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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2011-03-07 - 2011-04-28
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))
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Chemical name: monoethyleneglycol, polybutyleneoxidediamine
- Molecular formula: C10H24N2O2
- Appearance: clear liquid
- Lot/batch No.: HSMX20110211-0184
- Receipt date: 2011-02-15
- Purity: 90%
- Storage condition of test material: in original container (plastic bottle with lid) at 4°C
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Sludge: two city sewage plants (Jiangqiao WWT plant and Shidongkou WWT plant) and one industry sewage plant (Sino French water)
Water: two rivers (Suzhou River, Yangtze River), one lake (Dianshan Lake), one estuary (Yangtze River estuary)
Soil: one farm, one garden and one wetland from Qinpu County
- Culturing: All samples mixed and after removing floating particles and debris cultured at 25°C and adjusted to pH 7.0. Ca. 1/3 of supernatant removed every day and replaced by equal volume of synthetic sewage.
- Age of culture used for inoculation: 2 months
- Preparation of inoculum for exposure: Sample of sludge washed twice with mineral medium (sludge separated by centrifuging for 20 min at 4000 rpm). Small amount of sludge dried (105°C, 1 h) to determine dry weight (5.99%). Wet sludge suspended in mineral culture medium to obtain concentration of 2 g dw/L. This suspension was used as inoculum.
- Initial cell/biomass concentration: 100 mg dw/L.
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: standard mineral medium made up of KH2PO4, K2HPO4, Na2HPO4.2H2O, NH4Cl, MgSO4.7H2O, CaCl2, and FeCl3.6H2O in ultra pure water.
- Additional substrate: none
- Test temperature: 23.1-24.8°C
- pH: adjusted to 6.51-7.93
- pH adjusted: yes, before start of testing
- Suspended solids concentration: 100 mg dw/L at start of testing
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: automated closed-system oxygen consumption measuring apparatus (BOD-meter), with magnetic stirrer
- Number of culture flasks/concentration: 2+1 for blank control, 3+3 for test substance group, 1 for toxicity control, 2 for procedure control, 1+1 for abiotic control (the additional flasks are used for zero-time HPLC analysis).
- Measuring equipment: BOD-meter (WTW Oxitop 110)
- Details of trap for CO2: absorbed using NaOH (placing 2 NaOH pellets in the rubber sleeve of the bottle)

SAMPLING
- pH measured at the start and end of incubation.
- Nitrite and nitrate concentrations measured at end of incubation.
- Five bottles (blank control, test substance group, and abiotic control) were used for zero-time HPLC analysis.
- After 28 days, test suspensions of blank control, test substance group and abiotic control were taken for HPLC analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, inoculum added but no test substance
- Abiotic sterile control: yes, test substance added but no inoculum
- Toxicity control: yes, inoculum, test substance and reference substance added
- Procedure control: yes, inoculum and reference substance added
Reference substance:
benzoic acid, sodium salt
Test performance:
% degradation in toxicity control 41.81% after 14 days. This is higher than the toxicity inhibition level (> 25%), hence the test substance has no inhibition effect on the inoculum at the concentration of 30 mg/L.
Key result
Parameter:
% degradation (test mat. analysis)
Value:
3.88
Sampling time:
28 d
Details on results:
- Cumulative oxygen consumption level of 3 replicates of test substance group were 121.0, 106.0 and 117.0 mg/L (corrected for nitrification = 111.8, 96.4 and 105.5 mg/L), respectively, which is very similar to the mean cumulative oxygen consumption in blank controls (116.0 mg/L). Conslusion: no ultimate biodegradation occurred within 28 days.
- HPLC measurements: 30.52 and 30.96 mg/L (mean value) in test substance group and abiotic control, respectively, at the start of testing. After 28 days: 31.71 and 32.99 mg/L (mean value) in test substance group and abiotic control, respectively. Conclusion: no significant primary degradation after 28 days.
Results with reference substance:
% degradation of reference substance 63.35% and 64.13% after 7 and 14 days, respectively (requirements 40% and 65% after 7 and 14 days, respectively). Test can be considered valid.
Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
In this inherent biodegradability test performed according to OECD guideline 302C (modified MITI test), no significant ultimate or primary biodegradation of the substance was observed after 28 days of incubation using an initial test substance concentration of 30 mg/L with 100 mg dw/L of a mixed sludge (domestic and industrial WWTP, natural waters and soils). The toxicity control and procedure control indicated that the test can be considered valid.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Physical state: Clear colourless liquid
- Analytical purity: 97.8%
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 01 January 2004
- Storage condition of test material: at room temperature in the dark
- Other: specific gravity: > 0.94 g/cm3
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source: municipal sewage treatment plant 'Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands
- Storage conditions: under continuous aeration
- Preparation of inoculum for exposure: sludge allowed to settle (30-90 min), liquid decanted for use as inoculum
- Concentration of sludge: 3 g/L in concentrated sludge from treatment plant
- Initial cell/biomass concentration: not reported (10 mL of decanted fluid added per L of mineral medium)
- Before testing, mixture of mineral medium and inoculum aerated with CO2 free air overnight to purge the system of CO2.
Duration of test (contact time):
28 d
Initial conc.:
12 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium with 10 mL of solution A and 1 mL of solutions B, C and D in 1 L (made up with Milli-RO water (tap water purified by reverse osmosis). Solution A = 8.5 g KH2PO4, 21.75 g K2HPO4, 67.2 g Na2HPO4.12H2O and 0.5 g NH4Cl in 1 L Milli-Q water. Solution B = 22.5 g MgSO4.7H2O in 1 L Milli-Q water. Solution C = 36.4 g CaCl2.2H2O in 1 L Milli-Q water. Solution D = 0.25 g FeCl3.6H2O in 1 L Milli-Q water.
- Test temperature: 21.9-23.4°C
- pH: 7.4-8.5
- pH adjusted: no
- Pretreatment of incubation medium: aeration overnight with CO2-free air prior to testing.

TEST SYSTEM
- Culturing apparatus: 2-L glass brown coloured bottles, three CO2-absorbers connected in series to exit air line of each test bottle
- Number of culture flasks/concentration: 2 (for blank controls and treatment), 1 (for positive control and toxicity control)
- Method used to create aerobic conditions: aeration with CO2-free air
- Measuring equipment: see analytical methods
- Details of trap for CO2 and volatile organics if used: 3 CO2-absorbers in series per test bottle, containing 100 mL 0.0125 M Ba(OH)2

SAMPLING
- Sampling frequency: titrations every 2nd or 3rd day during first 10 days, every 5th day thereafter until the 28th day (last day: bottles + 1 mL concentrated HCl and aerated overnight to drive off CO2 present in test suspension - final titration therefore on day 29)

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 bottles containing only inoculum
- Positive control: 1 bottle containing reference substance and inoculum
- Toxicity control: 1 bottle containing test substance, reference substance and inoculum

STATISTICAL METHODS:
none reported
Reference substance:
other: sodium acetate
Preliminary study:
none
Test performance:
Reference substance degraded 73% within 14 days (mininum required = 60%).
Total CO2 release in blank at end of test did not exceed 40 mg/L (22 mg CO2/L).
Difference of duplicate values for % degradation was always less than 20.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
1 - 3
Sampling time:
28 d
Remarks on result:
other: average = 2%
Results with reference substance:
73% degradation within 14 days (86% within 28 days)
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
This ready biodegradability study investigated biodegradation of the test compound according to the OECD 301B guideline (CO2 evolution test). Activated sludge from a municipal waste water treatment plant was exposed to a single concentration of 12 mg TOC/L during 28 days. All vessels were aerated with CO2-free clean air and any CO2 formed was trapped in Ba(OH)2. The results from the test indicate only 1-3% biodegradation (average = 2%) of the test substance after 28 days, implying that the substance is not readily biodegradable. The results of this study are valid and reliable (Klimisch 1).

Description of key information

In both a ready biodegradation (key study) and inherent biodegradation (supporting study), no significant breakdown of the substances was observed.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information