Chemosphere, ​Vol. 28, No. 4, pp.

787-800, 1994 Pergamon

Elsevier Science Lid Printed in Great Britain ​0045-6535/94 $6.00+0.00
0045-6535(94)E0014-K
THE INTERPRETATION OF CEC L-33-T-82 BIODEGRADABILITY TEST DATA t
N.S. BATTERSBY*, P.A. FIELDWICK, T. ABLITT, S.A. LEE, and G.R. MOYS
Shell Research Limited,
Sittingbourne Research Centre,
Sittlngbourne, Kent ME9 8AG, U.K.
(R~vedin GermanyllOc~ber1993;~cepted5 November1993)
ABSTRACT
The CEC L-33-T-82 biodegradability test has become a de facto standard
industry method for assessing the biodegradabilities of oil products and is
stipulated in national legislation (Portugal), an ICOMIA international
standard and labelling schemes for environmentally-compatible lubricants. In
this paper we report data which indicate that the CEC test should be looked
upon as a method for assessing "primary" biodegradation, as defined by the
Organisation for Economic Co-operatlon and Development (i.e. "the alteration
in the chemical structure of a substance, brought about by biological
action, resulting in the loss of a specific property of that substance").
INTRODUCTION
The CEC L-33-T-82 biodegradability test was originally developed to assess
the biodegradabilities of two-stroke outboard engine oils in water [i].
However, it is now used in numerous laboratories in Europe and the USA to
assess the biodegradabilities of a wide variety of lubricant base fluids,
additives and formulated products. CEC test data are also specified in "eco-
labelling" schemes [2, 3], an international standard [4] and national
legislation [5] for environmentally-compatible oil products.
tCopyright ©1994 Shell Research Ltd.
* To whom correspondence should be ​addressed.
787
788 ​In some respects the CEC test is similar to OECD and EC "ready

biodegradability" tests which are often used to assess the

biodegradabilities of compounds and formulated products [6, 7]. The test oil

is the nominal sole source of carbon and energy in a mineral salts medium

inoculated with unacclimated micro-organisms from a sewage treatment plant.

Incubation is at 25 °C and the course of biodegradation is followed over a

number of weeks. However, "ready biodegradability" tests determine the

extent of "ultimate" blodegradatlon (i.e. conversion to carbon dioxide,

water, blomass and inorganic salts) by measuring the loss of test substance

as dissolved organic carbon (DOC), or the extent of respiration during its

biodegradation (i.e. net 02-uptake or C02-evolutlon expressed as a

percentage of the theoretical maximum for complete blodegradation). In

contrast, the CEC test measures blodegradation as the loss of infra-red

(I-R) absorbance in the range 2 920-2 940 cm "I in a l,l,2-trlchloro-

trlfluoroethane extract [I]. Absorbance in this region of the I-R spectrum

is due to C-H stretch in -CH 2- groups and compounds in the extract

containing these groups will be detected.

It could ​be ​argued that for oil products extensive loss of this absorption
 band is indicative of "ultimate" biodegradation and some authors have used

CEC test results to label oils as "readily biodegradable" according to the

OECD definition [8]. In addition, the German "Blue Angel" environmental

labelling scheme [2] allows the use of either CEC test or OECD "ready

biodegradability" test data. Recently, positive empirical [9] and

statistical [I0] correlations have been reported between blodegradatlon in

the CEC test and "ready biodegradability" test scores. In this paper we

describe studies on the CEC extraction procedure that help to explain why

for many oils there is a good correlation between biodegradation in the two

types of test and why sometimes this relationship does not hold.

MATERIALS AND METHODS

Chemicals

CEC reference oils dl-lsotridecyl adipate (DITA, RL 130) and white oll

Enerpar M 2632 (RL Ii0) were gifts from Unlchema Chemic, The Netherlands.

Octadecane, l-octadecanol, stearlc acid, eicosane, l-eicosanol, eicosanoic

acid, prlstane, methylmalonlc acid, succlnlc acid, adipic acid, tridecanoic

acid, l,lO-decanedlcarboxylic acid, hexadecanedioic acid and suberic acid

were purchased from Aldrich Chemical Company and had purities of 96-99%.

Oleic acid, llnoleic acid, linolenic acid, n-caproic acid, capric acid,

caprylic acid, ll-cis-eicosenolc acid and glutaraldehyde were obtained from

Sigma Chemical Company and were 95-100% pure. Eruclc acid and 3-trans-

hexenoic acid were purchased from Pfaltz and Bauer (90% and 97% pure,
789

respectively), and 2-trans-octenoic acid (90-95%) from Janssen Chimica.

Common and systematic names of the above compounds can be found in Table i.

Uranyl acetate, sodium citrate and lead nitrate (used to prepare lead

citrate), l,l,2-trichlorotrifluoroethane (99.9% pure) and sodium acetate

were obtained from Merck Limited, and carbon tetrachloride (I-R

spectroscopic grade) from Fluka Chemika-BioChemika. TAAB epoxy resin was

purchased from TAAB Laboratories and osmium tetroxide from Agar

Laboratories. iso-Trldecanol (ex. butylene trimer and propylene tetramer)

and rapeseed oil were supplied by Shell Group companies.

Extraction efficiency of the CEC procedure

Replicate 500 ml flasks containing 150 ml uninoculated medium were prepared as described in [I]. Substances soluble in
l,l,2-trichlorotrifluoroethane
were added to duplicate flasks from stock solutions (1.5 g/10 ml solvent) to

give a final concentration of 50 mg/l. Eicosanoic acid, l-eicosanol, suberic

acid and l-octadecanol were added from saturated solutions in the solvent.

Reference solutions of each compound, to determine extraction efficiency,

were also prepared by adding 50 ~i stock solution to 25 ml extraction

solvent. Dicarboxylic acids (insoluble in l,l,2-trichlorotrifluoroethane or

carbon tetrachloride) were added directly to the test medium (50 mg/l).

Duplicate flasks containing uninoeulated medium only (blanks) were also set

up.

After mixing, each flask was extracted with l,l,2-trichlorotrifluoroethane

(carbon tetrachloride used for adipie acid, methylmalonic acid and succinic

acid) as described in [I]. The maximum I-R absorption at a wavenumber of

2 930 ± i0 cm "I in each extract was measured using a Perkin-Elmer 881 Infra-

Red Spectrophotometer. Extraction efficiency was calculated as: (mean

absorption test flasks - mean absorption blanks)/absorption reference

solution x 100%.

Effect of the CEC extraction procedure on bacterial ​cells

Replicate flasks containing inoculated mineral salts medium, and inoculated

medium amended with either 50 mg/l DITA (± HgCI2) or 2% w/v sodium acetate

were prepared as described in [i]. The inoculum was coarse-filtered final

effluent from Sittingbourne Sewage Treatment Works (STW) and

contained ~ 106 colony-forming units/ml (Oxoid TTC dip slide). Initially

and after incubation at 25 °C for seven days, duplicate flasks were

extracted with carbon tetrachloride and the I-R absorbance of the extracts

(blanks and DITA) determined as described above. Samples of the aqueous

phase after extraction, and the aqueous phase of unextracted flasks were
790

Table ​I. Oils and compounds studied

Name Carbon no.(a)

3-trans-Hexenolc acid 6:1 Methylmalonlc acid 4:0, Succinic acid (butanedioic acid)
(13c)
Rapeseed oli (b) Enerpar M 2632 (CEC RL II0) (c) DITA (di-isotridecyl adlpate, CEC RL 130) 32 Erucic
acid (docosenolc acid) 22:1 Eicosane 20 l-Eicosanol 20 Eicosanoic acid 20:0 ll-cis-Eicosenoic acid 20:1
Pristane (2,6,10,14-tetramethylpentadecane) 19 Octadecane 18 Stearic acid (octadecanoic acid) 18:0
(llc)
l-Octadecanol 18 Oleic acid (9-cis-octadecenoic acid) 18:1 Linoleic acid (9,12-cis-octadecadienoic acid)
18:2 Linolenic (9,12,15-cis-octadecatrlenoic acid) 18:3 Hexadecanedloic acid 16:0, iso-Trldecanol (ex.
but. trlmer and prop. tet.) 13 Trldecanole acid 13:0 l,lO-Decanedicarboxylic acid I0:0, Caprlc acid
(decanoic acid) i0:0 Capryllc acid (octanolc acid) 8:0 Suberlc acid (octanediolc acid) 8:0,
2-trans-Octenolc acid 8:1 Caproic acid (hexanoic acid) 6:0 Adiplc acid (hexanedioic acid) 6:0,
(9c) (9c,12c) (9c,12c,15c)
 dlcarboxyllc acid
dlcarboxylic acid (3t)
dicarboxyllc acid dicarboxylic acid
dicarboxylic acid

dicarboxylic acid (2t)

Carbon number values for fatty acids are in the form - number of carbon atoms in acid : number of unsaturated centres (position of
unsaturated centre and whether ci___~s or trans) and are taken from [ii].

A vegetable oll wldely-used in the oleochemical industry which typically contains 97% w/w triglycerides of olelc (61%), linoleic
(22%), llnolenic (10%), elcosenoic (1%), and other fatty acids. Oils from earlier varieties of Brassica napus or B. camDestris
contained high levels of eruclc acid (up to 50%) and less oleic acid [12].

A lubricating "white oil" produced by the drastic refining of crude oil, solvent extraction and treatment with sulphurlc acid and alkali.

taken for electron microscopy (see below).

Electron microscopy

Samples of the aqueous phase were centrifuged at 23 000 x g for i0 minutes.

The cell pellets were fixed in 2.5% w/v glutaraldehyde in 0.1 M phosphate

buffer (pH 7.2) for three hours, washed twice in phosphate buffer

(15 minutes each) and then left overnight in fresh buffer at 4 °C. Samples

were post-flxed in 2% w/v osmium tetroxide in phosphate buffer for two

hours, washed twice in the same buffer, dehydrated through a graded series
791

of alcohols to propylene oxide, and then infiltrated with and embedded in

TAAB epoxy resin. Ultrathln (60 nm) sections were cut on a LKB ultratome

III, stained with uranyl acetate and lead citrate [13], prior to examination

in a JEOL 100 CX electron microscope at 60 kV.

Modified Sturm "ready biodegradability" test

The "ultimate" biodegradabilities (mlneralisation to CO2) of DITA, adipic

acid and iso-tridecanol (produced from either butylene trimer or propylene

tetramer) in the modified Sturm test were determined according to [7].

Duplicate units containing 3 1 medium inoculated with i % v/v coarse-

filtered supernatant of homogenised activated sludge (Canterbury STW) were

used. The test substance concentration was 20 mg/l. DITA and the is_.__qo-

tridecanols had a low water solubility and were added as emulsions as

described in [I0].
 RESULTS AND DISCUSSION

The CEC ​test - a​ n assessment ​of "primary" or "ultimate" biodegradability ?

The CEC test measures biodegradatlon as the loss of I-R absorbance

(C-H stretch in -CH 2- groups) in a l,l,2-trichlorotrifluoroethane extract.

Extraction of organic compounds is aided by ultrasonication, acidification

and salting-out of the spent medium. If unmodified oil accumulated by micro-

organisms or persistent biodegradation intermediates fail to extract into

the solvent, then this will be measured as "biodegradatlon". Alternatively,

if compounds extract into the solvent but have few methylene groups, then

the low I-R absorbance of the ​extract w

​ ill result in a high "biodegradation"

score being calculated. These are some of the potential problems with the

CEC ​test ​that could lead to biodegradability being ​overestimated a​ nd which

distinguish it from "ready biodegradability" tests which measure ​"ultimate"

biodegradation as a loss of dissolved organic carbon or an increase in net

respiration. The aim of this study was to determine whether the CEC

procedure measures simply the extent of parent material removal or more

extensive biodegradation.

There are several reports of micro-organisms accumulating unmodified oil in

Intracytoplasmic inclusions [see 14 for review]. In this study, wastewater

treatment bacteria incubated for seven days with DITA (31% biodegradation)

contained membrane-bound inclusions in the cytoplasm (Figures IA-C).

However, these may not have contained the ester as similar inclusions were

observed in cells grown on acetate (not shown). It can be seen from Figures

ID and IE that the CEC extraction procedure disrupted the cell wall, cell

membrane and the inclusion bodies, and any accumulated, unmodified oil would

have been released into the medium and extracted.

792
 Figure 1. Electron mtcrographs of bacteria incubated for seven days with di-isotridecyl adipate (DITA) before (A-C) and after (D-E) the CEC
extraction procedure. (IN - inclusion; CW - cell wall; CM - cytoplasmic membrane; ​LM - ​limiting membrane of inclusion; DB - dense body).
793

Initial microbial attack on vegetable oils (predominantly fatty acid

triglycerides) is an enzyme-catalysed hydrolytic cleavage to glycerol and

fatty acids (Figure 2). Glycerol is rapidly metabolised by phosphorylation,

conversion to the central metabolite glyceraldehyde-3-phosphate and

glycolysis. Liberated fatty acids (saturated and unsaturated) are

metabolised by the ~-oxidation cycle in which acetate units are sequentially

removed from the monocarboxylic acid and fed into central metabolic pathways

[15].

This study used rapeseed oil as a model vegetable oil as it is the base

fluid for many environmentally-compatible oil products [8,16,17]. Extraction

efficiencies for its main fatty acids and their sequential degradation

products are shown in Figure 3 (some of the compounds tested were not

intermediates of ~-oxidation but the closest commercially available). The

carbon number cut-off for the CEC extraction procedure appeared to be ~C8,

with C6 saturated and unsaturated fatty acids being poorly extracted. Such

short-chain, linear monocarboxylic acids are known to undergo rapid

"ultimate" biodegradation [18] and would be unlikely to accumulate in the

test medium.

Mineral oils used as lubricants consist primarily of iso-paraffins and

alicyclic structures containing one or more rings [19]. Relatively little is

known about the routes of oxidation for alicyclic compounds, although

pathways for cyclohexane biodegradation have been described [20]. Most of

the research on the biochemistry of hydrocarbon degradation has

concentrated on n-paraffins where initial microbial attack by monooxygenase

systems results in the conversion of the n-alkane to the corresponding

alcohol (Figure 2). Further oxidation is via the aldehyde to the

carboxylic acid which is then metabolised by ~-oxidation. Less is

known about the metabolism of iso-paraffins, although ~-oxidation can

proceed if there is a free (i.e. unsubstituted) ~-carbon available [20, 21].

In this study we used octadecane, eicosane and pristane as model

linear and branched paraffins, respectively. It can seen from Figure 3 that

n-paraffins, and most of the resulting long-chain fatty acids and alcohols,

were recovered from the aqueous phase by >90%. In many cases the extraction

efficiency was >100% due to solvent loss during extraction giving a

concomitant increase in concentration compared with the reference

solutions. However, in general upper extraction efficiencies did not exceed

110%. The high figures for l-eicosanol and l-octadecanol probably reflect
 errors in dosing that may have arisen due to the use of saturated solutions.

None of the branched, long-chain fatty acids formed during pristane

794
~ 0 I​ I ​" S-CoA

OH 0 ​
II
n-Paraffins I

~ o o ​II II
~ 0 2 + NADH + H +
S-CoA ​+ o ​II

0​ ​ acid triglycerides
I Fatty
~°​ ~ ,​ ~,
OH
+​f​ OH ​
OH ​
OH

o o​ ~ ​~
Enzymic I hydrolysis

0​
II

0 ​II
​ ​
​/ OH --I~ ~-oxidation ~'~

Th~.~ + A~ + CoASH

Figure 2. Biodegradatlon pathways for B-parafflns and vegetable oils.

795
biodegradation were available commercially but were assumed to be

extractable based on the data shown in Figure 3. Methylmalonic acid,

followed by succinic acid, are formed towards the end of pristane oxidation

pathway [21] and were not extracted. However, succinate is rapidly

metabolised by most bacteria via the tricarboxyllc acid cycle [15].

It can be seen from the above that for oils composed of vegetable esters, or

those containing a high proportion of n-paraffins or regularly branched is___oo-

paraffins, blodegradation intermediates which are not extracted into the

solvent phase in the CEC test are likely to be short-chain monocarboxyllc

acids which are readily metabolised by most bacteria. In other words, a high

biodegradation score in the CEC test for such products should be ​concomitant

with extensive "ultimate" biodegradation. This is consistent with the

statistical relationship between CEC scores and mineralisation to CO 2

reported in an earlier paper [I0].

Di-isotridecyl adipate ​(DITA) - a high ​CEC test score ​but ​low "ultimate"

biodegradability

An outlier from the published correlation between the CEC and modified Sturm

tests was the CEC reference oil DITA (dl-isotridecyl adipate, CEC RL 130),

which had a CEC score of 93% but a low biodegradability in the Sturm of only

31% ThCO 2 [10]. DITA is a dicarboxylic ester ("di-ester") produced from

adipic acid and iso-tridecanol. The C13 alcohol is usually based on

tetrapropylene or tributylene feedstocks [22] and is produced by the

catalytic addition of carbon monoxide and hydrogen to the olefin double bond

in the "oxo" process [20].

As with vegetable esters, initial microbial attack on DITA is likely to be

hydrolysis to yield the dicarboxylic acid and alcohol. It can be seen from

Figure 4 that in the modified Sturm "ready biodegradability" test DITA and

the two iso-tridecanols were biodegraded slowly over the 28 day incubation

period, with DITA being mineralised by 32% and the two iso-tridecanols by

28%. In contrast, adipic acid was rapidly and extensively (72%) mineralised.

C13 alcohols produced by the "oxonation" of tetrapropylene or tributylene

are a mixture of alcohols, less than 5% of which are linear, with complex

branching of the alkyl chain and the presence of ​quaternary-substituted

carbon atoms [20]. This tends to block the E-oxidation pathway and the low
 biodegradability of surfactants based on these alcohols is well known [20].

If a modified Sturm score for DITA is calculated as a weighted average based

on the scores for adipic acid and is__~o-tridecanol shown in Figure 4, and

their relative contribution to the carbon content of the ester, then the

value of 36% ThCO 2 is close to that measured. The poor "ultimate"

796
Rapeseed ​oil
Erudc ac~l
11 -c-Eicosenoic acid
Oletc acid
Capdc acid
Caprylic acid
Caproic acid
Unoleic add
2-t-Octenoic acid
Unolenic acid
3-t-Hexenoic acid
DITA
is o-Tddecanol (BT)
iso-Tridecanol tl~T)
Tridecanoic acid
Adipic acid
Enerpar M 2632
Eicosane
1 -Eicosanol
Eicosanoic acid
Octadecane
1 -Octadecanol
CO

J​
~i~i/ii!~iii!i!ii i/iiil/~i!i!~!!~iiiiiiiiiiiii ii/i~i~i~iiii~i/ii/i~ iiiiiiiiii~i/ii//i//!/iiii~ii~iiiii!iiiiiiii~ii ​ m ​Hexadecanedioic 1,10-Decanedic
Methylmalonlc Suodnlc Subedc Stearic Pdstane
acid
acid add
acid
add
acid

il ​ Dicarbo~lic acids ​
I

iII
I
I
20 40 60 80
100 120 ​Extraction efficiency (%)
Figure 3. Recovery of esters, a mineral oil, paraffins and putative biodegradation intermediates from aqueous test media using the CEC extraction
procedure.
797

10(1
 .... iso-Tridecanol iso:rridecanol ​DITA ​Adtptc aaa ​~--utylene b

.................. ,

..... 2 ................................ ; ​1
0​
i​ E

0 7 14 ~ 28
~me(days)

Figure 4. Blodegradation curves for the mineralisatlon of dl-isotrldecyl

adipate (DITA) and its hydrolysis products in the OECD modified

Sturm ​test.

biodegradability of DITA therefore appears to be due to its branched alcohol

component.

Figure 3 shows that is___oo-tridecanol would be extracted from the aqueous phase

in the CEC test. Therefore, the low I-R absorbance found with DITA in the

CEC test must be as a consequence of the conversion of the alcohol to non-

extractable metabolites. The likely candidates are highly-branched

dicarboxylic acids. These would be formed as follows: oxidation of the

alcohol produces the corresponding C13 monocarboxylic acid (see Figure 2).

Beta-oxidation would proceed, if at all, only to a limited extent because of

branch points. Such "blocked" acids would also be subject to ~-oxidation

resulting in the formation of ~, ~-dicarboxylic acids [20, 21]. The results

in Figure 3 show that dicarboxylic acids up to C16 are not extracted from

the aqueous phase by the CEC procedure and would therefore not contribute to

the I-R absorbance, resulting in a high apparent biodegradation score.

However, such branched-chain acids are known to be resistant to further

microbial attack [18] which would account for the low score in the modified

Sturm CO2-evolution test. These difficulties in the oxidation of DITA may

be the reason why, in this laboratory, the di-ester is removed by only

20-25% after seven days incubation in the CEC test compared with >80% for

n-paraffins, trlglycerides and rapeseed oil-based products.

798 ​The lack of agreement between the CEC and "ultimate" biodegradability tests

can be expected to be a problem with all highly-branched chemicals. This

applies not only to branched-chaln esters but also to branched and cyclic

paraffins. The above discussion serves to highlight the danger in using a

CEC test result as a surrogate measure of "ultimate" biodegradability - even

if for many oils there is a good positive correlation between the two.

CONCLUSIONS

l) ​Partial blodegradation of certain oils may give rise to non-extractable

metabolltes which are poorly degradable and in these cases the CEC ​test

gives an ​overestimation ​of ​biodegradability.

2) ​For this reason the CEC test cannot be considered to be test of "ready
biodegradability" as defined by the OECD and EC [6, 7], and data on the

"ultimate" biodegradability of oll products should be generated using

the appropriate OECD/EC methods.

3) ​In our opinion, the CEC test should be looked upon as a method which
measures the "primary" biodegradation of an oil; "primary"

biodegradatlon being defined as "the alteration in the chemical

structure of a substance, brought about by biological action, resulting

in the loss of a specific property of that substance" [6, 7].

4) ​A suitable analogy for the use of CEC test data is the area of
surfactant biodegradability where the standard tests measure "primary"

biodegradation [23], legislation requires ~80% "primary" blodegradatlon

[24, 25] and where extensive "primary" biodegradation of many products

is accompanied by substantial "ultimate" blodegradatlon [20].

ACKNOWLEDGEMENT

The authors would llke to thank Dr Rob Lyne for helpful comments.

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799

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[3] Environmental Labellin~ in the Nordic Countries. Position Report,

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[4] ICOMIA 27-92: Lubricatin~ Oil for 2-Stroke Cycle Outboard Engines

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