(1-methylethylidene)bis(4,1-phenyleneoxy-3,1-propanediyl) bismethacrylate

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:

09 - 12 Jun 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study under GLP, analytical confirmation of test substance concentration, unstable test substance concentrations in media.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2015

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

Test material

Test material information

Constituent 1

Reference substance name:

Procrylate

IUPAC Name:

Procrylate

Details on test material:

- Name of test material (as cited in study report): MTDID 9115
- Substance type: pure active substance
- Physical state: clear colorless to yellowish liquid
- Analytical purity: 96%
- Expiration date of the lot/batch: 24 Nov 2015
- Storage condition of test material: At room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and controls, at t = 0hr, 24hr, and 72 hrs
- Sampling method: Duplicate 1-mL samples were taken. At t = 0 hr, samples were taken from stock solutions. At t = 24 hours, duplicate 1-mL samples were taken from a flask set up for this purpose. At t = 72 hr, replicates were pooled at each concentration before taking samples.
- Sample storage conditions before analysis: in freezer.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substance at 100 mg/L in test medium was stirred for two days with a magnetic stirrer. The resulting emulsion was left to settle overnight (~17 hours). Subsequently, the clear and colourless Water Soluble Fraction (WSF) was collected and used as the limit test concentration.
- Controls:Blank test medium
- Evidence of undissolved material: Test solution was clear and colorless

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
.
- Method of cultivation: Stock cultures were maintained in M1 medium (Nederlandse Praktijk Richtlijn no. 6505).
NaNO3, 500 mg/l
K2HPO4∙3H2O, 52 mg/l
MgSO4∙7H2O, 75 mg/l
Na2CO3∙10H2O, 54 mg/l
C6H8O7∙H2O, 6 mg/l
NH4NO3, 330 mg/l
CaCl2∙2H2O, 35 mg/l
C6H5FeO7∙xH2O, 6 mg/l
H3BO3, 2∙9 mg/l
MnCl2∙4H2O, 1.81 mg/l
ZnCl2, 0.11 mg/l
CuSO4∙5H2O, 0.08 mg/l
(NH4)6Mo7O24∙4H2O, 0.018 mg/l

ACCLIMATION
Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2 according to OECD 201) at a cell density of 1e+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:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/L as CaCO3

Test temperature:

21.3 - 22.5 °C

pH:

8.0 - 8.2

Nominal and measured concentrations:

Nominal: Control, 100 mg/L
Measured (Time weighted average):

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass container, 50 mL fill volume
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 95.6E+04 cells/mL
- No. of vessels per concentration (replicates): 6 (a parallel rangefinding test had triplicate vessels with 1.0% or 10% dilutions of the WSF in M2 medium). An additional flask was set up for the 24-hr concentration measurement. Algae-free test flasks were also assembled.
- No. of vessels per control (replicates): 6. An additional flask was set up for the 24-hr concentration measurement.

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard medium (M2) prepared in reverse osmosis purified water
- Ca/mg ratio: 1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: Fluorescent (TL-D) lamps with a light intensity within the range of 81 to 85 μE/(m²∙s). Test vessels were placed randomly and randomly repositioned every day.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber to determine inoculum density. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength = 20 mm).
- Appearance: at the end of the test, microscopic examination was done on the test concentration closest to the EC50 to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: yes, done in parallel to limit test
- Test concentrations: Control, 1.0% and 10% dilutions of WSF
- Results used to determine the conditions for the definitive study: no

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.73 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: No significant effect at highest concentration tested

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.73 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: No significant effect at highest concentration tested

Details on results:

- Exponential growth in the control: yes (Table 2, 3)
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to highest concentration when compared to the control.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: yes
- Other: Test substance concentration declined throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.3 mg/L. Historical range for the reference substance at the contract lab lies between 0.82 and 2.3 mg/L
- Other: Reference substance toxicity assay conducted 92 days prior to test substance.

Reported statistics and error estimates:

Growth rate data were normally distributed by Shapiro-Wilk's test, p(W) = 0.145 > 0.05. Variance of growth rate data were homogeneous by Levene's test, p(F) = 0.724 > 0.05. The NOEC was determined by Williams multiple sequential t-test. No significant effect on growth rate was found using undiluted WSF
.

Any other information on results incl. tables

Table 2, Individual cell densities in the Procrylat algal toxicity test
Time-weighted average concentration (mg/L)	Replicate	Time
		0 h	24 h	48 h	72 h
Control	1	1.0	4.422	21.592	89.398
	2	1.0	4.198	22.147	91.991
	3	1.0	4.453	24.474	94.586
	4	1.0	4.142	23.911	100.971
	5	1.0	4.102	22.765	93.731
	6	1.0	4.208	25.863	103.173
Mean:		1.0	4.3	23.5	95.6
Std.Dev.:		0.0	0.1	1.6	5.3
CV:		0.0	3.5	6.8	5.6
0.73	1	1.0	7.765	21.389	86.122
	2	1.0	8.527	20.807	84.010
	3	1.0	5.041	23.972	93.745
	4	1.0	7.198	19.614	88.489
	5	1.0	6.181	26.675	92.982
	6	1.0	8.606	25.126	106.234
Mean:		1.0	7.2	22.9	91.9
Std.Dev.:		0.0	1.4	2.7	8.0
CV:		0.0	19.3	12.0	8.7

 

Table 3, Growth rates (1/day) in the Procrylat algal toxicity test
Time-weighted average concentration (mg/L)	Replicate	Interval
		0-24 h	24-48 h	48-72 h	0-72 h
Control	1	1.487	1.586	1.421	1.498
	2	1.435	1.663	1.424	1.507
	3	1.494	1.704	1.352	1.517
	4	1.421	1.753	1.440	1.538
	5	1.411	1.714	1.415	1.513
	6	1.437	1.816	1.384	1.545
Mean:		1.447	1.706	1.406	1.520
Std.Dev.:		0.034	0.078	0.032	0.018
CV:		2.400	4.600	2.300	1.200
		CV = 10.8% for all section-specific control growth rates
0.73	1	2.050	1.013	1.393	1.485
	2	2.143	0.892	1.396	1.477
	3	1.618	1.559	1.364	1.514
	4	1.974	1.002	1.507	1.494
	5	1.821	1.462	1.249	1.511
	6	2.152	1.071	1.442	1.555
Mean:		1.960	1.167	1.392	1.506
Std.Dev.:		0.208	0.274	0.086	0.028
CV:		10.600	23.500	6.200	1.900

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72-h NOEC (growth rate) of Procrylat to Pseudokirchneriella subcapitata was 0.73 mg/L based on time-weighted average concentration (OECD 201), in a limit test done with a water soluble fraction made at a loading rate of 100 mg/L. No effect was seen during the test.

Executive summary:

Toxicity of Procrylat to the freshwater alga Pseudokirchneriella subcapitata was assessed according to OECD201. A water soluble fraction was made at a loading rate of 100 mg/L, with analytical determination of actual exposure concentrations. Analytically determined concentrations declined throughout the test. Therefore, time-weighted average (TWA) concentrations were used to calculate effect concentration. No effect was observed in the test. The 72-hour NOEC (growth rate) was 0.73 mg/L (TWA). The 72-hour EC50 was >0.73 mg/L.

The study was conducted according to internationally accepted test guidelines and in accord with GLP criteria. Test substance concentrations were confirmed analytically. Test substance concentrations were not stable during the test. In addition, the results showed considerable variability. Both difficulties are to be expected given the low concentrations necessary to determine the test substance's toxicity, and its tendency to adsorb to surfaces including the multiplying cells. The study is deemed reliable with restrictions. It is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.