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Environmental fate & pathways

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:
08 November 2017 - 07 january 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. QA statement)
Remarks:
The test facility was operating in compliance with the OECD principles of Good Laboratory Practice. Date: 28/11/2017
Specific details on test material used for the study:
- Physical appearance: clear liquid
- Storage conditions: at room temperature
- Stable under storage conditions until: 30 March 2019 (retest date)
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 8 November 2017 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and the filtrate maintained on continuous aeration in a temperature controlled room at approximately 21 °C prior to use.
Duration of test (contact time):
60 d
Initial conc.:
1 mg/L
Based on:
test mat.
Initial conc.:
3.29 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Mineral medium
The mineral medium used in this study was that recommended in the OECD Guidelines.
The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

Preliminary work
The average volume of a completely filled 250-300 mL Biological Oxygen Demand (BOD) bottle was found to be 280 mL. This was calculated by measuring the volume of reverse osmosis water required to completely fill fifteen separate BOD bottles.

Test item preparation:
Following the results of the preliminary solubility work conducted and recommendations of the International Standards Organisation (ISO 10634, (1995)), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in inoculated culture media. Using this method allows extremely small amounts of test item to be added to each bottle, the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
A nominal amount of test item (100 mg) was dissolved in 10 mL of acetone to give a 100 mg/10 mL solvent stock solution. Aliquots (28 μL) of this solvent stock solution were dispensed onto 20 separate filter papers* and the solvent allowed to evaporate to dryness for approximately 15 minutes. A test item coated filter paper was then placed into 20 separate Biological Oxygen Demand (BOD) bottles prior to the addition of inoculated mineral medium to give a final concentration of 1.0 mg/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
A test concentration of 1.0 mg/L was employed in the study as the Theoretical Oxygen Demand (ThOD) of the test item was calculated to be 3.29 mg O2/mg (see Annex 3). Hence, if complete degradation of the test item occurred, the oxygen depletion in the test vessels would be 3.29 mg O2/L and as such de-oxygenation of the test media would not occur.
As it was not a requirement of the Test Guidelines, no analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP Compliance Statement and is considered not to affect the purpose or integrity of the study.

Reference Item preparation:
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium and an aliquot (18 mL) dispersed in a final volume of 6 liters of inoculated mineral medium to give a test concentration of 3.0 mg/L. A filter paper* with 28 μL of acetone added and evaporated to dryness for approximately 15 minutes was added into 20 separate BOD bottles in order to maintain consistency between the test and reference item vessels. Each bottle was then filled with the 3.0 mg/L reference item solution.
The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.
Toxicity Control:
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the study.
Aliquots (28 μL) of the solvent test item stock solution were dispensed onto eight separate filter papers* and the solvent allowed to evaporate to dryness for approximately 15 minutes. A test item coated filter paper was then placed into eight separate Biological Oxygen Demand (BOD) bottles. An aliquot (3.0 mL) of the sodium benzoate stock solution (see Section 3.3.3) was dispersed in a final volume of 3 litres of mineral medium. The BOD bottles containing the test item coated filter papers were then filled with the inoculated mineral medium containing sodium benzoate to give a final test concentration of 1.0 mg test item/L and 1.0 mg sodium benzoate/L.

Preparation of Test system:
The following test preparations were prepared and inoculated in 250 mL Biological Oxygen Demand (BOD) bottles (darkened glass) with ground glass stoppers:
a) An inoculum control, twenty vessels, consisting of inoculated mineral medium plus a filter paper*.
b) The procedure control containing the reference item (sodium benzoate), twenty vessels, in inoculated mineral medium plus a filter paper to give a concentration of 3.0 mg/L.
c) The test item on a filter paper*, twenty vessels, in inoculated mineral medium to give a concentration of 1.0 mg/L.
d) The test item (1.0 mg/L) on a filter paper* plus the reference item (1.0 mg/L), eight vessels, in inoculated mineral medium to act as a toxicity control.
The reference item concentration was reduced to 1.0 mg/L in the toxicity control to prevent complete deoxygenation of the test media occurring.
A filter paper with 28 μL of acetone added was allowed to evaporate to dryness and added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
Test media a to d were inoculated with sewage treatment micro-organisms at a rate of 1 drop of inoculum per liter.
The pH of all of the above test preparations were measured using a Hach HQ40d Flexi handheld meter prior to being transferred by siphon to BOD bottles, which were firmly stoppered to exclude all air bubbles. Sufficient bottles were prepared to allow a single oxygen determination per bottle with duplicate bottles for each test preparation at each sampling occasion.
The BOD bottles were incubated in darkness in a temperature controlled water bath at temperatures of between 20 and 21 °C.

Assessment:
Dissolved oxygen concentrations in duplicate samples from the control, reference and test item series were determined, on Days 0, 2, 5, 7, 9, 14, 21, 28, 42 and 60 by means of a YSI 54A dissolved oxygen meter and BOD Probe. Dissolved oxygen concentrations in duplicate samples from the toxicity control series were determined on Days 0, 14, 28 and 60 only.
The dissolved oxygen depletion for each replicate flask for the test item, reference item and toxicity control for each time period is calculated as follows:
Oxygen depletion = (Mto - Mtx) - (Mbo - Mbx) mg O2/L
Where:
Mto = oxygen concentration in test flask at time 0
Mtx = oxygen concentration in test flask at time x
Mbo = mean oxygen concentration of inoculated control flasks at time 0
Mbx = mean oxygen concentration of inoculated control flasks at time x
The oxygen depletion for the inoculated control (blank) flasks is calculated as follows:
Blank oxygen depletion = Mbo - Mbx
The percentage biodegradation for each replicate test item, reference item and toxicity control flask was calculated as follows:
% biodegradation = dissolved oxygen depletion/(concentration of test substance x ThOD (or COD)) x100
Mean % biodegradation = (% biodegradation of replicate 1 + % biodegradation of replicate 2) / 2

Major Computerized Systems:
The following computerized systems were used in the study:
Shimadzu TOC TOC measurement
Delta control system Building management
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
8
Sampling time:
14 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
9
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
9
Sampling time:
60 d
Details on results:
The oxygen depletion of the inoculated control did not exceed 1.5 mg O2/L after 28 days, the residual oxygen concentration in the test bottles remained at 7.55 mg O2/L or greater after 28 days in all test vessels and the difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20% in all vessels thereby satisfying the validation criteria.
The test item attained 9% biodegradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.
Extension of the test from 28 days to 60 days did not result in any increase in biodegration.
Variation in biodegradation rates on different sampling days was considered to be due to normal biological variation in respiration rates between the inoculum control and the test item vessels.
The toxicity control attained 27% biodegradation after 14 days, 31% biodegradation after 28 days and 30% biodegradation after 60 days therefore confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. The slight decrease in biodegradation between Days 28 and 60 was considered to be due to normal biological variation in respiration rates between the inoculum control and the toxicity control vessels.
Results with reference substance:
The reference item, sodium benzoate, attained 81% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 79% biodegradation was attained with 72% biodegradation being attained after 60 days. The slight decrease in biodegradation between Days 14, 28 and 60 was considered to be due to normal biological variation in respiration rates between the inoculum control and the procedure control vessels. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

1. Dissolved oxygen demand

        Dissolved Oxygen (mg O2/L)                           
        Day
     Test series  0  2  5  7  9  14  21  28  42  60
  a) Inoculum control R1   8.70  8.60  8.25  8.20 8.10   8.20  7.80  8.10  7.50  7.20
  R2  8.70  8.60  8.05  8.15  8.20  8.20  7.70  7.80  7.30  7.30
 b) Procedure Control R1  8.65  5.30  4.90  4.25  4.80  4.05  3.70  3.80  3.80  3.70
  R2  8.65  5.25  4.85  4.25  4.00  4.15  3.50  4.10  3.70  3.55
 c) Test Item R1  8.65  8.35  7.90  7.80  8.05  7.95  7.50  7.70  7.00  6.90
  R2   8.70  8.30  7.90  7.70  7.55  7.90  7.40  7.55  7.10  7.00
 d) Toxicity control R1  8.65  -  -  -  -  6.80  -  6.30  -  5.80
  R2  8.65  -  -  -  - 6.80   -  6.30  -  5.60

2. Oxygen Depletion and Mean Percentage Biodegradation Values

                                 days  
       Test series  2  5  7  9  14  21  28  42  60  
 a) Inoculum Control  Mean O2 depletion (mg O2/L)    0.100  0.550  0.525  0.550  0.500  0.950  0.750  1.300  1.450  

b) Procedure Control 

  O2 depletion (mg O2/L)

 R1

 3.250

 3.200

 3.875

 3.300

 4.100

 4.000

 4.100

 3.550

 3.500

 

 

   O2 depletion (mg O2/L)

 R2

 3.300

 3.250

 3.875

 4.100

 4.000

 4.200

 3.800

 3.650

 3.650

 

 

 % Biodegradation (mean)

 

 66

 65

 77

 74

 81

 82

 79

 72

 72

 

  c) Test Item

  O2 depletion (mg O2/L)

 R1

 0.200

 0.200

 0.325

 0.050

 0.200

 0.200

 0.200

 0.350

 0.300

 

 

   O2 depletion (mg O2/L)

 R2

 0.300

 0.250

 0.475

 0.600

 0.300

 0.350

 0.400

 0.300

 0.250

 

 

  % Biodegradation (mean)

 

 8

 7

 12

 10

 8

 9

 9

 10

 9

 

  d) Toxicity Control

  O2 depletion (mg O2/L)

 R1

 -

 -

 -

 -

 1.350

 -

 1.600

 -

 1.400

 

 

   O2 depletion (mg O2/L)

 R2

 -

 -

 -

 -

 1.350

 -

 1.500

 -

 1.600

 

 

  % Biodegradation (mean)

 

 -

 -

 -

 27

 -

 31

 -

 30

 

3. pH values of the test Preparations on day 0

 Test preparations

 pH

 Inoculum Control

 7.2

 Procedure Control

 7.4

 Test item

 7.4

 Toxicity Control

 7.4

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The registered substance attained 9% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.
Extension of the test from 28 days to 60 days did not result in any increase in biodegration.
Executive summary:

This study was performed to assess the ready biodegradability of the registered substance in an aerobic aqueous medium. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301D, "Ready Biodegradability; Closed Bottle Test” referenced as Method C.4-E of Commission Regulation (EC) No. 440/2008, and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 Paragraph (o).

The registered substance, at a concentration of 1.0 mg/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 20 and 21 °C for 60 days. At the request of the Sponsor the study was extended from 28 to 60 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the registered susbtance was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in inoculated mineral media. Using this method allows extremely small amounts of substance to be added to each bottle, the registered substance is evenly distributed throughout the test medium and the surface area of substance exposed to the test organisms is increased thereby increasing the potential for biodegradation. The degradation of the registered substance was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The registered substance attained 9% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.

Extension of the test from 28 days to 60 days did not result in any increase in biodegration.

Description of key information

OECD Guideline 301D, GLP, key study, validity 1:

9% biodegradation after 28 days and 60 days (inoculum: activated sludge)

Under the test conditions, the percentage biodegradation of the test item did not reach 60% in 14 day-window, so the test item can be considered as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

An OECD 301 D tests was performed to assess the ready biodegradability of the registered substance in an aerobic aqueous medium. The registered substance, at a concentration of 1.0 mg/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 20 and 21 °C for 60 days. At the request of the Sponsor the study was extended from 28 to 60 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the registered susbtance was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in inoculated mineral media. Using this method allows extremely small amounts of substance to be added to each bottle, the registered substance is evenly distributed throughout the test medium and the surface area of substance exposed to the test organisms is increased thereby increasing the potential for biodegradation. The degradation of the registered substance was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The registered substance attained 9% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.

Extension of the test from 28 days to 60 days did not result in any increase in biodegration.