Disodium L-cystinate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-07-19 to 2011-11-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cystine

EC Number:

200-296-3

EC Name:

Cystine

Cas Number:

56-89-3

Molecular formula:

C6H12N2O4S2

IUPAC Name:

cystine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity test date: >98.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

Sampling and analysis

Analytical monitoring:

no

Remarks:

Substance specific analysis of the test item in the test medium was not performed. Instead, the dissolved organic carbon (DOC) was measured as an indiciator of the presence of the test item in the test medium.

Details on sampling:

- Concentrations: control (test water without test item) and 100 mg test item/L (nominal)
- Sampling method: Substance specific analysis of the test item in the test medium was not performed. Instead, the dissolved organic carbon (DOC) was measured as an indiciator of the presence of the test item in the test medium.
- Sample storage conditions before analysis: All samples were analyzed immediately after sampling without storage
- Duplicate samples: duplicate samples (without algae) were taken from freshly prepared test medium of the single test concentration and the control at test start.
- No 72-hour stability samples were taken, since the measurement of carbon content is not adequate method to determine the stability or instability of the parent test molecule in the test medium.

Test solutions

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: due to low solubility of test item in test water, a dispersion with the loading rate of 100 mg/L was prepared. 30.0 mg of the test item was dispersed in 300 mL of test water, stirring intensely for 3 hours at room temperature in the dark. After the 3-hour stirring period, the dispersion of the test item (the test item particles were visible) was filtered through a membrane filter (pore size 0.45 um) and the undiluted filtrate was used as test medium.
- Eluate: not specified
- Differential loading: not specified
- Controls: test water without test material
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): none
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): test item particles were visible after 3-hours stirring but was filtered through a membrane filter.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum, green algae
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen); cultivated at Harlan Laboratories under standardized conditions according to the test guidelines
- Age of inoculum (at test initiation): inoculum culture was set up 4 days before start of exposure
- Method of cultivation: cultivated under the test conditions; the inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

ACCLIMATION
- Acclimation period: not specified
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not specified

CONTROL
- Positive control: Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions as well as evaluate the algal quality. The result of the latest positive control test performed in April 2011 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.3 mg/L (Harlan Laboratories Study D25712), range of the 72-hour EC50 for the growth rate from 2000 to 2011: 0.71-1.7 mg/L)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

24, 48, 72 hours

Test conditions

Hardness:

0.15 mmol/L (=15 mg/L as CaCO3)

Test temperature:

23 °C

pH:

8.1-8.5 (control); 7.9-9.0 (test item)

Dissolved oxygen:

not reported

Salinity:

not applicable

Conductivity:

not reported

Nominal and measured concentrations:

100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type (delete if not applicable): closed (covered with a glass dish)
- Material, size, headspace, fill volume: flasks were filled to 15 mL of test solution
- Aeration: not specified
- Renewal rate of test solution (frequency/flow rate): not specified
- Initial cells density: 10,000 cells/mL (nominal)
- Control end cells density: not specified
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, followed guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing.
- Total organic carbon: 0.271 mg DOC/L (control)
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH measured at start and end of test; water temperature and appearance recorded daily

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, pH of test water was adjusted to about 10 to enhance the solubility of the test item in the test water as far as possible
- Light intensity and quality: mean 6700 Lux (range 6000 to 7300 Lux, measured at 9 places in the experimental area)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter (Coulter Counter, Model ZM)
- Growth rate, yield, NOEC, LOEC

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: limit test according to test guidelines due to low solubility of test item
- Range finding study: yes
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Unusual cell shape: none
- Colour differences: none
- Flocculation: none
- Adherence to test vessels: none
- Aggregation of algal cells: none
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, the presence of the test item in the test medium of the single test concentration could be confirmed and the measured DOC-values in the test item treatment correspond to about 98 mg test item/L (based on a C-content of L-Cystine of 30%).
- Effect concentrations exceeding solubility of substance in test medium: 72-hour NOEC was 100 mg/L and the value might be higher but was not tested

Reported statistics and error estimates:

The test item had no statistically significant inhibitory effect on the growth of the algae during the test period of 72 hours at the loading rate of 100 mg/L (results of Student t-tests, one-sided smaller, α = 0.05)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a 72-hour static test conducted according to the OECD Guideline 201 (2006), the test item, L-Cystine, had no acute toxic effects on the green algal species Pseudokirchneriella subcapitata up the its solubility limit in the test water.

Executive summary:

The influence of the test item, L-Cystine, on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006), C.3 (1992) and the Commission Regulation (EC) No 761/2009, C.3.

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the algae up to the solubility limit of the test item in the test water.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared. A control group (test water without test item) was tested in parallel.

Since a filtrate was tested and no substance-specific analytical measurements were performed, the biological results are based on the loading rate of the test item.

At the loading rate of 100 mg/L, the algal growth (average growth rate and yield) was not inhibited (but enhanced) compared to the control after 72 hours of test duration. The EC50 and LOEC were both >100 mg test item/L and the NOEC was ≥100 mg test item/L. In conclusion, the test item, L-Cystine, had no acute toxic effects on the test organism up to its solubility limit in the test water under the present test conditions.