### BODIS t est

Final report

Report Num ber

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

limi ted 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 compli ance Page 3

1. Abstract Page 4

2. Introduction Page 5

3. Material and methods

3.1 Test ing 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 proce dure Page 7

3.8 Archiving Page 7

4. Results

4.1 Ultimate oxygen demand Page 8

4.2 Biod e gradability results Page 8

Appendices: Tables I and II

Figure I

Quality assurance declaration

P age 2

### Certificate o f 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)

Pag e 3

### 1. Abstract

The aerobic biodegradability of was determine d 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 %

a nd 78 % B OD 28 

### /C OD with a mean value of 67% BOD 28 

### /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 O 2 

### /g calculated from the molecular structure

, a

mean biodegradability rate of 60% BOD 28 

### / BOD T is obtained

.

Biodegradability i n 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)

Pag e 4

### 2. Introduction

The German Chemical Act (Chemikaliengesetz) requires that substances undergo production-volumebased tests to determine their toxicological and ecotox icological properties. Thi s includes

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

transformed by biochemi cal reactions

, especially the metabolic processes of microorganisms

. The end

products of the reactio n are either other compounds (metabolites) o r, in the case of complete

mineralization, CO 2 

### and H 2 

### O.

The BODIS t est used to determine biodegradability is a modification of the closed bottle test

, an

international ly 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 t esting facility

Testing laboratory

Laboratory manager

Study director

Practical implementation

3.2 Test method

BOD test for insoluble substances (BODIS)

Determ ination 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 sludg e/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

substan ce (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 O 2 

### measurements (B OD p arameter s ).

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

are tested as reference substan ces

.

P age 5

### 3.4 Evaluation criteria

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

biodegrada tion 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 b io degradable 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

Substan ce

Chemi cal name, s ynonyms

Manufacturer

Batch No.

Substan ce 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.

Pag e 6

### 3.7 Test procedure

The biodegradability evaluation was performed usi ng BODIS Test Preparation No. 137.

Test day Date

Start of pre -incubation Feb. 09, 1995

Start of test 0 Feb. 16, 1995

1 st 

### intermediate measurement 7 Feb. 23, 1995

2 nd 

### intermediate measurement 14 Mar. 02, 1995

3 rd 

### 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

Ret ained 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 s ubstan ce 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

.

P age 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 substan ce

.

The individual values were scattered between 2108 und 2815 mg O 2 

### / g substan ce

. Furthermore , black

flakes were observed after the reaction

, which may indicate incomplete oxidation

.

The average COD dete rmined from all the individual raw data values was 2487 mg O 2 

### /g s ubstan ce

.

The ultimate oxygen demand of the reference substance sodium acetat e trih ydrat e was calculated from

the molecular structure (B OD T = 470 mg O 2 

### /g substance ).

4.2 Biodegradability r esults

The individual results are listed in Tables I (reference substance) and II (test substance)

. Figure I i s a

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

P age 8Table I

Biodegradability evaluation

BOD TEST for ins oluble 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 substa nce/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

Li quid 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 mater ial 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 O 2 /l] b) 3.80 8.17 8.44 8.75

c) 3.73 8.05 8.38 8.59

Cs = [mg O 2 /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 = O 2 concentration at time t Ur

= relative oxygen uptake

Cbt = idem for blank {Ur = total O 2 uptake over n days

Cs = O 2 saturation value (at 20 °C 

### ) OC = oxygen capacity of flasks

Remarks:



### P age 9Table II

Biodegradability evaluation

BOD TEST f or insoluble substances (BODIS) Date: Feb. 16, 1995

Report No.

Test No.

Sub stance 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] 19 6

. 5 196.5 196.5

Air volume [ml] 98.3 97. 9 98.3

Total ox ygen 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 O 2 /l] b) 6.88 6.96 7.40 8.01

c) 7.34 7.44 7.52 7.97

Cs = [mg O 2 /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 = O 2 concentration at time t Ur

= relative oxygen uptake

Cbt = idem for blank {Ur = total O 2 uptake over n days

Cs = O 2 saturation value (at 20°C 

### ) OC = oxygen capacity of flasks

Remarks:



### Pag e 10

## Biodegradability in the BODIS test

[Graph from German text ]

% Abbau = % degradation

Versuchszeit in Tagen = Testing time in days

Natriumacetat -3- hyd rat = Sodium acetate trihydrate

Figure I



### P age 11

### QUALI TY ASSURANCE DECLARATION

Report number:

Study director :

Title :

BODIS t est

It is hereby confirmed that this final report provides an accurate de scription 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.

Inspe ction/check dates and com munication to study director and ma nager 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 , d ire ct additions

Mar. 16, 19 95

Oxygen me asurement

, data transfer

Jun. 07 , 19 95

Final report

[Signature] June 8, 1995

Qualit y assurance Date

Einig

Pag e 12