Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

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:

15th-18th October, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

EC Number:

944-271-1

Cas Number:

2305048-54-6

Molecular formula:

not applicable for multi-constituent.

IUPAC Name:

Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Maplewood, Lot# 653940
- Expiration date of the lot/batch: Feburary 2020
- Purity test date: 26th February 2019
- Purity: 100% (UVCB)
- Physical state: yellowish to reddish liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in a dark, ventilated


cabinet in the original container.
- Stability under storage conditions: Stable
- Stability under test conditions: Stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Concentrations: 0 (blank control) and 100 mg/L
- Water samples (5.0 mL) were taken from the approximate midpoint of each test concentration vessel before initial exposure (0 hours), at 24 hours, and at the termination of the experiment (72 hour). Samples for the initial time point were taken from the intermediate vessels before division into replicates. At 24 hours and 72 hours, replicate solutions from each respective concentration were composited after sampling. Three quality control samples, prepared in AAP medium at test substance concentrations similar to the treatment level range, were also run during each sampling interval and analyzed with the study samples.
- Sample storage conditions before analysis: Analysis was run immediately after sampling. Sub-samples of the test solutions were collected at each sampling interval and stored frozen as archive samples.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: The stock solution (100 mg/L) was prepared as a water-accommodated (WAF) by adding the test substance directly to the total volume of dilution water in a glass beaker. The solution was clear and colorless with visibly oily, white globules on the surface of the solution and the bottom of the test vessel. The solution was covered with foil and mixed overnight using a magnetic stir plate and teflon coated stir bar. After overnight mixing and a 30 minute settling period, the globules remained. The WAF was then siphoned from the middle of the solution and was clear and colorless with no undissolved material. This 100% WAF was used as the exposure solution.
- Controls:Test medium without test substance.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): UTEX The Culture Collection of Algae at the University of Texas, Austin, Texas (maintained in stock culture at Smithers), strain 1648
- Age of inoculum (at test initiation): Four days since previous transfer
- Method of cultivation: Growth medium: Algal Assay Procedure (AAP) medium, prepared with sterile deionized source water. pH 7.5, continuous illumination, 23 °C. Agitation at a continuous rate of 100 rpm on orbital shaker.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

22-23 °C

pH:

7.2-7.7

Conductivity:

95-110 uS/cm

Nominal and measured concentrations:

Nominal: 0 (blank control), and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL glass flasks covered with stainless steel caps
- Type: open / closed: Closed (gas exchange permitted)
- Aeration: shaking at continuous rate of 100 rpm
- Material, size, headspace, fill volume: Fill volume was 100 mL
- Initial cells density: 10,000 cells/mL
- Control end cells density: 1,461,250 cells/mL in blank (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes. AAP medium prepared with sterile, carbon filtered deionized water source.
- Detailed composition if non-standard medium was used: 25.5 mg/L NaNo3, 12.16 mg/L MgCl2.6H2O, 4.41 mg/L CaCl2.H2O, 14.7 mg/L MgSO4.7H2O, 1.368 mg/L K2HPO4.3H2O, 15.0 mg/L NaHCO3, 185.5 ug/L H3BO3, 1.88 ug/L Na2SeO4, 415.4 ug/L MnCl2.4H2O, 3.270 ug/L ZnCl2, 1.43 ug/L CoCl2.6H2O, 0.012 ug/L CuCl2.2H2O, 7.26 ug/LNaMoO4.2H2O, 160 ug/L FeCl3.6H2O, 300 ug/L Na2EDTA.2H2O.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium as stated above.
- Total organic carbon: 0.55 mg/L (October 2019)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: Premira VitaLux fluorescent bulbs. Light intensity ranged from 4500-5400 lux at test initiation, PAR throughout experiment ranged 60-72 uE/m^2/S

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Using a Beckmen Coulter Multisizer 4e Particle Analyzer. Duplicate counts of duplicate samples were done on each replicate every 24 hours. The average of the four counts was reported. Observations on health of algal cells were made of alternating replicates of control or treatments by microscopy in a hemacytometer.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0.2, 1.0, 10, 50 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Zinc chloride

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: Cells were healthy and normal in appearance
- Effect concentrations exceeding solubility of substance in test medium: at each observation interval, no difference from initial time with respect to precipitation and material adhered to sides of the flasks.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- EC50: 0.079 mg Zn/L
- A 72 hour acute toxicity test of zinc chloride was done to evaluate the health and sensitivity of this strain. The EC50 was 0.079 mg/L with 95% CI 0.066 to 0.088 mg/L. The historical mean of controls since March 2005 was 0.086 mg/L.
- Reference test run: 21st-24th May 2019 (144 days before definitive test)(.

Reported statistics and error estimates:

Statistics run: Variance Ratio F test for homogeneity, Shapiro-Wilk's Test for normality, and William's Multiple Comparison Test to determine if treatment data were statistically similar to the controls. All calculations were done within CETIS v1.9 software (Tidepool Scientific Software, McKinleyville, CA).

Any other information on results incl. tables

Table 2. 72-Hour Exposure of Pseudokirchenriella subcapitata to MTDID 49922 - Cell Density and Observations.

Nominal Loading Rate (mg/L)	Replicate	Cell Densities at 24 hours (cells/mL)	Cell Densities at 48 hours (cells/mL)	Cell Densities at 72 hours (cells/mL)
Control	A	45548	150150	507800
	B	47511	150250	500000
	C	43363	134450	478050
	D	45055	157750	471800
	E	43193	145050	476550
	F	42110	126150	478650
	Mean (SD)	44463 (1958)	143967 (11653)	485475 (14682)
100	A	30306	94720	389750
	B	28990	82769	324950
	C	30630	88775	372100
	D	31825	84567	355100
	E	26563	78103	327350
	F	33985	96752	373950
	Mean (SD)	30378 (2509)	87614 (7191)	357200 (26488)

Cells exposed to the treatment level tested and the control were observed to be normal

Table 3. 72-Hour Exposure of Pseudokirchenriella subcapitata to MTDID 49922 - Growth Rate Acceptance Criteria Data

	Growth rate: 0-24 Hour (1.10 days)	Growth rate: 24-48 Hour (0.936 days)	Growth rate: 48-72 Hour (0.925 days)	Growth rate Overall: 0-72 Hours, 3.01 days (SD)	% CV of Daily Growth Rate
Control: Rep A	1.38	1.27	1.32	1.32 (0.05)	3.92
Control: Rep B	1.42	1.23	1.30	1.32 (0.09)	7.14
Control: Rep C	1.33	1.21	1.37	1.30 (0.08)	6.51
Control: Rep D	1.37	1.34	1.18	1.30 (0.10)	7.59
Control: Rep E	1.33	1.29	1.29	1.30 (0.02)	1.77
Control: Rep F	1.31	1.17	1.44	1.31 (0.13)	10.31
100 mg/L: Rep A	1.01	-	-	1.24
100 mg/L: Rep B	0.97	-	-	1.18
100 mg/L: Rep C	1.02	-	-	1.22
100 mg/L: Rep D	1.05	-	-	1.21
100 mg/L: Rep E	0.89	-	-	1.18
100 mg/L: Rep F	1.11	-	-	1.22
Mean % CV section-by-section growth rate					6.21%
Mean (SD) of overall 0-72 hour growth rates					1.31 (0.01)
% CV of 0-72 hour overall growth rate					0.77%

The % inhibition for 0.72 hours for the 100 mg/L nominal loading rate was 8%. This growth rate inhibition is statistically significant, however, it did not reach 10% effect. Thus it is not considered biologically relevant.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

control biomass increase >16x (49x), CV for section-by-section g rowth rates <35% (6.21%), CV for average specific growth rates <7% (0.77%)

Conclusions:

72 hour EC10 > 100 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).
72 hour EC50 > 100 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).

Executive summary:

The 72 hour EC50 and EC10 of MTDID 49922 to Pseudokichneriella subcapitata was determined in a limit test according to OECD 201 guidelines. Due to low solubility, water accommodated fractions (WAFs) were used. WAFs made at loading rates of 100 mg/L and a blank control were run using 6 replicates per concentration and no dispersant. No biologically relevant impairment of growth rate nor abnormal cells were observed throughout the test when compared to the control. A loss of material was noted in the study, indicating the test substance was not maintained in solution. An EC50 >100 mg/L and EC10 > 100 mg/L (nominal concentrations) were determined.

Study was conducted under international guidelines and was compliant with GLP criteria. However, due to the inability to mainatin the test substance in solution, this study is considered reliable with restrictions. The result is considered suitable for use in Risk Assessment, Classification & Labeling, and PBT analysis.