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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Laboratories Ltd.

Test material

Constituent 1
Test material form:
solid: crystalline
Details on test material:
- Name of test material: Thymidine

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: A mixed population of sewage treatment micro-organisms was obtained on 26 March 2012 from the aeration stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed two times by settlement and resuspension in mineral medium to remove any excessive amounts of DOC that may have been present. A subsample of the washed sewage sludge was then removed and the suspended solids concentration determined.
This inoculum was used for preparation of the replicate inoculum control, procedure control and test item vessels and the single toxicity control vessel.
A further sub-sample of the washed sewage sludge (approximately 200 mL) was sterilized by autoclaving (120 °C, 15 minutes) in order to prevent any viable organisms surviving that may degrade the test item. The sterilized inoculum was used for preparation of the abiotic control and abiotic test vessels. Sodium azide was also added to the abiotic vessels in order to prevent any other viable micro-organisms surviving that may degrade the test item.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
40 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines.
- Additional substrate: None
- Solubilising agent: None
- Test temperature: 21 to 23°C
- pH: 7.4
- pH adjusted: yes (if necessary)
- Suspended solids concentration: 30 mg suspended solids/L
- Continuous darkness: yes
- Other: Constantly shaken at 100 rpm

TEST SYSTEM
- Culturing apparatus: 2 liter glass conical flasks each containing 1 liter of solution
- Number of culture flasks/concentration:
a) An inoculum control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final test concentration of 20 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final test concentration of 40 mg carbon/L.
d) The test item plus sodium benzoate, one replicate, in inoculated mineral medium to give a final test concentration of 60 mg carbon/L to act as a toxicity control.
e) An abiotic control, one replicate, consisting of mineral medium plus sterilized inoculum poisoned by the addition of 10 mL of a 10 g/L sodium azide solution.
f) The test item, one replicate, in mineral medium plus sterilized inoculum to give a final test concentration of 40 mg carbon/L poisoned by the addition of 10 mL of a 10 g/L sodium azide solution to act as an abiotic test vessel.
- Measuring equipment: Samples were analyzed for DOC using a Shimadzu TOC-V CPH TOC analyzer
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: Yes

SAMPLING
- Sampling frequency: Sampling on days 0, 1, 3, 6, 9, 14, 21 and 28
- Sampling method: Samples (30 mL) were removed from all test vessels and filtered through 0.45 um Gelman Acrocap filters (approximately 5 mL discarded)

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: Yes
- Abiotic sterile control: Yes
- Abiotic test: Yes
- Procedure control: Yes
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the test, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.
Test performance:
The results of the degradation test are considered valid if in the same test the reference item attains 70 % degradation within 14 days.
The test item may be considered to be readily biodegradable if 70 % degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10 %.
The test is considered valid if the difference of the extremes of replicate values of the removal of DOC at the time the plateau is reached, at the end of the test, or at the end of the 10-Day window is less than 20 %.
The toxicity control should attain 35 % degradation by day 14 for the test item to be considered as non-inhibitory.
% Degradationopen allclose all
Parameter:
% degradation (DOC removal)
Value:
97
Sampling time:
9 d
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
28 d
Details on results:
DOC values for the test item, procedure control, toxicity control, abiotic test and inoculum control vessels are shown in table 1 (see below). Percentage degradation values are shown in table 2.
The test item attained 99% degradation after 28 days and in the abiotic test vessel a 19 % loss in dissolved organic carbon was attained after 28 days.
The test item still attained the 10-Day window criterion, whereby 70 % degradation must be attained within 10 days of the degradation rate exceeding 10 %. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301-A.
The toxicity control (test item plus sodium benzoate) attained 98 % degradation after 14 days and 99 % degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms at the test concentration employed in the study.
The reference item, sodium benzoate, attained 99 % degradation after 14 days and 100 % degradation after 28 days, thereby confirming the suitability of the inoculum and culture conditions.
The pH values did not fall below 7.9 in any test vessel after 28 days of the study. Dissolved oxygen concentrations remained at approximately 6.3 mg O2/L or above in all culture vessels.

BOD5 / COD results

Results with reference substance:
Biodegradation of the reference substance was 99 % after 9 days and 100 % after 28 days.

Any other information on results incl. tables

Table 1: Dissolved Organic Carbon (DOC) Values on Each Sampling Occasion as  DOC (mg C/L)

Vessel  Day 
0 1 3 6 9 14 21 28
Inoculum Control  R1 0.64 0.62 0.52 0.87 0.68 0.63 0.81 1.17
R2  0.56 0.46 0.58 0.73 0.8 0.68 0.61 0.89
Mean  0.6 0.54 0.55 0.8 0.74 0.66 0.71 1.03
Procedure Control  R1 19.79 0.19 0.51 1.1 0.9 0.79 0.77 1.16
R2  19.34 0.21 0.53 1.2 0.81 0.81 0.69 0.87
Mean  19.57 0.2 0.52 1.15 0.86 0.8 0.73 1.02
Test Item  R1 38.36 36.74 17.36 2.59 1.83 1.71 1.4 1.41
R2 37.44 35.75 16.75 2.55 1.65 1.61 1.57 1.35
Mean  37.9 36.25 17.06 2.57 1.74 1.66 1.49 1.38
Toxicity Control  57.58 36.96 4.76 3.56 2.24 1.89 1.84 1.73
Abiotic Control  3.72 3.66 3.68 3.16 2.55 2.21 1.95 2.35
Abiotic Test  40.19 40.03 40.74 38.28 33.7 32.69 30.22 31.87

Table 2: Percentage Degradation Values

Degradation (%)*
Day  Procedure Control  Test Item Toxicity Control Abiotic Test 
1 102 4 36 0
3 100 56 93 0
6 98 95 95 4
9 99 97 97 15
14 99 97 98 16
21 100 98 98 22
28 100 99 99 19

* Corrected for control levels of DOC

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 99 % degradation after 28 days and in the abiotic test vessel a 19 % loss in dissolved organic carbon was attained after 28 days. The test item still attained the 10-day window criterion, whereby 70 % degradation must be attained within 10 days of the degradation rate exceeding 10 %. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301-A.
Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media following EU Method C.4-A, OECD Guideline 301-A (Assessment of Ready Biodegradability; DOC Die Away Test) and OPPTS Guideline 835.3110. The test item was exposed to activated sewage sludge micro-organisms at a concentration of 40 mg C/L with mineral medium in the dark at temperatures between 21 to 23 °C for 28 days. The degradation of the test item was assessed by the determination of Dissolved Organic Carbon (DOC) removal. Control cultures with inoculum and the reference item, sodium benzoate, together with a toxicity control, abiotic control and abiotic test vessel were used for validation purposes. The test item attained 99 % degradation after 28 days and in the abiotic test vessel a 19 % loss in dissolved organic carbon was attained after 28 days. The test item still attained the 10 -day window criterion, whereby 70 % degradation must be attained within 10 days of the degradation rate exceeding 10 %. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301-A.