Long-chain alkenyl amide

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:

15 August 2017 to 24 August 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 refrigerated until transported to ISI and kept refrigerated by ISI until analysis was conducted. All samples were analysed within 24 to 96 hours of the sample collection date.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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 dilution water and without the use of any solubilising agent. Due to the low solubility of the test material and its adhesive nature, stock solutions were prepared in 1-L glass aspirator bottles as follows. The test material was weighed out onto a pre-weighed plastic weigh boat containing a monolayer of glass beads. The test material remained attached to the surface of the glass beads while both the glass beads and the test material were transferred to the glass aspirator bottle leaving none of the test material in the plastic weigh boat. The solution was then stirred for approximately 96 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 solution was then settled for approximately 1 hour. The first ~ 100 mL of solution removed from each glass aspirator was disposed of. Individual test solutions were prepared by addition of the appropriate amount of stock solution to dilution water to achieve the desired concentrations for testing. All tests (range-finding and definitive) included a negative control consisting of dilution water with no test material added.

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 (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

ACCLIMATION
- 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:

yes

Total exposure duration:

48 h

Hardness:

200 mg/L as CaCO3

Test temperature:

20 ± 2 °C

pH:

8.3-8.5

Nominal and measured concentrations:

Nominal: 1000 mg/L
Measured (Time-weighted mean): 54 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker, 100 mL
- Material, size, headspace, fill volume: 75 mL
- 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: 1.0, 10, 100 and 1000 mg/L
- Concentrations were prepared by adding 0.5, 5, 50, and 500 mL of a nominal 1000 mg/L stock solution, respectively into a 500 mL volumetric flask and making this up to volume in 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 a nominal 1000 mg/L concentration of the test material (i.e., the highest concentration tested). As such, the definitive test was conducted as a Limit Test at a single nominal concentration of 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

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, measured concentrations at the beginning and end of the test were similar, indicating that the test material was stable over the period of the test. Nominal concentrations of 1000 and 100 mg/L achieved TWM concentrations of 20.0 and 1.50 mg/L, respectively (i.e., approximately 1.5 to 2% of the nominal concentration.
- Immobility: The range-finding test met all the test validity criteria (i.e., immobility ≤ 10% in the control; dissolved oxygen concentration at test termination ≥ 3 mg/L in all control and test vessels). 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. Ten percent (i.e. 1 out of 10) of the test organisms exposed to a TWM of 20 mg/L were considered to be immobilised during the first 24 hours, but 0% of these organisms were considered to be immobile at test termination. None of the test organisms exposed to any of the lower concentrations including the controls were observed to be immobile during the 48 hour test period. The range-finding test indicated that the 48-h EC50 for immobility was greater than 1000 mg/L (i.e., the highest concentration tested). Based on these results, the definitive test was conducted as a Limit Test at a single concentration of the test material (i.e., a nominal 1000 mg/L solution prepared).

DEFINITIVE TEST RESULTS
- 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. The TWM concentration of 54 mg/L achieved from preparation of a nominal 1000 mg/L solution of the test material used for the definitive test was 2.7 times higher that than TWM of 20 mg/L that was achieved for the range-finding test. In the definitive test, the spike recovery was lower than the accepted level (80 to 100%). Sample preparation procedures (e.g., stirring/settling time and mixing velocity) were similar for the range-finding and definitive tests; therefore, differences in these TWM concentrations were considered to be primarily related to the low solubility of the test material.
- Immobility: The definitive tests 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 there was no (0%) immobility of daphnids exposed to a nominal 1000 mg/L solution of the test material.
- The 48-hour NOEC and LOEC, and the 24 and 48-hour EC50s could not be calculated statistically, since the testing was conducted at a single nominal loading rate of each of these two test materials and daphnid immobility was not observed in either test or control treatments. Thus, the 48-hour NOEC and LOEC for immobility of daphnids were reported as 1000 mg/L and >1000 mg/L, respectively and the 24 and 48-hour EC50s were reported as >1000 mg/L.

VALIDITY CRITERIA
All of the validity criteria for the definitive tests were met as listed below:
• 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 (20) reference tests. In conjunction with the definitive test, the 48-hour LC50 for the Daphnia magna reference toxicant test conducted on August 15, 2017 was 6.0 g/L.

Reported statistics and error estimates:

- Statistical analysis of the toxicity test data was not required, since the test was conducted as a Limit test and there was no immobility (0%) of daphnids exposed to either the test material or the controls.
- Although analytical concentrations were verified, due to the nature of the test material (UVCB), measured concentrations were not necessarily representative of the whole substance. Therefore, test endpoints were expressed in terms of nominal concentrations.

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	48 Hours
1000	17.42d	22.87e	20.0
100	1.47	1.52	1.50
10	0.15	N/Af	0.15g
1	<MDL	N/A	<MDL
0 (Negative Control)	<MDL	<MDL	<MDL

d Average of duplicate analyses. The Relative Percent Difference (RPD) = 3.8 %.

e Average of duplicate analyses. RPD = 7.8 %.

f Not analysed.

g Based on a single measurement (not a TWM).

 

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
1000	20.0	10	0
100	1.50	0	0
10	0.15*	0	0
1	<MDL	0	0
0 (Negative Control)	<MDL	0	0

* Based on a single measurement (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 Hours	48 Hours
1000	71.4	40.2	54
0 (Negative Control)	<MDL	<MDL	<MDL

 

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

Nominal Concentration (mg/L)	Immobility (%)
	T= 24 hours	T= 48 hours
1000	0	0
0 (Negative Control)	0	0

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 24 and 48-hour EC50 based on results of the definitive test were reported to be > 1000 mg/L, exceeding the OECD limit of 100 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 1000 mg/L and > 1000 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 (i.e., immobility ≤ 10% in the control; dissolved oxygen concentration at test termination ≥ 3 mg/L in all control and test vessels). Test solution preparation included continuous stirring for approximately 96 hours, followed by a settling period of approximately 1 hour. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results were expressed in terms of nominal concentrations only.

Results of the range-finding test indicated that the 48-h EC50 for immobility was greater than the 1000 mg/L (i.e., the highest nominal concentration tested). As such, the definitive test was conducted as a Limit Test based on testing a nominal 1000 mg/L solution of the test material prepared in the same fashion as described for the range-finding test. Results of the definitive test showed that there was no immobility of daphnids in the study.

Under the conditions of this study, the 24 and 48-hour EC50 based on results of the definitive test were reported to be > 1000 mg/L, exceeding the OECD limit of 100 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 1000 mg/L and > 1000 mg/L, respectively.

Description of key information

Under the conditions of the study, the 24 and 48-hour EC50 based on results of the definitive test were reported to be > 1000 mg/L, exceeding the OECD limit of 100 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 1000 mg/L and > 1000 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 000 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 (i.e., immobility ≤ 10% in the control; dissolved oxygen concentration at test termination ≥ 3 mg/L in all control and test vessels). Test solution preparation included continuous stirring for approximately 96 hours, followed by a settling period of approximately 1 hour. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results were expressed in terms of nominal concentrations only.

Results of the range-finding test indicated that the 48-h EC50 for immobility was greater than the 1000 mg/L (i.e., the highest nominal concentration tested). As such, the definitive test was conducted as a Limit Test based on testing a nominal 1000 mg/L solution of the test material prepared in the same fashion as described for the range-finding test. Results of the definitive test showed that there was no immobility of daphnids in the study.

Under the conditions of this study, the 24 and 48-hour EC50 based on results of the definitive test were reported to be > 1000 mg/L, exceeding the OECD limit of 100 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 1000 mg/L and > 1000 mg/L, respectively.