Reaction mass of bis(2-ethylhexyl) adipate and cyclohexyl 2-ethylhexyl adipate and dicyclohexyl adipate

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

Study period:

24 - 27 Oct 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: DIN 38412, part 9

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 500 mg/L stock solution was preparted using 200 mg/L Cremophor as dispersant. After stirring of 17h the test solution was still an emulsion. This emulsion was tested.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Cremophor
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 200 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Test solutions were emulsions.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISMS
- Strain: SAG 86.81
- Method of cultivation: temperature: 20°C, stock culture 10000 cells/mL, cultivation in 250 ml Erlenmeyer flasks, test volume 100 mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20 °C

pH:

not specified

Nominal and measured concentrations:

Nominal: 7.812, 15.625, 31.25, 62.5, 125, 250 and 500 mg/L

Details on test conditions:

TEST SYSTEM
- Fill volume: 10 mL
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not specified
- Culture medium different from test medium: no

EFFECT PARAMETERS MEASURED: Biomass was recorded after 0, 24, 48 and 72h exposure time.
- Determination of cell concentrations: fluorimeter

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

398.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Validity criteria fulfilled:

yes

Conclusions:

A 72h acute toxicity test with Scenedesmus subspicatus revealed that DEHA is not acutely harmful to freshwater algae up to the saturation limit in water.

Executive summary:

The study was performed according to DIN guideline 38412, part 9 using Scenedesmus subspicatus as test organism. A 500 mg/L stock solution was preparted using 200 mg/L Cremophor as dispersant. After stirring of 17h the test solution was still an emulsion. This emulsion was tested. Nominal test concentrations between 7.8 and 500 mg/L were tested. The 72h-EC50 value is determined to be > 500 mg/L. An 72h-EC20 value is determined to be 398.6 mg/L. Since the effect concentration is greater than the solubility of DEHA in the test medium, it can be concluded that DEHA is not harmful to freshwater algae up to the saturation limit in water.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Data for Bis(2-ethylhexyl) adipate can be used to confirm the key study, since the three constituents are structurally very similar being diesters of adipic acid with either two cyclohexyl- (CAS 849-99-0, 25%, Sub 1), two 2-ethylhexyl- (CAS 103-23-1, 25%, Sub 2) or one cyclohexyl- and one 2-ethylhexyl-group (CAS 77357-98-3, 50%, Sub 3). This structural similarity leads to similar physico-chemical properties (log Pow: Sub1 = 5.9; Sub 2: 8.94; Sub 3: 7.02; water solubility: sub 1/2/3: < 0.1 mg/L) and subsequently to similar behavior (Sub 1/2/3: predicted to be readily biodegradable) and toxicity pattern in the environment. In addition, Bis(2-ethylhexyl) adipate is substance is considered to be a suitable read across substance, since it shows the highest log Pow value of all substances.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Reference 3

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: #30869
- Expiration date of the lot/batch: 26 Nov 2019
- Purity: 99.8% area-% (GC)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance is a poorly water-soluble mixture; therefore, a water accommodated fraction (WAF) was prepared using a liquid-liquid saturator technique following general guidance provided in OECD 23. A glass aspirator bottle (2-L) with a bottom side-outlet attached to a stopcock was filled with 2-L test media. The test substance was homogenized by shaking the container before use. The test solution was prepared separately (differential loading) by directly adding test substance to test medium. 204 μL test substance (considering density = 0.9802 g/cm3) were pipetted carefully with a positive displacement pipette (Gilson Microman E
M1000E) on the water surface. The bottle was closed, and the solution was stirred slowly at approx. 100 rpm on a magnetic stir plate for approx. 2 days. The undissolved test substance remained on the surface and the required volume of the saturated solution (approx. 1.5 L) was removed from the bottom of the bottle. The aqueous fraction was inspected visually for the presence of any undissolved test substance by observing the scattering of a laser light through the test solution (Tyndall effect). The aqueous fraction of the test solution is considered the water saturated fraction (WSF) in test media.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The stock solution appeared colorless-clear.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Source (laboratory, culture collection): obtained from SAG (Collection of algal cultures, Institute of Plant Physiology, University of Göttingen, Germany)
- Age of inoculum (at test initiation): A stock algal culture is maintained continuously at the test facility.

ACCLIMATION
- Acclimation period: Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

-

Test temperature:

23.3 – 23.5°C

pH:

start: 7.9 - 8.0
end: 7.9 - 9.3

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal: 100 mg/L as loading rate WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks plugged with gas permeable silicone sponge caps containing 100 mL test solution
- Initial cells density: 0.5E4 cells/mL
- Control end cells density: 106E4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was continuously measured during the whole test period in a separate deionized water filled flask. Light homogeneity was evaluated by measuring light intensity at 5 locations within the incubation area at the start and end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH value of the test medium was 7.9 and was adjusted to pH 8.1 by the addition of 1M NaOH.
- Photoperiod: permanent illumination
- Light intensity and quality: mean 6761.9 lux
- shaking: continuously

EFFECT PARAMETERS MEASURED:
- Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm) and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density. The TOC (Total organic carbon) values of the test concentrations and the control were analyzed at the start and the end of the exposure as an additional water quality parameter from uninoculated samples. The pH was measured at the start and the end of the test.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorphenol

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

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. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Any stimulation of growth found in any treatment: The growth inhibition of the 100 mg/L loading rate treatment was -0.9%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50(72h) = 2.884 mg/L
- The ErC50 of the reference substance 3,5 Dichlorphenol should be in the range: ErC50 = 0.9
– 4.4 mg/L after 72 hours

As TOC was slightly higher in test group 100 mg/L (loading rate; 0.4 mg TOC/L) than in the control (0.3 mg TOC/L) and test substance was directly pipetted on the water surface and lower phase of WAF was used for the test, it can be stated that test substance was present.

Validity criteria fulfilled:

yes

Conclusions:

The test substance had no observable inhibitory effect on Pseudokirchneriella subcapitata growth up to its saturation limit in test media and under test conditions. There were no compound-related phytotoxic effects.

Description of key information

EL50(72h) > 100 mg/L (loading rate WSF, nominal) for Pseudokirchneriella subcapitata (OECD 201)

NOELr(72h) >= 100 mg/L (loading rate WSF, nominal) for Pseudokirchneriella subcapitata (OECD 201)

Additional information

One study investigating the toxicity of the test substance to freshwater algae is available. The key study was performed according to GLP and OECD guideline 201 using Pseudokirchneriella subcapitata as test organism (BASF 2019). The test substance is a poorly water-soluble mixture; therefore, a water accommodated fraction (WAF) of a 100 mg/L loading rate was prepared using a liquid-liquid saturator technique following general guidance provided in OECD 23. Concentration control analyses were not performed, because a specific method for analyses in the required concentration range is not available. No growth rate inhibition was observed in the treatment in comparison to the control. Thus, the EL50 is determined to be > 100 mg/L as loading rate WSF and the NOELr is determined to be >= 100 mg/L as loading rate based on nominal concentrations. Based on this result, the test substance is considered to be not harmful to freshwater algae up to the saturation limit in water.

This result is confirmed by available data for Bis(2-ethylhexyl) adipate (CAS 103 -32 -1), which is one of the three main components of the multiconstituent substance. This study can be used to confirm the key study, since the three constituents are structurally very similar being diesters of adipic acid with either two cyclohexyl- (CAS 849-99-0, 25%, Sub 1), two 2-ethylhexyl- (CAS 103-23-1, 25%, Sub 2) or one cyclohexyl- and one 2-ethylhexyl-group (CAS 77357-98-3, 50%, Sub 3). This structural similarity leads to similar physico-chemical properties (log Pow: Sub1 = 5.9; Sub 2: 8.94; Sub 3: 7.02; water solubility: sub 1/2/3: < 0.1 mg/L) and subsequently to similar behavior (Sub 1/2/3: predicted to be readily biodegradable) and toxicity pattern in the environment. In addition, Bis(2-ethylhexyl) adipate is substance is considered to be a suitable read across substance, since it shows the highest log Pow value of all substances. The supporting study was performed according to DIN guideline 38412, part 9 using Scenedesmus subspicatus as test organism (BASF 1989). A 500 mg/L stock solution was preparted using 200 mg/L Cremophor RH40 as dispersant. After stirring of 17h the test solution was still an emulsion. This emulsion was tested. Nominal test concentrations between 7.8 and 500 mg/L were tested. The 72h-EC50 value is determined to be > 500 mg/L. An 72h-EC20 value is determined to be 398.6 mg/L. Since the effect concentration is greater than the solubility of DEHA in the test medium, it can be concluded that DEHA is not harmful to freshwater algae up to the saturation limit in water.