Escherichia coli dehalogenase catalyst

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14th November 2018 to 12th December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Unknown 6

IUPAC Name:

Unknown 6

Constituent 2

Reference substance name:

Unknown 7

IUPAC Name:

Unknown 7

Constituent 3

Reference substance name:

Unknown 5

IUPAC Name:

Unknown 5

Constituent 4

Reference substance name:

Unknown 4

IUPAC Name:

Unknown 4

Constituent 5

Reference substance name:

Unknown 3

IUPAC Name:

Unknown 3

Constituent 6

Reference substance name:

Unknown 2

IUPAC Name:

Unknown 2

Constituent 7

Reference substance name:

Unknown 1

IUPAC Name:

Unknown 1

Test material form:

solid

Specific details on test material used for the study:

- Source and lot/batch No.of test material: 4898318
- Expiration date of the lot/batch: 1 December 2019
- Purity test date: Not specified
- Storage condition of test material: ≤ -18°C during the test duration

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Source of inoculum/activated sludge:
Secondary effluent (after sandfilter) from the municipal wastewater treatment plant Breisgauer Bucht was used as inoculum (0.1 mL per litre). The treatment plant clarifies predominantly domestic wastewater in the region of Freiburg and has a capacity of 600,000 inhabitant equivalents. Sampling date of the secondary effluent was 12 November 2018. The inoculum was aerated and incubated in darkness until test begin at the temperature range of 21.0 – 21.8°C.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
A: Potassium dihydrogenphosphate KH2PO4 8.50g
Dipotassium hydrogenphosphate K2HPO4 21.75g
Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40g
Ammonium chloride NH4Cl 0.50g
are dissolved in demineralised water and made up to 1 litre.
B: Calcium chloride dihydrate CaCl2*2H2O 36.4 g
is dissolved in demineralised water and made up to 1 litre.
C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5g
is dissolved in demineralised water and made up to 1 litre.
D: Iron (III) chloride hexahydrate FeCl3 * 6H2O 0.25g
is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre.
For preparation of the mineral medium 1 ml of solution (A) is mixed with 800 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre. The medium was aerated and held in darkness for 24 h (temperature: 22.3 – 23.0°C). After 24 h the pH was 7.2.
- Additional substrate: None
- Solubilising agent (type and concentration if used): None
- Test temperature: 20-24°C (+/-1°C)
- pH: The pH of the test item vessels at the beginning of the test was 7.2 and at the end of the test the pH was between 6.7 and 6.8.
- pH adjusted: no
- CEC (meq/100 g): N/A
- Aeration of dilution water: The medium was aerated.
- Suspended solids concentration: N/A
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Not specified
- Number of culture flasks/concentration: 17
- Method used to create aerobic conditions: Aquarium pump for aeration
- Method used to create anaerobic conditions: N/A
- Measuring equipment:
Multi-parameter Instrument inoLab® Multi 9430, WTW, Weilheim
FDO-Oxygen Electrodes, WTW, Weilheim
pH-Electrodes SenTix and SenTix 940, WTW, Weilheim
Precision balance BP 221S, Sartorius AG Göttingen
Analytical balance BP 221S, Sartorius AG Göttingen
Thermometer with min/max-display
The COD of the test item was determined via LCK-cuvette-test from Hach for photometric analysis with dichromate according to ISO 6060-1989 and DIN 38409-H41-H44.
- Test performed in closed vessels due to significant volatility of test substance: Closed vessels due to test method not due to volatility of the substance.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: None

SAMPLING
- Sampling frequency: On days 7, 15, 21 and 28 at least duplicate bottles were removed for determination of dissolved oxygen and pH.
- Sampling method: The zero-time bottles were analysed immediately after filling using the electrode method. If the oxygen concentrations in duplicate bottles differed more than 0.3 mg/L, another measurement was performed to confirm data. At the end of the test dissolved oxygen concentration in all remaining bottles was measured.
- Sample storage before analysis: Not specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: A glass aquarium was filled with 6 L mineral medium and 0.6 mL of inoculum was added. 17 bottles were filled.
- Abiotic sterile control: None
- Toxicity control: A glass aquarium was filled with 5 L mineral medium. Then 24 mL of the test item stock solution and 12 mL of the reference item stock solution and 0.6 mL of the inoculum were added. The volume was made up to 6 litres. 17 bottles were filled
Reference substance: A sodium acetate stock solution of 2.0 g/L was prepared. A glass aquarium was filled with 5 L mineral medium. Then 12 mL of the stock solution and 0.6 mL of the inoculum were added. This corresponds to a concentration of 4 mg/L reference substance. The volume was made up to 6 litres. 17 bottles were filled.

STATISTICAL METHODS:
None

EVALUATION OF DATA
From this the biological oxygen demand (BOD) after x days is calculated as follows:
BODx (mg O2/ mg test item) = mg O2/l (oxygen uptake test item) - mg O2/l (oxygen uptake blank)/ mg test item pro litre
so the biological degradation is calculated as follows: % degradation = BODx/COD * 100
Test items with an oxygen depletion > 60% of the ThOD or COD after 28 d are "readily biodegradable" according to OECD 301. This has to be reached within 10 days after the end of the lag-phase (when degradation reaches 10% of ThOD or COD) = 10-day-window.
If in a toxicity test, containing both the test substance and a reference compound, less than 25% (based on ThOD or COD) occurred within 14 days, the test substance can be assumed to be inhibitory.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

Not applicable

Test performance:

Criteria met for the validity of the study:
The maximal deviations for the parallel oxygen measurements at the end of the test were The percentage degradation of the reference item met the criterion for biological degradation after 14 days.
The oxygen concentration in the test bottles never fell below 0.5 mg/L.
Oxygen depletion in the blanks never exceed 1.5 mg O2/L

The test is valid.

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

75.3

Sampling time:

28 d

Details on results:

Test item
The degradation of the test item was calculated on the base of COD. The test item reached a degradation level of 75.3% within 28 days without reaching a degradation plateau. On day 15 the variation of the oxygen measurements was higher than 0.3 mg/L, therefore a sampling of more than two bottles was performed. On day 28 the oxygen of all remaining bottles was measured. Here two measurements were regarded as outliers, because the oxygen was clearly higher than in the other measurements.
The pH of the test item vessels at the beginning of the test was 7.2 and at the end of the test the pH was between 6.7 and 6.8.
On day 15 the degradation level of the test item was 49.4% and therefore still below 60%. The 10-d-window was not met.

Toxicity control
In the toxicity control the degradation extent was 54.3% within 7 days, therefore the test item has no inhibitory effect on the inoculum. The pH in the toxicity control vessels was 7.2 at the beginning of the test and 6.6 at the end of the test.

Reference item
The reference compound sodium benzoate reached the pass level for ready biodegradability within 14 days. The pH in the reference vessels was 7.2 at the beginning of the test and 6.8 to 6.9 at the end of the test.

Blank
On day 28 the oxygen depletion in the blanks was 0.3 mg/L. The pH of the blanks was between 7.1 – 7.2 at the beginning and at the end of the test.

The temperature range was 20.4 to 22.5°C throughout the whole study. Therefore the temperature range of +/- 1°C was slightly exceeded but has no influence on the overall result of the study

BOD5 / COD results

Results with reference substance:

The reference compound sodium benzoate reached the pass level for ready biodegradability within 14 days. The pH in the reference vessels was 7.2 at the beginning of the test and 6.8 to 6.9 at the end of the test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

Escherichia coli dehalogenase catalyst reached a degradation extent of 75.3% within 28 days. The 10-d-window was not met.
The degradation of the toxicity control after 7 days was 54.3%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

Executive summary:

A Closed Bottle Test according to OECD 301 D (July 1992) was conducted in order to investigate the ready biodegradability of Escherichia coli dehalogenase catalyst.

Escherichia coli dehalogenase catalyst reached a degradation level of 75.3% within 28 days. The 10 day window was not met.

The degradation of the toxicity control after 7 days was 54.3%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

The test met the validity criteria.