3-acetyl-6-methyl-2H-pyran-2,4(3H)-dione

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source chemical is the sodium salt of the target. As such, both the source and the target are anticipated to behave similarly in aqueous systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Dehydroacetic acid, sodium salt [EC 224-580-1; CAS 4418-26-2]. Composition provided in TMI.
Target: Dehydroacetic acid [EC 208-293-9; CAS 520-45-6]. Purity/impurity profile as described in Section 1.2

3. ANALOGUE APPROACH JUSTIFICATION
The source chemical and the target are structurally identical apart from the sodium ion in the source replacing the hydrogen on the parent acid target. This results in an increase in the water solubility of the source (231 g/L at 20 °C; OECD 105, shake flask method) vs the target (> 0.5 g/L and < 2 g/L; similar to OECD 105, preliminary test). Thus, short term ecotoxicology studies performed with the source are expected to represent a worse-case for the target due to the increased exposure of the organisms to the substance in solution.

4. DATA MATRIX
Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

23.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 11.3 -31.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 14.3 - 69.9 mg/L

Details on results:

- Exponential growth in the control (for algal test): Yes, cell growth increased 200-fold (specific growth rate 1.76 d^-1).
- Observation of abnormalities: No.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- ErC50 (72 h): 0.435 mg/L, 95% confidence interval 0.414 - 0.458 mg/L (potassium dichromate)

Reported statistics and error estimates:

ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 h were calculated by sigmoidal dose-response regression. NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOECL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The Shapiro-Wilk Test was used to test for normal distribution of data. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value is 0.05.

ANALYTICAL RESULTS

The measured concentrations of the test item were between 92% and 98% of the nominal value at the start of the exposure (0 h) and between 94% and 97% of the nominal value at the end of the exposure (72 h). This indicates that the test item concentration was successfully maintained throughout the duration of the test.

Since the measured test item concentrations were within ± 20% of the nominal concentration, the effect concentrations are based on nominal concentrations.

BIOLOGICAL RESULTS

The test substance was found to inhibit the growth of the freshwater green alga P. subcapitata after 72 h. The derived NOEC values for both inhibition of growth rate and yield after 72 h were 10.0 mg/L, respectively.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 - 27 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation EC No. 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 1.00, 3.16, 10.0, 31.6, and 100 mg/L (nominal)
- Sampling method: Samples were collected in fresh test solutions ( 0 h) and in old test solutions (72 h).
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler), if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the solution was homogenous.
- Controls: Yes, consisting of test media without test item incubated under the same conditions as the test.
- Evidence of undissolved material: The test media were clear throughout the exposure phase.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular freshwater green alga
- Strain: SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): A 3 d old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 h per day. The culture medium was the nutrient medium Z, according to Lüttge et al. 1994.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21.5 °C (mean)

pH:

0 h: 8.09 (control), 7.49 - 7.85 (treatments)
72 h: 9.45 (control), 7.88 - 9.10 (treatments)

Nominal and measured concentrations:

Control, 1.00, 3.16, 10.0, 31.6, and 100 mg/L (nominal)
< LOQ, 0.943, 3.05, 9.61, 30.5, and 93.6 mg/L (measured after 72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks.
- Type: Sealed with cotton wool plugs.
- size, headspace, fill volume: Size: 250 mL; Fill volume: 100 mL
- Initial cell density: 6305 cells/mL
- Control end cell density: 1263285 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Same as test: OECD medium was used as dilution water.
- Intervals of water quality measurement: The pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes.
- Photoperiod: 24 h light/d
- Light intensity and quality: 120 µE*m^-2*s^-1
- Other: The flasks were positioned randomly on a rotary shaker (shaking at approximately 70 rpm) and repositioned daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Chlorophyll-a fluorscence measured by fluorometer (Microplate Reader Chameleon V, excitation at 436 nm, emission at 685 nm) at 0, 24, 48 and 72 h
- Other: Microscopic evaluation of cell morphology at the start and end of exposure (72 h).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16 (√10)
- Range finding study: Yes, preliminary non-GLP range-finding test.
- Test concentrations: 1, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, 88% growth rate inhibition was observed at a nominal concentration of 100 mg/L test item after 72 h.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

23.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 11.3 -31.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 14.3 - 69.9 mg/L

Details on results:

- Exponential growth in the control (for algal test): Yes, cell growth increased 200-fold (specific growth rate 1.76 d^-1).
- Observation of abnormalities: No.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- ErC50 (72 h): 0.435 mg/L, 95% confidence interval 0.414 - 0.458 mg/L (potassium dichromate)

Reported statistics and error estimates:

ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 h were calculated by sigmoidal dose-response regression. NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOECL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The Shapiro-Wilk Test was used to test for normal distribution of data. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value is 0.05.

ANALYTICAL RESULTS

The measured concentrations of the test item were between 92% and 98% of the nominal value at the start of the exposure (0 h) and between 94% and 97% of the nominal value at the end of the exposure (72 h). This indicates that the test item concentration was successfully maintained throughout the duration of the test.

Since the measured test item concentrations were within ± 20% of the nominal concentration, the effect concentrations are based on nominal concentrations.

BIOLOGICAL RESULTS

The test substance was found to inhibit the growth of the freshwater green alga P. subcapitata after 72 h. The derived NOEC values for both inhibition of growth rate and yield after 72 h were 10.0 mg/L, respectively.

Description of key information

ErC10 (72 h) = 23.9 mg/L (nominal, P. subcapitata, OECD 201, RA-A CAS 4418 -26 -2)

ErC50 (72 h) = 32.1 mg/L, (nominal, P. subcapitata, OECD 201, RA-A CAS 4418 -26 -2)

Key value for chemical safety assessment

EC50 for freshwater algae:

32.1 mg/L

EC10 or NOEC for freshwater algae:

23.9 mg/L

Additional information

There is no study available investigating the effects of the substance on the growth of freshwater microalgae. However, data is available from a GLP study on a structural analogue (DHA-Na, CAS 4418 -26 -2), which was conducted according to the OECD guideline 201. Due to the high chemical and structural similarity of the two substances as well as the higher water solubility of the source substance (worst-case), the results of this study are considered a justified estimate of the potential hazard of the target substance.

In this study, P. subcapitata was exposed to 5 nominal concentrations of the source substance ranging from 1.0 – 100 mg/L for 72 h in a static test under controlled conditions. Negative controls were run in parallel and the source substance concentrations were analytically verified by HPLC-DAD at the beginning and end of exposure.

The test media were clear throughout the exposure period. The measured concentrations of the source substance in fresh media (0 h) ranged from 92 – 98% of the nominal values and from 94 – 97% of the nominal values in aged media (72 h). This indicates that the concentration of the source substance was successfully maintained throughout the duration of exposure. Since the measured concentrations were within the required ± 20%, all effect values are based on the nominal source substance concentrations.

The source substance was found to inhibit the growth of the microalgae. However, no morphological abnormalities were observed. The derived NOErC was 10.0 mg/L and the LOErC was 31.6 mg/L. The corresponding effect concentrations and respective 95% confidence intervals are an ErC10 (72 h) value of 23.9 mg/L (11.3 – 31.5 mg/L) and an ErC50 (72 h) value of 32.1 mg/L (14.3 – 69.9 mg/L).