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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2017 to 21 December 2017
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Due to the low solubility of the test material and its adhesive nature, a nominal 3000 mg/L stock solution of the test material was prepared 96 hours in advance of test initiation.
- The stock solution was prepared by weighing 3.0143 g of the test material into a 1000 mL aspirator bottle and making this up to volume in Millipore MilliQ™ water. The test material was weighed out onto a pre-weighed plastic weigh boat containing a monolayer of glass beads. The test material remained attached to the surface of the glass beads, while both the glass beads and the test material were transferred to the glass aspirator bottle leaving none of the test material in the plastic weigh boat. This stock solution was stirred for approximately 96 hours and allowed to settle for one hour.
- The first ~ 25 mL of stock solution was removed from the upper portion of the solution via the top of the glass bottle and discarded. Approximately 200 mL of the stock solution was removed and transferred into a clean 250 mL beaker in the same fashion for use in preparing the test solutions. The COD of the test material was 1.061 mg O2/mg of test material. A 6-L volume of the test solution with a COD of 4.0 mg/L was prepared by pipetting 45.715 mL of stock solution into a 6-L Erlenmeyer flask and making this up to volume using inoculated mineral medium. This test solution was dispensed into BOD bottles (i.e., one set of duplicate bottles for each sampling period).
Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: secondary effluent was collected from the Guelph Wastewater Treatment Plant (Guelph, Ontario). The Guelph Wastewater Plant treats sewage of predominantly domestic origin.
- The secondary effluent was continuously aerated during transport to, and storage at the testing laboratory, and then pre-conditioned for testing. During pre-conditioning, the secondary effluent was maintained at test temperature (22 ± 2°C) prior to test initiation. The secondary effluent was settled for approximately 30 minutes prior to the clear supernatant being decanted for use as the inoculum.
Duration of test (contact time):
35 d
Initial conc.:
3.77 mg/L
Based on:
test mat.
Initial conc.:
4 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium: The composition is equivalent to that described in OECD Guideline 301 D (OECD, 1992), with the exception that Na2HPO4·2H2O is replaced by the appropriate amount of Na2HPO4.
On the day prior to test initiation, 28 L of mineral medium was prepared and the dissolved oxygen, pH and temperature were confirmed prior to use in the test. The dissolved oxygen concentration was 8.5 mg/L. The pH was within the required range (7.4 ± 0.2) and did not require adjustment. Temperature of the mineral media was also within the required range (22 ± 2°C).
- pH adjusted: not required
- Suspended solids concentration: 5 mL/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Glass, with ground glass stoppers and plastic caps (BOD bottles), 300 mL.
- Number of culture flasks/concentration: 2 bottles for inoculated control, reference, inhibition and test series; 1 bottle for the non-inoculated control
- Measuring equipment: Oxygen electrode
- Test performed in closed vessels: yes

SAMPLING
- Sampling frequency: Days 0, 4, 7, 11, 14, 28 and 35

CONTROL AND BLANK SYSTEM
- Non-Inoculated and Inoculated Blanks: A single non-inoculated control and duplicate inoculated control test vessels (BOD bottles) were established for each of the sampling periods. The pH of the mineral medium was measured prior to inoculation. The temperature of the mineral medium was monitored to ensure that it was within the required temperature range of 22 ± 2°C. The control bottles were treated similarly to the test bottles, except that no test material was added. Oxygen content of these bottles was measured in the same fashion as the test, reference and inhibition series.
- Reference Series (Procedural Control): A reference series was conducted in parallel with the test material series, to verify that the test system was working appropriately and that the reference substance by itself was not inhibitory to sewage bacteria. The reference substance, KHP with a COD of 1.161 mg/mg KHP, was tested at a concentration of 4 mg/L COD under the same conditions and with the same sampling intervals used in the test material series. A stock solution of KHP with a nominal COD of approximately 1000 mg/L (equivalent to 854 mg KHP/L) was prepared by weighing 0.854 g of the reference substance into a 1-L volumetric flask and making this up to volume in Millipore MilliQ™ water. The stock solution was hand mixed immediately prior to testing. The final COD of the stock solution was estimated to be 991.5 mg/L (based on the average of two samples). A 6-L volume of the reference solution with a final COD of 4.0 mg/L was prepared by pipetting 24.205 mL of stock solution into a 6-L Erlenmeyer flask and making this up to volume using inoculated mineral medium. This solution was dispensed into fourteen test vessels (i.e., one set of duplicate test vessels for each sampling period).
- Inhibition Series (Toxicity Control): An inhibition series was conducted in parallel with the test series, to determine whether the test material was inhibitory to sewage bacteria (i.e., the test material can be assumed to be inhibitory if less than 25% degradation occurs in this test within 14 days). The inhibition test utilised both the test material and the reference substance. The concentrations of the test material and reference substance used for the inhibition series were the same as in the test and reference series (i.e., 4 mg/L COD). As such, 45.715 mL of the test material stock solution and 24.205 mL of the reference substance stock solution were added into a 6-L Erlenmeyer flask and made up to volume using inoculated mineral medium. This solution was then dispensed into fourteen test vessels (i.e., one set of duplicate test vessels for each sampling period).

STATISTICAL METHODS:
The BOD was calculated as follows:
BOD = [(mg O2/L uptake of test material – mg O2/L uptake of blank) / mg test material/L in vessel ] = mg O2/mg test material
The amount of oxygen consumed within the test system was compared to the COD of the test material and the percent (%) degradation was calculated as follows:
% biodegradation at time t = (BOD (mg/L) at time t (days) / COD (mg/L)) X 100%
Using this method, the extent and rate of biodegradability was determined based on the cumulative uptake of molecular oxygen.
Reference substance:
other: Potassium Hydrogen Phthalate (KHP)
Test performance:
The definitive test met the test validity criteria described below:
- The reference substance achieved > 60% degradation within 14 days of test initiation.
- The difference of extremes of replicate values of the removal of the test material at the plateau, at the end of the test or at the end of the 10-day window, as appropriate, was less than 20%.
- Inoculated control (nutrient blank) did not exhibit more than 1.5 mg/L reduction in dissolved oxygen concentration in 28 days.
- The residual dissolved oxygen concentration in the test vessels did not fall below 0.5 mg/L.
- The toxicity control achieved > 25% degradation in 14 days
Key result
Parameter:
% degradation (O2 consumption)
Value:
6.2
Sampling time:
35 d
Parameter:
% degradation (O2 consumption)
Value:
10
Sampling time:
28 d
Details on results:
CONFIRMATION OF THE TEST MATERIAL STOCK SOLUTION
- The concentration of the test nominal 3000 mg/L stock solution of the test material was reported to be 494.61 mg/L. This result is the average of triplicate runs of the sample. Relative Percent Difference of 2.5%.
- In the control the concentration of test material was < MDL (method detection limit).

CHEMICAL OXYGEN DEMAND OF THE TEST MATERIAL
- The mean COD of the test material was 1.061 mg O2/mg test material.

NITRATE AND NITRITE ANALYSIS
- Dissolved oxygen values for the test solutions on days 4, 7, 11, 14, and 35 of the test were not corrected for interference by nitrification because it was negligible (i.e., resulting in a correction of < 0.1 mg/L). However, the dissolved oxygen concentrations for T-1 (7.8 mg/L) and T-2 (7.9 mg/L) were corrected by a 0.2 mg/L gain in oxygen to the system due to nitrification.

READY BIODEGRADABILITY OF THE TEST MATERIAL
- Reference Series (Procedural Control): The reference material achieved 87.5 and 90.0 % degradation after 14 and 35 days respectively, thereby meeting the pass criteria of ≥ 60% degradation within 14 days. Therefore, the test system was acceptable for the determination of biodegradability.
- Inhibition Series (Toxicity Control): The toxicity control test achieved 45.0 and 46.9% degradation after 14 and 35 days, respectively. Results of the inhibition test showed that Inhibition of the microbial population was not observed (i.e., % degradation exceeded the validity criteria of ≥ 25% degradation in 14 days).
- Test Material Series (Test Suspension): The test duration was extended beyond 28 days to Day 35 due to an extended lag phase where acclimation of the bacteria to the test material occurred late during the test. The test material achieved 10.0 and 6.2 % degradation after 28 and 35 days, respectively.
Results with reference substance:
The reference material achieved 87.5 and 90.0 % degradation after 14 and 35 days respectively, thereby meeting the pass criteria of ≥ 60% degradation within 14 days. Therefore, the test system was acceptable for the determination of biodegradability.

Table 1: Summary of Results

Series

Duration (Days)

Percent biodegradation (average of two replicates)

Reference substance

14

87.5

35

90.0

Toxicity control

14

45.0

35

46.9

Test material

28

10.0

35

6.2

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of this study the test material is not readily biodegradable.
Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301D using the closed bottle test, under GLP conditions.

The test duration was extended beyond 28 days to Day 35 due to an extended lag phase where degradation of the test material remained below 10% after 14 days.

The low solubility of the test material was enhanced by increasing the nominal concentration used to prepare the stock solution and by providing an extended period (approximately 93 hours) of continuous mixing at a rate sufficient to maintain a vortex of 10 to 35 %. A measured concentration of 494.61 mg/L was achieved by preparing a nominal 3000 mg/L stock solution of the test material.

The concentration of the test material in the mineral medium was 3.77 mg/L. The corresponding Chemical Oxygen Demand (COD) was 4.0 mg/L. The quality of the test system was confirmed through the use of a reference substance (potassium hydrogen phthalate) and all of the criteria for test validity were met.

The test material achieved 10 and 6.2 % degradation after 28 and 35 days, respectively.

Under the conditions of this study the test material is not readily biodegradable.

Description of key information

Under the conditions of the study the test material is not readily biodegradable.

Key value for chemical safety assessment

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

Additional information

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301D using the closed bottle test, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test duration was extended beyond 28 days to Day 35 due to an extended lag phase where degradation of the test material remained below 10% after 14 days.

The low solubility of the test material was enhanced by increasing the nominal concentration used to prepare the stock solution and by providing an extended period (approximately 93 hours) of continuous mixing at a rate sufficient to maintain a vortex of 10 to 35 %. A measured concentration of 494.61 mg/L was achieved by preparing a nominal 3000 mg/L stock solution of the test material.

The concentration of the test material in the mineral medium was 3.77 mg/L. The corresponding Chemical Oxygen Demand (COD) was 4.0 mg/L. The quality of the test system was confirmed through the use of a reference substance (potassium hydrogen phthalate) and all of the criteria for test validity were met.

The test material achieved 10 and 6.2 % degradation after 28 and 35 days, respectively.

Under the conditions of this study the test material is not readily biodegradable.