Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol]and propylidynetrimethanol and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Dec 2020 - 25 Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study to GLP standards with no deviations

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: For this calibration, a stock solution of the test item with a concentration of 1000 mg/L in demineralised water was prepared. By dilution of this stock solution or intermediate dilutions, the following calibration standards were prepared in demineralised water: 0.5 / 1 / 2 / 4 / 7 / 11 / 16 / 22 mg/L.
- Sampling method: QC samples (11 mg/L)
- Sample storage conditions before analysis: closed vessel at room temperature (17.3 –23.0 °C).

Vehicle:

yes

Remarks:

demineralized water

Details on test solutions:

The test item was melted in a water bath (max. 80 °C) until it was liquid to ensure homogeneity. This method was chosen to guarantee homogeneity. A stock solution containing 100.0 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments (1 / 3.2 / 10 / 32 mg/L nominal) were prepared by dilution of this stock solution with algal medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1C until the algal cell density was approximately 105 to 106 cells/mL.

ACCLIMATION
- Culturing media and conditions:The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.TEST ORGANISM
- Common name: Unicellular freshwater algae
- Strain: SAG Strain 86.81
- Source (laboratory, culture collection): MBM Science Bridge GmbH
- Age of inoculum (at test initiation):
- Method of cultivation: The algae were kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 4 days under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 21.5 – 22.0 °C). The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of the culture was determined.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not stated

Test temperature:

21.2 – 22.2 °C

pH:

7.6-8.2

Dissolved oxygen:

Not stated

Salinity:

Not stated

Conductivity:

Not stated

Nominal and measured concentrations:

1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration
The concentrations to be tested are based on the result of a non GLP pre-test
Nominal concentrations were used based on analytical monitoring results.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks total volume 65 ml
- Type: open (covered with perforated plastic foil acting as a stopper)
- Initial cells density: 2.00 x 10^3 cells/mL
- Control end cells density: 5.2 x 10^5 cells per mL
- No. of vessels per concentration (replicates): 3 vessels, each filled with 45 ±1 mL test solution and algae 1 vessel, filled with 45 ±1 mL test solution without algae for analytical determination
- No. of vessels per control (replicates): 6 vessels, each filled with 45 ±1 mL algal medium and algae 1 vessel filled with 45 ±1 mL algal medium without algae for analytical determination

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralised water enriched with minerals but without algae

OTHER TEST CONDITIONS
- Light intensity and quality: intensity approximately 4440 - 8880 lux
- Photoperiod: continuous illumination
-Shaking: Constantly shaken for 72 hours
Temperature: 21.2 – 22.2 °C

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9, purity of ≥ 99.0 %) was used as positive control in a separate reference test (Laus GmbH 202007R301) with a 72h ErC50 and EyC50 of 1mg/l and 0.35mg/l, repsectively

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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): were no abnormalities detected in any of the control or test cultures at 72 hours.
- Any stimulation of growth found in any treatment:Only the lowest concentration showed a slight inhibition, but this can be neglected, as all
other concentrations showed no toxicity
- Any observations: No observations were made which might cause doubts concerning the validity of the study outcome.

Results with reference substance (positive control):

ErC50 (0 to 72 hour) : 0.65mg/L - 1.10 mg/L
EyC50 (0 to 72 hour) : 0.21 mg/L - 0.66 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®).

Two experiments were performed. In the 1st experiment both validity criteria (coefficient of variation of the daily growth rates and coefficient of variation of average growth) were not met. Therefore, the experiment was aborted and repeated under the same conditions as the first experiment.

 

Test Two:

 

The cell concentration in the blank control increased by a factor of 260 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

 

The mean coefficient of variation for section by section specific growth rate for the control and solvent control cultures was 34% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

 

The coefficient of variation for average specific growth rate over the test period (0 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EyC50 (cell density/biomass) and ErC50 (growth rate) were determined to be >100 mg/L.

Executive summary:

In a 72 hour GLP acute toxicity study, cultures of Desmodesmus subcapitus were exposed to HB TMP at nominal concentrations of 0 (control), 1, 3.2, 10, 32, and 100 mg/L under flow-through conditions in accordance with the OECD guideline 201 (Version 23 March 2006). 

Two experiments were performed. In the 1st experiment, both validity criteria (coefficient of variation of the daily growth rates and coefficient of variation of average growth) were not met. Therefore, the experiment was aborted and repeated under the same conditions like the first experiment. The results of the 1st experiment are not stated, but will be kept together with the other raw data in the GLP archive of the test facility.

There were no compound related phytotoxic effects.

Based on the nominal test concentrations, both the 72-hour E_yC₅₀ (cell density/biomass) and E_rC₅₀ (growth rate) were determined to be >100 mg/L. 

No deviations were present that could have had a significant impact on the scientific purpose or integrity of the study. This toxicity study is classified as acceptable and satisfies the guideline requirements for toxicity to aquatic algae and cyanobacteria toxicity study.  

 

Results synopsis 

Test organism: Desmodesmus subcapitus

Test type: Flow-through                     

72 hr EC₅₀: > 100 mg/L (biomass and growth rate                            

Endpoint(s) effected: Biomass, growth rate

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out inaccordance with OECD 201 and GLP standards were maintained throughout.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

Samples were taken from control vessels and test vessels at 0h and 72 h

Vehicle:

no

Details on test solutions:

The test solution was prepared on the day of test initaition. Both the test item and distillate water were heated to 80oC. Then 1000 mg of the test product, equal to 690 mg of the test item, was added to 1000 ml of distillate water and growth media.

Test organisms (species):

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

Details on test organisms:

The test organsim was Pseudokirchneriella subcapitata. The inoculum was taken from a laboratory culture (NIVA CHL 1), which originates from NIVA, Oslo, Norway. A qualification test on the alga culture was made on the 19th of June 2012 to confirm the quality of the test organism.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

No information provided

Test temperature:

21-24 ±2oC

pH:

7.7-7.9

Dissolved oxygen:

No information provided

Nominal and measured concentrations:

Nominal test concentration: 690 mg/l

Details on test conditions:

See methods below

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

690 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 690 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The 72-hour NOEC was 690 mg/l and the 72-hour EC50 was found to be >690 mg/l.

Reported statistics and error estimates:

Average specific growth rate (µ) for a specific period was calculated as the logaritmic increase in the biomass from the equation for each for each single vessel and control treatment:

µ=(ln cells/ml 72 hrs - ln cells/ml 0 hrs)/3 (time in days)
For control and treatment groups a mean value for growth rate along with variance estimates was calculated. The specific growth rate for each test concentration was then relatd to the average growth rate of the controls:

%Ir = (µc - µr)/ µc * 100

Where:
%Ir = Percent inhibition in average specific growth rate
µc = mean value for average specific growth rate (µ) in control group
µ = average specific growth rate (µ) for treatment replicate

Table 1: 72 -hour NOEC and ErC-values in mg/l based on nominal concentrations of test item.

	72 h NOEC mg/l	72 h ErC10 mg/l	72 h ErC50 mg/l
Average specific growth rate,EC-values	690	>690	>690

Table 2: pH and conductivity measured in samples without algae at the start and end of test.

Concentration	Start (0h) pH	End (72h) pH	Start (0h) Conductivity (µS/cm)	End (72h) Conductivity (µS/cm)
Control	7.9	7.7	160	170
690 mg/l	7.8	7.7	250	250

Table 3: Calculated response variables for the treament group and the control group (mean ± coefficient of variation) after 72 hours exposure to the test item HB TMP.

Concentration	72h Specific growth rate Mean ± CV (%)
Control	1.62 ± 3.3
690 mg/l	1.61 ± 2.5

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour NOEC of HB TMP was found to be 690 mg/l and the 72-hour EC50 was determined to be >690 mg/l.

Executive summary:

A 72-hour algal growth inhibition study was carried out according to OECD 201 guidelines. Test concentrations were created at a nominal concentration of 690 mg/l. The test organism used was Pseudokirchneriella subcapitata. Test vessels were incubated on a shaking table at approximately 100µE m^-2s^-1continuous illumination at a temperature of 21-24±2oC.

The pH was measured in all replicates at test initiation and again at 72 hours. Samples for cell counts were taken at 0, 24, 48 and 72 hours. As a measurement of biomass the number of cells per ml was counted by microscope. The effect of the test item on the growth of algae was determined by average specific growth rate.

The 72 -hour NOEC was determined to be 690 mg/l, while the 72 -hour ErC50 was determined to be >690 mg/l. These results were based on nominal concentrations.

Description of key information

In a 72 hour GLP acute toxicity study, cultures of Desmodesmus subcapitus showed to have a 72 -hour E_yC₅₀ (Cell density/biomass) and E_rC₅₀ (growth rate) greater than 100 mg HB TMP/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

In a 72 hour GLP acute toxicity study, cultures of Desmodesmus subcapitus were exposed to HB TMP at nominal concentrations of 0 (control), 1, 3.2, 10, 32, and 100mg/L flow-through conditions in accordance with the OECD guideline 201 (Version 23 March 2006).  Two experiments were performed. In the 1st experiment, both validity criteria (coefficient of variation of the daily growth rates and coefficient of variation of average growth) were not met. Therefore, the experiment was aborted and repeated under the same conditions like the first experiment. The results of the 1st experiment are not stated, but will be kept together with the other raw data in the GLP archive of the test facility. There were no compound related phytotoxic effects. Based on the nominal test concentrations, both the 72-hour E_yC₅₀(cell density/biomass) and E_rC₅₀(growth rate) were determined to be greater than 100 mg/L.