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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:
12 Jun 2017 - 04 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted July 17, 1992
Qualifier:
according to guideline
Guideline:
other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium", (1995)
GLP compliance:
yes
Specific details on test material used for the study:
Test item information
Identification: Styrax resinoid oil
Appearance: Orange viscous liquid
Batch: K17 031-1
Purity/Composition: 100% UVCB
Test item storage: At room temperature
Stable under storage conditions until: 02 February 2019

Additional information
Test Facility test item number: 208399/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name: Resinoid obtained from the gum of Liquidambar styraciflua by extraction with solvent
CAS number: 8046-19-3
Molecular structure: UVCB
Molecular formula: UVCB
Molecular weight: UVCB
Highly reactive to water: Not indicated
Volatile: Not indicated
Solubility in water: Insoluble
Stability in water: Not indicated
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment: The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (44 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.

Reason for selection The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.
Duration of test (contact time):
28 d
Initial conc.:
30.93 other: ml
Based on:
test mat.
Remarks:
weighted amount ( Bottle A) The test item was tested in duplicate at a target concentration of 15.5 mg/L, corresponding to 12 mg TOC/L.
Initial conc.:
31.25 other: ml
Based on:
test mat.
Remarks:
weighted amount ( Bottle B) The test item was tested in duplicate at a target concentration of 15.5 mg/L, corresponding to 12 mg TOC/L.
Details on study design:
TEST PROCEDURE AND CONDITIONS

Test duration: 28 days for the inoculum blank and test item (last CO2 measurement on day 29). 14 days for the positive and toxicity control (last CO2 measurement on day 15). During the test period, the test media were aerated and stirred continuously.

Test vessels: 2 litre brown coloured glass bottles. Milli-RO water Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

Stock solutions:
of A) 8.50 g KH2PO4 mineral components 21.75 g, K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.

Mineral medium 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water. Barium hydroxide 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands),
stored in a sealed vessel to prevent absorption of CO2 from the air.

Synthetic air (CO2 < 1 ppm)1 A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination The test media were excluded from light.

PREPARATION OF BOTTLES

Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).

Preparation: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

DETERMINATION OF CO2

Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).

Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator. On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 15 (positive and toxicity control) and on day 29 (remaining vessels).

Theoretical CO2 production: The theoretical CO2 production was calculated from the results of the TOC-analysis.

MEASUREMENTS AND RECORDINGS

pH: At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.

Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.
Reference substance:
acetic acid, sodium salt
Remarks:
Purity: 99.1%
Test performance:
The relative biodegradation values calculated from the measurements performed during the test period revealed 38% and 20% biodegradation of Styrax resinoid oil (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, more than 25% biodegradation occurred within 14 days (56%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
38
Sampling time:
29 d
Remarks on result:
other: Test Item (Bottle A)
Key result
Parameter:
% degradation (CO2 evolution)
Value:
20
Sampling time:
29 d
Remarks on result:
other: Test Item (Bottle B)
Details on results:
Acceptability of the Test
1. The positive control item was biodegraded by at least 60% (85%) within 14 days.
2. The difference of duplicate values for % degradation of the test item was always less than 20 (≤ 18%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (50.6 mg CO2 per 2 litres of medium, corresponding to 25.3 mg CO2/L).
4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test
medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L). Since all criteria for acceptability of the test were met, this study was considered to be valid.
Results with reference substance:
The positive control item was biodegraded by at least 60% (85%) within 14 days.

 

Day

Test Item (Bottle A)

Test Item (Bottle B)

Cumulative

CO2(mg)

Biodegradation1)

(%)

Cumulative

CO2 (mg)

Biodegradation1)

(%)

1

0.0

0

0.0

0

4

3.5

4

1.9

2

6

8.1

9

4.7

5

8

8.1

9

6.2

7

11

12.6

14

9.2

10

15

17.1

20

12.0

14

18

27.8

32

14.4

16

22

30.7

35

16.5

19

25

31.5

36

17.2

19

292)

33.1

38

17.4

20

292)

33.1

38

17.6

20

292)

33.1

38

17.7

20

 

1) Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 87.2 mg CO2/2L.

2) Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

 

Validity criteria fulfilled:
yes
Remarks:
See details on result
Interpretation of results:
not readily biodegradable
Conclusions:
The relative biodegradation values calculated from the measurements performed during the test period revealed 38% and 20% biodegradation of Styrax resinoid oil (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met and is thus considered not readily biodegradable.
Executive summary:

The ready biodegradability of Styrax resinoid oil was investigated in a study conducted in accordance with OECDTG 301B (carbon dioxide evolution test (modified Sturm test)) and GLP. Since Styrax resinoid oil was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2 -litres test bottles containing medium with microbial organisms and mineral components. The relative biodegradation values calculated from the measurements performed during the test period revealed 38% and 20% biodegradation of Styrax resinoid oil (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10 -day window) was not met. In the toxicity control, Styrax resinoid oil was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. In conclusion, Styrax resinoid oil was designated as not readily biodegradable.

Description of key information

The ready biodegradability ofStyrax resinoid oilwas investigated in a study conducted in accordance with OECDTG 301B (carbon dioxide evolution test (modified Sturm test)) and GLP.Since Styrax resinoid oil was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2 -litres test bottles containing medium with microbial organisms and mineral components. The relative biodegradation values calculated from the measurements performed during the test period revealed 38% and 20% biodegradation of Styrax resinoid oil (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10 -day window) was not met. In the toxicity control, Styrax resinoid oil was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. In conclusion, Styrax resinoid oil was designated as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

[Type of water: freshwater]