DL-alanine

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1988-02-01 to 1988-02-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, used for read-across

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See Chapter 13 for Read Across document

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

(1984)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was weighed out and each portion was dissolved in 1 L of dilution water.
- Eluate: The diluation water used was DSWL, prepared from groundwater.
- Controls: DSWL (negative control)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Source: M.B. Ruysbroek b.v., Noordvliet 159, NL-Maassluis
- Length at study initiation (length definition, mean, range and SD): 2.1 ± 0.2 cm
- Weight at study initiation (mean and range, SD): 0.07 ± 0.02 g
- Feeding during test: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Hardness, expressed as CaCO3: 210 mg/L

Test temperature:

25 ± 1 °C

pH:

Control medium: Beginning of test: 8.1 to 8.2. End of test: 7.8 to 8.0
320 mg/L test substance: Beginning of test: 7.6 to 7.9. End of test: 7.9 to 8.0
560 mg/L test substance: Beginning of test: 7.5 to 7.8. End of test: 7.9 to 8.1
1000 mg/L test substance: Beginning of test: 7.4 to 7.7. End of test: 7.8 to 7.9
1800 mg/L test substance: Beginning of test: 7.3 to 7.5. End of test: 7.7 to 7.8
3200 mg/L test substance: Beginning of test: 7.2 to 7.4. End of test: 7.6 to 7.7

Dissolved oxygen:

Control medium: Beginning of test: 8.1 to 8.4 mg/L. End of test: 7.3 to 7.8 mg/L
320 mg/L test substance: Beginning of test: 8.3 to 8.5 mg/L. End of test: 7.2 to 7.7 mg/L
560 mg/L test substance: Beginning of test: 8.1 to 8.5 mg/L. End of test: 7.5 to 7.9 mg/L
1000 mg/L test substance: Beginning of test: 8.0 to 8.5 mg/L. End of test: 7.4 to 7.8 mg/L
1800 mg/L test substance: Beginning of test: 8.1 to 8.5 mg/L. End of test: 7.2 to 7.8 mg/L
3200 mg/L test substance: Beginning of test: 8.1 to 8.5 mg/L. End of test: 7.2 to 7.7 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations of test substance: 320, 560, 1000, 1800, 3200 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 2000 ml
- Aeration: slightly aerated
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater from NL-Linschoten. medium prepared by adding several salts.
- Metals: << 1 mg/l
- Chlorine: 2.72 mml/l
- Na+: 1.19 mmol/l
- K+: 0.20 mmol/l
- Ca2+: 1.36 mmol/l
- Mg2+. 0.73 mmol/l
- SO42-: 0.73 mmol/l
- HCO3-: 1.39 mmol/l
- Ca/Mg ratio: 1.86
- Culture medium different from test medium: yes
- Intervals of water quality measurement: Once prior to beginning for the above paramters

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : LC50 after 24 h, 48 h, 72 h and 96 h; LC 100, NOEC (mortality), NOEC (swimming behaviour) after 96 h.

TEST CONCENTRATIONS
- Test concentrations: 320, 560, 1000, 1800, 3200 mg/l

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks:

(= 96h LC0)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks:

(swimming behaviour)

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight: none
- Other biological observations: none
- Mortality of control: no
- 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

Validity criteria fulfilled:

yes

Conclusions:

DL-Methionine did not show acute toxicity effects to fish in a test according to OECD 203. LC 100 > 3200 mg/L, NOEC (mortality; swimming behaviour) >= 3200 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1990-10-15 to 1991-04-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, used for read-across

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was weighed out and each portion was dissolved in 1 l of dilution water.
- Eluate: The diluation water used was DSWL, prepared from groundwater.
- Controls: DSWL (negative control)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): visually

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Source: m.B. Ruysbroek b.V., Noordvliet 159, NL-Maassluis
- Length at study initiation (length definition, mean, range and SD): 2.3 +/- 0.1 cm (SD)
- Weight at study initiation (mean and range, SD): 0.1 +/- 0.02 g (SD)
- Feeding during test: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Hardness, expressed as CaCo3: 217 mg/l

Test temperature:

24.2 to 25.1 °C

pH:

Control medium: Beginning of test: 7.9. End of test. 7.6 to 7.8
1 g/l test substance: Beginning of test: 7.4 to 7.5. End of test: 7.5 to 7.8

Dissolved oxygen:

Control medium: Beginning of test: 8.0 to 8.6 mg/l. End of test. 7.6 to 8.3 mg/l.
1 g/l test substance: Beginning of test: 8.6 to 8.8 mg/l. End of test: 7.3 to 8.3 mg/l.

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal test concentration: 1 g/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass beakers
- Type: open
- Material, size, headspace, fill volume: glass, 2000 ml
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater from NL-Linschoten. medium prepared by adding several salts.
- Metals: << 1 mg/l
- Chlorine: 2.79 mml/l
- Na+: 1.33 mmol/l
- K+: 0.24 mmol/l
- Ca 2+: 1.43 mmol/l
- Mg2+. 0.74 mmol/l
- SO42-: 0.74 mmol/l
- Ca/Mg ratio: 1.93
- Culture medium different from test medium: yes
- Intervals of water quality measurement: Once prior to beginning for the above paramters

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light - 8 h dark with transition periods of 30 minutes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : LC50, NOEC after 96 h.
Condition: Swimming behaviour, colour, respiratory function, any other morphological or visual behavioural criterion after 24 h, 48 h, 72 h and 96 h.

TEST CONCENTRATIONS
- Test concentrations: 1.0 g/l

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 g/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Exact LC50 could not be calculated because there was no mortality at the only concentration tested.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 1 g/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Exact NOEC could not be calculated because there were no adverse effects at the only concentration tested.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 1 g/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks on result:

other: Exact NOEC could not be calculated because there were no adverse effects at the only concentration tested.

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight: none
- Other biological observations: none
- Mortality of control: no
- 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

Validity criteria fulfilled:

yes

Conclusions:

L-valine did not show any adverse effects including mortality in a test according to OECD 203 at a concentration of 1 g/L.

Description of key information

The predicted LC50 for short-term fish toxicity (96h) of DL-alanine is 26300 mg/L. The value was estimated by QSAR prediction based on trend analysis.

For Risk assessment and classification a short term toxicity to fish (96h) for DL-Alanine of >100 mg/L will be used as a realistic worst-case.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Marine water fish

Marine water fish

Effect concentration:

100 mg/L

Additional information

No experimental data for DL-alanine with regard to fish toxicity is available.

No toxicity at the limit concentration was observed in acute toxicity studies with daphnia (according to OECD202) and algae (according to OECD201) indicating that the substance has a very low / no hazard potential for aquatic organisms. Further, as the substance (in protein) is a basic component of fish food it is very unlikely that fish in an OECD203 study exposed to limit concentrations (100 mg/L) of L-alanine show any adverse effect. Therefore and for animal welfare reasons is seems sufficient to use an in silico approach to predict the LC50 for short-term fish toxicity (96h). An estimation by QSAR based on trend analysis results in a LC50 (96h) of L-alanine of 26300 mg/L.

Toxicity to fish (LC50) as predicted from Ecosar v0.99g is 383000 mg/L (Ecological Categorization Results from the Canadian Domestic Substance List, 2006).

The very low toxicity of proteinogenic amino acids to fish is further supported by two OECD203 studies with L-valine and D/L-methionine. Read-across was applied because of similar physico-chemical characteristics, a similar toxicological/ecotoxicological profile and the structural similarity of these amino acids to L-alanine. Brachydanio rerio exposed to 1000 mg/L L-valine in a limit study showed no signs of toxicity. The same was true if this species was exposed to D/L-methionine. Here Brachydanio rerio showed no signs of toxicity even at the highest concentration tested (3200 mg/l).

Another supporting study indicates that L-alanine does not cause any impact on the behaviour of fish.