Long-chain alkenyl amide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 November 2017 to 21 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Samples (approximately 150 mL) of the 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), after 48 hours and at test termination for analysis.
- All samples were refrigerated until transported to ISI and kept refrigerated by ISI until analysis was conducted. Samples collected at the beginning and end of the test were analysed within 24 hours of sample collection, whereas samples collected after 48 hours were analysed two days after sample collection.

Vehicle:

no

Details on test solutions:

- Duplicate test solutions of 1000 mg/L (nominal concentration) were prepared by direct addition of the test material in the test water and without the use of any solubilising agents. Due to the low solubility of the test material, the duplicate test solutions were prepared in 20 L glass aspirator bottles, stirred for approximately 4 days 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 approximately 1 hour prior to being dispensed into the test vessels.
- The first ~ 1000 mL of solution removed from each glass aspirator was disposed via the bottom port of the glass aspirator.
- The test included a procedural control (prepared in the same fashion as the test solution, except that no test material is added) and a negative control (i.e., 100% dilution water with no test material added).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Species: Oncorhynchus mykiss
- Age at study initiation: juvenile
- Weight/ Length: 0.41 ± 0.21 g wet weight; 3.64 ± 0.49 cm fork length

ACCLIMATION
- Acclimation period: The fish were acclimated to the laboratory conditions for at least twelve days prior to use in tests. 14-day acclimation took place in groundwater, there was < 5% mortality in 7 days prior to use in tests.
- Type and amount of food during acclimation: Commercial trout pellets. Feeding rate = 1 to 4% wet weight of fish

FEEDING DURING TEST
- No feeding took place during the test or in the preceding 24 hours before testing

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

200 mg/L as CaCO3

Test temperature:

15 ± 2°C

pH:

7.2 - 8.1

Dissolved oxygen:

8.5 - 9.6 mg/L

Nominal and measured concentrations:

Nominal: 1000 mg/L
Measured: 10.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 L plastic pail with Teflon liner
- Fill volume: 15 L
- Aeration: 6.5 ± 1 mL/L/min. Dissolved oxygen > 60% of saturation in controls.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The source of water used for holding and testing rainbow trout was moderately hard groundwater (water hardness ~350 mg/L as CaCO3, pH 7.8 - 8.1) from an aquifer in Aberfoyle, Ontario, Canada. Prior to use, the water was filtered through a 20-micron cellulose-acetate filter and diluted with Reverse Osmosis (RO) water to a hardness of 240 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. Water quality parameters (including pH, dissolved oxygen, conductivity and temperature) necessary for the survival of the test organisms were monitored and documented on a regular basis.
- Intervals of water quality measurement: Water quality parameters (including dissolved oxygen, pH, and conductivity) were measured daily in all test solutions. Dilution 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: Ambient laboratory illumination, 100 to 500 lux

EFFECT PARAMETERS MEASURED: For all test solutions and controls, fish mortality was recorded after 24, 48, 72, and 96 hours of exposure. Test organisms were considered dead if there was no evidence of opercular or other activity and did not respond to subsequent gentle prodding.

DEFINITIVE TEST
- The test material was assessed for acute toxicity to rainbow trout based on a static, 96 hour acute test. The definitive test was conducted as a single concentration Limit Test at a nominal concentration of 1000 mg/L.
- The individual 1000 mg/L test solutions (n = 2 replicates; A, B) were prepared by adding 18.0425 g (replicate A) and 18.0050 g (replicate B) of the test material into separate 20 L glass aspirator bottles labelled, replicates A and B, respectively, with 18 L of dilution water. A total of twenty fish (i.e., 2 replicates each with 10 fish) were exposed to each test and control treatments under static test conditions.

Reference substance (positive control):

yes

Remarks:

Potassium chloride

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

DEFINITIVE TEST RESULTS
- Confirmation of Exposure Concentrations: Nominal, measured and Time-Weighted Mean (TWM) concentrations of the test material determined on samples collected at the beginning (0 h), middle (48 h) and end (96 h) of the test are provided in Table 1. Measured concentrations of the nominal 1000 mg/L test solutions from replicates A and B collected after 48 and 96 hours were similar (i.e., within a factor of 1.3), but differed by a factor of approximately 2 for samples collected at the beginning of the test. Measured concentrations were generally higher at all times in replicate A relative to B. A TWM concentration of 10.0 mg/L was calculated based on an average of the two replicates.
- Mortality: The definitive test met all of the test validity criteria. A summary of the test results expressed in terms of the percent fish mortality observed in the test after 24, 48, 72 and 96 hours is summarised in Table 2. As can be seen in Table 2, no (0%) trout mortality was observed in the 1000 mg/L test solutions, or the procedural and negative controls. Additionally, there was no evidence of abnormal behaviour. The LC50s, NOEC and LOEC could not be calculated statistically, since the testing was conducted at a single nominal concentration of 1000 mg/L, and rainbow trout mortality was not observed in either test or control treatments. Thus, the 96 hour NOEC and LOEC for rainbow trout mortality was reported as 1000 mg/L and > 1000 mg/L, respectively and the 24, 48, 72 and 96-hour LC50s were reported as > 1000 mg/L.

Results with reference substance (positive control):

- Potassium 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.
- The 96-hour LC50 for the rainbow trout reference toxicant test associated with the definitive test was 3.277 g/L.

Reported statistics and error estimates:

- Statistical analysis of the mortality data for determination of the test endpoints was not required, since the test was conducted at a single nominal concentration of 1000 mg/L and no mortality or abnormal behaviour was observed in either the test or control solutions.
- Analytical concentrations were verified to check for stability of the test material in water, 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 here in terms of nominal concentrations only.

Sublethal observations / clinical signs:

Table 1: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material During the Definitive Test

Nominal Concentration (mg/L)	Measured Concentration (mg/L)			Time-Weighted Mean Concentration (mg/L)
	0 Hours	48 Hours	96 Hours
1000A	23.03	8.57c	11.28	10.0
1000B	11.04d	6.38e	8.09f
0 (Procedural Control) A	< MDL	< MDL	< MDL	< MDL
0 (Procedural Control) B	< MDL	< MDL	< MDL

c Average of duplicate analyses. The Relative Percent Difference (RPD) = 6.4%

d Average of duplicate analyses. RPD = 3.0%

e Average of duplicate analyses. RPD = 1.3%

f Average of duplicate analyses. RPD = 0.9%

Method Detection Limit (MDL) = 1.5 mg/L

 

Table 2: Mortality of Rainbow Trout After 24, 48, 72 and 96 Hours Exposure to the Test Material During the Definitive Test

Nominal Concentration (mg/L)	Time-Weighted Mean Concentration (mg/L)	Percent Mortality
		24 hours	48 Hours	72 Hours	96 Hours
1000	10.0	0	0	0	0
0 (Procedural Control)	0 (Procedural Control)	0	0	0	0
0 (Negative Control)	0 (Negative Control)	0	0	0	0

 

TEST VALIDITY CRITERIA

The range-finding and definitive tests met the test validity criteria:

- Mortality and impairment of the control fish ≤ 10%.

- Constant conditions were maintained throughout the test.

- Dissolved oxygen concentration were ≥ 60% of the air saturation value at the temperature of 15 ± 1°C throughout the test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, based on visual examination of the results of the definitive test, the 24, 48, 72, and 96 hour LC50s and the LC0 were reported to be > 1000 mg/L, exceeding the OECD limit of 100 mg/L. The NOEC and LOEC were reported to be 1000 mg/L and > 1000 mg/L, respectively.

Executive summary:

The acute toxicity of the test material to fish was determined in accordance with the standardised guideline OECD 203, under GLP conditions.

Based on discussions with the Sponsor, the definitive test was conducted as a Limit Test at a single nominal concentration of 1000 mg/L (i.e., exceeding the concentrations determined be toxic to algae). Further testing would only be required in the event that mortality of rainbow trout exposed to the limit test concentration was significantly different as compared to the controls.

Analytical concentrations were verified in this study, 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 are expressed here in terms of nominal concentrations only.

The definitive test, met all test validity criteria.

There was no mortality of rainbow trout exposed to the nominal concentration of 1000 mg/L. Additionally, there was no evidence of abnormal behaviour.

Under the conditions of this study, based on visual examination of the results of the definitive test, the 24, 48, 72, and 96 hour LC50s and the LC0 were reported to be > 1000 mg/L, exceeding the OECD limit of 100 mg/L. The NOEC and LOEC were reported to be 1000 mg/L and > 1000 mg/L, respectively.

Description of key information

Under the conditions of this study, based on visual examination of the results of the definitive test, the 24, 48, 72, and 96 hour LC50s and the LC0 were reported to be > 1000 mg/L, exceeding the OECD limit of 100 mg/L. The NOEC and LOEC were reported to be 1000 mg/L and > 1000 mg/L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1 000 mg/L

Additional information

The acute toxicity of the test material to fish was determined in accordance with the standardised guideline OECD 203, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based on discussions with the Sponsor, the definitive test was conducted as a Limit Test at a single nominal concentration of 1000 mg/L (i.e., exceeding the concentrations determined be toxic to algae). Further testing would only be required in the event that mortality of rainbow trout exposed to the limit test concentration was significantly different as compared to the controls.

Analytical concentrations were verified in this study, 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 are expressed here in terms of nominal concentrations only.

The definitive test, met all test validity criteria.

There was no mortality of rainbow trout exposed to the nominal concentration of 1000 mg/L. Additionally, there was no evidence of abnormal behaviour.

Under the conditions of this study, based on visual examination of the results of the definitive test, the 24, 48, 72, and 96 hour LC50s and the LC0 were reported to be > 1000 mg/L, exceeding the OECD limit of 100 mg/L. The NOEC and LOEC were reported to be 1000 mg/L and > 1000 mg/L, respectively.