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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 26 jul 2006 to 29 nov 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standardised guideline and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: from wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland).
- Laboratory culture: no
- Storage conditions: during holding, the sludge was aerated at room temperature.
- Storage length: no data
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was
decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g
(±10%) dry material per liter. During holding, the sludge was aerated at room temperature until use. Prior to use, the sludge was first thoroughly
mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted
activated sludge, defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.
- Concentration of sludge: 30 mg/L
- Water filtered: no
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:
- Composition of medium: The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:

a) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution was 7.4.

b) MgSO4 x 7H2O 22.50 g/L

c) CaCl2 x 2H2O 36.40 g/L

d) FeCl3 x 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter

To obtain the final test water, 10 mL of stock solution a) and 1 mL each of stock solutions b and d were combined and made up to 1000 mL with
purified water. The pH was adjusted from 8.0 to 7.4 with a diluted hydrochloric acid solution.

- Additional substrate: no
- Solubilising agent : no
- Test temperature: 22°C, maintained with a built-in thermostat and checked once per week
- pH: prior to the start, the pH was measured in each test flask before the addition of the activated sludge inoculum and, if necessary, adjusted with a dilution NaOH solution (for test item flasks, the pH were adjusted from 7.1 to 7.2). At the end of incubation, the pH was measured again in each test flask (from 7.3 to 7.7).
- pH adjusted: yes
- Aeration of dilution water: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: test flasks: 500-mL Erlenmeyer flasks
- Number of culture flasks/concentration: 8: 2 for test substance (101 mg/L), 2 for inoculum control (0 mg/L of TS), 2 for reference (100 mg/L of reference), 1 abiotic control (101 mg/L of TS) and 1 toxicity control (100 mg/L of TS)
- Method used to create aerobic conditions: incubated under continuous stirring. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution.
- Measuring equipment:
Apparatus: The test flasks (500-mL Erlenmeyer flasks, labeled with the necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT D12 (Voith GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily
reading at least on each working day.
Principle: Electro-chemical analysis process: the biodegradation process consumes the dissolved oxygen in the liquid and generates CO2. The CO2
is adsorbed by soda lime and the total pressure decreases in the airtight test flasks. The pressure drop is detected and converted into an electrical
signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate
solution.

SAMPLING:
- Sampling frequency: measurements have been made, without sampling, each day from day 0 to day 28, except at day 24.
- Sampling method: no sampling

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: 1
- Adsorption control: 0
- Toxicity control: 1
- other: procedure control: 2

STATISTICAL METHODS: no data
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
81
Sampling time:
28 d
Remarks on result:
other: 10-d window criteria fulfilled
Parameter:
% degradation (O2 consumption)
Value:
72
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
2
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
2.5
Sampling time:
10 d
Details on results:
The biochemical oxygen demand (BOD) of Paradimethoxybenzene in the test media significantly increased from about Exposure Day 18 until test
termination after 28 days. At the end of the 28-day exposure period, the mean biodegradation of Paradimethoxybenzene amounted to 81%. Moreover, the pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThOD in a 10-day window within the 28-day period of the test, was reached.
Consequently, Paradimethoxybenzene was found to be biodegradable under the test conditions within 28 days.
In the toxicity control, containing both Paradimethoxybenzene and the reference item sodium benzoate, Paradimethoxybenzene had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 100 mg/L (see table below).
Results with reference substance:
In the procedure controls, the reference item sodium benzoate was degraded by an average of 89% on Exposure Day 14, and reached an average
biodegradation of 94% by the end of the test (Day 28), thus confirming suitability of the activated sludge.
Validity criteria fulfilled:
yes
Remarks:
The O2 demand of the inoculum control was 3-4 mg O2/L (<60), the degradation rates of the two test flasks containing the test item deviated by 13% at the end of the test (<20%), the degradation of the reference item reached 64% at day 4 (60% at day 14)
Interpretation of results:
readily biodegradable
Conclusions:
readily biodegradable
Executive summary:

The test item Paradimethoxybenzene was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to EU Commission Directive 92/69 EEC, C.4-D (1992) and OECD Guideline for Testing of Chemicals No. 301 F (1992).

The percent biodegradation of the test item was calculated based on the theoretical oxygen demand (ThOD) of 2.20 mg O2/mg test item.

The biochemical oxygen demand (BOD) of Paradimethoxybenzene in the test media significantly increased from about Exposure Day 18 until test termination after 28 days. At the end of the 28-day exposure period, the mean biodegradation of Paradimethoxybenzene amounted to 81%. Consequently, Paradimethoxybenzene was found to be biodegradable under the test conditions within 28 days. Moreover, the pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThOD in a 10-day window within the 28-day period of the test, was reached.

Consequently, Paradimethoxybenzene was found to be readily biodegradable under the test conditions within 28 days.

No degradation of the test item occurred in the abiotic control under the test conditions within 28 days.

In the toxicity control, containing both Paradimethoxybenzene and the reference item sodium benzoate, Paradimethoxybenzene had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 100 mg/L.

In the procedure controls, the reference item sodium benzoate was degraded by an average of 89% on Exposure Day 14, and reached an average biodegradation of 94% by the end of the test (Day 28), thus confirming suitability of the activated sludge.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described concerning inherent biodegradability, but not GLP.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
no
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, industrial (adaptation not specified)
Details on inoculum:
Inoculum:
Mixture of activated sludge from the sewage treatment plant of the Hoechst AG at Frankfurt am Main, Höchst with activated sludge from the sewage treatment plant at Hoechst AG, site Kelsterbach in a rtaio 1 to 2.

Type of inoculum:
Industrial sewage sludge

Preparation of the suspension:
The standard procedure in 1988 was: washing the sediment from activated sewage sludge two times by centrifuging and resuspending in mineral medium.

Amount of the inoculum used: 12 g/L of wet centrifuged sludge were filled into the batch.
Duration of test (contact time):
28 d
Initial conc.:
200 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: corresponds to the requirements of the test directive DIN 38 412vPart 25 (1984)
- Test temperature: Testing was conducted in a room, which normally was thermostatically controlled at 21°C +/- 1°C, but measuring of the temperature during the test period is not documented in the laboratory manuscript.
- pH: 7.8
- pH adjusted: no
- Suspended solids concentration: approximately 1000 mg dry mass/L.
- Continuous darkness: no

TEST SYSTEM
- Culturing apparatus: 3-L beakers with magnetic stirrers, through which air was bubbled. The final volume of the batch was 2000 ml.
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: air bubbled
- Measuring equipment: no data
- Test performed in open system: yes

SAMPLING: the samples were filtered through folded paper filters

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, reaction medium with inoculum but without test or reference substance
- Abiotic sterile control: yes, batch with the test substance but without inoculum
- Toxicity control: yes (diethylene glycol at 271.6 mg DOC/L)

STATISTICAL METHODS: no data
Reference substance:
diethylene glycol
Remarks:
271.6 mg DOC/L
Parameter:
% degradation (DOC removal)
Value:
100
Sampling time:
12 d
Details on results:
Using methods for testing the biodegradability by analyzing the decrease of DOC it is distinguished between the total elimination and the biodegradation (100% value) the DOC value after 3 h minus the corresponding means of the blanks is used.
The elimination during the first 3 hours of the test remained below 10% and the following analyzes showed a total elimination of 98% of the test substance within 7 days in the test batch but also 76% in the abiotic control. The losses of DOC in the abiotic control were respected in the formula for the calculation of biodegradation:
BIODEGRADATION = [1-(100-E1) / (100 - AE)] * 100
E1 = calculated biodegradation in batch 1
AE = calculated elimination in the abiotic control.

With this formula at each time of measurement the biodegradation of 1,4-dimethoxybenzene is calculated on the basis of the remaining substance concentration in the abiotic control.
The results were confirmed by additionnally conducted COD control analyzes. The COD after 3 hours was 513 mg/L in the test batch and 673 mg/L in the abiotic control. After 12 days 31 mg/L in the test batch and 20 mg/L in the blank control, and 16 mg/L in the abiotic control were measured.
Results with reference substance:
100% degradation after 19 days.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
An indication for inherent and possibly even rapid biodegradability of 1,4-dimethoxybenzene may be derived from the results of the test. Due to the high abiotic elimination by volatility of the substance, the results of this test are not adequate for a certain evidence of this evaluation.
Executive summary:

In a study, (Clariant, 1990), the inherently biodegradability of 1,4-dimethoxybenzene was evaluated following the OECD 302B guideline (Zahn-Wellens test).

In the Zahn-Wellens test, biodegradation is determined by measuring the disappearence of Dissolved Organic Carbon (DOC removal). It specifies that the amount of degraded DOC within the test period of normally 28 days exceeds 70%

In this study, the percentage of biodegradation reached 100% after 12 days.

In these conditions, 1,4 -dimethoxybenzene is considered as inherently biodegradable.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Only one study was available and was selected as key study.

In this study (RCC, 2006), the test item Paradimethoxybenzene was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to EU Commission Directive 92/69 EEC, C.4-D (1992) and OECD Guideline for Testing of Chemicals No. 301 F (1992).

The percent biodegradation of the test item was calculated based on the theoretical oxygen demand (ThOD) of 2.20 mg O2/mg test item.

The biochemical oxygen demand (BOD) of Paradimethoxybenzene in the test media significantly increased from about Exposure Day 18 until test termination after 28 days. At the end of the 28-day exposure period, the mean biodegradation of Paradimethoxybenzene amounted to 81%. Consequently, Paradimethoxybenzene was found to be biodegradable under the test conditions within 28 days. Moreover, the pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThOD in a 10-day window within the 28-day period of the test, was reached.

Consequently, Paradimethoxybenzene was found to be readily biodegradable under the test conditions within28 days.