
		D	1.396
		E	1.437
		F	1.387
		Mean	1.407
0.0191	0.0085	A	1.458
		B	1.424
		C	1.428
		Mean	1.437
0.0610	0.023	A	1.447
		B	1.383
		C	1.427
		Mean	1.419
0.195	0.066	A	0.596
		B	0.429
		C	0.415
		Mean	0.480
0.625	0.18	A	0.501
		B	0.521
		C	0.736
		Mean	0.586
2.0	0.58	A	0.275
		B	-^a
		C	-^a
		Mean	0.275

All values are quoted to 3 decimal places.

^aIn the nominal 2.0 mg/L test concentration, the growth rates (0-72 hours) for replicates B and C were not calculable as (for replicate B) no cell particles were counted at 72 hours (negative cell particle densities were taken as zero) and (for replicate C) the number of cell particles counted at 72 hours were less than the inoculum value.

Mean growth rates over the test period:

Nominal concentration of Test substance (mg/L)	Mean measured concentration of Test substance (mg/L)	Mean growth rate/day (0-72 hours)	Mean growth rate/day 95% Cl	Percentage ofcontrol (%)
Culture medium control	0	1.41	1.38-1.44	-
0.0191	0.0085	1.44	1.39-1.48	102
0.0610	0.023	1.42	1.34-1.50	101
0.195	0.066	0.480 *	0.230-0.730	34
0.625	0.18	0.586 *	0.263-0.909	42
2.0	0.58	0.275 *	N/A	20

* Significant difference (p <0.05) from the control.

All biological measurements are quoted to 3 significant figures and percentages to the nearest integer.

Algal yeild:

Nominal concentration of Test substance (mg/L)	Mean measured concentration of Test substance (mg/L)	Replicate	Yield (10^4cells/mL)
			24 hours	48 hours	72 hours
Culture medium control	0	A	1.53	6.99	35.23
		B	0.84	5.95	27.93
		C	1.68	6.46	32.13
		D	1.83	7.83	30.63
		E	1.57	7.91	34.63
		F	1.38	6.08	29.83
		Mean	1.47	6.87	31.73
0.0191	0.0085	A	1.93	8.57	36.93
		B	1.95	8.12	33.33
		C	2.13	7.23	33.73
		Mean	2.00	7.97	34.66
0.0610	0.023	A	1.99	7.00	35.73
		B	1.61	6.48	29.33
		C	1.82	7.31	33.63
		Mean	1.81	6.93	32.90
0.195	0.066	A	0.97	0.46	2.35
		B	1.45	0.61	1.24
		C	0.83	0.82	1.17
		Mean	1.08	0.63	1.59
0.625	0.18	A	0.09	0.25	1.65
		B	0.01	0.42	1.78
		C	0.48	1.13	3.81
		Mean	0.19	0.60	2.41
2.0	0.58	A	0 [-0.57]	0 [-1.12]	0.60
		B	0 [-0.23]	0 [-1.01]	0 [-0.48]
		C	0 [-0.70]	0 [-0.27]	0 [-0.17]
		Mean	0.00	0.00	0.20

Yield values are quoted to 2 decimal places.

Where negative algal yields (shown in square parentheses) were calculated due to the subtracted blank particle density being higher than the test vessel particle density, these have been taken as zero (0) for calculation of the mean and statistical analysis.

Mean yeild over the test period:

Nominal concentration of Test substance (mg/L)	Mean measured concentration of Test substance (mg/L)	Mean yield (0-72 hours) (10^4cells/ml)	Mean yield (0-72 hours) 95% Cl	Percentage ofcontrol (%)
Culture medium control	0	31.7	28.8-34.7	-
0.0191	0.0085	34.7	29.8-39.6	109
0.0610	0.023	32.9	24.8-41.0	104
0.195	0.066	1.59 *	-0.0578-3.23	5
0.625	0.18	2.41 *	-0.596-5.42	8
2.0	0.58	0.200 *	-0.661-1.06	1

* Significant difference (p <0.05) from the control.

Mean yield values are quoted to 3 significant figures and percentages to the nearest integer.

Test solution pH measurements:

Nominal concentration of Test substance (mg/L)	Mean measured concentration of Test substance (mg/L)	pH
		0 hours	72 hours
Culture medium control	0	7.53	7.32
0.0191	0.0085	7.54	7.70
0.0610	0.023	7.57	7.71
0.195	0.066	7.56	7.48
0.625	0.18	7.57	7.40
2.0	0.58	7.56	7.36

Test incubator temperature:

Time (hours)	Current Temperature (°C)	Maximum Temperature (°C)	Minimum Temperature (°C)
0	22.2	-	-
24	22.2	22.6	22.1
48	21.9	22.3	21.8
72	22.1	22.2	22.0

Max/Min thermometer checked against a mercury thermometer (±0.2°C)

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, NOEC and LOEC values for the test substance for toxicity to freshwater green algae, were determined to be 0.057, 0.03, 0.046, 0.027, 0.023 and 0.066 mg/L respectively.

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, 'C18 -unsatd and C22 -unsatd. AAP EDM-ES' (active: 104%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and solvent control and triplicates of each concentration of the test substance were employed. Three replicate algal cultures, with a nominal cell density of approximately 0.5 x 10⁴cells/mL, were exposed to test substance at each nominal concentrations of 0.0191, 0.0610, 0.195, 0.625 and 2.0 mg/L in AAP medium for 72 h. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.483 × 104cells/mL and was used for growth calculations. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC method of analysis. The mean measured concentrations of the test substances were determined to be 0.0085, 0.023, 0.066, 0.18 and 0.58 mg/L; therefore the test results were expressed in terms of measured concentrations. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The ErC50 and ErC10 values for growth rate were found to be 0.057 and 0.03 mg/L respectively, whereas, EyC50 and EyC10 values for cell particle density were found to be 0.046 and 0.027 mg/L respectively. The NOEC and LOEC values were found to be 0.023 and 0.066 mg/L respectively for both growth rate and cell particle densities. The study results were considered to have fulfilled all the validity criteria. Under study conditions,the 72 h EC50 and EC10 values of the test substance for toxicity to freshwater green algae, were determined to be 0.057, 0.03 mg/L, based on growth rate, and 0.046, 0.027 mg/L (measured), based on biomass respectively. The NOEC value was determined at 0.023 mg/L (measured)(Scymaris, 2018).

Description of key information

Based on the results of the study, the 72 h EC50 and EC10 values of the test substance for toxicity to freshwater green algae, were determined to be 0.057, 0.03 mg/L, based on growth rate, and 0.046, 0.027 mg/L (measured), based on biomass respectively. The NOEC was determined at 0.023 mg/L (measured).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.057 mg/L

EC10 or NOEC for freshwater algae:

0.03 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, 'C18 -unsatd and C22 -unsatd. AAP EDM-ES' (active: 104%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and solvent control and triplicates of each concentration of the test substance were employed. Three replicate algal cultures, with a nominal cell density of approximately 0.5 x 10⁴cells/mL, were exposed to test substance at each nominal concentrations of 0.0191, 0.0610, 0.195, 0.625 and 2.0 mg/L in AAP medium for 72 h. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.483 × 104cells/mL and was used for growth calculations. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC method of analysis. The mean measured concentrations of the test substances were determined to be 0.0085, 0.023, 0.066, 0.18 and 0.58 mg/L; therefore the test results were expressed in terms of measured concentrations. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The ErC50 and ErC10 values for growth rate were found to be 0.057 and 0.03 mg/L respectively, whereas, EyC50 and EyC10 values for cell particle density were found to be 0.046 and 0.027 mg/L respectively. The NOEC and LOEC values were found to be 0.023 and 0.066 mg/L respectively for both growth rate and cell particle densities. The study results were considered to have fulfilled all the validity criteria. Under study conditions, the 72 h EC50 and EC10 values of the test substance for toxicity to freshwater green algae, were determined to be 0.057, 0.03 mg/L, based on growth rate, and 0.046, 0.027 mg/L (measured), based on biomass respectively. The NOEC value was determined at 0.023 mg/L (measured) (Scymaris, 2018).