2,2-dimethylpropane-1,3-diyl dibenzoate

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:

weight of evidence

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

SAMPLING SCHEDULE:
- Control: at 0, 24, 48 and 72 hours
- Test concentration/s: at 0, 24, 48 and 72 hours

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name : Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source : Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures : Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.
- Growth medium (OECD medium of OECD TG 201, annex 1) was used for the growth of the algae in the pre cultures and the preparation of stock and test solutions of the test item.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

21-24 °C

pH:

Control: 7.0 at start, 7.7 after 72 h
100 mg/L: 7.0 at start, 7.9 after 72 h

Dissolved oxygen:

No data

Salinity:

n.a.

Nominal and measured concentrations:

100 mg/L

Details on test conditions:

PRE-TREATMENT OF THE TEST ITEM:
- The dilution water was adjusted from pH 7.6 to pH 7.0.
- To produce the only test item concentration 200.1 mg of the test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 2 h on a magnetic stirrer
- Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7-12 μm
- The pH was measured to be 7.0
- 100 mL of the solution were taken and 0.633 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL.
- For each test item concentration and the control 6 replicates were prepared. All flasks were sealed with cotton stoppers

EXPOSURE CONDITIONS:
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers
- Test volume: 100 mL
- Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm; Temperature was measured and recorded daily
- Light intensity : A light intensity ranging from 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured; The light intensity was checked before the start
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced
- Experimental design : 1 test concentrations plus 1 control; 6 replicates per concentration, 6 replicates per control; Initial cell density in the test cultures approximately 5000 cells per millilitre; Additionally test concentration 100 mg/L without algae.
- Test item concentration/s : 100 mg/L
- Method of administration : direct weighing
- Criteria of effects : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.358 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.358 mg/L

Nominal / measured:

meas. (geom. mean)

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:

growth rate

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:

The EC 50 and EC 10 values were > 0.358. The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 0.40 % to 0.43 % of nominal values at 0 hours, from 0.33 % to 0.48 % of nominal values at 24 hours, from 0.38 % to 0.47 % of nominal values at 48 hours and from 0.21 % to 0.41 % of nominal values at 72 hours.

During the incubation time a second substance (3-hydroxy-2,2-dimethylpropyl benzoate) was formed by hydrolysis. This substance was also determined analytically and quantified via HPLC analysis.

 

Analysis 2,2-dimethylpropane-1,3-diyl dibenzoate

Test item concentration [mg/L]	HPLC values [mg/L]
	0h	24h	48h	72h
Control	--	--	--	< 0.0134
100	0.428 0.423	0.484 0.472	0.371 0.383	0.223 0.204
100 without algae	0.409 0.388	0.334 0.320	0.462 0.482	0.414 0.405

 

Analysis 3-hydroxy-2,2-dimethylpropyl benzoate

Test item concentration [mg/L]	HPLC values [mg/L]
	0h	24h	48h	72h
Control	--	--	--	< 0.0147
100	< 0.0147 < 0.0147	0.020 0.020	0.125 0.123	0.244 0.250
100 without algae	< 0.0147 < 0.0147	< 0.0147 < 0.0147	< 0.0147 < 0.0147	0.019 0.021

 

Validity criteria fulfilled:

yes

Remarks:

(- The cell density >16 within 72 hrs;- The mean coefficient of variation for section-by-section specific growth <35 %; - The coefficient of variation of the mean specific grotwh rate replicates in the control <7 %)

Conclusions:

The toxicity of 2,2-dimethylpropane-1,3-diyl dibenzoate to algae was measured during a period of 72 hours. No toxic effects against algae were observed at a limit test concentration of 0.358 mg/L under exposure conditions.

Executive summary:

A study was performed to assess the adverse effects of 2,2-dimethylpropan-1,3-diyl dibenzoate on the growth rate and the yield of the planktonic freshwater algal species Desmodesmus subspicatus over several generations. The test material had been previously purified (purity 99.6%) in order to avoid potential effects of impurities.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours Auxiliaries used to prepare the test media were an ultrasonic bath, magnetic stirrer and folded filters.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate, relative to control cultures grown under identical conditions. The results are expressed in terms of geometric mean measured concentrations. No toxic effects of 2,2-dimethylpropan-1,3-diyl dibenzoate against algae were observed at a limit test concentration of 0.358 mg/L under exposure conditions. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute algae study.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

SAMPLING SCHEDULE:
- Control: at 0, 24, 48 and 72 hours
- Test concentration/s: at 0, 24, 48 and 72 hours

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name : Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source : Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures : Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

21-24 °C

pH:

7.7-7.9

Dissolved oxygen:

No data

Salinity:

n.a.

Nominal and measured concentrations:

4.3 mg/L (n): after 72 h 0.021 mg/l
9.4 mg/L (n): after 72 h 0.026 mg/l
21 mg/L (n): after 72 h 0.046 mg/l
45 mg/L (n): after 72 h 0.256 mg/l
100 mg/L (n): after 72 h 0.280 mg/l
100 mg/L without algae (n): after 72 h 0.331 mg/l

Details on test conditions:

PRE-TREATMENT OF THE TEST ITEM:
- The dilution water was adjusted from pH 7.4 to pH 7.0.
- Direct weighings were prepared to produce the different test item concentrations
- To achieve 4.2, 9.5, 21.1, 45.0 and 100.3 mg/L were added to 1 litre of dilution water, treated for 1 h in an ultrasonic bath and stirred for 2 h on a magnetic stirrer
- Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7-12 μm
- The pH was measured and ranged between 6.9 and 7.0
- 100 mL of the solution were taken and 0.595 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL.

EXPOSURE CONDITIONS:
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers
- Test volume: 100 mL
- Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm; Temperature was measured and recorded daily
- Light intensity : A light intensity ranging from 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured; The light intensity was checked before the start
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced
- Experimental design : 5 test concentrations plus 1 control; 3 replicates per concentration, 3 replicates per control; Initial cell density in the test cultures approximately 5000 cells per millilitre; Additionally highest test concentration without algae
- Test item concentration/s : 4.3, 9.4, 21 ,45 and 100 mg/L
- Method of administration : direct weighing
- Criteria of effects : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.504 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.132 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.053 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: acc. Williams Multiple Sequential t-test Procedure

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.079

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: acc. Williams Multiple Sequential t-test Procedure

Details on results:

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 0.3 % to 1.5 % of nominal values at 0 hours, from 0.4 % to 1.6 % of nominal values at 24 hours, from 0.3 % to 1.5 % of nominal values at 48 hours and from 0.2 % to 0.6 % of nominal values at 72 hours.

Analytical results:

Test item

concentration

[mg/L] HPLC values [mg/L]

0 h *       24 h **       48 h       ** 72 h *

Control < 0.034       < 0.034 < 0.034       < 0.034

4.3       0.066       0.068       0.063       0.021

9.4       0.063       0.075       0.066       0.026

21        0.096       0.093       0.093       0.046

45        0.311       0.316       0.299       0.256

100       0.326       0.187 #    0.304       0.280

100 without

algae       0.354       0.364       0.271       0.331

Comments:

* Samples were taken on 2014-11-18 and on 2014-11-21 and stored deep frozen at -18 °C and were analysed on 2014-12-02

** Samples were taken on 2014-11-19 and on 2014-11-20 and stored deep frozen at -18 °C and were analysed on 2014-12-04.

# The value was classified as an outlier, because the recovery at 0, 48 and 72 hours of exposure and the recovery of the test item

concentration 100 mg/L without algal inoculum were higher. For further calculations the mean value at 0, 48 and 72 hours of the test

item concentration 100 mg/L were taken as a surrogate.

Validity criteria fulfilled:

yes

Remarks:

(- The cell density >16 within 72 hrs;- The mean coefficient of variation for section-by-section specific growth <35 %; - The coefficient of variation of the mean specific grotwh rate replicates in the control <7 %)

Conclusions:

The toxicity of 2,2-dimethylpropane-1,3-diyl dibenzoate to algae was measured during a period of 72 hours and the growth inhibition showed an ErC10 of 0.132 mg/L and an ErC50 of 0.504 mg/L. The NOEC was found to be 0.053 mg/L. The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 0.3 % to 1.5 % of nominal values at 0 hours, from 0.4 % to 1.6 % of nominal values at 24 hours, from 0.3 % to 1.5 % of nominal values at 48 hours and from 0.2 % to 0.6 % of nominal values at 72 hours.

Executive summary:

A study was performed to assess the adverse effects of 2,2-dimethylpropan-1,3-diyl dibenzoate on the growth rate and the yield of the planktonic freshwater algal species Desmodesmus subspicatus over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 4.3, 9.4, 21, 45 and 100 mg/L of 2,2-dimethylpropan-1,3-diyl dibenzoate dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, magnetic stirrer and folded filters.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate, relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Williams Multiple Sequential t-test Procedure.

The growth inhibition showed an ErC10 of 0.132 mg/L and an ErC50 of 0.504 mg/L. The NOEC was found to be 0.053 mg/L. The results are expressed in terms of geometric mean measured concentrations. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute algae study.

It was however noticed by checking the analytal monitoring data that beside the test item peak a second peak appeared. This peak eluted earlier in the reversed phase chromatography. The peak height was in the same order of magnitude as the test item. It was assumed that the impurity "3 -hydroxy-2,2 -dimethylpropyl benzoate" already present in the technical substance could be assigned to that peak (representative chromatograms are attached to this robust study summary). Later (outside this study) the impurity 3-hydroxy-2,2-dimethylpropyl benzoate was generated and the identity of the peak could be verified by co-chromatography. The original concentration of the impurity in the test material is 1.5% but the test solutions contained about the same concentrations of test item and impurity.

During pre-treatment (1 h ultrasonication, 2 h stirring and filtration) of the test item 2,2-dimethylpropan-1,3-diyl dibenzoate containing 3-hydroxy-2,2-dimethylpropyl benzoateas an impurity, 3-hydroxy-2,2-dimethylpropyl benzoate is enriched in ecotox tests in the water media due to the higher water solubility thus probably causing effects to algae.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without restriction

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

SAMPLING SCHEDULE:
- Control: at 72 hours
- Test concentration/s: at 0 and 72 hours

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name : Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source : Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures : Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.
- Growth medium (OECD medium of OECD TG 201, annex 1) was used for the growth of the algae in the pre cultures and the preparation of stock and test solutions of the test item.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

21-24 °C

pH:

Control: 7.1 at start, 8.6 after 72 h
1.9 mg/L: 7.1 at start, 8.7 after 72h
100 mg/L: 7.0 at start, 7.6 after 72 h

Dissolved oxygen:

No data

Salinity:

n.a.

Nominal and measured concentrations:

Samples of control and concentrations 4.3, 21 and 100 mg/L were analysed at 0h and 72h. Generally, the measured concentrations were in the range of nominal concentrations.
The high recovery of the test item concentration 4.3 mg/L at 0 and 72 hours of exposure could not be explained, especially because the documented weighing was 4.4 mg/L. As the recovery of the other two higher analysed test item concentrations 21 and 100 mg/L showed no significant difference to the nominal concentration the values of the test item concentration 4.3 mg/L were regarded as outlier and the results for the ECx determination were based on nominal concentrations.

Details on test conditions:

PRE-TREATMENT OF THE TEST ITEM:

- Prior to the test the dilution water was adjusted from pH 7.6 to pH 7.0.
- To produce the different test item concentrations 1.9, 4.3, 9.4, 21, 45 and 100 mg of the test item were added each to one litre of dilution water, treated for 1 hour in an ultrasonic bath and stirred for 2 hours on magnetic stirrers.
- Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7-12 μm
- The pH was measured to be between 7.0 and 7.1
- 100 mL of each solution was taken and 0.588 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL.
- For each test item concentration and the control 6 replicates were prepared. All flasks were sealed with cotton stoppers

EXPOSURE CONDITIONS:
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers
- Test volume: 100 mL
- Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm; Temperature was measured and recorded daily
- Light intensity : A light intensity ranging from 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured; The light intensity was checked before the start
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced
- Experimental design : 1 test concentrations plus 1 control; 6 replicates per concentration, 6 replicates per control; Initial cell density in the test cultures approximately 5000 cells per millilitre; Additionally test concentration 100 mg/L without algae.
- Test item concentration/s : 1.9, 4.3, 9.4, 21, 45 and 100 mg/L
- Method of administration : direct weighing
- Criteria of effects : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

measured concentrations = nominal conc.

Basis for effect:

growth rate

Remarks on result:

other: 6.5 – 9.0

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

measured concentrations = nominal conc.

Basis for effect:

growth rate

Remarks on result:

other: 1.7 – 3.4

Details on results:

The results are expressed in terms of nominal concentrations. Measured values verified the nominal concentrations.

Results of HPLC analysis

 

Analysis 3-hydroxy-2,2-dimethylpropyl benzoate

Test item concentration [mg/L]	HPLC values [mg/L]
	0h	72h
Control	not determined	< 0.0246
4.3	6.78	6.44
21	20.2	19.7
100	98.6	98.3

 

The high recovery of the test item concentration 4.3 mg/L at 0 and 72 hours of exposure could not be explained, especially because the documented weighing was 4.4 mg/L. As the recovery of the other two higher analysed test item concentrations 21 and 100 mg/L showed no significant difference to the nominal concentration the values of the test item concentration 4.3 mg/L were regarded as outlier and the results for the ECx determination were based on nominal concentrations.

 

Validity criteria fulfilled:

yes

Remarks:

The biomass parameter in controls 0 to 72 h must be>16. Found: 116. The mean of the replicate coefficients in the section-by-section growth rate was 13.1%. and thus <35%.. The coefficient of variation of the mean spec. growth rate replicates: 1.6% (<7%).

Conclusions:

The toxicity of 3-hydroxy-2,2-dimethylpropyl benzoate to algae was measured during a period of 72 hours and the growth inhibition showed an ErC10 of 2.6 mg/L and an ErC50 of 7.6 mg/L.

Executive summary:

A study was performed to assess the adverse effects of 3-hydroxy-2,2-dimethylpropyl benzoate on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 1.9, 4.3, 9.4, 21, 45 and 100 mg/L of 3-hydroxy-2,2-dimethylpropyl benzoate dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and a folded filter.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Williams Multiple Sequential t-Test Procedure.

 

The following values were determined for 3-hydroxy-2,2-dimethylpropyl benzoate [mg/L]:

ErC50 (0-72 h): 7.6

ErC10 (0-72 h): 2.6

Description of key information

The toxicity of 2,2-dimethylpropane-1,3-diyl dibenzoate to algae was measured during a period of 72 hours. No effects up to the limit of the water solubility were found.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.358 mg/L

EC10 or NOEC for freshwater algae:

0.358 mg/L

Additional information

Should read: ">0.358 mg/L"

The final assessment of the toxicity of 2,2-dimethylpropane-1,3-diyl dibenzoate to algae is based on a weight-of-evidence approach using the follow 3 studies:

 

1. Alga, Growth Inhibition Test with technical 2,2-Dimethylpropan-1,3-diyldibenzoat (Spoo-Kloeppel M, 2015):

In this study 2,2-dimethylpropane-1,3-diyl dibenzoate (purity 97.9 %) containing 1.5 % 3-hydroxy-2,2-dimethylpropyl benzoate as main impurity was used. In the study effects to algae were reported (72h-EC50: 0.5 mg/L). However, analytical monitoring showed that the impurity 3-hydroxy-2,2 -dimethylpropyl benzoate was significantly enriched in the water phase. 3-Hydroxy-2,2-dimethylpropyl benzoate has a rather good water solubility (>100 mg/L) compared to the solubility of the target substance 2,2-dimethylpropane-1,3-diyl dibenzoate which has a water solubility of 1.16 mg/L. It was assumed that the observed enrichment results from the pre-treatment (1 h ultrasonication, 2 h stirring and filtration) of the test item. Therefore, the test results were considered questionable and further studies were performed.

 

2. Alga, Growth Inhibition Test with purified 2,2-Dimethylpropan-1,3-diyl dibenzoate (Spoo-Kloeppel M, 2018):

In this study 2,2-dimethylpropane-1,3-diyl dibenzoate (purity 99.6 %) not containing 3-hydroxy-2,2-dimethylpropyl benzoate was used. In the study no effects to algae were reported (72h-EC50: >0.358 mg/L) up to the limit of the water solubility.

 

3. Alga, Growth Inhibition Test with the impurity 3-Hydroxy-2,2-dimethylpropyl benzoate (Spoo-Kloeppel M, 2016):

This study was performed with 3-hydroxy-2,2-dimethylpropyl benzoate, which constitutes the main impurity in the original sample. Monitoring analysis showed that 3-hydroxy-2,2-dimethylpropyl benzoate has a rather good water solubility as the recovery at a concentration of 100 mg/L was in the range of 100%. In the study effects to algae were reported (72h-EC50: 7.6 mg/L).

 

Conclusion:

Taking into account all these results, it becomes obvious that 2,2-dimethylpropane-1,3-diyl dibenzoate as such does not cause effects to algae. Effects are only observed by artefacts formed during preparation of the test solution. Especially the impurity 3-hydroxy-2,2-dimethylpropyl benzoate is artificially enriched during test solution preparation due to the higher water solubility.