Oleic acid, compound with 2-amino-2-methylpropan-1-ol (1:1)

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:

02 November 2017 to 12 November 2017

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:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Samples (approximately 150 mL) of the range-finding and definitive test solutions and controls were collected into Teflon® capped glass bottles (250 mL, no preservative).
- Samples were collected immediately prior to test commencement (0 hour before the addition of algae) and at test termination (72 hour, algae present, from pooled replicates) for analysis.
- The test material is stable in algal nutrient medium at room temperature for at least four days. As such, all samples collected for analysis by ISI were stored at room temperature overnight and transported to ISI the next day. Upon arrival at ISI, the samples were held at room temperature until analysis was conducted.

Vehicle:

no

Details on test solutions:

- Individual test solutions of the test material for both range-finding and definitive tests were prepared from stock solutions (1000 mg/L nominal concentration) prepared in algal nutrient media without the use of any solubilising agent.
- All stock and test solutions were corrected for purity of the test material (i.e., 98.75%). All stock solutions were prepared in 500 mL volumetric flasks, stirred for approximately 0.5 hours using a stir bar and stir plate. The stock solutions were then used to prepare the test concentrations for the range-finding test. For the definitive test, a sub-stock was prepared from the 1000 mg/L stock solution and was then used to prepare the test concentrations for the definitive test.
- The 1000 mg/L stock solution used for the range-finding test was prepared by adding 0.50633 g of the test material to 500 mL of algal nutrient media in a 500 mL glass volumetric and stirring for 0.5 h. The individual test solutions were prepared by adding an appropriate amount of the stock solution into each of two, 200 mL volumetric flaskS making this up to volume in algal nutrient media and then compositing two volumes into a 600 mL glass beaker. The 1000 mg/L stock solution used for the definitive test was prepared by adding 0.50634 g of the test material to 500 mL of algal nutrient media in a 500 mL glass volumetric and stirring for 0.5 h. A 100 mg/L sub-stock was prepared by removing 50 mL of the 1000 mg/L stock solution and mixing with algal nutrient media for a final volume of 500 mL. This sub-stock was stirred for 30 minutes and then used to prepare the test concentrations for the definitive test. The individual test solutions were prepared as noted above for the range finding test.
- All test solutions were stirred for 15 minutes using a stir bar and stir plate prior to being dispensed into the individual test vessels (i.e., 250 mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Source: University of Waterloo Culture Collection (CPCC 37)
- Age of inoculum (at test initiation): 3 – 7 days old and in exponential growth

CULTURES
- Cultures were aseptically transferred twice weekly (typically from 3 – 7 day old donors) and maintained in temperature and light controlled environments isolated from all testing. The axenic nature of the stock culture was verified by plating on Trypticase Soy Agar (TSA) and Plate Count Agar (PCA). Algal growth curves conducted semi-annually ensured that algae were in an exponential growth phase, suitable for testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 1 °C

pH:

7.5 ± 0.1

Nominal and measured concentrations:

Nominal: 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/L
Measured (TWA): N/A, N/A, 0.24, 0.35, 0.73, 1.43, 2.86 and 5.71 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Clear glass 250-mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs
- Material, size, headspace, fill volume: 50 mL
- Agitation: Continuous shaking of test vessels on a rotary shaker set at 100 rpm
- Initial cells density: 5 x 10^3 to 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water obtained from the University of Guelph was used in preparation of the nutrient medium for culture and testing of P. subcapitata. This water was free of particles, ions, organic molecules and microorganisms greater than 0.45 μm in diameter. Preparation of the medium was conducted according to Environment Canada. The nutrient medium was filter sterilised prior to use in cultures and in testing.
- Elemental composition of the test media: N 4.20 mg/L, Mg 2.65 mg/L, Ca 1.20 mg/L, S 1.91 mg/L, P 0.19 mg/L, K 0.47 mg/L, Na 11.00 mg/L, C 2.14 mg/L, B 32.44 µg/L, Mn 115.38 µg/L, Zn 1.57 µg/L, Co 0.35 µg/L, Cu 0.004 µg/L, Mo 2.88 µg/L and Fe 33.1 µg/L.
- Culture medium different from test medium: no
- Intervals of water quality measurement: At test commencement, pH was measured in the control and in each test solution. At the end of the test (72 hours), the pH was determined from the pooled replicate samples from the control and each test concentration.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous (24 hours)
- Light intensity and quality: Cool-white fluorescent, measured at the surface of the liquid in the flasks: 4590 - 6180 Lux for testing

EFFECT PARAMETERS MEASURED:
- For all replicates, cell counts were determined at approximately 24, 48, and 72 hours. Cell counts were conducted using a haemocytometer and a phase-contrast microscope (at 100 - 200 times magnification). Any changes in cell development or appearance, such as cell clumping, cell morphology, cell colour, cell shape, cell size, etc. (or lack thereof) were reported on the bench sheets. Any additional observations relating to the test solutions, such as sedimentation of the test solution, precipitation of cells, solution appearance / colouration or other abnormalities, were also recorded on the bench sheets.

TEST CONCENTRATIONS
- The test consisted of eight nominal concentrations of the test material including 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/L plus a negative control.
- Range finding study: The test consisted of four nominal concentrations of the test material including 0.1, 1, 10 and 100 mg/L plus a negative control (i.e., nutrient media with no added test material prepared the day of test initiation). For this test, two replicate 250 mL Erlenmeyer flasks, each containing 49 mL of the test solution (prepared in nutrient medium) and 1 mL of algal inoculum (5 x 10^3 to 1 x 10^4 algal cells/mL), were established for each concentration.

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % C.L: 1.04-1.17 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: cell yield

Remarks on result:

other: 95 % C.L: 0.56-0.65 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and cell yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and cell yield

Details on results:

RANGE-FINDING TEST
- Confirmation of the Exposure Concentrations: Nominal, measured and Time-Weighted Mean (TWM) concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 1. In some instances, test concentrations were less than the MDL at test end, so measured concentrations at 0 hours were used for endpoint determination. The test material was shown to be stable in algal nutrient media for at room temperature. Nominal concentrations of 100 mg/L achieved a TWM concentrations of 48.6 mg/L (i.e., approximately 49% of the nominal concentration).
- Results of Range-Finding Test: The range-finding test met all of the test validity criteria. Results of the range-finding test are summarised in Table 2. Test results are expressed as the percent inhibition of growth in terms of the effects on average specific growth rate and cell yield after 72 hours of exposure. The NOEC and LOEC were estimated to between 0.09 and 5.1 mg/L, respectively for both average specific growth rate and cell yield. In terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be between 0.95 and 5.1 mg/L, while for cell yield the same endpoints were estimated to be between < 0.09 and 5.1 mg/L.

DEFINITIVE TEST
- Confirmation of the Exposure Concentrations: Nominal, measured and TWM concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 3. One test concentration was less than the MDL at test end, so the measured concentration at 0 hours was used for endpoint determination. The TWM concentration of 5.7 mg/L was achieved from preparation of a nominal 10 mg/L solution of the test material used for the highest concentration of the definitive test. The spike recovery was 99.2 - 99.5 % which was acceptable (between 80 and 100%).
- Results of the Definitive Test: Results of the definitive test are summarised in Table 4. The test met all of the test validity criteria. Results of the definitive test showed that there were adverse effects on growth of algae in terms of average specific growth rate and cell yield at concentrations of the test material at and above 0.33 mg/L. The NOEC and LOEC were estimated to be 0.3 and 0.7 mg/L, respectively for both average specific growth rate and cell yield. In terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 0.52, 0.68 and 1.10 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 0.33, 0.40 and 0.60 mg/L, respectively.

TEST VALIDITY CRITERIA
All of the validity criteria for the range-finding and definitive tests were met as listed below:
- Biomass, as determined by cell number, in control test vessels increased by a factor of at least 16 times after 72 hours.
- The mean CV (%) for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the control cultures did not exceed 35%.
- The CV (%) of average specific growth rates during the whole test period (0-3 days) in replicate control cultures did not exceed 7%.

Results with reference substance (positive control):

- Sodium chloride was the reference toxicant used in this study.
- The 72-hour EC25 was compared to historical data and was deemed acceptable based on results falling within the 95% confidence interval (warning limit) calculated for the last fifteen reference toxicant tests.
- In conjunction with the definitive test, the 72- hour EC25 for the P. subcapitata reference toxicant test was 764 mg/L.

Reported statistics and error estimates:

- All endpoints for cell yield and average specific growth rate were generated using Time-Weighted Mean (TWM) concentrations calculated from measured concentrations of the test material at test initiation (t=0h) and test end (t=72h). In some instances, test concentrations were < MDL at test end, so endpoints were generated using measured concentrations at 0 hours.
- For all concentrations and the controls, average specific growth rate (0 to 3 days) and cell yield (72-hour) were calculated for each replicate and averaged. Percent inhibition (I) and Coefficient of Variation (CV; %) were also calculated for average specific growth rate and cell yield. For the control flasks, the section-by-section specific growth rates (e.g., 0-1, 1-2, and 2-3 days) and mean % CV were determined.
- All data satisfied the assumptions for normality and homogeneity of variance. For the range-finding test, the NOEC and LOEC endpoints for average specific growth rate and cell yield were determined using ANOVA/ Williams Multiple Comparison Test (CETIS, 2013) (α=0.05). The ECx range endpoints, for cell yield (EyCx) and average specific growth rate (ErCx), were estimated from the inhibition of the average response at each test concentration compared to the control using Linear Interpolation.
- For the definitive test, the NOEC and LOEC endpoints for average specific growth rate and cell yield were determined using ANOVA/Dunnett Multiple Comparison Test (CETIS, 2013) (α=0.05). Square root transformation was applied to cell yield data prior to ANOVA, in order to satisfy assumptions for normality and homogeneity of variance. Growth inhibition values were estimated from inhibition of average specific growth rate (ErCx) and cell yield (EyCx) compared to the control, and were calculated using Non-Linear Regression Analysis (CETIS, 2013). Poisson weighting was applied to cell yield data and Box-Cox weighting was applied to average specific growth rate data.

Table 1: Summary of the Nominal and Measured (at t = 0 hours) Concentrations of the Test Material in the Range-Finding Test

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Time-Weighted Mean Concentration (mg/L)
	0 Hours	72 Hours
100	91.95c	21.62d	48.6
10	9.81	2.21	5.1
1	0.95	<MDLe	0.95f
0.1	0.09	N/Ag	0.09f
0 (Negative Control)	<MDL	<MDL	<MDL

c Average of duplicate analyses. Relative percent difference (RPD) was 9.1%.

d Average of duplicate analyses. RPD was 2.5%.

e Method Detection Limit (MDL) = 0.08 mg/L.

f Based on a single measurement at 0 h (not a TWM).

g Not analysable as sample below MDL.

 

Table 2: Summary of Range-Finding Test Results Showing Percent Inhibition of Growth of P. subcapitata in Terms of Average Specific Growth Rate and Cell Yield After 72 Hours of Exposure to Test Material

Test Endpoint	Nominal Concentration (mg/L)/Time-weighted mean concentration (mg/L)
	0.1/0.09	1/0.95	10/5.1	100/48.6
% Inhibition in Average Specific Growth Rate (0 to 72 hours)	2.53	9.55	99.49	86.64
% Inhibition in Cell Yield (72 hour)	12.74	39.86	99.91	99.51

Table 3: Summary of the Nominal and Measured Concentrations of the Test Material in the Definitive Test Solutions.

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Time-Weighted Mean Concentration (mg/L)
	0 hrs	72 hrs
10.0	9.79c	2.96d	5.71
5.00	4.71	1.57	2.86
2.50	2.42	0.76	1.43
1.25	1.16	0.42	0.73
0.625	0.54	0.21	0.35
0.313	0.24	N/A	0.24f
0.156	N/A	N/A	N/A
0.0781	N/A	N/A	N/A
0 (Negative control)	<MDL	<MDL	<MDL

c Average of duplicate analyses, Relative Percent Difference (RPD) = 3.7%.

d Average of duplicate analyses, RPD = 18.1%.

f Based on a single measurement at 0 h (not a TWM).

MDL = 0.08 mg/L

NA = not analysed as below MDL

 

Table 4: Summary of Definitive Test Results Showing Percent Inhibition of Growth of P. subcapitata in Terms of Average Specific Growth Rate and Cell Yield After 72 Hours of Exposure to the Test Material. 

Nominal Concentration (mg/L)	Time-Weighted Mean Concentrations (mg/L)	% Inhibition on Average Specific Growth Rate (0 to 72 hours)	% Inhibition on Cell Yield (72 hour)
10.0	5.71	93.25	99.66
5.0	2.86	73.72	98.39
2.5	1.43	54.87	95.05
1.25	0.73	18.69	63.60
0.625	0.35	0.28	1.71
0.313	0.24a	1.40	7.58
0.156	N/A	0.24	1.52
0.0781	N/A	-0.28c	-1.01
0 (Negative control)	<MDL	0.00	0.00

a Based on a single measurement at 0 h (not a TWM).

c A negative value for % inhibition indicates stimulation as compared to the control.

Table 5: Effects of the Test Material on Growth Inhibition (Average Specific Growth Rate and Cell Yield) of P. subcapitata Based on a 72-Hour Static Exposure

Endpoint	ECxa (95% Confidence Limits) (Nominal; mg/L)			NOEC (mg/L)	LOEC (mg/L)
	EC10	EC20	EC50
Average Specific Growth Rate (0 to 72 hours)	0.52 (0.48 - 0.56)	0.68 (0.64 - 0.71)	1.10 (1.04 - 1.17)	0.3	0.7
Cell Yield (72 hour)	0.33 (0.26 - 0.38)	0.40 (0.35 - 0.45)	0.60 (0.56 - 0.65)	0.3	0.7

a Effective concentration causing a specified x % inhibition.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 1.10 and 0.60 mg/L, respectively.

Executive summary:

The potential of the test material to cause toxicity to aquatic algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions.

The test material was investigated by testing on the freshwater green alga, Pseudokirchneriella subcapitata under static test conditions for 72 hours. A range-finding test was performed to determine suitable concentrations for the definitive test.

Both the range-finding and definitive tests met the validity criteria.

Results were generated using Time-Weighted Mean (TWM) concentrations calculated from measured concentrations of the test material at test initiation and test end. In some instances, test concentrations were less than the Method Detection Limit (MDL) at test end, so endpoints were generated using measured concentrations at 0 hours.

From the range-finding test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be in the range of 0.95 - 5.1 mg/L. For cell yield, the EC10, EC20 and EC50 were estimated to be between <0.09 and 5.1 mg/L. The NOEC and LOEC from the range-finding test were estimated to be between 0.95 and 5.1 mg/L, respectively for average specific growth rate and between 0.09 and 0.95 mg/L, respectively for cell yield.

From the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 0.52, 0.68 and 1.10 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 0.33, 0.40 and 0.60 mg/L, respectively. The NOEC and LOEC were estimated to be 0.3 and 0.7 mg/L, respectively for both average specific growth rate and cell yield.

Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 1.10 and 0.60 mg/L, respectively.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

 

Description of key information

Under the conditions of the study, the 72 h EC50 for the specific growth rate and cell yield were 1.10 and 0.60 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.1 mg/L

EC10 or NOEC for freshwater algae:

0.3 mg/L

Additional information

The potential of the test material to cause toxicity to aquatic algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was investigated by testing on the freshwater green alga, Pseudokirchneriella subcapitata under static test conditions for 72 hours. A range-finding test was performed to determine suitable concentrations for the definitive test.

Both the range-finding and definitive tests met the validity criteria.

Results were generated using Time-Weighted Mean (TWM) concentrations calculated from measured concentrations of the test material at test initiation and test end. In some instances, test concentrations were less than the Method Detection Limit (MDL) at test end, so endpoints were generated using measured concentrations at 0 hours.

From the range-finding test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be in the range of 0.95 - 5.1 mg/L. For cell yield, the EC10, EC20 and EC50 were estimated to be between <0.09 and 5.1 mg/L. The NOEC and LOEC from the range-finding test were estimated to be between 0.95 and 5.1 mg/L, respectively for average specific growth rate and between 0.09 and 0.95 mg/L, respectively for cell yield.

From the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 0.52, 0.68 and 1.10 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 0.33, 0.40 and 0.60 mg/L, respectively. The NOEC and LOEC were estimated to be 0.3 and 0.7 mg/L, respectively for both average specific growth rate and cell yield.

Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 1.10 and 0.60 mg/L, respectively.