Registration Dossier

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
Study period:
03 November 2010 to 08 December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with OECD Guideline 301B "Ready Biodegradability; CO2 Evolution Test". This study was performed in compliance with UK GLP standards (SI 1999/3106 as amended by SI 2004/0994)).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
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:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: 3-trifluoromethylaniline
Description: Extremely pale yellow liquid
Purity: 99.66 %
Batch Number: 101009
Date Received: 25 October 2010
Storage Conditions: Room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 08 November 2010 for the aeration stage of the Severn Trent Water plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
The activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 degrees celsius and used on the day if collection. Determination of the suspended solids content of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105 degrees celsius for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.3 g/L prior to use.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
ThIC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The reference item (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg C/L.
c) the test item, in duplicate, in inoculated medium to give a final concentration of 10 mg C/L.
d) The test item plus the reference item in inoculated culture medium to give a final concentration of 20 mg C/L to act as a toxicity control (one vessel only).

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at approximately 21 degrees celsius, in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2,400 mL of culture medium and 20.9 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the volume in all the vessels adjusted to 3 L by the addition of culture medium.

The culture vessels were sealed and C)2-free air was bubbled through the solution at a rate of approximately 40 mL/min and stirred continuously by magnetic stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb) granules.

The CO2 produced by degradation was collectged in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

Samples were taken from the control, reference and test item first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessels on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, reference and test item and on Day 0 for the toxicity control.

The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analysed for CO2 immediately.

On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

Samples (30 mL) were removed from all culture vessels on Day 0 and from the control, reference and test item culture vessels on Day 28 for DOC analysis.

The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
17.1 mg/L (10 mg C/L)

Results and discussion

Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigation work indicated that the test item did not absorb to activated sewage sludge.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
4
Sampling time:
28 d
Details on results:
The total CO2 evolution in the control vessels on Day 28 was 30.59 mg/L and therefore satisfied the validation criterion given in the OECD Test Guideline. The IC content of the test item suspension in the mineral medium at the start of the test was below 5 % of the TC content and hence satisfied the validation criterion given on the OECD Test Guideline. The difference between the values for CO2 production at the end of the test for the replicate vessels was <20 % and hence satisfied the validation criterion given in the OECD Test Guideline.

The test item attained 4 % degradation after 28 days and therefore can not be considered to be readily biodegradable in accordance with OECD Guideline 301B.

The toxicity control attained 31 % degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 74 % degradation after 14 days and 75 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Analysis of the test media from the test item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC) gave percentage degradation values of 90 % and 87 % respectively for the test item replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from the inorganic carbon analysis. This was considered to be due to adsorption of the test item to the glassware and/or activated sewage sludge and the subsequent removal by centrifugation prior to DOC analysis of the test samples. The results obtained from the preliminary investigation work indicated that the test item did not absorb to the activated sewage sludge. However, the samples taken from this work were samples immediately after the test item stock solution had been inoculated and therefore, it was considered that over the test period of 28 days the test item could have adsorbed to the activated sewage sludge and/or the glassware.

Sodium benzoate attained 100 % and 97 % degradation respectively for Replicates R1 and R2 calculated from the results of the DOC analysis. The degradation rates calculated from the results of the DOC analysis were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occuring.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 74 % degradation after 14 days and 75 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Table 1. Inorganic Carbon Values on Each Analysis Occasion

  Control(mg IC)     SodiumBenzoate(mg IC)       Test Item(mg IC)       ToxicityControl(mg IC)   
   R1  R1  R2  R2  R1  R1  R2  R2  R1  R1  R2  R2  R1  R1
 Day  Abs 1  Abs 2  Abs 1  Abs 2  Abs 1  Abs 2  Abs 1  Abs 2  Abs 1  Abs 2  Abs 1  Abs 2  Abs 1  Abs 2
0  1.05  0.93  0.93  0.93  1.05  1.05  0.93  1.05  1.05  0.93  1.05  1.05  1.05  1.28
2  5.68  -  7.31  -  21.69  -  21.92  -  6.61  -  5.57  -  19.95  -
6  14.19  -  15.92  -  38.06  -  38.87  -  15.80  -  18.57  -  33.22  -
8  13.07  -  14.33  -  32.34  -  32.91  -  14.45  -  14.10  -  30.27  -
10  17.44  -  16.19  -  39.33  -  42.07  -  18.58  -  18.36  -  36.94  -
14  18.25  -  18.59  -  38.08  -  42.95  -  20.51  -  16.66  -  37.17  -
21  21.52  -  21.63  -  48.90  -  48.78  -  24.11  -  21.07  -  -  -
28   23.97  -  26.10  -  50.51  -  50.40  -  29.01  -  23.29  -  -  -
29  22.04  2.44  23.05  2.20  44.65  2.32  44.65  3.13  26.28  2.32  21.38  2.20  -  -

Table 2. Percentage Biodegradation Values

 Day  % Degradation Sodium Benzoate  % Degradation Test Item  % Degradation Toxicity Control
 0  0  0  0
 2  51  0  22
 6  78  7  30
 8  63  2  28
 10  80  34
 14  74  1  31
 21  91  3  -
 28  85  4  -
 29  75  4  -

Table 3. Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 28

   Day 0    Day 28    
 Test Vessel  mg C/L  % of Nominal Carbon Content  mg C/L  % of Initial Carbon Concentration  % Degradation
 Sodium Benzoate 10 mg C/L - R1 9.98  100   -0.07  <Control  100
 Sodium Benzoate 10 mg C/L - R2 11.24   112  0.31  3  97
 Test Item 10 mg C/L - R1  9.69  97  1.00  10  90
 Test Item 10 mg C/L - R2  9.72  97  1.25  13  87

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: Not readily biodegradable
Conclusions:
The test item attained 4 % degradation after 28 days and therefore can not be considered to be readily biodegradable in accordance with OECD Guideline 301B.