Calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl)aminoacetate]

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:

08 May to 11 May 2018 (definitive test)

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:

The temperature of the test area was briefly below the minimum (21.0 °C) for this type of test on one occasion, however this deviation was not considered to have impacted or affected the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SOAP005-2
- Expiration date of the lot/batch: 15 October 2022
- Purity: min. 95%
- Purity test date: Not reported


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and control
- Sampling method: At the start of the test (0 h) samples for chemical analysis were taken from the remaining test media and at 24, 48 and 72 h test media from replicates of each treatment group were pooled and the samples were taken from the pooled test media.
- Sample storage conditions before analysis: Samples were kept frozen until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was prepared at 100 mg/L by dispersing 50.6 mg of test item in 500 mL of OECD medium. To disperse the test item, the solution was shaken and treated with ultrasound for 20 minutes. An aliquot (81 mL) was diluted to 1000 mL with the medium to give the stock at 8.10 mg/L. This stock was then diluted (333 ml made up to 1000 ml) with medium to give the next concentration. The remaining stocks were prepared by serial dilution in the same way.
- Controls: OECD medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP)
- Age of inoculum (at test initiation): Stock algae was in the exponential growth phase
- Method of cultivation: A primary culture was established by the addition of approximately 0.1 ml of the algal stock to 100 mL of OECD medium contained in a 250 mL conical flask. The culture was incubated for ca. 72 hours under conditions similar to the test.

ACCLIMATION
- Acclimation period: approximately 72 hours
- Culturing media and conditions (same as test or not): The same as test
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

Test start: 22.6 - 22.9 °C
Test end: 21.6 - 22.6°C

pH:

Test start: 6.88 - 7.13
Test end: 7.68 - 7.81

Dissolved oxygen:

Not reported.

Salinity:

N/A

Nominal and measured concentrations:

Nominal: 0.1, 0.3, 0.9, 2.7 and 8.1 mg/L
Geometric mean measured: 0.05, 0.12, 0.379, 0.582 and 3.87 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile conical flasks
- Type (delete if not applicable): The test vessels were stoppered with air permeable stoppers in order to reduce evaporation and prevent dust and microorganisms from entering the solutions.
- Material, size, headspace, fill volume: Aliquots (100 mL) of the solutions were dispensed into sterile conical flasks with a total capacity of 250 mL.
- Aeration: The test vessels were placed on an orbital platform shaker and continually agitated at ca. 140 rpm in order to facilitate gaseous exchange.
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 5E3 cells/mL
- Control end cells density: approx. 5.11E5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH and temperature were determined in each of the bulk solutions and the media prior to starting the test and in a single vessel from each concentration at the end of the test period.


OTHER TEST CONDITIONS
- Adjustment of pH: The initial pH of the test media was measured and then adjusted to pH 8.1 ± 0.1, as required, using 1M NaOH or HCl
- Photoperiod: continuous illumination
- Light intensity and quality: 7840 – 7910 (mean 7858) lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The cell biomass was determined by sampling every 24 hours (± 1 hour).
- Determination of cell concentrations: Cell biomass was determined by cell counts using a haemocytometer.

- Other: Microscopic observations were made of the appearance of the cell cultures during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.0
- Range finding study: yes
- Test concentrations in the range finder test: 100, 10, 1.0 and 0.1 mg test item/L
- Results used to determine the conditions for the definitive study: The results of the range finder test showed that there was 8% inhibition of biomass at 0.1 mg test item L-1, at 1.0 mg test item L-1, 30% inhibition was observed and at 10 and 100 mg test item L-1, 100% inhibition of yield was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate, test conducted on 26 April 2018

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence intervals: 0.870 - 1.29 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.241 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence intervals: NA - 0.564 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence intervals: 3.34 - 4.02 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence intervals: 0.5 - 2.92 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.12 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.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.379 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.671 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals: 0.295 - 0.877 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.337 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals: 0.255 - 0.373 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals: 1.19 - 1.84 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals: 0.426 - 0.498 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.379 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): In the 0.1 and 0.3 mg/L treatments cells appeared normal. In the 0.9 mg/L treatment there were some small cells, in the 2.7 mg/L treatment there were small cells, swollen cells and abnormal shaped cells and in the 8.1 mg/L treatment there were small and abnormal shaped cells.

Results with reference substance (positive control):

The results of the most recent reference test (Study No. ENV-18-012, 26 April 2018), with potassium dichromate, showed that P. subcapitata had an EC50 for specific growth rate of 2.17 mg/L. The EC50 for the inhibition of yield (biomass) was 0.819 mg/L. These results indicated that the methods used to culture and test P. subcapitata were acceptable.

Reported statistics and error estimates:

Statistical analysis was carried out using CETIS™ v 1.9.7.2 software. The NOEC and LOEC values based on growth rate and yield (biomass) were estimated using a Dunnett multiple comparison test. The replicates for each control and treatment group were pooled before analysis then non-linear regression model with the best fit to the specific data set was applied. To calculate the ECx values for growth rate a 3 Parameter Cumulative log-normal (Probit) test was used and for biomass a 3 Parameter Log-logistic test was used.

The NOEC was defined as the test concentration below the lowest concentration that resulted in a statistically significant effect. Statistical significance was defined as a p-value ≤ 0.05. Where no statistical significance is seen at any of the treatment groups the NOEC was taken to be the highest concentration tested.

Where possible the ECx and their 95% confidence intervals were calculated using regression analysis.

Table 1: Summary of specific growth rate data

Nominal test concentration (mg/L)	Mean average specific growth rate/day (0 to 72 h)	Mean % inhibition (0 to 72 h)
Control	1.539	0
0.1	1.519	1.3
0.3	1.548	-0.6
0.9	1.457	5*
2.7	0.949	38*
8.1	0.321	80*

*statistically significantly different from the control (p = ≤0.05).

Table 2. The cell counts and percentage of yield inhibition

Nominal test item conc. (mg/L)	Number of cells (cells / mL )
	0 Hours	72 -hour mean cell count	72-hours mean yield	Mean percentage inhibition of yield
Control	5,000	511,875	506,875	N/A
0.1		483,333	478,333	6
0.3		523,333	518,333	-2
0.9		404,167	399,167^a	21
2.7		86,296	81,296^a	84
8.1		13,148	8,148^a	98

N/A = not applicable.

^a Statistically significantly different from the control (p = ≤0.05).

Table 3: Analytical recoveries of calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate]

	Analytical recoveries expressed as test item mg/L
Nominal test item conc. (mg/L)	T0 hours		T72 hours		Geometric mean (mg/L)	Geometric mean as % nominal
	mg/L	% Nominal	mg/L	% Nominal
0	0.009^a	NA	0.023^a	NA	NA	NA
0.1	0.013^b	13	0.091^b	91	0.0500^b,c	50^c
0.3	0.042^b	14	0.287	96	0.120^c	40^c
0.9	0.206	23	0.699	78	0.379	42
2.7	0.654	24	0.518	19	0.582	22
8.1	5.089	63	2.948	36	3.87	48

NA = not applicable. The limit of quantification (LOQ) for calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate] was 0.1 mg/L. The limit of detection for the method was 0.002 mg/L.

^acalcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate] was detected in the controls however this was < LOQ.

^bThese results were below the LOQ of the analytical method. 

^cAs the measured values for these samples were below the LOQ of the analytical method, the final geometric mean has been calculated using a value of half the LOQ. 

Validity criteria fulfilled:

yes

Remarks:

Specific growth rate and factor increase for controls were 1.54 and 102.4, respectively (>16); mean CoV section by section specific growth rates controls was 15.5% (<35%); CoV average specific growth rates in replicate controls at 72 hours 3.8% (<7%)

Conclusions:

Based on nominal concentrations, the 72-hour ErC50 and EyC50 for the green algae, P. subcapitata, were 3.67 mg/L and 1.48 mg/L, respectively. The 0 to 72-hour NOEC for growth rate and biomass was 0.3 mg/L and the 0-72 hour LOEC for growth rate and biomass was 0.9 mg/L. Based on geometric mean concentrations, the 72-hour ErC50 and EyC50 were 1.21 mg/L and 0.460 mg/L respectively. The 0 to 72-hour NOEC for growth rate and biomass was 0.120 mg/L and the 0-72 hour LOEC for growth rate and biomass was 0.379 mg/L.

Executive summary:

A study was performed according to the OECD 201 guideline to assess the effects of the test item, calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl)aminoacetate], on the growth of the green alga Pseudokirchneriella subcapitata (Dickinson 2019).

The cells were exposed to five nominal concentrations of calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate], 0.100, 0.300, 0.900, 2.70 and 8.10 mg/L for 72 hours under static conditions. The test item concentrations were analysed using a LC-MS MRM method and the geometric mean concentrations were 0.05, 0.120, 0.379, 0.582 and 3.87 mg/L. As analytical recoveries were outside the range of 100% ± 20% of nominal, study endpoints were expressed in terms of both nominal concentration and geometric mean measured concentrations. All validity criteria for the definitive test were met. The data were statistically analysed with CETIS v1.9.2.7 statistical software package.

The study was conducted according to the OECD guideline and is GLP-compliant, therefore is reliable without restrictions and is considered as suitable as a key study for this endpoint.

Based on nominal concentrations, the 72-hour ErC50 and EyC50 for the green algae, P. subcapitata, were 3.67 mg/L and 1.48 mg/L respectively. The 0 to 72-hour NOEC for growth rate and biomass was 0.3 mg/L and the 0-72 hour LOEC for growth rate and biomass was 0.9 mg/L.

Based on geometric mean concentrations, the 72-hour ErC50 and EyC50 were 1.21 mg/L and 0.460 mg/L respectively. The 0 to 72-hour NOEC for growth rate and biomass was 0.120 mg/L and the 0-72 hour LOEC for growth rate and biomass was 0.379 mg/L.

Description of key information

Based on nominal concentrations, the 72 hour ErC50 and EyC50 for the green algae, P. subcapitata, were 3.67 mg/L and 1.48 mg/L respectively. The 72-hour NOEC was 0.3 mg/L and the 72 hour LOEC was 0.9 mg/L, based on growth rate and yield. Based on geometric mean concentrations, the 72-hour ErC50 and EyC50 were 1.21 mg/L and 0.460 mg/L respectively. The 0 to 72-hour NOEC for growth rate and biomass was 0.120 mg/L and the 0-72 hour LOEC for growth rate and biomass was 0.379 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.21 mg/L

EC10 or NOEC for freshwater algae:

0.12 mg/L

Additional information

A study was performed according to the OECD 201 guideline to assess the effects of the test item, calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl)aminoacetate], on the growth of the green alga Pseudokirchneriella subcapitata (Dickinson 2019).

The cells were exposed to five nominal concentrations of calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate], 0.100, 0.300, 0.900, 2.70 and 8.10 mg/L (geometric mean concentrations: 0.05, 0.120, 0.379, 0.582 and 3.87 mg/L) for 72 hours under static conditions.

Based on nominal concentrations, the 72-hour ErC50 and EyC50 for the green algae, P. subcapitata, were 3.67 mg/L and 1.48 mg/L respectively. The 0 to 72-hour NOEC for growth rate and biomass was 0.3 mg/L and the 0-72 hour LOEC for growth rate and biomass was 0.9 mg/L. Based on geometric mean concentrations, the 72-hour ErC50 and EyC50 were 1.21 mg/L and 0.460 mg/L respectively. The 0 to 72-hour NOEC for growth rate and biomass was 0.120 mg/L and the 0-72 hour LOEC for growth rate and biomass was 0.379 mg/L.

The study is a guideline study and is GLP-compliant, therefore is reliable without restrictions and is considered as suitable as a key study for this endpoint.