2-Hydroxyethylmethacrylate, reaction products with proyleneoxide and Phthalic anhydride

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

other: U.S. EPA Series 850 – Ecological Effects Test Guidelines OCSPP Guideline 850.4500 (2012)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Purity: 100%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the active substances. At test initiation, samples were collected from each test concentration and control solution prior to distribution into test chambers. At exposure termination, the biological replicates from each respective test concentration and negative control solution were pooled and then sampled, and the single abiotic control replicate was also sampled. All samples were collected in glass vials and were acidified with 10% phosphoric acid upon collection. Two sets of samples were collected at each interval. One set was processed immediately for analysis and the second set was stored refrigerated for possible future analysis. Samples collected at the 72-hour sampling interval were centrifuged for five minutes at 14500 rpm, to remove algae prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
A primary stock solution was prepared at a nominal concentration of 120 milligrams per litre (mg/L) by dissolving 0.1200 g of the test substance in 1000 mL freshwater AAP medium. The stock was inverted at least twenty times, sonicated for thirty minutes and was stirred with a Teflon®-coated stirbar and magnetic stirplate for thirty minutes to mix. The stock appeared clear and colourless. No precipitates were observed. The 120 mg/L primary stock continued stirring while all subsequent dilutions were made. Additional test solutions were prepared at nominal concentrations of 7.5, 15, 30 and 60 mg/L by diluting 31.25, 62.5, 125 and 250 mL of the 120 mg/L stock, respectively, to a final volume of 500 mL in freshwater AAP medium. All test solutions appeared clear and colourless and were otherwise unremarkable at the time of preparation. Aliquots of the freshwater AAP medium were used for the negative control solution. The solutions appeared normal, with no particulates or surface-slicks observed, at test termination.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM: Original algal cultures were obtained from the University of Toronto Culture Collection in 2000, and were used to initiate a culture at Wildlife International, Easton, MD. Algal cells used in this test were obtained from Wildlife International cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test initiation. The negative control organisms were expected to exhibit exponential growth over the 72-hour exposure period.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.9 - 23.9°C

pH:

7.2 - 8.3

Salinity:

freshwater

Nominal and measured concentrations:

Nominal test concentrations: 0, 7.5, 15, 30, 60 and 120 mg/L
Mean, measured concentrations in biotic test solutions: 0, 7.0, 14, 28, 55 and 113 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks plugged with foam stoppers. Each flask contained 100 mL of test or negative control solution, and was labelled with the project number, concentration, and replicate.
- Initial cells density: In order to achieve an initial cell density of approximately 10000 cells/mL at test initiation, 1.0 mL of the inoculum was added to each biotic replicate.
- Control end cells density: 1489803 cells/mL
- No. of organisms per vessel: at test end 1.490e8
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: 7.5 ± 0.1 with 0.1N sodium hydroxide and 10% hydrochloric acid
- Photoperiod: light 24 hr
- Light intensity and quality: mean intensity 4,528 ± 117.8 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Test samples were collected from each replicate of the test concentrations and negative control for the determination of algal cell densities. Samples were collected at approximately 24-hour intervals during the 72-hour exposure. Cells were enumerated on the same day as sample collection. Prior to counting, the sample solutions were sonicated for 0.5 minutes to ensure the cells within each sample were homogeneously distributed. Cell counts for the samples collected during the exposure phase were performed using an electronic particle counter (Beckman Coulter® Z Series)
Samples were pooled within their respective treatments, and sub-samples were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or colour). Cells in the replicate test chambers also were assessed for aggregation, flocculation, or adherence of the cells to the test chamber.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

68 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 63 to 72 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

23 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 19 to 29 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

25 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 21 to 31 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Cells in the negative control and all of the treatment concentrations were normal in size, shape, and colour. There was no evidence of flocculation, aggregation or adherence to the test chambers in any group.
- 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

Reported statistics and error estimates:

The calculation of cell densities, area under the growth curve, growth rates, yields and percent inhibition values, as well as all statistical analyses, were conducted using SAS Version 8.2. Yield was calculated for each treatment and control replicate at 72 hours as the final cell density after 72 hours of exposure minus the nominal initial cell density. Specific growth rate was calculated for each replicate of the control and test concentrations at each 24-hour interval of exposure. The 72-hour area under the growth curve, growth rate and yield data were evaluated for normality and homogeneity of variance (α = 0.01) using the Shapiro-Wilk’s and Levene’s tests, respectively. Since the data were normally distributed with homogeneous variances, the treatment groups were compared to the negative control using Dunnett’s test (α = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC relative to each parameter at 72 hours. Inhibition values were calculated for each test concentration as the percent reduction in area under the growth curve, growth rate or yield relative to the negative control replicates. Area under the growth curve, growth rates and yields were analysed to estimate ECx values (i.e., the theoretical test concentrations that would produce an x% reduction in a variable of interest relative to the negative control) at the 72-hour exposure interval. ECx values for area under the growth curve, growth rate and yield and their corresponding 95% confidence limits, were calculated using non-linear regression at 72 hours of exposure.

Any other information on results incl. tables

At the end of the 72-hour exposure period, cell density increased in the negative control by at least a factor of 16, the coefficient of variation for the average specific growth rates in the control replicates was 3.2%, and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the control replicates was 35%. Cell density in the control replicates increased at an exponential rate over the 72-hour exposure period.

To determine if the test substance had an algistatic or algicidal effect, recovery was initiated for the mean, measured concentrations of 28, 55 and 113 mg/L. In the recovery phase, it was demonstrated that the effects of the test substance were algistatic (reversible) rather than algicidal at concentrations less than or equal to 113 mg/L based on greater than a 16X increase of healthy cells within four days.

Mean, Measured concentration (mg/L)	Mean 72-hour area under the growth curve (cells/mL)	% Inhibition  ^a
Negative control	24,489,156	--
7.0	22,228,086	9
14	16,147,392*	34
28	10,512,177*	57
55	3,818,034*	84
113	331,422*	99
a Relative to the negative control. * Treatment group mean is significantly different from negative control(Dunnett’s test, p< 0.05).
Mean, Measured concentration (mg/L)	Mean 0-72 hour growth rate	% Inhibition ^a
Negative control	0.0695	--
7.0	0.0684	2
14	0.0642*	8
28	0.0586*	16
55	0.0443*	36
113	0.0123*	82
a Relative to the negative control. * Treatment group mean is significantly different from negative control(Dunnett’s test, p< 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

72-hour EbC50 = 23 mg/L
72-hour ErC50 = 68 mg/L
72-hour NOEC = 7.0 mg/L
72-hour EyC50 = 25 mg/L

Executive summary:

Pseudokirchneriella subcapitata was exposed to five concentrations of the test substance and evaluated for effects on area under the growth curve, growth rate and yield according to OECD Guideline 201. Based on mean, measured concentration (7.0, 14, 28, 55 and 113 mg/L), the E_bC₅₀, E_rC₅₀ and E_yC₅₀ values were estimated to be 23, 68 and 25 mg/L, respectively. Dunnett’s test indicated that mean area under the growth curve, mean growth rate and mean yield were significantly reduced (p<0.05) from the negative control group at the mean, measured concentrations of 14, 28, 55 and 113 mg/L. Consequently, the 72-hour NOEC value for all endpoints was determined to be 7.0 mg/L.