Isoleucine, N-(1-oxohexadecyl)-

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 October 2018 - 05 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was carried out according to GUIDELINE OECD 201 (2006, corrected 28 July 2011)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

GUIDELINE OECD 201 (2006, corrected 28 July 2011)

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
- Solubility and stability of the test substance in the solvent/vehicle: the stability (non-GLP)
was checked for reference and test material 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 and according to the OECD 23, the choice of a solvent was necessary to solubilize the test item, at the concentration lower than 0.1 ml.L-1. So, a stock solution of test item was prepared in dimethyl sulfoxide. Test solutions were prepared by dilution of this solution.

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 (nominal concentration) correspond to 2.56 mg/L (geometric mean concentration)
- Sampling method: Test item solution and control were sampled and immediately analysed at 0h, 24h, 48h, and 72h.
- 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:Dilution of the stock solution prepared by dissolving 501 mg of test item in 10 mL of dimethyl sulfoxide(DMSO), concentration of stock solution = 50 g/L
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):Dimethyl Sulfoxide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): according to the OECD monograph 23, the solvent concentration was not exceeded 0.1 mL.L-1 in the test and control solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green Algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): CCAP (Culture Collection of Algae and Protozoa),
reference CCAP 1405/1.
- Method of cultivation: A culture was prepared from the stock culture and maintained in an exponential growth by successive transfers every 3 to 4 days.

ACCLIMATION
- Culturing media and conditions (same as test or not):
Medium: OECD medium
Temperature: 23 ± 2°C
Light: continuous illumination between 4440 and 8880 lux
pH: 7,80
Any deformed or abnormal cells observed: No abnormal appearance of the algae at the start and at the end of the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Microscopic observation:
No abnormal appearance of the algae at the start and at the end of the test.

Test temperature:

0h (23.75 -24.25 (°C)) ; 24h (24.13 - 24.31 (°C)) ; 48h (23.06 - 24.31 (°C)) ; 72h ( 23.31 - 24.25 (°C))

pH:

pH : 0h Control (7.8) Solvent control (7.69) Test material at [0.1] (7.67)

pH : 72 h Control (8.67) Solvent control ( 8.66) Test material at [0.1] (8.63)

Nominal and measured concentrations:

Limit test concentration: 5 mg/L in nominal concentration and 2.56 mg/L geometric mean concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume:Glass Erlenmeyer flasks of 250 mL. .
- Initial cells density: 25 000 cells per millilitres.
- Control end cells density: 3 203 333 cells per millilitres.
- No. of organisms per vessel: 25 000 cells per millilitres.
- No. of vessels per concentration (replicates):6 replicates for each
treatment

GROWTH MEDIUM
- Standard medium used: yes (OECD test medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: It was reconstituted with chemical reagents and ultra-pure water

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Photoperiod: Continuous illumination
- Light intensity and quality: Between 4440 and 8880 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beckman coulter Multisizer 3

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Justification for using less concentrations than requested by guideline: The toxicity was observed under the limit of solubility during the range finding-test
- Range finding study: 16.33 µg/L; 285.87 µg/L and 2770.53 µg/L
- Test concentrations: 5 mg test item.L-1.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate results: Starting date :18 May 2018 CEt50 = 1,07( mg.L-1) with 95% confidence limits between 0,94 – 1,2 (mg.L-1)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 2.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 2.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 2.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Based on yield rate and on day by day geometric mean analysed concentrations, NOEC (72h) was 2.56 mg test item.L-1

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 2.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormal appearance of the algae at the start and at the end of the test.

Results with reference substance (positive control):

Potassium dichromate results:
Starting date :18 May 2018
The concentration of the reference substance dissolved in test medium that results in an 50% reduction in growth of the test organism within an exposure period of 50 h is 1,07 (mg.L-1) CEt50 with 95% confidence limits between 0,94 – 1,2 (mg.L-1)

Reported statistics and error estimates:

The statistical evaluation (NOEC - No Observed Effect Concentration) was conducted with R
software. For 72-hour growth rate and yield, since data were consistent with a normal
distribution (Shapiro-Wilk test) and the variance homogeneity was confirmed (Bartlett test),
data were analysed for significant differences compared to the control using the parametric
ANOVA (p ≤ 0.05;)

Range finding test results :

There were no significant differences between growth and yield rates in the solvant control and all test material treatement after 72h (ANOVA, p ≤ 0.05), thus, for both growth and yield rates, the NOEC was 2770.531 μg test material.L ^-1 and both the EC₅₀ and the LOEC were estimated to be > 2770.531μg test material.L^-1.Then a limit test at the highest rate tested for preliminary test was performed for the definitive test.

Details of growth algae results are available in the attached report.

Validity criteria:

After 72 hours:

	Actual value	Reference value	Compliance
Growth multiplication factor in the control within the 72-hour test period	128	> 16	Yes
Coefficient of variation of the average specific growth rates in then control during the whole test period	1.45	< 7	Yes
Mean coefficient of variation for section-by-section specific growth rates (days 0 -1 , 1-2 and 2 -3) in the control	21.42	< 35	Yes

Validity criteria are met.

 

Quality Control :

	Actual value	Reference value	Compliance
Recovery at 20 (%)	101	70 -120	Yes
Recovery at 80 (%)	100	70 -120	Yes
Within reproducibility relative standard deviation (recovery at 20 (%)	7.4	< 20	Yes
Within reproducibility relative standard deviation (recovery et 80(%)	6.1	< 20	Yes
Recoveries difference (%) between 20% and 80%)	1	< 20	Yes

The analytical series are valid since the measured values are included in the reference interval.  

Analytical results:

Treatment ID		C01	C02	C03
Target concentration [μg test item/L]		0 (control)	0 (solvent control)	5000
	0h	<LOQ	<LOQ	4966.8
	Deviation from target rate[%]	NA	NA	-0.7
	24h	<LOQ	<LOQ	3814.0
Analysed concntration[μg test item/L]	Deviation from target rate[%]	NA	NA	-23.7
	48h	<LOQ	<LOQ	1751.9
	Deviation from target rate [%]	NA	NA	-65.0
	72h	<LOQ	<LOQ	1246.4
	Deviation from target rate[%]	NA	NA	-75.1
Geometrical mean [μg test item/L		NA	NA	2552.2

NA = not applicable. LOQ = limit of quantification = 0.5 µg active substance.L-¹ 

Analysis of the results were based on the geometric mean concentrations after 72h. The geometric mean concentration at the tested concentration was higher than the solubility of the test item (1.5 mg.L^-1 according the OECD 105).

Validity criteria fulfilled:

yes

Remarks:

The growth multiplication factor in the control within the 72-hour test period was higher than 16 (actual value: 128), the coefficient of variation of the average specific growth rates in the control was less than 35% (actual value: 21.42% after 72h).

Conclusions:

The effects of DL-Isoleucine, N-(1-oxohexadecyl) diluted in OECD test medium at the rates of
5 mg test item.L-1 to Pseudokirchneriella subcapitata were investigated under laboratory
conditions.
The study is valid since the growth multiplication factor in the control within the 72-hour test
period was higher than 16 (actual value: 128), the coefficient of variation of the average specific
growth rates in the control was less than 7% (actual value: 1.45%) and the mean coefficient of
variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control was
less than 35% (actual value: 21.42% after 72h).

The geometric mean concentration at the tested concentration was higher than the solubility of the test item (1.5 mg.L-1 according the OECD 105).
Based on growth rate and on day by day geometric mean analysed concentrations, the
NOEC72h was 2.56 mg test item.L-1 and both the LOEC72h and the EC50-72h were > 2.56 mg test
item.L-1.
Based on yield rate and on day by day geometric mean analysed concentrations, NOEC72h
was 2.56 mg test item.L-1 and both the LOEC72h and the EC50-72h were > 2.56 mg test item.L-1.

Executive summary:

The objective of this study was to assess the effects of test item DL-Isoleucine, N-(1-

oxohexadecyl) on the growth of Pseudokirchneriella subcapitata over a period of 72 hours

according to OECD 201 Guideline. Pseudokirchneriella subcapitata is a freshwater green

algae that is recommended by the OECD Guideline 201.

Since the test item is poorly soluble under the test conditions and according to the OECD

monograph 23, the choice of a solvent was necessary to solubilize the test item, at the

concentration lower than 0.1 ml.L-1. So, a stock solution of test item was prepared in dimethyl

sulfoxide. Test solutions were prepared by dilution of this solution.

Test item quantification was performed according to a validated analytical method (final report

RRCo-000155_01) 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 at 0, 24,

48 and 72 hours.

According to the range-finding-test, no toxicity was observed under the limit of solubility. So, a

test limit at 5 mg.L-1 was performed.

The effects of DL-Isoleucine, N-(1-oxohexadecyl) diluted in OECD test medium at the rates of

5 mg test item.L-1 to Pseudokirchneriella subcapitata were investigated under laboratory

conditions.

The study is valid since the growth multiplication factor in the control within the 72-hour test

period was higher than 16 (actual value: 128), the coefficient of variation of the average specific

growth rates in the control was less than 7% (actual value: 1.45%) and the mean coefficient of

variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control was

less than 35% (actual value: 21.42% after 72h).

Based on growth rate and on day by day geometric mean analysed concentrations, the

NOEC72h was 2.56 mg test item.L-1 and both the LOEC72h and the EC50-72h were > 2.56 mg test

item.L-1.

Based on yield rate and on day by day geometric mean analysed concentrations, NOEC72h

was 2.56 mg test item.L-1 and both the LOEC72h and the EC50-72h were > 2.56 mg test item.L-1.

The geometric mean concentration at the tested concentration was higher than the solubility

of the test item (1.5 mg.L-1 according the OECD 105).

Description of key information

The objective of this study was to assess the effects of test item DL-Isoleucine, N-(1-

oxohexadecyl) on the growth of Pseudokirchneriella subcapitata over a period of 72 hours

according to OECD 201 Guideline.Since the test item is poorly soluble under the test conditions and according to the OECD monograph 23,it was solubilized in dimethyl sulfoxide at the concentration lower than 0.1 ml.L-1

Based on growth rate and on day by day geometric mean analysed concentrations, the

NOEC_72h was 2.56 mg test item.L-1 and both the LOEC_72h and the EC50_72h were > 2.56 mg test

item.L-1.

Based on yield rate and on day by day geometric mean analysed concentrations, NOEC_72h

was 2.56 mg test item.L-1 and both the LOEC_72h and the EC50-_72h were > 2.56 mg test item.L-1

.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.56 mg/L

EC10 or NOEC for freshwater algae:

2.56 mg/L