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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-06 - 2017-10-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.9 (Biodegradation: Zahn-Wellens Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3200 (Zahn-Wellens / EMPA Test)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss GLP Monitoring Authorities, Date of decision 2015-11-16
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge, predominantly domestic, non-adapted
Details on inoculum:
The study was performed with aerobic activated sewage sludge from the aeration stage of the local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland), which treats predominantly domestic sewage.
No pre-adaptation of the inoculum to the test item was done.

Preparation of Inoculum
The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in tap water and finally in mineral medium. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.
Based on this determination, a calculated amount of wet sludge was suspended in mineral medium to obtain a concentration equivalent to 4 g dry material per liter. During the holding period of three days prior to use, the sludge was aerated at room temperature. Prior to use, the dry weight was determined again. Based on this determination defined amounts of the activated sludge suspension were added to mineral medium to obtain a final concentration of 300 mg dry material per liter. The ratio between inoculum and test item (based on the mean DOC measured) was 3.0:1 (therefore within the requested range of 2.5:1 to 4:1).
Duration of test (contact time):
28 d
Initial conc.:
>= 200.2 - <= 200.6 mg/L
Based on:
test mat.
Initial conc.:
>= 100.1 - <= 101.1 mg/L
Based on:
DOC
Remarks:
measured on Day 0 +3h and corrected for mean inoculum control
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
See also section below "Any other information on materials and methods incl. tables"

TEST CONDITIONS
- Composition of medium: according to guideline
- Additional substrate: no
- Solubilising agent (type and concentration if used): not used
- Test temperature: 22 °C
- pH: start 7.4 (adjusted from 9.9); end 6.9-7.1, see Table 3
- pH adjusted: yes
- Aeration of dilution water: by orbital platform shaker at 130 rpm
- Aerobic condition, i.e dissolved oxygen concentration: 7.9-8.4 mg O2/L, see Table 4
- Suspended solids concentration: 300 mg/L
- Continuous darkness: diffuse light

TEST SYSTEM
- Culturing apparatus: 2000 mL Erlenmeyer glass vessels, cleaned with alcoholic hydrochloric acid, rinsed with ultrapure water and dried. The test vessels were filled to a volume of 1000 mL. Each vessel was loosely covered with an aluminum cap to reduce losses by evaporation. The test vessels were incubated in an incubator (i.e. Multitron Pro from Infors AG, Bottmingen, Switzerland) with automatic temperature control and equipped with an orbital platform shaker.
- Number of culture flasks/concentration: 2 for Test item, 2 for Procedure control, 2 for Inoculum control and 1 for Toxicity control.
- Method used to create aerobic conditions: orbital platform shaker, at approximately 130 rpm.
- Measuring equipment: TOC infrared gas analyzer equipped with an automatic sampler (i.e. vario TOC cube from Elementar Analysensysteme GmbH, Langenselbold, Germany)

SAMPLING
- Sampling frequency: Test item and inoculum control: Exposure Day 0 (0 and 3 hours after the addition of the test chemical), 4, 7, 11, 14, 21, 27 and 28. Procedure control and toxicity control: Exposure Day 0 (0 and 3 hours), 4, 7, 14, 27 and 28.
- Sampling method: For the DOC analyses one sample of approximately 10 mL was taken from each sampled test vessel per sampling occassion. Prior to sampling, water evaporation losses were determined by weighing the vessels and were compensated by adding purified water. Deposits on the test vessels were resuspended. Samples were filtered through a 0.45 µm filter. The first 2-3 mL of the filtrate were discarded. Thereafter, the samples were immediately analyzed for DOC.
- Sample storage before analysis: direct analysis

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Abiotic sterile control: no
- Toxicity control: 1 replicate
- Procedure control (reference substance): 2 replicates

Reference substance:
diethylene glycol
Preliminary study:
Solubility Pre-Experiment
Prior to test start, the solubility of the test item in mineral medium (approximately at the concentration expected to be used in the test) was checked, and the test item was found to be soluble at a concentration of 184.5 mg/L. The measured dissolved organic carbon content (DOC) of this solution was 0.500 mg C/mg test item.
Test performance:
No unusual observation.
Key result
Parameter:
% degradation (DOC removal)
Remarks:
mean
Value:
6
Sampling time:
28 d
Remarks on result:
other: The test item was found to be not biodegradable (6 %) after 28 days of exposure to activated sludge under the conditions of the conducted test.
Details on results:
In the test vessels containing the test item JEFFAMINE® EDR-176 in inoculated mineral medium, the mean concentration of dissolved organic carbon (DOC) measured on Day 0 at 0 h was 101 mg/L. The mean DOC concentration was 101 mg/L after 3 h of incubation, indicating that no adsorption of the test item to the sludge occurred in this time.

According to the guidelines the DOC values measured on Day 0 after 3 h of incubation are considered the starting concentration of DOC and are used for the calculation of the biodegradation.

The mean concentration of DOC varied between 94 and 101 mg/L over the exposure period of 28 days and thus was not significantly different from the initial mean value of 101 mg/L (Day 0 after 3 h). Expressed as percentage DOC removal, mean values in the range from 0 to 6 % were noted. The mean biodegradability of the test item was calculated to be 6 % at the end of the 28-days exposure period.

Consequently, the test item JEFFAMINE® EDR-176 was not biodegradable under the test conditions within 28 days.
Results with reference substance:
In the procedure controls, average biodegradation of the reference item diethylene glycol was 99 % by Exposure Day 14, thus fulfilling the validity criterion (i.e. 70 % degradation by Exposure Day 14).

Toxicity control

In the toxicity control, containing both the test substance (corresponding to 48 % of total added) and the reference item diethylene glycol (corresponding to 52 % of total added) in inoculated mineral medium, the initial concentration of 194 mg/L measured on Day 0 decreased to 96 mg/L on Day 14. Biodegradation amounted to 50 % within 14 days of exposure. By the end of the test (Exposure Day 28), average biodegradation was 52 %.

Thus the test substance was not inhibitory to activated sludge at the tested concentration of 201 mg/L because degradation was >35 % within 14 days.

Measurement of pH and Dissolved Oxygen Concentration

The pH was measured in each test vessel on Day 0, before the addition of the inoculum, and found to be in the range of 7.4 to 9.9. Where necessary, the pH was adjusted to pH 7.4 using a diluted sulfuric acid solution. Further during the test, the pH was measured at each sampling interval. Adjustment of the pH was not necessary during the exposure period.

TABLES

The tabulated values represent rounded results obtained by calculation using the exact raw data.

   

Table 1     Dissolved Organic Carbon (DOC) Concentration Measured in the Test Vessels

 

DOC [mg/L]*

DOC [mg/L]*

DOC [mg/L]*

DOC [mg/L]*

DOC [mg/L]*

DOC [mg/L]*

DOC [mg/L]*

DOC [mg/L]*

DOC [mg/L]*

DOC [mg/L]*

 

Test item

Test item

Test item

Procedure control

Procedure control

Procedure control

Inoculum control

Inoculum control

Inoculum control

Toxicity
control

Time [days]

Replicate No. 1

Replicate No. 2

Mean#

Replicate No. 1

Replicate No. 2

Mean#

Replicate No. 1

Replicate No. 2

Mean#

Replicate No. 1#

 

 

 

 

 

 

 

 

 

 

 

0 (0 h)

102.6

104.1

100.6

98.9

98.4

95.9

2.9

2.7

2.8

195.4

0 (3 h)

102.8

103.8

100.6

98.4

97.7

95.4

2.8

2.6

2.7

194.0

4

102.4

104.3

100.8

90.7

89.5

87.6

2.5

2.6

2.6

189.1

7

102.4

103.2

100.6

23.4

20.8

19.9

1.7

2.8

2.3

101.2

11

100.0

100.7

98.8

--

--

--

1.6

1.6

1.6

--

14

99.9

100.7

98.4

3.6

2.7

1.3

1.9

1.9

1.9

96.3

21

99.1

101.4

98.5

--

--

--

1.8

1.7

1.8

--

27

95.8

98.0

95.1

5.7

3.4

2.7

1.9

1.8

1.9

91.6

28

94.9

97.3

94.2

4.9

3.2

2.2

1.9

1.9

1.9

92.5

*         Mean values of triplicate measurements per sample.

#         Values corrected for the mean inoculum control.

--         Not determined.

 

 

Table 2     Biodegradation of the Test Item JEFFAMINE®EDR-176 and the Reference Item Diethylene glycol

 

Percentage Biodegr.#

Percentage  Biodegr.#

Percentage Biodegr.#

Percentage Biodegr.#

Percentage Biodegr.#

Percentage Biodegr.#

Percentage Biodegr.#

 Time

Test item

Test item

Test item

Reference item

Reference item

Reference item

Toxicity control

[days]

Replicate No. 1

Replicate No. 2

Mean

Replicate No. 1

Replicate No. 2

Mean

Replicate No. 1

0 (3 h)

0

0

0

0

0

0

0

4

0

-1*

0

8

8

8

3

7

0

0

0

78

80

79

48

11

2

2

2

--

--

--

--

14

2

2

2

98

99

99

50

21

3

1

2

--

--

--

--

27

6

5

6

96

98

97

53

28

7

6

6

97

99

98

52

#          Corrected for the inoculum control.

--         Not determined.

*         Negative value due to higher DOC-removal in the inoculum controls than in the test vessels with test item.

 

 

Table 3     pH of the Test Solutions during the Test

 

pH

pH

pH

pH

pH

pH

pH

Time

Test item

Test item

Procedure control

Procedure control

Inoculum control

Inoculum control

Toxicity control

[days]

Replicate1

Replicate 2

Replicate1

Replicate 2

Replicate1

Replicate 2

Replicate 1

 

 

 

 

 

 

 

 

0 (0 h)

9.9 / 7.4*

9.9 / 7.4*

7.4

7.4

7.4

7.4

9.9 / 7.4*

0 (3 h)

7.3

7.3

7.4

7.4

7.4

7.4

7.3

4

7.2

7.2

7.0

7.0

7.2

7.2

7.0

7

7.1

7.1

6.7

6.7

7.1

7.1

6.9

11

7.1

7.1

--

--

7.1

7.1

--

14

7.1

7.1

6.6

6.6

7.0

7.1

7.0

21

7.1

7.1

--

--

7.0

7.0

--

27

7.0

6.9

7.0

7.1

7.1

7.0

6.8

28

6.9

6.9

7.1

7.1

7.1

7.1

6.6

*         Before / after adjustment.

--         Not determined.

 

 

Table 4     Dissolved Oxygen Concentrations of the Test Solutions during the Test

 

Dissolved Oxygen Concentr. [mg O2/L]

Dissolved Oxygen Concentr. [mg O2/L]

Dissolved Oxygen Concentr. [mg O2/L]

Dissolved Oxygen Concentr. [mg O2/L]

Dissolved Oxygen Concentr. [mg O2/L]

Dissolved Oxygen Concentr. [mg O2/L]

Dissolved Oxygen Concentr. [mg O2/L]

Time

Test item

Test item

Procedure control

Procedure control

Inoculum control

Inoculum control

Toxicity control

[days]

Replicate1

Replicate 2

Replicate1

Replicate 2

Replicate1

Replicate 2

Replicate 1

 

 

 

 

 

 

 

 

0 (0 h)

8.3

8.4

8.4

8.4

8.4

8.4

8.4

0 (3 h)

8.3

8.3

8.3

8.3

8.4

8.4

8.3

4

8.4

8.4

8.3

8.3

8.4

8.4

8.3

7

8.4

8.4

8.0

8.0

8.4

8.4

8.3

11

8.3

8.4

--

--

8.3

8.3

--

14

8.4

8.3

7.9

7.9

8.3

8.3

8.2

21

8.2

8.2

--

--

8.1

8.2

--

27

8.2

8.3

8.0

8.0

8.2

8.1

8.2

28

8.4

8.4

8.4

8.4

8.4

8.4

8.4

--         Not determined.

 

Validity criteria fulfilled:
yes
Remarks:
The results are considered to be valid since the DOC of the reference item diethylene glycol in the procedure control was removed by 99% within 14 days (criterion: at least 70% within 14 days):
Interpretation of results:
not inherently biodegradable
Conclusions:
The test item was found to be not biodegradable (6 %) after 28 days of exposure to activated sludge under the conditions of the conducted Zahn-Wellens/EMPA test.
This valid test was performed according to the OECD Guideline for Testing of Chemicals,
No. 302 B (1992), the Part C.9 of Commission Regulation (EC) No 440/2008, and the US EPA OPPTS 835.3200 (1998).
Executive summary:

The test item was investigated for its potential (inherent) ultimate biodegradability in a Zahn-Wellens/EMPA test over 28 days, based on the OECD Guideline for Testing of Chemicals, No. 302 B (1992), Commission Regulation (EC) No 440/2008, Part C.9 and US EPA OPPTS 835.3200 (1998).

 In the test vessels containing the the test substance in inoculated mineral medium the mean concentration of dissolved organic carbon (DOC) varied between 94 and 101 mg/L over the exposure period of 28 days. This was not significantly different from the initial mean DOC concentration of 101 mg/L measured on Day 0 (starting value after 3 hours of exposure). Expressed as percentage DOC removal, mean values in the range from 0 to 6 % were noted. The mean biodegradability of the test item was calculated to be 6 % at the end of the 28-days exposure period.

Consequently, the test item was not biodegradable under the test conditions within 28 days.

No removal was observed during the first three hours of exposure which is an indication that the test item did not adsorb on activated sludge.

In the procedure controls, average biodegradation of the reference item diethylene glycol was 99 % by Exposure Day 14, thus fulfilling the validity criterion (i.e.70 % degradation by Exposure Day 14).

In the toxicity control, containing both the test substance and the reference item diethylene glycol, in inoculated mineral medium the initial DOC decreased by 50 % within 14 days of exposure. Thus, the test substance had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 201 mg/L.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-04-20 - 2005-05-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 590 = JEFFAMINE EDR 176
- Molecular formula: C8H20N2O2
- Physical state: liquid
- Analytical purity: 99%
- Lot/batch No.: 4N501
- Expiration date of the lot/batch: 30 March 2006
- Storage conditions of test material: at room temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge freshly obtained from a municipal sewage treatment plan: Waterschap de Maaskant, 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Storage conditions: under continuous aeration until further treatment
- Storage length: not reported
- Preparation of inoculum for exposure: before use the sludge was allowed to settle during 40 minutes and the liquid was decanted for use as inoculum at the amount of 10 mL/L of mineral medium
- Concentration of sludge: 4.2 g/L suspended solids (information obtained from treatment plant)
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
22 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST SOLUTIONS
- A weighed amount of 999.9 mg of XTJ 590 was dissolved in Milli-RO water and made up to 1000 mL
- Stock was clear and colourless solution
- Aliquots of 44 mL of this stock solution were added to the test medium

TEST CONDITIONS
- Dilution water: tap-water purified by RO (Milli-RO) and passed over activated carbon and ion exchange cartridges (Milli-Q)
- Composition of medium: according to guideline
- After adding test medium (without test substance) and inoculum test solutions were aerated with synthetic air to purge the system of CO2
- Test temperature: continuously monitored in a vessel with water in the test room and varying between 21.8 and 22.7 °C
- pH: adjusted prior to start of test to 7.5-7.6, varying between 7.6 and 8.0 on day 28
- pH adjusted: yes, with 1M HCl
- Aeration of dilution water: yes, with synthetic air and continuous stirring
- Continuous darkness: yes
- Test solutions were continuously stirred during the test to ensure optimal contact between the test substance and the test organisms

TEST SYSTEM
- Culturing apparatus: 2-L all-glass brown coloured bottles
- Number of culture flasks/concentration: 2 bottles, containing test substance and inoculum
- Method used to create aerobic conditions: sparging with air
- Measuring equipment: produced CO2 trapped in Ba(OH)2 bottles was determined by means of titration
- Details of trap for CO2 and volatile organics if used:
* 3 barium hydroxide (0.0125 M Ba(OH)2) bottles (100 mL) connected in series to the exit air line of each test bottle
* CO2 reacts with Ba(OH)2 and precipitates out as barium carbonate
* The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M HCl, phenolphthalein (1% solution in ethanol) was used as pH indicator
* Each sampling occasion the nearest CO2 absorber was removed for titration, the others were moved one position closer to the test bottle and a new one was added at the end
- On day 28 pH was measured and 1 mL of concentrated HCl (37%) was added to each bottle. Bottles were aerated overnight to drive off CO2 present in test suspension. Final titration was made on day 29

SAMPLING
- Sampling frequency: CO2 concentrations were determined on days 2, 5, 7, 14, 19, 23, and 27

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 bottles, containing only inoculum
- Abiotic sterile control: none
- Toxicity control: 1 bottle, containing test substance, reference substance and inoculum
- Positive control: 1 bottle, containig reference substance and inoculum
Reference substance:
acetic acid, sodium salt
Test performance:
- The positive control substance was degraded by at least 60% (68%) within 14 days.
- The difference of duplicate values for % degradation was always < 20.
- The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (23 mg/L).
- In the toxicity control more than 25% degradation occurred within 14 days (37% based on ThCO2), indicating that the test substance does not inhibit microbial activity at the test concentration used.
Parameter:
other: % of ThCO2
Value:
4
Sampling time:
29 d
Remarks on result:
other: mean of 2 bottles
Details on results:
- Theoretical CO2 production (ThCO2) for test item was calculated as 2.00 mg CO2/mg.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In this Modified Sturm test using non-adapted sludge from a municipal waste water treatment plant treating predominantly domestic sewage, the test substance showed only 4% biodegradation (mean of 2 bottles, based on % ThCO2). The test substance did not inhibit microbial activity at the concentration used in the test and the system was demonstrated to be healthy. The test substance is therefore concluded not to be readily biodegradable under the conditions of this test. The results of the test can be considered reliable.
Executive summary:

Determination of "ready Biodegradability": Carbon dioxide (CO2) evolution test (modified Sturm test) with the test substance.

The study procedure was based on EEC directive 92/69,C.4C, December 1992, OECD guideline N§ 301B July 17, 1992 and ISO Standard 9439 (1999).

The theorical CO2 production (ThCO2) of the test substance was calculated to be 2.00 mg CO2/mg.

The test substance was a clear colourless liquid with a purity of 99%. The tets item was tested in duplicate at 44 mg per 2 litres, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula.

The study consisted of 6 bottles:

- 2 blank controls (no test material)

- 2 test bottles (test item 22 mg/L)

- 1 positive control (sodium acetate, 40 mg/l) and

- 1 Toxicity control (test item, 22 mg/l; plus sodium acetate, 40 mg/l)

Since the test item was easily soluble in water the test media were prepared using a stock solution of 1g/l in Milli-RO water. A weighed amount of 999.9 mg of test iem was dissolved in Milli-RO water and made up to 1000 ml. The stock was clear and colourless solution. Aliquots of 44 ml of the stock solution were added to the test medium, containing the microbial organisms, of test substance bottles A and B and the toxicity control. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the etst organisms.

The relative degradaton values calculated from the measurements performed during the test period revealed no significant degradation of the tets item.

In the toxicity control, the ets item 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, the test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Description of key information

In 2005, one Ready biodegradbility test CO2 Evolution test (OECD 301B) was performed to evaluate its ready biodegradability in aerobic aqueous medium with microbial activity by inoculation with the supernatant of activated sludge. The test substance showed only 4% biodegradation (mean of 2 bottles, based on % ThCO2). The test item is therfore not readily biodegradable under the condition of this test.

The test item was investigated for its potential (inherent) ultimate biodegradability in a Zahn-Wellens/EMPA test over 28 days, based on the OECD Guideline for Testing of Chemicals, No. 302 B (1992), Commission Regulation (EC) No 440/2008, Part C.9 and US EPA OPPTS 835.3200 (1998).

The mean biodegradability of the test item was calculated to be 6 % at the end of the 28-days exposure period.

Consequently, the test item was not biodegradable under the test conditions within 28 days.

 In conclusion, the tets item is not readily biodegradable and no inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information