Methyl L-threoninate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The toxicity of the molecule to aquatic organisms is expected to be a result of the structure of the substance and the bioavailability (water solubility, Pow and vapor pressure) of the substance. Substances with similar structures and bioavailability will therefore show the same toxicity to aquatic organisms. Further information on read across to L-TEE using the analogue approach can be found in the data matrix table below attached as background material and in section 13.

Justification for type of information:

Data on target substance is not available. Thus, read-across has been applied using data drom the source substance L-Threonine Ethyl Ester (L-TEE). See further read-across justification attached as background document and in section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007.08.21 - 2007.08.24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemical No. 201 "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". ISO International Standard 8692 "Water quality - Freshwater algal growth inhibition test with unicellular green algae.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Siegfried Ltd., Switzerland; lot. TSC9153(NN); batch. 0706H016(Siegfried)
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 50-55°C
- Boiling point: 180.7-202.1°C
- Water solubility (under test conditions): > 4,000 g/L (20ºC)
OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: 97+/-3% of the ThOD after 28 days

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: -20ºC

Analytical monitoring:

yes

Remarks:

TOC analyses were not carried out in compliance with the GLP principles

Details on sampling:

Concentrations: Subsamples from control (as background determination) and from the test concentrations 500 mg/L, 1,000 mg/L and 2000 mg/L
- Sampling method: samples taken from flasks with test solution but without algae (blanks) at the initiation and the termination of the test.

Vehicle:

no

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: The test substance was disolved in test medium. The test medium was prepared from Millipore water according to ISO 8692 standard.

Test organisms (species):

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

Details on test organisms:

(clone: NIVA, CHL 1). The algae was received from NIVA, Norway and cultured at DHI under the test conditions prior to the start of the
test.

Test type:

other: continuous shaking

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

cell density at beginning of the test and after 24, 48 and 72 ± 2 hours

Hardness:

24 mg CaCO3/L

Test temperature:

23.1 ± 0°C (measured)

pH:

7.9-8.1 (start)
7.6-8.8 (end)

Variation start-end < 1 pH unit

Dissolved oxygen:

no data

Salinity:

NA

Nominal and measured concentrations:

Nominal
concentration Sampling time Theoretical organic carbon content Actually measured organic carbon content
(mg TOC/L) (mg TOC/L)
Control 0 hours 0 <2*
Control 72 hours 0 <2*
500 mg/L 0 hours 245 220
500 mg/L 72 hours 245 231
1,000 mg/L 0 hours 490 427
1,000 mg/L 72 hours 490 444
2,000 mg/L 0 hours 979 919
2,000 mg/L 72 hours 979 847

Details on test conditions:

The algae were incubated for approx. 72 hours under continuous shaking (approx. 120 RPM)

TEST SYSTEM
- Test vessel: The test was performed in glass flasks with wide neck and a total capacity of 250 mL, each containing 100 mL of test solution
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 100mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: The cell density was approx. 9,000 cells/mL at test start, measured in the control by use of a Beckman Coulter MultisizerTM 3 Coulter Counter®.
- Control end cells density:
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):6
- No. of vessels per vehicle control (replicates): NA

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture medium different from test medium: No
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality:
Constant illumination from a panel of fluorescent light with anintensity of approx. 60-120 μmol × m-2 × sec-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-The cell density was approx. 9,000 cells/mL at test start, measured in the control by use of a Beckman Coulter MultisizerTM 3 Coulter Counter®. The density was measured in the control mixture as in-vivo fluorescence at the beginning of the test and after 24, 48 and 72
± 2 hours of incubation in triplicate test flasks and the six controls. The fluorescence was measured by use of a Turner TD-700 Laboratory Fluorometer.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7, Merck 4864, Batch No. K30614564 227)ko½

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2 000 mg/L

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1 118 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 2 000 mg/L

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

500 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

>= 1 000 mg/L

Basis for effect:

growth rate

Results with reference substance (positive control):

potassium dichromate (K2Cr2O7, Merck 4864, Batch No. K30614564 227)
NOEC EC10 EC50
0.2mg/L 0.34 mg/L(0.27-0.40) 0.80mg/L (0.73-0.86)

Reported statistics and error estimates:

VKI (1999): TOXEDO Ver. 1.5. Program for statistical estimation of EC values,
based on experimental data from ecotoxicological assays.

US-EPA (1989): Short term methods for estimating the chronic toxicity of effluents
and receiving waters to freshwater organisms. Computer program: Dunnett’s
procedure in the analysis of data from short term toxicity tests with aquatic organisms.
US-EPA, Cincinnati, Version 1.1.

no

Validity criteria fulfilled:

yes

Conclusions:

A NOEC of 500 mg/L for the growth rate was determined. EC10 and EC50 were 1,118 mg/L and > 2,000 mg/L, respectively

Executive summary:

The test substance L-TEE was tested according to the OECD 201, which is further specified in the ISO International Standard 8692.

L-TEE was tested at the following nominal concentrations: 0 (control); 20; 50; 100; 200; 500; 1,000 and 2,000 mg/L. The growth of the algae during 72 hours was followed by measuring the fluorescence at the beginning of the test and every 24 hours. In order to verify the actual test concentrations, samples of the test solutions were collected for chemical analysis. The actual test concentrations were verified by analysis of the Total Organic Carbon (TOC) content.

The test results can be summarised as follows:

• The TOC analyses performed on subsamples from the three highest test concentrations at the initiation and termination of the test, respectively, showed a recovery of the test substance in the range of 86-94%. As it is thus demonstrated that the concentration of the test substance was satisfactorily maintained within ± 20% of the nominal value, the obtained results are based on the nominal test concentrations.

• At a concentration of 20-500 mg/L, no effect was observed on the growth rate of Pseudokirchneriella subcapitata. Significant inhibition of the growth of Pseudokirchneriella subcapitata was observed at concentrations from 1,000-2,000 mg/L.

• NOEC for the growth rate was 500 mg/L. EC10 was 1,118 mg/L and EC50 was >2,000 mg/L.

Data source

Reference

Reference Type:

other: Body responsible for the test

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemical No. 201 "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". ISO International Standard 8692 "Water quality - Freshwater algal growth inhibition test with unicellular green algae.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl L-threoninate

EC Number:

222-158-1

EC Name:

Methyl L-threoninate

Cas Number:

3373-59-9

Molecular formula:

C5H11NO3

IUPAC Name:

Methyl (2S,3R)-2-amino-3-hydroxy-butanoate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): L-threonine methyl ester
- Physical state: white crystalline powder
- Analytical purity: 99.8%

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 50-55°C
- Boiling point: 180.7-202.1°C
- Water solubility (under test conditions): > 4,000 g/L (20ºC)
OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: 97+/-3% of the ThOD after 28 days

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

no data

Test solutions

Vehicle:

no

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: The test substance was disolved in test medium. The test medium was prepared from Millipore water according to ISO 8692 standard.

Test organisms

Test organisms (species):

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

Details on test organisms:

(clone: NIVA, CHL 1). The algae was received from NIVA, Norway and cultured at DHI under the test conditions prior to the start of the
test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

23.1 ± 0°C (measured)

pH:

7.9-8.1 (start)
7.6-8.8 (end)

Variation start-end < 1 pH unit

Dissolved oxygen:

no data

Salinity:

NA

Nominal and measured concentrations:

Nominal
concentration Sampling time Theoretical organic carbon content Actually measured organic carbon content
(mg TOC/L) (mg TOC/L)
Control 0 hours 0 <2*
Control 72 hours 0 <2*
500 mg/L 0 hours 245 220
500 mg/L 72 hours 245 231
1,000 mg/L 0 hours 490 427
1,000 mg/L 72 hours 490 444
2,000 mg/L 0 hours 979 919
2,000 mg/L 72 hours 979 847

Details on test conditions:

The algae were incubated for approx. 72 hours under continuous shaking (approx. 120 RPM)

TEST SYSTEM
- Test vessel: The test was performed in glass flasks with wide neck and a total capacity of 250 mL, each containing 100 mL of test solution
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 100mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: The cell density was approx. 9,000 cells/mL at test start, measured in the control by use of a Beckman Coulter MultisizerTM 3 Coulter Counter®.
- Control end cells density:
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):6
- No. of vessels per vehicle control (replicates): NA

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality:
Constant illumination from a panel of fluorescent light with anintensity of approx. 60-120 μmol × m-2 × sec-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Other: The cell density was approx. 9,000 cells/mL at test start, measured in the control by use of a Beckman Coulter MultisizerTM 3 Coulter Counter®. The density was measured in the control mixture as in-vivo fluorescence at the beginning of the test and after 24, 48 and 72
± 2 hours of incubation in triplicate test flasks and the six controls. The fluorescence was measured by use of a Turner TD-700 Laboratory Fluorometer.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7, Merck 4864, Batch No. K30614564 227)ko½

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2 000 mg/L

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

500 mg/L

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1 118 mg/L

Basis for effect:

growth rate

Details on results:

L-TME holds the same structure as L-TEE except that L-TME contains a methyl alkyl-side group to the ester bond and L-TEE an ethyl alkyl-side group. Both substances are characterized by a high water solubility, a low log Pow and low vapor pressure. Due to the structure similarity and the similarity in physico-chemical properties determining the bioavailability of the substances L-TEE and L-TME, the toxicity of L-TME to aquatic algae and cyanobacteria is expected to be the same or very close to the toxicity of L-TEE.

Results with reference substance (positive control):

potassium dichromate (K2Cr2O7, Merck 4864, Batch No. K30614564 227)
NOEC EC10 EC50
0.2mg/L 0.34 mg/L(0.27-0.40) 0.80mg/L (0.73-0.86)

Reported statistics and error estimates:

VKI (1999): TOXEDO Ver. 1.5. Program for statistical estimation of EC values,
based on experimental data from ecotoxicological assays.

US-EPA (1989): Short term methods for estimating the chronic toxicity of effluents
and receiving waters to freshwater organisms. Computer program: Dunnett’s
procedure in the analysis of data from short term toxicity tests with aquatic organisms.
US-EPA, Cincinnati, Version 1.1.

Any other information on results incl. tables

no

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A NOEC = 500 mg/L for the growth rate was determined for L-TEE. EC10 and EC50 were 1,118 mg/L and > 2,000 mg/L respectively
L-TME holds the same structure as L-TEE except that L-TME contains a methyl alkyl-side group to the ester bond and L-TEE an ethyl alkyl-side group. Both substances are characterized by a high water solubility, a low log Pow and low vapor pressure. Due to the structure similarity and the similarity in physico-chemical properties determining the bioavailability of the substances L-TEE and L-TME, the toxicity of L-TME to aquatic algae and cyanobacteria is expected to be the same or very close to the toxicity of L-TEE.

Executive summary:

By use of read across to the analogue substance L-TEE, the acute toxicity of L-TME to aquatic algae, EC50 (72 hours) is considered to be > 2 000 mg/L and the NOEC 500 mg/L.

The test substance L-TEE was tested according to the OECD 201, which is further specified in the ISO International Standard 8692. L-TEE was tested at the following nominal concentrations: 0 (control); 20; 50; 100; 200; 500; 1,000 and 2,000 mg/L. The growth of the algae during 72 hours was followed by measuring the fluorescence at the beginning of the test and every 24 hours.

In order to verify the actual test concentrations, samples of the test solutions were collected for chemical analysis. The actual test concentrations were verified by analysis of the Total Organic Carbon (TOC) content. The test results can be summarised as follows:

The TOC analyses performed on subsamples from the three highest test concentrations at the initiation and termination of the test, respectively, showed a recovery of the test substance in the range of 86-94%. As it is thus demonstrated that the concentration

of the test substance was satisfactorily maintained within ± 20% of the nominal value, the obtained results are based on the nominal test concentrations.

At a concentration of 20-500 mg/L, no effect was observed on the growth rate of Pseudokirchneriella subcapitata. Significant inhibition of the growth of Pseudokirchneriella subcapitata was observed at concentrations from 1,000-2,000 mg/L.

NOEC for the growth rate was 500 mg/L for L-TEE. EC10 was 1,118 mg/L and EC50 was >2,000 mg/L.

L-TME holds the same structure as L-TEE except that L-TME contains a methyl alkyl-side group to the ester bond and L-TEE an ethyl alkyl-side group. Both substances are characterized by a high water solubility, a low log Pow and low vapor pressure. Due to the structure similarity and the similarity in physico-chemical properties determining the bioavailability of the substances L-TEE and L-TME, the toxicity of L-TME to aquaticalgae and cyanobacteriais expected to be the same or very close to the toxicity of L-TEE.