[No public or meaningful name is available]

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:

2nd-5th June, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2,2'-(1,3-phenylenebis(oxy))diethanol with 2-(phenoxymethyl)oxirane and 2-isocyanatoethyl methacrylate

Cas Number:

1431303-59-1

Molecular formula:

Not assigned for UVCB substance.

IUPAC Name:

Reaction products of 2,2'-(1,3-phenylenebis(oxy))diethanol with 2-(phenoxymethyl)oxirane and 2-isocyanatoethyl methacrylate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M, 6252020
- Expiration date of the lot/batch: September 2021
- Purity test date: 21st October 2019
- Purity: 100% (MTDID 58376), 14.22% ER-1GP IEM, 32.71% ER-2GP IEM, 29.91% ER-3GP IEM, 15.59% ER-4GP IEM

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
- Sampling method: Water samples were taken from the approximate midpoint of each test concentration vessel before initial exposure, at 24 hours, and at the termination of the experiment (72 hour). Samples for the initial time point were taken from the remaining WAF after division into replicates. At 24 hours and 72 hours, replicate samples from each respective concentration were composited before aliquotting. Aliquots (10 mL) were taken from the composited exposure solutions and directly transferred to disposable glass vials containing 10 mL acetonitrile (to a final composition of 50/50 acetonitrile/AAP medium, (v/v). Samples were then diluted into the calibration standard range with 50/50 acetonitrile/AAP medium (v/v) prior to analysis. Three quality control samples, prepared in diluent water at test substance concentrations similar to the treatment level range, were also run during each sampling interval and analyzed with the study samples. In addition, uninnoculated flasks containing WAF were sampled at 24 and 72 hours to assess the impact of algal biomass on test substance concentrations.
- Sample storage conditions before analysis: Analysis was run immediately after sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was melted in the original container in a water bath (70 °C) for 45 minutes. The primary stock solution (100 mg/L) was prepared as a water-accomodated fraction by adding the aliquot of test substance directly onto a glass slide and then suspending the slide within the preparation vessel containing AAP medium. The solution was mixed overnight with a magnetic stir plate and Teflon-coated stir bar, and left to settle for 10-minutes. The solution was clear and colorless with no undissolved material except what was on the glass slide and a globule on the stir bar. The WAF was siphoned from the mixing vessel, avoiding the surface and bottom of the vessel and stir bar. The WAF was clear and colorless with no visible undissolved material. The 100% WAF solutions was used as the exposure solution.
- Controls: Test medium maintained under the same conditions but without test substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The primary stock solution was clear and colorless with visible undissolved material on the glass slide and stir bar. After siphoning, all exposure solutions were clear and colorless with no visible undissolved 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, strain 1648
- Age of inoculum (at test initiation): Four days since previous transfer
- Method of cultivation: Maintained in stock culture at Smithers. Growth medium: Algal Assay Procedure (AAP) medium, prepared with sterile deionized source water. pH 7.5, continuous illumination of 61-70 uE/m²/S, 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:

7.1-7.8

Conductivity:

87-110 uS/cm

Nominal and measured concentrations:

Nominal: blank control and 100 mg/L
Measured: see Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL glass flasks covered with stainless steel caps
- Type: open / closed: Closed (gas exchange permitted)
- Material, size, headspace, fill volume: Fill volume was 100 mL
- Aeration: 100 RPM agitation on orbital shaker
- Initial cells density: 10,000 cells/mL
- Control end cells density: 779,000 cells/mL in blank
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: No. 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 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/LNa MoO4.2H2O, 160 ug/L FeCl3.6H2O, 300 ug/L Na2EDTA.2H2O
- 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 5700-6600 lux at test initiation, PAR throughout experiment ranged 62-78 uE/m^2/S

EFFECT PARAMETERS MEASURED :
- 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: control, 1.0, 10.0, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

zinc chloride (ZnCl2)

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

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

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: During the exposure period all control and exposed cells were healthy and normal in appearance.
- Adherence to test vessels: None reported
- 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. Test substance concentrations were not stable over the exposure period, see Table 1.

Results with reference substance (positive control):

Results with reference substance valid: Yes
- EC50: 0.14 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 0.14 mg/L with 95% CI: 0.13 to 0.15 mg Zn/L. The historical mean =0.096 mg/L with a range of 0.053 to 0.14 mg Zn/L, March 2005 to present.
- Reference test run: 29th May- 2nd June, 2020 (three days before initiation of toxicity 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. An Equal Variance Two-Sample t-Test was used to determine the NOEC/LOEC datapoints. 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 58376- 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	33700	230000	716000
	B	34800	202000	679000
	C	37000	179000	759000
	D	36200	149000	663000
	E	36600	220000	1010000
	F	32600	169000	854000
	Mean (SD)	35100 (1770)	191000 (31000)	779000 (130000)
100	A	38000	210000	774000
	B	34400	186000	878000
	C	30300	153000	825000
	D	35500	231000	928000
	E	38800	191000	827000
	F	33400	221000	924000
	Mean (SD)	35100 (3100)	199000 (28300)	859000 (61200)

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 58376- Growth Rates and Acceptance Criteria Data

Control	Growth rate: 0-24 Hour (1.01 days)	Growth rate: 24-48 Hour (1.01 days)	Growth rate: 48-72 Hour (0.989 days)	Growth rate Overall: 0-72 Hours, 3.01 days (SD)	% CV of Daily Growth Rate
Rep A	1.21	1.89	1.15	1.42 (0.41)	29.21
Rep B	1.24	1.74	1.22	1.40 (0.29)	20.96
Rep C	1.30	1.55	1.46	1.44 (0.13)	9.01
Rep D	1.28	1.40	1.51	1.39 (0.12)	8.32
Rep E	1.29	1.77	1.54	1.53 (0.24)	15.62
Rep F	1.17	1.63	1.64	1.48 (0.27)	17.99
Mean % CV section-by-section growth rate	-	-	-	-	16.90%
Mean (SD) of overall 0-72 hour growth rates	-	-	-	-	1.44 (0.05)
% CV of 0-72 hour overall growth rate	-	-	-	-	3.65%
100 mg/L Rep A	1.33	-	-	1.44	-
Rep B	1.22	-	-	1.49	-
Rep C	1.10	-	-	1.47	-
Rep D	1.26	-	-	1.50	-
Rep E	1.34	-	-	1.47	-
Rep F	1.20	-	-	1.50	-
Mean (SD)	1.24 (0.09)	-	-	1.48 (0.02)	-

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

control biomass increase >16x (78x), CV for section-by-section growth rates <35% (16.9%), CV for average specific growth rates <7% (3.65%)

Conclusions:

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

Executive summary:

The 72 hour EC50 and EC10 of ERGP IEM to Pseudokirchneriella subcapitata was determined according to OECD 201 guidelines in a limit test. Nominal concentrations of 100 mg/L and a blank control were run with six replicates per treatment level. No inhibition was observed and growth enhancement was not statistically significant.  An EC50 of >100 mg/L, and an EC10 >100 mg/L (nominal concentrations) were determined.

The study was well-documented, followed an international standard method and was GLP compliant. The study is considered reliable without restrictions. The results from this study are considered suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.