Methacrylic acid, monoester with propane-1,2-diol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report):Methacrylic acid, monoester with propane 1,2diol; HPMA
- Physical state: liquid
- Analytical purity: 99.1%
- Lot/batch No.: 0347403
- Storage condition of test material: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 10.0, 18.0, 32.0, 56.0, and 100 mg/L
- Sampling method: At the beginning of the exposure test, 2.0 mL portions of the test solutions taken from the three replicate vessels were mixed, which was used as analytical sample. At the end of exposure (72hr), 2.0 mL portions of the test soluitons taken from the three replicate vessels were mixed and centrifuged to separate cells (3000rpm, l0min.). The supernatant was used as the analytical sample. A 0.75 mL portion of the analytical samples was taken and an equal amount of acetonitrile was added to perform HPLC analysis. The concentrations of the test substance in the test solutions were determined by comparing the peak areas with that of standard solutions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The medium for test solutions was regulated at 23 *2"C in incubator. The stock solution was prepared and test solutions were made by diluting this stock solution with medium The control test solution contained - only medium. The stock solution was prepared at the beginning of exposure. The appearance of each test solution was observed and recorded when it was prepared.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No suspension, precipitation or oily substance was found in all test solutions. All test solutions were colorless and transparent.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
Unicellular green algae
Pseudokirchneriella subcapitata
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): During the pre-culture period, exponential growth of algae was observed.
- Method of cultivation: subculture in Gorham medium under sterile condition. Subculture has been confmed to be sterile with bacteria test every six months.

ACCLIMATION
- Acclimation period: During the pre-culture period, exponential growth of algae was observed
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.5 to 23.9 °C

pH:

7.7-7.8 at the beginning of the study; 9.7-10.1 at the end of the study

Nominal and measured concentrations:

0 (Control), 10.0, 18.0, 32.0, 56.0, and 100 mg/L-nominal
0 (non-detectable), 9.73, 17.7, 31.2, 54.7 and 97.2 mg/L -measured (day 0)

Details on test conditions:

TEST SYSTEM
- Test vessel: Iwaki, 300 mL Erlenmeyer flask Iwaki (with air-permeable silicon stopper)
- Type (delete if not applicable): open
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 1x10e4 cells/mL., initial loading
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes; OECD Medium

TEST MEDIUM / WATER PARAMETERS
Nutrient salts Concentration (mg/L)
MnCl2. 4H20 0.415
FeC13 . 6H20 0.08
CoC12 6H20 0.0015
CuC12 - 2H20 0.00001
NH4C1 15
NaHCO3 50
MaSO4 . 7H20 15
ZnCl2 0.003
Na2EDTA . 2H2O 0.1
NazMo04.2H2O 0.007
CaC12 2H2O 18
KH2PO4 1.6
MgCl2. 6H20 12
H3B03 0.185
- Culture medium different from test medium: No; same

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: 4000 lux (+/- 20%, at the surface of the test solutions), continuous (400-700nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :The color of the test solutions and cultures were observed with the naked eye at 0, 24 and 48 hours after the start of exposure. At the end (72 hours) of exposure, besides visual observation of cell color, the cell shapes of algae were observed through the microscope.
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: The cell concentration in each flask was determined at 24, 48 and 72 hours after the start of exposure by an electronic particle couinter (CDA-500)..


TEST CONCENTRATIONS
The following concentrations were selected as nominal concentrations of the definitive test based on the range finding study:
Control, 10.0, 18.0, 32.0, 56.0 and 100 mg/L (100mg/L is ceiling.); factor: 1.8

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 97.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: in the highest doses the test conc. maintaniend within 80 % of the measured initial conc.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 97.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: in the highest doses the test conc. maintaniend within 80 % of the measured initial conc.

Details on results:

By the microscopic observation at the end of exposure, neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with the control. The cell concentration in teh control increased by a factor of 314 (average value) during the 72-hour exposure, indicating that the growth in the controls was normal under the conditions of the test. The cell concentration of each test level had a tendency to decrease as concentration increased (dose-dependent); however, no significant difference in growth rate was observed.

Results with reference substance (positive control):

Growth inhibition test of a reference substance (K2Cr2O7, guaranteed reagent) has been conducted every six months. 72hour 50% growth inhibition concentration by comparison of areas under the growth curves (EbCSO) since November 1996 was as follows: average +/- standard deviation = 0.428 +/- 0.0728 mgL, n=15

Reported statistics and error estimates:

The EC50 values and associated 95% confidence limits could not be determined by least squares linear regression analysis because the growth inhibition (%) at the maximum concentration level was less than 50%.

The NOEC values were determined by an analysis of variance (ANOVA), Williams test, subsequent to Bartlett test for homogeneity of variances. Statistical analyses were performed using Yukms Statlight #4 software (Yukms Corp., Tokyo) and all tests of significance were at alpha -0.05, except Bartlett test, which was at alpha -0.01.

The proportions of the measured concentrations to the nominal values in test solutions were 97-98% at the beginning of exposure and those proportions were 73-79% at the end of exposure. It is considered that the main cause of the decrease of the concentration was a chemical change resulting from algae since a peak appearing at nearly 5 minutes in each chromatogram seemed to be transformed from the substance.

Validity criteria fulfilled:

yes

Conclusions:

The algal ErC50 (72h, growth rate) and NOEC (72h, growth rate) were determined under OECD test guideline 201 to be both >97.2 mg/L, the highest concentration tested.

Executive summary:

A 72 -hour growth inhibition study with methacrylic acid, monoester with propane-1,2-diol toPseudokirchneriella subcapitatawas conducted under static conditions with 3 replicates inoculated at 10,000 cells/mL per test concentration exposed to nominal test concentrations of 0 (control), 10.0, 18.0, 32.0, 56.0, and 100 mg/L. Water samples were measured for analytical verification of test concentrations 0 and 72 hours. The proportions of the measured concentrations to the nominal values in test solutions were 97-98% at the beginning of exposure and were 73-79% at the end of exposure. In the highest doses the test concentrations maintained within 80 % of the measuerd initial concentration. It is considered that the main cause of the decrease of the concentration was a chemical change resulting from the addition of algae. The study results were based on the day 0 measured concentrations of non-detectable (control), 9.73, 17.7, 31.2, 54.7, and 97.2 mg/L. 

No significant inhibition in growth rate was observed at 72-hours; therefore, the 72-hour EC50 and NOEC based on growth rate were both >97.2 mg/L.

Description of key information

A freshwater algal toxicity test with Pseudokirchneriella subcapitata exposed to methacrylic acid, monoester with propane-1,2-diol and conducted under GLPs and following OECD Test Guideline 201 resulted in both a 72-hour growth rate EC50 (ErC50) and NOEC of >97.2 mg/l, the highest measured concentration tested.

Key value for chemical safety assessment

EC50 for freshwater algae:

97.2 mg/L

EC10 or NOEC for freshwater algae:

97.2 mg/L

Additional information

One freshwater algal study was available for methacrylic acid, monoester with propane-1,2-diol and considered reliable for assessment of this endpoint. The study was conducted with Pseudokirchneriella subcapitata and found to be of good quality and assigned a Klimisch score of one. The study was conducted for 72-hours, under static conditions, following OECD Test Guideline 201, and under GLPs. The study concluded that the 72-hour growth rate EC50 (ErC50) and NOEC were both >97.2 mg/L, the highest measured concentration tested.