Pyrolytic sugar

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:

Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

uncritical deviation: Temperature range was 19.6 – 20.9 °C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range this is considered as uncritical concerning the outcome of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

yes

Remarks:

see OECD 301 B remark

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: DIN EN ISO 10634: Water quality – Preparation and treatment of poorly water soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium, adopted Oct. 2018

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,6-anhydro-β-D-glucose

EC Number:

207-855-0

EC Name:

1,6-anhydro-β-D-glucose

Cas Number:

498-07-7

Molecular formula:

C6H10O5

IUPAC Name:

(1R,2S,3S,4R,5R)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol

Constituent 2

Reference substance name:

Glycollaldehyde

EC Number:

205-484-9

EC Name:

Glycollaldehyde

Cas Number:

141-46-8

Molecular formula:

C2H4O2

IUPAC Name:

2-hydroxyacetaldehyde

Constituent 3

Reference substance name:

Organic acids

IUPAC Name:

Organic acids

Constituent 4

Reference substance name:

Ketones

IUPAC Name:

Ketones

Constituent 5

Reference substance name:

Phenols

IUPAC Name:

Phenols

Constituent 6

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Constituent 7

Reference substance name:

Oligomers of sugars and anhydrosugars

IUPAC Name:

Oligomers of sugars and anhydrosugars

Test material form:

liquid: viscous

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from a biologic sewage treatment plant was used as inoculum. The chosen plant treats mostly domestic sewage. The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, 67435 NW-Lachen-Speyerdorf.
- Pretreatment: The sludge was filtrated through a cloth, washed with test medium (2x) and resuspended in test medium. It was then aerated until use.
- Concentration of sludge: The dry matter was determined to contain 4.70 g of suspended solids/L.
- Initial cell/biomass concentration: 25.0 mg/L

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

41.6 mg/L

Based on:

TOC

Remarks:

test 1

Initial test substance concentration 2

Initial conc.:

41.5 mg/L

Based on:

TOC

Remarks:

test 2

Initial test substance concentration 3

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The medium was freshly prepared. 10 mL of solution a were mixed with 800 mL water, then 1 mL of solutions b, c and d were added and filled up to 1 L with H2O demin (volumes were adapted to final volume needed in the test)
Solution a: Potassium dihydrogen phosphate (KH2PO4) 8.5 g, Dipotassium hydrogen phosphate (K2HPO4) 21.75 g, Disodiumhydrogen phosphate dihydrate (Na2HPO4*2H2O) 33.4 g, Ammonium chloride (NH4Cl) 0.5 g, H2O demin. ad 1000 mL, The pH was 7.4.
Solution b: Calcium chloride dihydrate (CaCl2*2 H2O) 36.4 g , H2O demin. ad 1000 mL.
Solution c: Magnesium sulphate heptahydrate (MgSO4*7H2O) 22.5 g, H2O demin. ad 1000 mL
Solution d: Iron(III) chloride hexahydrate (FeCl3*6H2O) 0.25 g, Di-sodium-ethylene diaminetetraacetate dihydrate (Na2EDTA*2H2O) 0.4 g, H2O demin. ad 1000 mL
- Additional substrate: Sodium Hydroxide 0.25 M solution, used for trapping of emitted carbon dioxide and NaOH 1.5 M solution, used for scrubbing of purified air; Mercury Chloride used for poisoning of abiotic flasks; Barium Hydroxide solution, used for checking the purified air (saturated solution ,1:3 diluted); Hydrochloric Acid 2 M solution, used for driving off dissolved CO2 on day 28; Acetone used for dissolving the test item
- Test temperature: 19.6 – 20.9 °C without direct lighting
- pH: 7.5
- Suspended solids concentration: 4.7 g/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.
Magnetic stirrers were used to prevent deposition of inoculum.
The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.

- Test vessel: 2000 mL-SCHOTT-flasks were used as test vessels, 1500 ml volume, 100 mL scrubber flasks as absorbent vessels
- Number of culture flasks/concentration: 2 Test flasks, containing test item, mineral medium and inoculum;

- Measuring equipment: IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena.

SAMPLING
From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2 (on day 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2 (see also chapter 8.3.1).
On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 Blank Controls flasks, containing mineral medium and inoculum;
- Abiotic sterile control: 1 Abiotic control flask, containing test item, mineral medium and HgCl2;
- Toxicity control: 1 Toxicity control flask, containing test item, positive control, mineral medium and inoculum
- Other: 2 Positive control flasks, containing positive control, mineral medium and inoculum; 2 Apparatus blanks flasks, containing mineral medium only.

Reference substance

Reference substance:

aniline

Results and discussion

Test performance:

the degradation of the positive control was ≥ 60% within 7days

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

56.8

Sampling time:

29 d

Remarks on result:

other: mean test flask 1 and 2

BOD5 / COD results

Results with reference substance:

the degradation of the positive control was ≥ 60% within 7days

Any other information on results incl. tables

% degradation:

day	positive control 1	positive control 1	Test 1	Test 2	Mean	Abiotic control	Toxicity control
2	1.6	1.2	7.8	6.7	7.3	1.5	4.7
4	2.9	3.4	18.0	17.0	17.5	1.4	12.0
7	42.8	63.4	35.0	36.0	35.5	2.3	49.8
9	57.0	77.0	37.8	37.8	37.8	2.0	52.3
11	81.4	86.5	40.0	41.9	40.9	2.1	57.9
14	88.5	90.3	45.3	46.0	45.7	1.7	65.6
18	85.9	83.4	46.1	42.2	44.1	2.0	62.8
23	86.6	85.8	43.3	45.3	44.3	1.9	60.7
29	103.6	101.5	56.1	57.4	56.8	1.7	74.9

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item Pyrolytic Sugar is considered as “not readily biodegradable“. Degradation of the positive control surpassed the pass level of 60 % after 8 days.
The following data were determined for the test item Pyrolytic Sugar:
- 10-day-window: day 3-13
- degradation at the end of 10-day-window: 44 %
- degradation at the end of the test: 57%
- pass level following guideline: 60 % at the end of 10-day-window for pure substances respective 60 % at the end of the test for mixtures
Because the test item is a mixture the 10-day-window does not have to be taken into account. As degradation missed the pass level of 60% in the course of the test, Pyrolytic Sugar is considered as not readily biodegradable, within 28 days.

Executive summary:

The test item was tested using a concentration of nominally 20 mg organic carbon/L Pyrolytic Sugar in test medium following OECD 301B and EU-Method C.4 -C. All the validity criteria were met.

Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation reached 1.7 %. Both replicates of the test item showed very good correspondence.

Degradation values of more than 100 % were calculated. This is due to the fact that the measurement errors of test flasks and control flasks add up when using the equation proposed in the guideline.

If degradation in the toxicity flask is below 25 % after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 65.6 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 41.4 mg/L”.

Ready biodegradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10 %.
The 10-day-window began on day 3, at its end, 44 % degradation were reached, missing the pass level of 60 % given in the OECD guideline.

Because the test item is a mixture the 10-day-window does not have to be taken into account. As degradation missed the pass level of 60% in the course of the test, Pyrolytic Sugar is considered as not readily biodegradable, within 28 days.
The degree of biodegradation reached 57 % after 28 days.

As no plateau was reached at the end of the test duration, it can be assumed that the 60 % degradation can be exceeded with a prolonged test duration.

No observations were made which might cause doubts concerning the validity of the study outcome.

The result of the test can be considered valid.