e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 977–981

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A new biological test of water toxicity–yeast

Saccharomyces cerevisiae conductometric test

Jaroslava Dolezalova, Lubomira Rumlova ∗

Central Military Hospital – Military Faculty Hospital Prague, U Vojenské nemocnice 1200, 169 02 Praha 6,
Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: This new biological test of water toxicity is based on monitoring of specific conductivity
Received 27 March 2014 changes of yeast Saccharomyces cerevisiae suspension as a result of yeast fermentation activity
Received in revised form inhibition in toxic conditions. The test was verified on ten substances with various mecha-
30 September 2014 nisms of toxic effect and the results were compared with two standard toxicity tests based
Accepted 10 October 2014 on Daphnia magna mobility inhibition (EN ISO 6341) and Vibrio fischeri bioluminescence inhi-
Available online 18 October 2014 bition (EN ISO 11348-2) and with the results of the S. cerevisiae lethal test (Rumlova and
Dolezalova, 2012). The new biological test – S. cerevisiae conductometric test – is an express
Keywords: method developed primarily for field conditions. It is applicable in case of need of imme-
Biological test diate information about water toxicity. Fast completion is an advantage of this test (time
Water toxicity necessary for test completion is about 60 min), the test is simple and the test organism –
Saccharomyces cerevisiae dried instant yeast – belongs among its biggest advantages because of its long-term storage
Specific conductivity life and broad availability.
© 2014 Elsevier B.V. All rights reserved.

frequently used for testing. These tests require continuous

1. Introduction
Water pollution by various chemical substances and unknown
products of their mutual reaction or decomposition is very
common. Water may be contaminated by incorrect handling of
chemicals and waste, but also in a deliberate terrorist attack.
Number of easily accessible toxic substances abused for this
purpose is large.
Modern methods of chemical analysis may determine very
small amounts of water contaminants. Chemical analysis, in
fact, does not detect the toxicity of a sample of water, but only
the presence and concentration of selected toxic substances.
Non-specific biological toxicity tests may indicate the pres-
ence of a broad spectrum of chemical contaminants, including
mixtures and products of their interactions.
The freshwater fish Brachydanio rerio (EN ISO 7346) and
the cladoceran Daphnia magna (EN ISO 6341) are the most
breeding of the test organisms in prescribed physiological con-
ditions. Testing time is 24–96 h. This time span is too long
when it is necessary to get immediate information about water
toxicity in case of environmental accidents, wastewater tox-
icity monitoring, drinking water control, etc.
The only standard rapid biological test used worldwide is
bacterial bioluminescence test using the bacteria Vibrio fischeri
(EN ISO 11348-2). The lyophilized bacteria can be stored for a
long time and they are applicable to the measurement when-
ever, without long time of preparation. However, this test has
several limiting factors (pH interference, colouration and tur-
bidity of samples, etc.).
For non-specific detection of toxic substances it is neces-
sary to use different methods of bioassay, which complement
each other in spectrum of detected substances and in indi-
cation sensitivity. Therefore, we focused on research of new

∗
Corresponding author. Tel.: +420 973 208 559; fax: +420 224 313 327.
E-mail address: lubomira.rumlova@centrum.cz (L. Rumlova).
http://dx.doi.org/10.1016/j.etap.2014.10.009
1382-6689/© 2014 Elsevier B.V. All rights reserved.
978 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 977–981
rapid biological water toxicity tests. As the test organism, we
selected yeast Saccharomyces cerevisiae, which meets all the
requirements of a biological object suitable for rapid toxicity
testing. Its big advantage is the possibility of long-term storage
in a dry state (18 months under normal storage conditions).
When water is added, the yeast immediately comes alive and
it is ready for use in toxicity test.
We have proposed several new bioassays using yeast. The
first of them was lethal test, based on detection of changes
in yeast viability after exposure to toxic substances solution
(Rumlova and Dolezalova, 2012).
In this paper we present a description and results of the
water toxicity conductometric test, which is based on the
monitoring of changes of specific conductivity in yeast S. cere-
visiae suspension as a result of yeast fermentation activity
inhibition in toxic conditions.
Ten toxic substances with different mechanism of action
were used to verify the proposed method. First of all, the rep-
resentatives of three groups of potentially militarily misused
substances were selected: atropine (mentally and physically
disabling substance), fenitrothion (organophosphate whose
basic structure is the same as of nerve agents) and potas-
sium cyanide (poisonous substance in general – inhibitor of
the respiratory chain). Aconitine (plant toxin), mercuric chlo-
ride and lead nitrate (representatives of heavy metals salts),
sodium sulfide (toxic and caustic agent, dangerous to aquatic
organisms), arsenic trioxide (protoplasmic poison), thallium
sulfate (nerve poison, easily misused by terrorists) and phe-
nol (phenols from industrial wastes are a common source of
pollution of natural waters) were the other tested toxic sub-
stances.
We compared the results of the conductometric test with
two standard toxicity tests based on D. magna mobility inhibi-
tion (EN ISO 6341) and V. fischeri bioluminescence inhibition
(EN ISO 11348-2) and with the results of the S. cerevisiae
lethal test, based on the detection of yeast viability changes
(Rumlova and Dolezalova, 2012).
2. Materials and methods
2.1. Chemicals
Aconitine 95%, arsenic trioxide 99.5%, atropine 95%, feni-
trothion 99.9%, mercuric chloride 99.5%, lead nitrate 95%,
potassium cyanide 99.9%, phenol 100%, sodium sulfide 98%
and thallium sulfate 98%. All the chemicals were purchased
from Sigma–Aldrich company.
Sucrose – common refined table sugar purchased at a
supermarket.
2.2. Test organism
Instant dehydrated baker’s yeast, Saf-Instant, from the com-
pany Lesaffre, France (S. cerevisiae Hansen) was used.
2.3. Devices
The portable conductometer Cond 3110 with conductivity
probe TetraCon 325 from WTW GmbH (Germany) was used.
2.4. S. cerevisiae conductometric test
20 ml of examined water were pipetted in five test tubes
and 2.0 g of sugar were added in each tube. Other five test
tubes with sugar solution without any toxic substances were
prepared as control samples. After dissolution, the specific
conductivity of the sugar solution was measured in all test
tubes.
1.0 g of yeast was added into each of the test tubes and
stirred well. After 30 min of exposure the specific conductivity
was measured again in each test tube. It was indispensably
necessary to keep the same temperature in all test tubes dur-
ing the test.
2.5. Data analysis
To eliminate conductivity differences of the water sample and
the control sample, it is necessary to adjust the measured
values of the specific conductivity. It means that the appro-
priate value of the specific conductivity of the sugar solution
is subtracted from the specific conductivity of yeast suspen-
sion. Then the average corrected specific conductivity of the
tested suspension and the control suspensions is calculated.
If the difference between the average corrected specific con-
ductivity of the tested suspension and the control suspension
is 10% or more, it is assumed that the tested water sample
contains a substance toxic to the test organism.
The data were analyzed by regression analysis. The EC50
(30 min) values were defined as the concentration of toxicants,
which cause a 50% change of the average corrected specific
conductivity in comparison with the average corrected specific
conductivity of the control sample.
From the repeated experiments and their statistical evalu-
ations (F-test and t-test, signification level of 0.05) it emerged
that an average corrected specific conductivity decrease about
10% is statistically significant and it represents the lowest tox-
icant concentration reliably detectable by this test.
3. Results
The EC50 (30 min) values for nine toxic substances obtained
from the S. cerevisiae conductometric test are shown in Table 1.
These values are compared with EC50 (15 min) values for V. fis-
cheri bacteria bioluminescence test (EN ISO 11348-2) and with
EC50 (48 h) values for D. magna test (EN ISO 6341) reported in the
literature, in the Material safety data sheets, Sigma–Aldrich
or in the test reports of the Czech accredited laboratory
EMPLA (2007). The EC50 (30 min) values are also compared with
EC50 (60 min) values for S. cerevisiae lethal test (Rumlova and
Dolezalova, 2012). The EC50 (30 min) value for the tenth test
substance – fenitrothion – is not listed in Table 1, because this
value is higher than the solubility of fenitrothion in water.
The lowest concentrations (EC10 30 min) of the ten toxic
substances that the new conductometric and the lethal test
can reliably detect are shown in Table 2. For comparison, toxic
and lethal doses of these toxic substances for man are also
mentioned, based on the data from Simeonova and Fishbein
(2004), Patočka (2004), Yoshida et al. (1987), Kolev and Batev
(1996) and from BIOTOX, HERA and IPCS INCHEM databases.
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 977–981 979

Table 1 – Comparison of EC50 values from S. cerevisiae conductometric and lethal test and two standard toxicity tests.
Toxicant S. cerevisiae S. cerevisiae lethal V. fischeri standard test D. magna standard
conductometric test, test, EC50 EN ISO 11348-2, EC50 test EN ISO 6341,
EC50 (30 min)a (mg l−1 ) (60 min)b (mg l−1 ) (15 min) (mg l−1 ) EC50 (48 h) (mg l−1 )
Aconitine 38.7 13.28 – –
Atropine 179.2 240 More than 1000c 422.4c
Fenitrothion Not possible 1.12 8.6c 0.04c
to determine
Pb2+ 558.1 490 88.4e 2.0f
Phenol 523.7 515.1 32e 12.0f
Hg2+ 110.1 69 0.046e 0.03f
KCN 42.4 0.3 13.8d 2.0f
Arsenic trioxide 187.2 102.1 43.6f 8.2f
Thallium sulfate 41.5 40.8 – 2.2f
Sodium sulfide 79.8 571.7 4.3f 7.1f
a
Results of this study.
b
Rumlova and Dolezalova (2012).
c
Environmental Laboratory EMPLA (2007).
d
PAN Pesticide Databaze.
e
Dutka and Kwan (1981).
f
Material safety data sheets, Sigma–Aldrich.

Nweke (2010) investigated effect of toxicity of metals to

4. Discussion
The yeast S. cerevisiae is a convenient model organism for eval-
uation of toxic effect on human cells and tissues. It is often
used in study of genotoxicity of different chemicals.
Several papers inform about using of S. cerevisiae to water
toxicity testing. For example Esteve et al. (2009) presented a
bioassay based on inhibition of S. cerevisiae metabolic activity
at level of adenosine-5-triphosphate synthesis, as compared
with standard toxicity tests with D. magna (EN ISO 6341) and
V. fischeri (EN ISO 11348). Three pesticides were tested. The
yeast biotest was more sensitive than V. fischeri test, but less
sensitive than the D. magna test.
S. cerevisiae using TTC-dehydrogenase activity inhibition test.
The assay confirmed the toxicity of metals to S. cerevisiae. The
time to obtain the results was more than 24 h.
Suppi et al. (2014) studied the toxicity mechanisms of the
silver nanoparticles. Toxicity was evaluated by growth inhibi-
tion in rich YPD medium and cell viability in deionized water.
Hosiner et al. (2014) determined the lowest observable
effect level and EC50 values of 10 metal ions on yeast growth,
which was monitored via optical density. The time to obtain
the results was more than 12 h.
The studies focused on monitoring of toxicants effects on a
fermentation activity of the yeast were the most important for
our study. For example Weber et al. (2000) observed changes

Table 2 – EC10 (30 min) values from S. cerevisiae conductometric and lethal test and the toxicological data for tested
substances.
Toxicant S. cerevisiae conductometric S. cerevisiae lethal test, EC10 Toxic human dose Lethal human
test, EC10 (30 min)a (mg l−1 ) (60 min)b (mg l−1 ) (mg) dose (mg)
Aconitine 14.7 1.45 0.5c 5c
Atropine 135 0.44 5d 100d
Fenitrothion Not possible to determine 0.024 – 429 mg kg−1 i
Pb2+ 8 7.9 – 500c
Phenol 55 23.2 – 1000c
Hg2+ 15 0.41 – 500e
KCN 12.5 0.003 50f 200d
Arsenic trioxide 16 8.5 50c 150c
Thallium sulfate 13 6.4 500g 1000g
Sodium sulfide 10 105 – 10,000h
a
Results of this study.
b
Rumlova and Dolezalova (2012).
c
IPCS INCHEM, International Programme of Chemical Safety.
d
Patočka (2004).
e
Kolev and Batev (1996).
f
Simeonova and Fishbein (2004).
g
BIOTOX.
h
HERA, Human and Environmental Risk Assessment.
i
Yoshida et al. (1987).
980 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 977–981

in the fermentation activity of the yeast S. cerevisiae. This was toxic or lethal doses for man. This is a very important rea-
done by measuring the CO2 production of yeast cells in sus- son for using of this test for non-specific detection of toxic
pension with toxic substances. Organic compounds, inorganic substances in water.
salts (especially of heavy metals), surfactants and plant pes- S. cerevisiae conductometric test is mostly less sensitive
ticides were tested. The authors compared the results with than our previously published S. cerevisiae lethal test (Rumlova
the data of assay using Tetrahymena pyriformis and found a and Dolezalova, 2012). However, the conductivity test is faster,
congruence of 90% in sensitivity of both tests. simpler and less labour-intensive.
Hrenovic et al. (2005) evaluated yeast toxicity test, based
on the inhibition of saccharose fermentation activity by the
S. cerevisiae. The authors used the standard toxicants (cop-
5. Conclusion
per sulphate, formaldehyde, sodium nitrite, sodium sulphite,
The conductometric test is a completely new biological
phenol and zinc sulphate). The toxicity of wastewater from
method for the rapid detection of toxicants in water. The test
pharmaceutical industry obtained from yeast test corresponds
is based on the monitoring of conductivity changes of yeast S.
sufficiently with the results obtained by standard method
cerevisiae suspension as a result of yeast fermentation activity
(inhibition of V. fischeri bioluminescence, inhibition of dehy-
inhibition in toxic conditions.
drogenase activity).
The assay was validated on ten toxic substances with dif-
Weber et al. (2006) presented a newly developed particle
ferent mechanisms of action. The test sensitivity is generally
contact assay that uses the fermentation performance of the
lower than the sensitivity of standard tests with bacteria V. fis-
yeast S. cerevisiae for assessment of toxic effects in sediments.
cheri and cladoceran D. magna, but it indicates the presence of
Yeast cells were cultivated in sediment samples and their
seven in ten tested toxic substances in water at significantly
resulting fermentation performance was continuously mea-
lower concentrations than toxic or lethal doses for man.
sured.
The S. cerevisiae conductometric test is very simple and
These works are based on the measurement of CO2 pro-
fast in comparison with above-mentioned standard tests. It
duction. The time to obtain the results was more than 24 h.
requires only the conductometer, the yeast S. cerevisiae, sugar
Because of looking for a quick method suitable for use in field
and minimal laboratory equipment. This new test can extend
conditions, we did not measure the amount of CO2 production.
the capabilities of non-specific integrated rapid detection of
The changes in fermentation activity of yeast we investigated
toxic substances in water as part of the range of biological
indirectly by measuring of specific conductivity of yeast sus-
toxicity tests.
pension.
All the above cited studies confirm the sensitivity of S.
cerevisiae to different groups of toxic substances. Most of the Conflict of interest
authors, however, worked with another toxicants than those
used in our study. Only Weber et al. (2000) published the EC20 The authors declare that there are no conflicts of interest.
(24 h) = 54 mg l−1 for Pb2+ and the EC20 (24 h) = 0.8 mg l−1 for
Hg2+ . The EC20 (30 min) value for Pb2+ determined by our con-
ductometric test was 85 mg l−1 and the EC20 (30 min) value Transparency document
for Hg2+ was 19 mg l−1 . These data demonstrate that the test
based on the measuring of the CO2 production changes has a The Transparency document associated with this article can
comparable or a higher sensitivity than our S. cerevisiae con- be found in the online version.
ductometric test, but the time to obtain the results was 23 h
longer in comparison with our test.
Acknowledgements
We verified our test on ten toxic substances with different
effects. The biggest toxic effect was from aconitine. Unfortu-
We thank the anonymous reviewers for their critical com-
nately, comparable data of the D. magna standard test (EN ISO
ments which improved the manuscript considerably. The
6341) and of the V. fischeri test (EN ISO 11348-2) for aconitine
experiments described in this work were carried out in compli-
are not available. The sensitivity of S. cerevisiae conductomet-
ance with the current law of the Czech Republic. The research
ric test to other toxicants is relatively low. The EC50 values
was founded by Ministry of Defense of the Czech Republic,
were higher in the S. cerevisiae test (Table 1) compared with
grant number 6330.
the two standard tests. The EC50 (30 min) value for fenitroth-
ion was not assessed, because it is higher than solubility of
fenitrothion in water. references
The sensitivity of the new test is illustrated by the EC10
(30 min) values determined for all examined toxicants, except
for fenitrothion (Table 2). From repeated experiments and their BIOTOX, http://www.biotox.cz/toxikon (accessed on 26.08.14).
Dutka, B.J., Kwan, K.K., 1981. Comparison on three microbial
statistical evaluation, it emerged that they represent the low-
toxicity screening tests with the Microtox test. Bull. Environ.
est concentration of toxicants that the test can reliably detect.
Contam. Toxicol. 27, 753–757.
Comparison of EC10 values with the toxic and lethal doses EN ISO 6341, 1998. Water Quality – Determination of the
for man is very important. Table 2 shows, that the new con- Inhibition of the Mobility of Daphnia magna Straus
ductometric test can indicate a presence of seven in ten toxic (Cladocera, Crustacea) – Acute Toxicity Test. European
substances in water at significantly lower concentrations than Committee for Standardisation, Brussels.
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 977–981
EN ISO 7346, 1996. Water Quality – Determination of the Acute
Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio
rerio Hamilton-Buchanan (Teleostei, Cyprinidae)]. European
Committee for Standardisation, Brussels.
EN ISO 11348-2, 1998. Water Quality – Determination of the
inhibitory effect of water samples on the light emission of
Vibrio fischeri (Luminescent Bacteria Test) – Part 2: Method
Using Liquid-Dried Bacteria. European Committee for
Standardisation, Brussels.
Environmental Laboratory EMPLA, Accredited Testing Laboratory
No. 1110. Test reports No. T 535/2007, T 536/2007, T 544/2007
(in Czech).
Esteve, K., Poupot, C., Dabert, P., Mieton, M., Milisic, V., 2009. A
Saccharomyces cerevisiae-based bioassay for assessing pesticide
toxicity. J. Ind. Microbiol. Biotechnol. 36, 1529–1534.
HERA, 2006. Human and Environmental Risk Assessment.
http://www.heraproject.com (accessed 24.08.14).
Hosiner, D., Gerber, S., Lichtenberg-Fraté, H., Laser, W., Schuler,
Ch., 2014. Impact of acute metal stress in Saccharomyces
cerevisiae. PLOS ONE 9, e83330,
http://dx.doi.org/10.1371/journal.pone.0083330,
http://www.plosone.org (accessed 26.08.14).
Hrenovic, J., Stilinovic, B., Dvoracek, L., 2005. Use of prokaryotic
and eukaryotic biotests to assess toxicity of wastewater from
pharmaceutical sources. Acta Chim. Slov. 52, 119–125.
IPCS INCHEM, International Programme of Chemical Safety.
http://www.inchem.org/documents/pims/chemical/inorglea
(accessed 24.08.14).
Kolev, S.T., Batev, N., 1996. Mercury. National Poisons Information
Service, Medical Toxicology Unit, London, http://www.
inchem.org/documents/ukpids/ukpids/ukpid27.html
(accessed 26.08.14).
981
Material safety data sheets, Sigma–Aldrich
http://www.sigmaaldrich.com (accessed 26.08.14).
Nweke, C.O., 2010. Effects of metals on dehydrogenase activity
and glucose utilization by Saccharomyces cerevisiae. Nigerian J.
Biochem. Mol. Biol 25, 28–35.
Patočka, J., 2004. Military Toxicology. Grada Publishing and
Avicenum, Prague (in Czech).
Rumlova, L., Dolezalova, J., 2012. A new biological test utilising
the yeast Saccharomyces cerevisiae for the rapid detection of
toxic substances in water. Environ. Toxicol. Pharmacol. 33,
459–464.
Simeonova, F.P., Fishbein, L., 2004. Hydrogen Cyanide and
Cyanides. Human Health Aspects. Concise International
Chemical Assessment Document 61. World Health
Organization, Geneva,
www.inchem.org/documents/cicads/cicads/cicad61.htm
(accessed 06.08.14).
Suppi, S., Kasemets, K., Kahru, A., 2014. Toxicity mechanisms of
coated and uncoated solver nanoparticles to yeast
Saccharomyces cerevisiae. Toxicol. Lett. 229 (Supplement),
S194–S195 (Abstracts).
Weber, J., Plantikow, A., Kreutzmann, J., 2000. A new bioassay
with the yeast Saccharomyces cerevisiae on aquatic pollution.
Umweltwiss. Schadst-forsch 12, 185–189.
Weber, J., Kreutzmann, J., Plantikow, A., Pfitzner, S., Claus, E.,
Manz, W., Heininger, P., 2006. A novel particle contact assay
with the yeast Saccharomyces cerevisiae for ecotoxicological
assessment of freshwater sediments. J. Soils Sediments 6,
84–91.
Yoshida, M., Shimada, E., Yamanaka, S., Aoyama, H., Yamamura,
H., Owada, S., 1987. A case of acute poisoning with
fenitrothion (Sumithion). Hum. Toxicol. 10, 403–406.