Onium compounds, morpholinium, 4-ethyl-4-soya alkyl, Et sulfates

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 - 24 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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)

Remarks:

The department of health of the government of the united kingdom, Brixham, UK

Test material

Test material information

Constituent 1

Reference substance name:

Onium compounds, morpholinium, 4-ethyl-4-soya alkyl, Et sulfates

EC Number:

263-167-0

EC Name:

Onium compounds, morpholinium, 4-ethyl-4-soya alkyl, Et sulfates

Cas Number:

61791-34-2

Molecular formula:

Not available for UVCB substance

IUPAC Name:

4-ethyl-4-(hexadecanoyloxy)morpholin-4-ium 4-ethyl-4-[(9E)-octadec-9-enoyloxy]morpholin-4-ium 4-ethyl-4-[(9E,12E)-octadeca-9,12-dienoyloxy]morpholin-4-ium triethyl sulfate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At 0 hour sampling occasion, an aliquot was removed from each of the excess dosing solutions (three aliquots for 0.025 mg/L) and an equivalent volume of tetrahydrofuran (THF) was added (resulting in a x2 dilution). The sample was mixed and an aliquot transferred to an LCMS vial for analysis. At 72 hour sampling occasion, an aliquot was removed from one test vessel from each concentration (three test vessels for 0.025 mg/L) and an equivalent volume of THF was added (resulting in a x2 dilution). The sample was mixed and an aliquot transferred to an LCMS vial for analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A primary stock concentrate of the test item, with a nominal concentration of 10 mg/L, was prepared by weighing a nominal 0.01 g of test substance (actual weight: 0.00998 g) and making up to 1000 mL volume with the culture medium AAP in a volumetric flask. The stock was then stirred for 12 minutes. The resultant stock was a clear and colourless solution 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.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium only
- Evidence of undissolved material: The test solutions were all observed to be clear and colourless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source: laboratory culture
- Strain: CCAP 278/4
- Age of inoculum (at test initiation): 3-day old culture of the alga in the exponential growth phase was used as inoculum for the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.6 - 22.2

pH:

7.30 - 8.22

Nominal and measured concentrations:

Nominal concentrations of control, 0.007, 0.013, 0.025, 0.05 and 0.1 mg/L.
Mean measured concentrations of control, 0, 0.0043, 0.016, 0.022 and 0.044 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass vessel
- Material, size, headspace, fill volume: conical flasks: 250 mL, headspace: 150 mL, fill volume: 100 mL
- Initial cells density: 0.5 x 10E04 cells/mL (One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.526 × 104 cells/mL. This value was used for growth calculations.)
- Control end cell density: 39.2 x 10E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP-medium)

TEST MEDIUM / WATER PARAMETERS
- according to guideline
- Intervals of water quality measurement: The pH of the control and test solutions was measured at the start of the test. At the end of the test the pH of one replicate vessel (containing algae) from each test concentration was determined.
The temperature of the incubator was measured daily. The light intensity was measured daily.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: 6000 lux, "cool-white" illumination

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: electronic particle counting, using a Coulter counter
- other: 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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes (non-GLP)
- Test concentrations: Culture medium control, 0.01, 0.1, 1.0, 10, and 100 mg/L.
- Results used to determine the conditions for the definitive study: 0.1 mg/L: Single algal cells are deformed and some appear to have clumped together. 1, 10 and 100 mg/L: Algal cells are deformed and appear to be clumped together. A 0.1 mg/L test concentration was selected as the highest concentration.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.021 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.019-0.028

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.016 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.0045-0.018

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the nominal 0.007 and 0.013 mg/L test concentrations appeared normal. Algal cells from the nominal 0.025 and 0.05 mg/L appeared small and deformed (very few cells were observed in the 0.05 mg/L concentration). No cells were observed in a sample from the 0.1 mg/L test concentration.

Reported statistics and error estimates:

The data for cell counts obtained at 24, 48 and 72 hours was entered into electronic data files and statistically analysed using CETIS, where appropriate procedures were applied to test for significant differences (p <0.05) between the control and test concentrations and determine EC50, EC20 and EC10 values and the associated 95% confidence intervals where appropriate. As the measured concentrations at 0 and 72 hours for the nominal 0.007 mg/L were below the limit of quantification (0.006 mg/L), it was not included in the statistical analyses as it could skew the ECX calculations.

Any other information on results incl. tables

The measured concentrations at the start of the study were 0-105% of nominal and at the end were 0-39% of nominal. On this basis the mean measured concentrations were used for the calculation of the effect values.

Table 1: Algal cell particle density

Nominal concentration (mg/L)	Mean measured concentration (mg/L)	Replicate	Algal cell particle density (10^4cells/mL)
			24 hours	48 hours	72 hours
Culture medium control	0	A	1.96	8.55	34.7
		B	2.18	9.91	39.6
		C	3.10	9.89	40.2
		D	1.74	9.30	39.3
		E	2.19	10.0	42.8
		F	1.89	10.1	38.7
		Mean	2.18	9.63	39.2
0.007	0	A	3.31	10.50	36.7
		B	3.23	8.50	40.6
		C	2.27	8.77	39.7
		Mean	3.07	8.60	39.0
0.013	0.0043	A	2.99	10.50	47.7
		B	2.23	8.50	43.4
		C	2.27	8.77	39.8
		Mean	2.50	9.26	43.6
0.025	0.016	A	2.62	3.60	17.7
		B	2.92	6.54	29.2
		C	3.66	8.00	36.0
		Mean	3.07	6.05	27.6
0.05	0.022	A	1.06	0 [-0.02]	2.38
		B	2.12	2.51	7.67
		C	1.96	1.11	3.21
		Mean	1.71	1.21	4.42
0.1	0.044	A	1.32	0 [-0.38]	0.120
		B	0.736	0 [-0.98]	0 [-0.12]
		C	1.02	0 [-1.48]	0 [-0.51]
		Mean	1.03	0.00	0.0400

Table 2: Results based on growth rate and yield

	Test item (mg/L)	95% Confidence Limits
NOECgrowth rate	0.016	N/A
LOECgrowth rate	0.022	N/A
ErC20	0.017	0.015-0.019
NOECyield	0.0043	N/A
LOECyield	0.016	N/A
EyC50	0.018	0.011-0.020
EyC20	0.011	0.0052-0.022
EyC10	0.0078	0.0047-0.018

Table 3: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	increased by a factor of 74.5	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	11%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.6%	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

please refer to "any other information on results incl. tables"