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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
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Identifier: EXP1200078
Appearance: Very dark brown (almost black) viscous liquid
Batch: E00275-350
Sample Expiration Date: end-2013
Purity:100%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial (adaptation not specified)
Details on inoculum:
Activated sludge was collected from the Cambridge Wastewater Treatment Facility,
Cambridge, Maryland on February 07, 2013. The Cambridge facility treats predominantly residential
wastes. The sludge was sieved using a 2-mm screen, adjusted to approximately 1,000 mg total
suspended solids (TSS)/L with mineral media and then aerated at test temperature until use. A total
suspended solids measurement and standard plate count were performed on the inoculum on the day
of test chamber preparation. Plates were incubated at 20±3ºC for approximately 48 hours.
Duration of test (contact time):
>= 28 d
Initial conc.:
10 mg/L
Based on:
other: elemental analysis
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
All chambers were aerated with CO2-free air for approximately 24 hours at a rate of approximately 55 mL per minute to
purge the systems of CO2. After the aeration period, the flow of CO2-free air was stopped, and three CO2 traps, each
containing approximately 100 mL of 0.5 N KOH, were connected to the exit air lines of each chamber. Sufficient volumes of reference substance stock solution to achieve a nominal concentration of 10 mg C/L were added to the appropriate chambers. Sufficient amounts of test substance silicone oil emulsions to achieve a nominal concentration of approximately
10 mg C/L were added to the appropriate chambers. 20 mL of silicone oil was added to the control and reference chambers.
The final volume within all chambers was brought up to 3000 mL by the addition of NANO¿pure water and the airflow
was restarted on the system.

The following were added to each chamber:
1) 2470 mL of NANO¿pure water
2) 3 mL calcium chloride solution (2.75%)
3 mL of ferric chloride solution (0.025%)
3 mL of magnesium sulfate solution (2.25%)
30 mL of phosphate buffer (pH 7.4)
3) A volume of activated sludge inoculum to achieve a final TSS of approximately 30 mg/L

The biodegradation test was started by bubbling CO2-free air through each of the test chambers
at a rate of approximately 55 mL per minute. The CO2 produced from the degradation of organic
carbon sources within the test chamber was trapped as potassium carbonate (K2CO3) in the KOH
solution and the amount of inorganic carbon in the trapping solution was measured at various
intervals during the study, using a Shimadzu Model TOC-VCSH carbon analyzer.

On days 2, 5, 8, 12, 15, 19, 22 and 26 the CO2 trap nearest the test chamber was removed and
analyzed for inorganic carbon. The two remaining traps were placed one position closer to the test
chamber and a new trap was placed on the end of the series.

On the 28th day of the test, an aliquot of the contents of each test chamber was removed and
the pH determined. The contents of all chambers were then acidified by the addition of 1 mL of
concentrated hydrochloric acid to drive off inorganic carbonate. All chambers were aerated overnight
and then a sample from each test chamber was removed for dissolved organic carbon (DOC) analysis
and the trapping solutions closest to the test chambers were analyzed for inorganic carbon.

The results of the inorganic carbon analyses of the CO2 traps were converted to mg CO2 produced using the following equation:
mg CO2 = result (mg C/L) X vol. of KOH (L) X 3.67 mg CO2/mg C

The Cumulative mg of CO2 for vessels which did not contain solubilizer were corrected for the amount of CO2 evolved by the control, using the following equation:
Cumulative mg CO2 produced = S mg CO2 test - mean S mg CO2 control

The Cumulative mg of CO2 for all vessels containing solubilizer were corrected for the amount of CO2 evolved by the solubilizer control, using the following equation:
Cumulative mg CO2 produced = S mg CO2 test - mean S mg CO2 solubilizer control

The percentage of theoretical carbon dioxide (%TCO2) evolved was calculated as follows:
% TCO2 = mg CO2 produced / [(mg of carbon in test) x (3.67 mg CO2 /mg Carbon)] X 100
Reference substance:
benzoic acid, sodium salt
Remarks:
Fluka; Lot Number: 1373379
Key result
Parameter:
% degradation (CO2 evolution)
Value:
19.1
St. dev.:
2.9
Sampling time:
28 d
Details on results:
The final mean percent biodegradation for EXP1200078 was 19.1% after 28 days.
EXP1200078 may not be considered readily biodegradable, since 60% TCO2 was not achieved.
Results with reference substance:
The viability of the inoculum and validity of the test were supported by the results of the
reference substance, sodium benzoate, from which an average of 98.5% of theoretical CO2 was
evolved. An average percent biodegradation of greater than 60% was achieved by Day 8, thereby
fulfilling the criteria for a valid test by reaching the pass level by Day 14.
Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
EXP1200078 may not be considered readily biodegradable, since 60% TCO2 was not achieved.
Executive summary:

EXP1200078 was screened for ready biodegradability with a valid study conducted according to OECD guideline. The result of that study showed that EXP1200078 was not readily biodegradable since only 19.1 ± 2.1% was acheived during the 28 day period.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 March 2014 to 26 September 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Version / remarks:
1981
GLP compliance:
yes
Specific details on test material used for the study:
Identification: EXP1200078
Description/Appearance: Very dark brown (almost black) viscous liquid
Batch / Lot / Notebook Ref: E00048-256
Sample Expiration Date: end-2014
Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
Activated sludge, surface soil and surface water were sampled from ten sites distributed in four districts throughout Nanjing city, such as Chengdong, Chengbei, Baguazhou and Jiangning. 1 L of the sludge, soil and water were collected and mixed thoroughly together. After removing floating matter, the mixture was allowed to stand and then the supernatant is filtrated through 0.22 µm Millipore filter. After that the filtrate was adjusted the supernatant to pH 7.0 with sodium hydroxide or phosphoric acid. Finally an appropriate volume of the filtered supernatant was transferred to a fill-and-draw activated sludge vessel and aerated for about 23.5 h. Batch No.: I20140310.

Thirty minutes after stopping the aeration, about one third of the whole volume of supernatant was discarded. Then an equal volume of the solution (pH 7.0) containing 0.1% each of glucose, peptone and potassium orthophosphate was added into the settled material and aerated again. This procedure was repeated once per day during one month.

Before use the mixture was allowed to stand, and the supernatant was removed. A small quantity of sludge was taken to be centrifuged (10000 rpm×10 min) and then weighed. Then the sludge was dried in the oven and weighed again in order to calculate the content of dry sludge was 10%. At last 10 g of centrifuged sludge was diluted 1 L with basal culture medium (BSM) to get activated sludge suspension with a concentration of 1000 mg/L (dry basis) Batch No.: I20140424
Duration of test (contact time):
28 d
Initial conc.:
ca. 9.83 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
TOC removal
Details on study design:
Appropriate amount of the test substance was added to #1 vessel designated as “abiotic”, then filled to a final volume of 300 mL with deionised water.
#2, #3 and #4 vessels of “test vessel” added appropriate amount of the test substance, respectively, then 30 mL of activated sludge suspension were added to every vessel and filled to a final volume of 300 mL with BSM. Concentration of activated sludge was 100 mg/L.

#5 vessel of “procedure control” added 30 mL stock solution of reference substance and 9 mL of activated sludge suspension, then filled to a final volume of 300 mL with BSM. Concentration of activated sludge and reference substance was 30 mg/L and 100 mg/L, respectively.
#6 vessel of “blank control” added 30 mL of activated sludge suspension, and then filled to a final volume of 300 mL with BSM. The concentration of activated sludge was 100 mg/L.

After that, assembled the equipment, checked that it is air-tight, began the stirrers, and started the measurement of oxygen uptake under conditions of darkness.
The temperature, the operation of the stirrer and recorder was checked daily. Any changes in colour of the contents of the vessels were recorded. The BOD for the six bottles were determined and recorded.
After the 28 days of testing, concentration of the test substance in the testing bottles was analysed.
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium benzoate Molecular formula: C6H5CO2Na Purity: = 99.5% Lot: 20130117 Supplier: Sinopharm Chemical Reagent Co., Ltd.
Test performance:
The level of biodegradation of the reference substance sodium benzoate was 74.8% after 7 days (> 40%), and 77.2% after 14 days (> 65%).
Key result
Parameter:
% degradation (TOC removal)
Value:
37.9
Sampling time:
28 d
Remarks on result:
other: Inherently biodegradable
Key result
Parameter:
% degradation (DOC removal)
Value:
31
Sampling time:
28 d
Remarks on result:
other: Inherently biodegradable
Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
The BOD results showed that biodegradation of the test substancewas 31.0% after 28 days. These results indicate that EXP1200078 was inherently biodegradable when compared to the standard.
Executive summary:

Study conducted which was similar to OECD 302 C with sodium benzoate as a reference substance. Under conditions of the study, sodium benzoate degraded > 65% by day 14 compared to EXP1200078 which degraded by only 31% by 28 days.

However, EXP1200078 was deemed to be inherently biodegradable.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A key GLP study conducted according to OECD 301B guideline determined that the registration substance does not meet the criteria of readily biodegradable. The supporting OECD 302C study demonstrates slow, inherent biodegradation.