Registration Dossier

Environmental fate & pathways

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:
29 March 2011 - 19 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
yes
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (ISO 1995) is to dissolve the test item in an auxiliary solvent prior to adsorption onto filter paper. High shear mixing was also applied to break up the filter paper containing the test item. Using this method 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.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: Experimental ALC20110128 Exp1
Description: off white powder
Storage conditions: room temperature in the dark
The integrity of supplied data relating to the identity, purity and stability of the test item was the responsibility of the Sponsor.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PRE-STUDY SOLUBILITY WORK
Data supplied by the Sponsor indicated that the test item was insoluble in water and pre-study solubility work confirmed that it was not possible to attain a solution of the test item by using the traditional methods of ultrasonication and high shear mixing.
In order to obtain the best testable dispersion of the test item, further pre-study solubility/dispersibility work was performed.

EXPERIMENTAL PREPARATION
Following results of the pre-study solubility work and the recommendations of the International Standards Organisation (ISO 1995), the test item was prepared as a solvent stock solution and an aliquot dispensed onto filter paper prior to high shear mixing and dispersion in culture medium. This method appeared to increase the dispersibility of the test item over the other methods and increased the surface area of the test item exposed to the test organisms.

An initial experiment was conducted at a concentration of 10 mg C/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days. These results indicated that, under the strict terms and conditions of the OECD Guidelines, the test item would be classed as exhibiting inhibitory effects.

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test item at a reduced concentration of 5 mg C/L was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.
An amount of test item (1000 mg) was dissolved in 10 mL of acetone to give a 1000 mg/10 mL solvent stock solution. An aliquot (198 µL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was then dispersed in approximately 400 mL of culture medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to dispersal in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 6.6 mg/L, equivalent to 5 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.

Control vessels were prepared containing a single filter paper per 3 litres of inoculated culture medium. Acetone (198 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 30 minutes. The filter paper was then dispersed in approximately 400 mL of culture medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated culture medium in order to maintain consistency between the control and test item vessels.

REFERENCE ITEM
For the purposes of the test, a reference item, sodium benzoate, was used. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in culture medium with the aid of ultrasonication for approximately 5 minutes. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated culture medium and the volume adjusted to 3 litres to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

A filter paper was added to each vessel in order to maintain consistency between the test and reference item vessels. Acetone (198 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was then dispersed in approximately 400 mL of culture medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated culture medium in order to maintain consistency between the reference and test item vessels.

TOXICITY CONTROL
For the purposes of the test, 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 sludge micro-organisms used in the test.

An aliquot (198 µL) of the test item solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was then dispersed in approximately 400 mL of culture medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to the test vessel. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume was adjusted to 3 litres to give a final concentration of 6.6 mg/L test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 15 mg carbon/L.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK

PREPARATION OF INOCULUM
The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out 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 deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C 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 3.0 g/L prior to use.
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
5 other: mg carbon/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
CULTURE MEDIUM
Solution a KH2PO4 8.50 g/L
K2HP04 21.75 g/L
Na2HPO4.2H20 33.40 g/L
NH4CI 0.50 g/L
pH = 7.4
Solution b CaCl2 27.50 g/L
Solution c MgSO4.7H20 22.50 g/L
Solution d FeCI3.6H20 0.25 g/L
To 1 litre (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

*Reverse osmosis purified and deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)

PREPARATION OF TEST SYSTEM
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium a filter paper
b) The reference item (sodium benzoate), in duplicate, in inoculated culture medium plus a filter paper to give a final concentration of 10 mg carbon/L.
c) The test item, on filter paper, in duplicate, in inoculated culture medium to give a final concentration of 5 mg carbon/L.
d) The test item on filter paper, plus the reference item in inoculated culture medium to give a final concentration of 15 mg carbon/L to act as a toxicity control (one vessel only).

A filter paper with acetone evaporated to dryness was added to the control and reference item vessels in order to maintain consistency between these vessels and the test item vessels.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at approximately 21°C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of culture medium and 30.0 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 mL/minute 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 de-gassed water.

SAMPLING AND ANALYSIS
CO2 analysis
Samples (2 mL) were taken from the control, reference and test item first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, reference and test item and on Day 0 for the toxicity control.
The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analysed for CO2 immediately.
On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-Vcsh TOC analyser. Samples (300 or 50 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
Samples (30 mL) were removed from the test item and toxicity control vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (approximately 5 mL discarded) prior to DOC analysis.
DOC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.
On Days 0 and 28 samples (30 mL) were removed from the control and reference item vessels and filtered through 0.45 µm Gelman AcroCap filters (approximately 5 mL discarded) .
The samples were analysed for DOC using a Shimadzu TOC-Vcph TOC Analyser. Samples (50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

pH MEASUREMENTS
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

EVALUATION OF DATA
Determination of carbon content
The theoretical amount of carbon present in the test item (C24H30O4) was calculated as follows:
[(No of C atoms x mol wt of C) / mol wt of test item] x 100% = [(24 x 12.011) / 382.5] X 100 = 75.36%

Thus for a concentration of 5 mg C/L (a total of 19.8 mg of test item in 3 litres) the total organic carbon present was 15 mg C.

The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated as follows:
[(No of C atoms x mol wt of C) / mol wt of sodium benzoate] x 100% = [(7 x 12.011) / 144.11] x 100 = 58.34%
Thus for a 10 mg C/L test concentration (a total of 51.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 30 mg C.


PERCENTAGE DEGRADATION
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, in the following equation (using the mean values for replicates R1 and R2 for the control, test and reference items):

ThCO2(= % degradation) = [(mg IC in test flask - mg IC in control) / mg TOC as test chemical] x 100%


The percentage degradation from the results of the DOC analysis, see Table 2, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.

Percentage Degradation = [1 - (mg DOC in test flask on Day 28 / mg DOC in test flask on Day 0)] x 100%

The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The mean inorganic carbon values for Replicates R1 and R2 on Day 28 are substituted into the equation.

Total CO2 evolution = mg IC in control x (100 / %C of CO2) x (1 / test volume)
= mg IC in control x (100 / 27.29) x (1 / 3)


STATISTICAL ANALYSIS
Statistical analysis of the Day 29 IC values for the control and test items 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).

VALIDATION CRITERIA
-The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60% degradation by Day 14.
-The test item may be considered to be readily biodegradable if 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
-The toxicity control (test item and sodium benzoate) should attain 25% degradation by Day 14 for the test item to be considered as non-inhibitory.
-The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20%.
-The total CO2 evolution in the control vessels at the end of the test should not normally exceed 40 mg/L medium.
-The IC content of the test item suspension in the mineral medium at the beginning of the test should be < 5% of the TC.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
An initial experiment was conducted at a concentration of 10 mg C/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days. These results indicated that, under the strict terms and conditions of the OECD Guidelines, the test item would be classed as exhibiting inhibitory effects.
Following the recommendations of the OECD Guidelines it was considered appropriate to reduce the test concentration employed in the study to 5 mg C/L in order to minimise the toxic effect that the test item exhibited on the sewage treatment micro-organisms used in the study. It was not possible to test at concentrations below 5 mg C/L as below this concentration it was not possible to distinguish between background CO2 evolution from the inoculum and CO2 evolution due to biodegradation using inorganic carbon analysis.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
29 d
Details on results:
Inorganic carbon values for the test item, reference item, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 4. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 2, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 3. The pH values of the test preparations on Day 28 are given in Table 5.

-The total CO2 evolution in the control vessels on Day 28 was 27.57 mg/I and therefore satisfied the validation criterion given in the OECD Test Guidelines.
-The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
-The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels with the exception of test item replicate R1. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test item attained 0% degradation after 28 days.

Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. However the toxicity control only attained 3% degradation after 14 days thereby confirming that the test item was showing an inhibitory effect to the sewage treatment micro-organisms used in the test.
Care should be taken in the interpretation of the results due to the inhibitory nature of the test item to the activated sewage sludge micro-organisms used in the study; however the inhibitory nature of the test item and its physico chemical properties precluded the use of an alternative OECD/EC ready biodegradability test method to assess the biodegradability of the test item.

Analysis of the test media taken from the reference item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 3, gave percentage degradation values of 91% and 100% respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 66% degradation after 14 and 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Table 1 Inorganic Carbon Values on Each Analysis Occasion

 

Day

Control (mg IC)

Sodium Benzoate (mg IC)

Test Item (mg IC)

Test Item + Sodium Benzoate Toxicity Control (mg IC)

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

3.15

1.75

3.50

1.87

4.20

2.10

3.50

2.10

3.50

1.75

4.20

2.10

2.22

1.75

2

5.92

-

3.94

-

21.11

-

18.44

-

6.96

-

4.06

-

11.02

-

6

12.69

-

9.69

-

34.37

-

28.14

-

12.57

-

9.34

-

15.46

-

8

16.51

-

12.96

-

37.84

-

30.04

-

14.45

-

11.12

-

18.00

-

10

16.53

-

14.71

-

40.01

-

31.23

-

15.84

-

13.34

-

18.13

-

14

14.96

-

17.79

-

37.97

-

34.57

-

16.43

-

13.60

-

17.68

-

21

19.27*

-

19.83

-

46.98

-

37.07

-

19.15

-

17.69

-

-

-

28

22.51

-

22.62

-

47.27

-

39.76*

-

20.39

-

20.61

-

-

-

29

19.93

4.41

21.60

3.48

44.20

3.83

36.30

4.87

20.82

3.48

19.37

3.48

-

-

R1 - R2 = Replicates 1 and 2

Abs = CO2 absorber vessels

*Duplicate sample analysed as the original sample was deemed to be anomalous

 

Table 2 DOC Values in the Culture Vessels

 

Test Vessel

DOC* Concentration

Day 0

Day 28

mg C/L

% of Nominal Carbon Content

mg C/L

% of Initial Carbon Concentration

% Degradation

Sodium Benzoate

10 mg C/L R1

9.75

98

0.89

9

91

Sodium Benzoate

10 mg C/L R2

9.69

97

< control

0

100

 *Corrected for control values

 

Table 3 Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation Test Item + Sodium Benzoate Toxicity Control

0

0

0

0

2

50

4

14

6

67

0

9

8

64

0

7

10

67

0

6

14

66

0

3

21

75

0

-

28

70

0

-

29*

66

0

-

- = No degradation result obtained due to toxicity control being terminated after 14 days

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

 

Table 4 Total and IC Values in the Culture Vessels on Day 0

Test Vessel

Total Carbon*

IC*

IC Content (% of TC)

Sodium Benzoate

10 mg C/L R1

9.80

0.04

0

Sodium Benzoate

10 mg C/L R2

9.99

0.28

3

Test Item 5

mg C/L R1

4.99**

0.07

1

Test Item

5 mg C/L R2

4.83**

-0.11

0

Test Item + Sodium benzoate Toxicity Control 15 mg C/L

 

15.10**

 

0.17

 

1

R1 - R2 = Replicates 1 and 2

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

**Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item and sodium benzoate where applicable.

 

 

Table 5 pH Values of the Test Preparation on Day 28

Test Vessel

pH

Control R1

7.5

Control R2

7.4

Sodium Benzoate 10 mg C/L R1

7.5

Sodium Benzoate 10 mg C/L R2

7.5

Test Item 5 mg C/L R1

7.4

Test Item 5 mg C/L R2

7.4

Applicant's summary and conclusion

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

The test item attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).