ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 5, 4.5-55 (1981)

A Correlation Study of Biodegradability Determinations with Various Chemicals in Various Tests’

II. Additional Results and Conclusions

P. GERIKE AND W. K. FISCHER

Department of Ecology. Henkel KGaA, Diisseldorf, Germany

Received February 29, 1980

A correlation study of biodegradability determinations was started previously by testing 44 compounds covering a range of physical, chemical, and biological properties in seven tests (the coupled units test, i.e., the modification of the OECD Confirmatory Test for the

application of the carbon analysis, the Zahn- Wellens, the Japanese MITI, the French AFNOR T 90-302, the Sturm, the Modified OECD Screening, and the Closed Bottle Test according to Fischer). The individual tests were characterized by comparing their re-

sults with those obtained in the others. Two further tests were included in the present part of the study: The U.S. EPA Activated Sludge Test and a model trickling filter. Furthermore, more in-depth characterizations of the various tests were achieved by transinoculation

experiments: Typically, if a compound was degraded in one test but failed in another, the first was used as a preacclimation procedure for the second. The concept of the OECD Expert Group “Degradation/Accumulation” of a set of tests for the determination of “ready

biodegradability” could be confirmed on an experimental basis.

1 .O. INTRODUCTION

In the first (Gerike, 1978) and second (Gerike and Fischer, 1979) papers of this series an effort was made to characterize various biodegradability tests by a largely two-dimensional effort: 44 compounds covering a range of physical, chemical, and biological properties were tested in seven tests:

-The Coupled Units Test, i.e., the modification of the OECD Confirmatory Test (OECD, 1976) for the application of unspecific analyses (Fischer et al., 19T75);

-the Zahn-Wellens Test (Zahn and Wellens, 1974); -the Japanese MIT1 Test (The Chemical Substances Control Law in Japan,

19:7q; -the French AFNOR T 90-302 Test (AFNOR, 1977) with a test duration of 42

days; -the Carbon Dioxide Evolution Test according to Sturm (Sturm, 1973) with

its preacclimation procedure; -the OECD Screening Test modified for the application of the carbon analysis

(Umweltbundesamt, 1976) with a 1Pday test duration; -the Closed Bottle Test according to Fischer (Fischer, 1963 and Fischer et al.,

1974). Conclusions regarding the individual tests were drawn by comparing their results

with those obtained in the others. This effort is being continued in the present part of the study with the same compounds as in the first but with only two tests:

1 Dedicated to Dr. Bruno Werdelmann on the occasion of his 60th birthday.

45 0147-6513/81/010045-11$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.

46 GERIKE AND FISCHER

-Only 20 of the 44 compounds had been examined previously in the AFNOR Test and it was felt that a good description of this method would require a few more biodegradability determinations.

-While this study was in progress the U.S. Environmental Protection Agency published its “Preliminary Draft Guidance for Premanufacture Notification” (U.S. Environmental Protection Agency, 1978). This draft guidance includes a shake flask test, the Modified OECD Screening Test, the Sturm Test, a BOD (biochemical oxygen demand) test, and an “Activated Sludge Test,” a combina- tion of the original surfactant “Semicontinuous Activated Sludge Test” (SCAS) of the American Soap and Detergent Association (SDA Subcommittee on Bio- degradation Test Methods, 1963, and a 1Pday die-away test of the kind of the Zahn-Wellens Test. Since no data elaborated in the EPA test have, to our knowledge, been published so far it seemed worthwhile to include it in the correlation study. Since it was suspected that the method would exaggerate considerably comparatively few biodegradability determinations were believed to characterize the test sufficiently.

Beyond this largely two-dimensional effort the present part of the study aims more into a third dimension, something which had already been started before: First, in some instances where the Closed Bottle Test had yielded lower results than the others the preacclimation procedure of the Sturm Test was put ahead of the first. Second, where the Coupled Units Test had yielded lower results than the Sturm or Zahn-Wellens Test the square wave feeding version of the first (Gerike et al., 1980) was run. This kind of effort constitutes the main part of this second part of the correlation study. Typically, if a compound was degraded in one test but failed in another, the first was used as a preacclimation step for the second. These experiments yielded a much more thorough characterization of the involved tests than did the simple comparison of test results.

A further method was also included: The application of the coupled units test principle to model trickling filters (Gerike et al., 1980) has yielded very interesting results. Because of its good model character the test was, besides the OECD Confirmatory Test, used as a further referee method. Essential differences between the two methods will be considered below.

The present paper requires, for good understanding, a knowledge of the previous publication of this series (Gerike and Fischer, 1979). To include all the pertinent points made previously would yield an all too voluminous manuscript. Especially the calibration of the tests by true environmental chemicals, whose fate in sewage treatment plants and receiving waters is known to at least some extent, has been reported in this previous paper while the present part constitutes largely a comparison of the tests among each other.

2.0. RESULTS AND DISCUSSION

2.1. AFNOR Test Results

Twelve compounds were, in addition to the 20 of the first series, tested. They are, together with the percentage DOC (dissolved organic carbon) removals after 28 and 42 days and the results of the inhibition test after 7 days, listed in Table 1 (the inhibition test accompanies the biodegradability test routinely):

BIODEGRADABILITY DETERMINATIONS 47

TABLE 1

RESULTS OBTAINED IN THE AFNOR T 90-302 TEST -

Percentage DOC removal

after Day Percentage inhibition

NO. Compound 28 42 after 7 days

1.1 3-Hydroxybenzoic acid 96 97 0 1.2 4-Nitrobenzoic acid 92 92 0 1.3 Terephthalic acid 66 66 0 1.4 Diethanolamine 97 98 0 1.5 N-Methylaniline 29 67 0 1.6 Pyridine 46 65 0 1.7 terl-Butanol 80 93 0 1.8 Sulfanilic acid 24; 28 68; 81 0 1.9 3-Aminophenol 13 22 0 1.10 1,3-Benzenedisulfonic acid 0 0 0 1.11 Pentaerythritol 0 12 0 I. 12 2-Chloroaniline 14 25 65

-

-The 3-hydroxy- and 4-nitrobenzoic acid, terephthalic acid, and diethanolamine (Nos. 1. I - 1.4 in Table 1) were highly degraded as they had been in the other screening tests (MITI, Sturm, OECD Screening, and Closed Bottle), except, in particular cases, in the MIT1 and Closed Bottle Tests.

-iV-Methylaniline, pyridine, rert-butanol, and sulfanilic acid (Nos. 1 .S- 1.8 in Table 1) had been poorly degraded in the MITI, OECD Screening, and Closed Bottle Tests but were highly degraded in the Sturm Test (with the exception of terr- butanol). These results show that the AFNOR Test is, except in cases of inhibitory or toxic materials, more forceful than the Closed Bottle, the Modified OECD Screening, and the MIT1 Test and more in one category with the Sturm Test. (“Forceful” is used here generally in the sense of how many compounds are degraded in a test, not the extent to which a particular compound is degraded.) The comparatively high degradation of sulfanilic acid -68 and 81% DOC-removal, respectively-is of special significance and will be discussed further below.

-3-Aminophenol, 1,3-benzenedisulfonic acid, and pentaerythritol (Nos. 1.9- I. 11 in Table 1) had not been degraded in the other screening tests either (except 3- aminophenol once in the Sturm Test). The same pertains to 2-chloroaniline (No, 1.12 in Ta’ble 1). It is noteworthy, though, that this last material alone, among the 32 compounds tested altogether, yielded a clear indication of inhibition in the accompanying inhibition test. (Inhibitory and even toxic materials tested formerly without an indication of inhibitory effects: 2- and 4-nitrophenol, hydroquinone, linear alkylbenzenesulfonate (LAS), benzyldimethyldodecyl-ammonium chloride (BDMDAC), and cetyltrimethylammonium bromide (CTAB).)

2.2. EPA .Activated Sludge Test Results

As has already been mentioned above, only “difficult” compounds which were not degralded or degraded inconsistently in the other tests were selected for a

48 GERIKE AND FISCHER

TABLE 2

RESULTS OBTAINED IN THE U.S. EPA ACTIVATED SLUDGE TEST

No. Compound Percentage

DOC removal

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

2.9 2.10 2.11 2.12

2.13 2.14 2.15

3-Aminophenol Diethyleneglycol Sulfanilic acid Linear alkylbenzenesulfonate (LAS) 1,3-Benzenedisulfonic acid Pentaerythritol o-Chloroaniline Benzyldimethyldodecylammonium chloride

(BDMDAC) Cetyltrimethylammonium bromide (CTAB) Tetrapropylenebenzenesulfonate (TBS) 3-Aminobenzenesulfonic acid (metanilic acid) cis ,cis ,cis- 1,2,3,4-cyclopentanetetracarboxylic

acid (cycloacid) Ethylenediaminetetraacetic acid (EDTA) Carboxymethylcellulose (CMC) Ethylene oxide-propylene oxide block polymer

(Pluronic)

98 96 98 98 87 98 98 84

91 80 0

15

18; 54 27 56

biodegradability evaluation in this test. The 15 substances chosen are listed in Table 2 together with the results obtained. They allow a full characterization of the method:

-Compounds judged inconsistently in the screening tests were all highly degraded in the Activated Sludge Test. These include such difficult compounds as sulfanilic acid and diethylene glycol which, as one of the few low molecular weight compounds, had often yielded results in the intermediate range (Nos. 2.1-2.4 in Table 2).

- 1,3-Benzenedisulfonic acid, pentaerythritol, and o-chloroaniline (Nos. 2.5-- 2.7 in Table 2) were degraded in the Activated Sludge Test but in none of the screening tests. 1,3-Benzenedisulfonic acid was eliminated only to the extent of 59% DOC removal in the Zahn- Wellens Test although highly in the coupled units test (No. 3.8 in Table 3). The o-chloroaniline result is of special significance and will be discussed further below.

-The highly bactericidal surfactant quaternary ammonium salts BDMDAC and CTAB (Nos. 2.8 and 2.9 in Table 2) were highly degraded or eliminated in the EPA Activated Sludge Test in spite of the comparatively high test concentration corresponding to 100 mg C/liter, i.e., 160 mg/liter of active material in case of CTAB. This shows clearly that the test is very insensitive to inhibitory and toxic effects.

-Most telling is the extent to which tetrapropylenebenzenesulfonate (TBS, No. 2.10 in Table 2) was degraded in this test: This compound, removed from the market in many countries because of its proved environmental incompatability, reached fully 80% DOC removal, accompanied by >98% MBAS removal. A method which classifies TBS as so well degradable certainly does not leave room for a sensible biodegradability classification of environmental chemicals anymore.

BIODEGRADABILITY DETERMINATIONS 49

-On the other hand, the low molecular weight compounds metanilic acid, EDTA, and cycloacid (Nos. 2.11-2.13 in Table 2) which had not been degraded in any of the other tests appeared non- or insufficiently degradable in this test also. The same pertains to the polymeric materials carboxymethylcellulose (CMC) and the ethylene oxide-propylene oxide block polymer Pluronic (Nos. 2.14 and 2.15 in Table 2).

2.3. Comparisons of Other Test Results

The results elaborated for a further and more in-depth characterization of the individual tests are listed in Table 3. Some of them which were already reported previously are included again in order to give as comprehensive as possible a picture. These old results are marked by a superscript asterisk.

The compounds and their biodegradabilities in the various methods are discussed successively together with the characterizations they yield of the tests.

2.3.1. Pyridine, N-methylaniline, and 3-aminophenol (Nos. 3.1-3.3 in Table 3) which ha.d formerly-among the screening tests-only been degraded in the Sturm Test were also degraded in the Modified OECD Screening Test when the inoculum was preacclimatized according to the Sturm Test procedure. On the other hand, these compounds did not yield to bacterial attack in the Closed Bottle Test although it was inoculated in the same fashion.

The 2chlorobenzoic acid (No. 3.4 in Table 3) had previously not been degraded in the Closed Bottle and the OECD Screening and inconsistently in the Sturm Test. When the Sturm and the OECD Screening Test were started with the very same inoculum and run with the same test concentration and duration of 28 days both turned out negative in this particular experiment as did the Closed Bottle Test which was inoculated in the same fashion. On the other hand, 3-chlorobenzoic acid (No. 3.5 in Talble 3) was highly degraded in the OECD Screening and the Sturm but not in the Closed Bottle Test. tert-Butanol displayed a very similar behavior (No. 3.6 in Table 3). From all these results it may, with caution, be concluded that there is no difference between the Sturm and the Modified OECD Screening Tests inherent in the systems. The Sturm Test clearly derives its relative forcefulness from its preacclimation procedure. On the other hand, the Closed Bottle Test is the most stringent even if the Sturm preacclimation procedure precedes the actual test.

2.3.2. Diethyleneglycol (No. 3.7 in Table 3) had often yielded results in the intermediate range, even in the coupled units test. It was degraded highly on the trickling filter after a working-in time of 18 days. This result characterizes the test as quite forceful, especially if the total organic load is comparatively low. However, because of its good model character conclusions regarding the degradation performance of actual trickling filters with respect to particular compounds can be drawn by comparing the respective operating parameters. The system itself and its operating parameters have been described (Gerike et al., 1980; Fischer et al., 1978; Gerike and Kurzyca, in preparation).

2.3.3. :1,3-Benzenedisulfonic acid and pentaerythritol (Nos. 3.8 and 3.9 in Table 3) had not been degraded in any of the screening tests (MITI, AFNOR, OECD Screening, Sturm, Closed Bottle). Even in the Zahn-Wellens Test 1,3- benzenedisulfonic acid reached only 59% DOC removal although, as mentioned above, 80% in the EPA Activated Sludge Test. It was degraded highly in the

TABL

E 3

RESU

LTS

OBT

AINE

D IN

VAR

IOU

S TE

STS

NO.

Comp

ound

OECD

Co

nfirm

atory

Test

(co

upled

un

its

test)

Squa

re

wave

- Co

ntinu

ous

feedin

g (%

DO

C (%

DOC

rem

oval

rem

oval

k to

I,,.

, )

k to

I.,,.

, )

Trick

ling

Filter

(co

upled

un

its

test)

(%

DO

C re

mov

al k

tol,i.

, )

Clos

ed

Bottle

Te

st

Test

us

ed

as

%

prea

cclim

a-

B 0

DTx,

tio

n ste

p

Mod

ified

OECD

Sc

reenin

g Te

st

% DO

C re

mov

al Te

st

used

as

aft

er

19

prea

cclim

a-

days

tio

n ste

p

Stur

m

Test”

’

% co

* Te

st

used

pro

duc-

% DO

C as

pre

ac-

tion

(28

rem

oval

clim

ation

days

) (28

da

ys)

step

3.1

F’yrid

ine

YY?

2*

3.2

N-M

ethyla

niline

10

2 *

4*

3.3

3-Am

inoph

enol

94+

4%

3.4

2-Chlo

roben

zoic

acid

Y3t

8*

3.2

3-Chlo

roben

zoic

acid

3Ot

8*

3.6

tcrt-B

utano

l

3.7

Dieth

ylene

glyco

l

33

e 4*

53

i- 13

*

0 0

- -

I

- 0*

9s

* 31

YY

+ 2

I*

6Y?8

* -

0 St

urm

29

* -

o*

32*

Stur

m

YOi-

3’ SO

* -

42*

31*

33*

Stur

m

Stur

m’

91

Stur

m

S8*

97

Stur

m

Stur

m

92

Stur

m

46*

92*

Stur

m

Stur

m

Stur

m

91 0”

Stur

m

Stur

m

0* 70*

31

22*

Stur

m

loo*

Stur

m

0 St

urm

93”

Stur

m

II5

100

sturm

3.8

3.9

3.10

3.11

I ,3-B

enze

nedi-

s.<

;fci,ii

-#

.id

84

+ 5O

92

i

2”

Penta

eryth

ritol

96

+ I*,

’ -

Sulfa

nilic

acid

7?

8” -3

-t 2

205

4’ IO

t

3’

195

5”

o-Chlo

roanili

ne

5+

s* 6

i 5”

23r

3” 16

t

4’

32

t 5’

- 10

9 OE

CD

Conf.

93

OE

CD

Conf.

Test

1 e

st

93i

1’ 10

9 Za

hn-

92

Zahn

-

Welle

ns

Welle

ns

86

e 9”

55

Stur

m

92

Stur

m

44

-t 12

k I

Zahn

- I6

Zahn

-

Welle

ns

Welle

ns

53

88

Zahr

- Welle

ns

66*

97’

Stur

m

0 0

zahn

- Welle

ns

* Re

sult

was

alrea

dy

previo

usly

repo

rted.

” Als

o te

st co

ncen

tratio

n an

d du

ration

as

in

Stur

m

Test

, the

sa

me

inocu

lurn

was

used

fo

r the

OE

CD

Scree

ning

and

the

Stur

m

Test

.

b Af

ter

a wo

rking

-in

perio

d of

4 we

eks.

c Af

ter

a wo

rking

-in

perio

d of

3 we

eks.

d Af

ter

a wo

rking

-in

perio

d of

25

days

. c

SWF

plus

6 hr

mea

n re

tentio

n tim

e.

‘Tot

al te

st du

ration

42

da

ys;

inocu

lation

wi

th

sludg

e fro

m

a po

sitive

EP

A Ac

tivate

d Slu

dge

Test

. p

Tota

l te

st du

ration

28

da

ys;

inocu

lation

fro

m

a po

sitive

St

urm

Te

st.

* To

tal

test

durat

ion

34

days

. ’ T

otal

test

durat

ion

45

days

: ino

culat

ion

with

slu

dge

from

a

posit

ive

Zahn

-Welle

ns

Test

. ’

Afte

r a

worki

ng-in

pe

riod

of ll-

I8 da

ys.

li To

tal

test

durat

ion

4 we

eks.

’ In

case

of

the

Stur

m

Test

as

a

prea

cclim

ation

ste

p on

ly the

ac

clim

atiza

tion

proce

dure

was

used

fo

r the

ino

culum

pre

parat

ion,

not

the

test

liquo

r. “I

All

Stur

m

Test

re

sults

co

nsist

of

a sin

gle,

final

CO,

deter

mina

tion

in ord

er to

avoid

the

er

ror

poss

ibilitie

s inh

erent

in the

an

alytic

al pa

rt of

this

test.

Th

e sa

me

perta

ins

to the

ev

aluati

ons

report

ed

previo

usly

(Ger

ike

and

Fisch

er,

1979

).

52 GERIKE AND FISCHER

coupled units test version of the OECD Confirmatory Test after a working-in period of 4 weeks. When an inoculum was drawn from this test the compound was also degraded in the Closed Bottle and the Modified OECD Screening Test proving that its degradation is just a matter of acclimatization.

Pentaerythritol displayed the same pattern of behavior. Using a positive Zahn-Wellens Test run (the compound had previously also been degraded in the coupled units test in an independent experiment without preacclimation) as a preacclimation step for the screening tests a high degradation was achieved in these also. This shows that the degradation of pentaerythritol does not require special conditions such as the presence of carbon dioxide as has been claimed by Ilic (1978a, b). As a matter of fact, only the activation of the Entner-Doudoroff metabolic pathway seems to be required (Robra, personal communication).

2.3.4. Sulfanilic acid (4-aminobenzenesulfonic acid, No. 3.10 in Table 3) had not been degraded in any of the screening tests except the Sturm Test. However, when the Closed Bottle and the Modified OECD Screening Test were started with an inoculum prepared according to the Sturm preacclimation procedure the compound was degraded highly in these tests also.

On the other hand, sulfanilic acid could not be degraded in either the conventional or square-wave feeding versions of the coupled units test (OECD Confirmatory Test) at either 3 or 6 hr mean retention time. As a matter of fact, when sludge which had been successfully acclimatized to sulfanilic acid in the EPA Activated Sludge Test was used to start the coupled units test the acclimatization was immediately lost and the compound was not degraded anymore. The same happened when the test was started with the liquor from a successful Sturm Test. This result must be of environmental significance.

But, at first very surprising, sulfanilic acid was highly degraded on the trickling filter after a working-in time of 25 days after the introduction of the compound. How can this difference be explained? There are some important differences between this type of sewage work and the activated sludge plant. The sludge age is one such essential difference, another which does not seem to be as commonly realized is that trickling filters display a load gradient, i.e., they are heavily loaded at the top but much less so at the bottom. Activated sludge plants, on the other hand, ideally constitute completely mixed systems where nutrient competition is really maintained at all times. This difference may result in different biodegradability evaluations in particular cases such as presented by sulfanilic acid. In particular, this could furnish an explanation why, in certain cases, trickling filter results may be closer to those of screening tests than to those obtained in the OECD Confirmatory Test since in the screening tests discussed here the test material constitutes the sole carbon source.

It is also noteworthy that sulfanilic acid attained 68 and 81% DOC removal, respectively, in the AFNOR Test. Perhaps this test is, with a duration of 42 days, already too long for a test for “ready biodegradability.” After 4 weeks the compound had attained only 24 and 28% DOC removal, respectively. It seems obvious that the choice of the proper test duration constitutes quite a precarious balancing act. Environmental monitoring data for this compound would obviously be of special value.

2.3.5. 2-Chloroaniline (No. 3.11 in Table 3) also turned out to be quite an enlightening example for biodegradability studies. This compound was only highly

BIODEGRADABILITY DETERMINATIONS 53

eliminated in the Zahn-Wellens Test (whereby true biodegradation was clearly suggested by the shape of the curve and it was furthermore ascertained that the elimination was not due to solubility problems), and the EPA Activated Sludge Test. All other results were negative, including those obtained in the OECD Confirmatory Test in the conventional or square-wave feeding versions either at a 3 or 6 hr mean retention time. The compound was even only degraded to the insufficient extent of (44 rt 12)% DOC removal on the rather forceful trickling filter at a concentration corresponding to 20 mg C/liter. Even at half that concentration the result was still negative. In addition, o-chloroaniline was not degraded in the OECD Confirmatory Test when it was started with the sludge from a successful Zahn-Wellens Test. This result again has to be of environmental significance. Furthermore, and contrary to the results obtained with sulfanilic acid, o-chloroaniline was not degraded in any of the screening tests to any appreciable extent when these were inoculated with bacteria drawn from a positive Zahn- Wellens Test.

3 .O. CONCLUSIONS

On the basis of the experimental results presented above and previously the individual tests may be characterized. Furthermore, a few general conclusions with regard to biodegradability testing requirements may be drawn.

3.1. Characterization of the Individual Tests

3.1 .I. The AFNOR T 90-302 Test with a 42-day test duration. According to our investigations the test would be characterized, in summary, as follows: It is quite a powerful test for noninhibitory compounds and might even exaggerate in certain cases, e.g., sulfanilic acid. On the other hand, the method seems to be overly sensitive toward inhibitory or toxic materials because of the comparatively high test concentration as has been discussed before. Finally, the inhibition test does not seem to fulfill its control function properly as has also been discussed before.

3.1.2. The EPA Activated Sludge Test. It can be stated that the U.S. EPA Activated Sludge Test turned out to exaggerate strongly with respect to the predictive value of biodegradability test results for the general environment. This had been expected.

3.1.3. The evaluations given previously for the other tests need, in our opinion, no major revision: The Closed Bottle Test, the Modified OECD Screening Test, and the MIT1 Test can be assumed to yield environmentally relevant positive results.

3.2. Principles for Biodegradability Testing

3.2.1. We feel that special preacclimation procedures should be excluded in the first step of biodegradability testing and the test duration ought to be restricted to 4 weeks. The reason for this conclusion is clearly shown by the example of sulfanilic acid.

In the light of all these results it appears that the OECD Expert Group “Degradation/Accumulation” has recommended a fail-safe set of tests for the determination of “ready biodegradability” (OECD, 1980), i.e.:

-th#e Japanese MIT1 Test,

54 GERIKE AND FISCHER

-the French AFNOR T 90-302 Test restricted to 4 weeks in its test duration, -the Sturm Test without the preacclimatization step, -the Modified OECD Screening Test extended to 4 weeks in its duration, -the Closed Bottle Test according to Fischer,

with the limits 60% DOC removal or 50% BODT or COz production, respectively.

No argument against this concept can be derived, in our opinion, from a consideration of the approximately 360 biodegradability determinations which were conducted altogether in the course of this investigation. To be sure, it can be expected that the tests may yield differing results for particular compounds; however, all positive results can be expected to be safe in an environmental sense according to present knowledge. Thus it would seem safe to recognize results from any of this group of tests or that the results from the individual tests could be mutually recognized.

It should be noted that the studies reported here employed, with only two exceptions, the 19-day version of the Modified OECD Screening Test. We feel sure, though, that a prolongation of this test to 28 days would not yield too high results, e.g., because of the intermediate 28-day results in the AFNOR T 90-302 Test (see Table 1).

3.2.2. Further measures such as preacclimation steps, longer test durations, and more forcible tests such as the Zahn- Wellens Test in its present form and the EPA Activated Sludge Test-classified by the OECD Expert Group as tests for “inherent biodegradability” -yield valuable additional information but should not be used when testing for “ready biodegradability.”

3.2.3. A consideration of the test results also shows that the coupled units test version of the OECD Confirmatory Test yields environmentally as safe biodegradability evaluations as all the screening tests for “ready biodegradability .” This pertains only to the continuous version of the test and not its square-wave feeding modification. The latter constitutes already a special condition which is not fulfilled by all although by many activated sludge plants. The value of this test has recently been considerably enhanced by a mathematical model describing the fate of recalcitrant, soluble, but strongly adsorbing compounds: It could be shown that this test is, beyond a certain base value, also a test for true biodegradation and not just elimination for all soluble compounds (Wierich and Gerike, in preparation).

ACKNOWLEDGMENTS We gratefully acknowledge the financial support of this research project by the German Ministries for

Research and Technology (BMFT) and of the Interior (BMI) under Grant 037307. We also wish to extend our appreciation to H. J. Grebe, W. Jasiak, G. Kurzyca, and K. Richterich, Department of Ecology, Henkel KGaA, who conducted the various tests.

REFERENCES AFNOR T 90-302 (1977). L’Association Francaise de Normalisation.

FISCHER, W. K. (1%3). Der “geschlossene Flaschentest,” eine einfache quantitative Methode zur

Bestimmung der biologischen Abbaubarkeit anionischer Detergentien und anderer Substanzen.

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