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Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21-Dec-2017 to 18-Jan-2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Source and lot/batch No.of test material: Lot 20160912
- Purity: 99.2%
- Expiration date of the lot/batch: 1-August-2018
- Storage condition of test material: at room temperature
- Stability under test conditions: test substance stable

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from the 'Waterschap Aa en Maas' municipal sewage treatment plant ('s-Hertogenbosch, The Netherlands), receiving predominantly domestic waste. The sludge was diluted slightly with mineral medium, and the concentration of suspended solids was determined to be 4.0 g/L as used for the test.
- Storage conditions: Continuous aeration until use.
- Storage length: Not reported ("freshly obtained")
- Preparation of inoculum for exposure: Sludge allowed to settle 30 minutes and liquid decanted for use as inoculum.
- Pretreatment: None
- Concentration of sludge: 10 mL/L mineral medium
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
20 mg/L
Based on:
organic carbon
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: Mineral medium as per method TG 310, water was purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges (Millipore, Bedford, MA, USA)
- Solubilising agent: none
- Test temperature: 19 - 20 °C
- pH: 7.7 at beginning of test
- pH adjusted: no
- Continuous darkness: yes
- Culturing apparatus: septum-sealed glass bottles, 160 mL capacity
- Number of culture flasks/concentration: triplicate bottles for days 1, 7, 14, and 21, five bottles for day 28, plus three parallel reserve vessels
- Method used to create aerobic conditions: Test was performed in sealed bottles with a headspace: medium ratio of 1:2 to provide a reservoir of oxygen adequate for the duration of the exposure.
- Measuring equipment: Equipment as detailed
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Details of trap for CO2 and volatile organics if used: Closed bottle test. Evolved CO2 was forced into the test medium by the addition of 1 mL 7M NaOH.
- Sampling frequency: Days 1, 7, 14, 21, and 28
- Sampling method: bottles sacrificed and analyzed as detailed
- Sterility check if applicable: None
- Inoculum blank: Yes, sampled at each time point
- Abiotic sterile control: yes, inoculum free sample additionally treated with 1 mL/L of a 10 g/L solution of HgCl2 (sampled on Days 1, and 28 only)
- Toxicity control: Yes, reference substance and MTDID 49220, each at 20 mg C/L (sampled on Days 1, 7, and 14 only)
- Positive control: Sampled on Days 1, 7, and 14 only
Reference substance
Reference substance:
Octan-1-ol (CAS No. 111-87-5)

Results and discussion

% Degradation
Key result
% degradation (inorg. C analysis)
ca. 0
Sampling time:
28 d
Remarks on result:
Not significantly different from blanks
Details on results:
Average inorganic carbon (IC) concentration in blanks (Table 1) on Day 28 was 0.8838 mg/L (SD, 0.181 mg/L). Average IC concentration in MTDID 49220 bottles (Table 2) on Day 28 was 0.8968 mg/L (SD, 0.045 mg/L), corresponding to 0% degradation.
The reference substance was 64% degraded by day 14 (Table 3). The toxicity control (Table 3) exhibited 29% degradation on Day 14, thus exceeding the criteria of greater than 25% degradation by Day 28, and thus MTDID 49220 was considered to have not inhibited microbial activity in this study.

Any other information on results incl. tables

Table 1, Individual concentrations of inorganic carbon (IC) in mg/L for the blank control during the biodegradation test.

Day Vessel A Vessel B Vessel C Vessel D Vessel E Mean Std. Dev. 
1 0.9454 1.022 0.8558 n.a. n.a. 0.9411 0.083
7 0.7469 0.8846 0.9757 n.a. n.a. 0.8691 0.115
14 0.8836 1.044 1.076 0.9720 1.222 1.040 0.126
22 1.082 0.8734 0.8760 n.a. n.a. 0.9438 0.120
28 3.091* 0.8108 0.7392 0.8372 1.148 0.8838 0.181

n.a. - not applicable (not measured)

* - outlier according to Dixon's Q-test; value is not used in further calculations

Table 2, Individual concentrations of inorganic carbon (IC) in mg/L for MTDID 49220 during the biodegradation test.

Day Vessel A Vessel B Vessel C Vessel D Vessel E Mean Std. Dev.  Percent Degradation
1 0.8749 1.124 1.085 n.a. n.a. 1.0280 0.134 0
7 1.120 1.034 1.062 n.a. n.a. 1.0720 0.044 1
14 1.237 1.286 0.8520 n.a. n.a. 1.125 0.238 0
22 1.474 0.9756 1.022 n.a. n.a. 1.1572 0.275 1
28 0.9761 0.8818 0.8759 0.8701 0.8801 0.8968 0.045 0

Table 3, Individual concentrations of inorganic carbon (IC) in mg/L for positive control (R), toxicity control (T), and abiotic control (A) during the biodegradation test.

Day Vessel A Vessel B Vessel C Vessel D Vessel E Mean Std. Dev.  Percent Degradation
R1 0.8717 1.072 1.261 n.a. n.a. 1.0682 0.195 1
R7 9.614 11.42 11.19 n.a. n.a. 10.7413 0.983 49
R14 16.1 13.08 14.38 14.39 10.99 13.788 1.897 64
T1 0.8454 1.198 1.206 n.a. n.a. 1.0831 0.206 0
T7 10.09 7.308 9.009 n.a. n.a. 8.8023 1.402 20
T14 10.98 15.02 12.29 12.10 13.60 12.7980 1.552 29
A1 0.9432 0.9523 0.7365 n.a. n.a. 0.8773 0.122 0
A28 0.6316 0.4465 0.7861 0.4906 0.4063 0.5522 0.156 0

Applicant's summary and conclusion

Validity criteria fulfilled:
less than 3 mg/L inorganic carbon was in blanks at end of test, reference substance greater than 60% degraded by day 14
Interpretation of results:
under test conditions no biodegradation observed
MTDID 49220 is not readily biodegradable, as determined according to OECD TG 310.
Executive summary:

Ready biodegradability of MTDID 49220 was studied in a test done according to OECD TG 310. MTDID 49220, octan-1-ol (reference substance), or both chemicals (toxicity control) were introduced to test bottles at 20 mg/L theoretical organic carbon (ThOC). Bottles also contained mineral medium inoculated with activated sludge from a predominantly domestic source. Blank bottles containing only inoculated medium were also prepared. Bottles were sacrificed at intervals by addition of 7M NaOH to draw evolved CO2 into the mineral medium. After equilibration, CO2 concentrations were measured using a carbon analyzer.

No degradation of MTDID 49220 was measured after 28 days of incubation. The blank bottles had < 3 mg/L inorganic carbon, and the reference substance was >60% degraded by day 14. The toxicity control exhibited sufficient degradation (>25% by 14 days) that MTDID 49220 was considered to have not inhibited microbial actitivity in this test. Since MTDID 49220 was not biodegraded in this test, MTDID 49220 is not readily biodegradable. This test was conducted under an internationally accepted guideline for volatile chemicals, and was in accord with GLP criteria. Therefore, this study is reliable without restrictions.