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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:
27 July 2017 to 31 August 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)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Guelph Wastewater Treatment Plant (Guelph, Ontario) which treats sewage of predominantly domestic origin.
- 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.:
4.72 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: 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 8.3 mg/L. The dissolved oxygen concentration was measured using an oxygen electrode on days 0, 4, 7, 11, 14, 28, and 35.
- Testing was conducted at 22 ± 2°C.
- pH: 7.4 ± 0.2
- pH adjusted: not required
- Inoculum concentration: 5 mL/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Glass 300 mL, with ground glass stoppers and plastic caps (BOD bottles)
- Number of culture flasks/concentration: For each sampling period (Day 0, 4, 7, 11, 14, 28 and 35): 2 bottles for inoculated control, reference, inhibition and test series; 1 bottle for the non-inoculated control
- A nominal 3,000 mg/L stock solution of the test material was prepared by weighing 3.0027 g of the test material into a 1000 mL aspirator bottle and making this up to volume in Millipore MilliQ™ water. This stock solution was stirred for approximately 23 hours and allowed to settle for one hour. The first 100 mL were removed from the bottom of the bottle and discarded. Another 500 mL were removed and used as the test stock. The COD of the test material was 0.848 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 56.940 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). The test series was conducted in parallel with the reference (KHP) series, non-inoculated and inoculated control series, and an inhibition series.
- Measuring equipment: Oxygen electrode

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

CONTROL AND BLANK SYSTEM
- A single non-inoculated control and duplicate inoculated control test vessels (BOD bottles) were established for each of the sampling periods. 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.
- 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, 56.940 mL of the test material stock solution and 24.170 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).
- 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 1.163 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/L² (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 993 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 24.170 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).

STATISTICAL METHODS:
- The BOD was calculated as follows:
BOD (mg O2/mg test substance) = (mg O2/L uptake of test material – mg O2/L uptake of blank) / mg test substance/L in vessel
- 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 item 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:
16.2
Sampling time:
35 d
Details on results:
CHEMICAL OXYGEN DEMAND OF THE TEST MATERIAL
The mean COD of the test material was 0.848 mg O2/mg test material.

CONFIRMATION OF THE TEST MATERIAL STOCK SOLUTION
The concentration of the test nominal 1000 mg/L stock solution of the test material was reported to be 496.9 mg/L.

READY BIODEGRADABILITY OF THE TEST MATERIAL
- Reference Series (Procedural Control): The reference material achieved 85% degradation in 14 days, thereby meeting the pass criteria of ≥ 60% within 14 days. Therefore, the test system was acceptable for the determination of biodegradability. Percent biodegradation after 35 days was 95%.
- Inhibition Series (Toxicity Control): Results of the inhibition test showed that Inhibition of the microbial population was not observed (i.e., > 25% degradation in 14 days). It should be noted that a large crack was observed for one of the two replicate test vessels on day 28, which resulted in a high (suspect) DO concentration (6.3 mg/L) relative to the other replicate (3.5 mg/L). The result was not used in any statistical analysis and the DO concentrations of the replicate test vessels on day 35 were not affected.
- Test Material Series (Test Suspension): The test material achieved 16.2% degradation over the 35-day exposure. It should be noted that 3 small air bubbles were observed in one of the two replicate test vessels on day 28. However, this did not appear to have any appreciable effect on the test results as the measured DO concentrations for the two replicates were similar (i.e., 7.2 and 7.3 mg/L). Additionally, the DO concentrations measured on day 35 were the same (i.e., 6.7 mg/L) for both replicates.

SUMMARY
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 concentration of the test material in the mineral medium was 4.72 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 16.2% degradation over a 35-day exposure.


Key result
Parameter:
COD
Value:
0.848 other: mg O2/mg test material
Results with reference substance:
The reference material achieved 85% degradation in 14 days, thereby meeting the pass criteria of ≥ 60 % within 14 days. Therefore, the test system was acceptable for the determination of biodegradability. Percent biodegradation after 35 days was 95%.

Table 1: Summary of Results

Duration (days)

Percent biodegradation (%)

Reference substance

Toxicity control

Test material

14

85.0

46.2

-

35

95.0

60.6

16.2

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test material was not readily biodegradable under the conditions of the test.
Executive summary:

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301D under GLP conditions.

The test involved exposure of an active microbial population (secondary effluent from a wastewater treatment plant) to the test material under constant temperature in the dark. The test is typically terminated on the 28th day; however, the test was extended by an additional 7 days since acclimation of the bacteria to the test material occurred late in the test. Initial oxygen demand for the test material was determined from the Chemical Oxygen Demand (COD) , the test material was evaluated at approximately 4.0 mg COD test material/L in a series of test chambers.

During the test, the bacteria utilise a nutrient solution (devoid of carbon) and the test material (the carbon source), consuming dissolved oxygen in the process. The dissolved oxygen from each biodegradation test vessel was measured with an oxygen electrode on a frequency adequate to track the degradation of the test material (i.e., on Days 0, 4, 7, 11, 14, 28 and 35). The amount of oxygen consumed within the test system was compared to the COD of the test material and the percent (%) degradation was calculated.

Under the conditions of the study the test material achieved 16.2% degradation over a 35-day exposure. The test material was therefore concluded to be not readily biodegradable. 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.

Description of key information

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

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301D 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 involved exposure of an active microbial population (secondary effluent from a wastewater treatment plant) to the test material under constant temperature in the dark. The test is typically terminated on the 28th day; however, the test was extended by an additional 7 days since acclimation of the bacteria to the test material occurred late in the test. Initial oxygen demand for the test material was determined from the Chemical Oxygen Demand (COD) , the test material was evaluated at approximately 4.0 mg COD test material/L in a series of test chambers.

During the test, the bacteria utilise a nutrient solution (devoid of carbon) and the test material (the carbon source), consuming dissolved oxygen in the process. The dissolved oxygen from each biodegradation test vessel was measured with an oxygen electrode on a frequency adequate to track the degradation of the test material (i.e., on Days 0, 4, 7, 11, 14, 28 and 35). The amount of oxygen consumed within the test system was compared to the COD of the test material and the percent (%) degradation was calculated.

Under the conditions of the study the test material achieved 16.2% degradation over a 35-day exposure. The test material was therefore concluded to be not readily biodegradable. 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.