BODIS test

Final report

Report Number 

May 1995 

This document is directed to USEPA pursuant the Toxic Substances Control Act (TSCA). This 

document is the property of the sponsor and may be used only for that purpose intended by the 

sponsor and as authorized under applicable law. Every other or additional use, exploitation, 

reproduction, publication or submission to other parties requires the written permission of the 

sponsor, except as otherwise required by applicable law (e.g., TSCA). The use of these 

studies/documents for regulatory, registration or other commercial purposes [including but not 

limited to Registration, Evaluation, Authorization and Restriction of Chemical Substances 

(REACH)], without the express written permission of the sponsor is unauthorized and strictly 

prohibited.
Contents

 Cover sheet Page 1

 Contents Page 2

 Statement of compliance Page 3

1. Abstract Page 4

2. Introduction Page 5

3. Material and methods

3.1 Testing facility Page 5

3.2 Test method Page 5

3.3 Test principle Page 5

3.4 Evaluation criteria Page 6

3.5 Test substance data Page 6

3.6 Reference substance Page 6

3.7 Test procedure Page 7

3.8 Archiving Page 7

4. Results

4.1 Ultimate oxygen demand Page 8

4.2 Biodegradability results Page 8

 Appendices: Tables I and II

 Figure I

 Quality assurance declaration

Page 2 
Certificate of compliance 

with principles of GLP

Test number: 

Title: BODIS test

Study director: 

I hereby certify that to the best of my knowledge and belief the study described in this report was 

conducted in conformity with the principles of good laboratory practice in accordance with the 

German Chemicals Act (ChemG) of July 25, 1994. 

Study director 

(Signature)

May 24, 1995 

(date)

Page 3 
1. Abstract

The aerobic biodegradability of was determined in the BODIS test, a screening test 

that is particularly suitable for the evaluation of test substances that are poorly soluble in water. Good 

biodegradability results were obtained for the test substance under these test conditions: between 57% 

and 78 % BOD28/COD with a mean value of 67% BOD28/COD. 

The rising curve indicates that increasing the length of the test may be expected to lead to higher 

biodegradability rates. 

The basic COD determination on the test substance led to very scattered measured values. 

Biodegradability rates based on COD should therefore be regarded with caution. 

Based on the ultimate oxygen demand of 2757 mg O2/g calculated from the molecular structure, a 

mean biodegradability rate of 60% BOD28/BODT is obtained. 

Biodegradability in the BODIS test

Test substance Test conc.

mg COD/l

%BOD/COD or %BODT after no. of days

7 14 21 28

100 19 39 55 67 

Reference substance 

sodium acetate 100 74 84 87 89 

Author: 

(Signature)

May 29, 1995 

(date)

Study director: 

(Signature)

May 29, 1995 

(date)

Page 4 
2. Introduction

The German Chemical Act (Chemikaliengesetz) requires that substances undergo production-volumebased tests to determine their toxicological and ecotoxicological properties. This includes 

biodegradability tests. Biodegradability is understood to mean the ability of a substance to be 

transformed by biochemical reactions, especially the metabolic processes of microorganisms. The endproducts of the reaction are either other compounds (metabolites) or, in the case of complete

mineralization, CO2 and H2O. 

The BODIS test used to determine biodegradability is a modification of the closed bottle test, an

internationally recognized test method for determining the ready biodegradability. 

The BODIS test is particularly suitable for testing organic compounds that are poorly soluble or 

insoluble in water. 

3. Material and methods 

3.1 Testing facility: 

Department

Head of testing facility

Testing laboratory 

Laboratory manager

Study director 

Practical implementation

3.2 Test method 

BOD test for insoluble substances (BODIS)

Determination of the biodegradability under aerobic conditions (BOD parameters) of substances that 

are poorly soluble in water 

3.3 Test principle 

Wide-neck reagent bottles of known volume are filled 2/3 full with inoculated (approx. 30 mg-dry 

activated sludge/l) mineral nutrient salt solution. After a week of pre-incubation (18-22 °C), the 

mineral nutrient medium is saturated with atmospheric oxygen, and the calculated amount of test 

substance (approx. 20 mg COD/bottle) is added. 

The closed bottles are then shaken on a round shaker at 18-22°C and the biodegradability is 

determined by regular O2 measurements (BOD parameters).

At the same time, sodium acetate and an inoculum blank containing only inoculated nutrient medium 

are tested as reference substances. 

Page 5 
3.4 Evaluation criteria

The test substance has good biodegradability if the measured rate is above 60%. A similar 

biodegradation result obtained in a closed bottle test would lead to the substance being classified as 

readily biodegradable.

The requirements of RAL-UZ 64 (ecolabel for readily biodegradable lubricants and forming oils) are 

considered to be met when the biodegradation results obtained for the base substances are above 80%. 

To ensure that the test has been performed correctly, sodium acetate is run as a reference substance in 

parallel with the test substance. If the biodegradability of the reference substance after 14 days is 

greater than 60%, the test is considered valid. 

3.5 Test substance data 

Client 

Reason for test Hazardous Substances Ordinance, water hazard 

class and labeling

Substance

Chemical name, synonyms 

Manufacturer 

Batch No. 

Substance No. 

Test No.

Purity 

Solubility in water

Volatility (RT)

100% active

insoluble

non-volatile

Color 

Consistency

yellow 

semi-solid

Production date 

Expiration date 

12.93 

12.96 

Further data 

3.6 Reference substance

Sodium acetate trihydrate for analysis (content at least 99.5%, Batch 21810, 

) was used as a reference substance.

Page 6 
3.7 Test procedure 

The biodegradability evaluation was performed using BODIS Test Preparation No. 137. 

Test day Date

Start of pre-incubation Feb. 09, 1995 

Start of test 0 Feb. 16, 1995 

1st intermediate measurement 7 Feb. 23, 1995 

2nd intermediate measurement 14 Mar. 02, 1995

3rd intermediate measurement 21 Mar. 09, 1995 

Final measurement 28 Mar. 16, 1995

Test concentration and introduction of test sample

The reference substance was introduced by adding an aliquot of the stock solution. 

The poorly water-soluble test sample was introduced by direct addition of a weighed amount from a 

glass carrier plate. 

Analysis 

Basic COD analyses are performed in the analytical laboratory.

The oxygen dissolved in the water was determined amperometrically using a WTW OXI 2000 oxygen 

meter. 

Inoculation 

Sewage sludge from the sewage treatment plant was used as the inoculating material. 

( is an activated sludge plant with a capacity of approx. 27,000 PE (population equivalents), 

achieving an average COD removal efficiency of more than 80%.)

Archiving

Retained samples of the test substance, the original copies of the test plan, the raw data and the final 

report, as well as all other documents, are kept in the archive. 

Records of the location of the test substance are kept in the central test substance management 

system. 

The raw data record for the reference substance is kept in the archive after the substance has been 

returned. 

Page 7 
4. Results 

4.1 Ultimate oxygen demand (BODT, COD)

The COD measured by the modified Kelkenberg method was used to determine the ultimate oxygen 

demand of the test substance. 

The individual values were scattered between 2108 und 2815 mg O2/g substance. Furthermore, black 

flakes were observed after the reaction, which may indicate incomplete oxidation. 

The average COD determined from all the individual raw data values was 2487 mg O2/g substance. 

The ultimate oxygen demand of the reference substance sodium acetate trihydrate was calculated from 

the molecular structure (BODT = 470 mg O2/g substance).

4.2 Biodegradability results

The individual results are listed in Tables I (reference substance) and II (test substance). Figure I is a 

graphical representation of the biodegradation of the test and reference substances. 

Page 8 
Table I

Biodegradability evaluation

BOD TEST for insoluble substances (BODIS)

Date: Feb. 16, 1995

Report No. 

Preparation No. 137

Sample No. 4-6 

Test/reference substance: Sodium acetate trihydrate

Theoretical oxygen demand: 0.470 mg ThOD/mg sample

Stock solution ===> 85070 mg test substance/I ====> 20.0 mg ThOD in 0.5 ml

Test flasks ====> No. 1 No. 2 No. 3

Flask volume [ml] 295.2 295.3 295.2

Liquid volume [ml] 197.0 197.0 197.0

Air volume [ml] 97.2 97.3 97.2

Total oxygen capacity [mg /flask] 29.0 29.0 29.0

Test material added => No. 1 No. 2 No. 3

Stock solution [ml] 0.5 0.5 0.5

or direct addition [mg]

Equivalent to ThOD/flask 20.0 20.0 20.0

Results ===> Data I incubation time ====> 7 14 21 28

Cbt (average n = 3) 8.51 8.76 8.62 8.80

a) 3.90 8.24 8.47 8.64

Ct = [mg O2/l] b) 3.80 8.17 8.44 8.75

c) 3.73 8.05 8.38 8.59

Cs = [mg O2/l] 9.20 9.20 9.20 9.20

 Cbt-Ct a) 0.50 0.06 0.02 0.02

Ur = ---------------- b) 0.51 0.06 0.02 0.01

 Cs c) 0.52 0.08 0.03 0.02

a) 0.50 0.56 0.57 0.59

{Ur b) 0.51 0.58 0.59 0.60

c) 0.52 0.60 0.62 0.64

a) 14.53 16.15 16.62 17.11

{Ur x OC b) 14.86 16.71 17.26 17.41

c) 15.06 17.29 18.03 18.69

a) 73 81 83 86

% oxidation b) 74 84 86 87

c) 75 86 90 93

% oxidation (average) 74 84 87 89

Ct = O2 concentration at time t Ur = relative oxygen uptake

Cbt = idem for blank {Ur = total O2 uptake over n days

Cs = O2 saturation value (at 20°C) OC = oxygen capacity of flasks

Remarks:

Page 9 
Table II

Biodegradability evaluation

BOD TEST for insoluble substances (BODIS)

Date: Feb. 16, 1995

Report No. 

Test No.

Substance No.

Preparation No. 137

Sample No. 10-12

Test/reference substance: 

Theoretical oxygen demand: 2.487 mg ThOD/mg sample or mg COD/mg sample

Stock solution ===> 0 mg test substance/I ====> 0.0 mg ThOD in 0.5 ml

Test flasks ====> No. 1 No. 2 No. 3

Flask volume [ml] 295.8 295.4 295.8

Liquid volume [ml] 196.5 196.5 196.5

Air volume [ml] 98.3 97.9 98.3

Total oxygen capacity [mg /flask] 29.3 29.2 29.3

Test material added => No. 1 No. 2 No. 3

Stock solution [ml] 0.0 0.0 0.0

or direct addition [mg] 9.01 8.87 8.56

Equivalent to ThOD/flask 22.4 22.1 21.3

Results ===> Data I incubation time ====> 7 14 21 28

Cbt (average n = 3) 8.51 8.76 8.62 8.80

a) 7.47 7.57 7.71 7.90

Ct = [mg O2/l] b) 6.88 6.96 7.40 8.01

c) 7.34 7.44 7.52 7.97

Cs = [mg O2/l] 9.20 9.20 9.20 9.20

 Cbt-Ct a) 0.11 0.13 0.10 0.10

Ur = ---------------- b) 0.18 0.20 0.13 0.09

 Cs c) 0.13 0.14 0.12 0.09

a) 0.11 0.24 0.34 0.44

{Ur b) 0.18 0.37 0.50 0.59

c) 0.13 0.27 0.39 0.48

a) 3.31 7.09 9.98 12.83

{Ur x OC b) 5.17 10.87 14.73 17.22

c) 3.73 7.92 11.41 14.04

a) 15 32 45 57

% oxidation b) 23 49 67 78

c) 17 37 54 66

% oxidation (average) 19 39 55 67

Ct = O2 concentration at time t Ur = relative oxygen uptake

Cbt = idem for blank {Ur = total O2 uptake over n days

Cs = O2 saturation value (at 20°C) OC = oxygen capacity of flasks

Remarks:

Page 10 
Biodegradability in the BODIS test

[Graph from German text] 

% Abbau = % degradation

Versuchszeit in Tagen = Testing time in days

Natriumacetat-3-hydrat = Sodium acetate trihydrate

Figure I

Page 11 
QUALITY ASSURANCE DECLARATION

Report number: 

Study director: 

Title: 

BODIS test

It is hereby confirmed that this final report provides an accurate description of the methods 

and procedure used and the observations made in accordance with the German Chemicals Act 

(ChemG) of July 25, 1994. 

The reported results accurately reflect the raw data. 

The report has been checked. 

Inspection/check dates and communication to study director and manager of testing facility: 

Feb. 03, 1995 

Test plan

Feb. 09, 1995 

 Preparation of mineral nutrient medium + activated sludge

Feb. 16, 1995 

Bottle volume, reference stock solution, direct additions

Mar. 16, 1995

 Oxygen measurement, data transfer

Jun. 07, 1995 

 Final report 

 [Signature] June 8, 1995

Quality assurance Date

 Einig

Page 12