1. 1813
Environmental Toxicology and Chemistry, Vol. 18, No. 8, pp. 1813–1816, 1999
᭧ 1999 SETAC
Printed in the USA
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Methods
A BEHAVIORAL TOXICITY TEST USING THE CILIATED PROTOZOAN
TETRAHYMENA THERMOPHILA. I. METHOD DESCRIPTION
GUY GILRON,*† SCOTT G. GRANSDEN,‡ DENIS H. LYNN,‡ JIM BROADFOOT,§ and RICHARD SCROGGINS࿣
†12 Pine Street, Erin, Ontario N0B 1T0, Canada
‡MicroVita Consulting, 23 Lyon Avenue, Guelph, Ontario N1H 5C5, Canada
§Broadfoot Consulting, R.R. 1, Midland, Ontario L4R 4K3, Canada
࿣Environment Canada, Method Development and Application Section, 3439 River Road South,
Gloucester, Ontario K1A 0H3
(Received 12 March 1998; Accepted 20 October 1998)
Abstract—A novel behavioral toxicity test using the ciliated protozoan Tetrahymena thermophila is described. Tests using a control
water (spring water) and two reference toxicant solutions (sodium chloride and guaiacol) yielded reproducible results, indicating
that the method has potential as a reliable toxicity test for use in industrial effluent monitoring.
Keywords—Tetrahymena thermophila Toxicity test Chemotaxis Guaiacol Behavior
INTRODUCTION
Microbial species, which are easily manipulated experi-
mentally, are increasingly being used as test organisms to eval-
uate the biological effects of chemical stressors on aquatic
ecosystems. Toxicity tests that have used bacteria, algae, pro-
tozoa, and microinvertebrates [1] are not only typically rapid
and sensitive, but also easily performed. The information
gained from the responses of representatives from these diverse
trophic levels has expanded the body of knowledge on the
ecological effects of anthropogenic contaminants on aquatic
ecosystems [1].
Ciliated protozoa are abundant and ubiquitous components
of aquatic ecosystems and act as important trophic interme-
diaries between microbial and macrobial components of aquat-
ic food webs, and as such are useful in evaluating water quality
and monitoring the environmental effects of industrial effluents
[2]. Studies that use the saprobity system [3] and artificial
substrates [4] to assess anthropogenic impacts on ciliate com-
munities typically require taxonomic expertise and are time-
consuming and labor-intensive. Studies that examine the ef-
fects of specific chemical toxicants on single species of ciliates
[2,5–7] are more rapid and do not require taxonomic expertise.
However, only one of these toxicity test methods measuring
the concentration–response relationships of ciliates to toxi-
cants has undergone an interlaboratory comparison [8,9]. None
of the test methods, to our knowledge, has examined the re-
sponse of ciliates to complex contaminant mixtures, such as
municipal or industrial effluents, specifically at sublethal re-
sponse levels.
In light of this, a new sublethal method [10] was developed
to measure the response of freshwater ciliates to environmental
toxicants. This test measures the chemotactic response (i.e.,
movement toward or away from a toxicant source) exhibited
by ciliates when they encounter chemicals in a test environ-
* To whom correspondence may be addressed
(ggilron@yahoo.com). The current address of G. Gilron is ESG In-
ternational, 361 Southgate Drive, Guelph, Ontario N1G 3M5, Canada.
ment [11]. The test system combines a simple apparatus, a
T-maze, to assess chemotaxis as previously used in cell phys-
iology studies by Van Houten and colleagues [11,12] and a
toxicological approach developed by Berk and colleagues [13–
15].
This paper describes the standard operating procedure for
this behavioral toxicity test using ciliates as test organisms.
To evaluate the test design, commercially available spring wa-
ter (as control dilution water) and two reference toxicants,
sodium chloride (a common reference toxicant for freshwater
species, and one previously tested using the T-maze test design
[11,12]) and guaiacol (a resin acid often detected in pulp mill
effluent), were used. The common freshwater ciliate, Tetra-
hymena thermophila (Oligohymenophorea; Tetrahymenidae),
was used to evaluate the effects of these toxicants.
MATERIALS AND METHODS
A summary of culture and test conditions is presented in
the Appendix. The main elements of the standard operating
procedure are described below. All solutions must be at room
temperature (20 Ϯ 2ЊC). Ambient laboratory lighting condi-
tions should be between 100 and 500 lux.
Culture medium preparation
Prepare a proteose peptone yeast extract (PPYE) medium
by adding dextrose (0.5 g), proteose peptone (2.0 g), and finally
yeast extract (2.0 g) to 400 ml of distilled water, and stirring
until the ingredients are dissolved. Autoclave in batch volumes
as required below.
Culture preparation
Forty-eight hours before the food deprivation pretreatment,
transfer, using sterile technique, 1 ml of stock T. thermophila
culture (ATCC 30382 Strain B-18684 [1975] or ATCC 30383
Strain B-18686 [1975], American Type Culture Collection,
Manasas, MD, USA) to 10 ml of sterile PPYE. After 24 h,
transfer, using sterile technique, the 10 ml culture to 50 ml of

2. 1814 Environ. Toxicol. Chem. 18, 1999 G. Gilron et al.
Fig. 1. T-maze toxitactic assay apparatus.
sterile PPYE in a 250-ml Erlenmeyer flask. At this time, the
corks (C8215-2TC, CANLAB, Mississauga, ON, Canada) for
the T-mazes should be soaked in commercially available spring
water (which is also used as dilution water in the test). After
an additional 24 h, centrifuge the 50 ml of PPYE culture in
four 15-ml centrifuge tubes at 1,200 rpm (300 g) for 3 min.
With a Pasteur pipette, remove the supernatant, ensuring that
the pellet of cells is not disturbed. Gently and completely
resuspend the four pellets of cells into one 15-ml centrifuge
tube and then top up to 15 ml with spring water. Repeat the
centrifugation process two times, removing the supernatant
between steps and resuspending the pellet each time with ap-
proximately 15 ml of spring water.
Food deprivation pretreatment
Transfer the rinsed and resuspended ciliates using a Pasteur
pipette into 35 ml of spring water (for a total of 50 ml) in a
250-ml Erlenmeyer flask and leave for 18 h. Repeat the above
centrifugation process with the 50 ml of food-deprived cells.
After one centrifugation, gently and completely resuspend the
four pellets of cells. Combine the pellets and resuspend in 10
ml of spring water. Determine the cell density and adjust to
400,000 cells/ml (Ϯ10%) using spring water.
Motility test for toxicant concentration range
For each of five test concentrations in a dilution series,
initially 100, 50, 25, 12.5, and 6.25%, perform the following
motility test. Place approximately 10 to 20 ␮l of test cells in
the bottom of each of five wells of a nine-well spot depression
slide (Corning 7220, Thomas Scientific, Swedesboro, NJ,
USA). Add the five test solutions in a 50:1 ratio (at the very
least). Immediately note the degree of cell motility as follows:
acceptable motility—more than one half of the cells are motile;
or unacceptable motility—at least one half of the cells are not
motile. After 5 min, assess cell motility again. The highest
concentration in which motility is acceptable will become the
highest concentration for the definitive 20-min test (see below).
If this is not the highest concentration in the dilution series,
this concentration should become the highest concentration for
the dilution series for the definitive 20-min test (see below).
If after 5 min acceptable motility is not observed in any di-
lution, establish a dilution series with the lowest concentration
as the top and repeat the 5-min tests until acceptable motility
is observed. After 20 min, again assess cell motility. The high-
est concentration of test solution in which T. thermophila ex-
hibit acceptable motility after 20 min will be the highest of
the five concentrations in the test dilution series. If after 20
min acceptable motility is not observed in any dilution, es-
tablish a dilution series with the lowest concentration as the
top and repeat the 20-min test until acceptable motility is ob-
served.
Test apparatus and design
A T-maze apparatus (LG9601-114, LG9680106-183050,
Lab Glass, Vineland, NJ, USA) is used in the implementation
of the test (Fig. 1). Stopcocks should be greased sparingly with
high-vacuum grease before use. The test design comprises
three replicate glass T-mazes, for each of five concentrations
in the dilution series and a control (spring water in both arms
of the maze) for a total of 18 mazes.
Exposure test
The stopcock of each T-maze (Fig. 1) is turned so that the
bore is in line with the third (upright) arm. The T-maze arms
should be labeled test and control and are then filled, one at
a time, with the respective solutions. Rinse all the corks with
fresh spring water immediately before these steps. First deliver
the test toxicant solution into one arm using a 14.61-cm Pasteur
pipette and cork the arm. Then fill the control arm with spring
water using a clean 14.61-cm Pasteur pipette and insert the
cork. To ensure that no air bubbles are caught in the arms, or
around the stopcocks, firmly (but gently) strike the T-maze
apparatus on the palm of the hand at a 45Њ angle. If necessary,
release any air bubbles, refill, and recork. Using a 22.86-cm
Pasteur pipette, the cells are then transferred from a homo-
geneous suspension at 400,000 cells/ml into the stopcock bar-
rel, filling to just above the level of the bore. Gently tap the
bottom of each T-maze apparatus to remove any air bubbles
from the stopcock barrel and top up, if necessary. After all
T-mazes bores are completely loaded, quickly turn all stop-
cocks to a level point, aligning them with the two arms of the
maze, so that the cells are able to migrate freely through the
arms. It is critical that the stopcock barrel be completely
aligned with the stopcock arms at this point. At the end of a
20-min exposure period, quickly turn all stopcocks to a closed
position (i.e., upright) to terminate the exposure test.
Cell enumeration
Immediately after the exposure test is completed, the arms
of the T-maze are emptied into counting tubes (e.g., small glass
test tubes, Coulter counter cuvettes). Using a 14.61-cm Pasteur
pipette, rinse each arm with a small volume of the test solution
that was just removed from that arm to ensure that all cells
will be removed. Enumerate the cells under ϫ400 magnifi-
cation, as follows: homogenize the cells in the counting tubes
by inverting the tubes or using a Vortex mixer; depending on
density, take 5- to 10-␮l samples from each counting tube and
add this to each of five wells of a polystyrene 96-well micro-
titer plate with flat wells (e.g., Corning 255880-96, Thomas
Scientific); and add 20 ␮l of dilution water and 10 ␮l of Lugol’s
iodine solution to each of the five wells. Add a 1 to 10 volume
of Lugol’s iodine to each counting tube to preserve cells in
the event that recounting is required. The total counts for test
or control microtiter wells must exceed 200 ciliates (to meet
a statistically precise estimate of cell density [16]). Record the
replicate counts on a standard bench sheet.
Data analysis
A toxitactic index (Itox) is then calculated for each T-maze
as follows:
T
I ϭtox
T ϩ C

3. Behavioral toxicity test using ciliates Environ. Toxicol. Chem. 18, 1999 1815
Fig. 2. Results from sodium chloride experiments. Mean Ϯ 1 SE,
dashed line ϭ no effect.
Fig. 3. Results from guaiacol experiments. Mean Ϯ 1 SE, dashed line
ϭ no effect.
where T is the mean number of ciliates in the test arm and C
is the mean number of ciliates in the control arm.
The Itox index is a measure of chemotaxis, and is therefore
a number between 0 and 1. An Itox greater than 0.5 indicates
positive chemotaxis, or attraction, whereas an Itox less than 0.5
indicates negative chemotaxis, or avoidance. Using standard
statistical software, a number of toxicity endpoints can be
calculated with the Itox data. Endpoints such as the concentra-
tion causing an effect in 50% of the population (IC50) or the
lowest-observed-effect concentration (LOEC) can be easily
calculated using these data. The IC50, for example, is a com-
mon endpoint that can be calculated by the linear interpolation
method [17] using a standard software package [18].
Test validity criteria
The test is invalid if the mean control Itox value is outside
the range 0.43 to 0.57, and total cell counts (control and/or
test arms added) are Յ200 cells. The first validity criterion
was determined based on an evaluation of the range of control
(spring water) Itox values determined (see below) and represents
one standard deviation of the mean control Itox value (Appen-
dix). The second criterion is based on statistical guidance re-
lating to the precision for counting unicellular organisms mi-
croscopically, as provided by Venrick [16].
Test method evaluation
The test method was evaluated by conducting experiments
with controls (i.e., spring water in both arms of the T-maze)
and with two reference toxicants. Sodium chloride was tested
using the concentration series 10,000 mg/L, 5,000 mg/L, 2,500
mg/L, 1,250 mg/L, 625 mg/L, and a control. Guaiacol was
tested using the concentration series 1.5 g/L, 0.75 g/L, 0.38
g/L, 0.19 g/L, 0.1 g/L, and a control.
RESULTS AND DISCUSSION
Control (spring water) treatments
An ANOVA was applied to the complete control data set
and indicated that T. thermophila showed no preference for
either arm of the T-maze. The mean Itox value was 0.50 with
an SD of Ϯ0.062 (n ϭ 33).
Sodium chloride (NaCl) treatments
Test organisms consistently moved toward (i.e., exhibited
an attraction to) this reference toxicant. The LOEC was cal-
culated as 2,500 mg/L with a mean Itox value at the LOEC of
0.66 (n ϭ 3) (Fig. 2). These results are comparable to endpoints
reported for invertebrates (e.g., Daphnia magna 48-h EC50
for immobility: 2,250–4,500 mg/L [19], 1,661–4,571 mg/L
[20]; Ceriodaphnia dubia 7-d IC50 for reproduction: 1,500
mg/L [BEAK International Ecotoxicity Laboratory, unpub-
lished data]); and fathead minnow 96-h LC50 [acute lethality]:
2,000 mg/L [21]).
Guaiacol treatments
Test organisms moved away from (i.e., exhibited an avoid-
ance reaction to) this reference toxicant. A reasonably good
concentration–response relationship was found, with an LOEC
of 380 mg/L (adjusted to pH 7.5) as the final endpoint and a
mean Itox value at the LOEC of 0.24 (n ϭ 3) (Fig. 3). This
value is much less sensitive than literature values for fish sur-
vival (e.g., rainbow trout acute 96-h LC50 [acute lethality]:
44 mg/L [22]).
CONCLUSION
This toxicity test has a number of important benefits. The
test method is relatively simple to conduct in comparison to
other ciliated protozoan toxicity testing techniques [5]. The
exposure duration to test organisms is only 20 min, so that
many replications can be performed in a fraction of the time
required to run a standard acute lethality test (i.e., 48 or 96
h). The test organisms grow rapidly (i.e., generation time ϭ
2–5 h) and are easily cultured in the laboratory. The test mea-
sures a behavioral response that provides an early indication
of sublethal effects.
Additional method validation work required to standardize
the test method has recently been conducted, including an
interlaboratory comparison (‘‘round robin’’) using reference
toxicants and pulp mill effluents and a sensitivity comparison
with other standard sublethal tests using pulp mill effluents.
This work will be presented in an upcoming publication.
Acknowledgement—We wish to thank J. Van Houten, C. Blaise, G.
Atkinson, T. Kovacs, and K. Holtze for valuable input and guidance
in various aspects of the study. S. Hattie, V. Olson, S. Knutson, and
M. O’Reilly provided valuable technical support. B. Zajdlik and two
anonymous reviewers provided insight and helpful comments on the
draft manuscript. The study was funded by the Research Advisory
Committee of the Ontario Ministry of the Environment, Environment
Canada (Centre St.-Laurent), and the Environment Canada Green Plan
Fund through the Environmental Innovation Program.
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4. 1816 Environ. Toxicol. Chem. 18, 1999 G. Gilron et al.
APPENDIX
Summary of test conditions for the T-maze toxitactic assay using
freshwater ciliates
Test condition Description
Test type Static, nonrenewal
Duration 20 min
Temperature 20 Ϯ 2ЊC
Lighting Ambient laboratory light in the range 100–
500 lux (diffuse, nondirectional lighting)
Test vessels T-maze apparatus (see Fig. 1)
Test volume 5 ml
Control water Commercially available spring water
Ciliates Tetrahymena thermophila; 400,000 cells/ml
(Ϯ10%) in initial culture
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No. replicates Three replicate mazes (at a minimum) at
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Feeding 18-h period of food deprivation in control
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Measurements Temperature, pH, hardness, conductivity,
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