Isoleucine, N-(1-oxohexadecyl)-

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 October 2018 to 24 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

GUIDELINE OECD 211 (2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch number 1700000153
- Expiration date of the lot/batch:25 January 2020
- Purity test date: January 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in darkness at a temperature of 20°C
- Stability under test conditions:The stability (non-GLP) was checked for reference and test items stock solutions after 8 days at - 20°C +/- 5°C. Recoveries (n=2) were higher than 90%.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Since the test item is poorly soluble under the test conditions, a stock solution in dimethyl sulfoxide was prepared at 50 g of test item.L-1

FORM AS APPLIED IN THE TEST (if different from that of starting material):The test material was applied as liquid after solubilization in dimethyl sulfoxide (DMSO)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Limit test at 5 mg.L-1
- Sampling method: Test item solution and control were sampled and immediately analysed.
- Sample storage conditions before analysis: no storage before analysis

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Since the test item is poorly soluble under the test conditions, a stock solution in dimethyl
sulfoxide (DMSO) was prepared at 50 g test item.L-1. Test solution was prepared by dilution of
this solution in the OECD test medium, every day.
- Controls: A solvent control was set up with 500 µL of DMSO diluted in 5000 mL of test medium, corresponding to the maximum quantity of DMSO used to dissolve the test item in the treated samples.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):organic solvent dimethyl sulfoxide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): a solvent control was set up with 500 µL of DMSO diluted in 5000 mL of test medium, corresponding to the maximum quantity of DMSO
used to dissolve the test item (for preparation of C03)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Strauss
- Strain/clone:Strauss clone 5
- Age at study initiation : between 6 and 24 hours old
- Method of breeding: The test organism was produced from parthenogenically reproducing brood female population. Daphnia are issued from the breeding lab. The juvenile stemming at least from the second litter were used to start the test.
- Source: Daphnia are issued from the breeding lab.
- Feeding during test
- Food type: Pseudokirchneriella subcapitata and Chlorella vulgaris suspensions .
- Amount: 0.1 mg of Carbon / daphnia / day.
- Frequency: Daily feeding

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Culture conditions:
Medium: Elendt M4
Temperature: 20 °C ± 2
Light/dark cycle: 16 h/8 h
pH: Between 6 and 9
Hardness (CaCO3): Between 140 and 250 mg.L-1

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

Hardness (CaCO3): Between 140 and 250 mg.L-1

Test temperature:

Temperature (°C):
03/10/2018 (20.1°C)
10/10/2018 (19.7°C)
17/10/2018 ( 19.8°C)
24/10/2018 (20.8°C)

pH:

[C01; C02; C03]:
03/10/2018 : Control (C01) = 7.8 ; Solvent Control (DMSO) (C02) = 8.0 ; 5 mg.L-1 (C03) = 7.9
10/10/2018 : Control (C01) =7.9 ; Solvent Control (DMSO) (C02) = 8.0 ; 5 mg.L-1 (C03) = 8.0
17/10/2018: Control (C01) =7.9 ; Solvent Control (DMSO) (C02) = 7.9 ; 5 mg.L-1 (C03) = 7.9
24/10/2018: Control (C01) =7.8 ; Solvent Control (DMSO) (C02) = 7.9 ; 5 mg.L-1 (C03) = 7.7

Dissolved oxygen:

Dissolved Oxygen (mg.L-1)
[C01 C02 C03]:
03/10/2018: Control (C01) = 9.1 ; Solvent Control (DMSO) (C02) = 9.0 ; 5 mg.L-1 (C03) = 8.5
10/10/2018: Control (C01) = 8.9 ; Solvent Control (DMSO) (C02) = 8.2 ; 5 mg.L-1 (C03) = 8.5
17/10/2018: Control (C01) = 8.1 ; Solvent Control (DMSO) (C02) =8.2 ; 5 mg.L-1 (C03) = 8.1
24/10/2018: Control (C01) = 8.4 ; Solvent Control (DMSO) (C02) = 8.0 ; 5 mg.L-1 (C03) = 8.3

Nominal and measured concentrations:

Limit test concentration: 5 mg/L in nominal concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass aquarium of 1.5 liters, filled to 1000 mL.
- Aeration: The test vessel was aerated during the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): The test was performed under continuous flow-through condition
- Renewal rate of test solution (frequency/flow rate):Test solution was prepared by dilution of this solution in the OECD test medium, every day.
- No. of organisms per vessel: 10 daphnia
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates):4 replicates
- No. of vessels per vehicle control (replicates):4 replicates


TEST MEDIUM / WATER PARAMETERS:
The test medium used was the OECD test medium (Elendt M4). It was reconstituted with chemical reagents and deionised water.The test medium was performed several times a week.


OTHER TEST CONDITIONS
- Photoperiod: A light:dark cycle of 16 hours light and 8 hours dark.
- Light intensity: Between 1000 and 1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
Range finding study:
- Spacing factor for test concentrations: 5
- Test concentrations: 200 µg/L ; 1000 µg/L ; 5000 µg/L (nominal concentration)
- Results used to determine the conditions for the definitive study: Limit test at 5 mg/L (nominal concentration)

Reference substance (positive control):

yes

Remarks:

POTASSIUM DICHROMATE RESULTS : Starting date : 19 September 2018.

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 5.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 5.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 5.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 5.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The total number of living offspring produced per parent animal alive was calculated for each
replicate. If a parent animal dead during the test in a replicate treated with the test item and if that mortality follows a dose response function, the parental mortality was considered as an effect of the test item and these replicates were not excluded from the analysis. Otherwise,
replicates with parental mortality were be excluded from the analysis.
The LOEC, the NOEC and the ECx were determined using the following data:
-The total number of living offspring per parent animal not accidentally or fortuitously dead at
the end of the test.
-The total number of living offspring per parent animal alive at the end of the test.
The NOEC (No Observed Effect Concentration) were determined by statistical test from replicate data on survival and reproduction.

Results with reference substance (positive control):

- Results with reference substance were valid:
- Relevant effect levels: 24-hour EC50 at 1.85 mg/L with 95% confidence limits (mg/L) between 1.753 – 1.948 (mg.L-1)
- Reference value: >= 0.6 mg/L and <= 2.1 mg/L
- Compliance: Yes

Reported statistics and error estimates:

The NOEC (No Observed Effect Concentration) were determined by statistical test from
replicate data on survival and reproduction. Statistical calculations were made using R
statistical software.
A Shapiro-Wilk test was used to assess data normality and Bartlett’s test for homogeneity of
variance:
Shapiro-Wilk results:
W :Control (C01) = 0.9256; Solvent Control (DMSO)(C02) = 0.8938; 5 mg.L-1(C03) = 0.9955
p-value : Control (C01) = 0.5686 ; Solvent Control (DMSO)(C02) = 0.4010; 5 mg.L-1(C03) = 0.9836
p-value > 0.05: data normality is met for all conditions.

Bartlett’s test results:
Bartlett's K-squared = 0.3691, df = 2, p-value = 0.8315
p-value > 0.05: homogeneity of variance is met.

To check if a significant difference exists between concentrations and control, a t-test was
performed: p-value > 0.05 No significant difference.
More statistical results are included in the area "any other information on results"

Range finding test:

Sixty juveniles per adult were not reached either in control and tested concentrations as indicated in the OECD 211 recommendations. Indeed, we observed that a part of adults used were male that directly affected the total juveline production along the test.Despite this result, this range finding test allows to conlude that the substance has no significant effet on Daphnia magna reproduction at the highest tested concentration.

So a definitive limit test at 5 mg/L was performed.

Geometric mean calculation:

At least three concentrations in a geometric series spaced by a constant spacing factor of 5 enables the choice of the appropriate limit concentration and the geometric means in μg test item/L were calculated. At a concentration of 200 μg test item/L the geometric mean was 83.70 μg test item/L , at 1000 μg test item/ L the geometric mean was calculated to be 448.52 μg test item/L and at the highest concentration 5000 μg test item/L the geometric mean was 3466.12 μg test item /L.

A significant deviation from the theoretical concentration was observed due to a decrease in the substance within 2 days before each medium renewal and the definitive test was performed under continual medium renewal.

The test substance concentration is not stable (i.e. outside the range 80 - 120% of nominal measured initial concentration), consideration was done for frequent medium renewal, so the test was performed under continuous flow-through condition.

A definitive limit test at 5 mg/L was then performed:

A stock solution in dimethyl sulfoxide (DMSO) was prepared at 50 g test item.L-1. Test solution was prepared by dilution of this solution in the OECD test medium, every day .

Reported statistics and error estimates

Endpoint determination:

• Reproduction

A Shapiro-Wilk test was used to assess data normality and Bartlett’s test for homogeneity of

variance:

Shapiro-Wilk results:

	W	p-value
C01	0.9256	0.5686
C02	0.8938	0.4010
C03	0.9955	0.9836

p-value > 0.05: data normality is met for all conditions.

Bartlett’s test results:

Bartlett's K-squared = 0.3691, df = 2, p-value = 0.8315

p-value > 0.05: homogeneity of variance is met.

To check if a significant difference exists between concentrations and control, a t-test was

performed:

	C01	C02
C02	0.74	-
C03	0.30	0.74

p-value > 0.05 No significant difference.

• Survival:

A Shapiro-Wilk test was used to assess data normality and Kruskal-Wallis test (no parametric

test) to check if a significant difference exists between concentrations and control:

Shapiro-Wilk results:

	W	p-value
C01	0.6298	0.001241
C02	0.6298	0.001241
C03	0.7286	0.023857

p-value < 0.05: data normality is not met for all conditions.

Kruskal-Wallis results:

Kruskal-Wallis chi-squared = 0.6875, df = 2, p-value = 0.7091

p-value > 0.05: No significant difference.

Validity criteria:

	Experimental value	Reference value		Compliance
Mortality in control	0 -10%	<	20%	Yes
Mean number of living offspring produced per adult	> 100	>=	60%	Yes
EC50 24h potassium dichromate	1.85 mg/L-1	>= <=	0.6 mg/L  2.1 mg/L	Yes

Validity criteria are met.

Analytical results

Date	Measured concentration (mg/L)
	C01	C02	C03
03/10/2018	0	0	4.93
04/10/2018	0	0	4.35
05/10/2018	<0.001	<0.001	7.01
08/10/2018	0	0	4.71
09/10/2018	0	0	4.41
10/10/2018	0	0	4.22
12/10/2018	0.00055	0.0007	4.46
15/10/2018	0	0	6.20
18/10/2018	0	0.00	5.70
22/10/2018	0	0.00	5.91
24/10/2018	0	0.00	5.95
Arithmetic Average	0.00	0.00	5.26

All results and subsequent calculations were based on arithmetic mean of mesures concentration.

Validity criteria fulfilled:

yes

Conclusions:

The objective of this study was to assess the effects of the test item on the reproductive output
of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012).

Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211.

Since the test item is poorly soluble under the test conditions, a stock solution in dimethyl
sulfoxide was prepared at 50 g of test item.L-1. Test solution were prepared by dilution of this
solution in the OECD test medium.
Test item quantification was performed according to a validated analytical method using a LC-MS/MS from Sciex (LC Sciex Exion AD and MS QTRAP 5500)
as mass spectrometry detection to measure DL-Isoleucine, N-(1-oxohexadecyl)
concentrations. As some of the measured concentrations were not within ± 20% of nominal or
initial concentrations, all results were based on mean measured concentrations.

The effects of the test item diluted in OECD test medium at the definitive measured
concentration of 5 mg.L-1 to Daphnia magna were investigated under laboratory conditions.


Results :

LC10 Survival (95% confidence limits) > 5.26 mg.L-1;
LC50 Survival > 5.26 mg.L-1;
EC10 Reproduction (95% confidence limits) > 5.26 mg.L-1;
EC50 Reproduction > 5.26 mg.L-1;
LC = lethal concentration
EC = effect concentration

NOEC Survival > 5.26 mg.L-1;
NOEC Reproduction > 5.26 mg.L-1;

The study is valid since:
• the mortality of the parent animals (female Daphnia) was not exceed 20% at the end
of the test.
• the mean number of living offspring produced per parent animal surviving at the end
of the test was ≥ 60.
• EC50 with reference item over a period of 24 hours must be between 0.6 and 2.1 mg.L1

Based on the reproduction rate and the survival rate, the EC50 and LC50 after a 21 days
exposure period were greater than 5.26 mg.L-1
The NOEC was estimated to be at 5.26 mg.L-1

Executive summary:

The objective of this study was to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012).

Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211.

Since the test item is poorly soluble under the test conditions, a stock solution in dimethyl sulfoxide was prepared at 50 g of test item.L^-1. Test solution were prepared by dilution of this solution in the OECD test medium.

Test item quantification was performed according to a validated analytical method using a LC-MS/MS from Sciex (LC Sciex Exion AD and MS QTRAP 5500) as mass spectrometry detection to measure DL-Isoleucine, N-(1-oxohexadecyl) concentrations. As some of the measured concentrations were not within ± 20% of nominal or initial concentrations, all results were based on mean measured concentrations.

The effects of the test item diluted in OECD test medium at the definitive measured concentration of 5 mg.L^-1 to Daphnia magna were investigated under laboratory conditions.

Based on the reproduction rate and the survival rate, the EC₅₀ and LC₅₀ after a 21 days exposure period were greater than 5.26 mg.L^-1. The NOEC was estimated to be at 5.26 mg.L^{-1 .} 

 

Description of key information

The objective of this study was to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012). 

Since the test item is poorly soluble under the test conditions, a stock solution in dimethyl sulfoxide was prepared  and test solutions were prepared by dilution of this solution in the OECD Elendt-M4 test medium. 

The effects of the test item diluted in OECD test medium at the definitive measured concentration of 5 mg.L-1 to Daphnia magna were investigated under laboratory conditions. 

Based on the reproduction rate and the survival rate, the EC50 and LC50 after a 21 days exposure period were greater than 5.26 mg.L-1 . The NOEC was estimated to be at 5.26 mg.L-1 .

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

5.26 mg/L