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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From March 10, 1997 to April 9, 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study
Justification for type of information:
Refer to the section 13 for details on the read across justification.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
; no abiotic control was taken as previous experience has shown that abiotic degradation under the test conditions does not occur; no toxicity control taken as test material was found to be non-toxic at tested concentration in a preliminary study.
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
; no abiotic control was taken as previous experience has shown that abiotic degradation under the test conditions does not occur; no toxicity control taken as test material was found to be non-toxic at tested concentration in a preliminary study.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Stage and source of inoculum/activated sludge: A mixed population of activated sludge micro-organisms from the aeration stage of the Severn Trent Water plc sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
- Pretreatment: Sample washed 3 times by settlement and resuspension in culture medium.
- Concentration of sludge: 49.7 mL of sludge added to each test vessel to give concentration of 30 mg suspended solids/L.
- Storage conditions: Sample maintained on aeration at 21 °C prior to use.
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 20 mg/L
Based on:
other: Elemental carbon
Initial conc.:
ca. 28.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium:
Solution A: KH2PO4 - 8.50 g/L; K2HPO4 - 21.75 g/L; Na2HPO4·2H2O - 33.40 g/L; NH4Cl - 0.50 g/L (pH = 7.4)
Solution B: CaCI2 - 27.50 g/L
Solution C: MgSO4.7H2O - 22.50 g/L
Solution D: FeCI3·6H2O - 0.25 g/L
Ion exchange and reverse osmosis treated tap water mixed with 1,000 mL of solution A and 100 mL each of solution B, C and D to give 100 L final volume.
- Test temperature: 21 °C
- pH: 7.2 - 7.4 on Day 28
- Agitation: Yes; by magnetic stirrer
- Aeration of dilution water: Using CO2 free air at 40 - 70 mL/min.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessel
- Number of culture flasks: 2 replicates each for test and reference substance
- Test performed in closed vessels: Yes (foiled)
- Trap for evolved CO2: Absorber vessels (no details reported)

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 sample taken
- Sample storage before analysis: Samples taken on Days 12 and 22 stored at -20°C for later analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (in duplicate)
- Abiotic sterile control: Not required
- Toxicity control: Not required

* After addition of 1 mL concentrated HCl on Day 28 to release remaining CO2
Reference substance:
benzoic acid, sodium salt
Remarks:
17.1 mg/L equivalent to 10 mg carbon/L; Batch no. 65H1093
Preliminary study:
Preliminary investigational work was carried out using the Activated Sludge Respiration Inhibition test method (OECD Guideline No. 209), which showed that test material did not inhibit the respiration of sewage sludge micro-organisms at the concentration employed in the test (see Table 3 in attached full study report for details).
Test performance:
None
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
ca. 97
Sampling time:
28 d
Details on results:
Degradation of the test material attained 10 % on Day 2 and 62 % on Day 10 of the study, thereby meeting the 10 d window validation criteria. The percentage degradation value for the test material increased to 100 % on Day 29 due to release of remaining CO2 by addition of HCl (see Table 1 for details).
Results with reference substance:
Sodium benzoate attained 97 % degradation after 28 d (also fulfilled 10 d window criteria) thereby confirming the suitability of the inoculum and test conditions. The percentage degradation value for the test material increased to 98 % on Day 29 due to release of remaining CO2 by addition of HCl (see Table 1 for details).

Table 1. % Biodegradation values of reference and test material at various time intervals

Day

% Degradation (Reference)

% Degradation (Test)

0

0

0

1

9

6

2

22

10

3

37

23

6

54

60

8

58

58

10

64

62

14

79

77

16

92

100

20

91

99

22

101

96

24

94

98

27

96

96

28

97

97

29

98

100

DOC analysis: The measured concentrations of DOC were 0 - 23 % of nominal on Day 0 and 0 - 34 % of nominal on Day 28. A marked decline in carbon concentration was also observed after centrifugation of the 200 mg/L stock dispersion used to prepare the test vessels. This effect was considered to be due to the test material being a fine homogenous dispersion and not a true solution at the concentrations employed in the study.

Validity criteria fulfilled:
not specified
Remarks:
All the criteria met but the total CO2 evolution in blank exceeded normal 40 mg/L value and initial IC content of one of the test vessel was greater than 5 % of TC
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the test substance was considered to be readily biodegradable
Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance, N-(2-hydroxypropyl) dodecamide, according to OECD Guideline 301 B (Modified Sturm test), in compliance with GLP. A dispersion of the test substance in a defined medium, corresponding to 20 mg carbon/L, was inoculated with activated sludge (30 mg suspended solid/L). The test vessels were aerated by the passage of carbon dioxide-free air and incubated under aerobic conditions for 28 days. The reference material used was sodium benzoate at a concentration of 10 mg carbon /L. Degradation was followed by determining the CO2 produced and trapped in CO2 absorber vessel via IC measurement at 1-4 day intervals until Day 28. On Day 28, HCI was added to flasks in order to eject all the remaining CO2 and a last analysis was carried out on Day 29. Sodium benzoate attained 97% degradation after 28 days (98% on Day 29), thereby confirming the suitability of the inoculum and test conditions. The test substance also attained 97% degradation after 28 days (100% on Day 29). Under the study conditions, the test substance was therefore considered to be readily biodegradable (Mead, 1997).

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 November 2, 2005 to December 1, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study
Justification for type of information:
Refer to the section 13 for details on the read across justification.
Reason / purpose:
reference to other study
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 read across substance, Amides, C8-18 (even-numbered) and C18-unsatd., N-(2-hydroxypropyl), 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).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

CO2 headspace test

A study was conducted to determine the ready biodegradability of the read across substance, amides, C8-18 (even-numbered) and C18-unsatd., N-(2-hydroxypropyl), 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).

CO2-evolution test

A study was conducted to determine the ready biodegradability of the read across substance, N-(2-hydroxypropyl) dodecamide, according to OECD Guideline 301 B (Modified Sturm test), in compliance with GLP. A dispersion of the test substance in a defined medium, corresponding to 20 mg carbon/L, was inoculated with activated sludge (30 mg suspended solid/L). The test vessels were aerated by the passage of carbon dioxide-free air and incubated under aerobic conditions for 28 days. The reference material used was sodium benzoate at a concentration of 10 mg carbon /L. Degradation was followed by determining the CO2 produced and trapped in CO2 absorber vessel via IC measurement at 1-4 day intervals until Day 28. On Day 28, HCI was added to flasks in order to eject all the remaining CO2 and a last analysis was carried out on Day 29. Sodium benzoate attained 97% degradation after 28 days (98% on Day 29), thereby confirming the suitability of the inoculum and test conditions. The test substance also attained 97% degradation after 28 days (100% on Day 29). Under the study conditions, the test substance was therefore considered to be readily biodegradable (Mead, 1997).