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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:
supporting study
Study period:
22/04/1999 to 20/05/1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was done according to OECD Guideline 301F recommendations and no deviation affecting the reliability of results have been observed.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 22 April 1999 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic
sewage
- Storage conditions: maintained on continuous aeration
upon receipt
- Storage length: not specified
- Preparation of inoculum for exposure: washed three times by settlement and resuspension in culture medium
- Pretreatment: none
- Concentration of sludge: 30 mg suspended solids/L
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: see below
- Test temperature: 21°C
- pH: 7.6
- pH adjusted: no
- Suspended solids concentration: 30 mg ss/L
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: vessels were placed in Camlab BOD cabinets
- Number of culture flasks/concentration: 1 or 2
- Method used to create aerobic conditions: not specified
- Method of preparation of test solution: An amount of test material (15.7 mg) was dispersed in culture medium and subjected to ultrasonic disruption for approximately 30 minutes prior to the addition of inoculum (15.7 mL).
- Measuring equipment: manometer scale of Camlab BOD apparatus
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: 50% v/v ethanolamine water solution
- DOC analysis apparatus: Shimadzu TOC-SOSOA TOC analyser
- DOC analysis method: Samples 27 and 22 pi were injected into the Total Carbon (TC) and Inorganic Carbon (TC) channels of the TOC analyser. Total carbon analysis is carried out at 680°C using a platinum catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

SAMPLING
- Sampling frequency: every day from day 0 to day 28
- Sampling method: not specified


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes


STATISTICAL METHODS: none
Reference substance:
aniline
Remarks:
(Sigma Lot No. 68H3441)
Preliminary study:
None
Test performance:
No data
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 54
St. dev.:
1.4
Sampling time:
28 d
Details on results:
Average percent degradation of test substance at day 7, 14, 28 were 45, 52, and 54% respectively, based on oxygen consumption.
Results with reference substance:
Degradation of aniline was 79% at day 7 and 87% at day 28 based on oxygen consumption, and 94% at day 28 based on DOC removal.

Table 5.2.1/2: Calculation table for the degree of degradation by BOD

Sample description ThOD
(mg O2/L)
Day 7 Day 14 Day 28
BOD
(mg O2/L)
Degradation
(%)
BOD
(mg O2/L)
Degradation
(%)
BOD
(mg O2/L)
Degradation
(%)
Mean degradation
(%)
Aniline plus inoculum
(100 mg/L)
  309 260 79 280 84 290 87 -
Test material plus inoculum (100 mg/L) R1 231 120 45 140 52 150 55 54
R2 231 120 45 140 52 145 53
Toxicity control plus inoculum (test material plus aniline 100 mg/L)   540 240 42 310 54 400 70 -
Control R1 - 16 - 19 - 22 - -
R2 - 14.5 - 21 - 23 -
R1 – R = Replicates 1 and 2
BOD Curves are shown in Figure 1
Degradation curves are shown in Figure 2
Degradation (%) - [BODtest - BODcontrol]/[ThOD] x 100%
ThOD of test material - 2.31 mg O2/mg

Table 5.2.1/3: Calculation table for the degree of degradation by DOC analysis

Sample description Theoritical carbon content (%) (A) Day 0 Day 28
DOC (mg C/L) DOC (mg C/L) Mean degradation (%)
Measured Corrected for mean control values (B) % Thoritical carbon content (C) Measured Corrected for mean control values Degradation (%) (D)
Aniline plus inoculum
(100 mg/L)
  77.38 75.06 72.65 94 8.94 4.71 94 -
Control R1 - 2.41 - - 4.5 - - -
R2 - - - - 3.96 - -
R1 – R = Replicates 1 and 2
(A) = Theoritical carbon content = [(No. Carbon atoms x atomic weight carbon)/molecular weight]
(B) = DOC corrected for mean control value = DOCmeasured- DOCcontrol
(C) = % Theoritical carbon content [B/A] x 100%
(D) = % Degradation = [1-(corrected DOCDay28/corrected DOCDay0)] x 100%
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance attained 54% degradation after 28 days calculated based on oxygen consumption and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No 301 F. However the test material has exhibited the potential for rapid degradation. The test material did not exhibit toxicity to micro-organisms at 100 mg/L under test conditions.
Executive summary:

A study was performed to assess the ready biodegradability of the test substance in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301 F, "Ready Biodegradability; Manometric Respirometry Test".

The test material was exposed to activated sewage sludge micro-organisms at a concentration of 100 mg/L with culture medium in sealed culture vessels in the dark at 21°C for 28 days. The degradation of the test material was assessed by the measurement of daily oxygen consumption. Control solutions with inoculum and the standard material, aniline, together with a toxicity control were used for validation purposes.

This study meets all the validity criteria mentioned in OECD Guideline 301F and was assessed to be reliable. The reference substance underwent 70% degradation in presence of test substance, therefore the test material did not exhibit toxicity to micro-organisms at 100 mg/L under test conditions.

The test material attained 54% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 F. However the test material has exhibited the potential for rapid degradation.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 01/02/2000 to 18/07/2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to the guideline without deviation, even though some information were not reported, such as the measurement of pH and the sampling method. These minor deficiencies do not affect the reliability of the study.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Theoritical oxygen demand (ThOD): 2.31 mg O2/mg
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
A freshly collected and a pre-exposed inocula were prepared.

Freshly collected inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Prospect Bay Wastewater Treatment Facility, Grasonville, Maryland
- Storage conditions: not mentionned
- Storage length: not mentionned
- Preparation of inoculum for exposure: The sludge was sieved using a 2 mm screen and
homogenized in a blender at medium speed for approximately 2 minutes and then allowed to settle for
approximately 30 minutes. The supernatant above the settled solids was removed, filtered through glass
wool and aerated for approximately 48 hours. After the aeration period, the inoculum was allowed to
settle for 30 minutes prior to the removal of a sample for DOC analysis. The inoculum then was
centrifuged for 15 minutes at 2500 rpm. The supernatant from each inoculum was decanted and aerated
overnight. Prior to use, the dissolved organic carbon and total suspended solids
concentrations were measured. In addition, a plate count was performed on inoculum. Plates were
incubated at 20±3°C. for approximately 48 hours.
- DOC of sludge: 10.6 mg C/L
- Initial cell/biomass concentration: 1.33 x 10^4 CFU/mL
- Water filtered: yes
- Type and size of filter used, if any: 2 mm

Pre-exposed inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Prospect Bay Wastewater Treatment Facility, Grasonville, Maryland
- Storage conditions: not mentionned
- Storage length: not mentionned
- Preparation of inoculum for exposure: After the pre-exposure period, equal volumes of sludge from each SCAS units were combined, homogenized in a blender at medium speed for approximately 2 minutes and then allowed to settle for approximately 30 minutes. The supernatant from above the settled sludge solids was removed, filtered through glass wool and then aerated for approximately 48 hours. After the aeration period, the inoculum was allowed to settle for 30 minutes prior to the removal of a sample for DOC analysis. The inoculum then was centrifuged for 15 minutes at 2500 rpm. The supernatant was decanted and aerated overnight. Prior to use the dissolved organic carbon (DOC) and total suspended solids (TSS) concentrations were measured. DOC samples were filtered using a 0.45llffi filter, acidified and sparged with nitrogen prior to analysis using a Shimadzu Model TOC-5000 Carbon Analyzer. TSS samples were filtered through tared glass fiber filters and the residue retained was dried at approximately 105°C and allowed to cool for at least 30 minutes iu a desiccator prior to weighing. In addition, a plate count was performed on inoculum. Plates were incubated at 20±3°C for approximately 48 hours.
- Concentration of sludge: 2500 mg/L
- DOC of sludge: 12.8 mg C/L
- Initial cell/biomass concentration: 3.25 x 10^3 CFU/mL
- Water filtered: yes
- Type and size of filter used, if any: 2 mm
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
EXPERIMENTAL DESIGN
The test contained two inoculum control groups, two reference groups, and two treatment groups. Each group contained ten replicate test chambers. The inoculum control chambers were used to measure the dissolved oxygen consumption of the inoculum and were not dosed with a carbon source. The reference chambers were dosed with sodium benzoate, a substance known to be biodegradable, at a concentration of 2 mg/L. The test chambers within the treatment group were used to evaluate the test substance at 2 mg A/L. Measurements of oxygen consumption were performed on two test chambers from the control, reference, and treatment groups on days 0, 7, 14,21 and 28.
The test was performed using: a) freshly collected inoculum and b) inoculum that was pre-exposed to the test substance for 31 days prior to start of the test. Pre-exposure was performed in two replicate semi-continuous activated sludge (SCAS) units.

TEST CONDITIONS
- Composition of medium: The test medium was a modified biochemical oxygen demand (BOD) test dilution water and was prepared using NANO®pure water as described in Protocol Appendix 1.
- Test temperature: 19 to 22°C
- pH of medium: 7.2
- pH adjusted: no data
- Aeration medium: The mineral medium was aerated for approximately 20 minutes and then allowed to stand overnight at test temperature.
- Suspended solids concentration (freshly collected inoculum): 0 mg/L
- Suspended solids concentration (pre-exposed inoculum): 7.7 mg/L
- Continuous darkness: no data


TEST SYSTEM
- Culturing apparatus: 300 mL BOD bottles
- Number of culture flasks/concentration: 10
- Method used to create aerobic conditions: not mentionned
- Measuring equipment: Orion Dissolved Oxygen Meter
- Test performed in closed vessels due to significant volatility of test substance
- Administration of test substance: volumetric addition. Dosing volume was calculated based on the density & purity of the test substance. The actual amount of test substance added was determined by weight verification of the calculated dosing volume. The average weight of three replicate aliquots of the calculated dosing volume was used as the actual amount of test substance added.

SLUDGE PRE-EXPOSURE
The activated sludge was collected from the Prospect Bay Wastewater Treatment Facility, Grasonville, Maryland. The sludge was sieved using a 2 mm screen and then settled for approximately 30 minutes. The supernatant above the settled solids were removed and the total suspended solids (TSS) concentration of settled sludge was measured. Based on the measured solids concentration, the sufficient amount of sludge and tap water to achieve 2500 mg/L in total test volume of 1.5 L was added to two semi-continuous activated sludge (SCAS) units.
The necessary volume of test substance to achieve 2 mg/L (based on a IL draw and fill volume), 10 mL of synthetic sewage, and adequate tap water to bring the volume up 1.5 L was added to SCAS units each day prior to the start of a 23.5-hour aeration period. At the end of the aeration period, the airflow was stopped and the sludge was settled for 30 minutes. One liter of effluent above the settled solids was drained from a port located at 500 mL mark. This cycle was repeated each working day for 3l-days.


SAMPLING
- Sampling frequency: every 7 day (days 0, 7, 14, 21, 28)
- Sampling method: not mentionned


CONTROL AND BLANK SYSTEM
- Inoculum blank: no information
- Abiotic sterile control: none
- Toxicity control: none


STATISTICAL METHODS:
No statistical method used beyond averages.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
None
Test performance:
No data
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 68.2
St. dev.:
4.6
Sampling time:
28 d
Remarks on result:
other: Freshly collected inoculum
Parameter:
% degradation (O2 consumption)
Value:
ca. 19.5
St. dev.:
6.1
Sampling time:
28 d
Remarks on result:
other: pre-exposed inoculum
Details on results:
Freshly Collected Inoculum
Average percent degradation at day 7, 14, 21, 28 are 0, 0, 6.5 and 68.2% respectively.

Pre-exposed Inoculum
Average percent degradation at day 7, 14, 21, 28 are 0, 0, 18.4 and 19.5% respectively.
Results with reference substance:
For the reference substance (sodium benzoate, ThOD 1.67 mg O2/mg) a percent biodegradation of greater than 60% was achieved by day 7, thereby fulfilling the criteria for a valid test.

Raw data are presented in Table 1 and Table 2 below. Percent degradation versus time is presented in Figures 1 and Figure 2.

Table 5.2.1/1: Freshly Collected Inoculum: Average Oxygen Uptake, Biochemical Oxygen Demand (BOD) and Percent Degradation Results

Test Chamber Conc. (mg A/L) Day DO (mg/L) DO Uptake Average Uptake BOD (mg/mg) Percent Degradation Average Percent Dagradation Standard Deviation
Control NA 0 8.6 NA NA NA NA NA NA
Control NA 0 8.5 NA NA NA NA NA NA
Control NA 7 8.5 0.1 0.1 NA NA NA NA
Control NA 7 8.5 0.1 NA NA NA NA
Control NA 14 7.8 0.8 0.5 NA NA NA NA
Control NA 14 8.4 0.2 NA NA NA NA
Control NA 21 7.9 0.7 0.7 NA NA NA NA
Control NA 21 7.9 0.7 NA NA NA NA
Control NA 28 8.4 0.2 0.2 NA NA NA NA
Control NA 28 8.4 0.2 NA NA NA NA
Benzoate 2 0 8.3 NA NA NA NA NA NA
Benzoate 2 0 8.5 NA NA NA NA NA NA
Benzoate 2 7 3.8 4.6 4.45 2.25 135% 130.2% 6.4%
Benzoate 2 7 4.1 4.3 2.1 126%
Benzoate 2 14 5 3.4 5.4 1.45 87% 146.7% 84.7%
Benzoate 2 14 1 7.4 3.45 207%
Benzoate 2 21 5.1 3.3 3.7 1.3 78% 89.8% 16.9%
Benzoate 2 21 4.3 4.1 1.7 102%
Benzoate 2 28 3.7 4.7 5 2.25 135% 143.7% 12.7%
Benzoate 2 28 3.1 5.3 2.55 153%
Test substance 2 0 8.7 NA NA NA NA NA NA
Test substance 2 0 8.6 NA NA NA NA NA NA
Test substance 2 7 8.6 0 0.05 -0.05 -2% 0.0% 3.1%
Test substance 2 7 8.5 0.1 0.05 2%
Test substance 2 14 8.2 0.4 0.4 -0.05 -2% -2.2% 0.0%
Test substance 2 14 8.2 0.4 -0.05 -2%
Test substance 2 21 7.8 0.8 1 0.05 2% 6.5% 6.1%
Test substance 2 21 8.5 1.2 0.25 11%
Test substance 2 28 5.5 3.2 3.35 1.5 65% 68.2% 4.6%
Test substance 2 28 5.2 3.5 1.65 71%

NA: Not Applicable

Table 5.2.1/2: Pre-exposed Inoculum: Average Oxygen Uptake, Biochemical Oxygen Demand (BOD) and Percent Degradation Results

Test Chamber Conc. (mg A/L) Day DO (mg/L) DO Uptake Average Uptake BOD (mg/mg) Percent Degradation Average Percent Dagradation Standard Deviation
Control NA 0 8.7 NA NA NA NA NA NA
Control NA 0 8.7 NA NA NA NA NA NA
Control NA 7 8.6 0.1 0.05 NA NA NA NA
Control NA 7 8.7 0 NA NA NA NA
Control NA 14 8.2 0.5 0.45 NA NA NA NA
Control NA 14 8.3 0.4 NA NA NA NA
Control NA 21 7.1 1.6 1.65 NA NA NA NA
Control NA 21 7 1.7 NA NA NA NA
Control NA 28 7 1.7 1.7 NA NA NA NA
Control NA 28 7 1.7 NA NA NA NA
Benzoate 2 0 8.5 NA NA NA NA NA NA
Benzoate 2 0 8.7 NA NA NA NA NA NA
Benzoate 2 7 4 4.6 4.5 2.275 136% 133.2% 4.2%
Benzoate 2 7 4.2 4.4 2.175 130%
Benzoate 2 14 5.3 3.3 3.25 1.425 85% 83.8% 2.1%
Benzoate 2 14 5.4 3.2 1.375 82%
Benzoate 2 21 4.2 4.4 4.5 1.375 82% 85.3% 4.2%
Benzoate 2 21 4 4.6 1.475 88%
Benzoate 2 28 3 5.6 5.4 1.95 117% 110.8% 8.5%
Benzoate 2 28 3.4 5.2 1.75 105%
Test substance 2 0 8.5 NA NA NA NA NA NA
Test substance 2 0 8.5 NA NA NA NA NA NA
Test substance 2 7 8.5 0 0 -0.025 -1% -0.5% 0.8%
Test substance 2 7 8.5 0 0 0%
Test substance 2 14 8.1 0.4 0.35 -0.025 -1% -2.2% 1.5%
Test substance 2 14 8.2 0.3 -0.075 -3%
Test substance 2 21 5.5 3 2.5 0.675 29% 18.4% 15.3%
Test substance 2 21 6.5 2 0.175 8%
Test substance 2 28 5.7 2.8 2.6 0.55 24% 19.5% 6.1%
Test substance 2 28 6.1 2.4 0.35 15%

NA: Not Applicable

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The final percent degradation of the test substance using freshly collected inocula was 68% (with the 10-day window). The test substance met the OECD criteria for readily biodegradability.
Executive summary:

The biodegradability of the test substance was evaluated using the Closed Bottle Test Method (OECD Guideline 301D). Degradation was followed by the analysis of dissolved oxygen over a 28-day test period. The dissolved oxygen uptake of the test solution was expressed as a percentage of the theoretical oxygen demand (ThOD) of the test substance. The test was performed using: a) freshly collected inoculum and b) inoculum that was pre-exposed to the test substance for 31 days. The test meets all the validity criteria of OECD Guideline 301D even though some information were not reported, such as measurement of pH and sampling method. However, these minor deviations are not deemed to affect the reliability of this study.

The final percent degradation of the test substance using freshly collected inocula was 68%. Furthermore, the test met the 10-day window since 6.5% degradation was observed at day 21 and 68% at day 28. Therefore, the test substance met the OECD criteria for readily biodegradability, including the 10-day window.

According to CLP criteria, the test substance is considered to be rapidly biodegradable.

Description of key information

OECD Guideline 301D, GLP, key study, validity 1:

68% degradation after 28 days, within the 10-day window, using freshly collected inoculum.

Readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Two studies are available to assess the biodegradation of the registered substance.

The first study, assessed as a reliable key study, was performed in an aerobic aqueous media, according to OECD Guideline 301D. The substance was exposed to activated sewage sludge (domestic, freshly collected) at a concentration of 2 mg/L. The degradation was assessed by the determination of the oxygen consumption. Control solutions with inoculum and the reference substance, benzoate, together with a toxicity control were used for validation purposes. Greater than 60% biodegradation of the substance was reached on Day 28, meeting the 10-day window as described in OECD guideline, and therefore should be considered to be readily biodegradable.

 

The second study, assessed as a reliable supporting study, was performed on the substance in an aerobic aqueous media, according to OECD Guideline 301F. The substance was exposed to activated sewage sludge micro-organisms at a concentration of 100 mg/L with culture medium in sealed culture at 21°C for 28 days. The degradation of the substance was assessed by the measurement of daily oxygen consumption. Control solutions with inoculum and the reference substance, benzoate, together with a toxicity control were used for validation purposes. The substance attained 54% degradation after 28 days, and therefore should not be considered to be readily biodegradable under this test conditions.

 

In conclusion, based on data available, the registered substance is considered to be readily biodegradable.