Applying whole-water samples directly to fish cell cultures in order to evaluate the toxicity of...

Water Research 36 (2002) 3727–3738

Applying whole-water samples directly to fish cell cultures inorder to evaluate the toxicity of industrial effluent

Vivian R. Dayeha, Kristin Schirmerb, Niels C. Bolsa,*aDepartment of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1

bDepartment of Chemical Ecotoxicology, UFZ-Centre for Environmental Research, Permoserstr, 15, 04318, Leipzig, Germany

Received 1 October 2001; accepted 1 February 2002

Abstract

Methodology was developed for presenting to fish cells in culture whole-water samples without extraction and used

to evaluate the toxicity to a rainbow trout gill cell line, RTgill-W1, of more than 30 whole-water samples collected from

a paper mill over approximately a year of operation. Presentation to cells was achieved by adding to water samples the

amounts of salts, galactose and sodium pyruvate, as solids, that were necessary to give concentrations and osmolality of

the basal growth medium, Leibovitz’s L-15. Cell viability was measured with three fluorescent indicator dyes: alamar

Bluet for metabolism, 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for membrane function, and

neutral red for lysosomal activity. Eighteen samples were tested with the Daphnia lethality bioassay and 11 of these

were toxic. None of these were judged cytotoxic to RTgill-W1. Sixteen samples were tested with the rainbow trout

lethality bioassay and only one was toxic. This sample was also the only sample that was cytotoxic to RTgill-W1.

Therefore, these methods for presenting water samples and measuring their cytotoxicity to RTgill-W1 are a promising

substitute for toxicity tests of industrial effluent with rainbow trout but not with Daphnia.r 2002 Elsevier Science Ltd.

All rights reserved.

Keywords: In vitro toxicity testing; Cell viability; Fish cell lines; Paper mill effluent; Whole effluent testing

1. Introduction

Animal cell lines have been considered periodically as

an alternate technology to the use of whole animals in

testing water quality [1]. Tests with cell lines could

potentially be less expensive but performed more

rapidly. They would also satisfy a societal desire to

reduce the use of whole animals in toxicity testing.

Two different strategies have been used to present

water samples to cells in culture [2,3]. One is to extract

organic contaminants from water in order to concen-

trate them, and to add the extracts to cultures in carrier

solvents, such as dimethyl sulfoxide (DMSO) [4,5]. The

other strategy is to make up culture medium with sample

water, which has been done in two different ways. One is

to add sample water to 2X tissue culture medium,

making the highest water sample concentration 50%

[1,3]. Alternatively, the sample water is added to the

solid (powdered) components of culture medium to

reconstitute complete growth medium [2,6]. In this way,

cells can be exposed to different concentrations of a

whole-water sample, including 100%.

Testing whole-water samples on cells offers several

important advantages, particularly for routine screening

of many environmental samples. Applying the whole

sample to cultures assures that little or no toxicant is lost

in any processing steps. As well, the total toxicity of the

sample, encompassing all potential synergistic, antag-

onistic and additive interactions, is measured. The cost

and time of testing is reduced because labor-intensive

extraction procedures with expensive organic solvents

are eliminated. Costs can also be lowered for other

*Corresponding author. Tel.: +1-519-885-1211x3993; fax:

+1-519-746-0614.

E-mail address: [email protected] (N.C. Bols).

0043-1354/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 4 3 - 1 3 5 4 ( 0 2 ) 0 0 0 7 8 - 7

reasons. The cost of shipping large volumes of effluent

water samples from distant sources to central toxicity

testing facilities is reduced because relatively small

volumes are needed for testing. Finally, whole-water

samples are more analogous to the protocol used to test

the toxicity of water to fish.

Although mammalian cell lines have been used to

monitor water quality [2–4,6,7], fish cell lines have

additional advantages. Fish rather than mammalian

cells are a more logical substitute for fish, which are used

to test water quality. Cell lines from fish are easier to

maintain than those from mammals, as they can be

grown without the need for an incubator or frequent

renewals of the culture medium [8]. In addition, fish cells

tolerate being maintained in culture for a day or two in a

simple exposure medium, which contains only salts,

pyruvate and galactose [9]. The simplicity favors

expression of cellular responses to toxicants because

many protective molecules such as antioxidants are

absent. As well, the medium is much less expensive than

complete cell culture medium.

The goals of this study were four fold. The first was to

develop a routine methodology for applying whole-

water samples in a concentration series to fish cell

cultures. As many biological processes that are studied

in vitro are sensitive to the quality of water [10], the

second goal was to check whether whole-water samples

that were not toxic to rainbow trout elicited cytotoxic

responses in cell cultures of a fish gill cell line, RTgill-

W1. The third goal was to determine whether RTgill-W1

cultures identified whole-water samples that had been

spiked with common organic toxicants, abietic acid and

pentachlorophenol, as being toxic. The final goal was to

evaluate the overall methodology by applying whole-

water samples taken over the span of 1 year of a paper

mill operation to RTgill-W1 cultures and measuring

their ability to cause cytotoxicity. These in vitro toxicity

results were compared with the results obtained on the

same whole-water samples by conventional in vivo

toxicity tests with rainbow trout and Daphnia magna.

2. Materials and methods

2.1. Cell line and culture media

RTgill-W1 is a cell line from gills of rainbow trout

(Oncorhynchus mykiss) [11], and available from the

American Type Culture Collection (ATCC # CRL

2523). Cells were cultured routinely in 75 cm2 culture

flasks at 211C in Leibovitz’s L-15 culture medium, with

10% fetal bovine serum (FBS) and 2% penicillin

streptomycin (100mg/mL streptomycin, 100 IU/mL pe-

nicillin) (Gibco/BRL, Burlington, ON). L-15 osmolality

ranges from 300 to 340mOsmkg�1, depending on the

lot. Exposure of RTgill-W1 cells to potential toxic

solutions was conducted at 211C in the dark for 24 h in

wells of 48 well Falcon plates (Becton Dickinson,

Franklin Lakes, NY). These microwell cultures were

initiated by removing cells from confluent flasks and

adding to each well 30,000 cells in 500mL L-15 with FBS.

Three days later the cultures were confluent and used.

2.2. Whole-water samples

Whole-water samples from an unidentified paper mill

were tested routinely by BEAK International (Bramp-

ton, ON) for toxicity, as required by Canadian Legisla-

tion for the pulp and paper industry [12]. Toxicity was

determined by exposing either rainbow trout or D.

magna to the samples according to proscribed proce-

dures [13,14]. The whole-water samples were of receiving

water (RW), secondary effluent (SEC), final effluent

(FE) and clearwater bypass (CWB). Within 48–72 h of

collection, portions (100–1000ml) of the whole-water

samples were transported to this laboratory in order to

evaluate their cytotoxicity. All in vitro testing was

conducted prior to any knowledge of the results of

toxicity testing with rainbow trout or D. magna.

2.3. Whole-water samples/ex

The whole-water samples were processed so that they

could be applied to fish cell cultures (Fig. 1). Usually,

this began immediately after they were received,

although in a few cases samples were stored in the dark

at 4–81C for 1 to 2 weeks prior to testing. Sample

processing began by determining the pH and osmolality,

measured with a Westcor 5001B vapor pressure osm-

ometer (Westcor, Logan, Utah). Osmolality was well

below the osmolality typically required by animal cells

(Table 1). For the 23 samples with osmolality values

below 90mOsmkg�1, solid amounts of salts, galactose,

and pyruvate were dissolved in the whole-water samples

to give the concentrations that are found in L-15. For

the 8 samples with osmolality values above

90mOsmkg�1, solid amounts of salts, galactose, and

pyruvate were dissolved in the whole-water samples to

give the concentrations that are 80% of those found in

L-15. The osmolality of these solutions was measured

and found to range between 290 and 360mOsmkg�1

(Table 1). Portions of these solutions, as well as

solutions of model toxicants (see below), were filtered

through a 0.2mm membrane filter (Gelman Sciences,

Ann Arbor, MI) in a glass- filter apparatus. These

solutions are referred to as unfiltered and filtered whole-

water samples/ex, where ex abbreviates exposure.

In seven cases, whole-water samples were chosen at

random and examined for microbial contamination by

standard methods agar. Whole-water samples were

diluted in water in a ratio of 1:9 respectively. 100mLwas distributed across the surface of the agar and

V.R. Dayeh et al. / Water Research 36 (2002) 3727–37383728

incubated at room temperature for 24 h after which

colonies were counted.

Whole-water samples/ex were applied to RTgill-W1

cell cultures over a range of concentrations (0%, 15%,

30%, 45%, 60%, 75%, 90%, and 100% (v/v)). The 0%

(control) was L-15/exposure (L-15/ex), which consisted

of L-15 salts, galactose and pyruvate dissolved in

commercial tissue culture water (Gibco/BRL). The

intermediate values (o100%) were obtained by diluting

whole-water samples/ex in L-15/ex. After exposures of

24 h, the viability of RTgill-W1 cultures was measured.

2.4. Model toxicants

Pentachlorophenol (98% purity) was purchased from

Sigma Chemical Co. (St. Louis, MO), abietic acid (85%

purity), from Acros Organics through Fisher Scientific

(Nepean, ON). The chemicals were dissolved in 100%

DMSO (BDH Inc., Toronto, ON) to yield stock

solutions in the following concentrations: pentachlor-

ophenol, 30mg/mL and abietic acid, 1mg/mL. Serial

dilutions of each compound were made in L-15/ex and

in whole-water sample/ex, and both serial dilutions were

tested for their effect on cell viability.

2.5. Cell viability

Three fluorescent indicator dyes, alamar Blue (Med-

icorp, Montreal, PQ), 5-carboxyfluorescein diacetate

acetoxymethyl ester (CFDA-AM) (Molecular Probes,

Eugene, OR) and neutral red (Sigma Chemical Co) were

used to evaluate cell viability in RTgill-W1 cultures. The

dyes were chosen because they were relatively easy to use

and inexpensive. A decline in alamar Blue and CFDA-

AM readings indicate respectively a reduction in cell

metabolism and a loss of cell membrane integrity. The

neutral red assay provides a measure of lysosomal

function. Alamar Blue and CFDA-AM were used as

recently described in a detailed step-by-step format [15].

The neutral red assay was done as described by Schirmer

et al. [9]. Fluorescence units were read with the

CytoFluor series 4000 microplate reader after 30min.

Fig. 1. Schematic of L-15/ex and whole-water sample/ex preparation. Tissue culture salts were dissolved into commercial tissue culture

water to give L-15/ex. L-15/ex was the control in all of the following figures. Tissue culture salts were dissolved in a whole-water sample

to give whole-water sample/ex. Filtration was done through a 0.2 mm Gelman filter. These solutions were used to dose confluent

monolayers of the rainbow trout gill epithelial cell line, RTgill-W1, in wells of 48 well microplates.

V.R. Dayeh et al. / Water Research 36 (2002) 3727–3738 3729

Excitation and emission wavelengths were 530 and

595 nm for alamar Blue, 485 and 530 nm for CFDA-

AM, and 530 and 645 nm for neutral red.

2.6. Data analysis

Raw data from cell viability assays were compiled and

analyzed using Excel (Microsoft Corporation, Red-

mond, WA). The values of the control water sample/ex

and spiked water sample/ex concentrations were ex-

pressed as a percentage of the average fluorescent

readings of the respective L-15/ex control. These values

were averaged for each concentration and their standard

deviations were calculated. The averages and standard

deviations were plotted using SigmaPlot 2000 (SPSS

Inc., Chicago, IL). EC50 values were determined using

the logistic function option in SigmaPlot for dose-

response curves falling below 50% of the control. To test

for significant difference of the experimental values from

the L-15/ex control a one-way ANOVA with Dunnett’s

test (a ¼ 0:05) was conducted in GraphPad Prism

(GraphPad Software Inc., San Diego, CA). These points

are indicated on the graphs with an asterisk (*). An

unpaired t-test (a ¼ 0:05) was conducted to determine if

the spiked water sample/ex yielded fluorescence units

significantly different from the non-spiked water sample/

ex control. These points are indicated on the graphs with

a cross (w).

Table 1

Physico-chemical characteristics of whole-water samples from a paper mill

Sample characteristics

No.a Typeb Appearancec pH (n ¼ 2) Raw osmolality

mOsmkg�1 (n ¼ 2)dAdjusted osmolality

mOsmkg�1 (n ¼ 2)eFiltered? Y/Nf

1 FE Cloudy, yellow 6.97 26.570.7 338.570.7 Y


3 FE Light yellow 7.05 38.570.7 345.574.9 Y


5 CWB Gray with ppte 7.30 56.574.9 352.071.4 N






11 FE Yellow 6.92 123.570.7 299.570.6 N

12 FE Light yellow 6.91 100.570.7 313.570.7 N

13 FE Cloudy, yellow 6.87 111.570.7 310.570.7 N

14 FE Cloudy, yellow 6.93 96.071.4 287.570.7 N

15 FE Light yellow 6.92 96.570.7 302.570.7 N

16 FE Light yellow 7.00 77.071.4 392.570.7 N

17 FE Light yellow 6.81 108.572.1 326.071.4 N

18 FE Gray with ppte 6.80 95.071.4 315.570.7 N

19 FE Light yellow 6.44 89.570.7 314.071.4 N

20 CWB Clear with ppte 6.77 86.071.4 321.570.7 N

21 FE Clear with ppte 6.53 62.572.1 32871.4 N

22 SEC Cloudy, yellow 6.66 50.570.7 34571.4 Y

23 CWB Clear with ppte 6.90 46.071.4 335.071.4 N

24 FE Clear with ppte 7.15 7071.4 329.570.7 N

25 FE Gray with ppte 7.01 78.570.7 321.570.7 N

26 SEC Cloudy, yellow 6.95 84.570.7 32671.4 N

27 FE Cloudy, yellow 6.69 9771.4 30971.4 N

28 CWB Clear with ppte 6.23 7471.4 341.572.1 Y & N

29 SEC Cloudy, yellow 6.81 6370.7 322.071.4 Y & N

30 CWB Clear with ppte 6.93 5571.4 317.072.1 Y & N

31 FE Gray with ppte 6.93 7070.7 330.071.4 Y & N

aWhole-water samples were obtained at different times from the same anonymous paper mill.bFE=final effluent; SEC=secondary effluent; CWB=clearwater bypass.cDescription of the whole-water sample appearance; ppte=precipitate.dOsmolality of the whole-water sample upon reception.eOsmolality of the whole-water sample after addition of salt constituents of L-15/ex.fFiltration through a 0.2 mm Gelman filter to remove any potential bacterial contamination.


3. Results

3.1. Paper mill whole-water sample characteristics

For most samples, pH values were between 6.8 and

7.0 (Table 1). Even the extremes, 6.23 and 7.30, would

not be expected to unduly stress fish cells in culture [16].

Although only seven samples were tested for microbes,

they were always present and their numbers varied 150

fold between sample types (Table 2). Filtering samples

through a 0.2mm filter completely eliminated contam-

ination. All 31 samples had an osmolality of less than

125mOsmkg�1, but addition of L-15 salts to these

samples brought the osmolality to between 290 and

360mOsmkg�1 (Fig. 1).

3.2. Effect of osmolality on cell viability

RTgill-W1 remained viable for at least 24 h in L-15/ex

in which the osmolality ranged from B150 to

400mOsmkg�1. Without the addition of L-15 salts to

the whole-water samples, viability was reduced by

B90% (Fig. 2 Panel A). Viability increased as the

osmolality was increased and reached or was higher than

the value of untreated whole-water samples by

150mOsmkg�1 (Fig. 2 Panel A). As the osmolality of

cultures was increased above the values in control

cultures (32979.6mOsmkg�1; n ¼ 31), viability showed

little sign of significant diminishment until

400mOsmkg�1 and only with neutral red (Fig. 2 Panel

B). Within the 150–400mOsmkg�1 range, some mea-

sures of cell viability were stimulated slightly in cultures

in which the osmolality was either higher or lower than

the control cultures. This stimulation was never more

than 25% (Fig. 2).

3.3. Effect of % whole-water sample on cell viability

As the concentration of common constituents in

whole-water samples could potentially have a detrimen-

tal effect on the viability of cells in culture but not on the

viability of whole organisms, increasing percentages of

whole-water samples, including receiving water, that had

been shown previously not to be toxic to Daphnia and

rainbow trout were applied to RTgill-W1 cultures for

24 h and cell viability measured. Cell viability remained

undiminished (Fig. 3), suggesting that false positives

were unlikely. Whole-water samples in many cases

increased readings in cell viability assays, most com-

monly with unfiltered samples and the alamar Blue

assay. Although the elevation did not consistently occur

at the same % of whole-water sample, the mean increase

for alamar Blue was 43%724 (n ¼ 16).

3.4. Effect of abietic acid-spiked whole-water samples on

cell viability

A 24 h exposure to increasing concentrations of

abietic acid in either non-filtered or filtered whole-water

sample caused a progressive decline in RTgill-W1

viability (Fig. 4). The shapes of dose-response curves

and the EC50 values were comparable for filtered and

non-filtered water samples (Table 3).

3.5. Effect of pentachlorophenol-spiked whole-water

samples on cell viability

A 24h exposure to increasing concentrations of

pentachlorophenol in either non-filtered or filtered

whole-water samples caused a progressive decline in

RTgill-W1 viability as measured with two indicator dyes,

Table 2

Microbial contamination of selected whole-water samples from a paper mill

Sample characteristics

Typea Appearanceb pH (n ¼ 2) Osmolality mOsmkg�1 Microbial population CFU/mLe

Raw (n ¼ 2)c Adjusted (n ¼ 2)d

SEC Light yellow 6.87 2870.7 30170.7 4� 10273� 101

SEC Cloudy, yellow 6.79 6470.7 32072.1 3.1� 10471� 103

CWB Clear with ppte 6.85 5770.7 31771.4 2.6� 10371� 102

CWB Clear with ppte 6.88 2471.4 30471.4 3� 10276� 101

FE Gray with ppte 6.93 7070.7 33071.4 2.4� 10372� 102

FE Cloudy, yellow 6.89 2970.7 30171.4 2� 10271� 101

RW Clear with ppte 6.88 5272.8 32971.4 1.1� 10275� 100

aSEC=secondary effluent; CWB=clearwater bypass; FE=final effluent; RW=receiving water.bDescription of the whole-water sample appearance; ppte=precipitate.cOsmolality of the whole-water sample upon reception.dOsmolality of the whole-water sample after addition of salt constituents of L-15/ex.eMicrobial colony forming units (CFU/mL) after 24 h enumerated using standard methods agar; the microbial population was

completely eliminated upon filtration of the whole-water sample through a 0.2mm filter.


alamar Blue and neutral red (Fig. 5 and Table 3). CFDA-

AM did not yield a dose response relationship in either the

non-filtered or filtered spiked water sample treatments.

3.6. Effect of paper mill whole-water samples on cell

viability

As RTgill-W1 cells could detect toxicants that had

been spiked into whole-water samples, 31 whole-water

samples taken over the span of 1 year of paper mill

operation were evaluated for toxicity with the fish cell

bioassay. Only one sample (# 28), which was clearwater

bypass (CWB), was toxic to RTgill-W1 (Fig. 6). The

dose-response curve for this sample was more distinct

when the sample had been filtered (Fig. 6 Panel B),

except for the neutral red assay, which no longer

registered a loss of viability.

3.7. Comparison of fish cell and D. magna bioassays

The toxicity of 18 whole-water samples from the

paper mill was evaluated with both D. magna and fish

cells. Eleven were toxic to D. magna, but none were toxic

to RTgill-W1 cells. Therefore, the fish cell bioassay was

not able to identify paper mill water samples that were

toxic to D. magna.

3.8. Comparison of fish cell and rainbow trout bioassays

The toxicity of 16 whole-water samples from the

paper mill was evaluated with both rainbow trout and

050100150200250300

% Whole-water sample0 20 40 60 80 100 120

% o

fL-1

5/ex

Con

trol

0

20

40

60

80

100

120

140

160

alamar BlueCFDA-AMNeutral Red

**

*

***

**

*

*

**

*

**

Osmolality (mOsmkg-1)

300 320 340 360 380 400 420

KCl (mg/mL)0 1 2 4 5 7 83 6

% o

fL-1

5/ex

Con

trol

0

20

40

60

80

100

120

140

160


* * *

* *

*

A

B

Osmolality (mOsmkg-1)

Fig. 2. Viability of RTgill-W1 cultures after 24 h in hypoosmo-

tic and hyperosmotic whole-water samples. In panel A, L-15/ex

was mixed with a whole-water sample to create a dosage range

from approximately 15% to 100% water sample (275–

50mOsmkg�1). In panel B, L-15/ex was mixed with KCl to

create a dosage range from approximately 320 to

400mOsmkg�1. Cell viability was assessed with alamar Blue

(closed circles), CFDA-AM (open squares), and neutral red

(closed triangles). The results were expressed as a percentage of

the readings in control cultures in L-15/ex alone. The values are

mean of five culture wells with standard deviation. Asterisks

denote values that were significantly different from control

(one-way ANOVA followed by Dunnett’s Test, a ¼ 0:05). Inpanel A the EC50 values were 103.7mOsmkg�1 for alamar

Blue, 86.27mOsmkg�1 for CFDA-AM and 109.2mOsmkg�1

for neutral red.

% Whole-water sample/ex

0 20 40 60 80 100 120

% o

f L-1

5/ex

Con

trol

0

20

40

60

80

100

120

140


******

Fig. 3. Viability of RTgill-W1 cultures after 24 h in a whole-

water sample that was not toxic to rainbow trout in 96 h

lethality test. Cell viability was assessed with three indicator

dyes as described in Fig. 2. Asterisks denote values that were

significantly different from the control (one-way ANOVA

followed by Dunnett’s Test, a ¼ 0:05).


the fish cells. Only one of these samples was toxic

to rainbow trout in the 96 h lethality test, and this

was the same sample (#28) that was toxic to RTgill-W1

cells. Therefore, the rainbow trout lethality test and

cell bioassay were in agreement for this type of

effluent.

4. Discussion

A methodology was developed for applying to fish

cells in culture whole-water samples with only a limited

number of preparatory steps, none of which included

extraction. The strengths, weaknesses, and future of

2D Graph 4

Abietic Acid (µg/mL)

2D Graph 4

Abietic Acid (µg/mL)

0

20

40

60

80

100

120

140

Non-Filtered

0 20 40 60 80 100 120

% o

fL-1

5/ex

Con

trol

0

20

40

60

80

100

120

140Filtered

0

20

40

60

80

100

120

140alamar Blue

CFDA-AM


0

20

40

60

80

100

120

140Neutral Red


% o

fL-1

5/ex

Con

trol

0

20

40

60

80

100

120

140Neutral Red

% o

fL-1

5/ex

Con

trol

0

20

40

60

80

100

120

140CFDA-AM

alamar Blue

****

* *

**

***

*

*

***

****

*

**

*

*

*****

*

*

*

**

*

†

†

†

† † † †

†

† †

*† † † †

† † †

†

†

†

† † † †

†

†

† †

† †

† † †

† †

†

† † † †

0 20 40 60 80 100 120

0 20 40 60 80 100 120 0 20 40 60 80 100 120

0 20 40 60 80 100 120 0 20 40 60 80 100 120

0 20 40 60 80 100 120 0 20 40 60 80 100 120

Fig. 4. Viability of RTgill-W1 cultures after 24 h in abietic acid. A non-toxic whole-water sample was spiked either with 100 mg/mL

abietic acid in DMSO (closed symbols) or with DMSO alone (open symbols) and either filtered (right panels) or not (left panels).

Abietic acid in L-15/ex was tested simultaneously (shaded symbols). Whole-water sample preparations were mixed in culture wells with

various volumes of L-15/ex in order to obtain a dose-range of abietic acid-spiked whole-water sample/ex (closed symbols) or

appropriate DMSO control (open symbols). Cell viability was assessed with three indicator dyes as described in Fig. 2. Asterisks denote

the % of abietic acid-spiked whole-water sample/ex that resulted in fluorescence units different from those in L-15/ex controls (one-

way ANOVA followed by Dunnett’s test, a ¼ 0:05). The w symbol indicates the % abietic acid-spiked whole-water sample/ex that

yielded fluorescence unit readings significantly different from DMSO-spiked control (unpaired t-test, a ¼ 0:05).


using the cell bioassay are discussed in the following

sections.

4.1. Sample preparation

Preparation of whole-water samples in L-15/ex has

several advantages over the use of complete culture

medium for applying whole-water samples to cells in

culture. The simplicity of this medium, which contains

only salts, pyruvate and galactose, favors expression of

cellular responses to any toxicants that might be present

because protective molecules, such as antioxidants, are

absent. The medium is much less expensive than

complete cell culture medium. As well, the amount of

L-15/ex components can be varied easily to account for

any big differences in the osmolality of whole-water

samples. Finally, the simplicity of the medium reduces

the growth of any microbial contamination during the

24 h of presentation of the whole-water sample to cells in

culture.

The elevation of the readings in the cell viability

assays by whole-water samples likely has multiple

causes, with the importance of each likely depending

on the particular sample. One cause is microbial

contamination from the water sample. This is particu-

larly true for alamar Blue. The microbes would be

difficult to remove completely from the microculture

wells and be carried over into the alamar Blue assay. In

the assay, microbes as well as fish cells would reduce the

dye. In this case, the elevation of alamar Blue readings

should increase as the % of whole-water sample

increases but not be observed with filtered whole-water

samples. However, with some samples, elevated alamar

Blue readings were observed in filtered samples and

the elevation did not increase with increasing sample

concentration. The explanation for this cannot be

microbes. Although osmolality raised alamar Blue

readings, this also was not the explanation for

these water samples because the osmolality

(mOsmkg�1) of water samples in L-15/ex (327721)

was very similar to the osmolality of tissue culture water

in L-15/ex (329710). Another possibility is that the

whole-water samples supply something that is missing in

tissue culture water and that stimulates metabolism of

the cells. A similar hypothesis has previously been put

forth by Schirmer et al. [5] who found exposure of fish

cells to a 90% oil refinery effluent to yield elevated

readings of alamar Blue fluorescence. Some candidate

chemicals that may be supplied are essential trace

minerals.

4.2. Spiked contaminants

Despite the elevation of the indicator dye readings

by some whole-water samples, the fish cell bioassay

was capable of detecting the organic toxicants, abietic

acid and pentachlorophenol (PCP), which could be

expected in paper mill effluent. When spiked into whole-

water samples, these compounds decreased the viability

of RTgill-W1 cultures in a dose-dependent manner.

However, the three viability assays responded slightly

differently to the two different classes of organic

toxicants. An impairment of cellular functions by abietic

acid was detected in whole-water samples with

each of the three indicator dyes, suggesting a general

cytotoxic mechanism. An impairment of cellular func-

tions by PCP in effluent was detected clearly with

two (alamar Blue and neutral red) of the three indicator

dyes, suggesting an interference with energy metabolism

but perhaps less of an impairment of plasma membrane

integrity. These results stress the importance of

using multiple endpoints to detect the presence of

toxicants.

Both abietic acid and PCP appeared to be as potent in

L-15/ex that had been made up in tissue culture water as

in L-15/ex that had been made up in either unfiltered or

filtered water samples. This suggests that the availability

of these contaminants to cells was not influenced by any

suspended matter in the water sample or by filtering the

water sample. The result also suggests that the quality of

the water did not alter the sensitivity of RTgill-W1 to

abietic acid and PCP. Thus, the cell bioassay could be

used to test paper mill samples for their toxic potential

in vitro.

Table 3

Summary of EC50 values for the chemical compounds tested in whole-water samples from a paper mill and in L-15/ex

Compound Indicator dye Non-filtered EC50 (mg/mL) Filtered EC50 (mg/mL) L-15/ex EC50 (mg/mL)

Abietic acid Alamar Blue 29 32 46.9

CFDA-AM 51 51 48.5

Neutral red 32 46 54.6

Pentachlorophenol Alamar Blue 2.72 1.27 2.29

CFDA-AM Not calculated Not calculated 2.30

Neutral red 2.39 2.67 2.49


4.3. Paper mill whole-water samples

The cell bioassay detected toxicant(s) in only 1 (# 28)

of the 31 whole-water samples from the paper mill. As

evaluated with alamar Blue and CFDA-AM, this sample

was toxic even after having been filtered. However, the

neutral red results were complex and difficult to explain.

Firstly, no cytotoxicity was detected with neutral red for

the filtered sample 28. Thus the toxicant(s) in the filtered

sample must be less damaging to lysosomes than to cell

membrane integrity or metabolic activity. Secondly, in

the unfiltered sample 28, cytotoxicity was more pro-

nounced with neutral red than with alamar Blue and

CFDA-AM. Although the expectation would be that

Pentachlorophenol (µ(µ g/mL)Pentachlorophenol g/mL)0 1 2 3 4 5 0 1 2 3 4 5

% o

f L-1

5/ex

Con

trol

0

20

40

60

80

100

120

140

0

20

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120

140

Non-Filtered

0 20 40 60 80 100 120 0 20 40 60 80 100 120

0 20 40 60 80 100 120 0 20 40 60 80 100 120

0 20 40 60 80 100 120 0 20 40 60 80 100 120

% o

f L-1

5/ex

Con

trol

0

20

40

60

80

100

120

140Filtered

0

20

40

60

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120

140alamar Blue

CFDA-AM


0

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140Neutral Red


% o

f L-1

5/ex

Con

trol

0

20

40

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140Neutral Red

CFDA-AM

alamar Blue

*

***

****

****

*

****

** *

**

*

**

**

***

* * *

* * * *

* *

*

† †

† †

†

† †

†

† † *† † † †

† † † † † †

†

† † † † †

† †

† † † †

† † †

† †

†

† † † †

Fig. 5. Viability of RTgill-W1 cultures after 24 h in pentachlorophenol (PCP). A non-toxic whole-water sample was spiked either with

4.4mg/mL PCP (closed symbols) or with DMSO alone (open symbols) and either filtered (right panels) or not (left panels). PCP in L-

15/ex was tested simultaneously (shaded symbols). Whole-water sample preparations were mixed in culture wells with various volumes

of L-15/ex in order to obtain a dose-range of PCP-spiked whole-water sample/ex (closed symbols) or appropriate DMSO control (open

symbols). Cell viability was assessed with three indicator dyes as described in Fig. 2. Asterisks denote % of PCP-spiked whole-water

sample/ex that resulted in fluorescence units different from those in L-15/ex controls (one-way ANOVA followed by Dunnett’s test,

a ¼ 0:05). The w symbol indicates the % PCP-spiked whole-water sample/ex yielded fluorescence unit readings significantly different

from DMSO-spiked control (unpaired t-test, a ¼ 0:05).


any microbial contamination would not interfere with

the neutral red assay because prokaryotic microbes lack

lysosomes, microbes could have caused lower neutral

red readings in at least two ways. They could have

metabolized the neutral red making less available for the

fish cells to accumulate or they could have produced

something that specifically impaired lysosomal func-

tions. An alternative explanation for the lower neutral

red readings in the unfiltered sample is the presence in

sample 28 of a filterable toxicant that is specific for

lysosomes. This problem with the neutral red assay and

sample 28 suggests two recommendations for future

screens of industrial effluent. Firstly, more than one

endpoint of cell viability should be tested. Secondly,

both filtered and unfiltered sample should be tested, if

possible.

4.4. Comparison of fish cell and D. magna bioassays

RTgill-W1 appeared less sensitive than D. magna to

toxicant(s) in the water samples as 11 samples were toxic

to D. magna but not to the gill cell line. This might be

because Daphnia are more generally sensitive to

toxicants or more sensitive to specific classes of toxicants

than cells in culture. In a study of 19 metals, D. magna

was more sensitive than the cells of the fathead minnow

cell line (FHM) [17]. This was most pronounced for very

toxic heavy metals, such as mercury and silver. D. magna

was also more sensitive to surfactants than hepatocytes

and gill epithelial cells from rainbow trout [18]. Thus in

vitro tests with RTgill-W1 or other fish cells are unlikely

to replace Daphnia tests, which are inexpensive, not

subject to criticism on ethical grounds, and the back-

bone of environmental safety testing.

4.5. Comparison of fish cell and rainbow trout bioassays

Despite the complexity of the neutral red results, the

fish cell bioassay identified the only sample that was

toxic to rainbow trout. However, the rainbow trout

appeared to have been more sensitive than the RTgill-

W1 cells to the toxicant(s) in sample 28. All 10 rainbow

trout died in the 96 h lethality test, whereas the viability

of the cell cultures was reduced at the most only by

about 55%. One possible explanation for this difference

is that the toxicant(s) require more time than the 24 h of

the in vitro tests to elicit their full toxicity. Exposing

RTgill-W1 cultures to samples for a longer period could

test this. However, as a routine practice, this is not

desirable because microbial contamination is more likely

to appear and overwhelm the fish cell cultures. Another

possibility is that the particular toxicant(s) in this sample

are more potent at the organismal level than the cellular

level. Toxicants that target specific organ systems, such

as the nervous system, might fit into this category.

The reason(s) why sample 28 was toxic to both cells

and rainbow trout is unknown and was not the focus of

this study. However, the normal industrial process was

not the source of toxicant(s) because sample 28 was from

the ‘clearwater bypass’. This is water that is used in

cooling of the plant and eventually will receive effluent,

but a sample of ‘clearwater bypass’ has not yet received

effluent. Therefore, the toxicant(s) could have arisen

from some temporary change in the piping such as a

high concentration of antiscalant.

Using fish cell lines to evaluate effluent toxicity has

several advantages over the standard rainbow trout 96-h

Non-Filtered


0 20 40 60 80 100 120

% o

f L-1

5/ex

Con

trol

0

20

40

60

80

100

120 alamar BlueCFDA-AMNeutral Red

**

***

*

*

****

***

** *

* * *

Filtered


% o

f L-1

5/ex

Con

trol

0

20

40

60

80

100

120


*

*

**

****

**

**

0 20 40 60 80 100 120

B

A

Fig. 6. Viability of RTgill-W1 cultures after 24 h in whole-

water sample 28 from the paper mill. The sample had been

filtered in Panel B but not in Panel A. Cell viability was assessed

with three indicator dyes as described in Fig. 2. Asterisks denote

the % of whole-water sample/ex that resulted in fluorescence

units different those in L-15/ex controls (one-way ANOVA

followed by Dunnett’s test, a ¼ 0:05).


lethality bioassay. One is cost. This method requires

milliliters instead of hundreds of liters of effluent, which

reduces the cost of shipping samples from distant

industrial sites to central testing facilities. Once estab-

lished as a routine procedure fish cell cultures could be

less expensive than the routine purchase of rainbow

trout. The exposure time is only 24 h as opposed to 96 h,

which could reduce the cost of labor but more

importantly allow decisions to be made more rapidly.

Another advantage is that the use of fish cell cultures

satisfies a societal goal to reduce the use of animals in

toxicity testing.

Yet, additional studies should be considered to further

show the reliability of the RTgill-W1 bioassay as a

replacement for the rainbow trout 96-h lethality test.

More samples that are toxic to rainbow trout need to be

evaluated in vitro, as well the feasibility of applying

whole water samples from different industries to fish

cells needs to be explored. Also how salts might

influence cytotoxicity could contribute to understanding

the reliability of the in vitro tests. Eventually, enough

results could be obtained to allow a correlation between

in vivo and in vitro toxicity to be established more

generally. Once this has been done, whole-water samples

could be applied to fish cells to routinely screen

industrial effluents for the purpose of quickly identifying

episodic events of high toxicity.

5. Conclusions

Based on the analysis of the results obtained from this

study, the following conclusions can be drawn:

(I) A methodology was developed for presenting to

fish cells in culture unextracted samples of

industrial effluent (whole-water samples).

(II) Exposure of the rainbow trout cell line, RTgill-

W1, to some whole-water samples from a paper

mill for 24 h caused no change in cell viability as

measured with three fluorescent indicator dyes:

alamar Blue for metabolism, CFDA-AM for

membrane integrity and neutral red for lysosomal

activity.

(III) However, the toxicity of whole-water samples

could be detected by these methods because

spiking these samples with abietic acid or penta-

chlorophenol did cause a dose-dependent decline

in cell viability.

(IV) When whole-water samples from over a year of

routine monitoring of a paper mill were evaluated

for their toxicity with RTgill-W1, rainbow trout,

and D. magna, some samples were toxic to D.

magna, but none of these were toxic to RTgill-W1.

(V) Only 1 of the 31 paper mill samples was toxic to

RTgill-W1, and this was also the only sample toxic

to rainbow trout.

(VI) These results suggest that presenting whole-water

samples to fish cells in culture can be used to

evaluate the toxicity of industrial effluents and can

serve potentially as a replacement for rainbow

trout but not D. magna in routine toxicity testing.

Acknowledgements

This research was supported by Center for Research

in Earth & Space Technologies (CRESTech). We would

like to thank Dennis G. Farara of BEAK International

Inc. and Dr. Jim Barker of the University of Waterloo

for their support and encouragement as well as Dr.

Ulysses Klee, Julie Schroeder and Serena Maki at

BEAK International Inc. for coordinating effluent

shipments from the paper mill and the helpful discus-

sions.

References

[1] Ahne W. Use of fish cell cultures for toxicity determination

in order to reduce and replace the fish tests. Zentralbl

Bakteriol Mikrobiol Hyg [B] 1985;180(5–6):480–504.

[2] Mochida K. Aquatic toxicity evaluated using human and

monkey cell culture assays. Bull Environ Contam Toxicol

1986;36(4):523–6.

[3] Richardson D, Dorris TC, Burks S, Browne RH, Higgins

ML, Leach FR. Evaluation of a cell culture assay

for determination of water quality of oil-refinery effluents.

Bull Environ Contam Toxicol 1977;18:683–90.

[4] Maruoka S. Estimation of toxicity using cultured mam-

malian cells of organic pollutants recovered from Lake

Biwa. Water Res 1978;12:371–5.

[5] Schirmer K, Tom DJ, Bols NC, Sherry JP. Ability of

fractionated petroleum refinery effluent to elicit cyto-and

photocytotoxic responses and to induce 7-ethoxyresorufin-

o-deethylase activity in fish cell lines. Sci Total Environ

2001;127:129–41.

[6] Kfir R, Prozesky OW. Detection of toxic substances in

water by means of a mammalian cell culture technique.

Water Res 1981;15:553–9.

[7] VanDoren SR, Hall MS, Frazier LB, Leach FR. A rapid-

cell culture assay of water quality. Bull Environ Contam

Toxicol 1984;32(2):220–6.

[8] Bols NC, Lee LEJ. Cell lines: availability, propagation and

isolation. In: Hochachka PW, Mommsen TP, editors.

Biochemistry and molecular biology of fishes, vol. 3.

Amsterdam: Elsevier Science, 1994. p. 145–9.

[9] Schirmer K, Chan AGJ, Greenberg BM, Dixon DG, Bols

NC. Methodology for demonstrating and measuring the

photocytotoxicity of fluoranthene to fish cells in culture.

Toxicol In vitro 1997;11:107–19.

[10] Wiemer KE, Anderson A, Stewart B. The importance of

water quality for media preparation. Hum Reproduction

1998;13:166–72.


[11] Bols NC, Barlian A, Chirino-Trejo M, Caldwell SJ,

Goegan P, Lee LEJ. Development of a cell line from

primary cultures of rainbow trout, Oncorhynchus mykiss

(Walbaum), gills. J Fish Dis 1994;17:601–11.

[12] Environment Canada. The development document for the

effluent monitoring regulation for the pulp and

paper sector. Queen’s Printer for Ontario, Environment

Ontario, 1989. p. 1–55.

[13] Environment Canada. Biological test method: acute

lethality test using rainbow trout. EPS 1/RM/9, Ottawa,

Canada, Environment Canada, Environmental Protection

Series, 1990. p. 1–51.

[14] Environment Canada. Biological test method: reference

method for determining acute lethality of effluents to Daphnia

magna. EPS 1/RM/14, Ottawa, Canada, Environment

Canada, Environmental Protection Series, 1990. p. 1–18.

[15] Ganassin RC, Schirmer K, Bols NC. Methods for the use

of fish cell and tissue cultures as model systems in basic and

toxicology research. In: Ostrander GK, editor. The

laboratory fish. San Diego, CA: Academic Press, 2000.

p. 631–51.

[16] Wolf K, Ahne W. Fish cell culture. Adv Cell Cult

1982;2:305–28.

[17] Dierickx PJ, Brendael-Rozen E. Correlation between the in

vitro cytotoxicity of inorganic metal compounds to

cultured fathead minnow fish cells and the toxicity to

Daphnia magna. Bull Environ Contam Toxicol

1996;57:107–10.

[18] Sandbaka M, Christianson I, Isomaa B. The acute

toxicity of surfactants on fish cells, Daphnia magna

and fish—a comparative study. Toxicol In vitro

2000;14:61–8.


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