DL-alanine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-23 - 2018-01-25 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

"OECD guidelines for testing of chemicals 202: Daphnia sp. Acute Immobilisation Test" (2004)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L and a control
- Sampling method: 12 ml samples were collected using disposable syringes, filtered (0.45 pm) and collected in glass vials. Three samples were taken from the fresh dilution at the start, and from three replicate vessels of the used solutions at the end of the test.
- Sample storage conditions before analysis: All samples were immediately frozen (-20°C) and kept frozen until analyses.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: At test start 50.9 mg of the test item DL-alanine was weighed and added to 500 ml of JP 4 medium. The test item is easily soluble in the medium.
- Controls: JP 4 medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test item is easily soluble in the medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: DM 04 02 K36
- Source: Test organisms are grown at the VITO laboratory facility.
- Feeding during test: no

ACCLIMATION
- Acclimation period: No acclimatisation necessary since JP 4 medium was used in the culture / breeding and in the test.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: For the test neonate organisms (max. 24 hours of age) were used. On 22/01/2018 (one day before test start) some parent organisms were separated in a breeding vessel and fed with enriched algae food. The youngsters that were born overnight were then used for this test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

19.5°C ± 0.1°C

pH:

7.97 - 8.01

Dissolved oxygen:

95 - 96%

Conductivity:

582 - 609 µS/cm

Nominal and measured concentrations:

A limit test was performed with the nominal test concentration of 100 mg/l DL-alanine. The concentrations in the three samples that were taken from the freshly prepared solution at the beginning of the test showed high variability, indicating that at the time of sampling the solution was not homogeneous (t(0h): 62.4, 109.2, 77.5 mg/L). The measurements from three individual replicates at the end of the test are much more identical, indicating that at the time the vessels were filled the solution was well homogenised (t(48h): 113.6, 101.1, 99.4 mg/L). The analytical results show an actual DL-alanine concentration of 105 mg/l at the end of the test, which is comparable to the intended 100 mg/l nominal test concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml glass vessels, with transparent lid
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: fill volume of 20 ml per vessel
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: JP4 medium was used as dilution medium (composition see "Any other information on material and methods including tables").
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: Light/Dark= 16h/8h
- Light intensity: not critical

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After 24 and 48 hours of exposure the mobility of the organisms was evaluated: the vessels were slightly shaken to provoke movement of the organisms, and the number of mobile organisms was registered.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Concentrations up to 1000 mg/l were tested.
- Results used to determine the conditions for the definitive study: The preliminary test (DAC 18 001) showed no adverse effects in none of the concentrations tested. The stability test showed that a nominal concentration of 100 mg/l of the test item is stable for more than 80 hours. Therefore, it was decided to perform a static limit test using an exposure concentration of 100 mg/l DL-alanine.

Reference substance (positive control):

yes

Remarks:

Periodically an acute daphnia test is performed with ZnCl2 as a reference item. The last test was performed in February 2018.

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

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No, all test organisms were active and mobile after 48 hours of exposure at the test concentration of 100 mg/L.
- Mortality of control: No, mean survival in the control replicates was 100%.
- Other adverse effects control: No.
- Abnormal responses: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- Relevant effect levels: historical range for EC50 (48h) = 1.04-2.35 mg/L ZnCl2, mean value 1.7 mg/L ZnCl2
- ECx: EC50 (48h) = 2.2 mg/L ZnCl2

Reported statistics and error estimates:

A statistical analysis was not necessary, as no immobilisation or any other adverse effects were observed.

Validity of the test

Validity criteria were met:

• Mean survival in the control replicates was 100% and met the validity criterion that not more than 10% of the daphnids should be immobilised.
• The dissolved oxygen concentration was ≥ 95% oxygen in both test conditions and met the validity criterion of at least 3 mg/l (appr. 30% of the air saturation at 20°C) during the test duration.

Test results

All test organisms were active and mobile after 48 hours of exposure at the test concentration (100 mg/L) and LC50 > 100 mg/L.

Conclusions

It is concluded that a concentration of 100 mg/L of the test item DL-alanine had no acute effect on the mobility of Daphnia magna and LC50 > 100 mg/L DL-alanine.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 202 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of DL-alanine towards daphnids.
Based on preliminary test results a limit test was performed with the nominal test concentration of 100 mg/l. 20 Daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised Daphnia were counted.
None of the concentrations showed toxicity and also none of the animals was immobilised in the blank control.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.
At the beginning and at the end of the test, the content of the test item in the test solution was determined using HPLC determination. The concentrations in the three samples that were taken from the freshly prepared solution at the beginning of the test showed high variability, indicating that at the time of sampling the solution was not homogeneous. The measurements from three individual replicates at the end of the test are much more identical, indicating that at the time the vessels were filled the solution was well homogenised. The analytical results show an actual DL-alanine concentration of 105 mg/l at the end of the test, which is comparable to the intended 100 mg/l nominal test concentration. Therefore, the determination of the biological results was based on the nominal concentration.
No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met.
The result of the test is considered valid.
The EC50 > 100 mg/L is above the limit value for classification as aquatic chronic Cat. 3, so DL-Alanine does not need to be classified according to Regulation 1272/2008 and amendments.

Executive summary:

The study was conducted under GLP according to OECD guideline 202 on the registered substance itself.

One valid experiment was performed.

Based on the results of a preliminary test the study was performed using a limit concentration of nominal 100 mg/L. Daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised Daphnia were counted.

None of the concentrations showed toxicity and also none of the animals was immobilised in the blank control.

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.

At the beginning and at the end of the test, the content of the test item in the test solution was determined using HPLC determination. The concentrations in the three samples that were taken from the freshly prepared solution at the beginning of the test showed high variability, indicating that at the time of sampling the solution was not homogeneous. The measurements from three individual replicates at the end of the test are much more identical, indicating that at the time the vessels were filled the solution was well homogenised. The analytical results show an actual DL-alanine concentration of 105 mg/l at the end of the test, which is comparable to the intended 100 mg/l nominal test concentration. Therefore, the determination of the biological results was based on the nominal concentration.

The following results were determined for the test item DL-Alanine (species: Daphnia magna).

48h-NOEC ≥ 100 mg/L
48h-LOEC > 100 mg/L
24h-EC50 > 100 mg/L
48h-EC50 > 100 mg/L

Description of key information

The toxic effect of the test item L-alanine to Daphnia magna was assessed in a static limit-test. The 48-hour NOEC was determined to be 100 mg test item/L. The 48-hour LOEC was determined to be > 100 mg test item/L and the 48-hour EC50 value was determined to be >100 mg test item/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

L-Alanine did not show short-term toxicity to Daphnia magna. It can be concluded that L-alanine does not exhibit short-term toxicity to aquatic invertebrates.