[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:

26 May 2020 - 29 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,10-decanediol dimethacrylate (CAS 6701-13-9) and 10-methacryloyl-oxy-decylphosphate

Molecular formula:

C18H30O4 and C14H27O6P

IUPAC Name:

Reaction mass of 1,10-decanediol dimethacrylate (CAS 6701-13-9) and 10-methacryloyl-oxy-decylphosphate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M, batch: 5674800

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature (15-25°C) in a ventilated cabinet, away from heat, acids, bases and amine in a container tightly closed.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: Preliminary monitoring showed that test item is not stable under the test conditions (measured concentration variation > 20%).

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Blank control, 0.1, 0.3, 1.0, 3.1 and 10 mg/L
- Sampling method: 10 mL of test media was transferred into 50 mL plastic centrifuge tubes for control and fortification. Sample fortification, if required, was carried out at this time. At least one untreated control and two controls fortified with a known amount of test item were analyzed in each analytical set to demonstrate acceptable performance of the method. After filling vessels (T0), the left-over test solutions were sampled, quantified, and validated. After 24, 48 and 72 hours the samples used for cell counts were pooled. The soltions were sampled (min volume 20 mL), quantified and validated.
- Sample storage conditions before analysis: Samples extracted same day as analysis. Stored under refrigerated conditions (4 ± 2°C).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
Test solutions were prepared by Water Accommodated fraction (WAF). Test concentrations of 1.0, 3.0, 10, 31 and 100 mg/L mg/L (loading rate level) were added directly to test media and mixed for 24 hours on an agitation table with a glass magnetic stirrer. The solutions were then centrifuged for 5 minutes at 3000 RCF. Following this, the aqueous phase (WAF) was drawn for testing. Test flasks were filled directly from the test solution containers immediately after preparation.
- Controls: Test medium without test substance or other additives.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): After centrifugation, solutions were clear with a film at the surface. However, the WAF was drawn off after this step.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Source: Culture at SGS France Laboratoire de Rouen, strain from SAG Gottingen collection (ID n° 61.81).
- Age of inoculum (at test initiation): 4 days
- Method of propagation: A pre-culture was inoculated 4 days before the beginning of the test with about 5000 cells per mL and cultured under the same conditions as the test. Algae cultures were maintained at exponential growth rate by adding aliquots of algal cultures to fresh medium each week. Microscopic examinations of each sub culture were conducted to validated strain purity

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.7-22.2 °C

pH:

7.9-8.3

Nominal and measured concentrations:

See Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: 125 mL, glass flasks. Capped with microbiology paper cap
- Test solution volume: 50 mg/L
- Aeration: Continuous shaking with magnetic stirrer
- Initial cells density: 9940 per mL
- Control end cells density 5.8E+05 cells per mL
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Detailed composition if non-standard medium was used: Demineralized water reconstituted with salts. The test media composition is consistent with standard “ASTM media”. Product is as follows: Ammonium chloride 15 mg/L, Magnesium chloride, 6H2O 12 mg/L, Calcium Chloride 2H2O 18 mg/L, Magnesium Sulphate 7H2O 15 mg/L, Potassium Dihydrogen Phosphate 1.6 mg/L, Iron (III) Chloride 6H2O 0.064 mg/L, EDTA disodium, 2H2O 0.1 mg/L, Boric acid 0.185 mg/L, Manganese Chloride, 4H2O 0.415 mg/L, Zinc Chloride 0.003 mg/L, Cobalt Chloride, 6H2O 0.0015 mg/L, Copper Chloride, 2H2O 0.00001 mg/L, Sodium Molybdate, 2H2O 0.007 mg/L, Sodium Hydrogen Carbonate 50 mg/L.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: continuous, with plant growth specific spectra fluorescent bulb, between about 4440 and 8880 Lux.

EFFECT PARAMETERS MEASURED : Growth rate and growth inhibition at 24, 48 and 72 hours

VEHICLE CONTROL PERFORMED: No vehicle
- Determination of cell concentrations: After 24, 48 and 72 hours, rowth rate and growth inhibition were measured via cells counted using a Malassez cell counter (1µL counting volume).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2x

RANGE-FINDING STUDY
- Test concentrations: Blank control, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: No
A range-finding test was performed under non-GLP conditions with three replicates per test concentration (10 and 100 mg/L) including controls, in order to determine the impact of test item on algal growth rate and yield. 1,10-Decandiol-dimethacrylate (1,10 D-D) and 10-Methacryloyloxy-decylphosphate (10 M-O-D) concentrations were measured at the beginning and at the end of the test. According to range finding test results, the selected loading levels for the final test were 1.0, 3.0, 10, 31, and 100 mg/L. However, an initial test done using these dilutions had unexpectedly high growth inhibition and the dilution range was lowered as a result. The preliminary monitoring showed that test item is not stable under the test conditions (measured concentration variation > 20%). Thus the final test would need to be conducted with solution renewal to maintain the exposure level. 1,10-Decandiol-dimethacrylate (1,10 D-D) and 10-Methacryloyl-oxydecylphosphate (10 M-O-D) concentrations were measured at the beginning and after 24, 48 and 72 h to determine exposure levels.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.718 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

CI: 0.639 – 0.840 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.175 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

CI: 0.126 – 0.236

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

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): None reported
- Any stimulation of growth found in any treatment: None reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Nothing reported
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

K2Cr2O7 sensitivity test run on 26-29 Nov 2019: 72hr EC50= 0.94 mg/L (95% CI:≥0.92 ≤1.46)

Reported statistics and error estimates:

Statistics run: ErC50 / ErC10 (based on growth inhibition rate), EyC50 / EyC10 (based on yield inhibition) and confidence limits were obtained by calculation according to Regtox® Macro (“Vindimian”) using the Hill model. NOEC and LOEC were estimated by statistic comparison using Statgraphics® software. Data homoscedasticity was checked by Levene test and data were compared by ANOVA. Tukey HSD test (Honestly Significant Difference, with p < 0.05) was used for pairwise comparisons of several treatment groups.

Any other information on results incl. tables

 Table 2. Cell counts at 24 – 48 and 72 h (cells/mL)

Loading rate (mg/L)	24 HRS	48 HRS	72 HRS
Control	28000	72000	690000
	29000	128000	500000
	25000	135000	570000
	34000	107000	550000
	40000	83000	660000
	26000	129000	545000
0.1	22000	91000	525000
	30000	99000	545000
	22000	104000	675000
0.29	25000	74000	327500
	28000	52000	310000
	19000	41000	212500
1.1	16000	21000	46000
	15000	21000	43000
	9000	36000	29000
3	18000	14000	21000
	16000	7000	16000
	11000	19000	12000
10.3	18000	24000	13000
	6000	16000	14000
	15000	10000	17000

Table 3. Specific Growth Rates

	24 hours	48 hours	72 hours	(0-72 H)	CV (%)
	(0-24H)	(24-48 H)	(48-72 H)
Control	1.00	0.944	2.26	1.39	52.9
Rep A	1.04	1.48	1.36	1.29	17.8
Rep B	0.894	1.69	1.44	1.34	30.2
Rep C	1.19	1.15	1.64	1.32	20.4
Rep D	1.35	0.729	2.07	1.38	48.6
Rep E	0.932	1.60	1.44	1.32	26.3
Mean				1.34	32.7
Std Dev				0.041
CV (%)				3.03
0.1 mg/L	0.871	1.39	1.78	1.34
0.29 mg/L	0.842	0.824	1.64	1.099
1.1 mg/L	0.255	0.665	0.428	0.447
3.0 mg/L	0.379	-0.177	0.258	0.155
10.3 mg/L	0.162	0.288	-0.072	0.126

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

control biomass increase >16x (59x), CV for section-by-section growth rates <35% (32.7%), CV for average specific growth rates <7% (3.0%)

Conclusions:

72 hour EC50 of 0.718 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).
72 hour EC10 of 0.175 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).
72 hour NOEC of 0.10 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).

Executive summary:

The 72 hour EC50 to Pseudokichneriella subcapitata was determined in a definitive test according to OECD 201 guidelines. Nominal concentrations of 0.1, 0.3, 1.0, 3.1, 10 mg/L (prepared as Water Accommodated Fraction) and a blank control were run using 3 replicates per concentration and no dispersant. An EC50 of 0.718 mg/L (nominal concentration) and EC10 of 0.175 mg/L (nominal concentrations) was determined. A NOEC of 0.1 mg/L (nominal concentrations) was also 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.