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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:
22 May 2018 to 1 Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Principles of method if other than guideline:
NA (study was performed according to guidelines).
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch: 99101243
Purity: 76.39%
Physical State/Appearance: Beige coloured paste
Expiry date: 23 November 2019
Storage: Room temperature in the dark under nitrogen
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Activated sewage sludge was obtained on 10 September 2018 from the the aeration stage of the Severn Trent Water Plc sewage treatment plant (Loughborough, Leicestershire, UK) which predominantly treats domestic sewage.

The activated sewage sludge sample was washed twice by settlement and re suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) present. The washed sample was then maintained on continuous aeration in the laboratory at approximately 21ºC and used on the day of collection. Suspended solids level determination of the activated sewage sludge was performed by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.9 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10.1 mg/L
Based on:
test mat.
Remarks:
equivalent to 5 mg carbon/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Mineral medium:
The mineral medium used followed the recommendations of the OECD Guidelines (see ‘Any other information on material and methods incl. tables’ for details).


Test item preparation:
An initial experiment conducted using a test item concentration equivalent to 10 mg carbon/L showed that the toxicity control vessel, containing both the test item and reference substance (sodium benzoate), attained <25% biodegradation after 14 days based on the combined theoretical CO2 yield. Compared to the performance of the reference substance without the test item, this result indicated that severely inhibited the microbial inoculum activity.

Further preliminary work was therefore conducted using a toxicity control vessel containing a test item concentration equivalent to 5 mg carbon/L and sodium benzoate at a concentration of 10 mg carbon/L, which was not shown to be inhibitory to the activated sewage sludge micro-organisms. Based on this result, the definitive test used an initial exposure concentration equivalent to 5 mg carbon/L.

Test item (30.3 mg) was dispersed into approximately 400 mL of mineral medium, with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium and the volume adjusted to 3 litres, giving a final concentration of 10.1 mg/L, equivalent to 5 mg carbon/L.


Reference item preparation:
The reference item, sodium benzoate was used to prepare procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving sodium benzoate directly in mineral medium and an aliquot (51.4 mL) of stock solution added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 litres, giving a final concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The flask containing sodium benzoate was inverted several times to ensure homogeneity of the solution.


Toxicity control:
A toxicity control containing the test item and sodium benzoate, was prepared by dispersing 30.3 mg of test item into approximately 400 mL of mineral medium, with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres, giving a final concentration of 10.1 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a combined total of 15 mg carbon/L.


Test system preparation:
The following test preparations were prepared and inoculated in 5 litre test culture vessels, each containing 3 litres of solution:
a) Inoculated control (in duplicate), consisting of inoculated mineral medium.
b) Procedure control containing sodium benzoate (in duplicate), in inoculated mineral medium, giving a final concentration of 10 mg carbon/L.
c) Test item (in duplicate), in inoculated mineral medium, giving a final concentration of 5 mg carbon/L.
d) Toxicity control (one vessel), containing test item plus sodium benzoate in inoculated mineral medium, giving a final concentration of 15 mg carbon/L.

Test vessels were inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. Tests were conducted in sealed culture vessels in the dark at temperatures between 22-24°C for 28 days.

Approximately 24 hours prior to addition of the test and reference items, vessels were filled with 2400 mL of mineral medium and 31.0 mL of inoculum and aerated overnight. On Day 0, test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to adjusting the volume of the vessels to 3 litres by the addition of mineral medium, which had been purged overnight with CO2 free air. The inoculum control vessels were prepared in a similar manner without the addition of test item or reference item.

Test vessels were sealed and CO2 free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer. The CO2 free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules. The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.


Test preparation assessments:
Appearance of test preparations were recorded on days 0, 7, 14, 21 and 28. The pH of test preparations was determined on day 0 and day 28 before acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.


Supplementary inhibition check:
Given the toxicity seen during the initial test, supplementary information was obtained by comparing IC production during the incubation between the inoculated control and the test item vessels, to check whether inhibition of microbial inoculum activity occurred during the test. Divergence in background respiration from the inoculated control may indicate disturbance of endogenous metabolic processes that may account for a negative biodegradation test outcome. To make this comparison statistical analysis of the Day 29 IC values for the inoculum control and test item vessels was carried out using a Student’s t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
Reference substance:
benzoic acid, sodium salt
Preliminary study:
A preliminary experiment demonstrated that a test item concentration equivalent to 10 mg carbon/L resulted in significant inhibition of the microbial inoculum, therefore a test item concentration equivalent to 5 mg carbon/L was used in the definitive test.
Test performance:
Total CO2 evolution in inoculum control vessels on day 28 was 27.98 mg/L. The inorganic carbon content of the test item suspension in the mineral medium at test initiation was 5% of the total carbon content. The difference between CO2 production values at the end of the test for the replicate vessels was <20%. As such, the study guideline validity criteria were satisfied and the study therefore considered valid.
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
Inorganic carbon (IC) analysis of samples from the first absorber vessels on day 29 showed an increase in all replicate vessels, with the exception of test item replicate 2 (which showed an IC decrease from 23.29 mg on day 28 to 23.16 mg on day 29). IC analysis of samples from the second absorber vessels on day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

Statistical analysis of day 29 IC values from control and test item vessels showed no statistically significant differences (P≥ 0.05) between the control and test item. The test item was therefore not considered to have toxic effects on the sewage sludge micro-organisms in the study. This was confirmed by results from the toxicity control, which attained 56% biodegradation after 14 days and 46% biodegradation after 28 days.

The test item attained 0% biodegradation after 28 days and is therefore considered not readily biodegradable.
Remarks on result:
not measured/tested
Results with reference substance:
Sodium benzoate attained 79% biodegradation after 14 days, with >60% degradation within a 10-day window, thereby confirming the suitability of the inoculum and test conditions.

Inorganic carbon values on each analysis occasion

Day Inorganic Carbon (mg IC)
Inoculum Control  Procedure Control Test Item  Toxicity Control
R1 R2 R1 R2 R1 R2 R1
Abs 1 Abs 2 Abs 1 Abs 2 Abs 1 Abs 2 Abs 1 Abs 2 Abs 1 Abs 2 Abs 1 Abs 2 Abs 1 Abs 2
0 1.05 1.05 1.05 1.75 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.17 1.05 1.17
2 4.18 - 5.92 - 22.62 - 15.43 - 5.92 - 5.22 - 18.68 -
6 6.23 - 10.03 - 32.29 - 24.11 - 9 - 9.11 - 30.68 -
8 11.24 - 10.78 - 34.63 - 24.42 - 8.94 - 10.09 - 34.86 -
10 11.4 - 13.23 - 39.56 - 28.27 - 9.81 - 10.71 - 36.25 -
14 14.39 - 15.87 - 44.77 - 33.09 - 12.13 - 12.92 - 40.35 -
21 17.13 - 16 - 40.67 - 38.87 - 15.66 - 16.45 - 38.42 -
28 22.18 - 23.63 - 48.16 - 44.58 - 22.18 - 23.29 - 43.34 -
29 24.05 2.2 25.49 2.09 51.21 2.09 48.21 2.09 24.72 1.39 23.16 2.09 45.36 2.09

R = Replicate

Abs = CO2 absorber vessel

Percentage biodegradation values

Day

Biodegradation (%)

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

47

3

30

6

67

6

50

8

62

0

53

10

72

0

53

14

79

0

56

21

77

0

49

28

78

0

45

29*

83

0

46

*Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Total and inorganic carbon values in the culture vessels on day 0

Test vessel

Total Carbon*
(mg/L)

Inorganic Carbon*
(mg/L)

IC Content
(% of TC)

Test Item
5 mg C/L R1

5.00**

-0.06

0

Test Item
5 mg C/L R2

4.79**

-0.15

0

R = Replicate

* Corrected for control values. Negative values are due to measured concentrations being less than control values

** TC value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

pH values of the test preparations on days 0 and 28

Test Vessel

pH

Day 0
Pre-Adjustment

Day 0
Post-Adjustment

Day 28

Inoculum Control R1

7.7

7.4

7.4

Inoculum Control R2

7.7

7.4

7.5

Procedure Control R1

7.7

7.4

7.5

Procedure Control R2

7.7

7.4

7.5

Test Item R1

7.7

7.4

7.5

Test Item R2

7.8

7.4

7.5

Toxicity Control

7.7

7.4

7.5

R = Replicate

Observations on the test preparations throughout the test period

Test Vessel

Observations on Test Preparations

Day 0

Day 7

Day 14

Day 21

Day 28

Inoculum Control

R1

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

 

R2

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Procedure Control

R1

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

 

R2

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Test Item

R1

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light pink/brown dispersion, no undissolved test item visible

Light pink/brown dispersion, no undissolved test item visible

R2

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light pink/ brown dispersion, no undissolved test item visible

Light pink/brown dispersion, no undissolved test item visible

Toxicity Control

 

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

Light pink/brown dispersion, no undissolved test or reference item visible

Light pink/brown dispersion, no undissolved test or reference item visible

R = Replicate

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item attained 0% biodegradation after 28 days and is therefore considered not readily biodegradable.
Executive summary:

The ready biodegradability of the test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, was assessed under aerobic conditions at a GLP accredited laboratory according to the OECD Guideline 301B, “Ready biodegradability; CO2 evolution test”.

 

In an initial experiment conducted using a test item concentration equivalent to 10 mg carbon/L, the toxicity control vessel, containing both the test item and reference substance (sodium benzoate), showed <25% biodegradation after 14 days, indicating that the test item had inhibitory effects on the microbial inoculum activity. The definitive test therefore exposed activated sewage sludge micro-organisms to a test item concentration equivalent to 5 mg carbon/L. Tests were conducted in sealed culture vessels in the dark at temperatures between 22-24°C for 28 days.

 

The reference substance attained 79% biodegradation after 14 days, with >60% degradation within a 10-day window, thereby confirming the suitability of the inoculum and test conditions. All other validity criteria specified by the OECD 301B Guideline were also met.

 

The test item attained 0% biodegradation (based on CO2 evolution) after 28 days and is therefore concluded to be not readily biodegradable.

Description of key information

The ready biodegradability of Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, was assessed under aerobic conditions at a GLP accredited laboratory according to the OECD Guideline 301B, “Ready biodegradability; CO2 evolution test” (Best, 2018).

 

In an initial experiment conducted using a test item concentration equivalent to 10 mg carbon/L, the toxicity control vessel, containing both the test item and reference substance (sodium benzoate), showed <25% biodegradation after 14 days, indicating that the test item had inhibitory effects on the microbial inoculum activity. The definitive test therefore exposed activated sludge micro-organisms with mineral medium to a reduced test item concentration equivalent to 5 mg carbon/L. Tests were conducted in sealed culture vessels in the dark at temperatures between 22-24°C for 28 days.

 

The reference substance attained 79% biodegradation after 14 days, with >60% degradation within a 10-day window, thereby confirming the suitability of the inoculum and test conditions. All other validity criteria specified by the OECD 301B Guideline were also met.

 

The test item attained 0% biodegradation (based on CO2 evolution) after 28 days and is therefore concluded to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information