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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to a well-documented method (Aerobic biodegradation in soils) without deviations that influence the quality of the results.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: Environmental Assessment Technical Assistance Document 3.12, Aerobic biodegradation in soils. US Food and Drug Administration, Washington DC, PB87-175345, 1987.
Principles of method if other than guideline:
Degradability of radiolabeled test substance was followed in laboratory microcosms simulating sludge-amended river sediment (and soils) during 12 weeks. Samples were analysed by LSC. Metabolites were characterised on TLC.
GLP compliance:
no
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural sediment
Details on source and properties of surface water:
not relevant
Details on source and properties of sediment:
Delware river
- Origin: Delawarer River location in central New Jersey, USA
- pH at time of collection: 6.7
- solids (%): 73.4% (after air drying)
- PO4 as P: < 2.0 mg/kg
- NH3 as N: 20 mg/kg
- Water holding capacity: 36%
Details on inoculum:
Sediment as indicated above is used as inoculum in the study, moistened to circa 75% water holding capacity with distilled water. No nutrients were added, no pH adjustment.
Abiotic controls were made by autoclaving during 1 hour and addition of mercuric chloride (500 mg/kg final concentration) = killed controls.
Duration of test (contact time):
12 wk
Initial conc.:
17 other: mg/kg dw
Based on:
test mat.
Initial conc.:
0.34 other: µCi per gram soil dw
Based on:
other: radioactivity
Parameter followed for biodegradation estimation:
radiochem. meas.
test mat. analysis
Details on study design:
The fate of 14C-OTNE in sediment was studied in microcosms according to protocols described in documents from the U.S. Food and Drug Administration of 1987. Samples were taken from the sediment of the Delaware River in central New Jersey, USA.
Sealed flasks with sediment spiked with 17 µg test substance/g sediment dw (17 mg/kg) were incubated at laboratory ambient temperature (22-23 ºC) for 12 weeks. Periodically the headspace was flushed for oxygen replenishment and the effluent gas was drawn through a train of scintillation fluids to capture volatile organics and CO2 to be determined by liquid scintilation counting. Periodically also flasks were sacrificed and exhaustively extracted with hexane/acetone. An aliquot of the solvent fraction was used for thin layer chromatography for analysis of the test substance and metabolites including CO2.

TEST SYSTEM AND TEST CONDITIONS
- Test system: 34 air-tight serum vials (160 ml) with 20 g river sediment and 3 screw top Erlenmeyer flasks (250 ml) with 50 g of river sediment.
- Composition of medium: sediment and distilled water, to 75% water holding capacity
- Additional substrate: no additional nutrients were supplied
- Test temperature: 22-23ºC
- pH: 6.7 (initial)
- pH adjusted: no
- Aeration of dilution water: headspace air refreshment during sampling times
- Continuous darkness

- Details of trap for CO2 and volatile organics if used:
universal scintillation fluid (Liquiscint) to trap volatile organic metabolites;
phenethylamine-containing scintillation fluid (14C-Oxosol) for CO2.
Measurements by Pharmacia LKB 1209 Rackbeta liquid scintillation counter.

SAMPLING
- Sampling frequency: day 0, week 1, 2, 3, 4, 6, 8, 12.
- Sampling method: low vacuum on system with air-tight serum vials to sequential traps with specific scintillation fluid for organic metabolites and CO2.

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: six serum vials were autoclaved for 1 hour and hen amended with mercuric chloride (500 mg/kg final concentration)

Test performance:
This test was one of a series where also sludge amended soil and agricultural soil was tested. The rapid CO2 evolution in the system showed that microorganisms capable of mineralising the test substance were indigenous of the systems. The rate of 14-CO2-evolution from the sediment was lower than from the other systems. No nutrient amendments were made and incubation was under aerobic, undisturbed conditions. This shows that the test system performed well. The total 14-C-mass balance in time was acceptable ranging from 76 to 84%.
Compartment:
other: water / sediment, material (mass) balance
Remarks on result:
other: Not relevant: test system was a slurry (75% water holding capacity)
% Degr.:
90
Parameter:
test mat. analysis
Sampling time:
3 wk
Remarks on result:
other: 10% of parent material remaining in the microcosm. Thus 90% primary degradation.
% Degr.:
> 99
Parameter:
test mat. analysis
Sampling time:
12 wk
Remarks on result:
other: 0.53% of parent material remaining in the microcosm. Thus primary degradation was almost complete.
% Degr.:
14
Parameter:
CO2 evolution
Sampling time:
3 wk
% Degr.:
31.4
Parameter:
CO2 evolution
Sampling time:
6 wk
% Degr.:
50
Parameter:
CO2 evolution
Sampling time:
12 wk
Remarks on result:
other: 38.6% of total recovery 78% in combustion / 46% with total recovery of radiolabel 59% in extracts.
Key result
Compartment:
sediment
DT50:
9.5 d
Type:
other: non-linear regression analysis
Remarks on result:
other: based on the amount of parent remaining at each time point throughout the 12 week time course
Other kinetic parameters:
pseudo-first order rate constant
Transformation products:
yes
Details on transformation products:
Results from TLC analyses on the soxhlet-extractable metabolites show that the parent migrated to 'region 5' whereas more polar metabolites were distributed in regions 1 to 4. During the first three weeks a significant portion of the total radioactivity was associated with these more polar metabolites. The majority migrated with the regions 3 and 4 (Rf = 0.38 - 0.56 and Rf 0.56 - 0.75, respectively) which is significantly separated from that of the parent in region 5 (Rf = 0.75 - 1.0).
In week 8 the percent total radioactivity associated with the polar metabolites began to decrease, indicating that the metabolites represent transient intermediates that can be further degraded into CO2, water soluble products and humic-bound products. The total percent of radioactivity recovered from the samples by soxhlet extraction in week 8 and 12 decreased to 4.8 and 2.8 % , whereas only 1.7% was recovered as the parent. Thus the half-life of the parent was estimated as 9.5 days (by non-linear regression analysis).
For the killed controls, the majority of the radioactivity migrated with the parent ("region 5"). The week 4 Killed control showed 97% of total radioactivity associated withthe parent whereas this was 84 % in week 12. This shows that there was very little abiotic loss of the parent substance over the 12-week time course.
Details on results:
The total C14-mass balance for the test systems was good for the other samples (soil) but was somewhat lower for the sediment test (57 - 73%). In the killed control 100% 14C-recovery was recorded. SInce the river sediment represented a different matrix than de other soils it was hypothesized that some of the radioactivity associated with the metabolites could not be recovered by the extraction procedure due to tight asdorption to the matrix. This was checked by a total sediment combustion study. This showed that the total 14-C mass balance ranged (in time) from 75.7 to 83.5% and it was concluded that a significant portion of the radioactivity was strongly bound to the sediment matrix and was not extractable by conventioanl solvents.
Results with reference substance:
no reference included

14 -C Mass balance analysis of test substance incubated in Delaware River Sediment

 

Measurement

Time of analysis

 

Killed – week (8 & 12)

Week 3

Week 6

Week 12

Starting Parent substance added (µg)

340

340

340

340

Final Parent substance measured in microcosm (µg)

228

33

7.9

1.8

%Parent substance remaining

67%

10%

2.4%

0.53%

 

 

 

 

 

14-C recovered as CO2

0%

14.0%

31.4%

44.7%

14-C recovered in aqueous fraction

9.6%

12.9%

4.7%

2.6%

14-C recovered in methanol extraction

41.0%

19.7%

4.9%

1.8%

14C-recovered in 2ndorganic extraction (hexane/acetone)

45.6%

16.7%

6.1%

2.5%

14-c recovered in base extraction (humic fraction)

3.5%

9.2%

9.4%

7.5%

TOTAL 14-C recovered

100%

73%

57%

59%

         

 

Combustion analysis of the incubated Delaware River Sediment (14-C Mass balance)

 

Measurement

Time of analysis

 

Killed – week 6

Week 4

Week 6

Week 8

Starting Parent added (dpm)

1.42E+07

1.42E+07

1.42E+07

1.42E+07

Final dpm measured in combusted sediment as CO2a

1.19E+07

8.17E+06

6.30E+06

5.56E+06

%radioactivityin sediment

83.5%

57.6%

44.4%

39.2%

Final dpm measured as CO2 evolved from biodegradationb

0.0

2.8E+06

4.45E+06

5.48E+06

14-C recovered as CO2

0%

19.7%

31.33%

38.6%

TOTAL 14-C recovered

83.5%

77.3%

75.7%

77.7%

a.Average value from triplicate combustion analyses

b.Average cumulative CO2 evolved from vials flushed at each time point

 

Thus, after 8 weeks, the mineralization (CO2) corrected for the total recovery was 50% after 8 weeks, whereas the other 50% was detected in the sediment. 

Validity criteria fulfilled:
yes
Remarks:
Study according to national Guideline. 14C-Mass balance was checked and recovery was found acceptable. Metabolites were characterised by retention rate. Killed control was included.
Conclusions:
Radiolabled OTNE in sediment is almost completely degraded after 3 weeks. After 12 weeks the mineralisation (CO2 evolution) is circa 50% and a range of more polar metabolites were found. After a lag time of approx. 7 days the initial rate of CO2 production was 0.8% /day. The half-life of the parent substance was estimated at 9.5 days.
Executive summary:

The fate of14C-OTNE in sediment was studied in microcosms according to protocols described in documents from the U.S. Food and Drug Administration of 1987. Samples were taken from the sediment of the Delaware River in central New Jersey, USA.

Sealed flasks with sediment spiked with 17 µg test substance/g sediment dw (17 mg/kg) were incubated at laboratory ambient temperature for 12 weeks. Periodically the headspace was flushed for oxygen replenishment and the effluent gas was drawn through a train of scintillation fluids to capture volatile organics and CO2to be determined by liquid scintilation counting. Periodically also flasks were sacrificed and exhaustively extracted with hexane/acetone. An aliquot of the solvent fraction was used for thin layer chromatography for analysis of the test substance and metabolites. The total 14C-mass balance was established for the parent and metabolites based on extraction as well as on combustion of the total matrix. .

After 8 weeks circa 50% was recovered as 14-CO2 and < 1% remained of the parent material. The TLC results showed that the test substance was degraded to a range of more polar fractions. The total recovery of radiolabel from the solvent extraction was below 80% for the river sediment but with total combustion 74 - 84% was recovered showing that a significant portion of the radioactivity was strongly bound to the sediment matrix.

After a lag time of approx. 7 days the initial rate of CO2 production was 0.8% /day. The half-life of the parent substance was estimated at 9.5 days.

Endpoint:
biodegradation in water and sediment: simulation tests
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 October 2004 - 5 September 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to a method equivalent OECD TG 314 (before this TG was available). It is reliable without deviations that influence the results. Reliability 2 is assigned because the study was not conducted in compliance with GLP.
Qualifier:
equivalent or similar to
Guideline:
other: OECD TG 314
Deviations:
yes
Remarks:
Guideline was not available at time of testing
Principles of method if other than guideline:
Simulation Tests to Assess the Biodegradability of Chemicals Discharged in Wastewater: treated effluent in the mixing zone of surface water,
Die-away studies with radio-labeled OTNE in river water. The degradation process was followed for 28 days using SPE extraction and Rad-TLC separation. Quantification by LSC.
GLP compliance:
no
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: river water supplemented with 4 mg activated sludge per liter
Details on source and properties of surface water:
River water was collected from the Choptank River in Denton, Maryland. pH = 6.7; Total plate count (after 48h at 20 +/ 3 ºC) 6.1 * 10^2 CFU/ml; Hardness 108 mg/l as CaCO3, Total organic carbon 6.7 mg C/l.
Details on source and properties of sediment:
Not relevant
Details on inoculum:
Activated sludge mixed liquor was collected from the Denton Wastewater Treatment Facility, Denton, Maryland. It was sieved through a 2 mm mesh.
Aliquots of river water were supplemented with activated sludge solids at 4 mg/l. It was mixed overnight open to ambient air.

The abiotic control was amended with 2.5% mercuric chloride buffer solution (to achieve 1 g/l) and the pH of the abiotic control was matched with KOH to 7.2 in the biotic system. It was stoppered with a foam plug.
Duration of test (contact time):
28 d
Initial conc.:
3 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
other: Characterisation of metabolites: Rad-TLC of solvent extracts showes distincts peaks with a range of Rf (defined as the ratio of the distances traveled by a spot and by the solvent)
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 2 liters
- Composition of medium: Riverwater supplemented with 4 mg activated sludge solids/l
- Additional substrate: none
- Solubilising agent (type and concentration if used): Test substance was dissolved in a surfactant solution consisting of 0.0011 g in 500 ul of NANOpure water. Dosing solution was prepared by adding 115 ul of test substance directly to the surfactant solution, mixing and bringing the volume up to 10 ml with NANOpure water. The measured activity and calculated concentration of the dosing solution were approx. 0.799 microCi/ml and 4.23 microgram/ml, respectively. Target test substance dosing concentration was 3 microgram/l. Surfactant was Alkyl ethoxylated sulfate provided by Procter and Gamble, identified as TSIN S10392.01; C14/C15; E2.2 Sulfate, 19% solution. Nominal concentration of dosing solution was 0.11 mg/ml 0.0209 mg a.i./ml. Calculated surfactant concentration in the test system was 78.7 microgram/l (1.49 ug a.i./l).
- Test temperature: 20 +/ 3 ºC
- pH: 7.2
- pH adjusted: no
- Suspended solids concentration: 4 mg activated sludge solids/l
- Continuous darkness: yes (dark vessels)
- Other: The required dose of test material was added beneath the surface of the liquid in the test vessel while the water was stirred gently on a magnetic stir plate. After vigourous mixing in the stoppered vessel, the first samples were removed.

TEST SYSTEM
- Culturing apparatus: Test vessels: Two 1-gallon amber glass jugs
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: water was mixed overnight open to ambient air
- Test performed in closed vessels due to significant volatility of test substance: Vessels were stoppered with a Polyurethane plug to assess the loss due to volatilisation

SAMPLING
- Sampling frequency: time: 1 min, 1, 3, 5, 9, 12, 24 hours, 2, 3, 4, 5, 6, 7, 14, 21, 28 days. Less samples for abiotic control. Removal of PUP to assess volatilisation at 12, 24 hours, days 2, 3, 4, 5, 6, 7, 14, 21, 28.
- Sampling method: After thorough mixing, samples were taken with wide-mouth pipettes. For direct LSC: 1 ml of river water; three 25 ml-samples for analytical characterisation;
- Sample storage before analysis: not indicated

CONTROL AND BLANK SYSTEM
- Inoculum blank: not included
- Abiotic sterile control: test medium amended with sufficient 2.5% mercuric chloride buffer solution to achieve a final solution of 1 g/l. pH was adjusted to 7.2 by approx. 2 ml of 1.5 N KOH.
- Toxicity control: not included
Key result
% Degr.:
ca. 50
Parameter:
other: Rad-TLC
Sampling time:
5 h
Remarks on result:
other: Primary degradation of parent (Rf 0.59 - 0.63)
% Degr.:
95
Parameter:
other: Rad-TLC
Sampling time:
7 d
Remarks on result:
other: Primary degradation of parent (Rf 0.59 - 0.63)
% Degr.:
ca. 100
Parameter:
other: Rad-TLC
Sampling time:
14 d
Remarks on result:
other: Primary degradation of parent (Rf 0.59 - 0.63)
Key result
Compartment:
entire system
DT50:
1 d
Type:
other: Disappearance of > 50% of parent substance, read from figures
Temp.:
20 °C
Other kinetic parameters:
pseudo-first order rate constant
Transformation products:
not specified
Remarks:
The transformation products were more polar than the parent substance.
Details on transformation products:
- Formation and decline of each transformation product during test:
Rad-TLC of the solvent extracts from the biotic treatment revealed five distinct peaks. The peak (Rf 0.59-0.63) associated with the parent material was present at 86.08% of the initial activity at the 5-minute sampling point and declined to 4.88% by day 7. A peak with an Rf range of < 0.00 represented an average of 9.17, 7.25 and 9.79 percent of the initial activity on days 14, 21 and 28, respectively. A second peak with an Rf range of 0.09 to 0.165 was detected throughout the study and ranged from approximately 19% to 56% of the initial activity. A peak with an Rf range of 0.16 to 0.25 was initially
detected at 8.56% of initial activity at the 3-hour interval and increased to an average of 27.85% on day 28. A peak with an Rf range of 0.24 to 0.33 was initially detected at 8.75% of initial activity at the 3-hour interval, increased to a maximum level of 34.16% by day 5 and was detected at 27.85% on day 28. The level of activity associated with the extracted solids was variable and ranged from 0.31 to 1.77%.
Evaporation of parent compound:
not specified
Volatile metabolites:
yes
Details on results:
EXTRACTABLE RESIDUES
- % of applied amount at day 0: > 99%
- % of applied amount at end of study period: 85.8 %

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: < 1% (remaining with the solids)
- % of applied amount at end of study period: circa 1% (remaining with the solids)

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: Gradually increasing from 0.5 % on day 1 to 10.42% on day 28. This value is taken from Table 2 on page 18 of the report. The tables in sunnary and text are not correct in stating that the percentage Mineralised was NA (not analysed).

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: up to 4.26 % at day 28.

Abiotic TREATMENT
- total recovery: 69% after 5 minutes to 56% at day 28.
- Volatilization: 34% at day 28 (caught in Polyurethane plug, 61% if corrected for recovery)
Results with reference substance:
no reference substance included

Distribution of radioactivity  5 min day 3 day 7  day 14   abiotic 5 min abiotic day 28
 14C-OTNE, 5 ug/l, river water + 4 mg AS/l             
 % as parent (corr. for extraction efficiency)  86.08 9.05 4.88 0.00  100  11.37 
% total metabolites  24.59 68.29 67.71  89.07  0.00  12.99 
 
   5 min 12 hours day 7 day 28 abiotic 5 min abiotic day 28 
 % mineralised @  NA   1.19  6.28  10.42  NA  NA
 % recovered in solvent extract  122.27 83.18  85.90   75.70  68.24  19.50
 % recovered in aqueous 0.34  1.34  6.54  10.09  0.27  0.45 
  % remaining with the solids  0.89 0.60  1.74  1.32  0.32  1.57 
% volatilised  NA 0.29  3.02  4.26  NA  34.12 
  Total recovery %  123.51 86.61  103.48  101.79  68.82  55.65 

@ Data in summary tables of the report are not correct. The figures are taken from report p. 18, table 2 , 3rd column

NA: not analysed

Validity criteria fulfilled:
yes
Remarks:
the mass balance for radioactivity is acceptable. Recovery stated in the newly developed TG 314 is more strict than in this test. Acc. to the TG 314, however, this should not be used as criterion for acceptance of the test.
Conclusions:
In the degradation process OTNE is transformed to metabolites with variable polarity and some CO2. After 14 days the parent material had disappeared completely. The 50% disappearance time for primary degradation was less then 2 days.
Executive summary:

A die-away study in river water was carried out with radio-labelled C14 -OTNE. The test was carried out at a nominal concentration of 3 µg/l in a sample of river water with activated sludge added at a level of 4 mg SS/l (to mimic conditions in the mixing zone). The disappearance of parent material and the formation of metabolites were determined with radio thin layer chromatography (Rad-TLC). The temperature was maintained at 20 + 3 ºC. Mass balances were made for the components in the test medium.

The parent substance disappears fast and four separate metabolites are formed in time at different rates and with higher polarity than the parent. The total radioactivity of metabolites rose from 25% after 5 minutes up to 89% on day 14. Circa 4% volatilised. After 28 days 10 % mineralisation to CO2 was detected. The DT50 (Disappearance 50) for primary degradation is circa 1 days. The kinetic assessment showed an initial first-order loss rate of 1.83/h and a second first-order loss rate of 0.022/h.

Description of key information

See below

Key value for chemical safety assessment

Half-life in freshwater:
1 d
at the temperature of:
20 °C
Half-life in freshwater sediment:
9.5 d
at the temperature of:
22 °C

Additional information

Die-away studies were carried out with radio-labelled C14 -OTNE in river water as well as in a sediment microcosm system. Both studies are reliability 2 as they were not carried out under GLP.

A die-away study in river water was carried out with radio-labelled C14 -OTNE (Wildlife, 2006). The test was carried out at a nominal concentration of 3 µg/l in a sample of river water with activated sludge added at a level of 4 mg SS/l (to mimic conditions in the mixing zone). The disappearance of parent material and the formation of metabolites were determined with radio thin layer chromatography (Rad-TLC). The temperature was maintained at 20 ± 3 ºC. Mass balances were made for the components in the test medium. The parent substance disappears fast and four separate metabolites are formed in time at different rates and with higher polarity than the parent. The total radioactivity of metabolites rose from 25% after 5 minutes up to 89% on day 14. Circa 4% volatilised. After 28 days 10 % mineralisation to CO2 was detected. The DT50 (Disappearance 50) for primary degradation is circa 1 days. The kinetic assessment showed an initial first-order loss rate of 1.83/h and a second first-order loss rate of 0.022/h.

The fate of 14C-OTNE in sediment was studied in microcosms according to protocols described in documents from the U.S. Food and Drug Administration of 1987 (Envirogen, 1999). Samples were taken from the sediment of the Delaware River in central New Jersey, USA. Sealed flasks with sediment spiked with 17 µg test substance/g sediment dw (17 mg/kg) were incubated at laboratory ambient temperature for 12 weeks. Periodically the headspace was flushed for oxygen replenishment and the effluent gas was drawn through a train of scintillation fluids to capture volatile organics and CO2 to be determined by liquid scintillation counting. Periodically also flasks were sacrificed and exhaustively extracted with hexane/acetone. An aliquot of the solvent fraction was used for thin layer chromatography for analysis of the test substance and metabolites. The total 14C-mass balance was established for the parent and metabolites based on extraction as well as on combustion of the total matrix. After 8 weeks circa 50% was recovered as 14-CO2 and < 1% remained of the parent material. The TLC results showed that the test substance was degraded to a range of more polar fractions. The total recovery of radiolabel from the solvent extraction was below 80% for the river sediment but with total combustion 74 - 84% was recovered showing that a significant portion of the radioactivity was strongly bound to the sediment matrix.

After a lag time of approx. 7 days the initial rate of CO2 production was 0.8% /day. The half-life of the parent substance was estimated at 9.5 days.