Ecotoxicology

https://doi.org/10.1007/s10646-019-02107-0

TECHNICAL NOTE

Evaluation of dimethyl sulfoxide (DMSO) as a co-solvent for toxicity

testing of hydrophobic organic compounds
1,2
Jakub J. Modrzyński ●
Jan H. Christensen
1 1
●
Kristian K. Brandt

Accepted: 3 September 2019

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Toxicity testing of hydrophobic compounds with low aqueous solubility remains challenging. Dimethyl sulfoxide (DMSO)
is widely used as a co-solvent for toxicity testing of hydrophobic chemicals, but it may modulate chemical toxicity patterns.
In this study, we critically evaluated the suitability of DMSO as a co-solvent for toxicity testing of hydrophobic organic
compounds in aqueous solutions. As the toxicity measure, we used growth inhibition of a natural bacterial community, and
the test toxicants included phenol, BTEX (benzene, toluene, ethylbenzene and xylene) and transformation products of
polycyclic aromatic hydrocarbons (PAHs). We found that dose-response curves for phenol were unaffected by DMSO
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concentrations up to 10% (v/v) and that DMSO (5% v/v) did not affect the degree of bacterial growth inhibition for any of
the other test compounds in short-term experiments (3.5 h). By contrast, marked co-solvent effects of DMSO were observed
in the long-term assay (25 and 27 h). We therefore conclude that DMSO has excellent co-solvent properties for short-term
(≤3.5 h) toxicity testing of sparingly water-soluble compounds and its application provides a simple, inexpensive approach
for screening of various environmentally relevant hydrophobic chemicals. Importantly, the use of DMSO allows for
generation of full dose-responses that may otherwise not be attained.
Keywords BTEX Ecotoxicity Hydrophobic organic compounds Leucine incorporation Soil bacterial community
● ● ● ● ●

Solubility

Introduction other molecular reagents for various biological applications

Use of a co-solvent can increase the effective water solu-
bility of hydrophobic test chemicals and is commonly
applied in (eco)toxicological investigations. In this way, full
dose-response curves can be obtained even for hydrophobic
test compounds that are only sparingly soluble in water
(Iizumi et al. 1998; Murado et al. 2011; Hafner et al. 2015).
Co-solvents are also frequently used to dissolve probes and
Supplementary information The online version of this article (https://
doi.org/10.1007/s10646-019-02107-0) contains supplementary
material, which is available to authorized users.
* Kristian K. Brandt
kkb@plen.ku.dk
1 solvents in (eco)toxicological tests: (i) the co-solvent may
Department of Plant and Environmental Sciences, University of
Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark
2 may interact with the toxicant tested, thereby skewing dose-
Department of Geochemistry, Geological Survey of Denmark and
Greenland (GEUS), Øster Voldgade 10, 1350 Copenhagen
Denmark
(Wiederschain 2011). Dimethyl sulfoxide (DMSO) is a
frequently used co-solvent for toxicity testing as it is mis-
cible with water and most organic liquids (Brayton 1986).
DMSO is a powerful aprotic solvent used as a drug solvent,
permeability enhancer, cryoprotectant, or anti-inflammatory
agent (Williams and Barry, 2004; Yu and Quinn, 1994). It
has been widely used as co-solvent in bioassays, including
toxicological studies, yet mostly at very low concentrations
(<0.1%) to prevent co-solvent artifacts (Ames et al. 1975;
Lee et al. 2013; Maron and Ames, 1983; Smith et al.
2013a). However, there are also many examples from the
literature, where higher DMSO co-solvent concentrations
(up to 10% v/v) have successfully been used for microbial
toxicity testing (e.g. Bundy et al. 2003; Dong et al. 2013;
Hafner et al. 2015; Negin et al. 2015).
There are two major concerns regarding use of co-
itself be toxic to the test organism(s) and (ii) the co-solvent
response curves for the test compound (Negin et al. 2015).
Provided that the co-solvent toxicity is only partial, the first

concern can be overcome by using the same co-solvent
concentrations in all samples, including controls. However,
the potential interactions between co-solvent and test che-
mical effects in the tested organisms is more problematic
and may compromise the use of DMSO for ecotoxicity
testing (Bundy et al. 2003; Hafner et al. 2015; Negin et al.
2015). Suitability of DMSO co-solvent properties was
previously tested for a series of amphiphilic pyrogallolar-
enes and three different antibiotics with two E.coli strains in
pure cultures. Despite some growth inhibition caused by
DMSO, the concentration range of 5–10% v/v was found
appropriate for bringing sparingly water-soluble chemicals
into aquatic solutions. However, the authors concluded that
DMSO showed different effects depending on the identity
of the organism and the antibiotic and suggested conducting
controls between DMSO and each of the individual com-
ponent in the experiment in order to avoid erroneous
inference of synergy (Negin et al. 2015).
DMSO have also been used as a co-solvent for tox-
icological studies of petroleum-derived compounds (PDCs)
(e.g. Bundy et al. 2003, 2004; Iizumi et al. 1998, Murado
et al. 2011). PDCs are toxic to cellular life forms and
constitute important environmental contaminants (Dawson
et al. 2007; Modrzyński et al. 2016; Prince and Douglas,
2010; Sikkema et al. 1995). Toxic PDCs include aromatic
substances such as benzene, toluene, ethylbenzene and
xylenes (collectively called BTEX) and polycyclic aromatic
hydrocarbons (PAHs, e.g. naphthalene), as well as various
transformation products like phenols and acids. Ecotox-
icological testing of these substances is often challenged by
their high hydrophobicity and low water solubility (Smith
et al. 2013b; Tsao et al. 1998, Ha et al. 2019), which may
make it impossible to obtain full dose-response curves
during short-term assays in water suspensions. This is
especially a challenge for microbial community-level toxi-
city testing, which must be performed as short-term assays
in order to avoid bias due to the microbial community
adaptation processes such as biodegradation of the test
compound or changed community composition (Brandt
et al. 2015).
Our objective was to assess applicability of DMSO as a
co-solvent for (eco)toxicological tests of PDCs by evalu-
ating the DMSO effect on the test organisms and possible
interactions between DMSO and test toxicants. Hence, we
assessed both the toxicity of DMSO alone, and the ability of
DMSO to modulate the toxicity of PDCs. Phenol was
chosen as a model PDC toxicant and used to obtain dose-
response curves at DMSO concentrations ranging from 0 to
10% v/v. BTEX and various polar naphthalene derivatives
were tested at their water saturation concentrations in the
presence (5% v/v) or absence of DMSO. Bacterial com-
munities extracted from unpolluted soil were used as the test
organisms because only little information is available on
J. J. Modrzyński et al.
how DMSO may modulate toxicity in complex bacterial
assemblages. Bacterial communities were also selected,
because they provide several crucial ecosystem services
(Brandt et al. 2015). Toxicity was assessed as inhibition of
bacterial growth activity as measured by the [3H]leucine
incorporation technique (Bååth et al. 2001), which has
recently been suggested as a sensitive and relevant micro-
bial ecotoxicity assay appropriate for environmental risk
assessment (Brandt et al. 2015). We hypothesized, that
although DMSO may itself be toxic, it will not interact with
the toxicity of hydrophobic and hydrophilic PDCs when
used in concentrations up 10% v/v.
Materials and methods
Highly diverse soil bacterial communities originated from
unfertilized agricultural soil (top 20 cm; sandy loam, pH
6.8, moisture 12% d.w.) sampled in April 2012 from a long-
term field trial (CRUCIAL experiment, University of
Copenhagen Experimental Farm, Taastrup, Denmark)
(Poulsen et al. 2013; Lekfeldt et al. 2014). Growth activity
was measured by [3H]leucine incorporation assay as
described previously (Bååth et al. 2001; Brandt et al. 2009)
with the modification that all incubations were carried out in
1.5-mL screw-cap glass vials with polytetrafluoroethylene-
coated septa (i.e. Teflon; Waters) to avoid contact with
plastic or rubber known to sorb the tested toxicants. MOPS
buffer (3-morpholinopropane-1-sulfonic acid, Sigma-
Aldrich; final conc. 10 mM, pH 6.8) was used for extraction
of soil bacteria for all tests with aromatic organic com-
pounds. Soil bacterial suspensions were preincubated with
the test chemicals in the dark at 22 °C for 0.5–24 h (see the
results for details) prior to addition of [3H]leucine (2.59
TBq mmol−1, 37 MBq mL−1, Amersham, Hillerød, Den-
mark) and unlabeled L-leucine to yield a final leucine
concentration of 200 nM and an added radioactivity of
6 kBq per vial. Leucine incorporation incubations were
carried out in a total volume of 1.6 mL (1.5 mL soil bac-
terial suspension +50 µL toxicant solution +50 µL [3H]
leucine solution) for 3 h, then stopped by transferring the
samples to 2-mL plastic microtubes containing 160 µl ice-
cold 50% trichloroacetic acid (TCA) to kill bacteria and
precipitate proteins. Samples amended with TCA prior to
addition of [3H]leucine served as negative (killed) controls.
[3H]leucine incorporated into precipitated proteins was
separated from the non-incorporated fraction by a series of
centrifugation and washing steps, re-dissolved in 1 M
NaOH, and measured by scintillation counting (Perkin-
Elmer, USA) (Modrzyński et al. 2016).
Solutions of phenol, benzene, toluene, ethylbenzene, m-
xylene, 1-naphthoic acid, 2-naphthoic acid, 1-naphthol and
1,6-naphthalenediol were prepared either from DMSO stock
Evaluation of dimethyl sulfoxide (DMSO) as a co-solvent for toxicity testing of hydrophobic organic. . .
solutions (the test chemical dissolved in DMSO) or from
saturated water stocks (i.e. deionized water with crystals).
The data for the test substances (including solubility) were
taken from ChemID website: http://chem.sis.nlm.nih.gov/
chemidplus.
Toxicity of the chemicals was assessed as inhibition of
bacterial growth, normalized to corresponding control
samples with the same amount of DMSO. One-way
ANOVA was used to determine effect of DMSO alone,
and two-way ANOVA was used to determine significant
interactions between DMSO level and PDC toxicity. For
phenol toxicity assessment, the data were log-transformed
(following a visual analysis of the data with ‘boxcox’
function from MASS package) and linear regression models
were made using ‘lm’ and ‘gls’ functions in R (R Devel-
opment Core Team, 2014). ANOVA and modeling of the
sigmoidal 3-parameter logistic curve fit to data were per-
formed in Sigma Plot 13.0.
Results and discussion
DMSO showed low short-term toxicity (0.5 h preincubation
+3 h incubation) to soil bacterial communities at con-
centrations up to 10% v/v (Fig. 1). Slightly higher toxicity
was observed for long-term assays (22 h preincubation +3 h
incubation), but only at DMSO concentrations above 5% v/
v (Fig. 1). DMSO concentrations between 1 and 5 % v/v
significantly inhibited bacterial growth (p < 0.05), but the
degree of inhibition did not exceed 20%. These results are
generally in line with a previous study by Negin and
Fig. 1 Effect of DMSO (% v/v) on growth of soil bacterial commu-
nities as measured by the [3H]leucine incorporation technique. DMSO
concentrations of 0, 1, 2.5, 5, 7.5, 10 and 20 % (v/v) were tested.
Preincubation time (i.e. contact time between bacteria and DMSO
prior to addition of [3H]leucine) was either 0.5 h or 22 h. Means ±
standard deviations (n = 3) are shown
colleagues, who tested growth inhibition of pure cultures of
Escherichia coli over 20-h incubation (Negin et al. 2015).
One discrepancy between the two studies was that DMSO
initially slowed E.coli growth (first 4 h), which was recov-
ered after about 20 h (Negin et al. 2015), whereas we
observed slightly higher toxic effect at the longer incubation
(Fig. 1). The difference can likely be attributed to the fact
that we used a complex soil bacterial community, whereas
Negin & co-workers used two specific E. coli strains as test
organisms. This suggests some variation in sensitivity to
DMSO among different microorganisms (Negin et al.
2015), thus, it may be advantageous to determine strain-
specific DMSO sensitivity when using pure cultures in a
DMSO-mediated toxicant assessment.
Full dose-response curves with up to 100% inhibition of
bacterial growth were obtained for phenol (Fig. 2 and
Supplementary Material, Fig. S1). Although DMSO par-
tially inhibited growth (Fig. 1; p < 0.001), the dose-response
curves were highly similar regardless of the DMSO con-
centration used (0–10% v/v). This indicates that DMSO did
not interact with phenol toxicity, which was confirmed by
two-way ANOVA using growth inhibition data for the 5 to
10 mM phenol concentration range (corresponding to 471
and 941 mg/L, respectively; 0.5 h preincubation;
p = 0.418).
The ability of DMSO to modulate PDCs toxicity was
also tested for BTEX compounds and several polar naph-
thalene derivatives (PAH transformation products). This
was performed by comparing growth inhibition in the pre-
sence or absence of DMSO (5% v/v) at the maximal water-
solubility concentration of each test compound (PDC water
saturation, ‘1 × S’). Growth inhibition did not depend on the
presence or absence of DMSO for the short-term assays
(0.5 h preincubation; Fig. 3). For BTEX compounds at the
saturation level, benzene showed the highest toxicity, fol-
lowed by toluene, ethylbenzene and m-xylene. This order
also reflects the water solubility of the PDCs (Table 1) and
thus the dose applied. The toxicity posed by polar naph-
thalene derivatives varied with the lowest toxicity for 2-
naphthoic acid and highest toxicity for the naphthols. As
expected, the toxic effects were more pronounced for all test
compounds when concentrations equivalent to 5-fold water
saturation were applied using DMSO as a co-solvent as
compared to the water saturation conditions (Fig. 3). This
demonstrates the ability of DMSO to solubilize and increase
bioavailability of PDCs (Supplementary Material, Figs. S2
and S3). For 1-naphthol and 1,6-naphthalenediol the rela-
tive growth rates were <1% already at water-solubility level
(data not shown), thus the effect of DMSO addition could
not be assessed.
In stark contrast to our data obtained from the short-term
assays (0.5 h preincubation; Fig. 3), growth inhibition was
not consistently observed for the long-term assays (24 h
 J. J. Modrzyński et al.

Fig. 3 Effect of PDCs on growth of soil bacterial communities in the

presence or absence of 5% v/v DMSO as measured by the [3H]leucine
incorporation technique following 0.5-h preincubation period (i.e.
contact time between bacteria and test chemicals prior to addition of
[3H]leucine). The growth rate was calculated relative to corresponding
controls with the same DMSO level. Means ± standard deviations (n
= 3) are shown. ‘1 × S’ is test compound concentration at water
saturation (Table 1); ‘5 × S’ is test compound concentration 5 times
higher than the water saturation. 1-NA, 1-naphthoic acid; 2-NA, 2-
naphthoic acid

Table 1 Concentrations at water saturation (mg L−1) for the

compounds used in this study
Compound name Solubility (mg L−1)a

Fig. 2 Effect of phenol on growth of soil bacterial communities at four Phenol 82 800b
concentrations of DMSO (% v/v) as measured by the [3H]leucine Benzene 1 790
incorporation technique. Preincubation time (i.e. contact time between
bacteria and test chemicals prior to addition of [3H]leucine) was either Toluene 526
0.5 h (a) or 24 h (b). The growth rate was calculated relative to cor- Ethylbenzene 169
responding controls with the same DMSO level. Means (n = 3) are m-xylene 161
shown. The error bars represent the average standard deviation for all
data shown on the panel. The line represents a 3-parameter logistic 1-Naphthoic acid 86
curve fit to data obtained for all four DMSO concentrations 2-Naphthoic acid 47
1-Naphthol 866
preincubation). Hence, marked co-solvent effects of DMSO 1,6-Naphthalenediol 165
on the degree of bacterial growth inhibition were observed a
Defined at 20 °C
in the long-term assay and several PDCs even stimulated b
Equals 879.8 mM
bacterial growth (Supplementary Material, Fig. S3). This
may be explained by the long-term response of the tested
bacterial communities, which may use the test compounds effects on sea urchin larvae growth, which however was not
as sources of carbon and energy (Modrzyński et al. 2016) or attributable to DMSO, but rather due to stimulatory action
in other ways adapt to the PDC presence, for instance, by of some oil components or hormesis (Murado et al. 2011).
preferential growth of resistant community members (Berg Therefore, DMSO should probably only be used for short-
et al. 2010; Lekfeldt et al. 2014; Brandt et al. 2015). Given term toxicity bioassays to avoid co-solvent effects.
the possibility for such complex community-level respon- The use of DMSO for toxicity testing also has other
ses, the effects of DMSO in the long-term assay should be limitations, even when no interactions between the co-
treated with caution. Similarily, a 48-h exposure to low solvent and test compound occur. Hence, the test com-
doses of DMSO fuel oil extracts showed slight stimulatory pound’s concentration is intentionally increased above its
Evaluation of dimethyl sulfoxide (DMSO) as a co-solvent for toxicity testing of hydrophobic organic. . .
water-solubility, which might compromise the direct trans-
lation of the results to environmental conditions. Moreover,
the use of co-solvents does not prevent losses (by degra-
dation or evaporation) of the test compounds from the test
solution. Passive dosing techniques, broadly acknowledged
as state-of-the-art for toxicity testing of hydrophobic com-
pounds, provide solutions for these issues (Thomas et al.
2015). Hence, passive dosing enables fairly constant
exposure to either nonvolatile (e.g. PAHs) or highly volatile
(e.g. BTEX) hydrophobic compounds (Smith et al. 2015;
Modrzyński et al. 2016); however it does not work for
hydrophilic substances. In contrast, the DMSO co-solvent
approach allows for parallel comparison of several organic
compounds varying in both volatility and water solubility
using the same experimental setup. Moreover, our approach
allows for obtaining full dose-response curves, which is
often not available with passive dosing (Smith et al. 2015;
Redman et al. 2017). Finally, using DMSO is more
straightforward and inexpensive, which makes it an attrac-
tive simple alternative for passive-dosing techniques, sui-
table for e.g. quick screening of a broad spectrum of
compounds and their mixtures (Iizumi et al. 1998; Subbarao
et al. 2006).
Collectively, our data indicate that DMSO can be safely
used as a co-solvent for short-term (≤3.5 h) bacterial growth
inhibition assays at concentrations up to 3.3–10% (v/v) as it
does not modulate the toxicity of PDCs, such as phenol,
BTEX and naphthalene derivatives. Similarly, Negin et al.
(2015) and Dong et al. (2013) concluded that DMSO con-
centrations up to 5–10% v/v are appropriate to enhance
solubility of chemicals otherwise insoluble in water.
Recently, DMSO has been used as a co-solvent for acute
toxicity test (24 h) with aquatic invertebrates exposed to a
set of petroleum-derived compounds, including benzene,
toluene, naphthalene and dibenzothiophene, yet in ranges
not exceeding their solubility (Ha et al. 2019). The authors
concluded that preparation of the test solutions using
DMSO had no effect on the test organisms, nor on the
concentration of the compounds. However, DMSO may not
work equally well for all toxicants as it may interfere with
the mode of toxic action for some compounds. DMSO has
for instance been reported to modify bacteriostatic effects of
antibiotics (Negin et al. 2015), scavenge reactive oxygen
species (ROS) and alter cellular membrane permeability
(Williams and Barry, 2004; Yu and Quinn, 1994). Due to
the synergistic interaction with antibiotics, it has been
suggested to conduct controls between each individual
compound and DMSO (Negin et al. 2015). Noteworthy, in
contrast to majority of antibiotics, the primary mode of
toxic action for PDCs is general narcosis, being a non-
specific effect (Sikkema et al. 1995; Smith et al. 2013b).
Therefore, although we cannot entirely exclude the possi-
bility for similar DMSO effects in environmental bacteria,
our work suggests that the aforementioned interferences are
unlikely to constitute any significant problem for toxicity
testing of PDCs and other narcotics using bacterial com-
munity growth inhibition.
Overall, DMSO has excellent co-solvent properties for
sparingly water-soluble compounds, and its application can
be a useful and affordable experimental approach for eco-
toxicological evaluation for both individual bacterial strains
and complex microbial assemblages. Due to its powerful
and universal solvent properties, DMSO enables generation
of full dose-response curves for hydrophobic test chemicals
even in short-term assays, which may otherwise not be
possible for compounds with limited water solubility. This
represents an advantage for a number of ecotoxicological
applications such as the analysis of solvent-generated
extracts from environmental matrices like soils or sedi-
ments (Dawson et al. 2007; Hafner et al. 2015; Subbarao
et al. 2006) or the detection of pollution-induced commu-
nity tolerance (Brandt et al. 2015; Lekfeldt et al. 2014).
Acknowledgements We acknowledge assoc. prof. Bo Markussen
(University of Copenhagen) for assistance and help in statistical ana-
lysis. Funding was obtained from University of Copenhagen via the
emerging elite research area ‘Environmental Chemistry and Ecotox-
icology’ headed by prof. Nina Cedergreen and from the Center for
Environmental and Agricultural Microbiology (CREAM) financed by
the Villum Foundation.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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