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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
Study period:
02 June 2015 to 15 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP in accordance with recognised guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
On five days of the study the temperature in a vessel containing water was recorded as being approximately 25 °C which is above the range of 22 ± 2 °C as stated in the study plan. Considered to have had no adverse effect on the study.
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
See above
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
yes
Remarks:
See above
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
-Physical state: pale yellow paste
- Analytical purity: 100% product
- Expiry date: 24 April 2017
- Storage condition of test material: Room temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
other: mixed population of activated sewage sludge micro-organisms
Details on inoculum:
- Mixed population of activated sewage sludge micro-organisms obtained on 15 June 2015 from the aeration stage of the Severn Trent Water Plc. Sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present.
- The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and used on the day of collection.
- Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel
- Filtration was continued for a further 3 minutes after rinsing the filter three successive time with 10 mL of deionised reverse osmosis water.
- The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to cool before weighing.
- The process was repeated until a constant weight was attained.
- The suspended solids concentration was equal to 3.2 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10 other: mg Carbon L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
MINERAL MEDIUM
- Mineral medium was that recommended in the OECD Guidelines (see Appendix 2, attached).

PRELIMINARY INVESTIGATIONAL WORK
- To investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, samples are taken for Dissolved Organic Carbon (DOC) analysis.
-The samples are either filtered or centrifuged to remove the sewage sludge solids
- Samples were analyzed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-Vcph TOC analyzer (see Appendix 3, attached).
- A nominal amount of test item (100 mg) was dissolved in mineral medium (1 L) to give a 100 mg/L stock solution.
- 2 samples were taken for DOC analysis; one untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter).
- A further nominal amount of test item (100 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 L.
- 2 samples were also taken for DOC analysis; one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and one after centrifugation at 4000 g for 15 mins.
- Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 30 mg ss/L and filtering or centrifuging as per test item samples.

TEST ITEM
-The test item was dissolved directly in mineral medium.
- A nominal amount (300 mg) of test item was dissolved in mineral medium with the aid of ultrasonication (20 minutes) and the volume was adjusted to 2 L to give a 150 mg/L stock solution.
- An aliquot (360 mL) of the stock solution was dispersed in inoculated mineral medium and the volume adjusted to 3 L to give a final concentration of 18.0 mg/L, equivalent to 10 mg carbon/L.
- The volumetric flask containing test item was inverted several times to ensure homogeneity of the solution.
-A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

REFERENCE ITEM
- Sodium benzoate (C6H5COONa) was used as the reference item in procedure control vessels.
- An initial stock solution of 1000 mg/L was prepared by dissolving the reference item in mineral medium with the aid of ultrasonication for approximately 10 mins.
- An aliquot (51.4 mL) of the stock solution was added to the test vessel containing inoculated mineral medium and the volume was adjusted to 3L to give a final test concentration of 17.1 mg/L (equivalent to 10 mg carbon/L).
- The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

TOXICITY CONTROL
- A toxicity control containing the test item and sodium benzoate was prepared in order to assess any toxic effect of the test item on sewage sludge micro-organisms.
- An aliquot of the test item stock solution (360 mL) was dispersed in inoculated mineral medium.
- An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3L to give a final concentration of 18.0 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

TEST SYSTEM
- The following were prepared and inoculated in 5L test culture vessels each containing 3L of solution:
(a) Inoculated control, in duplicate, consisting of inoculated mineral medium.
(b) Procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
(c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
(d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
- Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
- Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.
- The test was carried out in a temperature controlled room at 23 to 25 °C in the dark, which is above the range of 22 ± 2°C as stated in the study plan. This slight deviation was considered to have had no adverse effect on the study given that all validation criterion were met.
- Approximately 24 hours prior to addition of the test and reference items, the vessels were filled with 2400 mL of mineral medium and 28.1 mL of inoculum and aerated overnight.
- The test and reference items were added on day 0 and the pH of all vessels was measured using a Hach HQ40d Flexi handheld meter and adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution before the volume in each vessel was adjusted to 3L by the addition of mineral medium which had been purged overnight with CO2-free air.
- The test vessels were sealed and CO2-free air was bubbled through the solution in each vessel at a rate of 30 to 100 mL/min whilst stirring continuously using a magnetic stirrer.
- The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb) granules.
- The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH.
- The CO2 absorbing solutions were prepared using purified water.

EVALUATIONS
- The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

pH MEASUREMENT
- The pH of the test preparations was determined on Days 0 and 28 using a Hach HQ40d Flexi handheld meter prior to acidification with hydrochloric acid.
Reference substance:
other: sodium benzoate
Preliminary study:
PRELIMINARY INVESTIGATIONAL WORK
-Results from samples taken for DOC analysis from the preliminary work indicated that the test item did not absorb to filter matrices or to activated sewage sludge (see Appendix 3, attached).
- Therefore, samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.
Test performance:
DEFINITIVE TEST
- Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels are shown in Table 1 (attached).
- Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 (attached).
- Biodegradation curves are presented in Figure 1 (attached).
- Total and inorganic carbon values in the culture vessels on Day 0 are given in Table 3 (attached).
- The pH values of the test preparations on Days 0 and 28 are given in Table 4 (attached).
- Observations made on the contents of the test vessels are given in Table 5 (attached).

VALIDATION CRITERIA
- Total CO2 evolution in the inoculum control vessels on Day 28 was 32.83 mg/L and therefore satisfied the validation criterion given in the OECD test guidelines.
- The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3, attached) was below 5 % of the TC content and hence satisfied the validation criterion given in the OECD test guidelines.
- The difference between values for CO2 production at the end of the test for the replicate vessels was < 20 % and hence satisfied the validation criterion given in the OECD test guidelines.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
43
Sampling time:
28 d
Remarks on result:
other: Did not satisfy criteria for rapid biodegradability (> 60 % within 28 days)
Details on results:
BIODEGRADATION
- Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the test guidelines.
- Acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels.
- Any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
- The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels with the exception of test replicate 1 and the toxicity control vessel.
- Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
- The test item attained 43 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD guideline No. 301B.
- The toxicity control attained 65 % biodegradation after 14 days and 77 % biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.
Results with reference substance:
- Sodium benzoate attained 70 % biodegradation after 14 days and 86 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 43 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guidelines No. 301B.
Executive summary:

INTRODUCTION

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301B "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4 -C of Commission Regulation (EC) No 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph m).

 

METHODS

The test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 23 and 25 °C for 28 days.

 

The biodegradation of the test item was assessed by determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

RESULTS

The test item attained 43 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Study initiation date: Mar 29 2016 Experiment completion date: May 13 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
The test met the requirements of: Guidelines for Testing of Chemicals 301 D. Closed Bottle test. Paris: OECD, 1992.
Deviations:
no
Qualifier:
according to
Guideline:
other: HJ/T 153 -2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004
Version / remarks:
HJ/T 153 -2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004
Deviations:
no
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Degradation and Accumulation[M]. 2nd edition. Beijing: China Environment Press. 2013: 44-50.
Version / remarks:
CRC-MEP. The Guidelines for the Testing of Chemicals, Degradation and Accumulation[M]. 2nd edition. Beijing: China Environment Press. 2013: 44-50.
Deviations:
no
Qualifier:
according to
Guideline:
other: GB/T 21831-2008, Chemicals-Ready biodegradability Closed bottle test[S]: Beijin g: SAC, 2008.
Version / remarks:
GB/T 21831-2008, Chemicals-Ready biodegradability Closed bottle test[S]: Beijin g: SAC, 2008.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
EC Directive 2001/59, C.4-E, Closed Bottle Test. 0. J. L142, 2008.
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
Fate, Transport and Transformation Test Guidelines OPPTS 835.311 0. Ready Biodegradability. EPA 7 l 2-C-98-076, 1998.
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Remarks:
Samples of secondary effluent were obtained as the test inoculum from a sewage plant treating predominantly domestic sewage (Nanjing Chengdong Sewage Treatment Plant).
Details on inoculum:
Samples of secondary effluent were obtained as the test inoculum from a sewage plant treating predominantly domestic sewage (Nanjing Chengdong Sewage Treatment Plant). Aerobic conditions were maintained in the effluent by aeration from a laboratory supply of oil -free compressed air. (Batch No.: EW201604081).
On the day of the test, the test inoculum was prepared by filtering the sample of sewage effluent through a coarse filter, (discarding the first 200 mL) and collecting the remaining filtrates. This inoculum was maintained under aerobic conditions in the test area until use.
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
COD
Remarks:
The concentration of test substance was 3 mg/L, which equal to COD of 6.99 mg02/L.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
The BOD (mg 0 2/mg) of test and reference substance was calculated by dividing the blank-corrected level of oxygen consumption in the test or reference mixtures by the appropriate test concentration.
Details on study design:
Apparatus
BOD-Bottle (300 mL, blown, Jintan Jingbo Experiment Instrument Factory)
Incubator (MIR-253, Sanyo, Japan)
Analytical balance (MS105DU, METTLER TOLEDO, Switzerland) Water purification system (Milli-Q, Millipore, USA)
pH-meter (HQ40d, HACH, USA)
Dissolved oxygen meter (HQ40d, HACH, USA) COD analyzer (ET99718, lovibond, Germany) Transfer pettor (FINNPIPETTE, Thermo, USA).

Reagents
Deionized water: Resistivity = 18.2 MQ.cm
COD regent (0-150 mg/L) Lot: POSA
Supplier: Lovibond
Reagents for the medium were all analytical pure. Potassium di-hydrogen phosphate
Molecular formula: KH2P04 Purity: 99.5%
Lot: 14101112154
Supplier: Nanjing Chemical Reagent Co., Ltd.
Dipotassium hydrogen phosphate Molecular formula : K2HP04 Purity: 98.0%
Lot: 20130322
Supplier: Sinopharm Chemical Reagent Co., Ltd.
Disodium monohydrogen phosphate dihydrate Molecular formula : Na2HP0 4. 2H20
Purity: 99.5%
Lot: 20140710
Supplier: Sinopharm Chemical Reagent Co., Ltd.
Ammonium chloride Molecular formula: N CI Purity: 99.0%
Lot: 13082011297
Supplier: Nanjing Chemical Reagent Co., Ltd.
Calcium chloride Molecular formula: CaC12 Purity: 96.0%
Lot: 20130617
Supplier: Sinopharm Chemical Reagent Co., Ltd.
Magnesium sulphate heptahydrate Molecular formula: MgS0 4.7H20 Purity: 2: 99.0%
Lot: 14052811195
Supplier: Nanjing Chemical Reagent Co., Ltd.
Iron (III) chloride hexahydrate Molecular formula: FeCb.6H20 Purity: 2: 99.0%
Lot: 20131017
Supplier: Sinopharm Chemical Reagent Co., Ltd.
Concentrated hydrochloric acid Molecular formula: HCI Purity: 36.0 - 38.0%
Lot: 13032230133
Supplier: Nanjing Chemical Reagent Co., Ltd.
Sodium benzoate
Molecular formula: C6H5C0 2Na Purity: 2: 99.5%
Lot: 20141121
Supplier: Sinopharm Chemical Reagent Co., Ltd.
n-hexane
Molecular formula: C6H14 Purity: >=: 98.0%
Lot: K46764991524
Supplier: MERCK KGaA

Test Method
Preparation of the Test Solutions
Stock solution of the test substance (1064 mg/L): 0.0532 g of the test substance was dissolved into 50 mL with the n-hexane .
Stock solution of the reference substance (1001 mg/L): 1.0009 g of the reference substance (sodium benzoate) was dissolved into 1OOO mL with BSM.
Preparation of the Test Medium
The dilution medium (BSM) was prepared by adding 1 mL of each of the following stock solutions, prepared in pre-aerated deionized water to each litre of deionised water. Batch No.: BSM20160415.

Stock solution A g/L
KH2P04 (potassium dihydrogen phosphate) 8.50
K2HP0 4 ( dipotassium hydrogen phosphate) 21.75
Na2HP0 4. 2H20 (disodium monohydrogen phosphate dihydrate) 33.40
NH4Cl (ammonium chloride) The pH of this solution was 7.38 0.50

Stock solution B
CaCb (calcium chloride) 27.50
Stock solution C
MgS0 4. 7H20 (magnesium sulphate heptahydrate) 22.50

Stock solution D
FeCh.6H20 (iron (III) chloride hexahydrate) 0.25
Add 1 drop of concentrated hydrochloric acid per liter for storage.

Test Procedures
Four groups of "inoculum control", "test", "procedure control" and "toxicity control" were assigned in this test, with 2 replicates each. The test medium was aerated for 20 minutes and allowed to stand at the test temperature for at least 20 h before use. Dissolved oxygen of the mineral medium was measured to be 9.04 mg/L.
Add 20 mL of inoculum into 20 L BSM.
Firstly, 0.846 mL stock solution of test substance (1064 mg/L) was added to BOD-bottles of "test" and "toxicity control", with solvent was dried. Then, 600 µL of stock solution of sodium benzoate (1001 mg/L) was added to BOD-bottles of "procedure control" and "toxicity control"; then 0.846 mL n-hexan e was added into bottles of Inoculum control, with solvent was dried. Finally, BSM included inoculum was added to all BOD-bottles.
a) Inoculum control: containing inoculum only (1 mL/L);
b) Test 1: containing test substance (3 mg/L) and inoculum (1 mL/L);
c) Reference control: containing reference substance (2 mg/L) and inoculum (1 mL/L);
d) Toxicity control 1: containing test substance (3 mg/L), reference substance (2 mg/L) and inoculum (1 mL/L).
The BOD bottles were fully filled (Total Volume: 300 mL) with the aerated test medium using a hose which reached down to the bottom of the bottle to achieve adequate mixing. Each bottle was filled to ensure that no air bubbles were enclosed. Bottle numbers for each treatment groups were as follows: 4 bottles for toxicity control, 20 bottles for inoculum control, 20 bottles for test group, and 20 bottles for reference control.
The concentrations of dissolved oxygen in duplicate vessels of each ''test", "inoculum control", "procedure control" and ''toxicity control" group were determined after addition of the prepared solutions to the BOD bottles (0 d) and the remaining bottles were incubated at 20±1°C in darkness. Subsequent determinations of the concentration of dissolved oxygen (DO) in duplicate vessels for each group, except toxicity control, were made on 0, 2, 5, 7, 11, 14, 18, 21, 25 and 28 d.
The pH and temperature of the contents of each bottle were measured after the concentration of dissolved oxygen had been determined.
Determinations of DO, pH, and temperature in the "toxicity control" group were made at the start and after 14 days.

Analysis of COD
The COD was determined to substitute the ThOD which cannot be calculated as the test substance is mixture.
29.8 mg/Land 50 mg/L samples were prepared by adding 56 µLand 94 µL of stock solution of the test substance (1064 mg/L) into test tubes, with solvent was dried . Then 2 mL deionized water and COD reagents were added, and 3 parallel for each concentration. Blank control was prepared by above method expect for stock solution of the test substance substituted by solvent.
Heat all samples at 150°C for 120 minutes, and then determine the COD of samples after cool them to room temperature.

Validity of Test
The test is considered valid if:
a) The level of biodegradation of the reference substance achieves 60% or more within 14 days after the test starts, and
b) Following biodegradation of the test substance, the residual level of dissolved oxygen does not fall below 0.5 mg Oi/L, and
c) Oxygen consumption in inoculated blanks must not exceed 1.5 mg Oi/L after 28 days of incubation, and
d) Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%, and
e) Degradation of toxicity control is not less than 25% (based on ThOD) within 14 days.
Key result
Parameter:
% degradation (O2 consumption)
Value:
6.22
Sampling time:
28 d
Remarks on result:
other: The result indicated that biodegradation of the test substance was 6.22 % after 28-days
Details on results:
Test Results

ThOD or COD
Analytical Result of COD
COD results was shown in the attached table 4, COD of the test substance was 2.33 mg O2/mg based on the COD analysis.

Test Results
The attached Table 1 shows the temperature and pH during the 28-d test. The temperature was kept at 20±1°C and pH was kept at 6.69-7.13 (see attached Table 1). The test is valid because the biodegradation of the reference substance achieved 79.9 % within 14 days, achieving 60% within 14 days. Oxygen consumption in inoculated blanks was 1 .40 mg 0 2/L after 14 days of incubation, less than 1.5 mg 02/L. Degradation in the toxicity control was 28.0 % after 14 days, no less than 25% in 14 days. The result indicated that the test substance was not toxic to the inoculum. Additionally, the lowest level of dissolved oxygen in test substance group was 7.06 mg O2/L, which did not fall below 0.5 mg 02/L and the difference of biodegradation of the test item in the two replicates was less than 20%. Thus, all validity criteria listed were met. Therefore, the test is considered valid.
The attached Table 2 and Table 3 showed the concentrations of dissolved oxygen and the biodegradation data during the 28-d test, respectively. The result indicated that biodegradation of the test substance was 6.22 % after 28-days

Deviations
None.

Health & Safety
In order for PEAPC to comply with Law of the People's Republic of China on the Prevention and Treatment of Occupational Diseases 2001, and the current Control of Substances Hazardous to Health Regulations, it is a condition of undertaking the study that the Sponsor provides PEAPC with all information available to it regarding known or potential hazards associated with the handling and use of any substance supplied by the Sponsor to PEAPC. The Sponsor also complied with all current legislation and regulations concerning shipment of substances by road, rail, sea orair.
Such information in the form of a completed PEAPC test item data sheet must be received at PEAPC before the test item can be handled in the laboratory.

Maintenance of Records
All raw data arising from the performance of this study will remain the property of the Sponsor.
Records and documentation relating to this study (including the Study Protocol, raw data and a copy of the final report) will be maintained for a period of ten year from the date on which the Study Director signs the final report. Remaining samples will be retained by test facility in its archive for a period of one year from the date on which the Study Director signs the final repo rt. After this one year, if no request of sponsor on return or further retention of the materials, the retained samples will be disposed as hazardous waste process.
Test report in 3 copies with original signatures will be provided. 1 of them will be retained by test facility in its archive, and the remaining 2 will be sent to sponsor. The Quality Assurance records relevant to this study will also be archive
10 References
[l] HJ/T 153 -2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004
[2] CRC-MEP. The Guidelines for the Testing of Chemicals, Degradation and Accumulation[M]. 2nd edition. Beijing: China Environment Press. 2013: 44-50.
[3] GB/T 21831-2008, Chemicals-Ready biodegradability Closed bottle test[S]: Beiji ng: SAC, 2008.
[4] Guidelines for Testing of Chemicals 3010 . Closed Bottle test. Paris: OECD, 1992.
[5] EC Directive 2001/59, C.4-E, Closed Bottle Test. 0 . J. Ll42, 2008.
[6] Fate, Transport and Transformation Test Guidelines OPPTS 835.3110. Ready Biodegradability. EPA 712-C-98-076, 199 8.
Key result
Parameter:
COD
Value:
2.33 other: mg O2/mg
Remarks on result:
other: COD results was shown in the attached table 4, COD of the test substance was 2.33 mg O2/mg based on the COD analysis.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 6.22 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guidelines No. 301B.
Executive summary:

The study was conducted according to the guidelines of HJ/T 153-2004, "The Guidelines for the Testing of Chemicals, Degradation and Accumulation" (2nd edition) (Beijing: China Environmental Press. 2013), and with the reference to OECD Procedure 3010, Closed Bottle test etc. Under valid conditions, the ready biodegradability of the test substance was determined in a 28-day dissolved oxygen depletion using secondary effluent from a domestic waste water treatment plant.

The concentration of test substance was 3 mg/L, which equal to COD of 6.99 mg0 2/L. The concentration of inoculum in test system was 1 mL/L, and the tested concentration of sodium benzoate used as reference substance was 2.00 mg/L which equal to Th0 D NH3 of 3.34  mg02/L.

During the test, the temperature was kept at 20±1°C, pH was kept at 6.69-7.13. The test is valid because the biodegradation of the reference substance achieved 79.9 % within 14 days, achieving 60% within 14 days. Oxygen consumption in inoculated blanks was 1.40 mg 0 2/L after 28 days of incubation, less than 1 .5 mg 0 2/L. Degradation in the toxicity control was 28.0 % after 14 days, no less than 25% in 14 days. The result indicated that the test substance was not toxic to the in inoculum . Additionally, the lowest level of dissolved oxygen in test substance group was 7.06 mg 0 2/L, which did not fall below 0.5 mg 0 2/L and the difference of biodegradation of the test item in the two replicates was less than 20%. Thus, all validity criteria listed were met. Therefore, the test is considered valid.

Finally, the results showed that under the valid conditions, the average biodegradation rate of test substance was 6.22 % after 28-days.

Description of key information

In the key test Assessment of Ready Biodegradability; CO2 Evolution 41500916, The test item attained 43 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guidelines No. 301B.

Further in the supporting study Report for Ready Biodegradation  R2015NC066-03, The test item attained 6.22 %  biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guidelines No. 301B.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

INTRODUCTION

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301B "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4 -C of Commission Regulation (EC) No 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph m).

 

METHODS

The test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 23 and 25 °C for 28 days.

 

The biodegradation of the test item was assessed by determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

RESULTS

The test item attained 43 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.