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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Remarks:
publication
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
see 'Principles of method if other than guideline'
Principles of method if other than guideline:
- Test substance concentration used: approx. 30 mg ThOD/L (50-100 mg ThOD/L according to guideline)
- Bacterial density of: 80 mL STP effluent/L final solution (10+E8 CFU/mL according to guideline)
GLP compliance:
yes
Remarks:
(not applicable)
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
The inoculum for each experiment was freshly taken from the Lüneburg municipal STP.
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test was run for 28 d at 20±1 °C in the dark with gentle stirring. Oxygen consumption was recorded daily using an OxiTop control OC110 system (WTW, Weilheim, Germany), measuring the pressure decrease in the headspace (about 1/3 of bottle volume), while CO2 produced was removed by adsorption/absorption to NaOH pellets/conc. NaOH solution. The temperature was monitored daily; the pH was measured at days 0 and 28, was adjusted to 6.5-8 if necessary at day 0, and was in this range at day 28.
Key result
Parameter:
% degradation (O2 consumption)
Value:
71.2
St. dev.:
6.6
Sampling time:
28 d
Details on results:
Results from two replicates:
66.6 % and 75.9 % biodegradation

The theoretical oxygen demand ThOD was calculated under the assumption that sulfur is oxidized to SO42-.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the degree of biodegradation reached 71.2% after 28 days, therefore the test substance was considered to be readily biodegradable.
Executive summary:

A study was conducted to evaluate the ready biodegradability of the tests substance according to a modified OECD Guideline 301F (Manometric Respirometry). The test was run for 28 d at 20±1 °C in the dark with gentle stirring. Oxygen consumption was recorded daily using an OxiTop control OC110 system, measuring the pressure decrease in the headspace (about 1/3 of bottle volume), while CO2 produced was removed by adsorption/absorption to NaOH pellets/conc. NaOH solution. The theoretical oxygen demand ThOD was calculated under the assumption that sulfur is oxidized to SO42-. Under the study conditions, the degree of biodegradation reached 71.2% after 28 days, therefore the test substance was considered to be readily biodegradable (Rücker, 2017).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 07, 2000 to March 07, 2000
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
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
CAS No.: 79-42-5; EWG No.: 201-206-5; Batch No.: B99S5170; Purity: 98.3 %; Appearance: clear liquid; Odour: characteristic
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge:
Prior to use, a sample of activated sludge from the sewage plant at Waldems-Esch was washed twice with the mineral nutrient solution used in this test to estimate organic components and carbonates from the sludge. After resuspension in mineral nutrient medium the sludge was aerated by means of compressed humidified air for about four hours. Before use, the sludge was homogenized in a Waring blender at low speed for 2 minutes and then flitrated through a cotton filter which had been carefully rinsed with deionised water. The first 200 mL of filtrate were discarded.

Inoculum from soil:
A mixture of nearly 100 g soil of the Institut Fresenius area at Taunusstein was suspended in 1000 mL drinking-water and shaken at laboratory shaker for approximately 30 min. The suspension was filtered through a paper filter. The first 200 mL of filtrate were discarded.

Inoculum from surface water:
A sample of water from the Eschbach near Waldems-Esch was filtered through paper filter. 200 mL of filtrate were rejected.
Same volumes of three inocula were mixed together and used in concentration of 1 % of the final volume of the test solution on the preparation.
Duration of test (contact time):
28 d
Initial conc.:
>= 20 - <= 40 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
A known quantity of the test substance of about 20-40 mg DOC/L was diluted in a mineral nutrient solution. The test solution was inoculated with 10 mL/L of the mixed inoculum. The test solutions were aerated with moistened compressed air and stirred on the magnetic stirrer. The incubation was running through 28 d in the dark at a consistent temperature (22±2 °C). Biodegradation of the test is followed by DOC-determinations made at regular intervals and is expressed as per cent loss of DOC. DOC measurements were performed at days: t0, t1, t3, t6, t9, t13, t16, t20, t23, t27 and t28.

Application Method:
According to the DOC of the test substance, the amount of test substance to be applied was calculated to give a final concentration of about 20 to 40 mg DOC/L. For preparation of the test solutions, the following stock solutions of mineral nutrient salts were prepared:

Stock solutions:
Solution 1:
8.50 g KH2PO4
21.75 g K2HPO4
33.40 g Na2HPO4 x 2 H2O
0.50 g NH4Cl
The salts were diluted in 500 mL ultrapure water and filled up to 1000 mL with ultrapure water; the pH should be 7.4.
Solution 2: 22.5 g MgSO4 x 7 H2O/ L ultrapure water (Seral)
Solution 3: 36.40 g CaCl2 x 2 H2O/ L ultrapure water (Seral)
Solution 4: 0.25 g FeCl3 x 6H2O/ L ultrapure water (Seral)

The test solutions contained a total volume of 2000 mL. First the test and/or control substance was added to nearly 1000 mL of deionised water, thereafter 20 mL of solution l, 2 mL of the solutions 2 to 4 and 20 mL of the before prepared inoculum were added and filled up to 2000 mL with deionised water. Test solution l contains 242 mg test substance/2L and test solution 2 contains 252 mg test substance/2L. Into the test solutions with control substance in each flask 140 mg Na-benzoate/2L were weight in.
To determine a possible bacterial toxicity of the test substance one test solution “toxicity control” containing 140 mg Na-benzoate plus 251 mg test substance/2000 mL of the test solution was prepared in the same way.
The ''abiotic sterile control'' test solution for the determination of an possible abiotic degradation contained 250 mg test substance and 60 mg HgCl2 / 2000 mL. Further two test solutions “blank” were prepared containing only deionized water, solutions 1 to 4 and the inoculum in the same amounts as named before.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (DOC removal)
Value:
38
Sampling time:
28 d
Details on results:
The mean degradation value at day 28, was 38 %. A degradability of ≥70 % for "ready biodegradability" was not obtained after 28 days. Therefore the test substance must be considered to be "not readily biodegradable".
The control substance was degraded 98 % within 9 d. The threshold of "ready biodegradability" of ≥70 % was met within 3 d of incubation (85 %). Accordingly, the test is considered to be valid.
There was no bacterial toxicity detectable at the used concentration.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the study conditions, a mean degradation of 38 % after 28 d was observed. According to the test guideline the pass level for ready biodegradability is removal of 70 %. Therefore, the test substance is considered not readily biodegradable.
Executive summary:

A study was conducted to determine the test substance ready biodegradability in a DOC Die-Away test over a period of 28 days according to OECD Guideline 301A, in compliance with GLP. A known quantity of the test substance of about 20-40 mg DOC/L was diluted in a mineral nutrient solution. The test solution was inoculated with 10 mL/L of the mixed inoculum. The test solutions were aerated with moistened compressed air and stirred on the magnetic stirrer. The incubation was running through 28 d in the dark at a consistent temperature (22±2 °C). DOC measurements were performed at days: t0, t1, t3, t6, t9, t13, t16, t20, t23, t27 and t28. The control substance Na-benzoate was degraded by 98 % within 9 d. The threshold of "ready biodegradability" of 70 % was met within 3 d of incubation (85 %). Therefore, the test is considered to be valid. Under the study conditions, a mean degradation of 38 % after 28 d was observed. According to the test guideline the pass level for ready biodegradability is removal of 70 %. Therefore, the test substance is considered to be not readily biodegradable (Springer, 2000).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Study 1:

A study was conducted to determine the test substance ready biodegradability in a DOC Die-Away test over a period of 28 days according to OECD Guideline 301A, in compliance with GLP. A known quantity of the test substance of about 20-40 mg DOC/L was diluted in a mineral nutrient solution. The test solution was inoculated with 10 mL/L of the mixed inoculum. The test solutions were aerated with moistened compressed air and stirred on the magnetic stirrer. The incubation was running through 28 d in the dark at a consistent temperature (22±2 °C). DOC measurements were performed at days: t0, t1, t3, t6, t9, t13, t16, t20, t23, t27 and t28. The control substance Na-benzoate was degraded by 98 % within 9 d. The threshold of "ready biodegradability" of 70 % was met within 3 d of incubation (85 %). Therefore, the test is considered to be valid. Under the study conditions, a mean degradation of 38 % after 28 d was observed. According to the test guideline the pass level for ready biodegradability is removal of 70 %. Therefore, the test substance is considered to be not readily biodegradable (Springer, 2000).

Study 2:

A study was conducted to evaluate the ready biodegradability of the tests substance according to a modified OECD Guideline 301F (Manometric Respirometry). The test was run for 28 d at 20±1 °C in the dark with gentle stirring. Oxygen consumption was recorded daily using an OxiTop control OC110 system, measuring the pressure decrease in the headspace (about 1/3 of bottle volume), while CO2 produced was removed by adsorption/absorption to NaOH pellets/conc. NaOH solution. The theoretical oxygen demand ThOD was calculated under the assumption that sulfur is oxidized to SO42-. Under the study conditions, the degree of biodegradation reached 71.2 % after 28 days, therefore the test substance was considered to be readily biodegradable (Rücker, 2017).

Overall assessment:

It is assumed that structurally similar compounds should behave similarly under same 301 OECD test. However, published biodegradation data for substances containing both an acid or ester and a mercaptan functional group are in many cases contradictive (Rücker, et al. 2017).

Results of repeated biodegradation experiments for the same substance in the same test using inoculum from the same source may differ considerably. These findings are also supported by the relevant guidelines: “Realising that ready biodegradability tests may sometime fail because of the stringent test conditions, positive test results should generally supersede negative test results” (ECHA 2017, page 208; OECD 2006, page 3), and “When contradictory results in ready biodegradability tests are obtained the positive results could be considered valid irrespective of negative results, when the scientific quality of the former is good and the positive test results are well documented, …” (ECHA 2017, page 230).

Based on the above mentioned information, weight of evidence approach was taken to assess the biodegradability of the test substance. Following studies were taken into consideration (I) a study conducted according to OECD Guideline 301 A (Springer 2000) and (2) published data from the article (Rücker, et al. 2017), which provides results from OECD study 301 F.

In the study by Rücker, et al. 2017, 24 substances containing divalent sulfur, (i. e., mercaptocarboxylic acids, their esters, disulfides, sulfides and mercaptans) were tested in two standardised biodegradation tests, OECD 301 D (Closed Bottle Test, CBT) and OECD 301 F (Manometric Respirometry Test, MRT) in the same laboratory.

For most of the test substances strong differences between CBT (OECD 301 D) and MRT (OECD 301 F) results were observed with MRT test being more effective. For our substance of interest, MRT test biodegradation of 71.2 % in day 28 and a biodegradation of 12.4 % with CBT test were achieved. Based on the literature and experimental biodegradation information presented in the article, mercaptocarboxylic acids and their esters as a class are found to be either readily biodegradable or at least biodegradable to a significant extent.

Based on the weight of evidence approach, the test substance is concluded to be readily biodegradable.

 

ECHA, 2017: Guidance on information requirements and chemical safety assessments, Chapter R.7b: Endpoint specific guidance, June 2017,

OECD, 2006: Organisation for Economic Co-operation and Development, OECD Guidelines for the testing of chemicals, Revised introduction to the OECD Guidelines for testing of chemicals, Section 3, adopted 23 March 2006,

Rücker Ch., Mahmoud W. M. M., Schwartz D., Kümmerer K. Biodegradation tests of mercaptocarboxylic acids, their esters, re-lated divalent sulfur compounds, and mercaptans. Manuscript (24.11.2017).