Calcium di(octanoate)

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

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:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations
- Sampling method: At 24, 48 and 72 hours, media of each treatment group was pooled (10 mL from each vessel) and 2 x 15 mL samples were taken from the pooled media.
- Sample storage conditions before analysis: Samples were frozen until required for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was prepared at 100 mg/L by dispersing 80.3 mg of test item in 1000 mL of OECD medium. To disperse the test item, the solution was shaken and treated with ultrasound for 20 minutes. An aliquot (313 mL) was diluted to 1000 mL with OECD medium to give the stock at 25.0 mg/L. This stock was then diluted (333 mL made up to 1000 mL) with OECD medium to give the next concentration. The remaining stocks were prepared by serial dilution in the same way.
- Eluate: OECD test medium
- Differential loading: Yes
- Controls: Untreated test medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported during test. A formulation trial showed that the test item formed a hazy dispersion after shaking and treatment with ultrasound for 15 - 20 minutes.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP)
- Age of inoculum (at test initiation): 72 hours
- 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. After 72 hours the concentration of algal cells (per ml-1) was determined and two secondary cultures were prepared by inoculating 100 ml of OECD medium to give an initial start biomass of ca. 5 x 103 cell ml-1. These cultures were then incubated under conditions similar to the test for approximately 72 hours. This method of culture ensured that the algal cells were in an exponential growth phase when used in the study. One of these cultures was then used as the inoculum in the study. The initial algal biomass of this culture was 323,750 cell/ml.

ACCLIMATION
- Acclimation period: None
- Culturing media and conditions (same as test or not): Yes
- 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:

21.6 to 23.4 °C

pH:

6.97 to 7.83

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Nominal: 0.763, 2.44, 7.81, 25.0 and 80.0 mg/L
Geometric mean measured: 0.107, 0.161, 0.456, 1.18 and 4.65 mg/L

As the test item was unstable under the condition of the test, the geometric mean concentration for the exposure groups was calculated. This was calculated using a value that was half the LOD (0.14 mg/L) of the analytical method i.e. 0.07 mg/L in samples where the test item was not detected.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml conical flasks filled with 100 ml test medium
- Aeration: None. The test vessels were stoppered with air permeable stoppers and placed on an orbital platform shaker and continually agitated at ca. 140 rpm in order to facilitate gaseous exchange.
- Initial cells density: 5,000 cells/mL
- Control end cells density: Mean = 545,000 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: Distilled water
- 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
- Sterile test conditions: Yes; sterile conical flasks used
- Adjustment of pH: None
- Photoperiod: Continuous
- Light intensity and quality: 7450 to 7990 Lux (mean 7653 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Haemocytometer
- Chlorophyll measurement: No
- Other: Microscopic observations were made of the appearance of the cell cultures during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: Nominal concentrations of 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval 23.0 to 43.9 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval 1.35 to 1.88 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 1.01 to 9.97 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.223 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 0.139 to 0.312 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 5.09 to 17.6 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.489 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 0.368 to 0.618 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.763 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The NOEC for specific growth rate is below the limit of quantification of the analytical method.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.107 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Geometric mean concentration < LoD

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.161 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.08 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence interval: 3.43 to 4.86 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence interval: 0.212 to 0.368 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.763 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.107 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.984 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence interval: 0.721 to 1.29 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

< 0.107 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.763 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: The NOEC for yield nominal results is below the limit of quantification of the analytical method.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.107 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.161 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test):
- Unusual cell shape - At the nominal 2.44 and 7.81 mg/L concentrations some small and abnormal shaped cells were observed. At the nominal 25.0 and 80.0 mg/L concentrations small and abnormal shaped cells were observed, and at the nominal 80.0 mg/L concentration swollen cells were observed.
- Colour differences: None reported
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Other: During the test it was noted that at the nominal concentrations of 7.81 to 80.0 mg/L, there was significant bacterial growth. This was most pronounced in the groups at 25.0 and 80.0 mg/L. No significant bacterial contamination was noted in the controls. As this bacterial contamination was only observed in the test groups, the source of the bacteria was (possibly) the test item itself.
- Any stimulation of growth found in any treatment: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No observations reported
- Effect concentrations exceeding solubility of substance in test medium: No
- Analytical chemistry: At the start of the test (0 hours) the analytical recoveries of the test item ranged from 49% of nominal (at 0.763 mg/L) to 97% of nominal. Concentrations from 2.44 to 80.0 mg/L ranged from 80% to 97% of nominal. At 24 hours, test item was not measured at nominal concentrations of 0.763 and 2.44 mg/L and at nominal concentrations of 7.81 to 80.0 mg/L, recoveries were 17% to 82% of nominal. By 48 hours the test item was not detected in the concentrations at 0.763 to 25.0 mg/L and 2% of nominal was measured at 80.0 mg/L. By 72 hours the test item was not detected in any of the test groups. These results indicated that the test item was not stable under the conditions of the test. As the test item was unstable under the condition of the test, the geometric mean concentration for the exposure groups was calculated. This was calculated using a value that was half the LOD (0.14 mg/L) of the analytical method (i.e. 0.07 mg/L) in samples where the test item was not detected. All study results were reported in terms of the nominal concentrations and the geometric mean concentrations.
At 0.763 mg/L, the 0-hour analytical recovery was 0.378 mg/L. This result was confirmed with the analysis of the reserve sample. This result was below the LOQ of the analytical method, 0.54 mg/L. Attempts to decrease the LOQ of the method were unsuccessful. Therefore, as the value was measured but below the LOQ, it was reported and included in the calculations of the study end points. The Study Director acknowledges that this data point is below the limit of quantitation of the analytical method for the test item.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 2.17 mg/L for specific growth rate and 0.819 mg/L for yield (biomass)

Reported statistics and error estimates:

All study end points were statistically analysed in terms of the nominal calcium dioctanoate concentrations and the geometric mean measured concentrations.

For the EC10, EC20 and EC50 calculations a dose-response curve was fitted to the data – percentage inhibition vs log concentration. Where possible the EC10, EC20 and EC50 and the 95% confidence intervals were calculated using regression analysis.

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. The LOEC was defined as the lowest test concentration that has a statistically significant effect (p-value ≤ 0.05) when compared to the control.

Table 1: Cell counts (cells/mL)

Nominal test item conc. (mg/L)	Cells/mL						Mean %Iy
	0 hours	24 hours	48 hours	72 hours	72 -hours mean cell count	72 -hours mean yield
Control	5000	21667	142778	552500	545000	540000	N/A
		20000	108333	350000
		21667	117778	592500
		23889	124444	416250
		25000	187778	637500
		27778	140556	721250
0.763		18333	142222	670000	451667	446667	17
		24444	160556	415000
		18889	88333	270000
2.444		18889	110000	366250	340417	335417	38
		24444	146111	378750
		15556	101111	276250
7.81		13889	81667	170000	195000	190000	65
		19444	92222	207222
		21111	70000	207778
25.0		7778	30000	93333	87222	82222	85
		7778	37778	86111
		6111	34444	82222
80.0		5000	7778	7778	9444	4444	99
		4444	8333	10000
		5000	5000	10556

%Iy = percentage inhibition of yield. N/A = not applicable

Table 2: Summary of Specific Growth Rate Data

Nominal Test Item Conc. (mg/L)	Mean Average Specifc Growth Rate per Day (0 to 72 hours)	Mean % Inhibition (0 to 72 hours)
Control	1.554	0
0.763	1.478	4.9
2.44	1.404	9.7
7.81	1.220	22
25.0	0.953	39
80.0	0.209	87

Table 3: E_rC_x and E_yC_x, LOEC and NOEC dat

	Test Item Concentration (mg/L)
	Nominal	Geometric Mean Measured
Specific Growth Rate
ErC10	5.40 [1.01, 9.97]	0.223 [0.139, 0.312]
ErC20	11.0 [5.09, 17.6]	0.489 [0.368, 0.618]
ErC50	32.3 [23.0, 43.9]	1.60 [1.35, 1.88]
NOEC	0.763	0.107
LOEC	2.44	0.161
Yield
EyC10	< 0.763	< 0.107
EyC20	0.984 [0.721, 1.29]	< 0.107
EyC50	4.08 [3.43, 4.86]	0.280 [0.212, 0.368]
NOEC	0.763	0.107
LOEC	2.44	0.161

[ ] = 95 % confidence intervals

Table 4: Observations

Nominal Test Item Concentration (mg/L)	Summary Observations (24 - 72 hours)
Control	Cells appear normal
0.763	Cells appear normal
2.44	Normal cells, Some small and abnormal shaped cells
7.81	Some small and abnormal shaped cells, normal cells
25.0	Small and abnormal shaped cells
80.0	Small and abnormal shaped cells, swollen cells

Table 5: pH and temperature of the test media at the start and end of the test

Treatment Solution No.	Nominal Concentration (mg/L)	pH		Temperature (°C)
		0 hours	72 hours	0 hours	72 hours
1 (Control)	0	7.00	7.83	22.8	21.6
2	0.763	7.08	7.77	22.8	21.9
3	2.44	7.06	7.74	23.0	21.9
4	7.81	6.98	7.68	23.1	21.7
5	25.0	7.02	7.78	23.2	22.0
6	80.0	6.97	7.74	23.4	22.0

Table 6: Analytical recoveries and geometric mean calculations

Nominal Conc. (mg/L)	Analytical Recoveries
	T0 hours		T24 hours		T48 hours		T72 hours		Geometric mean (mg/L)
	mg/L	% Nominal	mg/L	% Nominal	mg/L	% Nominal	mg/L	% Nominal
0 (Control)	ND	NA	ND	NA	ND	NA	ND	NA	NA
0.763	0.378	<LOQ	0.0700	ND	0.0700	ND	0.0700	ND	0.107
2.44	1.96	80	0.0700	ND	0.0700	ND	0.0700	ND	0.161
7.81	6.61	85	1.33	17	0.0700	ND	0.0700	ND	0.456
25.0	24.7	99	16.2	65	0.0700	ND	0.0700	ND	1.18
80.0	77.8	97	65.3	82	1.31	2	0.0700	ND	4.65

ND = not detected, the concentration is below the LOD of the analytical method (0.14 mg/L).

NA = Not applicable

< LOQ = Less than the limit of quantification for the analytical method (0.56 mg/L)

Validity criteria fulfilled:

yes

Remarks:

Specific growth rate and factor increase in controls: 1.55 and 109, respectively (must be >0.92 and >16, respectively); CoV section specific growth rate in controls: 14.3% (must be ≤35%); CoV average specific growth rate at 72 h: 5.9% (must be ≤7%)

Conclusions:

The 72-hour ErC50 and ErC10 were calculated to be 32.3 mg test item/L and 5.40 mg test item/L, respectively, based on nominal concentrations and 1.60 mg test item/L and 0.223 mg test item/L, respectively, based on geometric mean measured concentrations. The 72-hour EyC50 and EyC10 were calculated to be 4.08 mg test item/L and <0.763 mg test item/L, respectively, based on nominal concentrations and 0.280 mg test item/L and <0.107 mg test item/L, respectively, based on geometric mean measured concentrations.

Executive summary:

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was determined in a study following OECD guideline 201. Algae were exposed to nominal concentrations of 0.763, 2.44, 7.81, 25.0 and 80.0 mg test item/L, equivalent to geometric mean measured concentrations of 0.107, 0.161, 0.456, 1.18 and 4.65 mg test item/L alongside a control.

At the start of the test the analytical recoveries of the test item ranged from 49% to 97% of nominal. At 24 hours, test item was not measured at nominal concentrations of 0.763 and 2.44 mg/L and at nominal concentrations of 7.81 to 80.0 mg/L, recoveries were 17% to 82% of nominal.  By 48 hours the test item was not detected in the concentrations at 0.763 to 25.0 mg/L and 2% of nominal was measured at 80.0 mg/L.  By 72 hours the test item was not detected in any of the test groups. As the test item was unstable under the condition of the test, the geometric mean concentration for the exposure groups was calculated.  This was calculated using a value that was half the LOD (0.14 mg/L) of the analytical method i.e. 0.07 mg/L in samples where the test item was not detected. All study results were reported in terms of the nominal concentrations and the geometric mean concentrations.

The 72-hour ErC50 and ErC10 were calculated to be 32.3 mg test item/L and 5.40 mg test item/L, respectively, based on nominal concentrations and 1.60 mg test item/L and 0.223 mg test item/L, respectively, based on geometric mean measured concentrations. The 72-hour EyC50 and EyC10 were calculated to be 4.08 mg test item/L and <0.763 mg test item/L, respectively, based on nominal concentrations and 0.280 mg test item/L and <0.107 mg test item/L, respectively, based on geometric mean measured concentrations.

The study is a GLP compliant, guideline experimental study and is available as an unpublished report. There are no restrictions and it is fully adequate for assessment.

Description of key information

The 72-hour ErC50 and ErC10 were calculated to be 32.3 mg test item/L and 5.40 mg test item/L, respectively, based on nominal concentrations and 1.60 mg test item/L and 0.223 mg test item/L, respectively, based on geometric mean measured concentrations. The 72-hour EyC50 and EyC10 were calculated to be 4.08 mg test item/L and <0.763 mg test item/L, respectively, based on nominal concentrations and 0.280 mg test item/L and <0.107 mg test item/L, respectively, based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.6 mg/L

EC10 or NOEC for freshwater algae:

0.223 mg/L

Additional information

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was determined in a study following OECD guideline 201 (Dickinson 2018). Algae were exposed to nominal concentrations of 0.763, 2.44, 7.81, 25.0 and 80.0 mg test item/L, equivalent to geometric mean measured concentrations of 0.107, 0.161, 0.456, 1.18 and 4.65 mg test item/L alongside a control.

At the start of the test the analytical recoveries of the test item ranged from 49% to 97% of nominal. At 24 hours, test item was not measured at nominal concentrations of 0.763 and 2.44 mg/L and at nominal concentrations of 7.81 to 80.0 mg/L, recoveries were 17% to 82% of nominal.  By 48 hours the test item was not detected in the concentrations at 0.763 to 25.0 mg/L and 2% of nominal was measured at 80.0 mg/L.  By 72 hours the test item was not detected in any of the test groups. As the test item was unstable under the condition of the test, the geometric mean concentration for the exposure groups was calculated.  This was calculated using a value that was half the LOD (0.14 mg/L) of the analytical method i.e. 0.07 mg/L in samples where the test item was not detected. All study results were reported in terms of the nominal concentrations and the geometric mean concentrations.

The 72-hour ErC50 and ErC10 were calculated to be 32.3 mg test item/L and 5.40 mg test item/L, respectively, based on nominal concentrations and 1.60 mg test item/L and 0.223 mg test item/L, respectively, based on geometric mean measured concentrations. The 72-hour EyC50 and EyC10 were calculated to be 4.08 mg test item/L and <0.763 mg test item/L, respectively, based on nominal concentrations and 0.280 mg test item/L and <0.107 mg test item/L, respectively, based on geometric mean measured concentrations.

The study is a GLP compliant, guideline experimental study and is available as an unpublished report. There are no restrictions and it is fully adequate for assessment.