Diundecyl phthalate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Test material was first evaluated for primary biodegradation in river water and also was studied with activated sludge using the Soap and Detergent Association 24-h semicontinuous procedure (Saeger, V. W., and E. S. Tucker. 1973) and modified feed (Soap and Detergent Association. 1969) in magnetically stirred glass vessels of 1.5-liter capacity.
- Short description of test conditions: see below
- Parameters analysed / observed: see below

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Activated sludge: Domestic sewage was obtained from a local treatment plant.

- Preparation of inoculum for exposure:
Activated sludge: Soap and Detergent Association 24-h semicontinuous procedure (Saeger, V. W., and E. S. Tucker. 1973).

Duration of test (contact time):

24 h

Initial conc.:

3.3 mg/L

Based on:

test mat.

Initial conc.:

13.3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Test temperature: room temperature (river water)
- pH: 7.5 (river water)
- pH adjusted: no
- Continuous darkness: yes
- Volume of test solution in flask, V: 1.5 l (activated sludge)

TEST SYSTEM
- Number of culture flasks/concentration:
Activated sludge: Magnetically stirred glass vessels of 1.5-liter capacity. Concentrations: 5 and 20 mg of DUP/24 h.


SAMPLING
- Sampling frequency and method:
Activated sludge: 50-ml samples of activated sludge mixed liquor were withdrawn after feeding and after 24 h of exposure.


CONTROL AND BLANK SYSTEM
Activated sludge:
-blank control: synthetic sewage medium without the addition of test compound.
- Reference controls: monobutyl phthalate (MBP), phthalic acid (PA), LAS and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl) ethane (p,p'-DDT).
MBP was tested at addition rate of 100 mg/24 h. Method of analysis: UV
PA was tested at addition rates of 50, 100 and 250 mg/24 h. Method of analysis: UV
LAS was tested at addition rate of 20 mg/24 h. Method of analysis: Colorimetric.
p,p'-DDT was tested at addition rate of 1 mg/24 h. Method of analysis: electron-capture gas chromatography.

Reference substance:

other: monobutyl phthalate (MBP)

Reference substance:

other: phthalic acid (PA)

Reference substance:

other: 1-phenyl dodecane-p-sulfonate sodium salt (dodecene-1 derived reference LAS 2)

Reference substance:

other: 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl) ethane (p,p'-DDT)

Key result

Parameter:

% degradation (test mat. analysis)

Value:

45

St. dev.:

11

Sampling time:

24 h

Remarks on result:

other: Addition rate 5 mg/24 h

Key result

Parameter:

% degradation (test mat. analysis)

Value:

29

St. dev.:

7

Sampling time:

24 h

Remarks on result:

other: Addition rate 20 mg/24 h

Details on results:

No significant effect on the suspended solids level was observed in the activated sludge test.

To verify that the disappearance of DUP was not due to volatilization, the off-gases from the test system were passed through a train of three hexane scrubbers during a complete cycle. No significant (<0.5%/cycle) volatility losses were observed.

Results with reference substance:

MBP: >99% at 100 mg/24 h,
PA: >99% at 50, 100 and 250 mg/24 h,
LAS: >99% at 20 mg/24 h,
p,p'-DDT: -7% ± 16 at 1 mg/24 h.

Validity criteria fulfilled:

not applicable

Interpretation of results:

inherently biodegradable

Conclusions:

Under experimental conditions DUP underwent rapid primary biodegradation in acclimated activated sludge.

Executive summary:

Primary biodegradability of DUP was assessed in acclimated activated sludge. The test material was studied with activated sludge using the Soap and Detergent Association 24-h semicontinuous procedure (Saeger, V. W., and E. S. Tucker. 1973) and modified feed (Soap and Detergent Association. 1969). Reference substances monobutyl phthalate (MBP), phthalic acid (PA), 1-phenyl dodecane-p-sulfonate sodium salt (dodecene-1 derived reference LAS 2) and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl) ethane (p,p'-DDT) were used for the activated sludge test. Also blank and sterile controls were run along with the assays. DUP was tested at additional rates of 5 and 20 mg/24 h in the activated sludge test. Samples of DUP were analysed by gas chromatography equipped with dual-flame ionization detector. In the semicontinuous activated sludge unit DUP showed degradation rates of 45% and 29% at addition rates of 5 and 20 mg/cycle, respectively. However, no significant effect on the suspended solids level was observed. The results of this study demonstrate that DUP undergoes rapid primary biodegradation in acclimated activated sludge.

Reference 2

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Test material was first evaluated for primary biodegradation in river water and also was studied with activated sludge using the Soap and Detergent Association 24-h semicontinuous procedure (Saeger, V. W., and E. S. Tucker. 1973) and modified feed (Soap and Detergent Association. 1969) in magnetically stirred glass vessels of 1.5-liter capacity.
- Short description of test conditions: see below
- Parameters analysed / observed: see below

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

(Unacclimated surface water (river water)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
River Water: obtained from the Mississippi River (St. Louis waterfront).

- Preparation of inoculum for exposure:
River water: 6-gallon (ca. 22.7-liter) supply of water was settled for 2 days.

Duration of test (contact time):

5 wk

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Test temperature: room temperature (river water)
- pH: 7.5 (river water)
- pH adjusted: no
- Continuous darkness: yes
- Volume of test solution in flask, V: 200 mL (river water)

TEST SYSTEM
- Number of culture flasks/concentration:
River water: After settling for 2 days, 200-ml portions of supernatant were withdrawn and added to 16-ounce (ca. 0.47-liter) screw-cap bottles. Four microliter portions of DUP ethanol solution (50 µg of DUP per µl) were injected into each bottle with a 10-µl Hamilton syringe (701 RN). Each bottle was sealed with a foil-lined cap, mixed by swirling, and stored in the dark at room temperature.


SAMPLING
- Sampling frequency and method:
River water: the total contents of a river water and the corresponding sterile river water bottle were periodically analyzed for residual ester. After carefully transferring the 200-ml water sample to a 250-ml separatory funnel, the bottle and sample were extracted with three successive 25-ml aliquots of hexane.


CONTROL AND BLANK SYSTEM
River water:
- Sterile control: included to verify that a decrease in the initial 1-mg/liter DUP level was due to biodegradation and not to some other physical or chemical factor.
- Reference control: 1-phenyl dodecane-p-sulfonate sodium salt (dodecene-1 derived reference LAS 2). River water and sterile river water samples were prepared with 4 mg of LAS per liter as an internal monitor of the biological activity. The LAS concentration was monitored in these samples via the standard methylene blue chloroform extraction method (American Public Health Association. 1971).

Reference substance:

other: 1-phenyl dodecane-p-sulfonate sodium salt (dodecene-1 derived reference LAS 2)

Key result

Parameter:

% degradation (test mat. analysis)

Value:

80

Sampling time:

5 wk

Remarks on result:

other: Complete evolution of degradation showed graphically below.

Details on results:

Remaining test material in primary biodegradation test with river water after 5 weeks: 20%.

To verify that the disappearance of DUP was not due to volatilization, the off-gases from the test system were passed through a train of three hexane scrubbers during a complete cycle. No significant (<0.5%/cycle) volatility losses were observed.

Validity criteria fulfilled:

not applicable

Interpretation of results:

inherently biodegradable

Conclusions:

Under experimental conditions DUP underwent rapid primary biodegradation in unacclimated river water.

Executive summary:

Primary biodegradability of DUP was assessed in unacclimated river water. The test material was evaluated for primary biodegradation in river water sample obtained from the Mississippi River (St. Louis waterfront) under room temperature and non-adjusted pH for a 5-weeks period. Reference substance 1-phenyl dodecane-p-sulfonate sodium salt (dodecene-1 derived reference LAS 2) was used for the river water test. Also blank and sterile controls were run along with the assay. DUP was tested at 1 mg/L in the river water sample. Samples of DUP were analysed by gas chromatography equipped with dual-flame ionization detector. DUP underwent reasonably rapid degradation in river water. No significant effect on the suspended solids level was observed. The results of this study demonstrate that DUP undergoes rapid primary biodegradation in unacclimated river water.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

(inoculum was pre-adapted to the test substance)

Qualifier:

according to guideline

Guideline:

other: Chemical fate test guidelines. EPA 560/6-82-003

Deviations:

not specified

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

other: Mixture of organically rich fresh soil, activated sludge (domestic) and raw domestic influent sewage

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The organically rich soil was collected just upland from a freshwater marsh in Berry Park, Syracuse, N.Y. Soil was obtained from a depth of ca. 10 cm and screened through a seive with 2-mm openings. Mixed liquor and raw influent sewage were obtained from the Meadowbrook-Limestone Treatment Plant, Fayetteville, N.Y. This facility treats only domestic wastes. Soil and sewage samples were refrigerated at 4°C and used within 48 h of collection.

- Preparation of inoculum for exposure: The acclimation medium was prepared by adding 1.0 g of organically rich fresh soil, 2.0 ml of fresh aerated mixed liquor obtained from an activated sludge treatment plant, and 50 ml of raw domestic influent sewage with 1 liter of mineral salts medium prepared similarly to that used by Gledhill (Gledhill, W. 1975). This medium was mixed for 15 min and filtered through glass wool. The filtrate was supplemented with 25 mg each of vitamin-free Casamino Acids and yeast extract (Difco Laboratories, Detroit, Mich.) per liter.
1 litre of acclimation medium in a 2-liter Erlenmeyer flask was inoculated with DUP concentration equivalent to 4 mg of carbon at the start of acclimation. The acclimation flasks were sealed and incubated in the dark on a Gyrotory shaker at 120 rpm and 22 ± 2ºC. An additional test compound equivalent to 8 mg of carbon was added on day 7 and again on day 11 during the 14-day acclimation period. At the end of the acclimation period, the contents of all of the acclimation flasks in a set (six test compounds included DUP) were pooled and filtered through glass wool to provide a common inoculum for the primary and ultimate biodegradation tests.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral salts medium was prepared by adding 1 ml each of solution 1 (NH4Cl, 35 g/liter; KNO3, 15 g/liter; K2HPO4 31420, 75 g/liter; NaH2PO4 * H2O, 25 g/liter), solution 2 (KCl, 10 g/liter; MgSO4, 20 g/liter; FeSO4 * 7H2O, 1 g/liter), and solution 3 (CaCl2, 5 g/liter; ZnCl2, 0.05 g/liter; MnCl2 * 4H20, 0.5 g/liter; CuCl2, 0.05 g/liter; CoCl2, 0.001 g/liter; H3BO3, 0.001 g/liter; MoO3, 0.0004 g/liter) to 1 liter of aerated distilled water.
- Test temperature: 22 ± 2ºC
- pH: 7.0 ± 0.2
- pH adjusted: no
- Suspended solids concentration:
- Continuous darkness: yes
- Volume of test solution in flask, V:

TEST SYSTEM
- Number of culture flasks/concentration: Four 2-liter Erlenmeyer flasks for DUP analysis and three 2-liter CO2 evolution flasks (Ace Glass Co., Vineland, N.J.) were used. On the day before beginning 900 ml of distilled water was added to each of the test flasks, which were then supplemented with 1 ml each of solutions 1, 2, and 3 (see composition above). To begin the tests, a measured weight (nominal 20 mg) of test compound and 100 ml of the pooled acclimation inoculum were added to the seven flasks.

- Measuring equipment: three 2-liter CO2 evolution flasks were equipped with a suspended reservoir containing barium hydroxide solution.

SAMPLING
- Sampling frequency and method: The contents of two of the Erlenmeyer flasks were immediately extracted for specific chemical analysis at time zero to determine the percent recovery.
Periodically during the test, base was removed by syringe from the suspended reservoirs for analysis. The appropriate sampling schedule was dictated by the rate of CO2 evolution, which was qualitatively judged from the amount of BaCO3 precipitate present in the absorber.
After sampling, the flasks were sparged for 5 min, and fresh base was added. DUP analysis was conducted on the contents of the remaining duplicate Erlenmeyer flasks at the time when the CO2 evolution results indicated that at least 50% ultimate biodegradation may have occurred. On day 28, the medium was acidified to pH 3 with 20% H2SO4 to convert residual carbonates to CO2. Base was removed for analysis on the next day, and the entire contents of the three CO2 evolution flasks were extracted for specific chemical analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: One 2-liter CO2 evolution flask with the content mentioned above (Test system) but without test compound.
- Reference control: One 2-liter CO2 evolution flask with the content mentioned above (Test system) but with 20 mg reference item instead of test compound.

STATISTICAL METHODS:
Primary degradation results are expressed as the percentage of initial DUP which was lost during the test period. Means and standard deviations were calculated when possible (i.e., when <99% primary degradation had occurred).

Ultimate biodegradation results are expressed as the percentage of theoretical CO2 evolution in each flask. Means, standard deviations, and 95% confidence intervals were also calculated. The CO2 evolution data from each flask were used to calculate an ultimate biodegradation rate constant by nonlinear regression analysis which fit the data to the model suggested by Larson (Larson, R. J. 1979). This analysis was conducted by a computer-based iterative procedure described previously (Werstiuk, N. H., and S. Banerjee. 1981). The individual rate constants calculated for each of the triplicate runs were used to calculate a mean rate constant and its standard deviation. The difference between mean values was determined by a comparison of the 95% confidence intervals. Mean values were judged to be statistically different if their 95% confidence intervals did not overlap.

Reference substance:

other: Glucose

Key result

Parameter:

% degradation (CO2 evolution)

Value:

76

St. dev.:

2

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Value:

68

St. dev.:

3

Sampling time:

21 d

Parameter:

% degradation (CO2 evolution)

Value:

57

St. dev.:

4

Sampling time:

14 d

Parameter:

% degradation (CO2 evolution)

Value:

42

St. dev.:

5

Sampling time:

9 d

Parameter:

% degradation (CO2 evolution)

Value:

30

St. dev.:

2

Sampling time:

7 d

Parameter:

% degradation (CO2 evolution)

Value:

3

St. dev.:

1

Sampling time:

3 d

Details on results:

Lag phase was determined to be 2.7 days(SD = 0.2)
Rate constant was determined to be 0.115 days-1 (SD = 0.024)
Half life was determined to be 6.17 days (SD = 1.17)
Primary biodegradation at day 28: >99%.

Results with reference substance:

Reference item reached biodegradation (based on CO2 evolution) of 92% (SD =15).
Lag phase was determined to be 0.35 days(SD = 0.70)
Rate constant was determined to be 0.227 days-1 (SD = 0.038)
Half life was determined to be 3.11 days (SD = 0.50)

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

Under experimental conditions used in a CO2 evolution test, the ultimate biodegradation of DUP attained 76% in 28 days. Thus, the substance is regarded, as a minimum, as inherently biodegradable and can be regarded as readily biodegradable even though failing the 10 -day window criterium.

Executive summary:

An acclimated shake flask CO2 evolution test similar to OECD TG 301B was used to study the biodegradability of 14 commercial phthalate esters (PAEs) including diundecyl phthalate (DUP). Both CO2 evolution (ultimate biodegradation) and loss of parent PAEs (primary biodegradation) were measured during a 28-day period. The inoculum was prepared from soil, sewage microorganisms, and test compound (set of six PAEs) in a 2-week acclimation period before the test initiation. 20 mg of DUP were added to four 2-liter Erlenmeyer flasks for DUP analysis (GC with FID) and three 2-liter CO2 evolution flasks (equipped with a suspended reservoir containing barium hydroxide solution), containing the inoculated medium, and the flasks were incubated in the dark with shaking. Furthermore, two 2-liter CO2 evolution flask were used in the same conditions for blank control (without test compound) and reference control (20 mg glucose).The ultimate biodegradation of DUP attained 76% (% of ThCO2). Primary biodegradation was >99% (% degraded). Thus, the substance is regarded, as a minimum, as inherently biodegradable and can be regarded as readily biodegradable even though failing the 10 -day window criterium.

Description of key information

Key study. Test method similar to OECD Guideline 301 B. The ultimate biodegradation of DUP attained 76% in 28 days. Thus, the substance is regarded, as a minimum, as inherently biodegradable and can be regarded as readily biodegradable even though failing the 10-day window criterium.

Supporting studies. Data from peer reviewed publication: Under experimental conditions DUP underwent rapid primary biodegradation in both unacclimated river water and acclimated activated sludge.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

Key study: An acclimated shake flask CO2 evolution test similar to OECD TG 301B was used to study the biodegradability of 14 commercial phthalate esters (PAEs) including diundecyl phthalate (DUP). Both CO2 evolution (ultimate biodegradation) and loss of parent PAEs (primary biodegradation) were measured during a 28-day period. The inoculum was prepared from soil, sewage microorganisms, and test compound (set of six PAEs) in a 2-week acclimation period before the test initiation. 20 mg of DUP were added to four 2-liter Erlenmeyer flasks for DUP analysis (GC with FID) and three 2-liter CO2 evolution flasks (equipped with a suspended reservoir containing barium hydroxide solution), containing the inoculated medium, and the flasks were incubated in the dark with shaking. Furthermore, two 2-liter CO2 evolution flask were used in the same conditions for blank control (without test compound) and reference control (20 mg glucose).The ultimate biodegradation of DUP attained 76% (% of ThCO2). Primary biodegradation was >99% (% degraded). Thus, the substance is regarded, as a minimum, as inherently biodegradable and can be regarded as readily biodegradable even though failing the 10 -day window criterium.

Supporting studies: Primary biodegradability of DUP was assessed in both unacclimated river water and acclimated activated sludge. The test material was first evaluated for primary biodegradation in river water sample obtained from the Mississippi River (St. Louis waterfront) under room temperature and non-adjusted pH for a 5-weeks period, and also was studied with activated sludge using the Soap and Detergent Association 24-h semicontinuous procedure (Saeger, V. W., and E. S. Tucker. 1973) and modified feed (Soap and Detergent Association. 1969). Reference substance 1-phenyl dodecane-p-sulfonate sodium salt (dodecene-1 derived reference LAS 2) was used for the river water test. Reference substances monobutyl phthalate (MBP), phthalic acid (PA), LAS and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl) ethane (p,p'-DDT) were used for the activated sludge test. Also blank and sterile controls were run along with the assays. DUP was tested at 1 mg/L in the river water sample and at additional rates of 5 and 20 mg/24 h in the activated sludge test. Samples of DUP were analysed by gas chromatography equipped with dual-flame ionization detector. DUP underwent reasonably rapid degradation in river water. In the semicontinuous activated sludge unit DUP showed degradation rates of 45% and 29% at addition rates of 5 and 20 mg/cycle, respectively. However, no significant effect on the suspended solids level was observed. The results of this study demonstrate that DUP undergoes rapid primary biodegradation in both unacclimated river water and acclimated activated sludge.