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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:
supporting study
Study period:
From November 2, 2005 to December 1, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study with the test substance was conducted according to ISO Guideline 14593 and can be considered comparable to a reduced version of OECD Guideline B. The following validity criteria of the OECD guideline were not met: 1. Inorganic carbon analysis done only on Day 28 for both reference and test substances, therefore the course of biodegradation was not followed throughout the study. 2. Toxicity control not used and no information was provided on inhibition of inoculum by the test substance.
Qualifier:
equivalent or similar to
Guideline:
ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))
Deviations:
yes
Remarks:
A reduced version of the test was performed, as a screening test, with inorganic carbon analysis on Day 28 only.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary effluent, from a laboratory treatment unit treating sewage from Newton Abbot sewage treatment works
- Pretreatment: On the day of the test the effluent was filtered and sparged with CO2 free air for at least 2 h at pH 6.5 ± 0.2, to remove any dissolved
carbon dioxide. After this time the effluent was neutralised, to pH 7.0 ± 0.2, and allowed to stand for 1 h.
- Concentration: 10 % of the final volume of test bottles
- Water filtered: Yes
- Type and size of filter used, if any: Whatman G/FC filter paper
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 19 mg/L
Based on:
ThIC
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: 85 mg of KH2PO4, 217.5 mg of K2HPO4, 334 mg of Na2HPO4.2H2O, 5 mg of NH4CI, 22.5 mg of MgSO4.7H2O, 36.4 mg of CaCl2.2H2O and 0.25 mg of FeCl3.6H2O per litre of deionised water
- Test temperature: 22 ± 2 °C
- pH: Initial - 7.0 ± 0.2; Final - 6.9 in the inoculum blank bottle, 6.6 in the reference substance bottle and 6.7 in the test material bottle.
- pH adjusted: Yes (initially)
- Other: Continuous shaking of bottles in orbital incubator

TEST SYSTEM
- Culturing apparatus: Septum sealed bottles with a nominal volume of 125 mL (each filled with 95 mL inoculated mineral medium, and made up to
100 mL with test or reference substance solution and/or water as applicable)
- Number of culture flasks/concentration: Triplicate bottles of each set (blank, reference and test bottles). Additional triplicate blank and reference substance test bottles for TOC analysis
- Measuring equipment and method: 1 mL of 10 M sodium hydroxide solution was added to the appropriate test bottles, and shaken for at least 1 h. Inorganic carbon (IC) was then measured using a Dohrman DC-190 carbon analyser. For IC analysis, this instrument acidifies the sample with 20 % phosphoric acid, purges with carrier gas oxygen (carbon dioxide free) and quantifies the resulting gas for entrained carbon dioxide using a non-dispersive infra-red detector.
- Test performed in closed vessels: Yes
- Other: TOC was analysed only for reference. The low solubility of test material made TOC analysis impractical.

SAMPLING
- Sampling frequency: Triplicate bottles of each set sacrificed at the end of the study (Day 28). Additional triplicate blank and reference substance test bottles were also sacrificed on Day 0 and were analysed for TOC.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes


Reference substance:
benzoic acid, sodium salt
Remarks:
Theoretical carbon concentration: 58 %; concentration used: 20 mg carbon/L (nominal)
Test performance:
None
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
ca. 74
St. dev.:
6.51
Sampling time:
28 d
Results with reference substance:
Reference substance attained a maximum level of biodegradation (based on evolved carbon dioxide) of 104 %. This is over 60 % degradation, as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms (See table 1 for details).

Table 1. Results of CO2 headspace biodegradability test

 

Blank

Reference substance

Test substance#

Day 0 stock

solution TOC (mg/L)

-

378

-

Day 0 test bottles TOC (mg/L)

3.04

21.8

-

3.65

22.7

-

2.45

9.31*

-

Mean (mg/L)

3.05

22.3**

-

Standard deviation

0.60

-**

-

Day 28 test bottles

IC (mg/L)

2.30

24.8

15.5

2.44

26.6

17.0

3.85

26.7

18.1

Mean (mg/L)

2.86

26.0

16.9

Standard deviation

0.86

1.07

1.31

Biodegradation*** (%)

-

98

67

-

106

74

-

107

80

Mean (%)

-

104

74

Standard deviation

-

4.93

6.51

*Anomalous value, not included in mean

**Mean calculated on two values only; standard deviation not calculated

***Biodegradation = (IC concentration in test bottle – Mean IC concentration in control) x 100 / Initial TOC in test bottle

#No Day 0 TOC measurements were made, due to the low aqueous solubility test material, so the nominal concentration of 19 mg C/L was used.

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the test substance was degraded by 74% after 28 d and was therefore considered to be readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance in the CO2 headspace test based on ISO Guideline 14593, in compliance with GLP. A reduced version of the test was performed, with inorganic carbon analysis on Day 28 only. A solution of the test substance in a mineral medium, corresponding to 19 mg carbon/L (nominal), was inoculated with secondary sewage effluent in septum sealed bottles, then shaken for 28 days. The reference material used was sodium benzoate at a concentration of 20 mg carbon /L (nominal). Sufficient bottles were prepared to allow triplicate samples of each set (blank, reference and test bottles) to be sacrificed at the end of the study. CO2 generated as a result of mineralisation of organic carbon arising from biodegradation was then measured using a Dohrman DC-190 carbon analyser. Triplicate blank and reference substance test bottles were also collected on Day 0 and analysed for total organic carbon (TOC). TOC was not determined for the test substance because of low solubility. The reference substance attained a maximum level of biodegradation (based on evolved CO2) of 104%. This is over 60% degradation, as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. Under the study conditions, the test substance was degraded by 74% after 28 d and was therefore considered to be readily biodegradable (Daniel, 2006).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From March 10, 1997 to April 9, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
read-across study
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details on the read across justification. The ready biodegradation study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge micro-organisms were obtained on March 10, 1997, from the aeration stage of the Severn Trent Water plc sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
- Preparation of inoculum for exposure: 100 mL samples were filtered through Whatman GF/A paper and dried at 105 °C.
- Pretreatment: Sample washed 3 times by settlement and resuspension in culture medium prior to suspended solids determination. Samples were maintained on aeration at 21°C prior to use.
- Concentration of sludge: 30 mg TSS/L
- Initial cell/biomass concentration:
- Water filtered: Yes
- Type and size of filter used, if any: Whatman GF/A paper
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Standard culture medium according to OECD 301B.
- Test temperature: 22±2°C
- pH: 7.4
- Aeration of dilution water: Yes, see below
- Suspended solids concentration: 30 mg TSS/L
- Continuous darkness: Yes, in darkness sheilded from laboratory lighting


TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels
- Number of culture flasks/concentration: Two for every conc.
- Method used to create aerobic conditions: Using CO2 free air at 30-100 mL/min
- Method used to create anaerobic conditions: Not applicable
- Other:
- Method of preparation of test solution: Direct dispersion in culture medium.
-Agitation: Yes, using magnetic stirrer


SAMPLING
- Sampling frequency: On Day 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29.
- Sampling method: 2 mL of samples were taken on the days specified above for IC analysis.
- Sample storage before analysis: Samples taken on Days 12 & 18 were stored at -20°C


CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: No, since a preliminary study performed on Empilan CME indicated that, the test material is non-toxic and does not inhibit the respiration of sewage sludge micro-organisms at the concentration employed in the test.
- Reference control: Yes


Reference substance:
benzoic acid, sodium salt
Remarks:
10 mg C/L (ThCO2 =58.34%; TOC = 30 mg C; total carbon = 51.4 mg)
Preliminary study:
The oxygen consumption rates (mg O2/L) for the inoculum blank was 0.43 for both replicates; for the test material it was 0.46 and 0.42 at 10 and 20 mg C/L respectively and for reference material it was 0.38 and 0.11 at 3.2 and 32 mg/L respectively. Hence, toxicity control was omitted from the study.
Test performance:
- The decrease of percentage degradation value for the test material from 99% on Day 28 to 98% on Day 29 due to the increase in inorganic carbon (caused by addition of HCl) in the control absorber vessels was greater than that in the standard material absorber vessels and this decrease is considered to be due to biological variation between the activated sewage sludge in the control and standard material vessels.
- Inorganic carbon analysis of samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
- Sodium benzoate attained 97% degradation after 28 d thereby confirming the suitability of the inoculum and test conditions.
- Toxicity control was omitted from the study, in light of results of a preliminary investigation of the test material. These results carried out using Activated Sludge Respiration Inhibition test method (OECD Guideline No 209), indicated that Empilan CME did not inhibit the respiration of sewage sludge micro-organisms at the concentration employed in the test.
- Low DOC values obtained for the samples taken from the test material culture vessels on day 0 are due to physical removal of the test material by the suspended solids during centrifugation. This effect is considered to be due to the test material being a fine homogenous and not a true solution at the concentrations employed in the study.
Parameter:
% degradation (CO2 evolution)
Value:
24
Sampling time:
3 d
Parameter:
% degradation (CO2 evolution)
Value:
62
Sampling time:
8 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
99
Sampling time:
28 d
Details on results:
- Percent degradation values: 0, 3, 9, 24, 54, 62, 69, 72, 92, 97, 98, 96, 99, 99 and 98 after 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 d.
- The measured concentrations of DOC was found to be 56% of nominal on Day 0 and 17 to 26% of nominal on Day 28.
Results with reference substance:
The reference material met the validity criteria for the test, greater than 60% biodegradation within 14 d, and within the 10 d window (Day 2 = 22%, Day 10 = 64%, Day 28 = 97% ThCO2).
- The increase in inorganic carbon in the first absorber vessels on Day 29 resulted in an increase in the percentage degradation value for the standard material from 97% on Day 28 to 98% on Day 29.
Validity criteria fulfilled:
yes
Remarks:
However, the data on replicate difference and the biodegradation data for control were not documented.
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, the test material undergoes 99% degradation within the 28 d period and therefore can be considered as readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the read-across substance according to OECD Guideline 301 B (CO2 evolution test). The test (20 mg C/L) and reference (sodium benzoate) substances were incubated with activated sludge and observed for degradation by measurement of the theoretical amount of carbon dioxide (ThCO2) over a 28 d period. The reference substance, sodium benzoate reached 10% biodegradation after Day 2 and > 60% after Day 10. It met the validity criteria established in the guideline for a reference substance. Biodegradation of the test substance on Day 28 equalled 99%. Under the study conditions, the test substance was considered to be readily biodegradable (Mead, 1997).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The study with the test substance was conducted according to ISO Guideline 14593 and can be considered comparable to a reduced version of OECD Guideline B. The following validity criteria of the OECD guideline were not met:

1. Inorganic carbon analysis done only on Day 28 for both reference and test substances, therefore the course of biodegradation was not followed throughout the study.

2. Toxicity control not used and no information was provided on inhibition of inoculum by the test substance.

However, a guideline-compliant study with the read across substance C8 -18 and C18 -unsatd. MEA is available and this study was therefore used for risk assessment purposes.

A study was conducted to determine the ready biodegradability of the read-across substance according to OECD Guideline 301 B (CO2 evolution test). The test (20 mg C/L) and reference (sodium benzoate) substances were incubated with activated sludge and observed for degradation by measurement of the theoretical amount of carbon dioxide (ThCO2) over a 28 d period. The reference substance, sodium benzoate reached 10% biodegradation after Day 2 and > 60% after Day 10. It met the validity criteria established in the guideline for a reference substance. Biodegradation of the test substance on Day 28 equalled 99%. Under the study conditions, the test substance was considered to be readily biodegradable (Mead, 1997).

A study was conducted to determine the ready biodegradability of the test substance in the CO2 headspace test based on ISO Guideline 14593, in compliance with GLP. A reduced version of the test was performed, with inorganic carbon analysis on Day 28 only. A solution of the test substance in a mineral medium, corresponding to 19 mg carbon/L (nominal), was inoculated with secondary sewage effluent in septum sealed bottles, then shaken for 28 days. The reference material used was sodium benzoate at a concentration of 20 mg carbon /L (nominal). Sufficient bottles were prepared to allow triplicate samples of each set (blank, reference and test bottles) to be sacrificed at the end of the study. CO2 generated as a result of mineralisation of organic carbon arising from biodegradation was then measured using a Dohrman DC-190 carbon analyser. Triplicate blank and reference substance test bottles were also collected on Day 0 and analysed for total organic carbon (TOC). TOC was not determined for the test substance because of low solubility. The reference substance attained a maximum level of biodegradation (based on evolved CO2) of 104%. This is over 60% degradation, as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. Under the study conditions, the test substance was degraded by 74% after 28 d and was therefore considered to be readily biodegradable (Daniel, 2006).