Applied Catalysis B: Environmental 69 (2007) 207–212

www.elsevier.com/locate/apcatb

Green approach for the preparation of biodegradable lubricant

base stock from epoxidized vegetable oil
Piyush S. Lathi *, Bo Mattiasson
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University,
P.O. Box 124, SE 221 00 Lund, Sweden
Received 10 April 2006; received in revised form 16 June 2006; accepted 20 June 2006
Available online 4 August 2006

Abstract
A novel process for the production of biodegradable lubricant-based stocks from epoxidized vegetable oil with a lower pour point via cationic
ion-exchange resins as catalysts was developed. This involves two steps, first, ring-opening reactions by alcoholysis followed by esterification of
the resultant hydroxy group in the first step.
The ring-opening reaction of epoxidized soybean oil with different alcohols such as n-butanol, iso-amyl alcohol and 2-ethylhexanol was carried
out in presence of Amberlyst 15 (Dry) as a catalyst; identity of products was confirmed by IR and NMR. Pour points of the products were observed
in the range of 5 to 15 8C. The hydroxy group of ring-opening product of n-butanol was further reacted with acetic anhydride in presence of
catalyst Amberlyst 15 (Dry), which was previously used to carry out ring-opening reaction by alcoholysis and identity of the resulting product was
confirmed by IR. Pour point of the resulting product was observed to be 5 8C.
# 2006 Elsevier B.V. All rights reserved.

Keywords: Bio-lubricants; Epoxidized soybean oil; Cationic ion-exchange resins

1. Introduction and nitrogen compounds with traces of a number of metals.

Renewable raw materials are going to play a very noteworthy
role in the development of sustainable green chemistry. They
offer a large number of possibilities for applications which can be
rarely met by petrochemistry [1]. Oils and fats of vegetable and
animal origin share the greatest proportion of the current
consumption of renewable raw materials in chemical industry
[2].
A steady increase in the use of eco-friendly consumer
products like lubricants has occurred as a result of strict
government regulation and increased public awareness for a
pollution free environment [3]. There are wide ranges of
lubricant base oils in current use which includes mineral oils,
synthetic oils, re-refined oils, and vegetable oils. Among these,
mineral oils are the most commonly used. They consist
predominantly of hydrocarbons but also contain some sulfur
* Corresponding author. Tel.: +46 46 222 73 63; fax: +46 46 222 47 13.
E-mail addresses: Piyush.Lathi@biotek.lu.se, lathips@yahoo.com,
lathips@gmail.com (P.S. Lathi).
0926-3373/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.apcatb.2006.06.016
Due to their inherent toxicity and non-biodegradable nature
they pose a constant threat to ecology and vast ground water
reserves [4]. Synthetic oils include others polyalphaolefins,
synthetic esters and polyalkylene glycols. Polyalphaolefins are
petrochemical derived synthetic oils that mostly resemble
mineral oils. Synthetic esters form a large group of products,
which can be either from petrochemical or oleochemical
origin. In the preparation of re-refined oil, oil undergoes an
extensive re-refining process to remove contaminants to
produce fresh base oil.
These concerns have resulted in an increasing interest in
vegetable oils with high content of oleic acid, which are
considered to be potential substitutes to conventional mineral
oil-based products [5,6]. Vegetable oil lubricants are preferred
not only because they are renewable raw materials but also
because they are biodegradable and non-toxic [7]. They also
acquire most of the properties required for lubricants such as
high index viscosity, low volatility and good lubricity and are
also good solvents for fluid additives. However, vegetable oils
have poor oxidative and thermal stability, which is due to the
presence of unsaturation [8]. This unsaturation restricts their
208 P.S. Lathi, B. Mattiasson / Applied Catalysis B: Environmental 69 (2007) 207–212
use as a good lubricant. Several attempts have been made to
improve their oxidative stability such as transesterification of
trimethylopropane and rapeseed oil methyl ester [9]; selective
hydrogenation of polyunsaturated C C bonds of fatty acid
chains [10] and conversion of C C bonds to oxirane ring via
epoxidation [11,12]. Among these, epoxidation received
special attention because it opened up a wide range of feasible
reactions that can be carried out under moderate reaction
conditions due to the high reactivity of the oxirane ring [13].
For instance, the epoxide can react with different nucleophiles
to produce mono-alcohols, diols, alkoxyalcohols, hydroxye-
sters, N-hydroxyalkylamides, mercaptoalcohols, aminoalco-
hols, hydroxynitriles, etc. [1,14].
Standard industrial production for epoxidation of vegetable
oil is based on in situ epoxidation, in which peracid is generated
by reacting acetic or formic acid with hydrogen peroxide in the
presence of strong mineral acids such as H2SO4 and H3PO4
[15]. The strong mineral acid leads to many side reactions, such
as oxiran-ring opening to diols, hydroxyesters, estolides and
other dimers formation. Furthermore, they cause equipment
corrosion and must be neutralized and also removed from the
end product.
Warwel and Klaas reported an alternative process that is
milder and more selective, wherein lipase is used to catalyze
the peracid formation from a fatty acid and hydrogen peroxide
[16]. This is a new and a very promising technique for
epoxidation of double bonds. It has several advantages over
the chemical catalysts such as: (i) mild reaction conditions,
(ii) formation of stable hydroperoxides directly from fatty
acid, i.e. no need for acetic or formic acid addition, (iii) high
region and stereoselectivity, (iv) significant suppression of
side reactions and (v) high conversion. In most of the recent
publications, volatile organic solvents were used as the
reaction media [17,18]. However recently Orellana-Coca
et al. from our department has developed the solvent free
chemo enzymatic process for the epoxidation of fatty acid
[19].
It was reported earlier that epoxidized unsaturated fatty
acids can be used as metal working fluids and lubricating
additives to eliminate corrosion from chlorine containing
compounds [20,21]. Adhvaryu and Erhan reported epoxidized
soybean oil as a potential source for high temperature
lubricants applications [12]. Various procedures have been
described for the preparation of alkoxyalcohols and hydro-
xylated fatty acids. Gast et al. reported that the product
obtained by ring-opening reaction of epoxidized fatty acid
esters followed by esterification of the resulting hydroxyl
group shows good performance for lower temperature
lubricant applications [22].
In most of these processes, sulfuric acid was used as a
catalyst for alcoholysis, whereas the other homogenous
catalysts such as p-toluenesulfonic acid, boron trifluoride,
and sodium methoxide were used for comparative studies. The
resulting hydroxy group via alcoholysis is further reacted with
acid anhydride in presences of pyridine [23–25]. In some
cases, the first step of the ring opening was performed by
addition of acetic acid, perchloric acid or formic acid, and in
the second step, i.e. esterification of the resulting hydroxyl
group with acid anhydride was done via pyridine [4,26,27].
Though these products come with the tag of green and
biodegradability, the reported ways are not eco-friendly and
green. Maybe therefore, these processes did not gain industrial
importance.
There were few reports in which cationic ion-exchange
resins such as Dowex 50W-X8, Duolite C 26, and Lewatit S 100
were used as an acylative ring cleavage catalyst for epoxidized
soybean oil [28]. These organic catalysts showed poor cleavage
efficiencies as compared to sulfuric acid. It may be due to the
microporous gel type nature of Dowex 50W-X8 and Lewatit S
100. Since they have no discrete pores, solute ions have to
diffuse through the particle to interact with the exchange site
which may cause the diffusional limitation and reduce the
overall efficiency.
The use of homogenous catalysts is very common in the
chemical and refinery industries, and those technologies
employ highly corrosive, hazardous and polluting liquid acids.
The present work deals with the preparation of lubricant-based
stock by using cationic ion-exchange resins, particularly, the
macroporous variety. It is very well documented that resins are
very versatile catalysts and are known for carrying out number
of reactions such as esterification, etherification, transalkyla-
tions, hydration and alkylation [29]. They also offer distinct
advantages over homogenous catalysts with respect to
corrosion, product recovery, selectivity, etc., both from the
standpoint of catalysis as well as engineering of reactions for
commercial purposes [30]. Hence, it is worthwhile to develop a
green and environmental friendly process based on macro-
porous resins with an emphasis on biodegradability of the
lubricant. As previously mentioned, the preparation of
lubricant-based stock from epoxidized vegetable oil involves
two steps, ring-opening reaction via alcoholysis followed by
esterification of the resulting hydroxy group through alcoho-
lysis. Novelty of the current work is that the same catalysts
without any treatment can be used for both the reactions, i.e.
ring-opening reaction via alcoholysis and also for esterification
of the resulting hydroxyl group.
2. Materials and methods
2.1. Materials
The epoxidized soybean oil was a gift sample from Akzo
Nobel Industrial Coatings Sweden, and was used as received.
Unless otherwise specified, all other chemicals including
alcohols, Amberlyst 15 (Dry) (% water content 1.5 m3/m3 of
resin, ionic form H+) and acetic anhydride were purchased from
Sigma and used without further purification.
2.2. Analytical methods
The oxirane value was determined titrimetrically with
perchloric acid [31]. The infrared (IR) spectra were recorded
on a Fourier transform IR 8300 from Shimadzu. All the
spectra’s were recorded by spreading the samples between
 P.S. Lathi, B. Mattiasson / Applied Catalysis B: Environmental 69 (2007) 207–212 209

NaCl plates. The spectra were recorded in transmittance mode.

The 1H and 13C NMR spectra were recorded qualitatively using
a Bruker ARX-400 spectrometer (Bruker, Rheinstetten,
Germany) at a frequency of 400 and 100 MHz, respectively,
using a 5 mm dual probe. For 1H and 13C experiments, sample
solutions were prepared in deuterated chloroform (CDCl3
99.8% D, Aldrich Chemical Company). Proton NMR spectra
were obtained from 16 co-added FIDs with a delay time of 1 s
and 13C experiment was carried out with NS = 5000, AQ
0.62 s, DW 019 ms, D1 = 0.1 s. Pour point measurement was
done using a Freezer Hetofrig CB 8 (ISO 3106). Kinematic
viscosity measurement was done at 40 8C by using a Schott-
Geräte CT 42 (ISO 3104) and dynamic viscosity measurement
was done at 28 and 40 8C by using a Bohlin Visco 88
Viscometer.

3. Experimental set-up

3.1. Ring-opening reaction

The experimental set-up consisted of a 3 cm i.d. fully baffled

mechanically agitated reactor of 150 cm3 capacity, with a four-
bladed pitched-turbine impeller. The entire reactor assembly
was immersed in a thermostatic oil bath maintained at a desired
temperature with an accuracy of 1 8C.
In a typical experiment, the reaction mixture consisted of
epoxidized soybean oil 1 (60 g, 0.285 mol of epoxy group) and
an alcohol. The reaction mixture was agitated at 70 8C for
15 min at a speed of 1000 rpm and 1.5% of (w/w of epoxidized
soybean oil) catalyst Amberlyst 15 (Dry) was added to initiate
the reaction to obtain the products 2a, 2b and 2c. Table 1
summarizes the reaction conditions. Clear samples were
withdrawn periodically and titrated to measure the concentra-
tion of epoxide.

3.2. Esterification of resulting hydroxyl group in the ring
opened product
The same experimental set-up mentioned in Section 3.1 was
used. The reaction mixture consisted of 50 g of product
obtained by ring-opening reaction of epoxidized soybean oil
with n-butanol (2a) and 50 mL of acetic anhydride. The
reaction mixture was agitated at 90 8C for 15 min at a speed of
1000 rpm and the catalyst which was used in the previous
reaction was added without any further treatment to initiate the
reaction to obtain product 3.
4. Results and discussion
4.1. Effect of alcohols on pour point and viscosity
For investigation of alkyl group effect on the low-temperature
properties, three different alcohols, namely n-butanol, iso-amyl
alcohol and 2-ethylhexanol were chosen [32,33]. The main
purpose for choosing the above three alcohols is that n-butanol is
a short straight chain alcohol, iso-amyl alcohol is a short branch
chain alcohol and 2-ethylhexanol is a medium branch chain
alcohol. Pour point values for the ring-opening product of the

Table 1
Reaction conditions
Product entry Alcohol Temperature (8C) Mole ratio Catalysts loading (%) Reaction time (h)
2ai n-Butyl alcohol 100 1:2 2 15
2aii n-Butyl alcohol 100 1:3 2 15
2b iso-Amyl alcohol 110 1:2 2 17
2c 2-Ethyl hexanol 120 1:2 2 24
3 Esterification of resulting hydroxyl 90 1:2 2 15
group in the ring opened product 2ai
210 P.S. Lathi, B. Mattiasson / Applied Catalysis B: Environmental 69 (2007) 207–212
Fig. 1. Comparison of the 1H NMR spectra of the epoxidized vegetable oil (1),
product of the ring-opening reaction of epoxidized vegetable oil with n-butanol
(2ai) and product of the esterification reaction of resulting hydroxyl group in the
product 2ai with acetic anhydride (3).
Fig. 2. Comparison of the 13C NMR spectra of the epoxidized vegetable oil (1), product of the ring-opening reaction of epoxidized vegetable oil with n-butanol (2ai)
and product of the esterification reaction of resulting hydroxyl group in the product 2ai with acetic anhydride (3).
respective alcohols were in the range 0 to 5, 5 to 10 and 10
to 15 8C. Thus, it was observed that as the chain length of
branched alcohol increased, it gives lower pour point. 2-
ethylhexanol, a branched medium chain alcohol, gives low pour
point values as compared to other alcohols.
The ring-opening reaction was monitored by epoxy titration
and stopped when the ring-opening reaction was 100%. The
structure of the product was confirmed from infrared spectra by
the disappearance of epoxy group at 822 cm 1 and appearance
of the hydroxy peak at 3450 cm 1. The reaction completion
was further confirmed by 1H NMR spectra. As summarized in
Table 1, different alcohols required different conditions to
complete the reaction. Longer or bulkier alcohols required
longer reaction time and higher temperature.
The retention of the triacylglycerol backbone is important
for maintaining the biodegradability of the vegetable oil and
this was confirmed by 1H NMR spectra given in Fig. 1. 1H
NMR measurement on epoxidized vegetable 1 indicates that
the epoxy group is present in the d 3.0–3.2 ppm region. The
methine proton of –CH2–CH–CH2 backbone at d 5.1–
5.3 ppm, methylene proton of –CH2–CH–CH2– glycerol’s
backbone at d 4.0–4.4 ppm, CH2 proton adjacent to two epoxy
group at d 2.8–3.0 ppm, –CH– proton of the epoxy ring at d
3.0–3.2 ppm, a-CH2 to C O at d 2.2–2.4 ppm, a-CH2 to
epoxy group at 1.7–1.9 ppm, b-CH2 to C O at d 1.55–
1.7 ppm, b-CH2 to epoxy group at d 1.4–1.55 ppm, saturated
methylene group at d 1.1–1.4 ppm and terminal –CH3 groups
at d 0.8–1.0 ppm region.
Compounds 2a and 3 retain most of the characteristic peaks
of compound 1 except the one at d 2.8–3.2 and 1.4–1.55 ppm
region, corresponding to hydrogen’s attached to the epoxy
groups and methylene groups adjacent to epoxy, respectively.
13
C NMR measurement on epoxidized vegetable 1 indicates
that the epoxy group is present in the d 56.9–58.5 ppm region.
 P.S. Lathi, B. Mattiasson / Applied Catalysis B: Environmental 69 (2007) 207–212 211

Fig. 3. Comparison of the infrared spectra of the epoxidized vegetable oil (1),
product of ring-opening reaction epoxidized vegetable oil with n-butanol (2a)
and product of esterification reaction of resulting hydroxyl group in the product
2a with acetic anhydride (3).

Compounds 2a and 3 retain most of the characteristic peaks of

compound 1 except the one at d 56.9–58.5 ppm region,
corresponding to carbon’s attached to the epoxy groups (Fig. 2).
This gives the confirmation that no epoxide group remains in
the products.
The resulting hydroxyl group in 2a was further reacted with Fig. 4. Dynamic viscosity study performed at 28 8C at different shear rates to
acetic anhydride to obtain the corresponding product 3. The observe its effect on the viscosity: (a) shear rate vs. viscosity and (b) shear rate
product was confirmed by IR spectra by the disappearance of vs. shear stress.
the hydroxyl peak at 3450 cm 1. IR spectra of 1, 2a and 3 are
given in Fig. 3. The reaction completion was further confirmed
by 1H NMR spectra by the presence of an additional peak at d
2.0 ppm for product 3.
Pour point becomes very important in cold weather
applications and in cylinder and packing lubricant with very
cold suction temperature. The pour points of 2a–2c and 3 were
measured by using a standard method mentioned in materials
and method and the results are shown in Table 2.
Viscosity is of paramount importance to a lubricant. The
kinematic viscosity of 2a–2c and 3 were measured and the
results are shown in Table 2. The product 2a was synthesized
with two different mole ratios of epoxidized oil to n-butanol
and it was found that the viscosity of 2aii is higher than that of
2ai. Product 2aii was prepared by using higher molar
concentration of n-butanol.
Dynamic viscosity of 2ai was measured at 28 and 40 8C at
different shear rates. Figs. 4b and 5b show that viscosity does
not change with an increasing shear rate, whereas the plots of
shear stress versus shear rate (Figs. 4a and 5a) show a straight

Table 2
Kinematic viscosity and pour point values
Serial no. Product Kinematic Pour point
entry viscosity (mm2/s) (8C)
1 2ai 232.7 > 5, <0
2 2aii 312.9 > 5, <0
3 2b 172.4 > 5, <0
4 2c 73.2 > 15, < 10 Fig. 5. Dynamic viscosity study performed at 40 8C at different shear rates to
5 3 350.8 > 5, <0 observe its effect on the viscosity: (a) shear rate vs. viscosity and (b) shear rate
vs. shear stress.
212 P.S. Lathi, B. Mattiasson / Applied Catalysis B: Environmental 69 (2007) 207–212
line passing through the origin, thereby indicating Newtonian
type fluid.
4.2. Biodegradability study
A biodegradability and toxicity study for the lubricants was
performed. It was observed that lubricants are non-toxic and
biodegradable. In toxicity studies following tests such as
toxicity screening, phytotoxicity [34] and algal toxicity [35]
were executed. In each case no-observed-effect-concentration
(NOEC); the highest concentration without adverse effects was
2000, 1000 and 1000 mg/L, respectively. In biodegradability
study, values for biochemical oxygen demand (BOD) and
chemical oxygen demand (COD) were measured. It has been
documented that if the ratio of BOD5/COD is around 0.5 and
higher it means compounds are easily biodegradable [36,37].
This value for the present prepared lubricant products is in the
range of 0.43–0.63, which shows that compounds are
biodegradable.
4.3. Reusability of catalyst
Reusability of Amberlyst 15 (Dry) was tested by
conducting four runs. After the reaction, the catalyst was
filtered and then refluxed with 50 mL of alcohol for 30 min to
remove any adsorbed material from the catalyst surface and
pores and dried at 110 8C after every use. It was observed that
there is only a marginal decrease in conversion. Some catalyst
was lost during filtration. There was no makeup catalyst added
during each reuse study. Thus, the catalyst was reusable up to
four times.
5. Conclusion
The current work has focused on the synthesis of bio-
lubricant from epoxidized vegetable oil and alcohol using solid
acid catalyst Amberlyst 15. This investigation showed that the
introducing branching on epoxidized soybean oils leads to
dramatic improvement of low pour point value. The ring-
opening reaction of epoxidized soybean oils with 2-ethylhex-
anol gives the low pour point value. It was observed that the
catalyst has excellent reusability and stability. Lubricants are
non-toxic and biodegradable.
Acknowledgements
The work is a part of the GREENCHEM program sponsored
by MISTRA (The Swedish Strategic Fund for Environmental
Research) and the authors thank the same for the financial
support. The authors are very grateful to Mrs. Birgit Vainio
(Binol BioSafe Oy Laboratory, Finland) for the measurement of
the viscosity and the pour point tests.
References
[1] H. Baumann, H. Buhler, H. Fochem, F. Hirsinger, H. Zobelein, J. Falde,
Angew. Chem. 27 (1988) 41–62.
[2] U. Biermann, W. Friedt, S. Lang, W. Luhs, G. Machmuller, J.O. Metzger,
M.R. Klaas, H.J. Schafer, M.P. Schneider, Angew. Chem. Int. Ed. 39
(2000) 2206–2224.
[3] I. Rhee, NLGI Spokesman 60 (1996) 28.
[4] A. Adhvaryu, Z. Liu, S.Z. Erhan, Indust. Crops Prod. 21 (2005) 113–119.
[5] S.J. Randles, M. Wright, Synthetic Lubric. 9 (1992) 145–161.
[6] S. Asadauakas, J.M. Perez, J.L. Duda, Lubric. Eng. 52 (1996) 877–882.
[7] N.S. Battersby, S.E. Pack, R.J. Watkinson, Chemosphere 24 (1992) 1998–
2000.
[8] R. Becker, A. Knorr, Lubric. Sci. 8 (1996) 95–117.
[9] E. Uosukainen, Y.Y. Linko, M. Lamasa, T. Tervakangas, P. Linko, J. Am.
Oil Chem. Soc. 75 (1998) 1557–1563.
[10] L.E. Johansson, S.T. Lundin, J. Am. Oil Chem. Soc. 56 (1979) 974–980.
[11] S. Sinadinovic-Fiser, M. Jankovic, Z.S. Petrovic, J. Am. Oil Chem. Soc. 78
(2001) 725–731.
[12] A. Adhvaryu, S.Z. Erhan, Indust. Crops Prod. 15 (2002) 247–254.
[13] A. Padwa, S.S. Murphree, ARKIVOC 3 (2006) 6–33.
[14] L.A. Rios, P.P. Weckes, H. Schuster, W.F. Hoelderich, Appl. Catal. A: Gen.
284 (2005) 155–161.
[15] Z.S. Petrovic, A. Zlatanic, C.C. Lava, S. Sindinovic-Fiser, Eur. J. Lipid
Sci. Technol. 104 (2002) 293–299.
[16] S. Warwel, M.R. Klaas, J. Mol. Catal. B: Enzyme 1 (1995) 29–35.
[17] G.D. Yadav, I.V. Borkar, AIChE J. 52 (2006) 1235–1247.
[18] G.D. Yadav, K. Manjula Devi, J. Am. Oil Chem. Soc. 78 (2001) 347–351.
[19] C. Orellana-Coca, U. Toernvall, D. Adlercreutz, B. Mattiasson, R. Hatti-
Kaul, Biocatal. Biotransform. 23 (2005) 431–437.
[20] S. Watanabe, T. Fujita, M. Sakamota, J. Am. Oil Chem. Soc. 65 (1988)
1311–1312.
[21] D. Tao, H.L. Zhu, Z.M. Hu, Lubric. Sci. 8 (1996) 397–407.
[22] L.E. Gast, C.B. Croston, W.J. Schneider, H.M. Teeter, Indust. Eng. Chem.
46 (1954) 2205–2208.
[23] H.M. Teeter, L.E. Gast, E.W. Bell, J.C. Cowan, Indust. Eng. Chem. 45
(1953) 1777–1779.
[24] H. Hwang, S.Z. Erhan, J. Am. Oil Chem. Soc. 78 (2001) 1179–1184.
[25] H. Hwang, A. Adhvaryu, S.Z. Erhan, J. Am. Oil Chem. Soc. 80 (2003)
811–815.
[26] B. Dahlke, S. Helbardt, M. Paetow, W.H. Zech, J. Am. Oil Chem. Soc. 72
(1995) 349–353.
[27] S.Z. Erhan, A. Adhvaryu, Z. Liu, US Patent 6,583,302 (2003).
[28] M.H. El-Mallah, F.A. Zaher, M.M. El-Hefnawy, Res. Indust. 34 (1989)
286–289.
[29] G.D. Yadav, P.H. Mehta, Indust. Eng. Chem. Res. 33 (1994) 2198–2208.
[30] M.M. Sharma, React. Funct. Polym. 26 (1995) 3–23.
[31] R. Dijkstra, E.A.M.F. Dahmen, Anal. Chim. Acta 31 (1964) 38–44.
[32] A. Özgulsun, F. Karaosmanoglu, M. Tuter, J. Am. Oil Chem. Soc. 77
(2000) 105–109.
[33] N. Dörmö, K. Belafi-Bako, L. Bartha, U. Ehrenstein, L. Gubicza, Bio-
chem. Eng. J. 21 (2004) 229–234.
[34] T. Essam, H. Zilouei, A. Amin Magdy, O. El Tayeb, B. Mattiasson, B.
Guieysse, Chemosphere 63 (2006) 277–284.
[35] Organization for Economic Cooperation and Development (OECD), Algal
Acute Toxicity Test. Test Guideline 203. OECD Guidelines for the Testing
of Chemicals, OECD, Paris, France, 1984.
[36] Organization for Economic Cooperation and Development (OECD),
Guidelines for Testing of Chemicals. Test 302 B: Zahn–Wellens/EMPA
Test 302 C: Modified MITI Test (II), OECD, Paris, France, 1993.
[37] G. Tchobanoglous, F.L. Burton, H.D. Stensel, Wastewater Engineering:
Treatment and Reuse, McGraw Hill, New York, 2003.