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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: sewage treatment simulation testing
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
Remarks:
Non-GLP studies conducted to a high standard.
Principles of method if other than guideline:
Effluent monitoring of waste water treatment plants receiving predominantly municipal effluent. Concentration of alcohols and alcohol ethoxylates were measured.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Twenty-four hour composite samples of influent and effluent were collected from each of the locations from three days. They were preserved with formalin at the time of collection. These were composited in proportion to flow.
% Degr.:
>= 98.4 - <= 100
Parameter:
other: BOD removal
Remarks on result:
other: removal within WWTPs (residence time not stated)
Details on results:
Influent (In), effluent (Eff) values in ug/l, and % removal of alcohols are indicated in the table below, with alcohol data considered in two groups. The State in which the WWTP is found is indicated by the usual 2-letter abbreviation.

C12-15 OH C16-18 OH
WWTP type In eff % in eff %
TX Lagoon 297 2 99.3 92.7 2.4 97.4

NJ Oxidation 249 0.7 99.7 181 0.8 99.6
Ditch

OH Rotating 157 0.1 100 77 0.07 99.9
biological
contactor

IA Trickling 499 2.0 99.6 354 2.3 99.4
filter

MO Trickling 532 4.9 99.1 315 9 97.3
filter

KS Lagoon 67.5 1.1 98.4 35.4 2.2 93.8

CA Activated 20.05 0.2 99.9 169 0.4 99.8
sludge

OR Activated 92.9 0.2 99.8 133 0.6 99.5
sludge

AZ Oxidation 702 0.3 100 394 0.5 99.9
ditch

Results for the carbon number groups are considered alongside each other to enable the context of every data point to be seen. In the overall interpretation of the data, the results have been used with those from other studies to determine the contribution of measured alcohol concentrations from various sources.
Validity criteria fulfilled:
not applicable
Conclusions:
A very high level of biodegradation and removal was demonstrated in a study of alcohol concentrations in influent and effluent of several wastewater treatment plants in a reliable study.
Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Good quality study but not conducted to GLP. Published paper based on a more complete unpublished study report.
Qualifier:
according to
Guideline:
OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Initial conc.:
4 mg/L
Based on:
test mat.
Details on study design:
Laboratory continuous activated sludge study.

Concentration: 4 mg/L of TS
Temperature: 20ºC
Hydraulic residence time (HRT) 6 h
Sludge retention time (SRT) 10 d

The feed to the sludge unit was of sterile synthetic sewage and AE concentrate and non-sterile tap water.

19 d acclimation was used, followed by 10 days of evaluation.

At the start the unit was seeded with sewage treatment plant (STP) activated sludge.

The unit was sampled several times per week, and the samples were analysed immediately.


Test performance:
Analytical recovery of the alcohols was high.

The results showed that the CAS unit was running in a similar way to a full scale STP.
% Degr.:
98.6
Parameter:
test mat. analysis
Sampling time:
30 d
Details on results:
Results are corrected for control values.

Alcohol Conc. in Conc. in % removal
effluent ng/L sludge µg/g
C12 18 0.6 98.6
C13 21 0.7 99.5
C14 5.5 0 99.6
C15 2.9 1.1 99.8
C16 1.6 0.01 99.5
C18 58 0.7 99.1
Total 130 2 99.4

Total elimination of alcohols, correcting for control: 97.4% of input
Total alcohols in waste sludge solids 2.0% of input
Total alcohols in suspended solids 0% of input
Total alcohols dissolved in effluent 0.7% of input

This shows that most of that which does not degrade (itself a small amount) is in the solids.
Conclusions:
A very high degree of removal of C12-18 alcohols from a test substance constituting alcohols as part of a mixed alcohol ethoxylate test substance was demonstrated in a 30-day test using a continuous activated sludge simulation methodology. The findings are reliable as part of a weight of evidence.
Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2005-03-30 to 2005-06-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline. It was not compliant with GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD 314B. Deviations, reliability, and validity evaluated against current OECD 314B (Oct. 3, 2008)
Deviations:
no
Principles of method if other than guideline:
Radiolabelled test material was dosed to freshly collected activated sludge in an open test system. Periodically subsamples were collected, lyophilised and extracted. The disappearance of parent and progression of metabolite formation and decay were monitored over time by thin layer chromatogrphy with radioactivity detection. Production of CO2 was determined by comparing total radioactivity in a bioactive treatment compared to that in an abiotic control using liquid scintillation counting (LSC).
GLP compliance:
no
Remarks:
study conducted before 2008
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on source and properties of surface water:
Not applicable
Details on source and properties of sediment:
Not applicable
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge mixed liquor was obtained from Fairfield Wastewater Treatment Plant (Fairfield, OH), which receives predominantly domestic wastewater.

- Storage length: None (radiolabeled test material was dosed to freshly collected activated sludge)

- Preparation of inoculum for exposure: Not reported

- Concentration of sludge: TSS of sludge at the time of collection is not provided in the report. However, the solids level of the mixed liquor suspended solids was adjusted to 2500 mg/L before use.

- Initial cell/biomass concentration: Not reported
Duration of test (contact time):
48 h
Initial conc.:
9.3 µg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
test mat. analysis
other: Biomass... (see attached file)
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: Approx. 1 L

- Composition of medium: Each 2 L test flask contained: 1 L activated sludge, 0.05 µM of dodecanol

- Test temperature: The test flasks were incubated at 20 ±2°C and gently mixed on shaker table.

- pH: 7

- Aeration of dilution water: No

- Suspended solids concentration: 2500 mg/L of TSS

- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 2 L flasks

- Number of culture flasks/concentration: One

- Method used to create aerobic conditions: Test was conducted in open test system.

- Measuring equipment: Mineralization to 14CO2 was determined indirectly by measuring the difference in total radioactivity between samples from the biotic and abiotic treatments.

- Test performed in open system: Yes

SAMPLING
- Sampling frequency: At 0.02, 0.08, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 24, 48, and 144 h

- Sampling method: At each sampling, each sludge mixture was mixed thoroughly and sampled as follows:
a) For determination of mineralization: Triplicate 1 mL samples of the mixed sludge were removed and placed into 20 mL glass scintillation vials containing 1 mL of 0.5% HCI. acidified samples were incubated overnight.
b) For analysis of parent, metabolite and radioactivity associated with solids: 25 mL of sludge was collected from each treatment and transferred to a 35 ml screwtop centrifuge tube and immediately frozen in a dry ice acetone bath. The frozen samples were stored at -80°C until lyophilization on a Virtis bench-top model 3.3L freeze dryer.Analysis of sample is discussed in ‘Details on analytical method’ section.

- Sample storage before analysis: Not specified.

CONTROL AND BLANK SYSTEM
- Inoculum blank: No

- Abiotic sterile control: Yes (Sludge was autoclaved and amended with mercuric chloride (1g/L) to serve as an abiotic control)

- Toxicity control: No

STATISTICAL METHODS: For kinetic analysis, the data describing the disappearance of parent were fitted to various equations using Jandel TableCurve 2D software, Version 4.0. Based on statistical considerations and the visual quality of the fit, the two-compartment first order decay model was used to fit all data.
Reference substance:
not required
Test performance:
No data
Compartment:
other: water, material (mass) balance
% Recovery:
104.8
% Degr.:
73.9
Parameter:
CO2 evolution
Remarks:
(Mineralization)
Sampling time:
48 h
% Degr.:
20.7
Parameter:
other: % of radioactivity associated with solids
Sampling time:
48 h
% Degr.:
0.8
Parameter:
other: % of radioactivity as parent
Sampling time:
48 h
% Degr.:
9.4
Parameter:
other: % of radioactivity as metabolite
Sampling time:
48 h
Compartment:
other: Activated domestic sludge (non adapted)
DT50:
0.37 min
Type:
other: First order
Remarks on result:
other: Half-life of primary degradation
Other kinetic parameters:
first order rate constant
Transformation products:
yes
No.:
#1
No.:
#2
Details on transformation products:
- Formation and decline of each transformation product during test: Dodecanoic acid reached a maximum level of 7.6% within 0.08 h, while more polar metabolites reached their maximum of 31.7% after 0.02 h. Concurrent with the loss of parent, there was the instantaneous appearance of fatty acids and polar metabolites, which peaked and subsequently declined.

- Pathways for transformation: Dodecanol degradation involved two pathways:
* oxidation of the alcohol to a fatty acid, which was beta oxidized to form carbon dioxide, and
* omega oxidation of the methyl group to yield dioic acids, which undergo beta oxidation from either direction.

- Other: The majority of the activity in abiotic control remained as parent, 6.9% of radioactivity was in the form of transformation products, which had chromatographic mobility similar to the two major metabolites observed in the biotic treatments.
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

TRANSFORMATION PRODUCTS: Please refer to the above section 'Details on transformation products'.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: Not reported

EXTRACTABLE RESIDUES
- % of applied amount at Day 0: 59.28% (parent and metabolite; sample time was 0.02 h)

- % of applied amount at end of study period: 10.17% (parent and metabolite; sample time was 48 h)

NON-EXTRACTABLE RESIDUES
- % of applied amount at Day 0: 4.83% in active flask (sample time was 0.02 h)

- % of applied amount at end of study period: 20.67% in active flask (sample time was 48h)

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 73.93% in bioactive test flask (sample time was 48 h). CO2 evolution was not analyzed for abiotic flask as majority of the radioactivity in the abiotic control was recovered primarily as intact parent. For details, please refer to ‘table 1’ and 'table 2'in the ‘Any other information on results incl. tables’ section.

VOLATILIZATION: No volatilization of test material was observed as the TLC analysis of the abiotic control revealed that the parent test material remained intact throughout the experiment.

Continuous activated sludge (CAS) study of Dodecanol (study # 45535)

Table 1: Biotic flask

Sampling time (h)

Parent and less polar metabolite* (%)

Water extracted (polar metabolite)

Associated with solids (%)

CO2 (%)

Mass balance

0.02

27.57

31.71

4.83

29.05

93.15

0.08

24.17

24.56

7.34

28.88

84.94

0.25

18.13

22.38

6.58

35.24

82.33

0.5

15.91

NA

44.65

39.32

99.88

1

15.34

14.38

9.49

37.56

76.78

1.5

11.46

11.47

11.28

40.46

74.67

2

13.76

15.3

8.44

39.99

77.50

3

12.67

10.77

11.74

44.9

80.07

4

10.4

9.63

10.33

45.39

75.76

6

10.48

11.9

9.63

46.69

78.7

24

8.18

4.37

7.86

53.72

74.13

48

6.72

3.45

20.67

73.93

104.77

144

5.99

3.62

26.81

72.09

108.50

* Extracted using methanol as solvent

 

 

Table 2: Abiotic flask

Sampling time (h)

Parent (%)

Metabolite (%)

Water extracted

Associated with solids (%)

CO2 (%)

Mass balance

48

78.4

6.7

0.2

0.6

NA

85.8

 

 

Table 3: Predicted removal of parent dodecanol from activated sludge as a function of the concentration of effluent solids

% Removed as a Function of Effluent Solids

Kd (L/kg)

0 mg/L

5 mg/L

20 mg/L

3002

99.85

99.85

99.84

 

Validity criteria fulfilled:
yes
Conclusions:
A reliable study conducted according to to generally accepted scientific principles determined the substance to achieve 74% mineralization, 20.7% was non-extractable (solids), 9.4% was metabolite, and 0.8% remained as parent after 48 hours. The rate constants for primary biodegradation and mineralisation in activated sludge were 113 and 11 h-1, respectively.
Executive summary:

A simulation of the biodegradation of Dodecanol in activated sludge was conducted under aerobic conditions in accordance with the OECD 314B guideline. A solution of radiolabeled Dodecanol (1-14C) was tested at 9.8 µg/L.  The inoculum was activated sludge obtained from Fairfield Wastewater Treatment Plant (Fairfield, OH), which receives predominantly domestic wastewater.

After 48 h, 74% was mineralized, 20.7% was non-extractable (solids), 9.4% was metabolite, and 0.8% remained as parent. The rate constants for biodegradation of Dodecanol in activated sludge were:

Primary biodegradation: 113 h-1

Mineralization: 11 h-1

This biodegradation simulation test satisfied the guideline requirements for the OECD 314 B simulation tests to assess the biodegradability of chemicals discharged in wastewater.

Description of key information

73.9% degradation over 48 hours.

Primary degradation is rapid with evidence of rates in the range 0.7 - 17 d- ¹ in the dissolved phase.

Key value for chemical safety assessment

Half-life in freshwater:
2.1 d
at the temperature of:
12 °C
Half-life in freshwater sediment:
0.6 d
at the temperature of:
12 °C

Additional information

In accordance with Column 2 of REACH Annex IX, the simulation test on ultimate degradation in surface water and the sediment simulation test (required in Sections 9.2.1.2 and 9.2.1.4 respectively) do not need to be conducted as the substance is readily biodegradable. Identification of degradation products (required in Section 9.2.3) is also not necessary because extensive mineralisation has been demonstrated. Measured degradation in sediment data are available for two analogous longer-chain length alcohols (described below). In view of the lower adsorption coefficient and higher water solubility of dodecan-1-ol compared to these longer chain length linear alcohols, for which simulation tests are available, it can be expected that biodegradation of dodecan-1-ol in similar substrates will be equivalent or higher.

It is notable that significant technical difficulties were encountered during method development for a recent study of adsorption/desorption (OECD 106, Wildlife, 2015) with the analogous substance decan-1-ol (CAS 112-30-1), using natural standard soils, in that it was not possible to detect sufficient substance and establish equilibrium in non-sterilised soil samples. Refer to the Additional information under Section 5.2.3 for a full description of the relevant findings. Half-lives in non-sterilised test soils were in the range approximately 15 minutes to 2 hours. The polar degradation product is most likely the corresponding carboxylic acid, though it was not definitively identified. The chromatograms show that decan-1-ol was effectively fully removed in all four soil types by the 24 h time point (in the case of 2 of the soil types, within 2 hours). Though sediments were not studied in this test, similar instability is to be expected and similar rates of degradation would be anticipated for dodecan-1-ol.

A simulation of the biodegradation of dodecanol in an activated sludge test was conducted under aerobic conditions in accordance with the OECD 314B guideline (Federle, 2005). A solution of radiolabelled dodecanol (1-14C) was tested at 9.3 µg/L. After 48 h, 73.9% was mineralized, 20.7% was non-extractable (solids), 5.9% was metabolite, and 0.8% remained as parent. The rate constants for primary biodegradation and mineralization of dodecanol in activated sludge were 113 h-1 and 11 h-1, respectively.

Discussion of trends in the Category of C6 -24 linear and essentially-linear aliphatic alcohols

Sediment simulation testing

The degradation of C14 linear alcohol in sediments was determined in two studies conducted in accordance with OECD 314 test method and using radiolabelled (14C) test substance. After 92 days, 76.5% mineralisation to CO2was obtained using sediment from Ohio River near Cincinnati, Ohio area (Federle T Wand Itrich N R, 2010a). After 149 days, 83.6% mineralisation to CO2was obtained using Lytle Creek sediments from Wilmington, Ohio (Federle T W and Itrich N R, 2010b).

The degradation of C18 linear alcohol in sediments was determined in a similar study, conducted in accordance with OECD 314 test method and using radiolabelled (14C) test substance (Itrich, 2010). After 60 days, 61.1% mineralisation to CO2was obtained using sediment from Ohio River, and 71.6% mineralisation to CO2after 60 days was obtained using Great Miami River sediments.

The radiochemical analytical results for sediment-associated and aqueous alcohols in these three sediment degradation studies indicatedthat there are two pools of substance, understood to represent the strength of adsorption of the alcohol to sediment particles, which degrade at different rates.

Activated sludge simulation testing

A simulation of the biodegradation of tetradecanol and hexadecanol in an activated sludge test using similar methods to the sediment studies described above, was conducted under aerobic conditions in accordance with the OECD 314B guideline (Federle, 2005).

 

A solution of radiolabelled tetradecanol (1-14C) was tested at 10 µg/L. After 48 h, 76.7% was mineralized, 21% was non-extractable (solids), 6.3% was metabolite, and 1.3% remained as parent. The rate constants for primary biodegradation and mineralization of tetradecanol in activated sludge were 86.5 h-1and 3.4 h-1, respectively.

 

A solution of radiolabelled hexadecanol (1-14C) was tested at 10.7 µg/L. After 48 h, 66.3% was mineralized, 17.1% was non-extractable (solids), 11.5% was metabolite, and 2.6% remained as parent. The rate constants for primary biodegradation and mineralization of hexadecanol in activated sludge were 103.4 h-1and 1.8 h-1, respectively.

 

The inoculum was activated sludge obtained from Fairfield Wastewater Treatment Plant (Fairfield, OH), which receives predominantly domestic wastewater. The disappearance of parent and progression of metabolite formation and decay were monitored over time by thin layer chromatography with radioactivity detection. Production of CO2was determined by comparing total radioactivity in a bioactive treatment compared to that in an abiotic control using liquid scintillation counting (LSC).

 

This biodegradation simulation test satisfied the guideline requirements for the OECD 314 B simulation tests to assess the biodegradability of chemicals discharged in wastewater.

 

Another OECD 314 test using activated sludge, and using radiolabelled (14C) test substance, indicates 95% mineralisation of C16 to CO2in 31 days (Federle, 1993).

Another reliable study conducted according to ISO 11733:1995 and comparable to OECD 303 determined 99.5% DOC removal in 30 days for pentadecanol (Battersby N J, Sherren A J, Bumpus R N, 1999).