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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:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Inhibition of CO2 evolution was observed in a separate study to determine the toxicity of the test item to activated sludge in a respiration inhibition test. The EC20 value was 52 mg/L. Therefore, the test item concentration in this study was selected to be 40 mg/L, which fulfills the test guideline recommendations.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: ARA Ergolz II, Füllinsdorf, Switzerland
- Laboratory culture: No. Inoculum was prepared and used on the day of collection
- Preparation of inoculum for exposure: Sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted
- Pretreatment: A homogenized aliquot of the final sludge suspension was weighed, dried, and the ratio of wet to dry weight was calculated. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 4 g dry material per liter. During holding, the sludge was aerated at room temperature until use.
- Initial cell/biomass concentration: Not applicable. Test water was inoculated with defined volumes of diluted activated sludge to give a final concentration of 30 mg dry material per liter.
Duration of test (contact time):
15 d
Initial conc.:
39.9 - < 40.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Medium recommended in test guidelines
- Additional substrate: no
- Test temperature: 21 to 23 °C
- pH: Start of test: 7.4
End of test (day 15):
Inoculum controls: 7.2
Sodium benzoate: 7.4
Test material: 7.4 and 7.5
Test material plus Sodium benzoate: 7.4
Abiotic control: 7.4
- Aeration of dilution water: Before test initiation
- Suspended solids concentration: 30 mg suspended solids /L
- Other: Test solution stirred continuously by magnetic stirrer

TEST SYSTEM
- Culturing apparatus: 2000 mL Erlenmeyer flask
- Number of culture flasks/concentration: Two each for control, test substance, and Sodium benzoate. One for toxicity control (test substance plus Sodium benzoate) and one for abiotic control
- Measuring equipment: TOC Analyzer equipped with an autosampler (Shimadzu TOC-5000A)
- Test performed in closed vessels due to significant volatility of test substance: no

SAMPLING
- Sampling frequency: Day 0 (treatment day), 2, 5, 7, 9, 14 and 15

CONTROL AND BLANK SYSTEM
- Inoculum blank: activated sludge medium without test substance
- Abiotic sterile control: 39.9 mg test material/L, poisoned with mercury dichloride at a concentration of 10 mg/L
- Toxicity control: 39.7 mg test material/L plus 50.0 mg Sodium benzoate/L
- Positive control: Sodium benzoate, 50.0 mg/L
Reference substance:
benzoic acid, sodium salt
Remarks:
50 mg/L
Key result
Parameter:
% degradation (DOC removal)
Value:
100
Sampling time:
5 d

 Dissolved Organic Carbon (DOC)

Day

Mean DOC (mg/L) [a] [b]

Mean DOC Sodium
Benzoate [c]

Mean % Degradation
Test Material [c]

% Degradation
Toxicity Control

% Degradation
Abiotic Control

0

30.0

15.4

45.5

17.2

2

0.6

12.6

15.4

16.9

5

0.1

-0.1

0.0

16.6

7

0.3

0.2

0.0

16.9

9

0.3

-0.2

1.1

17.1

14

-0.2

--

-0.2

17.5

15

n.a.

-0.9

n.a.

n.a.

[a] mean DOC values of at least triplicate measurements per sample

[b] Corrected for inoculum controls (except for abiotic control)

[c] Mean of duplicate flasks

-- = no data obtained due to a technical defect

n.a. = Not analyzed

 

Percentage Biodegradation Values [a]

(Based on mean DOC values of at least triplicate measurements per sample)

Day

Mean % Degradation

Sodium Benzoate [b]

Mean % Degradation

Test Material [b]

% Degradation

Toxicity Control

% Degradation

Abiotic Control

0

0

0

0

0

2

98

18

66

2

5

100

100

100

3

7

99

99

100

2

9

99

101

98

1

14

100

--

100

-2

15

n.a

106

n.a.

n.a.

[a] Corrected for inoculum controls (except for abiotic control)

[b] Mean of duplicate flasks

-- = no data obtained due to a technical defect

n.a. = Not analyzed

 

In the test flasks containing the test item MERGAL V615 and activated sludge (inoculum), the mean concentration of DOC (dissolved organic carbon) decreased from 15.4 mg/L (Day 0) to –0.1 mg/L (corrected for the inoculum control) at Day 5 (see Table 5 below). The process of biodegradation was completely finished within this short period of 5 days

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test material, ‘MERGAL V615’, is readily biodegradable.
Executive summary:

A GLP-compliant study was conducted to determine the ready biodegradability of ‘MERGAL V615’ according toOECD Guideline 301A and EU Commission Directive 92/69 EEC C.4-A. Activated sewage sludge micro-organisms were exposed to approximately 40 mg test material/L with culture medium in sealed culture vessels in the dark at 21° to 23 °C for 15 days. Initial pH was 7.4. 

 

In the test flasks containing the test item MERGAL V615 and activated sludge (inoculum) the mean concentration of DOC (dissolved organic carbon) decreased from 15.4 mg/L at Day 0 to –0.1 mg/L (corrected for the inoculum control) at Day 5. The process of biodegradation was completely finished within this short period of 5 days. 

The pass level for ready biodegradability, i.e. biodegradation of at least 70% removal of DOC in a 10-day window within the 28-day period of the test, was achieved. The 10-day window started at approximately Day 1. After 5 days, the DOC decrease already reached a plateau and amounted to 100%.  

In the abiotic control which contained the test item and poisoned medium, the DOC concentrations varied from 16.6 to 17.5 mg/L over the exposure period of 15 days and were not significantly different from the initial DOC concentration of 17.2 mg/L measured at Day 0. Thus, no significant abiotic elimination occurred under the conditions of the test. 

Furthermore, the observed 100% DOC removal within 5 days for MERGAL V615 is consistent with the ready biodegradability of formaldehyde at concentrations below the toxicity threshold. Also, the IUCLID data set for ethylene glycol cites a large number of studies that demonstrate ready biodegradability. These data confirm that the products of ‘MERGAL V615’ hydrolysis are readily biodegradable. 

Description of key information

The biodegradability of the test substance was investigated in a study on ready biodegradability according to OECD Guideline 301D (Closed-Bottle-Test). Degradation was found to be 72% and 71% at test substance concentrations of 7.05 and 4.38 mg/L, respectively. After 14 days the oxygen uptake was 66 and 60% of the theoretical value. All validity criteria were met within the study. Therefore, EGForm can be classified as readily biodegradable, fulfilling the 10-days window criterion.

In a second test according to OECD Guideline 301A (DOC Die-Away Test) degradation was complete after 5 days.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Data on formaldehyde

Ready and inherent biodegradability

The biodegradability of formaldehyde was investigated in 2 tests on ready biodegradability according to the OECD guidelines 301 C and D (cf. Doc III A7.1.1.2.1/01 and Doc III A7.1.1.2.1/02). In the MITI-I-Test (OECD 301 C) degradation after 14 days was found to be 91% of BOD/ThOD and 97% of TOC. Although the test substance concentration indicates that toxic effects can not be excluded (c.f. Doc II A4.2.1.4, Doc III A7.4.1.4), the degradation was nearly complete. In a Closed-Bottle-Test (OECD 301 D), the substance degraded to 90% of ThOD after 28 days. No data are reported referring to the 14-days window criteria. Due to the results of two tests according to OECD 301 C and D formaldehyde is expected to be readily biodegradable. This is in agreement with the OECD evaluation (OECD 2002).

Mechanism of biodegradation

The mechanism of formaldehyde degradation by Pseudomonas putida is initiated by a dismutation reaction, yielding formic acid and methanol as products. Degradation of the products began after exhaustion of formaldehyde in the medium.

Biological sewage treatment - Summary and Conclusion (Doc III A7.1.2.1)

The removal of formaldehyde in biological sewage treatment plants was studied in a lab-scale activated sludge unit (cf. Doc III A7.1.2.1.1). The test is comparable to OECD 303 A (Confirmatory Test). During the operation period of 160 days the influent concentrations were increased stepwise from 26 to 3168 mg/L. Removal of formaldehyde was calculated based on substance-specific and COD measurements. During the operation period, formaldehyde concentrations increased slightly when the influent concentrations were increased. Based on substance-specific measurements, high removal efficiencies of around 99.5% were maintained at all influent concentrations. However, ca. 18% of influent COD was present in effluent. The relative high COD content in the effluent can be explained by disproportionation of formaldehyde to methanol and formic acid. It was shown in previous experiments that degradation of methanol and formic acid began after exhaustion of formaldehyde in the medium. In the unit, ammonium was removed around 99.9%, indicating that there was no inhibition of nitrification. The hydraulic retention time (2.4 days) in the test unit is above the value (6 hours) proposed by the OECD guideline 303 A. Therefore, the resulting removal rate is assumed to probably overestimate removal in biological treatment plants.

The anaerobic degradation of formaldehyde was studied in a non-guideline batch test (cf. Doc III A7.1.2.1.2). Sludge from the anoxic chamber of the full-scale wastewater treatment plant of a resin-producing factory was used as inoculum. An initial formaldehyde concentration of 1360 mg/L was applied. The formaldehyde concentration decreased rapidly in the test medium, after 4 days the test substance was completely biodegraded. Simultaneously to formaldehyde removal, methanol and formic acid were formed as intermediate products. Degradation of both metabolites began after the exhaustion of formaldehyde in the medium.

In conclusion, in the wwtp simulation tests formaldehyde was removed to 99.5% under aerobic conditions. The test on anaerobic degradation reveals that formaldehyde is rapidly removed in anaerobic digester sludge.