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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study included enhancements of OECD 301F, since not strictly to guideline, reliability = 2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Principles of method if other than guideline:
Method adapted from OECD 301F, but using acclimated bacteria.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
PART 1 was to determine toxicity of the test material or potential toxic transformation products to the activated sludge microorganisms with a test medium prepared as outlined in OEDC guideline with non-acclimated sludge. A detailed description can be found in Appendix B.

PART 2 was to determine biodegradability with acclimated inoculum with the test material from Part 1 and supernatant from a Semi-Continuous Activated Sludge system (SCAS). The test medium was then prepared as outlined in the OECD 301F1 guideline.

PART 3 was prepared from the acclimated inoculum from Part 1 and from the acclimated inoculum from the SCAS units from Part 2.

PART 4 was prepared using the combined acclimated inoculum from Part 1 and from Part 2 with the addition of supernatant obtained from a petrochemical –contaminated soil sample

Duration of test (contact time):
60 d
Initial test substance concentration
Initial conc.:
ca. 50 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
PART 1
Test system without acclimated inoculum

Test Medium Preparation

Fresh activated sludge was used as the inoculum. The activated sludge was obtained from Somerset-Raritan Valley Sewage Authority in Bridgewater, on 3 September 2009, Day -1 of the test. This treatment facility was selected because it deals predominantly with domestic sewage as specified in the guideline. There were no known contaminants in the fresh activated sludge believed to be present at levels high enough to have interfered with this study.

Duplicate 10 ml aliquots of the activated sludge were filtered through pre-weighed Whatman 934-AH filters in a Buchner funnel and vacuum flask set up. The filters were placed in an aluminum pan and dried in an oven for one hour at 102°C. After cooling, the filters in a dessicator, the filters were re-weighed and the mean total suspended solids concentration was
determined to be 4.74g/L. The sludge was homogenized in a blender for two minutes at low speed. The homogenated sample was allowed to settle for fifty minutes, after which the supernatant was decanted (avoiding carry-over of sludge solids). An aliquot of the supernatant was used to determine microbial activity. The microbial activity was determined using the Easicult-TTC dip slides Lot No. 1291000. This was accomplished by removing the agar stick from the culturing tube, and dipping the agar into the supernatant aliquot. Excess supernatant was blotted off with a clean paper towel, and the agar stick was then placed back into the culture tube. The whole unit was placed into a dark incubator for 48 hours at 20 ± 2°. Based on comparison of the density of colonies growing on the agar with the model density chart provided by the supplier, the microbial activity was determined to be 105CFU/mL. The remaining decanted sludge supernatant was used for final preparation of the test medium on Day -1.

Twenty liters of glass distilled water were collected in a carboy. A quantity of 460 mL of glass distilled water was removed from the carboy before the addition of the mineral salt solutions. The following quantities of mineral salt solutions and inoculum were added to the carboy:

mL per liter of glass distilled water Solution Concentration
1 Magnesium Sulfate 2.75% (VWR, Lot# 7304)
1 Calcium Chloride 2.75% (VWR, Lot# 7319)
10 Phosphate Buffer pH 7.2 (VWR, Lot# 8091)
1 Ferric Chloride 0.025% (VWR, Lot# 7291)

The test medium was prepared one day before the test began. The test medium was aerated with carbon dioxide free air for approximately 24 hours before use.

There were no known contaminants in the solutions believed to be present at levels that may have interfered with the study. All solutions were refrigerated when not in use.

The test system was considered as the following in a flask (detail in table below):

Blank: 1 L test medium
Sodium Benzoate: 5 mL sodium benzoate stock solution, 1 L test medium
Test substance: 3 reps, 44.7, 53.5, 52.8 mg added to 1L test medium.
Toxicity control: 3 reps, 44.7, 53.5, 52.8 mg test substance, 5 mL sodium benzoate stock solution added to 1L test medium.

All test containers used in this study were uniquely identified as to appropriate composition, i.e.,Study #, Blank -1, 2, 3; MRD-08-196 - 1, 2, 3, etc. All glassware was washed with Chem-Solv® and then rinsed with glass distilled water to remove any residual organic carbon. All glassware was inspected to ensure cleanliness. The manometric cells were rinsed with two portions of acetone, filled with soapy water and allowed to stand for a few hours. The cells were then rinsed with glass distilled water followed by acetone then finally air dried.

The test substance, positive control substance, and toxicity control were tested at concentrations of 50.3 mg/L, 54.5 mg/L, and 107.95 mg/L, respectively. Sodium benzoate was administered to the respective test systems by adding 5.0 mL aliquot of 10000mg/L aqueous stock solution. The test substance was injected directly into the test flasks containing one liter of test medium with a syringe containing the test substance. The weight difference of the syringe was recorded as the weight of test substance added to the flask. Each flask was sealed immediately after addition of the test substance to avoid loss due to volatilization.

After assembly of the test systems, stirring was initiated, the equipment was checked to ensure no leaks were present, and the oxygen uptake measurements were initiated. No further attention was required other than printing the respirometer data and making daily checks during normal working hours to see that the correct temperature (22 ± 2°C) and adequate stirring were maintained.

PART 2
Test system with inoculum from SCAS units

Activated sludge was obtained from Raritan Valley Sewage Authority, in Bridgewater , NJ. The sludge was homogenized in a blender at low speed for approximately 2 minutes, An aliquot was removed and the microbial activity was determined using Easicult TTC slide Lot 4223701. The activated sludge was transferred to a 12 liter carboy, aerated and synthetic sewage feed was added to the stock daily.
On 19 September 2009, approximately 700 mls of activated sludge was removed and transferred to each of 1 liter graduated cylinders, aerated and feed 50mls of synthetic sewage. A total of three systems were set up. On 21 September 2009, an aliquot was removed and the microbial activity was determined using Easicult TTC slide. On 22 September 7009, approximately 400mls of activated sludge was removed for each unit and replaced with activated sludge from the 12 liter reservoir. Test material was added to each unit, 50mls of synthetic sewage feed and aerated. The procedure of removing 400mls, adding test material, synthetic sewage and aerating continued for 21 days until 13 October 2009.

The addition of test materials, collection of activated sludge, addition and preparation of synthetic sewage feed and Easicult TTC results can be found in the raw data.

Twelve liters of glass distilled water were collected in a carboy. A quantity of 276 mL of glass distilled water was removed from the carboy before the addition of the mineral salt solutions. The following quantities of mineral salt solutions and inoculum from the SCAS units were added to the carboy:

mL per liter of glass distilled water Solution Concentration
1 Magnesium Sulfate 2.75% (VWR, Lot# 8302)
1 Calcium Chloride 7.75% (VWR, Lot# 8204)
10 Phosphate Buffer pH 7.2(VWR, Lot# 8091)
1 Ferric Chloride 0.025% (VWR, Lot# 8338)

The test medium was prepared one day before the test began. The test medium was aerated with carbon dioxide free air for approximately 24 hours before use

The test system was considered as the following in a flask (detail in table below):

Blank: 1 L test medium
Sodium Benzoate: 5 mL sodium benzoate stock solution, 1 L test medium
Test substance: 3 reps, 54.1, 54.3, 47.0 mg added to 1L test medium.

The test substance, and positive control substance, were tested at concentrations of 51.8mg/L, 54.57 mg/L, , respectively. Sodium benzoate was administered to the respective test systems by adding 5.0 mL aliquot of 10000 mg/L aqueous stock solution. The test substance was injected directly into the test flasks containing one liter of test medium with a syringe containing the test substance. The weight difference of the syringe was recorded as the weight of test substance added to the flask. Each flask was sealed immediately after addition of the test substance to avoid loss due to volatilization.

After assembly of the test systems, stirring was initiated, the equipment was checked to ensure no leaks were present, and the oxygen uptake measurements were initiated. No further attention was required other than printing the respirometer data and making daily checks during normal working hours to see that the correct temperature (22 ± 1°C) and adequate stirring were maintained.

PART 3
Test system with acclimated inoculums from Part 1 and Part 2

Test Medium Preparation

All systems from Part 1 and Part 2 were terminated on 17 November 2009. Flasks containing MRD-08-196 and the blanks were filtered through 0.45u filter using a Millipore set-up. The biomass was scraped from the filters into 20ml of test medium.

Two liters of glass distilled water were collected in a carboy. The following quantities of mineral salt solutions and 20 mls of accumulated biomass were added to the carboy:

mL per liter of glass distilled water Solution Concentration
1 Magnesium Sulfate 2.25% (VWR, Lot# 8302)
1 Calcium Chloride 2.75% (VWR, Lot# 8204)
10 Phosphate Buffer pH 7.2 (VWR, Lot# 8091)
1 Ferric Chloride 0.025% (VWR, Lot# 8338)

The test medium was prepared one day before the test began. The test medium was aerated with carbon dioxide free air for approximately 24 hours before use

Preparation of the Test Systems

Blank: 1 L test medium
Sodium Benzoate: 5 mL sodium benzoate stock solution, 1 L test medium
Test substance: 3 reps, 47.4, 46.0, 46.7 mg added to 1L test medium.

The test substance, and positive control substance, were tested at concentrations of 46.53mg/L, 50 mg/L, respectively. Sodium benzoate was administered to the respective test systems by adding 5.0 mL aliquot of 10000 mg/L aqueous stock solution. The test substance was injected directly into the test flasks containing one liter of test medium with a syringe containing the test substance. The weight difference of the syringe was recorded as the weight of test substance added to the flask. Each flask was sealed immediately after addition of the test substance to avoid loss due to volatilization.

After assembly of the test systems, stirring was initiated, the equipment was checked to ensure no leaks were present, and the oxygen uptake measurements were initiated. No further attention was required other than printing the respirometer data and making daily checks during normal working hours to see that the correct temperature (72 ± 1°C) and adequate stirring were maintained.

PART 4
Test system with acclimated inoculums from Part 1, and Part 2 and soil inoculum


Soil was obtained from ExxonMobil Chemical Holland with possible contact with hydrocarbons and Vammar D10. 500 grams of soil was placed in a 4 liter carboy with the addition of 27 liters of glass distilled water and the following mineral salts

mL per liter of glass distilled water Solution Concentration
1 Magnesium Sulfate 2.25% (VWR, Lot# 8302)
1 Calcium Chloride 2.75% (VWR, Lot# 8204)
10 Phosphate Buffer pH 7.2 (VWR, Lot# 8091)
1 Ferric Chloride 0.025% (VWR, Lot# 8338)

The carboy was placed on a stirrer and aerated on 10 November 2009. On 13 November 2009, an oil ring had formed on the top, cloudy supernatant in the middle and the soil remained on the bottom. The middle cloudy supernatant was decanted into a 7 liter flask and aerated until needed.


Preparation of the Test Systems

The test system was considered as the following in a flask (detail in table below):

Test substance, or positive control substance
**Test Medium which was 100ml of the acclimated inoculum from Part 1 and Part 2, 100ml of the soil supernatant, 800 ml of test medium

Sodium Benzoate: 5 mL sodium benzoate stock solution, 1 L test medium
Test substance: 3 reps, 51.1, 49.8, 52.1 mg added to 1L test medium.

Sodium benzoate was administered to the respective test systems by adding 5.0 mL aliquot of 10000 mg/L aqueous stock solution. The test substance was injected directly into the test flasks containing one liter of test medium with a syringe containing the test substance. The weight difference of the syringe was recorded as the weight of test substance added to the flask. Each flask was sealed immediately after addition of the test substance to avoid loss due to volatilization.

After assembly of the test systems, stirring was initiated, the equipment was checked to ensure no leaks were present, and the oxygen uptake measurements were initiated. No further attention was required other than printing the respirometer data and making daily checks during normal working hours to see that the correct temperature (22 ± 1°C) and adequate stirring were maintained.





Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
Results shall only be presented from Part 4 - which consisted of acclimated inocula from Part 1 and Part 2, as well as contaminated soil inoculum.
Test performance:
The toxicity control was evaluated at mean concentration of 107.95 mg/L in Part 1 of the study only. The guideline states that a test substance will be considered inhibitory if the toxicity test systems, containing both the test and positive control substance, reach less than 75% biodegradation by Day 14. The toxicity control systems exceeded 75% by Day 3 therefore the test substance cannot be considered inhibitory at the test concentration.
% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
63
St. dev.:
2.41
Sampling time:
28 d
Remarks on result:
other: Acclimated inocula
Parameter:
% degradation (CO2 evolution)
Value:
81.53
St. dev.:
1.86
Sampling time:
60 d
Remarks on result:
other: acclimated inocula

Applicant's summary and conclusion

Interpretation of results:
inherently biodegradable
Conclusions:
Based on the results of an aerobic biodegradation study with acclimated inocula, Vammar D10 is inherently biodegradible in the aqueous environment (63% in 28 days) . The studies were performed following OECD 301F Manometric Respirometry Test guidelines modified to include acclimated inocula, at test material concentrations of approximately 50 mg/L. Results are based on ThOD of the test substance.