Bis(2-ethylhexyl) malate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7/11/2017 to 12/2/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

17 July 1992

Deviations:

no

Principles of method if other than guideline:

In addition, the following ISO standard was followed:
ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium", (1995).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) malate

EC Number:

260-070-5

EC Name:

Bis(2-ethylhexyl) malate

Cas Number:

56235-92-8

Molecular formula:

C20H38O5

IUPAC Name:

1,4-bis(2-ethylhexyl) 2-hydroxybutanedioate

Test material form:

liquid

Details on test material:

Identification: Dermol DOM
Appearance: Clear colourless to pale yellow liquid
Batch: P7677
Purity/Composition: >95%
Test item storage: At room temperature
Stable under storage conditions until: 31 December 2018 (retest date)
Additional information
Test Facility test item number: 209081/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Molecular structure: Not indicated
Molecular formula: C20H38O5
Molecular weight: 358.5

Specific details on test material used for the study:

Test Item and Reference Item

Test Item
Test item information
Identification:
Dermol DOM
Appearance: Clear colourless to pale yellow liquid
Batch: P7677
Purity/Composition: >95%
Test item storage: At room temperature
Stable under storage conditions until: 31 December 2018 (retest date)

Additional information
Test Facility test item number: 209081/A
Purity/Composition correction factor: No correction factor required
Organic carbon (wt%): 67.0%
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Diethylhexyl Malate
CAS number: 56235-92-8
Molecular structure: Not indicated
Molecular formula: C20H38O5
Molecular weight: 358.5
Highly reactive to water: Not indicated
Volatile: Not indicated
Solubility in water: Insoluble
Stability in water: Stable

Reference Item
Reference Item Information
Test Facility Study No. 20136811
Identification number RS186
Container E1
Identification Sodium acetate
CAS Number 127-09-3
Molecular formula CH3COONa
Molecular weight 82.03
Appearance White powder (determined at Charles River Den Bosch)
Batch AM1027668
Purity 99.5%
Storage conditions At room temperature. Stable under storage conditions until 30 June 2018
Supplier Merck KGaA, Darmstadt, Germany
Article number 1.06268
Certified Yes

Reference Item Concentration and Preparation of Test Solutions
A solution of sodium acetate was prepared by dissolving 799.41 mg in Milli- RO water and making this up to a total volume of 200 mL. Volumes of 20 mL from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/L).

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis or equivalent document was provided to the Test Facility and is presented in Appendix 3.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Source
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment
The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 4 g/L in the concentrated sludge. The magnetically stirred sludge was used as inoculum at the amount of 15 mL/L of mineral medium, leading to a concentration SS of 30 mg/L.

Reason for selection
The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

ca. 12 mg/L

Based on:

TOC

Remarks:

Target but substance insoluble.

Initial test substance concentration 2

Initial conc.:

ca. 18 mg/L

Based on:

test mat.

Remarks:

Target but substance insoluble.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

[Copy and paste study methods and conditions to this section]

Include information on:
- Test medium composition and conditions
- Test system
-
TEST CONDITIONS
- Composition of medium:
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:

Results and discussion

Test performance:

All deviations that occurred during the study have been authorized/acknowledged by the Study Director, assessed for impact, and documented in the study records. All study plan deviations and those SOP deviations that could have impacted the quality or integrity of the study are listed below.
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Test Procedure and Conditions
On nominal day 3 the aeration tubing for blank A became disconnected. The tubing was immediately reconnected to restore aeration. Evaluation: Such a short breakdown in aeration (<1 day) is not expected to influence the results. In addition, replicates or blank A and B are in good agreement. This deviation has no impact on the validity or outcome of the study.

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 39

Sampling time:

28 d

% Degradation 2

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 41

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

80 % degradation of reference substance achieved in 14 days

Any other information on results incl. tables

RESULTS

Theoretical CO₂Production

The ThCO₂of Dermol DOM was calculated to be 2.45 mg CO₂/mg.

The ThCO₂of sodium acetate was calculated to be 1.07 mg CO₂/mg.

If applicable the ThCO₂per test bottle are given in the subscript of the tables (seeAppendix 1).

Biodegradation

All data are presented inAppendix 1.The results of CO₂production and biodegradation in blank bottles, background bottles and each test bottle are listed inTable 2to 8.Table 9contains the comparison of biodegradation of Dermol DOM in bottles A and B. 

Figure 1 shows the curves for biodegradation of the two bottles with Dermol DOM, the positive control and the toxicity control.

Figure 1(attached) Biodegradation of Dermol DOM and Sodium Acetate in the Modified Sturm Test

The relative biodegradation values calculated from the measurements performed during the test period revealed 39% and 41% biodegradation of Dermol DOM (based on ThCO₂), for the

duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, more than 25% biodegradation occurred within 14 days (59%, based on ThCO₂). Therefore, the test item was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve (see also paragraph5.1).

Monitoring of Temperature and pH

The temperature recorded in a vessel with water in the same room varied between 22 and 23°C. The pH values of the different test media are presented inTable 1.

Table 1         pH Values of Different Test Media

Test medium:	At the start of the test:	On day 14:	On day 28:
Blank control (A)	7.5	-	7.5
Blank control (B)	7.5	-	7.5
Positive control	7.6	7.7	-
Dermol DOM (A)	7.6	-	7.5
Dermol DOM (B)	7.6	-	7.5
Toxicity control	7.6	7.6	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In conclusion, Dermol DOM was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The objective of the study was to evaluate the test item Dermol DOM for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge; Carbon dioxide (CO₂) evolution test (modified Sturm test).

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the ISO standard 10634, 1995.

Dermol DOM was a clear colourless to pale yellow liquid with a purity of >95%. The test item was tested in duplicate at a target concentration of 18 mg/L, corresponding to

12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO₂production (ThCO₂) of Dermol DOM was calculated to be 2.45 mg CO₂/mg.

The study consisted of six bottles: 

2 inoculum blanks (no test item), 

2 test bottles (Dermol DOM), 

1 positive control (sodium acetate) and 

1 toxicity control (Dermol DOM plus sodium acetate).

Since Dermol DOM was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, weighed amounts were added to watch glasses, which were added directly to the test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days for the inoculum blank and test item (last CO₂measurement on day 29) and 14 days for the positive and toxicity control (last CO₂measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed 39% and 41% biodegradation of Dermol DOM (based on ThCO₂), for the

duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, Dermol DOM was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, Dermol DOM was designated as not readily biodegradable.