Reaction mass of sulfonium, dodecylethyl[1-(2-methoxy-2-oxoethyl)-3-oxo-3-(pentyloxy)propyl]-, tetrafluoroborate(1-)(1:1) and sulfonium, dodecylethyl[3-methoxy-1-(2-methoxy-2-oxoethyl)-3-oxopropyl]-, tetrafluoroborate(1-)(1:1) and sulfonium, dodecylethyl[3-oxo-1-[2-oxo-2-(pentyloxy)ethyl]-3-(pentyloxy)propyl]-, tetrafluoroborate(1-)(1:1)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

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:

other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Singular 2 mL samples for possible analysis were taken from all test concentrations and the control (centre of test vessels) at t=0 h, t=24 h and t=48 h. Samples were stored in the freezer (≤ -15°C) until analysis.
On the day of analysis, the samples were defrosted at room temperature. The test samples at 100 mg/L were diluted 100-fold with M2-medium. Finally, all (pre-diluted) test samples were diluted 100-fold in 50/50 (v/v) methanol/water to obtain concentrations within the calibration range.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface). The batch of Potassium tetrafluoroborate tested was a white powder with a purity of 99.1% (w/w) and completely soluble in test medium at the concentrations tested. Preparation of test solutions started with the highest test concentration of 100 mg/L. No special treatment other than careful mixing was necessary to completely dissolve the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions in test medium. The final test solutions were all clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 1E+04 cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM:
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Initial cell density: 1E+04 cells/mL as determined using a microscope and a counting chamber

ACCLIMATION
- Acclimation period: 3 days.
- Culturing media and conditions: The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test temperature:

22.1-23.1°C

pH:

7.6 - 8.2

Nominal and measured concentrations:

- Nominal: 0.10, 1.0, 10 and 100 mg/L.
- Measured: Analysis of the samples taken from 100 mg/L showed a measured concentration of 116 mg/L at the start of exposure. The concentration remained stable during the 72-hour test period (96-101% of initial).

Details on test conditions:

TEST SYSTEM:
- Test vessel: 100 ml, all-glass, containing 50 mL of test solution
- Type: closed
- Initial cells density: 1E+04 cells/mL
- No. of vessels (replicates): 6 replicates of the control and 100 mg/L, 3 replicates of 0.10, 1.0 and 10 mg/L, 1 extra replicate of each test group for sampling purposes after 24 hours, 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM:
- Standard medium used: yes (M2)

OTHER TEST CONDITIONS:
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: 30 Watt "Cool White" TLD lamps with a light intensity within the range of 78 to 90 µE.m-2.s-1

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of Potassium tetrafluoroborate tested. All results were based on nominal concentrations despite the fact that analytical results showed a slightly higher concentration than nominal. This was justified due to the fact that procedural recovery samples at 100 mg/L showed recoveries in the same range of nominal. No EC50-values could be calculated because the test substance proved to be non-toxic (EC50 > maximum concentration tested).

Results with reference substance (positive control):

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.5 mg/L with a 95% confidence interval ranging from 1.1 to 2.1 mgL. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Statistical analysis of the data was not needed as the effects recorded were not significant (<10%).

Validity criteria fulfilled:

yes

Conclusions:

Pseudokirchnerella subcapitata exposed to 0.10, 1.0, 10 and 100 mg/L potassium tetrafluoroborate under specific conditions for 72 hours displayed no significant reduction of growth rate or inhibition of yield. The 72-h ErC50 and EyC50 were >100 mg/L and the NOErC was 100 mg/L (based on nominal concentrations).

Executive summary:

In order to test the toxicity of potassium tetrafluoroborate to freshwater alga, a GLP-compliant study was performed according to OECD TG 201. In this study, Pseudokirchnerella subcapitata were exposed to potassium tetrafluoroborate in a combined limit/range-finding test for 72 hours under static conditions. Alga, at an initial cell density of 1E+04 cells/mL, were exposed to 100 mg/L and a blank control in the limit test (6 replicates per test group) and to concentrations of 0.10, 1.0 and 10 mg/L in the range-finding test (3 replicates per test group). Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. Analysis of the samples taken from 100 mg/L showed a measured concentration of 116 mg/L at the start of exposure. The concentration remained stable during the 72-hour test period (96-101% of initial). All results were based on nominal concentrations despite the fact that analytical results showed a slightly higher concentration than nominal. This was justified due to the fact that procedural recovery samples at 100 mg/L showed recoveries in the same range, i.e. 115-116% of nominal. No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of potassium tetrafluoroborate tested. No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum concentration tested). Therefore, the 72-h ErC50 (growth rate) and EyC50 (biomass) values were determined at >100 mg/L (based on nominal concentrations); the NOEC value was determined at 100 mg/L for both parameters.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02-March-2018 to 16-March-2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Dodecyl methyl sulfide is used within this study as an analogous chemical to dodecyl ethyl sulfide, which is a hydrolysis product of MTDID 47403. Dodecyl ethyl sulfide is not commercially available.
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma-Aldrich, Batch Number MKBW6054V
- Physical state: colorless, liquid
- Quality Release Date: 12JAN2016
- Purity: 97.8%
- Storage condition of test material: room temperature

Analytical monitoring:

yes

Details on sampling:

- Concentrations: negative control and solutions containing 1.0, 10, and 100% of the saturated solution (SS) prepared at a loading rate of 100 mg/L (limit test).
- Volume: Control: 3 mL for the regular samples and 1.5 mL for quality control (QC) samples
- Sampling method: Duplicate samples for analysis, and duplicate reserve samples, were taken from the test vessels. Two extra samples were taken from the limit concentration (100% SS) for analytical QC, and two reserve QC samples were also taken. Samples were taken at test initiation (T = 0 h), T= 24 h, and test completion (T= 72 h).
- Sample storage conditions before analysis: Samples were transferred to the analytical laboratory at the test facility on the day of sampling for extraction then stored in a freezer (≤ -15°C) until analyses.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of dodecyl methyl sulfide tested was a colorless liquid which was not completely soluble in test medium at the initially prepared loading rate. Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of gentle magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one day. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: Blank only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: NIVA CHL1
- Source: in-house laboratory culture.
- Age of inoculum (at test initiation): Three days
- Method of cultivation: Stock cultures were started by inoculating growth (M1) medium with algal cells from a pure culture on agar. The suspensions were continuously aerated in a climate room at a temperature of 21-24°C and light intensity of 60 to 120 μE/m²/s when measured in the photosynthetically effective wavelength range (400-700 nm). Growth (M1) medium (Nederlandse Praktijk Richtlijn no. 6505) was formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition.
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 period: Three days before the start of the test, fresh M2 medium was inoculated at a density of 1E4 cells/mL and was kept under the same conditions used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3

Test temperature:

22 °C to 23 °C

pH:

8.1

Nominal and measured concentrations:

Nominal: 100 mg/L loading level (100% SS), 10% SS, 1% SS, Control (Blank)
Measured: Only the 100 mg/L (100% SS) loading level and the blank were analyzed. A quantifiable response was not measured at any concentration, but estimated concentrations were determined for some samples by extrapolation of the standard curve (See Table 1 for analysis results). Based on these estimated values, a maximum soluble concentration was conservatively estimated to be 0.03 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass vessels
- Fill volume: 50 mL
- Aeration: continuous shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 245E+04 cells/mL
- No. of vessels per concentration: Six replicates for limit concentration (100%SS) three replicates for 1.0% and 10% SS. One extra replicate for each test group for sampling purposes after 24 h of exposure. One or two replicates of each test concentration without algae.
- No. of vessels per control: Six replicates
GROWTH MEDIUM
- Standard medium used: Yes, OECD medium (M2)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Growth medium
- Culture medium different from test medium: No
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: TLD lamps with a light intensity within the range of 84 to 85 μE/(m²∙s).
Test vessels were placed randomly in the incubator, and repositioned daily.
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 680 nm using a spectrophotometer with immersion probe (pathlength = 10 mm) against an algal medium blank and the extra replicates for the treated solutions.
- Recording times: cell densities were recorded at 24-hour intervals in the control and limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Ten
- Justification for using less concentrations than requested by guideline: Limit test
RANGE-FINDING STUDY
- Combined limt/range-finding study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2CrO7)

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

Remarks on result:

other:

Remarks:

based on loading level of 100 mg/L, which is above the water solubility of dodecyl methyl sulfide.

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

Remarks on result:

other:

Remarks:

based on loading level of 100 mg/L, which is above the water solubility of dodecyl methyl sulfide.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.03 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

0.03 mg/L was estimated to be the maximum soluble concentration in the medium, and was outside the range of the standard curve.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

0.03 mg/L was estimated to be the maximum soluble concentration in the medium, and was outside the range of the standard curve.

Details on results:

Under the conditions of the study no biologically significant inhibition of growth rate was observed at a loading rate of 100 mg/L. 14% inhibition of yield was observed at a loading rate of 100 mg/L.
- Exponential growth in the control: yes.
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

Results with reference substance valid? yes
- EC50: 1.6 mg/L (growth rate). Historical range for the reference substance at the contract lab lies between 0.82 and 2.3 mg/L
- Other: The reference substance test was conducted within one month of testing dodecyl methyl sulfide.

Table 2, Growth Rate and Percentage Inhibition for the Total Test Period

Dodecyl Methyl Sulfide*	Mean	Std. Dev.	n	% Inhibition
Control	1.833	0.0194	6
1.0	1.827	0.0162	3	0.3
10.0	1.831	0.0307	3	0.1
100	1.783	0.0467	3	2.7**

* Percentage of saturated solution prepared at 100 mg/L loading rate

** Effect was statistically, but not biologically significant

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by a factor of > 16 (i.e. 245); mean CV for section-by-section sp ecific growth rate in the controls was < 35% (i.e. 14%); CV of average specific growth rates in the whole test period for controls was < 7% (i.e. 1.1%).

Conclusions:

The 72-hour EC10 and EC50 (growth rate) of dodecyl methyl sulfide to Pseudokirchneriella subcapitata exceeded a loading rate of 100 mg/L, which exceeded the maximum soluble concentration for dodecyl methyl sulfide in test medium. The test was conducted according to OECD TG 201.

Executive summary:

The 72-hour EC10 and EC50 of dodecyl methyl sulfide to Pseudokirchneriella subcapitata were examined in a static test conducted according to OECD TG 201. Dodecyl methyl sulfide is a surrogate for dodecyl ethyl sulfide (not commercially available), a hydrolysis product of MTDID 47403. Test solutions were prepared as the control (0 mg/L), and a 100 mg/L loading level (water soluble fraction). Due to the very low solubility of dodecyl methyl sulfide, analytical measurement failed to quantify the compound in any samples. Extrapolation of the calibration curve allowed estimation of the concentration in some samples (0 h, 24 h), leading to an estimated maximum soluble concentration of 0.03 mg/L in the test medium. The 72-hour EC10 and EC50 for growth rate were > 100 mg/L (nominal loading concentration), and therefore greater than the water solubility of dodecyl methyl sulfide in the test medium. The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable with restrictions and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis in a weight of evidence (WOE) approach.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

weight of evidence

Study period:

29 June 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Reason / purpose for cross-reference:

assessment report

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Additive toxicity model based on inverse sum of contributions

GLP compliance:

no

Remarks:

(Calculation method using GLP-compliant data)

Analytical monitoring:

not required

Test organisms (species):

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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

assuming 1:2:1 ratio of constituents

Basis for effect:

growth rate

Table 1, main scenarios used to establish EC50 values for dimethyl glutaconate (DMG) and dipentyl glutaconate (DPG)

Constituent	Mass fraction (%)	Molar EC50 (µM)	Mass EC50 (mg/L)	fraction * 1/EC50
Scenario a)
DMG	36.9	3.64	0.576	0.64
DPG	63.1	3.64	0.984	0.64
Scenario b)
DMG	36.9	183.8	29.07	0.0127
DPG	63.1	1.838	0.50	1.269

Table 2, Secondary scenarios used to establish EC50mix values for 1:2:1 DMG:MPG:DPG mixture

Constituent	Mass fraction (%)	Molar EC50 (µM)	Mass EC50 (mg/L)	fraction * 1/EC50
Scenario a)
DMG	18.5	3.64	0.576	0.32
MPG	50	3.64	0.780	0.64
DPG	31.5	3.64	0.984	0.32
EC50mix (mg/L)				0.78
Scenario b)i)
DMG	18.5	183.8	29.1	0.00635
MPG	50	183.8	39.4	0.0127
DPG	31.5	1.838	0.497	0.635
EC50mix (mg/L)				1.53
Scenario b)ii)
DMG	18.5	183.8	29.1	0.00635
MPG	50	18.38	3.94	0.127
DPG	31.5	1.838	0.497	0.635
EC50mix (mg/L)				1.30
Scenario b)iii)
DMG	18.5	183.8	29.1	0.00635
MPG	50	183.8	0.394	1.27
DPG	31.5	1.838	0.497	0.635
EC50mix (mg/L)				0.52

Table 3, EC50 mix value for MTDID 47403 (1:2:1 molar mixture)

Constituent	Mass fraction (%)	Molar EC50 (µM)	Mass EC50 (mg/L)	fraction * 1/EC50
DM Gluborat	22.4	183.8	87.57	0.0026
MP Gluborat	50	1.838	0.979	0.51
DP Gluborat	27.6	1.838	1.08	0.255
EC50mix (mg/L)				1.30

Validity criteria fulfilled:

not applicable

Conclusions:

The EC50 of MTDID 47403 to green algae was calculated as being 1.3 mg/L using an additive toxicity method.

Executive summary:

The EC50 of MTDID 47403 to green algae was calculated using an additive toxicity method. MTDID 47403 hydrolyzes to form a mixture of glutaconate diesters. EC50 values for each of the potential glutaconate diesters (dimethyl, methyl pentyl, and dipentyl) were derived using data from the study on toxicity of a dimethyl/dipentyl glutaconate mixture to Pseudokirchneriella subcapitata (reported elsewhere in this dossier). The most conservative assumption was that dipentyl glutaconate carried essentially all of the toxicity in the experiment, and the methyl pentyl glutaconate was as toxic as dipentyl. The EC50 values for each glutaconate, in molar units, was used to calculate an EC50 for the corresponding constituent of MTDID 47403. The same additive toxicity method was then used to determine an EC50 for MTDID 47403 assuming a 1:2:1 molar ratio. The EC50 thus determined was 1.3 mg/L. The study uses an acceptable calculation method and is deemed reliable with restrictions.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

7-May-2018 to 17-May-2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Dimethyl glutaconate (CAS# 5164-76-1)
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M 668975
- Expiration date of the lot/batch: October 2018
- Form of test material: light yellow liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry, well ventilated
- Stability under test conditions: Stable under recommended storage conditions

Dipentyl glutaconate (CAS# None)
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M AH 21/057
- Expiration date of the lot/batch: October 2018
- Form of test material: colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No special storage requirements
- Stability under test conditions: Stable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: aliquots of each control and each total nominal test item loading rate collected at test initiation and at test completion
- Sampling method: Aliquots from test initiation were collected from the bulk test solutions. Aliquots from test completion were collected from pooled volumes of each of the control and test solution replicates. In addition, samples were taken from the extra 72-hour (non-inoculated) vessels were collected.
- Sample storage conditions before analysis: Approximately 40 mL of each test solution and each control were collected in ~40 mL amber glass vials and stored refrigerated (2 °C to 8 °C) until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions were prepared as water accommodated fractions (WAF). The two test items were tested by adding both test items to each individual loading rate WAF vessel as a representation of the hydrolysis products of MTDID 47403. The test items were weighed separately and added directly to ~ 2.5 L (i.e., half volume) of nutrient media (dilution water) in a ~5 L glass aspirator bottle (WAF vessel), and the volume topped up to 5.0 L with nutrient media. The WAF control was prepared in a similar manner; however, no test items were added. Each solution was stirred slowly (~80 – 96 rpm, no vortex) using a magnetic stir bar and stir plate and covered to reduce contaminants. Stirring occurred for ~22 hours and no settling period was required.
WAF solutions were removed from the WAF vessel and collected in a glass holding vessel (600 mL glass beaker) by draining through a Teflon tubing fitted at the bottom third of the water column, discarding the first ~50 mL into an appropriate container.
- Controls: A negative control and a WAF control
- Evidence of undissolved material: At the time of preparation, the test item WAFs appeared clear and colourless with test item visible on the surface of the water. After the ~22 hour stirring period, there was no change in appearance. After collection, the test item WAF solutions appeared clear and colorless with no visible test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CPCC 37
- Source: In-house culture; original culture was obtained from Canadian Phycological Culture Center
- Method of cultivation: The starter culture was used to initiate new algae cultures using nutrient medium (similar to AAP-medium; USEPA)
NaNO3, 25500 μg/l
MgCl2∙6H2O, 10000 μg/l
CaCl2∙2H2O, 4420 μg/l
H3BO3, 186 μg/l
MnCl2∙4H2O, 416 μg/l
ZnCl2, 3.28 μg/l
CoCl2∙6H2O, 1.43 μg/l
CuCl2∙2H2O, 0.012 μg/l
Na2MoO4∙2H2O, 7.26 μg/l
FeCl3∙6H2O, 160 μg/l
Na2EDTA∙2H2O, 300 μg/l
MgSO4∙7H2O, 14700 μg/l
K2HPO4, 1044 μg/L
NaHCO3, 15500 μg/l
CULTURING
Algal culture medium was the same as the test medium. Culture used to inoculate test vessels was three days old and in exponential growth phase, and contained approximately 0.94E+06 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 °C

pH:

7.3 - 7.8

Conductivity:

91 - 92 µS/cm

Nominal and measured concentrations:

The test was conducted using a 1:1 stoichiometric mixture of dimethylgluconate and dipentylgluconate as the test material.
Total nominal loading rates: 0.12, 0.27, 0.57, 1.27, 2.80, 6.16, and 13.55 mg Test Item/L
Nominal loading rates for Dimethyl glutaconate: 0.04, 0.10, 0.21, 0.47, 1.03, 2.27, and 5.00 mg/L
Nominal loading rates for Dipentyl glutaconate: 0.08, 0.17, 0.36, 0.80, 1.77, 3.89, and 8.55 mg/L
Measured; see Table 3, 4

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL, all-glass, covered with foam stopper. Fill volume was 50 mL
- Agitation: Yes, during incubation the algal cells were mixed on a horizontal shaker at approximately 75 rpm.
- Initial cells density: 0.94E+04 cells/mL
- Control end cells density: 127.8E+04 cells/mL
- No. of vessels per concentration (replicates): 3 (plus one extra vessel at each loading rate for possible confirmatory analysis at 72 hours)
- No. of vessels per control (replicates): 6 (plus one extra vessel for possible confirmatory analysis at 72 hours)
GROWTH MEDIUM
- Standard medium used: Similar to AAP-medium (USEPA), defined in section Details on test organisms
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Millipore deionised water system (Millipore RiOs16; Millipore Milli-Q Academic)
- Culture medium different from test medium: No
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: continuous illumination with light intensity within the range of 5378 to 5788 lux.
EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Replicate cell counts were taken on aliquots from each flask. Cells were counted with a Cellometer X4HM Auto Counter set to count fluorescent algae-PKS cell type, with dilution factor of 1. The cell density in each replicate was determined by counting a minimum of four aliquots from each test vessel. Any unusual unusual appearance of the algal cells, including observed differences in cell size, was noted, if present.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2
- Range finding study: Yes
- Test concentrations: 0, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

zinc sulphate heptahdrate (CAS# 7446-20-0)

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.78 mg/L

Nominal / measured:

nominal

Conc. based on:

other:

Remarks:

concentration is the sum of the nominal concentrations of the two individual test substances

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Confidence limits 0.74, 0.87 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

other:

Remarks:

concentration is the sum of the nominal concentrations of the two individual test substances

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Confidence limits, 0.21, 0.40 mg/L.

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.27 mg/L

Nominal / measured:

nominal

Conc. based on:

other:

Remarks:

concentration is the sum of the nominal concentrations of the two individual test substances

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes. The CV of the mean specific growth rates during the whole test period in the control was 5.1%. The mean CV for section-by-section (days 0-1, 1-2. and 2-3) specific growth rate in the control replicates was < 35% (28.5%).
- Observation of abnormalities (for algal test): None observed
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test solutions with nominal concentrations of 1.27, 2.80 6.16, and 13.55 mg/L for the mixture, had initial measured concentrations of dipentyl glutaconate of 0.50, 0.71, 0.62, and 0.64 mg/L, respectively. These measured values indicate that dipentyl glutaconate was saturated in the test solutions. Additionally, in the final samples, the concentration of dipently glutacononate was below the limit of quantitation in all test solutions except one, where it was measured to be 0.15 mg/L. For dimethyl glutaconate, the measured concentrations generally agreed with nominal concentrations, and final measured concentrations were similar to the concentrations measured in the initial samples.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.062 mg Zn(+2)/L (95% Confidence limits, 0.033 mg/L to 0.081 mg/L) (cell yield). Historical range (± 2 Std. Dev.) for the reference substance at the contract lab lies between 0.034 mg/L and 0.114 mg/L.
- Other: Reference substance toxicity assay conducted within one month prior to the assay of dimethyl and dipentyl glutaconate.

Reported statistics and error estimates:

The EL50 and EL10 were calculated using linear interpolation. The NOEL was calculated using the Wilcoxon/Bonferroni Adjustment Test. A comparison of the negative control and the WAF control was performed using the Equal Variance Two-Sample t-Test.
Statistical calculations were done using CETIS statistical software v.1.9.2.4. (Tidepool Scientific Software, McKinleyville, CA, USA).

Table 5, Individual cell densities in the algal toxicity test (x10E+04 cells/mL)

Concentration (mg/L)	Replicate	Time
		0 h	24 h	48 h	72 h
0 (Neg Ctrl)	1	0.94	1.7	18.4	90.7
	2	0.94	4.6	16.9	143.5
	3	0.94	3.0	14.2	95.3
	4	0.94	2.9	20.5	166.5
	5	0.94	4.3	18.1	149.5
	6	0.94	4.3	19.8	121.5
Mean:		0.94	3.5	18.0	127.8
0 (WAF Ctrl)	1	0.94	2.3	18.9	97.0
	2	0.94	2.6	14.1	105.3
	3	0.94	3.5	15.6	117.0
	4	0.94	3.2	16.0	125.3
	5	0.94	2.3	14.4	136.5
	6	0.94	2.6	24.7	154.3
Mean:		0.94	2.8	17.3	122.5
0.12	1	0.94	2.3	19.5	112.0
	2	0.94	3.8	11.9	116.1
	3	0.94	2.6	17.5	133.0
Mean:		0.94	2.9	16.3	120.4
0.27	1	0.94	2.9	10.6	77.8
	2	0.94	2.6	11.5	114.0
	3	0.94	2.9	13.2	96.1
Mean:		0.94	2.8	11.8	96.0
0.57	1	0.94	1.8	5.0	35.0
	2	0.94	1.5	6.8	34.9
	3	0.94	1.2	4.8	34.1
Mean:		0.94	1.5	5.5	34.7
1.27	1	0.94	2.6	0.9	1.2
	2	0.94	0.6	0.6	2.1
	3	0.94	1.2	0.9	0.6
Mean:		0.94	1.5	0.8	1.3
2.80	1	0.94	0.3	1.2	1.5
	2	0.94	0.9	0.9	0.9
	3	0.94	0.3	0.9	0.9
Mean:		0.94	0.5	1.0	1.1
6.16	1	0.94	1.2	1.2	1.2
	2	0.94	1.1	0.0	0.6
	3	0.94	0.0	0.3	1.0
Mean:		0.94	0.8	0.5	0.9
13.55	1	0.94	0.9	0.0	1.3
	2	0.94	1.5	1.2	1.8
	3	0.94	0.9	0.3	0.7
Mean:		0.94	1.1	0.5	1.3

Table 6, Growth rates (1/day) and percent inhibition in the algal toxicity test

Concentration (mg/L)	Replicate	Interval				Inhibition (%)
		0 - 24 h	24 - 48 h	48 - 72 h	0 - 72 h
0 (Neg Ctrl)	1	0.61	2.37	1.59	1.52	N/A
	2	1.59	1.29	2.14	1.68	N/A
	3	1.15	1.56	1.90	1.54	N/A
	4	1.12	1.96	2.10	1.72	N/A
	5	1.52	1.43	2.11	1.69	N/A
	6	1.52	1.52	1.81	1.62	N/A
Mean:		1.25	1.69	1.94	1.63	N/A
Std. Dev.:		0.38	0.40	0.22	0.08	N/A
CV:		30.02	23.61	11.07	5.10	N/A
0 (WAF Ctrl)	1	0.91	2.09	1.64	1.54	5.1
	2	1.03	1.68	2.01	1.57	3.5
	3	1.31	1.49	2.02	1.61	1.3
	4	1.21	1.62	2.06	1.63	-0.1
	5	0.89	1.84	2.25	1.66	-1.9
	6	1.02	2.25	1.83	1.70	-4.4
Mean:		1.06	1.83	1.97	1.62	0.59
Std. Dev.:		0.17	0.29	0.21	0.06	3.48
CV:		15.93	15.92	10.67	3.50
0.12	1	0.91	2.12	1.75	1.59	2.2
	2	1.40	1.14	2.27	1.60	1.5
	3	1.01	1.91	2.03	1.65	-1.3
Mean:		1.11	1.72	2.02	1.62	0.77
Std. Dev.:		0.26	0.51	0.26	0.03	1.85
CV:		23.31	29.86	12.97	1.87
0.27	1	1.13	1.29	1.99	1.47	9.7
	2	1.02	1.49	2.29	1.60	1.8
	3	1.12	1.52	1.99	1.54	5.3
Mean:		1.09	1.43	2.09	1.54	5.60
Std. Dev.:		0.06	0.12	0.17	0.06	3.92
CV:		5.79	8.66	8.31	4.15
0.57	1	0.65	1.01	1.95	1.20	26.0
	2	0.49	1.48	1.64	1.20	26.1
	3	0.23	1.39	1.96	1.20	26.5
Mean:		0.46	1.29	1.85	1.20	26.20
Std. Dev.:		0.21	0.25	0.18	0.00	0.29
CV:		45.78	19.19	9.87	0.40
1.27	1	1.01	-1.08	0.32	0.08	95.1
	2	-0.44	0.00	1.26	0.27	83.4
	3	0.23	-0.26	-0.45	-0.16	109.9
Mean:		0.26	-0.45	0.37	0.06	96.11
Std. Dev.:		0.73	0.57	0.85	0.22	13.27
CV:		275.70	-126.40	228.18	340.72
2.80	1	-1.20	1.40	0.24	0.15	91.0
	2	-0.08	0.01	0.00	-0.02	101.5
	3	-1.23	1.17	0.02	-0.01	100.8
Mean:		-0.84	0.86	0.09	0.04	97.80
Std. Dev.:		0.65	0.75	0.13	0.10	5.88
CV:		-78.29	87.07	151.29	266.99
6.16	1	0.22	0.00	0.03	0.08	94.9
	2	0.17	N/A	N/A	-0.17	110.5
	3	N/A	N/A	1.24	0.01	99.5
Mean:		0.19	0.00	0.64	-0.03	101.62
Std. Dev.:		0.04	N/A	0.85	0.13	8.01
CV:		20.06	N/A	133.96	-494.89
13.55	1	-0.10	N/A	N/A	0.10	94.1
	2	0.45	-0.22	0.45	0.22	86.3
	3	-0.10	-1.06	0.80	-0.12	107.4
Mean:		0.08	N/A	N/A	0.07	95.95
Std. Dev.:		0.32	N/A	N/A	0.17	10.64
CV:		388.18	N/A	N/A	263.14

The mean CV for section-by-section specific growth rate in the control replicates was 28.5%.

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by a factor of > 16 (136); The mean CV for section-by-section specific growth rate in the controls was < 35% (28.5%); the CV of average specific growth rates in the whole test period for controls was < 7% (5.10%).

Conclusions:

A 1:1 stoichiometric mixture of dimethyl glutaconate and dipentyl glutaconate was evaluated for inhibition of the growth of Pseudokirchneriella subcapitata according to the OECD 201 guideline. Based on the nominal, total concentration of the mixture, the 72-hour ELr50 was 0.78 mg/L, and the 72-hour ELr10 was 0.34 mg/L.

Executive summary:

The 72-hour ELr10 and ELr50 of a 1:1 stoichiometric mixture of dimethyl glutaconate and dipentyl glutaconate to Pseudokirchneriella subcapitata were examined in a static test conducted according to OECD TG 201. The mixture is a surrogate for hydrolysis products of MTDID 47403, which are approximately a 1:2:1 mixture of dimethyl glutaconate, methyl pentyl glutaconate and dipentyl glutaconate. Test solutions were prepared as water accommodated fractions (WAFs) with total loading rates 0 mg/L (control), and at concentrations of 0.12, 0.27, 0.57, 1.27, 2.80, 6.16, and 13.55 mg/L (nominal).

Concentrations of the individual substances were also analyzed by LC/MS-MS. Measured concentrations of dimethyl glutaconate were generally in agreement with it's nominal concentrations and the intial and final were similar, however initial measured concentrations of dipentyl glutaconate appeared to reach the limit of solubility and were similar for the four highest loading rates. Additionally, in the final samples, dipentyl glutaconate concentrations were found to be below the limit of quantitation in all but one test substance loading level. Due to these differences in measured values, results expressed as the total, initial nominal concentration of the two test substances is appropriate.

Based on the nominal concentrations of the mixture, the 72-hour ELr50 was found to be 0.78 mg/L, and the 72-hour ELr10 was 0.34 mg/L. The test was conducted according to internationally accepted test guidelines and was GLP compliant. It has been assigned a reliability of Klimisch two (K2) because the test substances are only some of the hydrolysis products of MTDID 47403, and because of the uncertainty regarding exposure level to dipentyl glutaconate. It is reliable with restrictions and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.

Description of key information

MTDID 47403 hydrolyzes rapidly (T1/2 < 12 hrs) in water. Results from toxicity tests (OECD TG 201) with the hydrolysis products of MTDID 47403 indicate toxicity by one or more glutaconate diesters. The full set of glutaconate diesters was not tested. The estimated 72-h EC50 of MTDID 47403 was calculated using an additivity method while making several assumptions about glutaconate toxicity. The resulting EC50 was 1.3 mg/L as MTDID 47403.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.3 mg/L

Additional information

MTDID 47403 is subject to rapid hydrolysis, forming products of differential water solubility that may be subject to phase separation. Analysis after hydrolysis revealed five products; dimethyl glutaconate, methyl pentyl glutaconate, dipentyl glutaconate (produced as a mixture), dodecyl ethyl sulfide, as well as tetrafluoroboric acid. These materials are of differential solubility, so that testing on a hydrolyzate would lead to uncertainty as to exposure concentrations. The varying classes of hydrolysis products were instead tested in isolation. Short-term toxicity testing results for these products follow.

The mixed diester methyl pentyl glutaconate was not available for testing. The two symmetric diesters, dimethyl glutaconate and dipentyl glutaconate, were tested in combination at equimolar ratios, in a test conducted according to OECD 201. Unexpected toxicity was found for this mixture. Analytically determined concentrations of dimethyl glutaconate were stable over time, however dipentyl glutaconate was quantifiable in only one of the test levels. In addition, dipentyl glutaconate concentrations reached saturation at higher loading rates, although maximum effect had already been attained. Therefore, the effective loading rate was expressed as the sum of the nominal initial loading rates of the two esters, for a 72-hour EL50 of 0.78 mg/L and an EL10 of 0.34 mg/L. Since this result did not include the effects of methyl pentyl glutaconate, a simple additive toxicity model for mixtures was used to examine various scenarios. The model followed the formula (1/EC50mix) = (fractiona / EC50a) + (fractionb / EC50b) + *** + (fractionn / EC50n), where (EC50i) is the EC50 of the ith component of the mixture and (fractioni) is the contribution of the ith component on a mass basis. This model implicitly assumes no synergism and no antagonism between effects of the glutaconates and a similar mode of action. Two scenarios were initially considered: a) that dimethyl glutaconate and dipentyl glutaconate had approximately equal effect in the OECD 201 test on a molar basis, and b) that dipentyl glutaconate had 100 times the effect of dimethyl glutaconate on a molar basis. Possible EC50n values for the two diesters were entered iteratively into the calculation until the EC50mix converged on the measured EC50 for the mixture. These EC50 values were taken into the next calculation. A 1:2:1 molar mixture of the three diesters was then assumed. Under scenario a), methyl pentyl glutaconate was assumed to have an equal toxicity to the other two diesters. Under scenario b), further possibilities included i) that methyl pentyl glutaconate and dimethyl glutaconate had equivalent toxicity on a molar basis, ii) that methyl pentyl glutaconate had an intermediate (10x) effect, and iii) that methyl pentyl glutaconate and dipentyl glutaconate had equivalent molar toxicity. The lowest mixture EC50, 0.52 mg/L, was obtained in scenario b,iii), in which most of the toxicity is carried equally between dipentyl glutaconate and methyl pentyl glutaconate, and at a much greater level than dimethyl glutaconate. The equivalent loading rate of MTDID 47403 to attain a glutaconate diester concentration of 0.52 mg/L is 1.3 mg/L as MTDID 47403. This is the key value for further assessment.

Dodecyl ethyl sulfide is not commercially available, and therefore dodecyl methyl sulfide was used as a surrogate. The 72-hour EC10 and EC50 of dodecyl methyl sulfide to Pseudokirchniella subcapitata were examined in a static, limit test conducted according to OECD 201. Test solutions were prepared as the control (0 mg/L) and as a water soluble fraction at a loading rate of 100 mg/L. The 72-hour EC10 and EC50 (growth rate) exceeded the loading rate of 100 mg/L. Due to the very low solubility of dodecyl methyl sulfide, analytical measurement failed to quantify the compound in any samples. Extrapolation of the calibration curve allowed estimation of the concentration in some samples (0 h, 24 h), leading to an estimated maximum soluble concentration of 0.03 mg/L in the test medium. The study was well-documented, followed an international standard method, and was GLP compliant. The study is considered reliable with restrictions. The results from this study are considered suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.

The toxicity of tetrafluoroboric acid, to Pseudokirchneriella subcapitata was examined as potassium tetrafluoroborate in a rangefinding/limit test conducted according to OECD 201. Test solutions were prepared as the control (0 mg/L), 0.1 mg/L, 1 mg/L, 10 mg/L, and at 100 mg/L. No inhibition of growth rate was seen at the 100 mg/L loading rate. No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations. Analysis of the 100 mg/L samples showed a measured concentration of 116 mg/L at the start of exposure. The study followed an international standard method, and was GLP compliant; therefore, the test is considered reliable without restriction.