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:
3 October 1996 - 14 November 1996
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:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
MMBC
Appearance: off-white crystalline solid
Storage conditions: room temperature

The identity, strength and purity of the test material received and its stability under the storage conditions were the responsibility of the Sponsor.

Test concentrations quoted in this report refer to the test material as received; no allowance has been made for a purity of less than 100 %.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, adapted
Details on inoculum:
BACTERIAL INHIBITION TEST
Source: The sample of secondary effluent was obtained on the day of the test From Thorndon sewage treatment works, a trickling-filter plant that treats predominantly domestic waste.
Treatment: It was maintained under aerobic conditions in the laboratory until required and vacuum-filtered through a Whatman GFC filter paper immediately before use. The filtrate was used as the source of inoculum for the test (1 drop/litre test medium).

MODIFIED STURM TEST
Source: A sample of activated sludge was collected on the day before the test from Oakley STW, a sewage treatment works that treats predominantly domestic waste.
Treatment: Aliquots (25 mL) of a homogenised sample were filtered through dried (approximately 105 °C) and pre-weighed Whatman's GFC filter papers. The litters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was then determined and the volume required to give a solids level of 30 mg/L was then calculated.
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
other: Nominal test material concentration
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
BACTERIAL INHIBITION TEST
Four groups of four BOD bottles were filled with Mineral Salts Medium (MSM; see below), inoculum and test substance and/or sodium benzoate. Care was taken to avoid the introduction of air bubbles during preparation and transfer of media to bottles.

Group Additions to MSM
1 lnoculum
2 lnoculum + sodium benzoate (2 mg/L)
3 lnoculum + test material (10 mgC/L)
4 Inoculum + test material (10 mgC/L) + sodium benzoate (2 mg/L)

The results of a preliminary solubility trial showed that the test substance was insufficiently soluble in water to allow preparation of a suitable stock solution so an appropriate weight (4.8 mg) was added directly to BOD bottles. Test concentrations of the reference substance were prepared front an aqueous solution (1 g/L).
The concentrations of dissolved oxygen (DO) and the temperatures of the contents of duplicate vessels from each group were measured, using a YSI dissolved oxygen meter fitted with a self-stirring DO/temperature bottle probe, at the start of the test and alter incubation in darkness for five days at 20 °C.
The pH of each control, test and reference mixture was measured after oxygen and temperature measurements.

MODIFIED STURM TEST
Five test vessels (five-litre brown glass carboys) each containing mineral salts medium (MSMS; see below), and the bacterial inoculum at a concentration of 1 % were aerated overnight with air that had been passed through cylinders containing calcium chloride and Carbosorb AS followed by two bottles containing 0.025N barium hydroxide to remove CO2. Mixtures containing test substance or sodium benzoate were then prepared. In each case the volume prepared was three litres.
Vessel No. Contents
1 Inoculated MSMS
2 Inoculated MSMS
3 Inoculated MSMS + sodium benzoate (10 mgC/L)
4 Inoculated MSMS + test material (10 mgC/L)
5 Inoculated MSMS + test material (10 mgC/L)

The test substance was added as ultrasound-treated suspensions (51.3 mg/100 mL). The reference substance sodium benzoate was added as an aqueous stock solution (1.72 g/L; 1 g carbon /litre).

Following the addition of the test and reference materials to the test vessels, samples (100 mL) were removed for the determination of pH. Each vessel was then fitted with a stopper holding an air inlet tube reaching approximately 10 cm below the liquid surface and an air outlet lust below the stopper.

The vessels contents were continuously flushed for 29 days with treated air. The air outlet from each vessel was connected to three Drechsel bottles in series, each containing 0.025N barium hydroxide (100 mL).

The residual concentrations of barium hydroxide in the bottles nearest to the test vessels were determined at intervals by duplicate titration of 20 mL samples with hydrochloric acid (0.05N), using phenolphthalein indicator.

Following the removal of the first Drechsel bottle in a series, the second was connected to the test vessel, and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.

On Day 28 of the test, titrations were undertaken and samples (approximately 100 mL) were removed from each test vessel for pH determination. Concentrated hydrochloric acid (1 mL) was then added to each vessel to drive off inorganic carbon dioxide
The contents of the vessels were aerated overnight and the final titrations carried out on Day 29.
The rate of-air flow through the apparatus was determined at intervals during the test.
The temperature of the test area was recorded using a maximum-minimum thermometer.

PREPARATION OF MINERAL SALTS MEDIA
Dilution water
The dilution water used to prepare stock and test solutions of mineral salts and test substance was tap water that had been softened and treated by reverse osmosis (Elga Ltd. Prima 4 reverse osmosis unit) and then purified (Elga Ltd, UHP) to give a resistivity of 18 Megohm/cm.

Mineral salts media
The mineral salts medium (MSM) for the preliminary test was prepared by adding 1 mL of each of the following stock solutions to each litre of water required for the test.
The mineral salts medium (MSMS) for the final test was prepared by mixing 10 mL of stock solution with 800 mL dilution water and then adding 1 ml of solutions 2 to 4 to each litre of water required for the test.

Stock 1 g/L
Potassium dihydrogen phosphate 8.50
di-Potassium hydrogen phosphate 21.75
di-Sodium monohydrogen phosphate dehydrate 33.40
Ammonium chloride 0.50

Stock 2
Magnesium sulphate heptahydrate 22.50

Stock 3
Calcium chloride dehydrate 36.40

Stock 4
Iron (III) chloride hexahydrate 0.25

CALCULATIONS
Calculation of theoretical oxygen demand (ThOD)
ThOD (mgO2/mg) = 16[2C + 1 /2(H - CI) -O] / MW

where MW is the molecular weight of the material.
The equation assumes that carbon is mineralised to CO2, hydrogen to H2O and chlorine is reduced to HCl.


Biochemical oxygen demand (BOD)
The amounts of oxygen consumed in test and control vessels in the bacterial inhibition test were calculated as follows:
BODn = (Mt0 - Mta) - (Mb0 - Mbn)

where n = the number of days after the start of the test
Mtn = the mean oxygen level in bottles containing test substance at the start of the test.
Mta = the mean oxygen level in bottles containing test substance n-days after the start of the test.
Mb0 = the mean oxygen level in bottles containing inoculated mineral salts medium at the start of the test.
Mbn = the mean oxygen level in bottles containing inoculated mineral salts medium n-days after the start of the test.

The percentage degradation of sodium benzoate during the test was calculated in terms of its ThOD in the following way:
% degradation n = [BODn (mgO2/mg) after n days / ThOD (mgO2/mg)] x 100

In groups containing both sodium benzoate and MMBC the apparent BOD of sodium benzoate has been calculated by subtracting the mean oxygen consumption in vessels containing test material alone at 10 mgC/L from that of mixtures. The resulting BOD has been expressed in terms of ThOD of sodium benzoate.

Theoretical carbon dioxide production (TCO2)
The theoretical amount of CO2 that can be generated by a 2.9-litre mixture containing the test or reference substance at a concentration of 10 mgC/L was calculated in the following way:
TCO2 /mg carbon = MW of carbon dioxide / MW carbon = 3.67 mg CO2/mg

Test mixture TCO2 = Volume (2.91) x conc. (10 mgC/L ) x 3.67 = 106.4 mgCO2

CO2 Production
Carbon dioxide production by control, reference and test mixtures was calculated in the following way.

Since 1 mmol of carbon dioxide is produced for every 2 mmol of hydrochloric acid titrated, the amount of CO2 precipitated as BaCO2 in a Drechsel bottle containing 100 mL Ba(OH)2 is:
mgCO2 = [N x (Vs - Vt) / 2] x (100 / V) x MW(CO2)

where: N = normality of hydrochloric acid.
Vs = volume of hydrochloric acid used to titrate the stock solution of barium hydroxide.
Vt = volume of hydrochloric acid used to titrate test samples.
V = volume of sample titrated.
MW(CO2) = molecular weight of carbon dioxide.

On each occasion when CO2 levels were determined, the mean CO2 level in the control group was subtracted from that in reference or test groups to give the blank-corrected value. The cumulative production of CO2 in each group was calculated by adding successive estimates of blank-corrected CO2 production.

Estimates of the percentage degradation of test and reference substances at intervals during the test were calculated from blank-corrected cumulative CO2 production in the following way:

% degradation = (cumulative CO2 (mg) / TCO2 of test mixture) x 100
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
6
Sampling time:
28 d
Details on results:
Calculation of ThOD
Test substance (C9H9O2Cl MW = 184.5) = 1.73 mgO2/mg.

Bacterial inhibition assay
The mean concentrations of test substance in BOD bottles containing the material alone and with benzoate sampled on Day 5, calculated from the weights added at the start, were 16.1 and 17.5 mg (9.4 and 10.3 mgC/L) respectively.

The pH and temperature ranges of the contents of individual bottles are shown below:
Day 0 Day 5
Temperature °C 21.6 - 21.8 21.1 - 21.3
pH 6.8 - 7.5 6.2 - 7.4

The mean oxygen concentrations of control and test media, the BODs of test substance and sodium benzoate expressed as mgO2/mg and the extent of degradation expressed as a percentage of ThOD are given in Table 1.

Oxygen consumption in bottles containing test substance alone at 10 mgC/L was negligible (0.3 % of its ThOD) which indicates that the test substance was not degradable under the conditions of this preliminary test.

Oxygen was not consumed in bottles containing inoculated mineral salts medium alone at five days.

Modified Sturm test
The blank-corrected CO2 production and percentage degradation in reference and test mixtures at intervals during the test is given in Table 2.
Cumulative CO2 production in the controls (12.7 and 11.3 mgCO2) was within the acceptable range for this assay system (recommended maximum for a three litre culture = 120 mgCO2).

Cumulative CO2 production by the mixtures containing test substance at 10 mgC/L was equivalent to 6 % of the TCO2 (106.4 mgCO2).

The pH of test mixtures ranged between 7.6 and 7.7 at the start of the test and between 7.5 and 7.7 at the end.

The rate of air flow during the test ranged from 23 to 75 mL/minute. Temperatures of the test area ranged from 20.1 °C to 22.0 °C.

BOD5 / COD results

Results with reference substance:
Calculation of ThOD
Sodium benzoate (C7H5O2Na MW = 144) = 1.67 mgO2/mg.

Bacterial inhibition assay
Sodium benzoate was degraded to 69% of its ThOD after five days of incubation which indicates that the inoculum was viable and exerting normal biodegradative activity. In the presence of test material at 10 mgC/L, degradation of benzoate was essentially unaffected at 66% of its ThOD which indicates that the test material was not inhibitory to the bacterial inoculum.
Modified Sturm test
The degradation of sodium benzoate was rapid and achieved 67 % of its TCO2 after seven days and 91 % alter 29 days. These results confirm that the inoculum was viable and that the test was valid.

Any other information on results incl. tables

Table 1 Bacterial Inhibition Assay - Mean Oxygen Concentration

BOD (mgO2/mg) and Percentage Degradation in Test and Control Bottles

Test Group

Mean Oxygen Concentration (mgO2/L)

BOD (mgO2/mg) Day 5

% Degradation BOD:ThOD

Day 0

Day 5

Inoculated mineral salts

Sodium benzoate (2 mg/L)

test substance (10 mgC/L)

Sodium benzoate (2 mg/L) + test substance (10 mgC/L)

8.9

9.0

8.9

9.0

8.9

6.7

8.8

6.7

(0)

1.15

0.006

1.10

-

69

0.3 %

66

 The value in brackets gives the oxygen consumption (mgO2/L) of the control group.

 

Table 2 Modified Sturm Test

Blank-corrected cumulative CO2 Production and Degradation as a Percentage of TCO2 in Reference and Test Mixtures

Day

Reference

Test Substance (10 mgC/L)

Culture 1

Culture 2

CO2 (mg)

% TCO2

CO2 (mg)

% TCo2

CO2 (mg)

% TCO2

2

3

4

5

7

12

19

28

29

14.5

30.1

44.4

58.4

71.1

84.6

92.3

95.3

96.4

14

28

42

55

67

80

87

90

91

1.9

2.7

3.8

4.

4.6

5.4

6.0

6.0

6.0

2

3

4

4

4

5

6

6

6

1.9

2.7

3.8

4.6

4.6

5.4

6.0

6.0

6.0

2

3

4

4

4

5

6

6

6

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
Under the conditions of this test, the test substance cannot be considered to be readily biodegradable.
Executive summary:

The ready biodegradability of the test substance was assessed in accordance with standardised guidelines EU Method C.4 -C and OECD 301B in a CO2 evolution test (modified Sturm test). A five-day bacterial inhibition test was performed under the conditions of the closed bottle test (in accordance with standardised guidelines EU Method C.4 -E and OECD 301D). This showed that the test substance at a nominal concentration of 10 mgC/L did not inhibit degradation of the reference material sodium benzoate. In this preliminary test the test substance showed no significant evidence of biodegradation. In the modified Sturm test the test substance was added to two vessels containing inoculated mineral salts medium, to give a nominal test concentration of 10 mgC/L. Control vessels comprised two containing inoculated mineral salts medium alone and one containing inoculated mineral salts medium plus the reference material sodium benzoate (10 mgC/L). Test and control vessels were aerated for 29 days with air that had been treated to remove carbon dioxide. The carbon dioxide produced by each culture was trapped in a series of Drechsel bottles containing barium hydroxide which were connected to the outlet from each test vessel. The residual barium hydroxide was determined at intervals by titration. The pH of control, reference and test mixtures was measured at the start of the test and after 28 days. Under the conditions of the study, sodium benzoate was degraded by 67% after seven days and 91% after 29 days. Cumulative carbon dioxide production in controls after 29 days (12.7 and 11.3 mg CO2) was within the acceptable range for this assay system confirming that the inoculum was viable and that the test was valid. Cumulative CO2 production by mixtures containing test substance at 10 mgC/L was equivalent to 6% of the theoretical value. Substances are considered to be readily degradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within 10 days of the level achieving 40%. The test substance cannot therefore be considered to be readily biodegradable.