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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:
from 2017-11-19 to 2018-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MEP, P.R. China, the Guideline for the Testing of Chemicals, Degradation and Accumulation (the Second Edition), No. 301A "DOC Die-Away Test"
Version / remarks:
2013.9
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
A fresh sample of activated sludge was collected from a sewage treatment plant on November 22, 2017 as recommended by the OECD Guidelines for the Testing of Chemicals. The sample of activated sludge was collected from the aeration tank of Shenyang North Sewage Treatment Plant (No.258, Kunshan Xilu Huanggu District Shenyang City) prior to the test. It is a properly working treatment plant receiving predominantly domestic sewage.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Test temperature: 22+/-2
- pH: 7 - 8
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no, diffuse light

TEST SYSTEM
- Number of culture flasks/concentration: 2

SAMPLING
- Sampling frequency: day 0, 2, 4, 7, 10, 14, 19, 23, 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
- Other: positive control
Reference substance:
benzoic acid, sodium salt
Test performance:
The validity criteria were met.
Key result
Parameter:
% degradation (DOC removal)
Value:
< 3
Sampling time:
28 d
Details on results:
The substance reached a biodegradation level of ca. 2.8% (DOC removal) after 28 d in an OECD 301A guideline study. Based on these results, the test item is not readily biodegradable according to OECD criteria under the test conditions.
Results with reference substance:
The percentage degradation of reference item attained 98.7% after 2 days and 100.2% at the 14th day, which exceeded the 70% pass level. The test was considered valid.

In the toxicity control containing test item, reference item and inoculums, the percentage degradationbased on total DOC attained47.6%after 2 days and 48.7%at the 14th day, which exceeded 35%,indicating that the test item can be assumed to be not inhibitory to activated sludge under the conditions of the test.

In the adsorption control containing test item and sterilising agent, the percentage removal of test item was 2.8% at the 28 day, the test item can be assumed to be not adsorbed by activated sludge particles.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance reached a biodegradation level of ca. 2.8% (DOC removal) after 28 d in an OECD 301A guideline study. Based on these results, the test item is not readily biodegradable according to OECD criteria under the test conditions.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2019-08-22 to 2020-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge: The sludge, water and soil were sampled from 10 sites on 13th Aug. 2019.
- City sewage plant: Zhu Yuan Domestic Sewage Treatment Plant and LangXia Wastewater Treatment Plant in Shanghai, China Industry sewage plant: Sino
- French Water in Shanghai, China
- River: Suzhou River in Shanghai, China; Chuanyang River in Shanghai, China; Huangpu River in Shanghai, China; Changjiang estuary in Shanghai, China
- Lake: Dianshan Lake in Shanghai, China
- Sea: Hangzhou Bay in Shanghai, China
- Soil: farmland soil, wetland soil and garden soil in Shanghai, China

- Preparation of inoculum for exposure: A fresh sample of inoculum was collected from the culture tank cleaned and washed three times with mineral medium. The sludge was separated by centrifuging for 20 minutes at about 4000 rpm.
- Pretreatment: All samples were mixed by stirring in a single container for 1 h and then settled for 1 h. The supernatant was transferred to a culture tank after removing the floating foreign matter. The culture was aerated and adjusted to pH 7.0 ± 1.0. Approximately 1/3 of the whole volume of the supernatant was removed and an equal volume of synthetic sewage (1 g of glucose, 1 g of peptones and 1 g of monopotassium phosphate are dissolved in 1 litre of water and the solution is adjusted to pH 7.0 ± 1.0 with sodium hydroxide) was added to the remaining portion of the supernatant everyday.
- Concentration of sludge: 2 g suspended solids/L
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: According to OECD 302 C (1981)
- Additional substrate: no
- Solubilising agent: no
- Test temperature: 25.0 - 25.3 °C.
- pH: 7.0 ± 1.0
- pH adjusted: yes
- Aeration of dilution water: si
- Suspended solids concentration: 2 g suspended solids/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Brown bottles with a volume of 500 mL were used.
- Number of culture flasks/concentration: five bottles (one bottle of blank control group, three bottles of test substance group and one bottle of abiotic control group) were used for zero-time HPLC analysis. One bottle of each blank control group, test substance group and abiotic control group were used for initial DOC determination. At the end of the test, the test suspensions of blank control group, test substance group and abiotic control group (one bottle of blank control group, three bottles of test substance group and one bottle of abiotic control group) were taken for HPLC analysis. One bottle of blank control group, three bottles of test substance group and one bottle of abiotic control group were used for DOC determination.
- Measuring equipment:
Moisture meter (Mettler Toledo HG63, INS-026-01);
BOD-meter (WTW Oxitop 110 C, INS-013-01);
MJ-250I Incubator (YIHENG TEC, MJ-250I, INS-022-05);
WTW Multi 3430 water quality meter (WTW, Multi 3430, INS-087-02);
Analytical balance (METTLER TOLEDO XS 250DU, INS-009-01);
KIMO electyonic temperature and humidity meter (KIMO, KT-210-AO, INS-167-03) ;
KMM700 Series Multifunctional mixer (KENWOOD, KMM700, INS-139-01);
ME2002E/02 Electronic Balance (ME2002E/02, INS-144-01);
High Speed Refrigerated Centrifuge (BECKMAN COULTER J-26XP, INS-028-01);
Spectroquant® Pharo 300 Spectrophotometer (Merck,Spectroquant Pharo 300, INS-126-01);
Total Organic Carbon Analyzer (Jena Multi N/C 3100, Multiwin v4.09, INS-012-02).

BOD Determination:
BOD is determined by WTW Oxitop Operational procedure as follows:
1) Estimate the measuring range of the test substance to be analyzed.
2) Insert a magnetic stirrer bar into the bottle.
3) Place 2 sodium hydroxide pellets in the rubber sleeve.
4) Insert the rubber sleeve onto the bottle.
5) Screw on the OxiTop® measuring head tightly.
6) Start the measurement on the OxiTop® head.
7) Place the overflow measuring flask in the incubator for 28 days.
8) All the data of oxygen demand are recorded automatically by OxiTop® measuring head. Stirring, temperature and data, are periodically inspected. Final results are recorded after 28 days.
- pH measurement: at the start and end of incubation, the pH is measured of each flask.
- Test performed in closed vessels: yes
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: The CO2 was absorbed by sodium hydroxide, which results in a decrease of the total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer.

TEST SOLUTIONS
- Test substance preparation: The test substance was added by preparing stock solution. To prepare the stock solution A of test substance, 200.09 mg of test substance was dissolved in 200 mL mineral medium and the concentration of the stock solution was 1 g/L. To prepare the stock solution B of the test substance, 200.09 mg of the test substance was dissolved in 200 mL ultra pure water and the concentration of the stock solution was 1 g/L.
- Reference compund preparation: To prepare the stock solution of the reference compound, 400.04 mg of sodium benzoate was dissolved in 200 mL mineral medium and the concentration of stock solution was 2 g/L. The final concentration of reference compound in the test system was 100 mg/L.

SAMPLING
- Sampling frequency: At the start (0 h) and at the end of the test.
- Sampling method: To demonstrate the validity of the method, three aliquots of 100 µL of the working solution with concentration of 10.7 mg/L were added each to 900 µL of sludge suspension to obtain three spiked samples at 1.07 mg/L. Three aliquots of 30 µL of the working solution with concentration of 1070 mg/L were added each to 970 µL of sludge suspension to obtain three spiked samples at 32.1 mg/L. At the same time, one blank sample was prepared and analyzed. These solutions were analyzed by HPLC to determine the recovery and validate the precision (RSD) of method after the pretreatment of samples. The samples, sludge suspension spiked samples and sludge suspension blank and then mixing and homogenization. 1 mL sample solution was taken and centrifuged for 10 min at 13000 rpm, and the supernatants were taken and analyzed by HPLC after dilution by water to the calibration curve range for the sample with concentration above of the highest standard.
- Sample storage before analysis: The samples were stored in refrigerator (2 ~ 8 °C) after pretreatment if the samples were not was analyzed directly within 24 h.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
- Reference control: yes

Reference substance:
benzoic acid, sodium salt
Preliminary study:
The COD of three replicates of the test solution was determined by spectrophotometer using a COD kit whith a range of 25 to 1500 mg/L. The COD values of the three replicates in the test system were 27 mg/L, 28 mg/L and 30 mg/L with the average COD value of 28.33 mg/L.
Test performance:
The validity criteria were met. The average percentage of degradations of procedure controls were 76 % (> 40 %) and 89 % (> 65 %) after 7 and 14 days, respectively. It is concluded that the inoculum degradation activity meets the requirements of this test. The degradation percentage of toxicity control on day 14 was 67 %, which was higher than the toxicity inhibition level (> 25 %). The results of activated sludge respiration inhibition test showed that the test substance has no inhibitive effect on inoculum.

Key result
Parameter:
% degradation (DOC removal)
Value:
11
Sampling time:
28 d
Key result
Parameter:
% degradation (test mat. analysis)
Value:
14
Sampling time:
28 d
Details on results:
- Results of biodegradation determined by respirometer: The results of BOD indicated that the cumulative oxygen consumption level of three replicates of test substance group all were 120.00 mg/L. They were very similar as the mean cumulative oxygen consumption of blank controls (122.00 mg/L). All the results indicated that no mineralization occurred during the 28 days (the test substance could not be totally utilised by micro-organisms resulting in the production of carbon dioxide, water, mineral salts etc.). There was no biodegradability of the test substance based on the “OECD guidelines for the testing of chemicals: revised introduction to the OECD guidelings for testing of chemicals, section 3, 2006”. No oxygen consumption could be observed in the abiotic control on day 28, the calculation of the results showed that there was no aerobic abiotic degradation.
- DOC determination: The results of DOC determination showed that the DOC of inoculum control group (IB), test substance group (TS) and abiotic control group (AC) at zero-time were 0.567 mg/L, 8.32 mg/L and 7.66 mg/L, respectively. The DOC of IB was 0.555 mg/L, the average DOC of TS was 7.80 mg/L and the DOC of AC was 8.11 mg/L on 28th day.The DOC removal of test substance on 28th day was 11 %.
- Concentration analysis by HPLC: The percentage biodegradation of the test substance was 14 % on day 28 which was calculated based on the residual concentration of HPLC analysis.
The test substance was found to be non-inherent biodegradable under the present test conditions.
Results with reference substance:
The mean degradation percentage of the reference compound were 76 % and 89 % after 7 and 14 days, respectively.
Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
The study of inherent biodegradation of the test substance was performed according to OECD 302 C (1981) under GLP conditions. The mean percentage biodegradation of the test substance on the 28th day was 14% based on the calculated results of HPLC analysis and 11% based on DOC removal. The test substance was found to be non-inherent biodegradable under the present test conditions.
Executive summary:

The study of inherent biodegradation of the test substance was performed by the modified MITI test II (“Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals – Degradation and Accumulation, 2013, 302C, Inherent Biodegradability: Modified MITI Test II” and “OECD, Guidelines for the Testing of Chemicals, 1981, 302C Inherent Biodegradability: Modified MITI Test (II)”. The results of biochemical oxygen demand (BOD) indicated that the cumulative oxygen consumption level of three replicates of test substance group all were 120.00 mg/L. They were very similar as the mean oxygen consumption of blank controls (122.00 mg/L). The calculated percentage of biodegradation based on the BOD determination showed that this test substance is not inherently biodegradable. 

The mean percentage biodegradation of the test substance on the 28thday was 14% based on the calculated results of HPLC analysis. The test substance has not been shown to be inherently degradable under the present test conditions.

Description of key information

The substance reached a biodegradation level of ca. 2.8% (DOC removal) after 28 d in an OECD 301A guideline study. Based on these results, the test item is not readily biodegradable according to OECD criteria under the test conditions.

In addition, a study of inherent biodegradation of the test substance was performed according to OECD 302 C (1981) under GLP conditions. The mean percentage biodegradation of the test substance on the 28th day was 14% based on the calculated results of HPLC analysis and 11% based on DOC removal. The test substance was found to be non-inherent biodegradable under the present test conditions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information