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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-05 to 2018-04-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.9 (Biodegradation: Zahn-Wellens Test)
Version / remarks:
EC No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3200 (Zahn-Wellens / EMPA Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification: Jeffcat LE 30
CAS Number: 65286-55-7
Chemical Name: 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine
Physical state/Appearance: clear colorless liquid
Batch: DR74271215
Purity: 98.2%
Chemical Formula: C13H31N3O2
Label: Jeffcat LE 30 Lot no DR74271215 5,000L
Receipt Date: 25 January 2017
Expiry Date: 30 December 2018
Storage Conditions: room temperature, in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 19 March 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.
Duration of test (contact time):
28 d
Initial conc.:
167 mg/L
Based on:
test mat.
Initial conc.:
100 mg/L
Based on:
IC (inorganic carbon)
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Experimental Design and Study Conduct

Preliminary Investigational Work
During the test, samples are taken for DOC/TOC analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analyzed for DOC/TOC using a Shimadzu TOC-VCPH TOC analyzer.
A nominal amount of test item (261 mg) was dissolved in mineral medium (1000 mL) to give a 261 mg/L stock solution. Two samples were taken for analysis; one untreated (TOC) and one filtered (DOC) through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further nominal amount of test item (261 mg) was dissolved in mineral medium and inoculated at a concentration of 300 mg suspended solids (ss)/L prior to adjusting to a final volume of 1000 mL. Two samples were taken for DOC analysis; one after filtration through a Gelman 0.45 µm AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 300 mg ss/L and then filtering or centrifuging as per the test item samples.
For the inoculum, a mixed population of activated sewage sludge micro-organisms was obtained on 5 March 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Test Item Preparation
The test item was dissolved directly in reverse osmosis purified deionized water prior to dispersal in inoculated mineral medium to give the required test concentration.
A nominal amount of test item (1670 mg) was dissolved in reverse osmosis purified deionized water and the volume adjusted to 1 liter to give a 1670 mg/L stock solution. An aliquot (200 mL) of the 1670 mg/L stock solution was dispersed in inoculated mineral medium and the volume adjusted to 2 liters to give a final test concentration of 167 mg/L, equivalent to 100 mg Carbon/L.
The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the solution.
The control group was maintained under identical conditions but not exposed to the test item.
A test concentration of 100 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item Preparation
A reference item, diethylene glycol, was used to prepare the procedure control vessels. An initial stock solution of 2000 mg/L was prepared by dissolving the reference item directly in reverse osmosis purified deionized water. An aliquot (220 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 2 liters to give a final concentration of 220 mg/L, equivalent to 100 mg Carbon/L. The volumetric flask containing the reference item stock solution was inverted several times to ensure homogeneity of the solution.

Toxicity Control
A toxicity control, containing the test item and diethylene glycol was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An aliquot (200 mL) of the 1670 mg/L test item stock solution was dispersed in inoculated mineral medium along with an aliquot (220 mL) of the 2000 mg/L reference item stock solution. The volume was adjusted to 2 liters to give a final concentration of 167 mg test item/L, plus 220 mg diethylene glycol/L, equivalent to a final concentration of 200 mg Carbon/L.

Preparation of Test System
The following test solutions were prepared and inoculated in 3 liter glass test vessels each containing 2 liters of solution:
a) An inoculum control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (diethylene glycol), in duplicate, in inoculated mineral medium to give a final concentration of 100 mg Carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 100 mg Carbon/L.
d) The test item plus diethylene glycol, one replicate, in inoculated mineral medium to give a final concentration of 200 mg Carbon/L to act as a toxicity control.
Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.

Each test vessel was inoculated with the prepared inoculum at a final concentration of 300 mg suspended solids/liter in order to give an inoculum to carbon ratio of 3:1.
The test vessels were covered with polypropylene lids to reduce evaporation and incubated at measured temperatures of between 20 and 25 ”C in darkness for 28 days. The room temperature was measured as being between 20 and 22 ºC during this time. The test vessels were constantly aerated with compressed air via glass tubes and stirred constantly by a magnetic stirrer.


Water Quality Criteria
Immediately prior to sampling the temperature and dissolved oxygen concentration (by means of a Hach HQ40d Flexi handheld meter) of each vessel were recorded. The pH of each vessel was also determined using a Hach HQ40d Flexi handheld meter and adjusted to pH 7 to 8, where necessary, with sodium hydroxide or sulphuric acid solutions.

Reference substance:
diethylene glycol
Remarks:
Physical state/Appearance: Clear colorless liquid Batch: 1665885 Purity: >99% Expiry Date: 09 May 2019 Storage Conditions: room temperature
Preliminary study:
The results obtained from the samples taken for organic carbon analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item
Key result
Parameter:
% degradation (DOC removal)
Value:
36
Sampling time:
28 d
Details on results:
From DOC analysis the test item attained 36% biodegradation after 28 days.
OECD Guideline No. 302B does not give any definitive pass levels for test items, however the “Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3 (2006)” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralization may be regarded as evidence for ultimate inherent biodegradation.
The test item can therefore be considered to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.
The procedure control, diethylene glycol attained 99% biodegradation after 14 days and 100% biodegradation after 28 days, thereby confirming the suitability of the inoculum and culture conditions.
The toxicity control attained 55% biodegradation after 14 days and 70% biodegradation after 28 days, therefore confirming that the test item was not toxic to the sewage treatment micro-organisms used in the study.
The pH values did not fall below 6.98 in any test vessel during the study and were readjusted to pH 7 to 8 where necessary on sampling days. Aerobic conditions in the test vessels were maintained throughout the study as dissolved oxygen concentrations remained at or above
7.24 mg O2/L in all culture vessels.

Table1            Dissolved Organic Carbon (DOC) Values on Each Sampling Occasion

 

Vessel

DOC (mg Carbon/L)

Day

0 Hours

3 Hours

1

7

14

21

27

28

Inoculum Control

R1

2.44

1.92

1.72

3.19

4.35

4.29

3.49

4.04

R2

1.65

1.77

1.70

3.92

4.10

3.75

4.44

3.90

Mean

2.05

1.85

1.71

3.56

4.23

4.02

3.97

3.97

Procedure Control

R1

104.40

105.70

95.66

5.19

5.15

4.78

4.49

4.38

R2

104.00

102.90

93.67

4.36

5.60

4.88

4.52

4.53

Mean

104.20

104.30

94.67

4.78

5.38

4.83

4.51

4.46

Test Item

R1

91.34

98.90

85.48

91.11

92.43

63.48

63.23

64.04

R2

93.89

94.79

86.60

94.87

95.22

65.37

64.48

65.81

Mean

92.62

96.85

86.04

92.99

93.83

64.43

63.86

64.93

Toxicity Control

200.60

198.10

179.30

93.75

93.25

63.69

61.39

63.68

R= Replicate

Table2            Percentage Biodegradation 

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

1

9

11

10

7

99

6

54

14

99

6

55

21

99

36

70

27

99

37

71

28

100

36

70


Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
The test item attained 36% biodegradation after 28 days.
OECD Guideline No. 302B does not give any definitive pass levels for test items, however the “Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3 (2006)” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralization may be regarded as evidence for ultimate inherent biodegradation.
The test item can therefore be considered to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.

Executive summary:

The study was performed to assess the inherent biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 302B, “Inherent Biodegradability; Modified Zahn-Wellens/EMPA Test”, Method C.9 of Commission Regulation (EC) No. 440/2008, and the US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3200.

 Methods

The test item was exposed to activated sewage sludge micro-organisms at a concentration of 100 mg carbon/L with mineral medium in the dark at measured temperatures of between 20 and 25ºC for 28 days. The biodegradation of the test item was assessed by the determination of dissolved organic carbon removal. Control cultures with inoculum and the reference item, diethylene glycol, together with a toxicity control were used for validation purposes.

 Results

The test item attained 36% biodegradation after 28 days.

OECD Guideline No. 302B does not give any definitive pass levels for test items, however the “Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3 (2006)” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralization may be regarded as evidence for ultimate inherent biodegradation.

The test item can therefore be considered to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.

Description of key information

One study (Envigo, 2018) is included in this dossier and regarded as a key study (Klimisch score of 1). The biodegradability of the test substance was determined according to OECD 302B, EU Method C.9 and EPAOPPTS 835.3110. Based on DOC analysis the test item attained 6% biodegradation after 14 days and 36% biodegradation after 28 days exposure time. Between days 21 and 28 the biodegradation rate reached a plateau. OECD Guideline No. 302B does not give any definitive pass levels for test items, however the “Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3 (2006)” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralization may be regarded as evidence for ultimate inherent biodegradation. The test item can therefore be considered to have exhibited no evidence of ultimate biodegradation under the experimental conditions employed in this study.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information