Ecotoxicity studies of glycerol ethers in Vibrio fischeri: checking the environmental impact of glycerol-derived solvents

Article abstract of DOI:10.1039/c5gc00857c

The toxicities of a series of glycerol mono-, di-, and trialkyl ethers against Vibrio fischeri bacteria have been determined. A systematic study has been carried out and the possible structure-toxicity relationships have been discussed using different QSAR models. Inhibition of bioluminescence after 30 minutes of exposure shows relatively low toxicity of many of the glycerol derived chemicals studied. Results indicate that, as a general rule, the ecotoxicity increases with the length and number of substituents. However, if the size of the molecule increases, an extra substituent at position 2 makes the toxicity lower than that of the corresponding analogues.

Full text of DOI:10.1039/c5gc00857c

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Ecotoxicity studies of glycerol ethers in Vibrio fischeri: checking
the environmental impact of glycerol-derived solvents
Received 00th January 20xx,
Accepted 00th January 20xx
a
a,b
c
c
c
c
J. I. García,* E. Pires, L. Aldea, L. Lomba, E. Perales and B. Giner*
DOI: 10.1039/x0xx00000x
The toxicities of a series of glycerol mono-, di-, and trialkyl ethers against Vibrio fischeri bacteria have been determined. A
systematic study has been carried out and the possible structure-toxicity relationships have been discussed using different
QSAR models. Inhibition of bioluminescence after 30 minutes of exposure shows relatively low toxicity for many of the
glycerol derived chemicals studied. Results indicate, that, as a general rule, the ecotoxicity increases as the length and
number of substituents do. However, if the size of the molecule increases, an extra substituent at position 2, makes the
toxicity lower than that of the corresponding analogues.
www.rsc.org/
such as esters, acetals or carbonates. Some of these glycerol
ethers, namely those bearing fluoroalkyl chains, exhibited
especial physical-chemical features, in some way similar to
Introduction
The search for new solvents, coming from new sources and/or
able to provide special features (often known as neoteric
solvents), is a field of growing interest, especially in connection
with the possibility of using renewable raw materials to
produce harmless solvents, more respectful with the
environment than those derived from petroleum (the so-called
those displayed by some IL: high polarity, low vapour pressure
at room temperature, immiscibility both with hydrocarbons
and with water. The most prominent example of these
compounds is 1,3-bis(2,2,2-trifluoroethoxy)propan-2-ol, which
can be efficiently prepared from trifluoroethanol and
epichlorohydrin (a commodity chemical currently produced
1
green solvents). In this context, biomass-derived chemicals
1
2
from glycerol using the Solvay procedure). Other authors
have also recognized the special character of these new
have attracted a great deal of attention in the last years, in
connection with the development of the biorefinery concept.
Agricultural and some industrial activities are able to generate
huge amounts of raw materials, capable of being used to
1
9
fluorinated solvents.
Apart from this particular case, glycerol ethers have a high
potential for chemical diversity, given that mono-, di, and
trialkylation, either symmetrical or unsymmetrical at positions
1 and 3, leads to 1,2-diols, alcohols and trialkyl ethers. These
possibilities allow tuning important solvent properties, such as
polarity, hydrogen-bonding ability, hydrophobicity or viscosity.
Some QSPR studies have been conducted to develop models
able to predict some key physical-chemical properties of
2
produce commodity and fine chemicals. In this sense, glycerol
is one of the platform molecules that has received much
3
–5
attention in recent years. Glycerol appears as a concomitant
product in the production of biodiesel, amounting ca. 10%
weight of the total output. At present, the world production of
glycerol coming from vegetable oil transformations surpasses
2
million metric tons, so it constitutes a valuable starting point
2
0
glycerol ethers, prior to their synthesis.
to obtain bio-based chemicals, useful as, for instance,
6
–9
solvents.
Our research group
Concerning the greenness of glycerol-derived solvents, not
only their renewable origin must be taken into account, but
also other aspects of their life cycle, including their fate and
the consequences on the environment. Being solvents coming
from glycerol and short chain alkyl alcohols, which can in turn
be obtained from biomass to a large extent, low toxicity of
glycerol ethers is generally taken for granted. The pertinent
question arises, however, as to whether this family of solvents
can be considered environmentally benign or not, given the
lack of systematic experimental evidences on its toxicity and
ecotoxicity.
1
0–14
15–18
and others
have described the
synthesis and application as solvents of glycerol ethers. These
kinds of solvents have the additional advantage of being much
more chemically inert than other glycerol-derived solvents,
In this paper, we present our first results on the ecotoxicity
of a series of glycerol-derived ethers. In this case, we have
studied the inhibition of the bioluminescence of Vibrio fischeri
(
V. fischeri) bacteria. This bioindicator has been frequently
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21,22
used,
since it is a well-known organism and operating
protocols are standardized. Inhibition bioluminescence tests
2
offer robust, easy handling and cost-effective responses.
100
3
0
00
Finally, this study provides some clues on the Structure-
Toxicity relationships found in the studied solvent set (Fig. 1).
This solvent set, though not quite large, have been chosen to
cover a wide range of structural and physical-chemical
features, such as number of free hydroxyl groups, length and
ramification of the alkyl chains, hydrophobicity, solubility in
water, and presence of fluorinated groups, among others.
80
100
200
400
60
40
20
0
1
2
3
4
5
6
-
1
log c (mg·L )
Fig. 2 Dose-response curves for studied solvents for V. fischeri:
00, 400.
ꢀ
000,

100, ꢁ
2
ꢂ
As far as we know, there is no previous experimental
ecotoxicological information of any of the studied glycerol
derivatives. Regarding glycerol itself (000), there are no
experimental data of ecotoxicity in V. fischeri. However,
previous studies indicate that glycerol is of low toxicity
towards microorganisms; in a 16 hour test with Pseudomonas
putida no inhibition of bacterial growth was found at
-1 24,25
concentrations between 100 and 10,000 mg·L
and similar
Fig. 1 Structures and codes of the 20 glycerol derivatives included in this study.
results are found for other microorganisms such as Entosiphon
2
6,27
sulcatum or Clostridium sp.
conclusion that, overall, glycerol is of low toxicity to
This information supports the
Results and Discussion
microorganisms. In this study, EC50 of glycerol for V. fischeri is
-1
EC50 values obtained in the biotests using V. fischeri, with their
respective adjustable parameters and standard deviations are
gathered in Table 1. Furthermore, results are graphically
higher than 100,000 mg·L , indicating the low toxicity of this
chemical for this bioindicator.
In view of the results, ecotoxicity of the studied solvents is
higher than that of glycerol in all the cases. However, taking
into account the results obtained for the bioindicator V.
fischeri, only few solvents can be considered hazardous for the
‡
represented in Fig. 2–9.
Table 1 Experimental EC50 values for V. fischeri obtained for studied solvents with their
adjustable parameters for Eq. 1 and standard deviations.
28
environment. According to Passino and Smith classification,
just 404, 404t and 414 are slightly harmful, while 111, 104,
14, 444, 104i, 114i and 104t can be considered practically
harmless. The rest of the studied solvents (100, 200, 400, 101,
02, 103i, 3i03i, 3F03F, 3F13F) are clearly harmless for the
environment.
Although the action mechanism is still unidentified, it is
-
1
Solvent code
EC50 (mg·L )
108,421
21,052
4,240
941
a
b
SD
0
1
2
4
1
1
2
4
1
1
4
4
00
00
00
00
01
11
02
04
14
04
14
44
0.01544
0.01698
0.01333
0.00873
0.01795
0.01655
0.02520
0.00777
0.01967
0.02099
0.01066
0.01796
0.01553
0.01280
0.00980
0.02083
0.03629
0.02154
0.01006
0.02849
0.09568
0.05195
0.05534
0.02610
0.10270
0.10070
0.07998
0.01893
0.13460
0.06516
0.03351
0.04461
0.10940
0.03896
0.03596
0.05875
0.03283
0.04298
0.05688
0.06963
4.791
3.587
3.910
1.839
3.798
4.003
4.082
1.831
5.365
3.468
2.937
3.564
3.764
2.876
2.523
3.905
3.391
3.544
2.375
3.845
1
2
13,702
969
1,215
11
known that bacterial bioluminescence reactions are coupled to
the electron transport system in cellular respiration.
Consequently, bioluminescence reactions are indicative of
cellular metabolism; i.e., lower bioluminescence implies
decreased cellular respiration. Thus, the presence of the
studied solvents in the culture medium affects to a greater or
lesser extent, the cellular respiration by the modification of
both lipid and protein biosynthetic pathways and therefore,
453
464
58
473
1
1
03i
04i
2,188
142
3
i03i
14i
1,064
258
29
alters the bioluminescence emission.
1
In general, the alteration of the bioluminescence emission
and thus, the ecotoxicity, increases as the length of the radical
does. This trend can be easily observed in Fig. 2, where we
show the results obtained for the series 000-100-200-400 and
ecotoxicity for V. fischeri increases substantially with the
length of the substituent at position 1.
1
4
04t
04t
189
16
3
3
F03F
F13F
1,597
4,033
2
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100
100
3
F03F
1
00
80
3F13F
80
101
1
1
11
14
60
60
40
40
20
20
0
2.0
2.5
3.0
3.5
4.0
4.5
0
log c (mg/L)
1
2
3
4
5
6
log c (mg/L)
Fig. 6 Dose-response curves for studied solvents for V. fischeri: ꢀ 3F03F, ꢁ 3F13F.
Fig. 3 Dose-response curves for studied solvents for V. fischeri: ꢀ 100, ꢁ 101,
ꢂ 114.
ꢀ
111,
Fig. 3 shows the representation of the 100-101-111-114
series. Results indicate that toxicity increases with the length
of the alkyl chain and also with the number of substituents,
independently of the position of the radical. However, if the
size of the molecule increases, an extra substituent at position
100
2
00
80
202
2, makes the toxicity lower than the corresponding analogues.
Fig. 5, 6 and 8 exemplify this behaviour. For example, toxicity
60
for 404 is ca. 5 times higher than 414, while for 3F03F is ca. 2.5
‡
times higher than 3F13F. It is worth mentioning, that in the
40
case of the pairs 104-114 and 104t-114t when molecular size is
between that of 101-111 and 404-414, the ecotoxicological
behaviour is quite similar and the extra radical at position 2
seems to affect only slightly the toxicity (Fig. 7 and 8).
2
0
0
2
.5
3.0
3.5
4.0
4.5
5.0
log c (mg/L)
100
1
04
Fig. 4 Dose-response curves for studied solvents for V. fischeri: ꢀ 200, ꢁ 202.
8
0
114
414
The presence of a substituent at position 3, makes toxicity
for V. fischeri higher comparing with the corresponding
analogous solvent. This is the case of the pairs 100-101, 200-
60
40
202 and 400-404, whose behaviour is illustrated in Fig. 3, 4 and
respectively.
5
20
0
0
100
.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4
00
log c (mg/L)
80
404
4
4
14
44
Fig. 7 Dose-response curves for studied solvents for V. fischeri: ꢀ 104, ꢁ 114,
ꢀ
414.
60
40
100
1
03i
20
3i03i
80
1
1
04i
14i
0
60
40
0
1
2
3
4
log c (mg/L)
2
0
Fig. 5 Dose-response curves for studied solvents for V. fischeri: ꢀ 400, ꢁ 404,
444.
ꢀ
414,
ꢁ
0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
log c (mg/L)
Fig. 8 Dose-response curves for studied solvents for V. fischeri: ꢀ 103i, ꢁ 3i03i,
04i, ꢂ 114i.
ꢀ
1
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Finally, to analyse the effect of the structure of the
Regarding the comparison with ILs, it is interesting to note
substituents, we have studied derivatives with branched that ionic liquids have been catalogued as “green solvents” in
3
4,35
radicals. The obtained results are graphically represented in several occasions,
although the toxicity of a number of
Fig. 8 and 9. Once again, the higher the size of radicals, the them is quite high to V. fischeri and many other
3
6
higher the ecotoxicity, both in the case of iso- or tert- bioindicators. Our solvents are generally much eco-friendly
substituents.
from the ecotoxicity point of view than the selected ILs,
independently of the cation (Table 2); only in the case of ILs
with small substituents toxicity is comparable to the vast
majority of the studied glycerol-derived solvents.
100
1
04t
8
0
404t
However, it should be mentioned that the comparison
made regarding the toxicity of glycerol-derived chemicals and
some other solvents is limited to only one bioindicator, V.
fischeri, in this case. Furthermore, to completely assess the
greenness of the studied compounds, not only ecotoxicity data
have to be taken into account; the analysis and evaluation of
some other important properties such as bioavailability or
biodegradability would change this first approach. Finally, the
whole lifecycle of the product; production processes, uses and
applications, removal rate in depuration processes, final fate
and toxicity of degradation products are some parameters that
should be also taken into account to determine definitively the
environmental properties of the solvents.
60
40
20
0
0
1
2
3
4
log c (mg/L)
Fig. 9 Dose-response curves for studied solvents for V. fischeri: ꢀ 104t, ꢁ 404t.
In Table 2, V. fischeri ecotoxicity data of a selection of some
conventional solvents and ionic liquids (ILs) are shown for Quantitative Structure-Activity Relationships
3
0–33
comparative purposes.
Ecotoxicity of most of the studied
To carry out a deeper analysis of the quantitative relationships
between the molecular structure and the measured
ecotoxicity we use the same QSAR approaches previously
applied to some physical-chemical properties of a larger family
glycerol derivatives is smaller than that of some traditional
solvents, such as toluene or phenol. Smaller compounds (100,
200 or 101) show values of EC50 similar to those of low
molecular weight alcohols (methanol, ethanol or propan-2-ol)
or acetone, known for being innocuous for V. fischeri. Only
glycerol derivatives with large substituents (404, 414 and 404t
are as ecotoxic as o-xylene or phenol, which are solvents
traditionally known for being toxic for the environment.
20
of glycerol-derived solvents.
The first part of a QSAR study is to choose the quantitative
description of the molecular structure. To this end, we used
two different approaches: local structure descriptors and
global structure descriptors. In the first ones, each descriptor
indicates the presence or absence of a group of atoms at a
given molecular position. As in our previous study, we used the
Table 2. Several EC50 values for different traditional solvents in V. fischeri bioassay
during 30 minutes of exposure.
3
7
DARC-PELCO approach, following the definition shown in Fig.
0.
-
1
Molecular Solvents
EC50 (mg·L )
1
1
Methanol
Propan-2-ol
Ethanol
101,068
2
35,383
2
23,089
2
Acetonitrile
Acetone
21,172
1
19,311
1
Dichloromethane
Chloroform
Ethylene glycol
Benzene
2,532
1
1,199
1
621
1
108.
1
Toluene
32
1
Phenol
31
1
o-Xylene
9
Ionic Liquids
3
1
1
1
-Methyl-3-propylimidazolium tetrafluoroborate
-Pentyl-3-ethylimidazolium tetrafluoroborate
-Butyl-3-ethylimidazolium tetrafluoroborate
1850
4
350
3
151
4
1
1
1
1
1
-Butylpyridinium dicyanamide
98
3
-Heptyl-3-methylimidazolium tetrafluoroborate
-Ethyl-3-hexylimidazolium tetrafluoroborate
-Methyl-3-octylimidazolium tetrafluoroborate
-Hexyl-3-methylpyridinium bromide
74
3
38
Fig. 10 DARC/PELCO scheme used to describe the molecular structures of the glycerol-
3
7
derived solvents.
4
2
4
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The DARC/PELCO method is particularly suitable for prediction. In the case of eq. 1, the toxicity of solvent 444
studying families of compounds with a common chemical could not be predicted by this procedure, as the B coefficient
substructure, and is based on the exhaustive generation of all cannot be calculated without this toxicity value. Using the
possible substitution sites around the reference structure (F remaining 19 solvents a RCV = 0.83 was obtained, still quite
corresponding to the glycerol skeleton common to all reasonable for a biological response. Figure 11 plots the
ethers— and the evaluation of their contribution to the experimental values vs. those calculated with the MLR models.
property. In this definition we have incorporated the From the QSAR point of view, a positive sign of the
1
0
)
—
symmetry of the glycerol derivatives used, by assuming that regression coefficient in eq. 1 means that the presence of an
the contributions of groups occupying equivalent positions (i.e. atom in the corresponding position leads to a decrease in
those linked to carbons 1 and 3 of the glycerol moiety) will toxicity, whereas the converse is true for negative coefficients.
display the same influence on the property under study. Thus, enlargement of the substituents at positions 1 and 3
Previous studies have demonstrated that this simplification results in a progressive increase of toxicity, which is easier to
2
0
does not alter the results of the regression analyses.
The second approach to molecular codification is the use of A , B and C (–0.342, –0.399 and –0.600, respectively). On the
see by looking at the standardized regression coefficients for
2
2
2
3
8,39
topological parameters,
which are easily derived from the other hand, substitution at the central oxygen atom tends to
connectivity and adjacency matrixes of each compound. The decrease the toxicity of these compounds, although in a less
number of connected components of a graph is a topological clear manner (standardized coefficient for B = 0.241).
1
invariant that measures the number of structurally
independent or disjoint subnetworks. These parameters are
excellent descriptors of molecular size, shape and flexibility.
They are global parameters in the sense that the whole
molecular structure is condensed in a single number. The full
list of DARC-PELCO and topological descriptors used in the
QSAR analyses is available in the Supplementary Information.
The second part of the QSAR analysis consists in relating
the dependent variable (ecotoxicity) with the independent
ones (the molecular descriptors), through a quantitative
model, derived in most cases from a least squares fit. In our
case, we used Multiple Linear Regression (MRL) analysis,
whose detailed description has already been done
2
0
elsewhere.
We started by applying the MLR analysis to the DARC-
PELCO model. Given that not all the molecular descriptors
have to be statistically relevant to the final equation, we used
a stepwise procedure for variable selection. The final
regression equation derived was the following:
Fig. 11 Plots of predicted vs. experimental values of logEC50 as calculated through MLR
analysis using the DARC/PELCO model. Circles represent the predictions made with the
plain regression equation and crosses are the cross-validated predictions. Dashed and
dotted lines represent the least squares fit between both sets of data, respectively.
logEC50
.614(±0.192)
1)
=
4.828(±0.320)
+
1.064(±0.460)

B2 – 0.685(±0.123)
B1
–
0


A2 – 0.310(±0.0.082)




C2
(
N = 20, R = 0.93, RCV = 0.83,
F = 22.6 (F(4,15, 0,05) = 3.1)
σ(y) = 0.418
The application of the stepwise MLR procedure to the
same data set, using the topological descriptors as structural
variables led to the following equation:
As can be seen, the model fits fairly well the experimental
data of ecotoxicity (86% of the experimental variance is
v
logEC50 = –7.715(±2.710) – 1.455(±0.200)
JX
Bal + 0.553(±0.180)


χ
2
+
2
explained by the model, as indicated by the R determination
4
.779(±1.023)




HBDcount
(2)
coefficient), with a ratio observations/adjustable parameters =
N = 20, R = 0.91, RCV = 0.85,
F = 27.0 (F(3,16, 0,05) = 3.2)
σ(y) = 0.436
5
, which is quite reasonable. The standard error of the model
(0.42) is also close to the experimental value (0.54). However,
to have a better knowledge of the true predictive ability of this
equation, a cross-validation procedure was applied. In this
procedure, the toxicity value of each solvent is predicted by
the regression equation derived without using the
experimental data of that solvent, so that it becomes a pure
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logEC50 = 3.243(±0.112) – 0.992(±0.110)


logP
(3)
N = 17, R = 0.92, RCV = 0.88, σ(y) = 0.429
F = 81.9 (F(1,16, 0,05) = 4.5)
Fig. 12 Plots of predicted vs. experimental values of logEC50 as calculated through MLR
analysis using the topological descriptors. Circles represent the predictions made with
the plain regression equation and crosses are the cross-validated predictions. Dashed
and dotted lines represent the least squares fit between both sets of data, respectively.
Again, a fairly good fit is obtained (84% of the total
experimental variance explained), which is mostly kept in the
cross-validation analysis (RCV = 0.85). Figure 12 plots the
Fig. 13 Plot of logEC50 vs. logP. Only the solvents represented with circles have been
experimental values vs. those calculated with these MLR used to obtain the least-squares fitting line.
models. As can be seen, 444 displays the highest deviation in
In any case, it is always dangerous to use this kind of
correlation to drive conclusions about causality, because of the
cross-correlations that exist among different molecular
properties. For instance, in the case of logP in a rather
homogeneous molecular set as the considered in this work,
there are strong correlations of this property with other
molecular properties mainly related with molecular size, such
as molar volume (0.984) or solvent accessible surface (0.977).
It is clear that a deep knowledge of the mechanisms
responsible for the toxicity measured in V. fischeri, as well as
of the bioavailability of the aqueous solutions of the glycerol
derivatives would be necessary before assigning a clear
causality in the SAR analyses. Studies in this direction, as well
as the enlargement of the ecotoxicity studies to other
bioindicators are currently in progress in our groups and will
be reported in due course.
the predicted toxicity, a fact undoubtedly related to being the
sole structure studied with a long alkyl chain at position 2.
The interpretation of results is not straightforward in this
case, due to the global character of the topological descriptors.
The positive coefficient of the hydrogen bond donor count
reflects the fact that the more free hydroxyl groups has the
solvent the lower its toxicity. The other two topological indices
are mostly related to the molecular size, with some corrections
to atom electronegativities and valence, which mainly affect to
the fluorinated compounds. Looking at the standardized
JX
v
coefficients for Bal and χ2 (1.157 and –1.513, respectively) it
can be seen that molecular size increase roughly results in an
increase of toxicity, too.
We finally tested a classical approach in QSAR studies, i.e.,
the correlation between the measured toxicity and the
hydrophobicity of the compounds, as expressed by logP, the
4
0,41
Concerning the predictive ability of the QSAR equations
presented in this work in real-world situations, we have tried
to provide some clues in this direction by synthesizing and
testing a new compound, namely that coded as 114t (1-tert-
butoxy-2,3-dimethoxypropane). First results obtained in the
ecotoxicity measurements of this compound indicate a value
logarithm of the octanol/water partition coefficient.
3 plots both sets of values. As can be seen, there are clear
deviations from a linear behaviour in three solvents, namely
44 and the two fluorinated solvents. This indicates that
Figure
1
4
hydrophobicity alone cannot account for the variations in
toxicity obtained. In fact, it has also been shown that there is
not always a direct correlation between lipophilicity and the
adsorption, biodegradation rates and therefore, toxicity; the
persistence of the substance appears to be a key factor when
correlating the bioavailability of the substance and the
–1
of 1,735±3.64 mg·L , which fits well in the equations derived
with the 20 original solvents. Furthermore, if the new solvent
is included in the QSAR analyses, the derived equations show
little difference with the original ones, indicating their
robustness against the inclusion of new data. The
4
2–45
biological effect.
Leaving aside these three solvents, the
remaining seventeen show a good linear response:
6
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Pleas Ge rdeo e nn o Ct ah de j mu si ts mt r ya rgins
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ARTICLE
corresponding plots and equations can be found in the We have described for the first time the ecotoxicity of a series
Supplementary Information.
of glycerol alkyl ethers solvents (including glycerol itself), using
the bacteria Vibrio fischeri as bioindicator. Although all the
glycerol-derived structures display higher ecotoxicities than
glycerol, half of them can be considered harmless for
environment on the basis of this bioindicator, and only three
are clearly harmful following the Passino–Smith classification.
Experimental
Synthesis of the glycerol derivatives
Monoalkylated glycerol derivatives were obtained by ring These results support the often assumed greenness of these
opening of glycidol using the appropriate alcohol and catalytic bio-based solvents, as far as potential environmental damage
amounts of potassium hydroxide. Non-symmetric dialkylated is concerned, although more ecotoxicity studies representing
glycerol derivatives were obtained by reaction of glycidol different trophic levels, are necessary to have a whole view on
ethers with the appropriate alkoxide. Symmetric dialkylated this topic.
glycerol derivatives were obtained by reaction of
A comparison of the ecotoxicity towards V. fisheri between
epichlorohydrin with the appropriate potassium alkoxide. several traditional solvents and glycerol ethers indicates that
Finally, trialkylated glycerol derivatives were obtained from EC50 of some of our solvents is similar to low molecular weight
the corresponding dialkylated derivatives, by alkylation of the alcohols, traditionally known as no harmful for this bacteria.
central free hydroxyl group. Full experimental details on the Furthermore, in general terms, ecotoxicity of ionic liquids
synthetic procedures are available as Supplementary seems to be higher than glycerol derived solvents, especially
Information.
when length of substituents increases.
The QSAR studies point to a dependence of the toxicity on
alkyl chain size, manifest in the case of substitution at 1 and 3
positions of glycerol, but less clear in the case of substitution
at the central 2 position. An enlargement of the solvent set
seems necessary to establish with certainty the latter effect.
Ecotoxicity experiments
The evaluation of the ecotoxicity was based on the
determination of the inhibition of luminescence of V. fischeri
bacteria. The experiments were carried out in accordance with
the test conditions and the operating protocol of the V. fischeri
‡
acute toxicity test (UNE-EN-ISO 11348-3 2007). Details of the
4
experiments can be found elsewhere.
6
Acknowledgements
Trend analysis and quantitative structure–activity
relationship (QSAR) models were applied with the software
QSAR Toolbox 2.3 to determine the solvents concentrations to
be tested. Once the range of EC50 was bounded, at least 10
dilutions for each of the studied solvents were prepared using
Financial support from the Spanish MINECO (project CTQ2011-
2
8124-C02-01, the European Social Fund (ESF) and the
Gobierno de Aragón (Grupo Consolidado E11) is gratefully
acknowledged. GreenLife acknowledges financial support from
MINECO (CTQ2013-44867-P), FEDER and EEE53 SL. business
groups: Pinares de Venecia División Energética and Brial
(ENATICA) for support. Both business groups are committed to
sustainable developments through environmental respect.
2% NaCl as stock solution. The pH of the solutions was
adjusted to 7–7.5. Additionally, positive controls (phenol 42.5
-1
-1
mg·L and zinc sulphate, 2.2 mg·L ) and negative controls
4
7
were also tested. The lyophilized V. fischeri (strain NRRL-B-
1177) used were provided by Macharey-Nagel (ref. 945 006).
Bacteria were exposed to the toxicants for 30 min at 15 C.
1
º
Notes and references
Luminescence measurements were taken with a Biofix®
Lumi-10 luminometer (Macharey-Nagel) using Biotox B mode
‡
toxicity test used are expressed in mg
molar masses (M) of the solvents studied, this could be an issue
when comparing relative toxicities. Of course, in the case of
Concentration units in the UNE-EN-ISO 11348-3 2007 acute


–1
L . Given the different
(for acute measurements).
Obtained results have been fitted using the least squares
method using Eq. 4 to obtain the corresponding EC50 values compounds with similar M, these differences tend to be
and standard deviations (SD):
negligible. However, for the sake of completeness, we have
repeated al the QSAR analyses with EC50 toxicities expressed in
mM units, without noticeable changes in the conclusions
reached. The results of these analyses are available as
Supplementary Information.
ꢀ = 100/(1 + 10(ꢁꢂꢃꢄꢅꢆꢇꢈꢇ
%
(4)
where %I denotes
%
bioluminescence inhibition,
–
1
c concentration (in mg·L ) and a and b are adjustable
1
Handbook of Green Chemistry, 3 Volume Set, Green Solvents,
ed. P. T. Anastas, Walter Leitner, Philip G. Jessop, Wiley-VCH
Verlag GmbH, 2010.
parameters.
2
3
R. A. Sheldon, Green Chem., 2014, 16, 950–963.
M. Pagliaro, R. Ciriminna, H. Kimura, M. Rossi and C. Della
Pina, Angew. Chem. Int. Ed., 2007, 46, 4434–4440.
C.-H. Zhou, J. N. Beltramini, Y.-X. Fan and G. Q. Lu, Chem.
Soc. Rev., 2008, 37, 527–549.
B. Katryniok, H. Kimura, E. Skrzyńska, J.-S. Girardon, P.
Fongarland, M. Capron, R. Ducoulombier, N. Mimura, S. Paul
and F. Dumeignil, Green Chem., 2011, 13, 1960–1979.
4
5
Conclusions
This journal is © The Royal Society of Chemistry 20xx
Green Chemistry, 2015, 17, 1-3 | 7
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Green Chemistry
Page 8 of 9
View Article Online
DOI: 10.1039/C5GC00857C
ARTICLE
Journal Name
6
7
Y. Gu and F. Jérôme, Green Chem., 2010, 12, 1127–1138.
A. E. Díaz-Álvarez, J. Francos, B. Lastra-Barreira, P. Crochet
41 A. K. Ghose and G. M. Crippen, J. Chem. Inf. Comput. Sci.,
1987, 27, 21–35.
and V. Cadierno, Chem. Commun., 2011, 47, 6208–6227.
A. E. Díaz-Álvarez and V. Cadierno, Appl. Sci., 2013, 3, 55–69.
42 A. J. Hendriks, A. van der Linde, G. Cornelissen and D. T. H.
M. Sijm, Environ. Toxicol. Chem., 2001, 20, 1399–1420.
8
9
J. I. García, H. García-Marín and E. Pires, Green Chem., 2014, 43 A. J. Hendriks and A. Heikens, Environ. Toxicol. Chem., 2001,
, 1007–1033. 20, 1421–1437.
0 H. Garcia-Marin, J. C. van der Toorn, J. A. Mayoral, J. I. Garcia 44 S. Bayen, T. L. ter Laak, J. Buffle and J. L. M. Hermens,
and I. W. C. E. Arends, Green Chem., 2009, 11, 1605–1609. Environ. Sci. Technol., 2009, 43, 2206–2215.
1 L. Aldea, J. M. Fraile, H. Garcia-Marin, J. I. Garcia, C. I. 45 L. S. McCarty, Regul. Toxicol. Pharmacol., 2012, 63, 353–362.
16
1
1
Herrerias, J. A. Mayoral and I. Perez, Green Chem., 2010, 12
,
46 L. Lomba, S. Muñiz, M. R. Pino, E. Navarro and B. Giner,
Ecotoxicology, 2014, 23, 1484–1493.
4
2 J. I. Garcia, H. Garcia-Marin, J. A. Mayoral and P. Perez, Green 47 V. L. K. Jennings, M. H. Rayner-Brandes and D. J. Bird, Water
35–440.
1
1
1
1
1
1
1
1
2
2
2
2
Chem., 2010, 12, 426–434.
3 L. Aldea, J. I. Garcia and J. A. Mayoral, Dalton Trans., 2012,
41, 8285–8289.
4 M. De Torres, I. W. C. E. Arends, J. A. Mayoral, E. Pires and G.
Jimenez-Oses, Appl. Catal. A-Gen., 2012, 425, 91–96.
5 S. Queste, P. Bauduin, D. Touraud, W. Kunz and J.-M. Aubry,
Res., 2001, 35, 3448–3456.
Green Chem., 2006,
6 M. Sutter, W. Dayoub, E. Métay, Y. Raoul and M. Lemaire,
ChemCatChem, 2013, , 2893–2904.
8, 822–830.
5
7 M. Sutter, L. Pehlivan, R. Lafon, W. Dayoub, Y. Raoul, E.
Métay and M. Lemaire, Green Chem., 2013, 15, 3020–3026.
8 M. Sutter, W. Dayoub, E. Métay, Y. Raoul and M. Lemaire,
Green Chem., 2013, 15, 786–797.
9 L. Moity, M. Durand, A. Benazzouz, C. Pierlot, V. Molinier and
J.-M. Aubry, Green Chem., 2012, 14, 1132–1145.
0 J. I. García, H. García-Marín, J. A. Mayoral and P. Pérez, Green
Chem., 2013, 15, 2283–2293.
1 W. B. Arbuckle and J. E. Alleman, Water Environ. Res., 1992,
64, 263–267.
2 M. Salizzato, V. Bertato, B. Pavoni, A. V. Ghirardini and P. F.
Ghetti, Environ. Toxicol. Chem., 1998, 17, 655–661.
3 M. Farré, F. Arranz, J. Ribó and D. Barceló, Talanta, 2004, 62
,
5
4 Henkel KGaA, Rep. No 9400063, 1994.
5 OECD SIDS Initial Assessment Report, 2002.
6 G. Bringmann and R. Kühn, Water Res., 1980, 14, 231–241.
7 B. Dabrock, H. Bahl and G. Gottschalk, Appl. Environ.
Microbiol., 1992, 58, 1233–1239.
8 D. R. M. Passino and S. B. Smith, Environ. Toxicol. Chem.,
49–558.
2
2
2
2
2
2
3
3
3
1
987,
9 F. Onorati and M. Mecozzi, Chemosphere, 2004, 54, 679–
87.
0 K. M. Docherty and J. Charles F. Kulpa, Green Chem., 2005,
85–189.
6, 901–907.
6
7,
1
1 A. Romero, A. Santos, J. Tojo and A. Rodríguez, J. Hazard.
Mater., 2008, 151, 268–273.
2 J. Ranke, K. Mölter, F. Stock, U. Bottin-Weber, J. Poczobutt, J.
Hoffmann, B. Ondruschka, J. Filser and B. Jastorff, Ecotoxicol.
Environ. Saf., 2004, 58, 396–404.
3
3
3
3
3
3
3 M. Alvarez-Guerra and A. Irabien, Green Chem., 2011, 13
507–1516.
4 H. Zhao, S. Xia and P. Ma, J. Chem. Technol. Biotechnol.,
005, 80, 1089–1096.
5 S. Keskin, D. Kayrak-Talay, U. Akman and Ö. Hortaçsu, J.
Supercrit. Fluids, 2007, 43, 150–180.
6 R. J. Bernot, M. A. Brueseke, M. A. Evans-White and G. A.
Lamberti, Environ. Toxicol. Chem., 2005, 24, 87–92.
7 J. E. Dubois, Computer Representation and Manipulation of
Chemical Information, John Wiley & Sons, New York, 1974.
8 L. B. Kier and L. H. Hall, Molecular Connectivity in Structure-
Activity Analysis, Ed. Research Studies Press Ltd., New York,
,
1
2
1
985.
9 A. R. Katritzky and E. V. Gordeeva, J. Chem. Inf. Comput. Sci.,
993, 33, 835–857.
0 A. Leo, C. Hansch and D. Elkins, Chem. Rev., 1971, 71, 525–
16.
3
4
1
6
8
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Green Chemistry
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DOI: 10.1039/C5GC00857C
Table of contents entry
Most glycerol-derived ethers display low or null ecotoxicity
against Vibrio fischeri biomarker, supporting the low
environmental impact of this biobased solvent family.