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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 5, 1992- December 3, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
The test treatments were conducted using single replicates, guideline stipulates duplicate treatments.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): diethyl ester dimethyl ammonium chloride (E 4607.01)
- Substance type: pure act. ingr. substance
- Physical state: liquid, A 5 % dispersion (E 4607.01) of DEEDMAC was prepared for dosing the test system using the parent test chemical E 4429.01.
- Preparation of Dispersion solution : 5% aqueous dispersion of DEEDMAC made up in 3.7% HCl Solution (pH 2.6)
- Stability under test conditions: yes
- Storage condition of test material: rt

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The sludge was treated with a mechanical blender and allowed to settle for ~30 minutes. The supernatant was decanted from the settled sludge and used as inoculum at the rate of 10 ml/L of mineral media
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
DOC
Initial conc.:
20 mg/L
Based on:
other: measured TOC of stock solution
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
TOC removal
Details on study design:
A 301B screening test was run using the following test treattments:
single replicate of the reference compound, Aniline and a single 10 and 20 mg/L test material treatment (E 4607.01)

3L of media was prepared for each treatment as described in the OECD 301 guideline. The inoculum was added to media at a concentration of 10mlL.THe mixture was aerated with CO2 free air for 24 hr to purge the system of CO2. The media containers were connected to 100 ml BaOH2 trapping train. The train is operated by bubbling CO2 free air through the solutions.

The CO2 collected in the BaOH2 traps was titrated with 0.05 N HCL. At the end of the test, the pH of the media was measured and 1 ml of HCL was added to each flask to drive off inorganic carbonate. After overnight incubation, 100 ml sampels were removed for organic carbon analysis.

Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
76
Sampling time:
28 d
Remarks on result:
other: 10 mg/L conc.
Parameter:
% degradation (CO2 evolution)
Value:
79
Sampling time:
28 d
Remarks on result:
other: 20 mg/L conc
Parameter:
% degradation (TOC removal)
Value:
79
Sampling time:
28 d
Remarks on result:
other: 10 mg/L conc
Parameter:
% degradation (TOC removal)
Value:
81
Sampling time:
28 d
Remarks on result:
other: 20 mg/L conc.
Details on results:
The two test treatments (10 and 20 mg/L) reached > 60% CO2 production and met the 10 day window ready biodegradation criteria. Reaction products of C16-18/C18 unsaturated fatty acid with methyl diethanolamine, MeCl quaternized can be classified as readily biodegradable (see table below).
Results with reference substance:
Aniline (20 mg/L) reached >60% CO2 production by day 11. It passed the current test criteria for a reference substance, > 60% CO2 in < 14 days.

More details on results
Table: Netto CO2 production and % biodegradation of
MDEA-Esterquat C16-18 and C18 unsatd.


Day of test

10 mg/l

20 mg/l

mg CO2 cum.

netto

biodegrad.

%

mg CO2 cum.

netto

biodegrad.

%

1

- 0.42

- 0.6

0.31

0.2

4

0.0

0.0

2.29

1.7

5

5.04

7.5

6.62

5.0

7

15.14

22.6

29.81

22.3

8

24.55

36.7

49.19

36.8

11

36.99

55.3

71.35

53.3

14

40.79

61.0

82.42

61.6

18

45.19

67.6

92.27

69.0

22

47.99

71.8

99.91

74.7

28

50.74

75.9

106.02

79.3

The test chemical treatments were not conducted in replicate, only as a single analysis. Duplicate analysis and the deviation between these replicates is specified in the current form (1992) of the 301B guideline. However, 2 different concentrations were tested with a similar trend in the results which lends further support to the data. All other criteria of the Guideline were met.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
MDEA-Esterquat C16-18 and C18 unsatd. proved to be readily biodegradable (>60% biodegradation after 28 d) in a study conducted in accordance with the OECD TG 301 B, and in compliance with GLP standards.
Executive summary:

The biodegradation of MDEA-Esterquat C16-18 and C18 unsatd. was studied in accordance with the OECD TG 301 B, and in compliance with GLP standards for 28 d. MDEA-Esterquat C16-18 and C18 unsatd. was applied at 2 concentration of 10 and 20 mg/l. CO2 production was analysed at 0, 1, 4, 5, 7, 8, 11, 14, 18, 22, and 28 days of incubation.The two test treatments (10 and 20 mg/L) reached > 60% CO2 production and met the 10 -day window. Therefore MDEA-Esterquat C16-18 and C18 unsatd. fulfills the OECD criteria of ready biodegradability.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 5, 1993 - Dec 14, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed according to standard test procedure, i.e. ECETOC Anaerobic Biodegradation, Technical report number 28 (comparable to OECD 311}
Qualifier:
according to guideline
Guideline:
ECETOC Anaerobic Biodegradation (Technical Report No. 28)
GLP compliance:
yes
Specific details on test material used for the study:
10% dispersion of MDEA-Esterquat C16-18 and C18 unsatd
Oxygen conditions:
anaerobic
Inoculum or test system:
digested sludge
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Anaerobic digester fermenting sludge from a dairy and municipal waste water treatment plant.
- Laboratory culture: not applicable
- Method of cultivation: not applicable
- Storage conditions: 35°C in sealed flask
- Storage length: 10 days
- Preparation of inoculum for exposure: Digester sludge filtered through 10 mesh sieve and post digested in an Erlenmeyer flask at 35°C. After 10 day stabilization, sludge was "washed" to lower TIC. The sludge was centrifuged and the pellet resupsended in mineral salt medium. This was repreasted a second time. The resulting pellet was resuspended in mineral salt medium and diluted to required conc.
- Concentration of sludge: The final level of inoculum in the test is 4.2 g dry matter per liter
- Pretreatment: not applicable
- Initial cell/biomass concentration: 3.15% or 31.5 g/L
- Water filtered: yes/no - not applicable
- Type and size of filter used, if any: not applicable
Duration of test (contact time):
61 d
Initial conc.:
25 other: mg C/L
Based on:
other: theoretical carbon percentages
Initial conc.:
50 other: mg C/ L
Based on:
other: theoretical carbon percentages
Initial conc.:
75 other: mg C/ L
Based on:
other: theoretical carbon percentages
Initial conc.:
100 other: mg C/L
Based on:
other: theoretical carbon percentages
Parameter followed for biodegradation estimation:
CH4 evolution
Parameter followed for biodegradation estimation:
inorg. C analysis
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The test medium used was the same as that described in OECD 311 minus the oxygen indicator.
- Additional substrate: not applicable
- Solubilising agent (type and concentration if used):
- Test temperature: 35°C
- pH: 6.98
- pH adjusted: no
- CEC (meq/100 g): not applicable
- Aeration of dilution water: not applicable
- Suspended solids concentration: 4.2 gr dry matter / Liter
- Continuous darkness: not provided
- Other: Vials incubated in water bath and maintained at 35-36°C. Vials covered in water to maintain headspace temperature of 35°C


TEST SYSTEM
- Culturing apparatus: not applicable
- Number of culture flasks/concentration: six
- Method used to create aerobic conditions: not applicable
- Method used to create anaerobic conditions: not provided
- Measuring equipment: GC used for measurement of gas production
- Test performed in closed vessels due to significant volatility of test substance: No, Test vessels sealed to maintain anaerobic conditions
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: not applicable
- Other:


SAMPLING
- Sampling frequency: day 0, 3, 7, 14, 21 ,28, 42, 60, 61
- Sampling method: Syringe needle attached to flask
- Sterility check if applicable: not applicable
- Sample storage before analysis: not provided
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: no
- Other:


STATISTICAL METHODS:
Reference substance:
other: glucose
Preliminary study:
No inhibitive effects at concentrations 25, 50, 75, and 100 mg/L after 14 days. The 50 and 75 mg/L concentrations were carried forward into the full 60 day test. The 25 and 100 mg/L concentrations were terminated.
Parameter:
other: % Biodegradation (Total inorganic carbon in both gaseous and liquid phase
Value:
90.9
St. dev.:
9.5
Sampling time:
61 d
Remarks on result:
other: 50 mg C/L concentration
Parameter:
other: % Biodegradation (Total inorganic carbon in both gaseous and liquid phases
Value:
89.4
St. dev.:
3.5
Sampling time:
61 d
Remarks on result:
other: 75 mg C/L concentration
Results with reference substance:
The glucose reference substance gave normal results
Validity criteria fulfilled:
yes
Remarks:
reference material reached > 60% biodegradation
Interpretation of results:
other: highly biodegradable under anaerobic conditions
Conclusions:
The test substance E-4708.04 can be defined as "highly biodegradable" under the anaerobic conditions (digestor sludge) of the test.
Executive summary:

The biodegradation of MDEA-Esterquat C16-18 and C18 unsatd.  was studied under anaerobic conditions with anaerobic digester fermenting sludge from a dairy and municipal waste water treatment plant in accordance with the ECETOC Technical report number 28 (comparable to OECD 311), and in compliance with GLP standards for 61 d. MDEA-Esterquat C16-18 and C18 unsatd.  was applied at concentrations of 25, 50, 75, and 100 mg C/l. At test concentrations of 50 and 75 mg C/L the degradation (based on total inorganic carbon in both gaseous and liquid phases) was 90.9% and 89.4%, respectively.

The test substance MDEA-Esterquat C16-18 and C18 unsatd.  can be defined as "highly biodegradable" under the anaerobic conditions of the test.

Description of key information

readily biodegradable under aerobic conditions and fulfilling the 10-day window citeria: > 60% biodegradation based on CO2 (test concentrations: 10 and 20 mg/L)
highly biodegradable" under the anaerobic conditions: 90.9% and 89.4% based on TIC (test concentrations: 50 and 75 mg C/L)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Ready and inherent biodegradability tests

The biodegradation of MDEA-Esterquat C16-18 and C18 unsatd.  was studied in accordance with the OECD TG 301B and in compliance with GLP standards for 28 d. MDEA-Esterquat C16-18 and C18 unsatd.  was applied at 2 concentrations of 10 and 20 mg/L. CO2 production was analysed at day 0 and after 1, 4, 5, 7, 8, 11, 14, 18, 22, and 28 days of incubation. The two test treatments (10 and 20 mg/L) reached > 60% CO2 production and met the 10 -d window. Therefore MDEA-Esterquat C16-18 and C18 unsatd.  fulfills the OECD criteria of ready biodegradability.

Similar results were obtained with the closely related read-across substances MDIPA-Esterquat C16-18 and C18 unsatd. and MDIPA C18 unsatd.:

The biodegradation of MDIPA-Esterquat C16-18 and C18 unsatd. was investigated over a 28-day period in a CO2 Evolution Test according to EC method C.4-C (92/69/EEC) and OECD guideline 301 B. The test medium was inoculated with microorganisms from a digester of a sewage treatment plant mainly fed with municipal wastewater.

The test substance was added from a stock solution made from an aqueous dispersion (5.28% a.i. w/w) provided by the sponsor.The test solutions were aerated by the passage of CO2 free air at a controlled rate in closed flasks at 22 °C for 28 days. The rate of degradation was monitored by measuring the carbon dioxide produced over the 28-d period. The amount of carbon dioxide produced by the microbial population during biodegradation of the test item at a concentration of 20 mg/L, corrected for that derived from the blank inoculum run in parallel, was expressed as a percentage of the nominal DOC loading initially present.

The degradation of MDIPA-Esterquat C16-18 and C18 unsatd. in the static test was found to be 60 % after 28 days. Biodegradation within the 10-day-window was found to be 40 % on the basis of the values obtained by measurement of the trap solutions in the course of the study and calculated to be 50 % assuming that the saturation of CO2 in the media found after acid dissolving at test end is stable after a few days of incubation.

With 40 % after 28 days, a significant abiotic degradation occurred.

No inhibitory effects of the test item were observed (more than 25 % degradation occurred within 14 days) in the toxicity control.

Although the 10-day window criterion is not fulfilled, the test substance can be regarded as readily biodegradable as it is a UVCB substance representing a complex multi-constituent substance with structurally similar constituents.

The biodegradation of MDIPA Esterquat C18 unsatd. (98.8% a.i.)was investigated in a study conducted according to OECD Guideline 301 F, adopted 17th July 1992 (Ready Biodegradability: Manometric Respirometry Test) and EU Method C.4-D, 2008, over a period of 28 days using activated sludge sampled from a sewage treatment plant mainly fed with municipal wastewater as inoculum. The biodegradation rate was determined by measurement of O2 consumption. The test was performed with 24 and 100 mg/L test substance concentration.

With 0%, nitrification of test item originated nitrogen can be neglected. Therefore, mineralization of the test item was evaluated on the basis of ThOD NH3. The biodegradation of the test item after 28 days of incubation in the static test was found to be 60% (SD = 6.0 %) in the assays with 100 mg/L and 74% (SD = 0.0%) in the assays with 24 mg/L based on ThOD NH3. Biodegradation on the basis of ThODNH3 at the end of the 10-day-window was found to be 42% in the assays with 100 mg/L and 60% in the assays with 24 mg/L. The 10-day-window started on day 2 – 3, depending on the concentration. With 0%, there was no abiotic degradation of the test item noticeable within the 28 days of incubation. The biodegradation of the item mixture in the toxicity control was found to be 23% after 14 days of incubation. Thus, the demanded threshold value of 25% is undercut. Nevertheless, due to comparable absolute oxygen demand to the reference assays, and test assays surpassing the threshold value of 60% degradation within a 10-day-window, the test item can be identified as being non-toxic in a ready biodegradability test. In this test, MDIPA Esterquat C18 unsatd. was readily biodegradable.

Anaerobic biodegradation

The anaerobic biodegradation of MDEA-Esterquat C16-18 and C18 unsatd.  was studied during a period of 61 d using anaerobic digester fermenting sludge obtained from a dairy and municipal waste water treatment plant in accordance with the ECETOC Technical report number 28 (comparable to OECD 311), and in compliance with GLP standards. MDEA-Esterquat C16-18 and C18 unsatd.  was applied at concentrations of 25, 50, 75, and 100 mg C/L. At test concentrations of 50 and 75 mg C/L the degradation (based on total inorganic carbon both in gaseous and liquid phase) was 90.9% and 89.4%, respectively. The test substance MDEA-Esterquat C16-18 and C18 unsatd.  can be defined as "highly biodegradable" under the anaerobic conditions of the test.