Dimethyl adipate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: screening tests

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

1982-05-04 to 1982-10-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study, non-GLP

Justification for type of information:

This IUCLID for dimethyl adipate is compiled using data from the substance itself, a structurally related compound (dimethyl glutarate) and a mixture of dibasic esters (dimethyl adipate, succinate and glutarate). The toxicity of each of these substances is similar and the document attached justifies why data on the category members can be used to support the data gaps for dimethyl adipate.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3170 (Shake Flask Die-away Test)

Version / remarks:

Federal Register 44(53):16272-16285, March 16, 1979

Deviations:

yes

Remarks:

- with regard to recent (1998) standards e.g. different inoculum, incubation at 100 rpm only, no substance specific analytics (primary degradation not investigated), no equilibrium sorption coeffifient investigated

Principles of method if other than guideline:

The procedure selected for this study was the aerobic shake flask method as suggested by the United States Environmental Protection Agency. The method involved two phases: an acclimation period of 13 days during which the mixed microbial inoculum adapted or acclimated to the test substances; the second phase was a 28-day test period during which measurements were taken of the respired Carbondoxide and, if possible, subsamples were removed for the determination of the changes in the substrate concentrations.
Also, pH, temperature and dissolved oxygen were measured to assure adequate incubation conditions.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: Adapted composite of three sources mixed 4:2:1: sewage, domestic (adaptation not specified) : natural soil : digested sludge

Details on inoculum:

The inoculum was a composite of three sources mixed together at a ratio of 4:2:1, respectively.
(1) Sewage, domestic (adaptation not specified): The primary component was obtained from the Wilmington, Delaware, U.S.A. secondary waste treatment facility. Freshly collected sludge was filtered through a glass wool pad
(2) Natural soil: The soil was collected in the woods around Haskell Laboratory, Newark, Delaware 19711, U.S.A. and 300 g soil was suspended in 3 L chlorine-free water. The soil suspension was stirred for 1-1½ hours, allowed to settle for 1 hour and then filtered.
(3) Digested sludge: A third component of the inoculum was the continuously mixed microbial culture maintained for over 2 years in a biological digestor system (Horizon Activated Sludge Percolator) at Haskell Laboratory. The original source for this mixed community was a soil and sludge mixture.

Acclimation period: The basal medium was dispensed in 100 mL aliquots into 250 mL sterile plastics crew-cap erlenmeyer flasks and autoclaved. After cooling, the yeast extract was added. Transfers were made every 48 to 72 hours to fresh medium containing the same constituents. After inoculation with 1.0 mL of the prepared inoculum, the flasks were incubated on a gyrorotary water bath shaker at 25° C at a shaker setting of 3-3¾. The flasks were kept in the dark to prevent photodegradation of the test compounds and /or growth of photosynthetic microorganisms.

Duration of test (contact time):

28 d

Initial conc.:

5 mg/L

Based on:

DOC

Initial conc.:

10 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test Period:
On the 13th day following the initial inoculation of the acclimation flasks, and 48 to 72 hours after the last transfer, 10 mL aliquots were transferred to 1 L erlenmeyer flasks containing 500 mL of the appropriate medium.

TEST CONDITIONS
- Composition of medium: The basal medium, which contained no added organic carbon substrate, was composed of a phosphate buffer solution, NH4Cl, MgSO4, CaC12, FeCl3, a trace element solution, yeast extract, and ASTM Type II water.
- Additional substrate: no
- Test temperature: 22-23 °C
- pH: 5.4-6.9
- pH adjusted: no
- Continuous darkness: yes
- Other: Dissolved oxygen 4.0 to 6.5 mg/L; every third sampling period, 90 mL of sterile ASTM Type II water was added to each flask to maintain the proper liquid level.

TEST SYSTEM
- Culturing apparatus: All flasks were incubated on a gyrorotary shaker set at 100 rpm
- Number of culture flasks/concentration: 2, all flasks were prepared in duplicate.
- Method used to create aerobic conditions: The flasks were specially designed to allow filtered air to enter through an ascarite-glass wool plug.
- Measuring equipment: Dissolved oxygen (DO) was measured with a Dissolved Oxygen Meter and probe (Yellow Springs Instruments), the pH was taken with a combination electrode coupled to an Orion pH Meter; and the temperature was read from mercury thermometers in the room.
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The flasks were specially designed an had a side arm on which replaceable CO2 traps could be attached. On each sampling day, the KOH-containing bottle, attached to the side arm of each rest flask to trap respired CO2, was removed. A fresh bottle containing 2 mL of standard KOH was immediately replaced on the side arm of each 1 L Erlenmeyer flask. In order to assure aerobic conditions, all stopcocks were opened during the sampling periods (approximately 2 hours) and closed throughout the rest of the incubation period.


SAMPLING
- Sampling frequency: on days 1, 2, 5, 7, 9, 14, 16, 21, 24, 28
- Sampling method: The flasks were specially designed and had a sterile sampling port. On each subsequent sampling day, 30 mL were withdrawn from the sampling port: 10 mL were fixed in 30% formaldehyde and 20 mL were filtered through boiled Nuclepore® filters to remove the microbial cells.
- Sample storage before analysis: The filtrates were frozen.

CONTROL AND BLANK SYSTEM
- Inoculum blank: no
- Abiotic sterile control: yes
- Toxicity control: no
- Other:
The organic amendments to the basal medium were:
- test compound at nominal 5 or 10 mg organic carbon/L basal medium;
- a known biodegradable substance, linear alkylate sulfonates (LAS), at a nominal 5 or 10 mg organic carbon/L basal medium;
- a control flask containing only basal medium;
- a control flask which contained the high concentration of all organic substances but no deliberate inoculum;
- another control flask which contained a reference material which is only partially biodegradable, benzisothiazolin-3-one (in Proxel®, active ingredient BIT), also at nominal 5 or 10 mg organic carbon/L basal medium.


STATISTICAL METHODS:
- Outliner elimination: After titration of the samples, an additional 2 to 3 blanks were also titrated. The blanks were averaged and values not within 5% of the mean were discarded.

Reference substance:

other: known biodegradable substance: LAS (CAS 42615-29-2), linear alkylate sulfonates

Reference substance:

other: material which is only partially biodegradable: benzisothiazolin-3-one (in Proxel®, active ingredient BIT)

Parameter:

% degradation (CO2 evolution)

Value:

36

Sampling time:

21 d

Remarks on result:

other: 36% of the theoretical cumulative percent CO2 was respired by the mixed microbial system

Details on results:

Figure 1 (illustration attached) depicts the amount of carbon dioxide produced from the Dibasic Esters at nominal concentrations of 5 and 10 mg C/L. The data have been corrected for the carbon dioxide respired in the control flasks. The maximum rate of carbon dioxide respiration was achieved for the low concentration between days 1 and 2. Then there appeared to be a very slightly increased level of carbon dioxide production throughout the remainder of the study period. Maximal degradation of DBE at the higher concentration, however, continued throughout the first seven days following inoculation.
Based upon the respiration rates shown in Figure 1 (illustration attached) for the DBE, LAS and Proxel®, half-life for carbon dioxide production from DBE can be calculated (Lehinger 1975). At the high concentration of DBE the rate is 2.5 days while at the lower concentration the half-life rate is 3.2 days.
- Lehinger AL (1975). Biochemistry, 6th ed. Worth Publishers, Inc.

Results with reference substance:

Respiration from LAS degradation did not appear to be important until about day 15 (Figure 1, illustration attached); but from Figure 2 (in study report), it is apparent that the LAS was being consumed by the microbial population. This difference is most likely the result of the microflora incorporating the LAS carbon into microbial biomass. 36% of the theoretical carbon dioxide that could be respired was produced from the DBE supplied to the cultures while only 6 to 15% of the LAS were respired, and for Proxel® the respiration values were 20-21% (Figure 3, illustration attached). The changes in the amount of Proxel® present in the inoculated and control flasks are shown in Figure 4 (in study report).

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Executive summary:

The microbial degradation of Dibasic esters (DBE) was investigated according to the EPA procedures as published in the Federal Register 44(53):16272-16285, March 16, 1979, which is considered similar to EPA OPPTS 835.3170 (Shake Flask Die-away Test). Formal GLP standards did not apply. The study is considered valid and conclusive.

Dibasic esters were subjected to microbiological biodegradation testing using the shake flask method.

The DO (Dissolved oxygen) values throughout the study period ranged from 4.0 t o 6.5 mg/L indicating that an aerobic atmosphere was maintained.

The pH values fluctuated very little during the study staying within a 5.4 t o 6.9 range. These data indicate that environmental factors were not limiting the amount or rate of biodegradation of the various test substances.

The half-life for the high concentration of DBE was 2.5 days while at the low concentration the half-life was 3.2 days. In either case, 36% of the theoretical cumulative percent CO₂ was respired by the mixed microbial system.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Study Start date: 1993-06-09, Study termination date: 1993-07-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

This IUCLID for dimethyl adipate is compiled using data from the substance itself, a structurally related compound (dimethyl glutarate) and a mixture of dibasic esters (dimethyl adipate, succinate and glutarate). The toxicity of each of these substances is similar and the document attached justifies why data on the category members can be used to support the data gaps for dimethyl adipate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

Qualifier:

according to guideline

Guideline:

EPA OTS 796.3340 (Inherent Biodegradability: Modified SCAS Test)

Version / remarks:

(40 CFR 796.3340)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Both activated sludge mixed liquor and raw waste water were obtained from the Grand Glaize treatment plant of St. Louis (U.S.A.) Metropolitan Sewer District which is predominantly domestic sewage.
- Concentration of sludge: After adjustment to a suspended solids concentration of 2200 to 2800 mg/L, 270 mL of the mixed liquor was charged to 8 uniquely labelled borosilicate glass aeration chambers.
- Water filtered: yes
- Type and size of filter used: Prior to use, the mixed liquor was filtered through a 20-mesh stainless steel screen to remove large particulate.

Duration of test (contact time):

29 d

Initial conc.:

16 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: not reported
- Additional substrate: no
- Solubilising agent: no
- Test temperature: not reported
- pH: weekly
- pH adjusted:no
- Suspended solids concentration: 2200 to 2800 mg/L


TEST SYSTEM
- Culturing apparatus: glass aeration chambers
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: The aeration chambers were connected to a compressed air manifold via inlet tubes. The mixed liquor was aerated using a 24-hour cycle (72 hours on weekends) at a 0.5 cubic foot per hour flow rate (i.e. about 14 L/h, conversion factor 28.31685).
- Test performed in open system: yes

SAMPLING
- Sampling frequency: daily
- Sampling method: At the end of each cycle, aeration was stopped, the activated sludge allowed to settle, and 180 mL of supernate removed. Samples of the supernate from the control, dimethyl ester samples, and 4-nitrophenol units were saved for measurements. Raw sewage was then added to each chamber bringing the volume back to 270 mL. Aeration was resumed.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: no
- Other: Positive control, reference substance with known biodegradability

Reference substance:

other: 4-Nitrophenol (CAS 100-02-7 )

Test performance:

Total suspended solids (TSS) of the mixed liquor was measured during week 1.

Details on results:

The pH for each SCAS unit on a weekly basis summarized in Table 1. The TSS for each SCAS unit during week1 of the test are summarized in Table 2. The % DOC removal for each indicated SCAS unit and cycle are summarized in Table3.The data for % DOC are also shown in a bar graph in Figures 1 through 7 showing first the three individual constituents of DBE, DMA (CAS 627-93-0), DMG (CAS 1119-40-0), and DMS (CAS 106-65-0), then in Figure 4 Dibasic Esters (DBE,. CAS 95481-62-2, attached as illustration, called in the study report DME), and finally combinations of two of the constituents (i.e. DMA and DMG) in different ratios, going by the names of DME-2, and DME-3).

Results with reference substance:

100% DOC removal at day 4, see Table 3, plateau reached between day 2 and 3.

Table 1: pH measurements

Weeks of exposure	pH
	control	DMA	DMG	DMS	DME	DME-2	DME-3	4-Nitrophenol^a
1	8.73	8.53	8.61	8.67	8.54	8.62	8.75	8.69
2	8.70	8.55	8.56	8.65	8.59	8.66	8.71	8.54
3	8.75	8.52	8.54	8.69	8.61	8.65	8.78	8.57
5	8.55	8.59	8.49	8.55	8.56	8.47	8.51	8.55

Table 2: Total Suspended Solids

TSS [mg/L] of SCAS unit for week 1
control	DMA	DMG	DMS	DME	DME-2	DME-3	4-Nitrophenol^a
2720	2400	2760	2440	2400	2740	2600	2240

 

Table 3: Percent Dissolved Organic Carbon removal

Days of exposure	DOC removal [%]
	DMA	DMG	DMS	DME	DME-2	DME-3	4-Nitrophenol^a
	18 mg C/L	15 mg C/L	14 mg C/L	16 mg C/L	15 mg C/L	16 mg C/L	20 mg C/L
1	106	70	67	67	100	91	23
2	104	102	97	96	101	102	93
3	100	100	99	95	100	100	96
4	101	100	96	97	100	101	100
5	108	98	101	102	105	105	103
6	104	104	100	100		103	101
7	102	102	102	99	103	105	102
8	98	97	101	103	107	106	106
9	106	110	109	109	114	110	111
10	102	106	104	103	106	108	103
12	101	91	93	104	101	106	103
13	101	105	104	102	99	110	101
14	100	105	110	105	102	108	102
15	102	105	107	105	105	106	102
16	102	103	99	102	104	107	102
17	104	104	106	102	102	108	101
18	99	101	107	99	101	106	102
19	97	95	100	98	97	106	99
20	103	102	105	105	104	105	101
21	101	103	99	99	100	103	99
22	103	101	102	104	102	95	103
23	104	105	103	102	102	106	105
24	104	103	107	103	106	101	104
25	105	108	116	107	110	102	106
26	97	99	105	101	101	105	101
27	100	103	102	96	102	103	99
28	103	105	106	103	103	103	103
29	103	103	104	103	103	103	102
Average for days 4 - 29	102	102	104	102	103	104	102

^apositive control with readily biodegradable reference substance

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Under the conditions of the test DBE and its single constituents were found inherently biodegradable.

Executive summary:

Biodegradation screening of Dibasic Esters (DBE), called in the study report DME, its three constituents DMA (CAS 627-93-0), DMG (CAS 1119-40-0), and DMS (CAS 106-65-0), and combinations of two to them (i.e. DMA and DMG) in different ratios, DME-2, and DME-3, were carried out using a 29 day semi-continuous activated sludge (SCAS) test. The study was performed according to OECD TDG 302A and 40 CFR 796.3340 standards under GLP conditions and is considered valid and conclusive.

The samples were tested at a dose level of about 15 mg C/L giving 102, 102, 102, 104, 103, and 104 % DOC removals for DBE, DMA, DMG, DMS, DME-2, and DME-3, respectively. These high DOC removals indicate fast mineralization of DBE as a mixture and of the individual constituents. These materials meets the OECD TGD 302A and the EPA (40 CFR 796.3340) criteria for ultimate biodegradability of greater than 70 % DOC removal.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1990-10-23 to 1990-11-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This IUCLID for dimethyl adipate is compiled using data from the substance itself, a structurally related compound (dimethyl glutarate) and a mixture of dibasic esters (dimethyl adipate, succinate and glutarate). The toxicity of each of these substances is similar and the document attached justifies why data on the category members can be used to support the data gaps for dimethyl adipate.

Qualifier:

according to guideline

Guideline:

ISO 7827 (Evaluation in an Aqueous Medium of the "Ultimate" Aerobic Biodegradability of Organic Compounds - Method by Analysis of Dissolved Organic Carbon (DOC))

Qualifier:

equivalent or similar to guideline

Guideline:

other: AFNOR T90-312 (Nov 1985)

Deviations:

yes

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Inflow water from the municipal sewage water treatment plant of St Germain au Mont D'Or, France
- Storage conditions: The inoculum was used within one day after sampling.
- Preparation of inoculum for exposure: The inoculum was filtered and centrifuged. The precipitate was suspended in ringer solution 1/4.
- Concentration of sludge: The inoculum suspension represtented 1% of the assay medium.
- Initial cell/biomass concentration: 10^5 to 10^6 cells/mL measured by optical density
- Water filtered: yes
- Type and size of filter used, if any: fiber glass

Duration of test (contact time):

29 d

Initial conc.:

40 mg/L

Based on:

DOC

Initial conc.:

105.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation:

inorg. C analysis

Parameter followed for biodegradation estimation:

TOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: Bi-distilled water was used for the preparation of solutions. Its DOC was less < 4 mg/kg (mg C/L)
KH2PO4: 85.000 mg
K2HPO4: 217.500 mg
NH4Cl: 25.000 mg
MgSO4 * 7 H2O: 22.5 00mg
CaCl2 * 2 H2O:36.400 mg
FeCl3 * 6 H2O: 0.250 mg
MnSO4 * H2O: 0.030 mg
H3BO3: 0.057 mg
ZnSO4 * 7 H2O: 0.043 mg
(NH4)6Mo7O24 * 4 H2O: 0.036 mg
C10H12N2O8Fe(III)Na: 0.100 mg
Double distilled water: ad 1000 g

TEST SYSTEM

SAMPLING
- Sampling frequency: days 4, 11, 18, 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: FB1 and FB2: Assay with inoculum only (blind control)
- Abiotic sterile control: FA: Assay with test item only (abiotic biodegradation control)
- Toxicity control: FI: Assay with test item, reference susbstance and inoculum
- Other:
FC: Assay with reference substance of known biodegradability and inoculum (activity control)
FE1 and FE2: Assay with test item and inoculum (2 replicates)

Reference substance:

acetic acid, sodium salt

Remarks:

PROLABO lot 83059

Parameter:

% degradation (DOC removal)

Value:

97

Sampling time:

28 d

Remarks on result:

other: plateau reached after 4 days

Parameter:

% degradation (DOC removal)

Value:

100

Sampling time:

4 d

Results with reference substance:

FC (positive control)
% biodegradation measured (on day)
0% (0)
100% (4)
93%(11)
98% (18)
100% (29)

The percentage of biodegradation in the activity control (FC) was ≥ 35% at day 7

Table 1: Biodegradation kinetics

Time [days]	FE1 (Assay 1)	FE2 (Assay 2)	FC Reference	FI (Inhibition control)	FA (Sterile assay)
0	0	0	0	0	0
4	100	100	100	93	0
11	94	93	93	94	0
18	97	98	98	96	0
29	96	99	100	98	0

 

Table 2: Organic Carbon Levels

Time [days]		FB1 (Empty 1)	FB2 (Empty 2)	FE1 (Assay 1)	FE2 (Assay 2)	FC (Reference)	FI (Inhibition control)	FA (Sterile assay)
0	CT	-0.04	0.19	44.47	47.15	40.49	80.48	41.12
	CM	3.47	1.74	2.25	2.53	3.63	2.75	1.82
	CO	0.00	0.00	42.23	44.62	36.87	77.72	39.30
	%
4	CT	3.13	6.33	4.21	4.65	13.30	15.72	55.09
	CM	2.27	2.69	1.95	2.30	11.41	8.14	0.63
	CO	0.85	3.64	2.25	2.36	1.89	7.58	54.46
	%			99.99	99.76	100.97	93.14	-32.86
11	CT	0.87	0.69	4.46	4.56	13.09	15.39	56.02
	CM	1.77	1.80	2.13	1.59	10.60	10.60	1.11
	CO	0.00	0.00	2.33	2.97	2.49	4.79	54.90
	%			94.48	93.34	93.24	93.84	-39.71
18	CT	0.50	0.42	4.22	3.59	13.20	14.93	53.09
	CM	1.80	1.88	2.88	2.91	12.47	12.08	1.57
	CO	0.00	0.00	1.34	0.68	0.73	2.85	51.52
	%			96.82	98.47	98.03	96.33	-31.09
29	CT	1.51	1.25	4.53	3.73	6.80	11.26	50.50
	CM	2.45	1.87	3.04	3.07	7.20	9.62	1,66
	CO	0.00	0.00	1.49	0.66	0.00	1.64	48,84
	%			96.48	98.53	100.00	97.88	-24.27

CT = TOC [mg/L]

CM = Inorganic Carbon [mg/L]

CO = DOC [mg/L] = dissolved organic carbon

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item was found 97% biodegradable and the 10-day window was met.

Executive summary:

The biodegradability of the dissolved test item, Dibasic esters (DBE) exposed in the test medium to aerobic micro-organisms as only source of carbon and energy was followed. The test was performed according to AFNOR T90-312 or ISO 7827 under GLP conditions. The study is considered valid and conclusive as described below:

The test item concentration in the test vessel was corresponding to 40 mg C/L. The DOC was measured in intervals allowing following the curve of biodegradation up to 28 days. The curve reached a plateau at day 4. Thus the 10 -day window condition was met. The percentage of biodegradation of the test item DBE is 97% under the conditions of the study. Therefore, the test item is considered as readily biodegradable.

In the abiotic control, containing the test substance and mineral medium only, no elimination was noted after 28 days of exposure (based on DOC measurements). Thus, no abiotic elimination occurred under the test conditions.

The reference substance was more than 80% biodegraded within seven days of exposure, thus confirming the suitability of the activated sludge. In the toxicity control, containing the test substance, the reference substance and activated sludge (inoculum), the biodegradation was > 35% within 7 days. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration.

Description of key information

In a ready biodegradability test, the test item was found 97% biodegradable and the 10-day window was met.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Justification for Read Across

Attached at section 13 is a category justification that explains the rationale for using data on a mixture of Dimethyl Esters (DBE), and Dimethyl Glutarate, to supplement the available data on Dimethyl Adipate.

Three reliable studies on biodegradability in water are available. The first study (Sechaud P., 1990) has been selected as key study as it has been performed according to ISO guideline and the GLP principles. The second study (O'Reilly JV and Trehy ML, 1993) has been performed according to the SCAS method and has been selected as supporting study as the substance fulfilled the criteria for ultimate biodegradability. The third study (Litchfield CD, 1982) performed with a pre-adapted inoculum showed 36% of ultimate biodegradation and half lives of 2.5 and 3.2 days.

In the key study, the biodegradability of the dissolved test item, Dibasic esters (DBE) exposed in the test medium to aerobic micro-organisms as only source of carbon and energy was followed. The test was performed according to AFNOR T90-312 or ISO 7827 under GLP conditions. The test item concentration in the test vessel was corresponding to 40 mg C/L. The DOC was measured in intervals allowing following the curve of biodegradation up to 28 days. The curve reached a plateau at day 4. Thus the 10 -day window condition was met. The percentage of biodegradation of the test item DBE is 97% under the conditions of the study. Therefore, the test item is considered as readily biodegradable.

In the abiotic control, containing the test substance and mineral medium only, no elimination was noted after 28 days of exposure (based on DOC measurements). Thus, no abiotic elimination occurred under the test conditions.

The reference substance was more than 80% biodegraded within seven days of exposure, thus confirming the suitability of the activated sludge.In the toxicity control, containing the test substance, the reference substance and activated sludge (inoculum), the biodegradation was > 35% within 7 days. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration.