DL-alanine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-22 - 2018-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

"OECD guidelines for testing of chemicals 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test " (2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Analytical purity: 99.9%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 200, 100, 50, 25 and 12.5 mg/L DL-alanine
- Sampling method: For HPLC Analyses 12 mL samples were collected using disposable syringes, filtered (0.45 µm) and collected in glass vials. Samples were taken from the fresh dilutions at the start, and from one replicate 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: The test item was dissolved in OECD medium directly.
- Controls: Algae OECD medium was used as a control.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CODE CCAP: 278/4 - batch 2803/03
- Source (laboratory, culture collection): Test organisms are grown at the VITO laboratory facility.
- Age of inoculum (at test initiation): Algae that were used for the current test were transferred from the breeding medium to the OECD mineral medium on 18/01/2018 (4 days before the start of the test).
- Method of cultivation: Cultivation of the algae was performed using Jaworski medium as breeding medium. No further details on cultivation are specified.

ACCLIMATION
- Acclimation period: Algae that were used for the current test were transferred from the breeding medium to the OECD mineral medium on 18/01/2018 (4 days before the start of the test).
- Culturing media and conditions (same as test or not): Cultivation of the algae was performed using Jaworski medium as breeding medium. No further details on cultivation are specified.
- Any deformed or abnormal cells observed: not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test temperature:

23°C ± 2°

pH:

7.85 - 7.98 at test start
9.07 - 10.36 at test end

Nominal and measured concentrations:

Nominal: 200, 100, 50, 25 and 12.5 mg/L
Measured: As expected from the preliminary tests the test concentrations show a significant decrease during the test.
The actual Alanine concentration for the highest nominal test concentration (200 mg/L) was however >100 mg/L during the entire test duration (time weighted mean value = 164 mg/L).
t(0h): 217, 106, 50, 25 and 12.7 mg/L
t(72h): 112 and 22.8 mg/L for the two highest test concentrations. The recovery in the other test concentrations was below the reporting limit.

Details on test conditions:

TEST SYSTEM
- Test vessel: disposable 500 ml vessels
- Type (delete if not applicable): closed with cotton air permeable stoppers
- Material, size, headspace, fill volume: fill volume: 100 ml medium or test dilution and 100 µl algal inoculum
- Aeration: no
- Initial cells density: appr. 7700 cells/ml
- No. of vessels per concentration (replicates): 3 for test dilutions
- No. of vessels per control (replicates): 6 for controls and 2 abiotic controls

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algae OECD medium was used as a control and dilution medium (composition see "Any other information on materials and methods incl. tables")
- Culture medium different from test medium: Yes. Breeding medium is Jaworski medium, while OECD mineral medium was used for the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: not specified
- Photoperiod: continuous light
- Light intensity and quality: appr. 3000 lux
- Other: continuous shaking (100 rpm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biomass after 24, 28 and 72 h of exposure.
- Determination of cell concentrations: At the start, a dilution series of the algae is measured both by Coulter counter (providing the number of cells in each dilution) and by Fluoroskan (providing a fluorescent signal). The correlation between both signals was then used during the rest of the test to recalculate the biomass from the Fluoroskan signal.
Daily fresh samples (800 pl) were taken from each test vessel and transferred to a 48 well plate. The fluorescence in each well was measured using a plate reader Fluoroskan and recalculated to the number of cells (biomass).
The mean fluorescent signal measured in the abiotic vessels is considered as blank signal and is used to correct fluorescent signals in the biotic compartments.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes. The preliminary test showed no adverse effects when concentrations up to 1000 mg/l were tested.
- Test concentrations: 200, 100, 50, 25 and 12.5 mg/l DL-alanine and a control
- Results used to determine the conditions for the definitive study: The stability test showed a decrease of almost 40% of the nominal concentration within 72 hours. To ensure that at least in one treatment the actual test concentration would remain above 100 mg/l during the entire test duration, the stated range of concentrations was tested.

Reference substance (positive control):

yes

Remarks:

Periodically an algal growth inhibition test is performed with ZnCl2 as a reference item. The last test was performed in February 2018.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 164 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 164 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 164 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not specified
- Any stimulation of growth found in any treatment: Slightly stimulatory effects) were seen on either growth rate or yield. These stimulatory effects were not taken into account for further evaluation, as the effects are small and not concentration related.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the obtained result is within the historical range (0.08 - 0.17 mg/L: mean value 0.12 mg/L).
- EC50 (72h) = 0.10 mg/L ZnCl2

Reported statistics and error estimates:

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

pH values

pH values at the start and the end of the test are listed in table 3. The pH values increased in all test conditions. pH increase is positively correlated with the growth rate. According to the OECD guideline the pH increase should not exceed 1.5 units, to avoid pH effects on the toxicity of the test item. In the current test, the increase was <1.5 in the two highest test concentrations.

pH values in the different test conditions at the start and at the end of the test:

Nominal concentration [mg/L]	pH		Increase
	t = 0 h	t = 72 h
200	7.85	9.07	1.22
100	7.90	9.24	1.34
50	7.94	9.46	1.52
25	7.93	10.03	2.10
12.5	7.92	10.28	2.36
0	7.98	10.36	2.38

pH increased by more than 1.5 units in the controls. This has no adverse effects on the growth pattern of the controls as can be concluded from the linear growth curve and the fulfilled validity criteria (§7.3).

Validity of the test

In controls:

a) The biomass should increase at least 16x from 0-72 hours, i.e. a minimum value for a specific growth rate of 0.92/day. This was the case: biomass increased 75.7 x, specific growth rate was 1.44/day.

b) The coefficient of variance (CV) for the mean specific growth rate for the individual days (days 0-1, 1-2, 2-3) < 35 %. This was the case: CV was 21.9 %.

c) The coefficient of variance for the specific growth rate for day 0 - 3 for replicates < 7 %. This was the case: CV was 0.57 %.

Test results

As the test concentrations decreased below reporting limit in the three lowest test concentrations, and <100 mg/L in the nominal 100 mg/L solution, only the results at the highest nominal test concentration are relevant to evaluate possible effects on the algae at an actual exposure concentration of >100 mg/L (nominal concentration of 200 mg/L , time-weighted mean measured concentration was 164 mg/L). In this treatment the pH shift is < 1.5 as required by OECD.

No inhibitory effects (but slightly stimulatory effects) were seen on either growth rate or yield in this test concentration (nor in the other different test concentrations). These stimulatory effects were not taken into account for further evaluation: the effects are small and not concentration related.

Because no inhibitory effects could be observed at the highest test concentration it can be concluded for both growth parameters:

NOEC ≥ 164 mg/L (i.e. time-weighted mean measured concentration)

LOEC > 164 mg/L (i.e. time-weighted mean measured concentration)

EbC50/ErC50 >164 mg/L (i.e. time-weighted mean measured concentration)

% inhibition of the growth rate by the test item DL-Alanine in the different test concentrations (negative values indicate stimulation):

% inhibition growth rate
concentration (mg/L)	200	100	50	25	12.5	0
Replicate 1	-3.79	-4.28	-1.80	-3.03	-1.70	-0.44
Replicate 2	-3.05	-4.72	-0.66	-2.87	-1.97	-0.52
Replicate 3	-2.02	-4.57	-2.99	-2.63	-2.02	-0.30
Replicate 4						0.01
Replicate 5						0.23
Replicate 6						1.01
mean	-3.0	-4.5	-1.8	-2.8	-1.9	0.0
SD	0.9	0.2	1.2	0.2	0.2	0.6

% inhibition of the yield by the test item DL-Alanine in the different test concentrations (negative values indicate stimulation):

% inhibition biomass (Yield (%Iy))
concentration (mg/L)	200	100	50	25	12.5	0
% inhibition	-17.2	-25.5	-11.5	-16.6	-11.8	0.0

 

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 201 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 algae.
The study was performed with 5 test concentrations of nominal 200, 100, 50, 25 and 12.5 mg/L. Incubation time (test organism Pseudokirchneriella subcapitata) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours using a plate reader Fluoroskan and recalculated to the number of cells (biomass) by the use of a correlation between Coulter counter (providing the number of cells in each dilution) and Fluoroskan (providing a fluorescent signal) that was set up at test start with a dilution series of algae. Growth rate µ and the yield were determined from the cell number at the respective observation times. No significant inhibition of algal growth was observed for both parameter.
At the start and the end of the test, the content of the test item was determined using HPLC. As expected from the preliminary tests the test concentrations show a significant decrease during the test. The actual Alanine concentration for the highest nominal test concentration (200 mg/lL was > 100 mg/L during the entire test duration (time weighted mean value = 164 mg/L). The determination of the results was based on the time weighted mean values.
The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.
pH increased by more than 1.5 units in the blank controls. This has no adverse effects on the growth pattern of the controls as can be concluded from the linear growth curve and the fulfilled validity criteria.In the two highest test concentrations the increase of pH was <1.5.
All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid
As no significant inhibition was observed up to and including the highest test concentration of 164 mg/L (time-weighted mean measured concentration), the EC50 for the parameter growth rate and yield was determined to be > 164 mg/L. Accordingly, DL-alanine does not need to be classified as hazardous to the environment, neither acute nor chronic.

Executive summary:

The study was conducted under GLP according to OECD guideline 201 on the registered substance itself.

One valid experiment was performed.

The study was performed with 5 test concentrations of nominal 200, 100, 50, 25 and 12.5 mg/L. Incubation time ( test organism Pseudokirchneriella subcapitata) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours using a plate reader Fluoroskan and recalculated to the number of cells (biomass) by the use of a correlation between Coulter counter (providing the number of cells in each dilution) and Fluoroskan (providing a fluorescent signal) that was set up at test start with a dilution series of algae. Growth rate µ and the yield were determined from the cell number at the respective observation times. No significant inhibition of algal growth was observed for both parameter.

At the start and the end of the test, the content of the test item was determined using HPLC. As expected from the preliminary tests the test concentrations show a significant decrease during the test. The actual Alanine concentration for the highest nominal test concentration (200 mg/L) was > 100 mg/L during the entire test duration (time weighted mean value = 164 mg/L). The determination of the results was based on the time weighted mean values.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results were determined for the test item DL-alanine:

Endpoint	NOEC	LOEC	EC50
Growth rate	≥ 164 mg/L	> 164 mg/L	> 164 mg/L
Yield	≥ 164 mg/L	> 164 mg/L	> 164 mg/L

* Values are based on time-weighted mean measured concentrations of DL-alanine

 

Based on the obtained results DL-alanine does not need to be classified as hazardous to the environment, neither acute nor chronic.

Description of key information

No effect at a concentration of 1000 mg/L test item.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

The performed GLP main experiment did not produce suitable results. In the only treatment group of nominal 500 mg test item/L, a high turbidity occurred in the replicates containing algae. This turbidity was caused by a contamination with microorganisms which could not be inhibited performing the test under sterile conditions. Due to the turbidity and/or interfering effects with the contamination the algae growth in the only treatment group was reduced and the low inhibitions allowed in a limit test were exceeded.

In accordance with Annex XI No. 1.2 there is sufficient weight of evidence from several independent sources of information leading to the assumption/conclusion that the substance does not cause aquatic toxicity to algae. Despite of that the described limit test was performed.

Although the main part of the above mentioned limit test did not produce reliable results the non-GLP pre-experiment indicated that no effect was caused by the test item itself up to 1000 mg test item/L. A cell count of the cell density at this concentration showed no inhibition in the cell number in comparison to the control cell density. Therefore the completed part of the study confirmed the assumption that L-alanine does not cause toxicity to aquatic algae and cyanobacteria.

This is not surprising. L-alanine is a naturally occurring essential amino acid which is omnipresent in nature. It is a basic metabolit and building block of all living organisms and cells and is also a nutrient for algae. Thus significant toxicity to algae can be excluded.