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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
Study period:
Between 11 January 1995 and 9 February 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The work described was performed in compliance with the UK Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Severn Trent Water Plc sewage Treatment Plant at Belper, Derbyshire, UK

- Laboratory culture:
Not specified.

- Method of cultivation:
Not specified.

- Storage conditions:
The sample of activated sewage sludge was maintained on continuous aeration upon receipt.

- Storage length:
Not recorded

- Preparation of inoculum for exposure:
A sample of 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.

- Pretreatment:
Not applicable

- Concentration of sludge:
Final concentration of 30mg suspended solids (ss)/l.

- Initial cell/biomass concentration:
Not recorded

- Water filtered: no

- Type and size of filter used, if any:
Not applicable.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
23.5 mg/L
Based on:
other: Analysis of the concentration of the test material in the test solutions were not appropriate to the Test Guideline.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
Modified Sturm Test
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
CaCl2 : 27.50 g/l

Solution c
MgSO4.7H2O: 22.50 g/l

Solution d
FeCl3.6H2O : 0.25 g/l

To 1 litre (final volume) of purified water* is added the following volumes of solutions a - d.

10ml of Solution a
1 ml of Solution b
1ml of Solution c
1 ml of Solution d

* Ion exchange and reverse osmosis treated tap water.

- Additional substrate:
No

- Solubilising agent (type and concentration if used):
Not recorded

- Test temperature:
21 Deg C

- pH:
pH of culture medium : 7.4

- pH adjusted:
no

- CEC (meq/100 g):
Not recorded

- Aeration of dilution water:
The CO2-free air was produced by sparging compressed air through the following series:

i) Three 500 ml Dreschel bottles filled with 350ml 10 N NaOH
ii) One 500 ml Dreschel bottle filled with 350ml 0.025 N Ba(OH)2
iii) One empty 500 ml Dreschel bottle to prevent liquid carry-over to the test vessels.

- Suspended solids concentration:
30mg suspended solids (ss)/l

- Continuous darkness:
yes

- Other:


TEST SYSTEM
- Culturing apparatus:
The test solutions were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution.

- Number of culture flasks/concentration:
1) A control, in duplicate, consisting of inoculated culture medium.
2) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final test concentration of 10 mg carbon/l.
3) The test material, in duplicate, in inoculated culture medium to give a final test concentration of 20 mg carbon/l.
4) The test material, in duplicate, in incolated culture medium at a concentration of 20 mg carbon/l, poisoned by the addition of 10ml of 10 g/l mercuric chloride solution to act as an abiotic control (one vessel only).
5) The test material plus the standard material in inoculated culture medium to give a final concentration of 30mg carbon/l to act as a toxicity control (one vessels only).

- Method used to create aerobic conditions:
The culture vessles were sealed and CO2-free air bubbled through the solution at a rate of 40 ml/minute and stirred continuously by magnetic stirrer.

- Method used to create anaerobic conditions:
Not applicable.

- Measuring equipment:
Ionics 555 or Dohrmann DC-190 TOC Analyser.

- Test performed in closed vessels due to significant volatility of test substance:
Not applicable.

- Test performed in open system:
No

- Details of trap for CO2 and volatile organics if used:
The CO2 produced by degradation was collected in two 500ml Dreschel bottles containing 350ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

- Other:


SAMPLING
- Sampling frequency:
Samples (2 ml) were taken from the first CO2 absorber vessel on days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29.
The second absorber vessel was sampled on days 0 and 29.

- Sampling method:
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.

- Sterility check if applicable:
Not applicable.

- Sample storage before analysis:
Samples were either analysed immediately or stored at 4 deg C until analysed. The agreed protocol states that samples would be stored at approximately -20 deg C until analysis. This minor deviation is not considered to affect the integrity of the study results.

- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank:
A control, in duplicate, consisting of inoculated culture medium

- Abiotic sterile control:
Not applicable.

- Toxicity control:
Preliminary investigational work to assess any toxic effect of the test material on sewage sludge micro-organisms following the method described in OECD Guideline No. 209 "Activated Sludge Respiration Inhibition Test" could not be performed due to the insoluble nature of the test material. Therefore a toxicity control (Alkane 2 and Sodium benzoate) was included in the study to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the study.

- Other:


STATISTICAL METHODS:
Calculation of Carbon Content:
The test material, Alkane 2, contains 85.11% carbon (data supplied by the Sponsor) and so for a concentration of 20mg C/l (ie a total of 70.5mg) the total organic carbon present was 60mg C.

The theoretical amount of carbon present in the standard material, sodium benzoate (C6H5.COONa) was calculated as follows:-

No. of C. atoms x mol.wt of C. / mol.wt of sodium benzoate x 100%

= 7 x 12.011 / 144.11 x 100 = 58.34%

Thus for a 10mg C/l test concentration (ie a total of 51.4mg) the total organic carbon present for sodium benzoate was 30 mg C.

Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values given in Table 1 in the following equation:
The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation.

%ThCO2 (=% degradation) = mg IC in test flask - mg IC in control / mg TOC as test material x 100%
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
An Initial stock solution of 1000 mg/l was prepared by direct dispersion in culture medium and a 51.4 ml aliquot added to the test vessel to give a final test concentration of 17.1 mg/l, equivalent to 10 mg carbon/l.

Results and discussion

Preliminary study:
Not applicable.
Test performance:
The OECD/EEC test guidelines describe the use of an optional uninoculated abiotic control. However, in this study an inoculated abiotic control was used. Some CO2 evolution was observed (see Table 1), but experience has shown that abiotic degradation cannot be detected from CO2 evolution or in this case distinguished from other processes. The percentage abiotic degradation could not be calculated due to the lack of suitable control for correction. The results of the study show little degradation (biotic or abiotic) and the lack of an abiotic control did not affect the determination of ready biodegradability.

The total CO2 evolution in the control vessels was 36.96 mg/l, and therefore satisfied the validation criterion for the test which states that the total CO2 evolution in the inoculated blanks should not exceed 40 mg/l at the end of the test.

The difference between the replicate values of production of CO2 for the control, standard and test material vessels at the end of the test were 1, 2 and 1% respectively and therefore satisfied the validation criterion for the test which states that the difference between the extremes of replicate values of production of CO2 should be less than 20%.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
4
Sampling time:
28 d
Details on results:
Inorganic carbon values for test material, Alkane 2, standard material, sodium benzoate and control vessels at each sampling occasion are given in Table 1. Percentage biodegradation of the test and standard materials is given in Table 2 and the biodegradation curves for the test and standard materials are presented in Attachment 1.

Alkane 2 attained 4% degradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.

The results of the inorganic carbon analysis of both absorber vessels on day 29 confirmed that no significant amounts of CO2 were present in solution in the culture vessels as inorganic carbonate, and that there was no significant carry-over of CO2 into the second absorber.

The toxicity control (Alkane 2 and sodium benzoate) attained 26% degradation after 14 days and 33% degradation after 28 days thereby confirming that the test material, Alkane 2, was non-inhibitory to the sewage sludge micro-organisms used in the study.

Total Carbon (TC) and Inorganic Carbon (IC) analysis of the test media from the test material culture vessels on day 0 prior to the addition of the test material showed that the Inorganic Carbon (IC) content in the test material suspensions was less than or equal to 3% of the theoretical Total Carbon *TC) content. The validation criterion of the test which states that the IC content of the test material suspensions in the culture media must be less than 5% of the TC on day 0 was therefore satisfied.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 93% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

The degradation of sodium benzoate was 69% on day 14 and therefore satisfied the validation criterion for the test which states that the standards must attain >60% by day 14.

Any other information on results incl. tables

Table 1: Inorganic Carbon Values on each Sampling Occasion
DAY Control (mg IC) Sodium benzoate (mg IC) Alkane 2 (mg IC) Alkane 2 Abiotic control (mg IC) Alkane 2 plus sodium benzoate Toxicity control (mg IC)
R1 R2 R1 R2 R1 R2 R1 R1
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 Abs 1 Abs 2
0 2.1 1.63 0.7 2.1 1.05 1.75 1.75 1.4 1.05 1.4 0.7 1.75 0.35 0 1.4 1.05
1 3.13  -   1.39  -   6.96  -   8  -   2.2  -   1.39  -   0.7  -   6.61  -  
2 7.96  -   4.15  -   18.57  -   19.38  -   6.81  -   4.84  -   3.11  -   18.45  -  
3 12.38  -   7.91  -   24.54  -   28.32  -   9.63  -   6.88  -   3.44  -   23.05  -  
6 18.92  -   9.69  -   26.91  -   34.2  -   9.46  -   7.29  -   2.74  -   32.15  -  
8 22.44  -   17.79  -   31.05  -   42.16  -   19.83  -   8.27  -   3.06  -   39.44  -  
10 24.11  -   20.62  -   38.87  -   45.07  -   22.65  -   23.32  -   4.06  -   43.15  -  
12 27.44  -   30.35  -   47.15  -   47.15  -   28.56  -   34.61  -   3.36  -   51.85  -  
14 28.39  -   28.06  -   48.76  -   48.87  -   29.28  -   34.29  -   5.34  -   51.88  -  
16 31.1  -   31.54  -   52.57  -   52.46  -   33.2  -   33.2  -   5.87  -   52.35  -  
18 35.64  -   35.75  -   60.94  -   61.05  -   36.08  -   35.64  -   7.04  -   55.55  -  
20 37.06  -   35.97  -   62.32  -   63.2  -   38.05  -   39.03  -   10.93  -   57.51  -  
22 37.6  -   36.29  -   62.81  -   65.42  -   37.82  -   38.69  -   10.76  -   58.25  -  
24 37.58  -   36.94  -   64.15  -   65.02  -   37.91  -   38.99  -   11.02  -   60.91  -  
27 35.96  -   37.25  -   63.97  -   64.18  -   39.18  -   39.61  -   10.63  -   63.86  -  
28 37.12  -   36.8  -   64.32  -   65.49  -   39.36  -   39.89  -   10.77  -   66.56  -  
29 37.31 2.32 37.21 2.44 66.88 3.02 66.99 2.9 39.43 2.2 39.85 1.85 10.81 0.58 68.79 1.04

R1 - R2 = Replicates Abs = CO2 absorber vessel

Table 2: Percentage Biodegradation Values

Day % Degradation Sodium benzoate % Degradation Alkane 2 % Degradation Toxicity control Alkane 2 plus Sodium Benzoate
1 17 0 5
2 43 0 14
3 54 0 14
6 54 0 20
8 55 0 21
10 65 1 23
12 61 4 26
14 69 6 26
16 71 3 23
18 84 0 22
20 87 3 23
22 91 2 24
24 91 2 26
27 92 5 30
28 93 4 33
29* 101 4 35

* Day 29 values corrected to include any carry-over of C)2 detected in absorber 2

Table 3: Total Carbon (TC) and Inorganic Carbon (IC) Values in the culture vessels on Day 0

Test Vessel TC (mg C/l) IC (mg C/l) TC added as test material (mg C/l) Theoretical TC (mg C/l) % IC
Alkane 2 20 mg C/l R1 7.22 0.08 20 27.22 <1
Alkane 2 20 mg C/l R2 6.78 0.74 20 26.78 3
Alkane 2 Abiotic control 20 mg C/l 6.91 0.32 20 26.91 1
Alkane 2 plus sodium benzoate Toxicity control 30 mg C/l 5.78 0.51 20 35.78 1

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
The test material, Alkane 2, attained a total of 4% degradation during the test and therefore cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.
Executive summary:

1. Methods

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines or Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4 -C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and US Code of Federal Regulations Title 40 Part 796, Section 3260.

2. Procedures

The test material was exposed to sewage sludge micro-organisms at a concentration of 20mg C/l with culture medium in sealed culture vessels in the dark at 21 Deg C for 28 days. The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the stardard material, sodium benzoate, together with an abiotic control and toxicity control were used for validation purposes.

3. Results

The test material attained a total of 4% degradation during the test and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.