L-Ascorbic acid, 3-O-ethyl-

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:

25 - 28 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Test material

Test material information

Constituent 1

Reference substance name:

(5R)-5-[(1S)-1,2-dihydroxyethyl]-4-ethoxy-3-hydroxy-2,5-dihydrofuran-2-one

EC Number:

617-849-3

Cas Number:

86404-04-8

Molecular formula:

C8H12O6

IUPAC Name:

(5R)-5-[(1S)-1,2-dihydroxyethyl]-4-ethoxy-3-hydroxy-2,5-dihydrofuran-2-one

Details on test material:

- Name of test material (as cited in study report): Ethyl ascorbic acid
- Physical state: powder
- Expiration date of the lot/batch: 21 Apr 2018
- Stability under test conditions: stable
- Storage condition of test material: in freezer at < -15 °C desiccated

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 0.1, 1.0, 10, 100 mg/L
- Sampling method: Samples were taken after 0, 24 and 48 h. 2 mL were taken from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples taken at the beginning of the study and after 24 h were stored in a freezer until analysis whereas samples taken at the end of the study were analysed immediately. At the end of exposure, the replicates were pooled at each concentration before sampling. Additionally, reserve samples of 2.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The highest test concentration of 100 mg/L was prepared by adding appropriate amount of the test substance to test medium followed by vigorous mixing. The lower concentrations were prepared by subsequent dilution of this stock solution.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material: no, test solutions were clear and colorless

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24 °C. The stock culture medium was M1 whereas M2-medium was used as the pre-culture medium. 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21 - 23 °C

pH:

8.1 - 8.2 (control)
7.4 - 7.8 (100 mg/L)

Nominal and measured concentrations:

nominal: control, 0.10, 1.0, 10, 100 mg/L
measured (0 h): < LOQ (control), 76.7 - 83.7 mg/L (100 mg/L nominal)
measured (24 h): < LOQ (control), 79.4 - 85.0 mg/L (100 mg/L nominal)
measured (72 h): 0.012 (control), 81.2 - 84.2 mg/L (100 mg/L nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Material, size, headspace, fill volume: glass, 100 mL, headspace: 50 mL, fill volume: 50 mL
- Aeration: continuous shaken
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 216.3 E+04 cells/mL (mean)
- No. of vessels per concentration (replicates): 6 (for the highest test concentration), 3 (for the remaining test concentrations)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water
- Culture medium different from test medium: same as test (for pre-culture)
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test in the control and the highest test concentration. Temperature was measured continuously.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 91 - 94 μE m-2 s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions. Cell densities were measured every 24 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (tested in a separate study)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 81 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- 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
- EC50: 1.5 mg/L (95% CI: 1.4 - 1.6 mg/L)

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test, alpha=0.05, one-sided, smaller). No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum concentration tested). The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Percentage inhibition of growth and yield after 72 h of exposure

Nominal concentration	% inhibition of growth	% inhibition of yield
Control	-	-
0.10	-.04	-2.2
1.0	0.0	0.3
10	0.5	2.7
100	1.4	7.1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes