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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2017 to 08 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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) and at test termination (48 hour, from pooled replicates) for analysis.
- All samples were stored at room temperature until transported to ISI and kept refrigerated by ISI until analysis was conducted.
- All samples from the range-finding and definitive tests were analysed within 24 hours of sample collection.

Vehicle:

no

Details on test solutions:

- All stock and test solutions were corrected for purity of the test material (i.e., 98.75%) and prepared in dilution water without the use of any solubilising agent.
- Preliminary testing conducted by ISI showed that a stable solution of the test material was achieved after approximately 24 hours of mixing, followed by a settling period of approximately 24 hours. As such, stock solutions were prepared in advance of the test in 1 or 2-L glass aspirator bottles and stirred for approximately 24 hours (at a rate sufficient to maintain a vortex between approximately 10 - 35% of the solution depth) using a stir bar and stir plate. The solutions were settled for 24 hours or longer if required to allow sufficient settling of the test material to occur.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna STRAUS
- Source: The organisms were originally obtained from the Ministry of the Environment and Climate Change (Toronto, Ontario, Canada), the organisms were cultured continuously from a population that has been actively reproducing in the testing laboratory since 1988.
- Age: First instar, neonate (<24 hours old)
- Feeding during test: no

CULTURE CONDITIONS
- Test organism acclimation was not required, since test organisms were cultured under conditions identical to those of the test.
- Type and amount of food: Once/day: yeast, Cerophyll broth, Trout Chow and Pseudokirchneriella subcapitata algae

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

200 - 210 mg/L as CaCO3

Test temperature:

20 ± 2°C

pH:

8.3 - 8.4

Nominal and measured concentrations:

Nominal: 27.5, 55, 110, 220 and 440 mg/L
Measured: 7.0, 13.3, 27.3, 55.3 and 112.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker, 100-150 mL
- Material, size, headspace, fill volume: 75 mL (min)
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Loading rate: 15 mL/daphnid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The source of water used for D. magna culturing and testing was moderately hard groundwater (~350 mg/L as CaCO3) obtained from an aquifer in Aberfoyle, Ontario, Canada. For culture and testing purposes with D. magna, the groundwater was filtered through a 20-micron cellulose-acetate filter, diluted with Reverse Osmosis (RO) water to achieve a hardness of ~200 mg/L (as CaCO3). A continuous supply of oil-free compressed air was provided to bring the pH and concentrations of dissolved oxygen and other gases into equilibrium with air. The concentration of dissolved oxygen in the water was maintained at > 80% of the air saturation value.
- Alkalinity: 130 mg/L (as CaCO3)
- Ca/mg ratio: 1.86:1
- Intervals of water quality measurement: Water quality parameters (including dissolved oxygen, pH, and conductivity) were measured in the control and all test solutions at the beginning and end of the test. Temperature was monitored and recorded daily. Water hardness was measured at the beginning of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light, 8-hour dark (with 30 minute transition period)
- Light intensity: 400 to 800 lux (Ambient laboratory illumination)

EFFECT PARAMETERS MEASURED:
- For all test and controls, daphnid immobility was determined after 24 and 48 hours.
- A test organism was considered to be immobile if it was unable to swim within 15 seconds following gentle agitation of the test solution and/or unable to free itself after gentle tapping of the test vessel.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Concentrations were prepared by adding 0.04, 0.4, 4, and 40 mL of a nominal 1000 mg/L stock solution, respectively into a 400 mL volumetric flask and making this up to volume with dilution water.
- A total of 10 daphnids (i.e., 2 replicates with 5 daphnids each) were exposed to each test level under static test conditions (i.e., no renewal of the test solution). All test organisms were transferred to fresh test and the negative control solutions using a pipette and bulb.
- Results used to determine the conditions for the definitive study: Results of the range-finding test indicated that the 48-hour EC50 for daphnid immobilisation was greater than the TWM concentration of 18.9 mg/L (or 100 mg/L nominal concentration), the highest concentration tested. As such, the definitive test was conducted as a multiple concentration test (at nominal concentrations exceeding 100 mg/L). Nominal concentrations of 27.5, 55, 110, 220 and 440 mg/L were prepared by adding 13.8, 27.5, 55, 110, and 220 mL of a nominal 1000 mg/L stock solution, respectively into a 500 mL graduated cylinder and made up to volume with dilution water.

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

43.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % C.L: 36.4-51.5 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

35.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % C.L: 30.4-40.0 mg/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

27.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

55.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE FINDING TEST
- Confirmation of 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. Measured concentrations were below nominal concentrations due to the low solubility of the test material. However, initial and final measured concentrations were within 20% of each other.
- Immobility: The range-finding test met all of the test validity criteria. The percentage immobility of daphnids exposed to the test material during the range-finding test along with the nominal and TWM concentrations are given in Table 2. A TWM of 18.9 mg/L (i.e., the highest concentration tested) resulted in only 10% immobility of daphnids. As such, the definitive test was conducted as a multiple concentration test that included nominal concentrations in excess of 100 mg/L (nominal concentration) to achieve TWM concentrations up to or exceeding 100 mg/L.

DEFINITIVE TEST
- Confirmation of 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. A TWM concentration of 112.1 mg/L (corresponding to 440 mg/L nominal concentration) was the highest concentration tested. Initial and final measured concentrations were within 20% of each other. The spike recovery was 93.2 to 99.6% which was acceptable.
- Immobility: The definitive test met all of the test validity criteria. A summary of the test results expressed in terms of the percent daphnid immobility observed in the test after 24 and 48 hours is summarised in Table 4. Results of the definitive test showed that TWM concentrations of the test material ≥ 27.3 mg/L caused immobility of daphnids within the 48 hour test period. The 24 and 48-hour EC50s (and 95% confidence intervals) for immobility of daphnids were reported to be 43.1 (36.4 - 51.5) and 35.3 (30.4 - 40.0) mg/L, respectively. The 48-hour NOEC and LOEC for immobility of daphnids were reported to be 27.3 and 55.3 mg/L, respectively.

TEST VALIDITY CRITERIA
All of the validity criteria for the definitive tests were met:
- Immobility of the controls did not exceed 10%.
- Dissolved oxygen concentrations at the end of the test were ≥ 3 mg/L in the control and test solutions.

Results with reference substance (positive control):

- Sodium chloride was the reference toxicant used in this study. The LC50 was compared to historical data and was deemed acceptable based on results falling within the 95% confidence interval (warning limit) calculated for the last twenty reference tests.
- In conjunction with the definitive test, the 48-hour LC50 for the Daphnia magna reference toxicant test was 5.9 g/L.

Reported statistics and error estimates:

- Test results from the range-finding test were based on visual examination of the data, since no immobility of daphnids was observed at any concentration.
- All endpoints were estimated (range-finding test) or generated (definitive test) using Time-Weighted Mean (TWM) concentrations calculated from measured concentrations of the test material at test initiation (t=0h) and test end (t=48h) Estimates of the 24 and 48-hour EC50s from the definitive test results were calculated using Linear Regression (MLE) (CETIS, 2013). The NOEC and LOEC from the definitive test data were calculated using Fisher Exact/Bonferroni-Holm Test (CETIS, 2013).

Table 1: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material for the Range-Finding Test

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Time-Weighted Mean Concentration (mg/L)
	0 Hours	72 Hours
100	18.95d	18.76e	18.85
10	1.86	1.76	1.81
1	0.18	<MDLf	0.18g
0.1	N/A	N/A	<MDL
0 (Negative Control)	<MDL	<MDL	<MDL

d Average of duplicate analyses. The Relative Percent Difference (RPD) = 2.8%.

e Average of duplicate analyses. RPD = 4.8%.

f MDL = 0.08 mg/L

g Based on a single analysis (not a TWM)

N/A = not analysed

 

Table 2: Immobility of D. magna After 24 and 48 Hours Exposure to the Test Material During the Range-Finding Test

Nominal Concentration (mg/L)	Time-Weighted Mean (mg/L)	Immobility (%)
		T= 24 hours	T= 48 hours
100	18.85	0	10
10	1.81	0	0
1	0.18*	0	0
0.1	N/A	0	0
0 (Negative Control)	<MDL	0	0

* Based on a single measurement at t=0 hours (not a TWM).

 

Table 3: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material for the Definitive Test

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Time-Weighted Mean Concentration (mg/L)
	0 hrs	72 hrs
440	113.53d	110.69e	112.1
220	56.69	53.92	55.3
110	28.46	26.08	27.3
55	13.63	13.05	13.3
27.5	7.06	6.97	7.0
0 (Negative control)	<MDL	<MDL	<MDL

d Average of duplicate analyses, Relative Percent Difference (RPD) = 2.1%.

e Average of duplicate analyses, RPD = 1.6%.

MDL = 0.95 mg/L

 

Table 4: Immobility of D. magna After 24 and 48 Hours Exposure to the Test Material During the Definitive Test

Nominal Concentration (mg/L)	Time-Weighted Mean Concentration (mg/L)	Immobility (%)
		T = 24 hours	T = 48 hours
440	112.1	100	100
220	55.3	50	95
110	27.3	30	20b
55	13.3	0	0
27.5	7.0	0	0
0 (Negative control)	<MDL	0	0

b Not significantly different from control response (α= 0.05).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, based on results of the definitive test, the 24 and 48-hour EC50s (95% Confident Limits) for the test material were reported to be 43.1 (36.4 - 51.5) and 35.3 (30.4 - 40.0) mg/L, respectively. The 48-hour NOEC and LOEC for immobility of daphnids were 27.3 and 55.3 mg/L, respectively.

Executive summary:

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202, under GLP conditions in the daphnia immobilisation test.

The range-finding and definitive tests met all the test validity criteria. Test results were based on measured concentrations, or more specifically, Time-Weighted Mean (TWM) concentrations.

Results of the range-finding test indicated that the 48-h EC50 for immobility of D. magna was >18.9 mg/L (or 100 mg/L nominal concentration), the highest concentration tested. As such, the definitive test was conducted as a multiple-concentration test that included nominal concentrations in excess of 100 mg/L (nominal concentration) to achieve TWM concentrations up to or exceeding 100 mg/L.

Under the conditions of this study, based on results of the definitive test, the 24 and 48-hour EC50s (95% Confident Limits) for the test material were reported to be 43.1 (36.4 - 51.5) and 35.3 (30.4 - 40.0) mg/L, respectively. The 48-hour NOEC and LOEC for immobility of daphnids were 27.3 and 55.3 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, based on results of the definitive test, the 24 and 48-hour EC50s (95% Confident Limits) for the test material were reported to be 43.1 (36.4 - 51.5) and 35.3 (30.4 - 40.0) mg/L, respectively. The 48-hour NOEC and LOEC for immobility of daphnids were 27.3 and 55.3 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

35.3 mg/L

Additional information

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202, under GLP conditions in the daphnia immobilisation test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The range-finding and definitive tests met all the test validity criteria. Test results were based on measured concentrations, or more specifically, Time-Weighted Mean (TWM) concentrations.

Results of the range-finding test indicated that the 48-h EC50 for immobility of D. magna was >18.9 mg/L (or 100 mg/L nominal concentration), the highest concentration tested. As such, the definitive test was conducted as a multiple-concentration test that included nominal concentrations in excess of 100 mg/L (nominal concentration) to achieve TWM concentrations up to or exceeding 100 mg/L.

Under the conditions of this study, based on results of the definitive test, the 24 and 48-hour EC50s (95% Confident Limits) for the test material were reported to be 43.1 (36.4 - 51.5) and 35.3 (30.4 - 40.0) mg/L, respectively. The 48-hour NOEC and LOEC for immobility of daphnids were 27.3 and 55.3 mg/L, respectively.