Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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:

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:

Aliquots of the samples from the biological test were directly analysed by HPLC and UV/VIS-detection (range of the injection volume: 100 μL, depending on the expected concentration).
The test item consists of three components. Only two components can be detected. The results are reported as sum of the two components.

Vehicle:

no

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations:
To produce the Water Accommodated Fraction (WAF) 100.5 mg of the test item were added to 1 litre of dilution water and treated for 60 sec. at 8000 rpm with an ultra turrax and afterwards stirred for 24 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 and an aseptic filter Sartobran 150 Sterile Capsule with a pore size 0.45 + 0.2 μm. The pH was measured to be 7.7.
100 mL of the solution were taken and 0.495 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For the only test item concentration and the control 6 replicates were prepared. All flasks were sealed with cotton/cellulose stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scene-desmus 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 – 120 µE. x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantumflux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using amicrocell- 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 and dilution water:
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

Test temperature:

21 - 24°C

pH:

7.9 at 0 hour
9.1 - 9.6 at 72 hour

Nominal and measured concentrations:

nominal concentrations in the range finding test: 1.0, 10 and 100 mg/L
nominal concentrations in the main test: 100 mg/L

Details on test conditions:

Exposure conditions:
- Test vessels : 300 mL Erlenmeyer flasks with cotton/cellulose stoppers, test volume: 100 mL
- Culturing apparatus: Light chamber 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.
- 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 on 2012-02-28.
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test
flask for measurements, which were not replaced.
- Experimental design: 1 test concentration plus 1 control, 3 replicates per concentration, 3 replicates per control
Initial cell density in the test cultures approximately 5000 cells per millilitre
- Test item concentration/s: 100 mg/L
- Method of administration: direct weighing
- Duration of exposure: 72 hours
- 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.

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

No toxic effects against algae were observed at a limit effective loading of 100 mg/L, corresponding to a measured concentration of < 0.131 mg/L, which reflects the maximum water solubility under exposure conditions.
The results are expressed in terms of Effective Loadings (EL). After 0 and 72 hours the values were below the quantification limit of the HPLC analysis (0.131 mg/L). The substance consists of several components, therefore a Water Accommodated Fraction (WAF) was used to test effects at a limit effective loading rate of 100 mg/L.

Validity criteria fulfilled:

yes

Remarks:

(The factor of biomass parameter 16; -The mean of the replicate coefficients of variation in the section-by-section growth < 35%; - The coefficient of variation of the mean specific growth rate replicates in the control < 7%.)

Conclusions:

The acute toxicity of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' to alga (Desmodesmus subspicatus) was tested according to OECD guideline 201 ' Alga, Growth Inhibition Test'. No toxic effects against algae were observed at a limit effective loading of 100 mg/L, corresponding to a measured concentration of < 0.131 mg/L, which reflects the maximum water solubility under exposure conditions.

Executive summary:

A study was performed to assess the adverse effects of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' on the growth rate (= rate of increase in cell density with time) 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 limit test concentration of nominally 100 mg/L of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer, a folded filter and an aseptic 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.

No toxic effects against algae were observed at a limit effective loading of 100 mg/L, corresponding to a measured concentration of < 0.131 mg/L, which reflects the maximum water solubility under exposure conditions.

The results are expressed in terms of Effective Loadings (EL). After 0 and 72 hours the values were below the quantification limit of the HPLC analysis (0.131 mg/L).

'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' consists of three components, therefore a Water Accommodated Fraction (WAF) was used to test effects at a limit effective loading rate of 100 mg/L.

Description of key information

The acute toxicity of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' to alga (Desmodesmus subspicatus) was tested. No toxic effects against algae were observed at a limit effective loading of 100 mg/L, corresponding to a measured concentration of < 0.131 mg/L, which reflects the maximum water solubility under exposure conditions.

Additional information

A study was performed to assess the adverse effects of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' on the growth rate (= rate of increase in cell density with time) 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 limit test concentration of nominally 100 mg/L of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer, a folded filter and an aseptic 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.

No toxic effects against algae were observed at a limit effective loading of 100 mg/L, corresponding to a measured concentration of < 0.131 mg/L, which reflects the maximum water solubility under exposure conditions.

The results are expressed in terms of Effective Loadings (EL). After 0 and 72 hours the values were below the quantification limit of the HPLC analysis (0.131 mg/L).

'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' consists of three components, therefore a Water Accommodated Fraction (WAF) was used to test effects at a limit effective loading rate of 100 mg/L.