Disodium L-cystinate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-02 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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%

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations: Negative control, 100 mg/L
- Sampling method: Duplicate samples were taken from the freshly prepared test medium of the single test concentration at the start of the test and at test medium renewal. All samples were anlayzed immediately after sampling without storage.
- Sample storage conditions before analysis: not specified

Test solutions

Vehicle:

not specified

Details on test solutions:

Due to the low solubility of the test item in the test water, a dispersion with the loading rate of 100 mg/L was prepared. At the start of the test and at test medium renewal, 30.05 mg respectively 30.02 mg of test item was dispersed in 300 mL of test water stirring intensely for 3 hours at room temperature in the dark.
The stirring period of 3 hours was chosen based on a pre-experiment (non-GLP) which showed that the content of dissolved organic carbon (DOC) in the undiluted filtrate could not be significantly increased prolongating the stirring time to 96 hours. After 3 hours stirring, the content of DOC in the test medium was 28.36 and 29.13 mg/L. After 96 hours, 29.96 and 29.54 mg DOC/L were measured. Thus, the stirring period of 3 hours was applied in the definite test.
After the 3-hours stirring period, the dispersion of the test item (test item particles visible) was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm) and the undiluted filtrate was used as test medium. The test medium was prepared just before the start of the test (= start of exposure).
The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Straus, clone 5
- Source: University of Sheffield / UK and bred at Harlan Laboratories
- Age of parental stock (mean and range, SD):
- Feeding during test: no
- Food type: algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen) or a mixture of the algal suspension and a commercial fish diet (Tetra Min Hauptfutter, supplied by TETRA-Werke).
- Amount: Not specified
- Frequency: 3 times per week

Study design

Test type:

semi-static

Water media type:

not specified

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

2.5 mmol/L, 250 mg/L as CaCO3

Test temperature:

21 - 22 °C

pH:

8.0 - 9.0

Dissolved oxygen:

8.2 - 8.6 mg/L

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

100 mg test item/L (nominal) and control

Details on test conditions:

TEST SYSTEM
- Test vessel: 100-mL glass beakers
- Material, size, headspace, fill volume: filled with 50 mL with test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Aeration: Prior to start of study, the test water was aerated until oxygen saturation was reached. During test period, test water was not aerated.
- Renewal rate of test solution (frequency/flow rate): after 24 hours to keep the concentration of the test item in the test medium as constant as possible during the test period of 48 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000
- Culture medium different from test medium: no
- Intervals of water quality measurement: at the start and end of each test medium renwal period, the pH values, dissolved oxygen concentrations, water temperature were determined at each treatment.

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to 9.0 to enhance the solubility of the test item in the test water was much as possible.
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period was used
- Light intensity: Between approx. 485 to 640 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mobility at 24 and 48 hours, NOEC, EC0, EC50, EC100

VEHICLE CONTROL PERFORMED: no - control was test water without test item

RANGE-FINDING STUDY
- Test concentrations: not specified
- 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:

48 h

Dose descriptor:

EC0

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No abnormalities observed

Reported statistics and error estimates:

Not reported

Any other information on results incl. tables

BIOLOGICAL RESULTS: In the control and at the loading rate of 100 mg/L, no immobilized test organisms were observed during the test period of 48 hours. Since an undiluted filtrate was tested and no substance specific analytical measurements could be performed, the biological results are based on the loading rate of the test item.

 

EFFECT CONCENTRATIONS: The 48-hour NOEC and 48-hour EC0 of L-Cystine to Daphnia magna were determined to be at least the loading rate of 100 mg/L. The 48-hour NOEC and 48-hour EC0 might even be higher, but concentrations above the loading rate of 100 mg/L were not tested, in accordance with the test guidelines. The 48-hour EC50 and the 48-hour EC100 were clearly higher than the loading rate of 100 mg/L. These values could not be quantified due to the absence of toxicity of L-Cystine at the loading rate of 100 mg/L.

 

VALIDITY: The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water). Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

At the start of the test and at test medium renewal, the dissolved organic carbon (DOC) content of the test medium of the single test concentration was determined to be 30.06 and 33.91 mg/L respectively. In the control, 0.173 and 0.536 mg DOC/L was measured. Thus, 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 100 mg test item/L (based on a C-content of L-Cystine of 30%).

The analyzed carbon contents of the standard samples used for the calibration of the system deviated by less than 5% from the nominal value of 50 mg C/L and, thus, the measurements were considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In an acute toxicity test according to the OECD guideline 202 Daphnia magna was exposed to 100 mg L-cystine/L and no toxic effects were observed.

Executive summary:

The 48-hr-acute toxicity of L-Cystine to Daphnia magna was studied under semi-static conditions in a limt test. Daphnids were exposed to control, and test chemical at nominal concentration of 100 mg/L at 24 and 48 hours. Mortality, immobilization and sublethal effects were observed daily.The 48-hour EC50 was at least 100 mg a.i./L. The 48-hr NOEC based on immobilization was at least 100 mg/L.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.