Volume 18 Number 16 21 August 2016 Pages 4315–4572
Green
Chemistry
Cutting-edge research for a greener sustainable future
www.rsc.org/greenchem
Themed issue: Molecular Design for Reduced Toxicity
ISSN 1463-9262
PAPER
Nicholas Gathergood, Klaus Kümmerer et al.
On the way to greener ionic liquids: identification of a fully mineralizable
phenylalanine-based ionic liquid
Green Chemistry
PAPER
Cite this: Green Chem., 2016, 18,
4361
On the way to greener ionic liquids: identification
of a fully mineralizable phenylalanine-based ionic
liquid†
Annette Haiß,a Andrew Jordan,b Janin Westphal,a Evgenia Logunova,a
Nicholas Gathergood*c and Klaus Kümmerer*a
Over the past few decades ionic liquids (ILs) are increasingly seen as an important building block of green
chemistry because of their specific properties as solvents, such as their potential for high recyclability, low
volatility, low flammability, low toxicity, and their potential for synthesis from renewable resources.
However, avoiding persistent or toxic cation/anion fragments is also urgently needed. In the best case
they should be fully mineralizable by microorganisms after their release into the aquatic environment. The
fragments fostering this can be determined by biodegradation studies, and the employment of identified
readily biodegradable building blocks presents an innovation in the targeted design of green environmentally friendly ILs. The aim of this study was to improve the data-platform for the design of completely
mineralizable ILs. Therefore the ready biodegradability of seven phenylalanine-based ILs and three nonionic related compounds was investigated with a modified Closed Bottle test based on OECD guideline
301D. Liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) analysis was
used to identify the chemical structures of products resulting from incomplete biodegradation and transformation. Two kinds of degradation pathways were observed: the hydrolysis of an ethyl ester group or
the hydrolysis of an amide bond and biodegradation of the released phenylalanine ethyl ester. Both
degradation pathways resulted in persistent transformation products (TPs) with the exception of IL (4), a
pyridinium substituted phenylalanine derived IL and the non-ionic deanol derivative (2a). IL (4) was ultimately biodegraded in the CBT after 42 days without leaving any TP. The biodegradation of compound
Received 12th February 2016,
Accepted 7th June 2016
(2a) was 78% after 42 days but resulted in a TP, which was readily biodegradable in a further CBT after a
DOI: 10.1039/c6gc00417b
lag phase of 3 weeks, respectively. Even if both compounds were not “readily biodegradable” in the sense
of the OECD guideline, particularly IL (4) can be proposed as a basic structure for sustainable and green
www.rsc.org/greenchem
ILs (benign by design) with the aim of optimizing its degradation rate further.
1.
Introduction
In the past two decades ionic liquids (ILs) have seen an intensive evolution in research and application. As solvents with a
large liquid range and the possibility of adjusting their properties for a variety of industrial processes they are attractive
for a wide range of applications, from materials science to
electrochemistry and from catalysis to medicinal chemistry.1,2
a
Institute of Sustainable and Environmental Chemistry, University of Lüneburg,
Scharnhorststraße 1, D-21335 Lüneburg, Germany.
E-mail: klaus.kuemmerer@uni.leuphana.de
b
School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland
c
Department of Chemistry, Chair of Green Chemistry, Tallinn University of
Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.
E-mail: nicholas.gathergood@ttu.ee
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6gc00417b
This journal is © The Royal Society of Chemistry 2016
ILs have according to their chemical nature as salts a very low
vapor pressure.3 They do not contribute to smog formation,
ozone depletion, and global climate change as do volatile
organic solvents,4 rendering them benign to the atmosphere.
However, new applications are anticipated for ILs that may
result in their introduction into the aquatic environment. The
effects of ILs on aquatic organisms and communities are
largely unknown. For several ILs eco-toxicological effects are
documented, e.g. imidazolium salts affected the growth of
freshwater algae (Selenastrum capricornutum), had, as well as
pyridinium-based ILs, significant antimicrobial activity to bacteria dependent on the alkyl-chain length,5 and reduced the
reproductive output of crustacea (Daphnia magna) similar to
phenol and ammonia.6 In view of the fact that ILs can be
easily synthesized in a huge variety it is not feasible to assess
toxicity and risk for all compounds in a timely and comprehensive manner. This makes it all the more important that ILs are
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Green Chemistry
Fig. 1 Chemical structure of the non-biodegradable 1-butyl-3-methylimidazolium bromide.8–10
fully mineralized in case of their introduction into the environment. Owing to their properties such as stability to a wide
range of chemicals as well as to high temperatures they are not
expected to be readily biodegradable.7 Indeed, second-generation ILs such as the 1-butyl-3-methylimidazolium class,
Fig. 1, are recalcitrant to biodegradation.8–10
Other ILs can only be classified as readily biodegradable
because of the biodegradable anions such as octyl sulphate
which increase biodegradation above the pass level, yet a nonbiodegradable cation can still remain, Fig. 2.9,11,12
Pyridinium ILs are, in general, biodegradable to a higher
extent than imidazolium ILs,10 particularly when bearing an
ester containing substituent at position 1 or 3.13 Several pyridinium cations are “readily biodegradable” in the sense of the
Fig. 2
OECD 301 guideline, e.g. 1-octyl-3-methylpyridinium (Fig. 3,
left).10 In general, “ready biodegradability” means that the biodegradation leads to a removal of dissolved organic carbon of
at least 70% or, in the case of respirometric methods, to an
oxygen consumption or CO2 production of 60% as a minimum
compared to the theoretical value for full mineralization.14
This is not to be confused with primary elimination, which is
also reported for several pyridinium ILs (Fig. 3, right).15
Thereby the term “primary elimination” refers only to the fact
that a decrease in compound concentration is stated without
giving data concerning the degree of biodegradation.
Nevertheless, analytical studies give important information
about microbial degradation pathways and the resulting transformation products (TPs). For pyridinium ILs, for example, it
was reported that depending on the microorganisms different
TPs were formed by p-hydroxylation of the pyridinium ring, a
cyclization reaction after oxidation of the alkyl chain or by
ester hydrolysis, some of which accumulated in the medium
with Rhodococcus rhodochrous.15 Furthermore, studies with the
pure strain R. rhodochrous underline that even one microorganism can transform the same compound in different ways
(Fig. 4).15
Readily biodegradable imidazolium octyl sulphate IL according to the OECD test guideline.9
Fig. 3 Readily biodegradable 1-octyl-3-methylpyridinium cation (left)10 and ethoxycarbonylmethylpyridinium cation (right) which was readily biodegradable with activated sludge13 but led to an accumulating transformation product (TP), the carboxymethylpyridinium cation, with a pure strain
of Rhodococcus rhodochrous.15
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This journal is © The Royal Society of Chemistry 2016
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Paper
Fig. 4 Transformation products (TP) resulting from the biodegradation of the 1-(3-hydroxypropyl)pyridinium cation by activated sludge and a pure
strain of Rhodococcus rhodochrous, accumulating TP framed. Modified illustration from ref. 15.
This demonstrates, notwithstanding the fact that single
specialists are not widespread and have a low environmental
abundance, the diversity of potential TPs which may be formed
by different microorganisms in the environment and which
could have an environmental profile i.e. stability, polarity, fate
and toxicity completely different from the original IL.15,16
Therefore, in the sense of a sustainable chemistry the property to be fulfilled by sustainable ILs is not only biodegradability but fast and full mineralization after they are introduced
into the environment. Measuring only biodegradation by standardized test methods regarding endpoints such as oxygen
consumption does not reveal any detailed information on the
remaining TPs or on chemical processes. Therefore biodegradation testing combined with detailed analysis would allow a
deeper insight into the course of degradation of ILs. The
identification of TPs resulting from incomplete degradation of
parent cations and anions would give information on structure
elements of ILs which are recalcitrant or can be mineralized by
microorganisms. This information is required for the “benign
by design” concept16,17 supporting the rational design of environmentally friendly chemicals possessing not only the desired
properties for application but also easy and fast degradation
after use.2,17–19
To put the benign by design concept into practice for
ILs, currently available knowledge can be used.12,18,19 ILs
which are structurally similar to biomolecules additionally
promise high biodegradability if enzymes of low substrate specificity are present in sufficient amounts.20 Furthermore, biodegradability screening of a series of L-phenylalanine derived ILs,
This journal is © The Royal Society of Chemistry 2016
which are the result of the design lessons learned from previous
generations of ILs, can be included in the study.21
The aim of the present study was to combine this knowledge to apply the benign by design approach to ILs. Therefore,
the biodegradability of several L-phenylalanine derived ILs and
tertiary amino analogues was investigated using the OECD
301D test to gain more knowledge of easily microbial biodegradable structures for the targeted design of rapidly and
completely biodegradable ILs of the future. For this purpose
high-resolution Orbitrap MSn was also employed.
2. Materials and methods
We investigated the biodegradation and formation of TPs of
seven L-phenylalanine ethyl ester derived ILs (IL (1)–IL (7)) of
four different types: prolinium, choline, imidazolium and pyridinium derivatives with bromide as the counter ion (for structures see Fig. 5). Additionally three structurally related nonionic compounds ((1a), (2a) and (3a)) were included to investigate the impact of the positive charge on the nitrogen for biodegradability. The synthesis of IL (1)–IL (7) and compounds
(1a)–(3a) is reported by Gathergood et al.22 in the accompanying paper as a part of this overall benign by design strategy.
2.1
Biodegradation testing
The ready biodegradability of the ten L-phenylalanine ethyl
ester derivatives (IL (1)–IL (7) and (1a)–(3a)) was investigated
using a modified version of the OECD 301D test guideline.14
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Fig. 5
Green Chemistry
Structures of the L-phenylalanine ethyl ester ILs and non-ionic derivatives of four different types.
The Closed Bottle test (CBT) was performed as described elsewhere in detail23 with the only variation to the OECD guideline
being that the biological oxygen consumption in the closed
bottles was measured with an optode oxygen sensor system
(Fibox 3 PreSens, Regensburg, Germany), which is based on
the physical principle of dynamic luminescence24,25 and
allows measuring the oxygen demand without opening the
bottles. Thus it is possible to measure more compounds
within one run. Furthermore this method allows for better
reproducibility.25
Each CBT consisted of four different test series each of
which was run in duplicate with two bottles, respectively. The
“blank series” contained only mineral medium and inoculum.
The “quality control” was prepared additionally with readily
biodegradable sodium acetate to monitor the activity of the
microorganisms. The “test series” included, besides medium
and inoculum, the test compound as the only carbon source
while the “toxicity control” contained both sodium acetate and
the test compound. The initial concentration of each compound corresponded to a theoretical oxygen demand (ThOD)
of approximately 5 mg L−1. Nitrification was not taken into
account. All test bottles were inoculated with 60 µl L−1 of the
waste water effluent of the municipal sewage treatment plant
(STP) in Lüneburg (Abwasser, Grün und Lüneburger Service
GmbH, Germany; 250 000 population equivalents). The inoculum was taken from the STP right before the start of each test.
Biodegradation values are given as the average of the values
obtained in the two bottles of one run.
In total three independent CBTs were performed. The first
test (CBT I) included all compounds and ran over 28 days. The
second test (CBT II) was performed over 42 days and included
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those compounds whose biodegradation curve in the first test
did not reach the plateau phase in order to assess long term
adaptation of the microorganisms. Samples were taken from
both CBTs at the beginning and at the end of each test and
stored at −20 °C for later LC-MSn analysis. After the structures
of the resulting TPs were proposed by LC-HRMS analysis and
the TPs have been synthesized by the Gathergood group,22 a
third CBT (III) was used to investigate the further biodegradability of these TPs.
According to the test guideline, a compound is classified as
“readily biodegradable” if measured oxygen consumption
exceeds 60% of the maximum theoretical value (ThOD) needed
for 100% mineralization of the test compound. This pass value
has to be reached within 10 days after 10% ThOD is reached
and within the test period of 28 days. Toxicity was assessed by
using toxicity controls which contained both the test substance
and the readily biodegradable sodium acetate each at a concentration of approximately 5 mg L−1 ThOD. If biodegradation
in these bottles is less than 25% within 14 days, the test substance is assumed to be inhibitory.14
2.2
Mass spectrometry
Samples from the beginning and the end of the CBTs (after
28 d (CBT I) and 42 days (CBT II)) were taken to determine
primary elimination of the phenylalanine ethyl ester derivatives and to identify TPs via liquid chromatography/high
resolution mass spectrometry (LC-Orbitrap HRMS).
2.2.1 Materials. LC-MS grade acetonitrile was purchased
from VWR (VWR International GmbH, Darmstadt, Germany).
The aqueous buffer solutions were prepared using ultrapure
water 18.2 MΩ cm (Ultra Clear UV TM, Barsbüttel, Germany)
This journal is © The Royal Society of Chemistry 2016
Green Chemistry
and formic acid for the analysis (Merck KGaA, Darmstadt,
Germany).
2.2.2 Instrumentation. The separation was performed on a
Dionex Ultimate 3000 UHLPC, equipped with a solvent rack
(SRD-3600), a binary pump (HPG-3400-RS), an auto-sampler
(WPS-3000-TRS), and a column oven (TCC-3000) coupled with
a LTQ Orbitrap XL mass spectrometer (Thermo Scientific,
Dreieich, Germany) equipped with a HESI source. The mass
accuracy of the used mass spectrometer was found to be better
than 1 ppm as also confirmed by other studies26 and allows
therefore for the determination of the exact mass of TPs.
Based on structural information available for the parent compound and the accurate mass of TPs (including mass losses),
accurate sum formulas for the TPs were given from which the
probable TP structures could be derived. After TP synthesis by
the Gathergood group,22 TP structures were confirmed by a
comparison of their retention times, accurate mass and fragmentation patterns with those of the TPs from CBT samples.
2.2.3 LC-MS method. Due to the high polarity of the analytes, normal phase chromatography was used. Normal phase
chromatography offers the opportunity to separate small polar
compounds effectively on a polar stationary phase. The separations were carried out on a Hypersil Gold CN (100/3 mm,
3 µm, Thermo Scientific, Dreieich, Germany) equipped with
an HILIC guard column (Macherey-Nagel, Düren, Germany),
and the later confirmation of the proposed structures was
carried out on a Nucleodur CN (125/3 mm, 3 µm, MachereyNagel, Düren, Germany). Separation performances of both
columns were comparable. The column was kept at 30 °C with
a flow rate of 400 µL min−1 using a gradient of acetonitrile and
0.05% formic acid in water (v : v). The gradient started with
97% acetonitrile, held for 1 min, decreased to 60% over
11 min, held for 3 min, and then increased back to the initial
percentage of 97% within 3 min followed by 7 min re-equilibration time.
The mass spectra were acquired in positive centroid mode
with a resolving power of 30 000. The parameters for the
heated electrospray source for sheath and auxiliary gas flow
rates were 15 and 5 arbitrary units, respectively. The spray
Paper
voltage and the vaporizer temperature were set at 4 kV and
200 °C, the capillary voltage and the capillary temperature
were set at −9 V and 275° and the tube lens voltage was kept
at 100 V.
3. Results and discussion
3.1
Biodegradation
None of the L-phenylalanine derived derivatives (IL (1)–IL (7)
and (1a)–(3a)) can be classified as “readily biodegradable”
according to the OECD guideline. The only compound that
reached the ready biodegradability threshold of 60% within 28
days was IL (4) with 63% biodegradation in CBT I that was performed in summer. This result could not be repeated in CBT
II, which was carried out in winter and where the biodegradation values were lower in total (Table 1).
However, as the microorganisms required more time to
reach this pass level than 10 days after 10% ThOD was reached
(Fig. 6), even IL (4) is not “readily biodegradable” in the strict
sense of the OECD requirements.14
Concerning toxicity, none of the investigated phenylalanine
derivatives (IL (1)–IL (7) and (1a)–(3a)) were toxic against the
degrading bacteria since after 14 days the degradation in the
toxicity controls was more than 25% (at least 40%; shown
exemplarily for IL (4) in ESI, Fig. S1†). This is consistent with
these compounds not having high toxicity to 8 bacteria and 12
fungi strains reported in the accompanying paper.22
Test prolongation. In CBT II prolonged up to 42 days the biodegradation of IL (4) and the two non-ionic phenylalaninebased derivatives (2a) and (3a) increased within the prolongation time by roughly a factor of 1.7, respectively (Table 1,
Fig. 7). After 42 days approximately 60% of compound (3a),
and more than 75% of (2a) and IL (4) were biodegraded,
demonstrating for (3a) inherent biodegradability and for the
latter two compounds inherent, ultimate biodegradability.27
Such long-term adaptation processes connected with an
increased biodegradability have been observed for several
other ILs.20,28 This indicates that the responsible bacteria
Table 1 Biodegradation rates (average of duplicates) in % ThOD of the L-phenylalanine derived ILs and related non-ionic derivatives (expanded with
“a”) in the Closed Bottle tests (CBTs) after 28 and 42 days; grey boxes mark biodegradation values close to or over 60%
a
One of the two bottles was leaking. b Not valid (biodegradation values in the replicates differ by more than 20%).
This journal is © The Royal Society of Chemistry 2016
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Fig. 6 Biodegradation of L-phenylalanine derived ILs and related non-ionic derivatives (expanded with “a”) in CBT I in comparison to the quality
control (sodium acetate). Biodegradation values are the average values of duplicates.
Fig. 7 Biodegradation of L-phenylalanine derived ILs and related non-ionic derivatives (expanded with “a”) in the prolonged CBT II. Biodegradation
values are the average values of duplicates.
needed more time than that given in the standardized OECD
tests to reach a sufficient cell density which results in the biodegradation of a xenobiotic above 60%. Probably they belong
to rare taxa (specialists) with longer generation time and lower
abundance. Cell density is one essential factor for the
threshold for ready biodegradability to be reached or not,
since some ILs that failed biodegradability assays could be
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quickly biodegraded by IL pre-adapted consortia.29 In that
study the specific IL caused a significant change in the
microbial composition probably due to the selective growth of
certain strains. However, it is noteworthy that preadaptation of
microorganisms in biodegradability testing can be done for
research purposes but is not within the scope of the OECD test
guidelines.14 Furthermore, according to the benign by design
This journal is © The Royal Society of Chemistry 2016
Green Chemistry
principle the mineralization should be quite rapid under
normal conditions to allow the compounds to be mineralized
in a reasonable time even under unfavorable environmental
conditions.
In the case of compounds (1a), (2) and (5) the plateau
phase was reached around day 30 ((2), (5)) or the degradation
remained quite low (<20%) even after 42 days ((1a)) so that a
further test prolongation would not result in higher biodegradation values. These results indicate that the biodegradation
of phenylalanine derivatives, even if they are structurally very
similar such as (2)/(2a) or (4)/(5), could not be increased in
general by prolongation of the incubation time. Therefore,
small structural variations seem to make the difference
whether the molecule fits into the active center of a cleaving
enzyme or not.
All in all, the data received by test prolongation give
more insight into the biodegradation behavior of the bacteria
and into which of the phenylalanine-based compounds were
biodegradable in principle to a higher extent. However, seen
from the aspect of sustainability, the aim of green ILs should
be that these compounds are rapidly and fully mineralized by
generalists and not just by specialist degraders that may be
present in the environment in a few places under specific circumstances and in low abundance only.
Non-ionic derivatives versus ILs. Compounds (1a), (2a) and
(3a) are the non-ionic related derivatives which correspond to
the ILs (1), (2) and (3). Substance (1a) is comparable to IL (1),
where the N-methylation of the proline subunit causes a positive charge on the nitrogen. In (2a) the nitrogen in the deanol
(2-(dimethylamino)ethanol) side chain is only tertiary-substituted in contrast to IL (2), which is a quaternary ammonium
(choline) derivative with a positive charge on the nitrogen. In
(3a) the imidazole is mono-substituted with a phenylalanine
ethyl ester while in IL (3) the attachment of a second phenylalanine ethyl ester causes a positive charge on one nitrogen at
the five membered aromatic ring (for structures see Fig. 5).
For (1a), only in CBT II, valid biodegradation results (10%)
were obtained. For the comparable IL (1) the biodegradation
in CBT I was more than two times as high (24%) but as, in
general, the biodegradation values were higher in CBT I ( performed in summer) than in CBT II ( performed in winter),
these values should not be overrated. On the basis of these
limited results it is impossible to predict whether the positive
charge of nitrogen in proline derivatives has a positive or negative effect on biodegradability. In the case of the deanol derivatives the tertiary ammonium compound (2a) was at least two
to four times better biodegradable than the quaternary aliphatic ammonium derivative IL (2), where only an ethyl ester
was eliminated. This indicates that the positive charge of the
nitrogen in the choline or the stereochemistry of the quaternary substituted nitrogen hindered the cleavage of the amide
bond and degradation of phenylalanine ethyl ester and had a
negative effect on biodegradability, which is a fact described
also in the literature.18 On the contrary, no difference in biodegradation was found for the imidazole derivatives IL (3) and
(3a). In both compounds the amide bond was cleaved and the
This journal is © The Royal Society of Chemistry 2016
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phenylalanine ethyl ester was eliminated, resulting in biodegradation values of 40% after 28 days.
In summary, according to these results it is not possible to
give a general statement whether a positively charged nitrogen
has a positive or negative effect on biodegradability.
Summary of biodegradation results. None of the L-phenylalanine derived compounds (IL (1)–IL (7) and (1a)–(3a)) was
“readily biodegradable” according to the OECD guideline.14
The microorganisms needed either a greater time frame within
the 28 days test period to pass the 60% threshold (IL (4), more
than 10 days after reaching 10% of degradation) or they
needed, in general, more than 28 days ((2a), (3a)). Biodegradation was probably accomplished by specialized rare bacterial
groups, as is known for many xenobiotics,30 which needed,
based on their low abundance and high generation time, more
time for adaptation. Nevertheless, test prolongation increased
biodegradability for three compounds up to 59% (3a), 76%
(IL (4)) and 78% (2a) after 42 days, respectively, showing at
least for the latter two compounds that ultimate degradation is
possible, in principle.27
3.2
Analysis
At the end of the CBTs, samples were analyzed by LC-HRMS to
determine the primary elimination of the phenylalanine-based
compounds and to identify the resulting TP structures
(Table 2). After the synthesis of TPs their biodegradability was
investigated in CBT III.
Transformation products and their biodegradability. No TP
was found for IL (4). For all other phenylalanine-based compounds TPs were identified. Their confirmed structures are
given in Table 2, while non-confirmed isomeric structures are
represented in brackets.
Two different degradation pathways were observed for the
series of compounds screened: ester bond cleavage and amide
bond cleavage. For L-phenylalanine-based ILs (1), (2) and (7), it
was observed that only the ester group was hydrolyzed, leaving
behind an amino acid IL TP. Ester bond cleavage resulted in
low biodegradation rates, limited to approximately 30% ThOD,
after 28 days. For the TPs of IL (1) and IL (7) two possible
isomers of the same m/z were initially proposed for each IL.
MS2 fragmentation could not contribute to a more precise
structural elucidation because the subsequent fragmentation
of both TPs (isomers) results in elimination of the second
ester group, which gives the same identical fragment making
precise identification impossible (see Fig. 8).
Therefore, for IL (1) and IL (7) the synthesis of both TP
isomers was necessary to compare their analytical characteristics (accurate mass, retention time, and MS2 and MS3 fragmentation pattern) with those of the TPs resulting from the
CBT and to find out which isomer was, in fact, the degradation
product. The resulting TPs from ester bond cleavage were nonbiodegradable (<20% for TP-(1) and TP-(2)) or only slightly biodegradable (<40% for TP-(7ii)) in CBT III.
For compounds (1a), (2a), (3), (3a), (5), and (6), biodegradation occurred through cleavage of the amide bond with complete degradation of the phenylalanine ethyl ester (Fig. 9). The
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Table 2 Structures of the L-phenylalanine derived compounds and their transformation products (TPs) at the end of CBT I and II. Primary elimination (PE) of the mother substances is given below its structures; the abbreviation PEE stands for phenylalanine ethyl ester
Compound no.
Separation of
Identified sum formulas and TP structures
(non-confirmed isomers in brackets)
Ethanol
Sum formula: C21H31O5N2Br
m/z [cation, calculated]: 391.2228
m/z [cation, acquired]: 391.2234
Mass error: 0.6 mmu
Sum formula: C19H27O5N2
m/z [calculated]: 363.1914
m/z [acquired]: 363.1922
Mass error: 0.8 mmu
Epimerisation of the stereogenic centre is
assumed not to occur
PEE
Sum formula: C20H28O5N2
m/z [M + H+, calculated]: 377.2071
m/z [M + H+, acquired]: 377.2076
Mass error: 0.5 mmu
Sum formula: C9H15O4N
m/z [M + H+ calculated]: 202.1074
m/z [M + H+ acquired]: 202.1077
Mass error: 0.3 mmu
Ethanol
Sum formula: C17H27O4N2Br
m/z [cation, calculated]: 323.1965
m/z [cation, acquired]: 323.1957
Mass error: 0.8 mmu
Sum formula: C15H23O4N2
m/z [calculated]: 295.1652
m/z [acquired]: 295.1660
Mass error: 0.8 mmu
PEE
Sum formula: C16H25O4N2
m/z [M + H+, calculated]: 309.1809
m/z [M + H+, acquired]: 309.1817
Mass error: 0.8 mmu
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Sum formula: C5H12O3N
m/z [calculated]: 134.0812
m/z [acquired]: 134.0815
Mass error: 0.3 mmu
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Table 2
Paper
(Contd.)
Compound no.
Separation of
Identified sum formulas and TP structures
(non-confirmed isomers in brackets)
PEE
Sum formula: C29H35O6N4Br
m/z [cation, calculated]: 535.2551
m/z [cation, acquired]: 535.2560
Mass error: 0.6 mmu
Sum formula: C18H22O5N3
m/z [calculated]: 360.1554
m/z [acquired]: 360.1561
Mass error: 0.5 mmu
PEE
Sum formula: C16H19O3N3
m/z [M + H+, calculated]: 302.1500
m/z [M + H+, acquired]: 302.1508
Mass error: 0.8 mmu
Sum formula: C5H6O3N2
m/z [M + H+ calculated]: 127.0502
m/z [M + H+ acquired]: 127.0505
Mass error: 0.3 mmu
None detected
Sum formula: C18H21O3N2Br
m/z [cation, calculated]: 313.1547
m/z [cation, acquired]: 313.1555
Mass error: 0.8 mmu
PEE
Sum formula: C19H23O4N2Br
m/z [cation, calculated]: 343.1652
m/z [cation, acquired]: 343.1657
Mass error: 0.5 mmu
This journal is © The Royal Society of Chemistry 2016
Sum formula: C8H10O3N
m/z [calculated]: 168.0655
m/z [acquired]: 168.0661
Mass error: 0.6 mmu
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Table 2
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(Contd.)
Compound no.
Separation of
Identified sum formulas and TP structures
(non-confirmed isomers in brackets)
PEE
Sum formula: C20H26O3N3Br
m/z [cation, calculated]: 356.1969
m/z [cation, acquired]: 356.1976
Mass error: 0.5 mmu
Sum formula: C9H13O2N2
m/z [calculated]: 181.0972
m/z [acquired]: 181.0977
Mass error: 0.5 mmu
Ethanol
Sum formula: C21H25O5N2Br
m/z [cation, calculated]: 385.1758
m/z [cation, acquired]: 385.1764
Mass error: 0.6 mmu
Sum formula: C19H21O5N2
m/z [calculated]: 357.1445
m/z [acquired]: 357.1451
Mass error: 0.6 mmu
Fig. 8 Biodegradation of IL (7) as an example of ester bond cleavage and the isomer structures of the two potential TPs, TP-(7i) on top and TP-(7ii)
at the bottom, of which the latter could be confirmed by LC-HRMS after TP synthesis.
significance of the cleavage of the amide bond is that, of the
two TPs formed, the amino acid can undergo complete mineralization, therefore achieving regularly higher levels of biodegradation than when just the ester is cleaved. The remaining
TP is a carboxylic acid derived from the head group as has
4370 | Green Chem., 2016, 18, 4361–4373
been demonstrated by LC-MS, which were independent of its
head group being not further biodegradable in CBT III (<20%).
The only exception was TP-(2a). Although the microorganisms
needed an adaptation time of 3 weeks, TP-(2a) proved to be
readily biodegradable (see the next section). Also worthy of
This journal is © The Royal Society of Chemistry 2016
Green Chemistry
Fig. 9
Paper
Biodegradation of IL (3) as an example of amide bond cleavage.
note is that none of the parent compounds was seen to
undergo ester hydrolysis; the TPs detected at the end of
the biodegradation screening were solely amide cleaved
products.
The most biodegradable compounds. IL (4) and the nonionic derivatives (2a) and (3a) were the most biodegradable
compounds with biodegradation rates of approximately 60%
or more after test extension (in the same order 76%, 78%
and 59%). While biodegradation of both non-ionic derivatives resulted in TPs after 42 days (for structures see
Table 2), IL (4) was the only compound where no TP was
found by LC-MS analysis, not even after 28 days. Analysis
showed that the parent compound was primarily eliminated
to 73% after 28 days and completely transformed after 42
days. Together with the fact that after 42 days 76% biodegradation was found, an ultimate degradation of IL (4) can be
assumed as the remaining fraction of about 25% is expected
to be assimilated by biomass or present as products of biosynthesis.27 Probably also for IL (4) a microbial attack of the
amide bond occurred releasing phenylalanine ethyl ester and
(1-carboxymethyl)pyridinium, which were both ultimately biodegraded by the bacteria (Fig. 10).
This assumed microbial pathway is supported by our own
experimental data showing that not only phenylalanine ethyl
ester but also (1-carboxymethyl)pyridinium bromide is readily
biodegradable in the CBT (88% after 28 days or 69% considering nitrification). The findings are in accordance with the literature where, on the one hand, a ready biodegradability of
80% is described13,31 for the chemical precursor 1-(2-ethoxycarbonylmethyl)pyridinium bromide in a CO2 Headspace test
including a ring cleavage. On the other hand, the similar compound (1-hydroxymethyl)pyridinium iodide was readily biodegradable in the Manometric Respirometry test14 to 65 ± 10%.28
Therefore, IL (4) was the only test compound which was fully
mineralized after 42 days in the CBT.
For the non-ionic derivatives (2a) and (3a) CBT III underlines that the biodegradation of compound (3a) results in nonbiodegradable imidazole acetic acid (10% biodegradability in
CBT III), while an ultimate biodegradation of (2a) is possible,
in principle, even if it took more than 42 days: after 42 days
100% of compound (2a) was primarily eliminated (Table 2),
but not completely biodegraded as shown by TP-(2a), which
was still detectable at the end of CBT II, but has been proved
to be readily biodegradable after a lag-phase of 3 weeks in CBT
Fig. 10 Fully mineralizable IL (4), for which an amide bond cleavage is assumed resulting in ultimate biodegradable phenylalanine ethyl ester and
carboxylic acid residue ((1-carboxymethyl)pyridinium bromide).
This journal is © The Royal Society of Chemistry 2016
Green Chem., 2016, 18, 4361–4373 | 4371
Paper
Green Chemistry
III (to 100% or 75% considering nitrification). It is worthy of
note that a biodegradation value of 78% after 42 days combined with a detectable TP is not in line with the above cited
OECD guideline that assumes ultimate biodegradation for substances with biodegradation values >60% as the remaining
fraction, up to 40% of the substance, can be assumed to be
assimilated by the biomass or be present as products of
biosynthesis.27
Further studies are planned to examine the biodegradability
of new ILs synthesized based on the structure of IL (4) and
known quantitative structure–activity relationships (QSAR) to
further pursue the aim of producing sustainable and green ILs
which are rapidly and fully mineralized in the aquatic environment. The findings of this study and the future studies will
also aim for the development of more general rules on how to
improve the biodegradability of ILs in the future.
4.
Acknowledgements
Summary and conclusion
The results of the present study expand the data platform
upon which readily/ultimately biodegradable ILs can be
designed (“benign by design”), an important building block of
sustainable chemistry. An essential tool in this respect is to
combine biodegradability investigations with LC-HRMS analysis to have complete information about the biodegradability of
molecule parts. What is more, the example of compound (2a)
demonstrates that even high biodegradation values (78% after
42 days) did not mean it is mandatory that the whole compound has been mineralized or assimilated as TP-(2a) was the
remaining TP after 42 days. Furthermore, the results of this
study underline that the combination of ionic head groups
with readily biodegradable biomolecules did not necessarily
lead to an increase in biodegradability or degradation of the
whole compound. The investigated proline and choline
derived phenylalanine ethyl ester ILs were less biodegradable
than imidazole derivatives and pyridinium derived ILs as only
an ester cleavage and mineralization of ethanol occurred. In
contrast, an amide bond cleavage and mineralization of the biomolecule were stated for imidazole derivatives and pyridinium
derived ILs with the exception of IL (7). Therefore, imidazole
and pyridinium head groups are to be preferred. Nevertheless,
for five of the seven compounds, even amide bond cleavage led
to persistent carboxyl acid TPs derived from the head groups,
while for two compounds inherent, ultimate biodegradability
was stated with IL (4), a pyridinium substituted phenylalanine
derived compound, as the only IL. Full mineralization of IL (4)
took 42 days. Although IL (4) was not readily biodegradable to
OECD criteria it could be proposed as a basic structure for the
(benign) molecular design of green ILs. From this starting
point additional structures could be constructed by modification of the substituents according to the rules for improved
biodegradability to give the molecule the required properties.
But as learnt from the presented studies, even small variations
in the molecular structure could be crucial in deciding
whether a compound is or is not biodegraded. Thus it is a
major challenge to give reliable predictions or even derive
general rules for biodegradability of this class of compounds.
However, the aim of the design of a green IL (4)-derivative
should be to improve its degradation rate further because with
respect to sustainability it is advisable that ILs of the future
are rapidly and fully biodegraded after their introduction into
the environment everywhere and not just under special
conditions.
4372 | Green Chem., 2016, 18, 4361–4373
The authors (NG, AJ) would like to thank the EPA in Ireland
for financial support. This project has also received funding
from the European Union’s Seventh Framework Programme
for research, technological development and demonstration
under grant agreement No. 621364 (TUTIC-Green). We also
thank COST Actions CM1206 and TD1203 for their contribution to this study. We acknowledge Hannah Prydderch for
her contributions to TP synthesis and Dr Andrew Kellett for
his assistance with the project at Dublin City University.
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