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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:
Experiment start date - 21 October 2020; Experiment end date - 10 March 2021.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
See "Any other information on materials and methods incl. tables"
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
yes
Remarks:
See "Any other information on materials and methods incl. tables"
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
yes
Remarks:
See "Any other information on materials and methods incl. tables"
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: 4-Hydroxycoumarin
CAS Number: 1076-38-6
Appearance/Physical State: Beige powder
Batch Number: HCN20002
Purity: > 99 % w/w
Expiry Date: 26 September 2022
Storage Conditions: Room temperature, in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic (adaptation not specified)
Details on inoculum:
A mixed population of sewage treatment micro-organisms was obtained on 9 February 2021 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
Solution a : KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L
NH4Cl 0.50 g/L

pH = 7.4

Solution b CaCl2.2H2O 36.40 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeCl3.6H2O 0.25 g/L
(In order to avoid having to prepare solution (d) immediately before use, one drop of concentrated HCl per liter was added as a preservative).
Prior to the start of the test, the purified water was aerated for approximately 20 hours at the test temperature to give a dissolved oxygen concentration of approximately 9 mg O2/L.
To 1 L (final volume) of purified water* was added the following volumes of solutions a - d:

10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d
The pH of the mineral media was measured to be pH 7.5.

- Test temperature: 18 and 21 ºC
- pH: 7.7 - 7.8
- Aeration of dilution water: Aeration
- Continuous darkness: no. The test was conducted in diffuse light.

TEST SYSTEM
- Culturing apparatus: 500 mL glass bottles
- Number of culture flasks/concentration:
a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control plus a filter paper*.
b) Two replicate bottles containing inoculated mineral medium plus a filter paper*and the reference item, aniline, at a concentration of 100 mg/L to act as the procedure control.
c) Three replicate bottles containing the test item on filter paper* containing inoculated mineral medium at a concentration of 100 mg/L.
d) Two replicate bottles containing the test item on filter paper* at a concentration of 100 mg/L in inoculated mineral medium plus the reference item, aniline, at a concentration of 100 mg/L to act as toxicity control vessels

- Method used to create aerobic conditions: Aeration
- Measuring equipment: CES Respirometer for BOD measurement. Shimadzu TOC-VCPH (Software Version 2.30): TOC measurement


CONTROL AND BLANK SYSTEM
- Inoculum blank: Mineral medium
- Toxicity control: Yes
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
23
Sampling time:
28 d
Parameter:
BOD5
Value:
20.8 mg O2/g test mat.
Results with reference substance:
Aniline (procedure control) attained 75% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 82% biodegradation was attained thereby confirming the suitability of the inoculum and test conditions.

Validation criteria and Biodegradation

- The mean BOD of the inoculated mineral medium (control) was 20.80 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

- The pH of the inoculated test item vessels on Day 28 ranged from 7.7 to 7.8 and hence satisfied the validation criterion given in the OECD Test Guidelines.

- The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

- The test item attained 23% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

- The toxicity control attained 79% biodegradation after 14 days and 83% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro‑organisms used in the test.

- Aniline (procedure control) attained 75% biodegradation after 14 dayswith greater than 60% degradation being attained in a 10-day window. After 28 days 82% biodegradation was attainedthereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 23% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
Executive summary:

Introduction

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110.

Methods

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro‑organisms with mineral medium in sealed culture vessels in diffuse light at temperatures between 18 and 21 ºC for 28 days. Following the recommendations of the International Standards Organisation (ISO 1995), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item was evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms was increased thereby increasing the potential for biodegradation.

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values for 28 days. Control solutions with inoculum and the reference item, aniline, and a toxicity control were used for validation purposes.

Results

The test item attained 23% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Aniline (procedure control) attained75% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days,82% biodegradationwas attained.

Description of key information

The test item attained 23% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

Introduction

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110.

Methods

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro‑organisms with mineral medium in sealed culture vessels in diffuse light at temperatures between 18 and 21 ºC for 28 days. Following the recommendations of the International Standards Organisation (ISO 1995), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item was evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms was increased thereby increasing the potential for biodegradation.

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values for 28 days. Control solutions with inoculum and the reference item, aniline, and a toxicity control were used for validation purposes.

Results

The test item attained 23% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Aniline (procedure control) attained75% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days,82% biodegradationwas attained.