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314 A. Zitova et al.

retardants. Of the 33 substances specified 11 have been identified as being priority

hazardous substances which are of particular concern, and are destined to be phased

out over a twenty year period. On the 17

of July 2006 the European Commission

adopted a proposal to expand this list of 33 to include an additional 8 compounds

(EU, 2006).

As a consequence of these new initiatives novel screening techniques capable of

sensitive and high throughput analysis while also limiting or replacing the use of

animal testing are required. One such technique developed by our group is respiro-

metric screening technology (RST) (Papkovsky 2005). RST-based assays operate

by means of fluorescence-based oxygen-sensitive probes (Papkovsky 2004) which

are simply added to test samples where the fluorescence of the probe is quenched

by sample dissolved oxygen in a nonchemical, reversible manner (collisional

quenching of fluorescence (Papkovsky 2004)). The predictable manner in which

the probe fluorescence changes in response to the changing concentration of oxygen,

allows oxygen consumption rates to be monitored. As molecular oxygen is a key

metabolite of all aerobic cells and higher organisms, alterations in cellular oxygen

uptake serve as useful markers of their metabolic status, viability, and responses

to various stimuli. The simple addition of the water-soluble probe to the sample

being tested allows for great flexibility of the assay setup, as the sample can sub-

sequently be pipetted into 96- or 384-well microtitre plates. The compatibility of

the oxygen probe with conventional fluorescent plate readers also allows for the

rapid and convenient parallel screening of multiple samples using a very simple

kinetic assay format.

Monitoring changes in respiration also fulfils the second criteria of these assays

in that it allows testing of sublethal toxic effects. One of the tensions in designing

toxicity testing strategies is between reducing animal use and suffering and regula-

tory needs for more information on a wider array of chemicals or more detailed

information on a smaller group of chemicals. Traditionally toxicity analysis is

measured based on the concentration of a samples that kills 50% of the animals

being tested called the LC

. However with certain organisms death can be difficult

to determine unequivocally. As a result other effects, such as pharmacological,

biochemical or physiological responses, which closely correlate with death such as

immobility, are measured instead. As with death for a measurement endpoint,

results can be analyzed by comparing percent effect for organisms exposed to the

toxicant and those not exposed to the toxicant allowing parameters such as the

, the median effective concentration, to be determined. In addition to the afore-

mentioned difficulty of ascertaining mortality in some cases when determining

lethal concentration analysis, effective concentration analysis also have the advan-

tage of being superior in terms of sensitivity as unsurprisingly a sublethal effective

will occur at a lower toxicant concentration then death (EPA, 1994).

To date RST has proven suitable with a wide range of model systems and

in vitro assays including the prokaryotes E. coli (O’Mahony and Papkovsky 2006)

and S. Pombe (O’Riordan et al. 2000), the eukaryotic cell lines (Alderman et al.

2004; Hynes et al. 2003; Hynes et al. 2005), and the marine organism Artemia

salina (O’Mahony et al. 2005) allowing these models to be used in the place of the

24 Biological Toxicity Testing of Heavy Metals 315

conventional animal models of toxicity. The current study uses these three different

groups of organisms to study their sensitivity and applicability for toxicity monitor-

ing. In particular the current study focuses on heavy metal toxicity as one of the

chemicals listed in the REACH program as well as a panel of wastewater samples

discharged from treatment plants as would be tested under the Water Framework

Directive. These samples are used to evaluate the sensitivity of different test organ-

isms and the effects of exposure time to toxicant on the assay sensitivity.

24.2 Experimental Section

24.2.1 Materials

Phosphorescent oxygen probe (type A65N-1) and low-volume sealable 96-well

microplates (MPU96-U1) were from Luxcel Biosciences, Ireland. Standard poly-

styrene 96-well plates, 20 ml cell culture flasks and 50 ml reagent tube were

obtained from Sarstedt (Ireland). Heavy mineral oil, sea salt, heavy metals salts

ZnSO

and CuSO

, dimethyl sulfoxide (DMSO), trypan blue, LB broth and RPMI-

1640 growth media were from Sigma-Aldrich (Ireland). All the other chemicals

and solvents were of analytical grade. Solutions of heavy metals and dilution of

environmental samples were prepared using Millipore grade water. Industrial

wastewater samples (discharged from WWT plants after treatment) were obtained

from EPA laboratory in Inniscarra, Co. Cork, Ireland.

24.2.2 Organism Culturing

Colonies of E.coli (DH5α MCR) were removed from solid agar and suspended in

100 ml LB broth in a 500 ml flask. Bacteria were grown at 37°C on a shaker, until

an OD600 of 0.5 was reached. Cells were enumerated using a Neubauer hemocy-

tometer (Assistant) and light microscope Alphaphot-2 YS2 (Nikon).

Jurkat cells (a human leukaemia T-cell line) were a gift from Professor Tom

Cotter (University College Cork, Ireland). The cells were cultured in RPMI 1640

medium supplemented with 10% (v/v) fetal bovine serum, 2 mM l-glutamine,

100 U/ml penicillin, and 100 µg/ml streptomycin. Typically, cells were cultured in

75 cm

suspension cell flasks (Sarstedt) at 37°C in humidified atmosphere of 5%

in air to a concentration greater than 1 × 10

cells/ml (stained using trypan blue

and counted under a microscope using a hemocytometer) prior to testing.

Artemia salina were hatched in artificial seawater (35 g/l) prepared from sea

salts (Sigma-Aldrich). After 48 h at 25°C under continuous illumination by a bulb

(no feeding required), Artemia molted to the instar II stage, a growth stage when

most toxicity testing is conventionally carried out on Artemia (Vanhaecke and

Persoone 1984).

316 A. Zitova et al.

24.2.3 Respirometric Measurement Setup

Two different platforms were utilised for respirometric measurements, both pro-

viding adequate sensitivity and performance with respect to the measurement of

respiration of test organisms and relative simplicity of assay setup and execution.

The first format employs standard 96-well plates (Sarstedt) which were used for

the cell-based assays. The second more sensitive format employs Luxcel sealable

microplates (Alderman et al. 2004) used for the Artemia-based assays. The

MPU96-U1 plates are made of clear polystyrene and contain wide and shallow

wells, 6 mm in diameter, 0.5 mm deep, actual volume ∼6 µl, which are surrounded

by rims and frames that help in sealing the samples, removing air bubbles and

excluding ambient oxygen. A small excess of sample (usually 10 µl) containing

test organisms, toxicant and oxygen probe is added to each well, which is retained

by capillary forces, and the oxygen uptake assay is initiated by applying the adhe-

sive sealing tape to the plate.

A65N-1 phosphorescent oxygen probe (Luxcel) was reconstituted in 1 ml of

water to give a 1 µM stock, which was stored in the dark at 4°C and used at the

working concentrations described below. Artemia salina assays conducted on

Luxcel plates required probe working concentration of 100 nM. Fluorescence

intensity measurements were carried on a prompt fluorescence reader, SpectraMax

Gemini (Molecular Devices), with excitation set at 380 nm and emission at 650 nm.

E.coli and Jurkat-cell-based assays conducted on 96-well plates required probe

concentrations of 3 nM. Time resolved fluorescence (TR-F) measurements were

carried out on a Genios Pro fluorescent plate reader (Tecan, Mänerdorf, Switzerland)

using standard set of filters of 380 and 650 nm, respectively, with delay and gate

times of 60 µs and 100 µs. In order to assess the effect of incubation time on the sen-

sitivity of the organisms to the test samples incubation times of 0 and 24 h were used.

Samples were added to test organisms in medium to give a resulting concentrations

of 6.4e + 4, 6.4e + 3, 6.4e + 2 µg/l of heavy metals, and wastewaters samples were

10, 100 and 1000 times diluted. Sets of control samples, particularly those contain-

ing test organisms, oxygen probe and no toxicant (100% of respiration) and test

organisms without the probe and toxicant (blank) were included in each experi-

ment. All concentration points and controls were run in quadruplicates. Protocols

for the individual model organisms are outlined below.

24.2.3.1 Artemia Salina

For 0 h and 24 h incubation-based toxicity assays Artemia salina were transferred,

using Pasteur pipettes (Volac, U.K.), into 1.5 ml Eppendorf tubes (Starstedt)

containing the toxicant diluted in seawater in addition to the appropriate

concentration of oxygen sensitive probe. Post incubation (0 and 24 h) the animals

were pipetted into the wells of the low volume Luxcel plates at a concentra-

tion of 5 animals per well (volume ∼10 µl). Assays were initiated by sealing the

24 Biological Toxicity Testing of Heavy Metals 317

microplate with Mylar sealing film and measuring the plate at constant temperature

of 30°C every 5 min over a period of 1–2 h.

24.2.3.2 Jurkat Cells and E. coli

For 0 h incubation, 135 µl sample of Jurkat cells in RPMI-1640 growth medium

(10

cells/ml) containing the oxygen probe were pipetted into the wells of a stand-

ard 96-well plate. 15 µl of toxicant stock(s) were added to the samples to produce

the required concentrations of toxicant (may vary for different wells). The sam-

ples were then sealed by pipetting 100 µl of heavy mineral oil on top of each and

monitored on a plate reader at 37°C as described above. For 24 h incubation cells

were split in RPMI-1640 growth medium and dispensed in 4.5 ml aliquots into

the 20 ml culture flasks (Sarstedt). 0.5 ml of various concentrations of test sample

were added to make up the appropriate concentrations of toxicants. Cells were

subsequently cultured for 24 h under standard conditions of 37°C, humidified

atmosphere of 5% CO

in air. Following the incubation period 150 µl of cell solu-

tions were added to the wells of standard 96-well plate together with appropriate

concentration of probe, sealed with 100 µl heavy mineral oil and monitored.

Assay with E. coli cells in LB broth resembled that conducted with Jurkat cells

for the 0 h incubation step. For the 24 h incubation, 10 ml of E.coli were added to

100 ml of LB Broth, and subsequently aliquoted in 9 ml volumes into 50 ml rea-

gent tubes (Sarstedt), each containing 1 ml of test compound at required concen-

tration. These samples were cultured at 37°C with shaking and then analyzed in

96-well plates as above.

24.2.4 Data Analysis

Measured time profiles of fluorescence were analysed to determine the initial

slopes of each sample. The slopes, which reflect oxygen rates by test organisms,

were calculated using the following formula:

slope

II tt

blank

−

()

−

()

121

where I

and I

= fluorescence signal at time points t

and t

(usually 10 and

20 min after start of monitoring, selected by operator) and I

blank

= signal without the

probe (when comparable with the signal). To assess respiration rates of test organ-

isms and alterations produced by toxicants, calculated slopes were compared to

those of untreated organisms (positive controls, 100% respiration) and to those

without organisms (negative controls). EC

values (i.e., the concentration of the

toxicant which caused a 50% decrease in the respiration, compared to untreated

organisms) were calculated using sigmoidal fits in OriginPro 7.5 G software.

Statistical analysis was conducted using a T-test with 95% confidence limits.

318 A. Zitova et al.

24.3 Results

24.3.1 Heavy Metal Toxicity

Typical respiration profiles of Artemia salina treated with heavy metal ion (Cu

in this

case) are shown in Fig 24.1a. Oxygen consumption for the treated animal samples is

significantly reduced, and samples without animals (negative controls) produce negli-

gible slope. Such slopes can be used to assess the rate of respiration of treated Artemia

compared to untreated animals. Figure 24.1b shows dose and time dependence of Cu

Fig. 24.1 (a) Respiration profiles of Artemia salina in 6.35e + 4 µg/l Cu

( ), negative (x) and

positive controls (◊), at 0 h incubation time, (top): (b) Effects of Cu

on Artemia respiration at 0 h

and 24 h exposure times, measured with 5 animals per well over 2 h, 30°C (bottom)

24 Biological Toxicity Testing of Heavy Metals 319

on respiration. When no pre-incubation is allowed, a significant effect on respiration is

only apparent above 6.4e + 3 µg/l with a T value of 5.3292. Whereas with 24 h animal

exposure to the toxicant the respirometric assay detects a significance difference at

concentrations below 6.4e + 3 µg/l of Cu

with a T value of 10.2365.

Increased sensitivity to heavy metals is also observed with E.coli and Jurkat

cells, Fig. 24.2. With a 0 h incubation period, E.coli was identified as the most sen-

sitive to Cu

followed by Artemia and Jurkat cell, giving EC

− 0h values of

Fig. 24.2 Effects of Cu

on different animals: Artemia salina (ϒ), Jurkat cells (⊄) and E.coli (∆),

at 0 h (top), and 24 h (bottom) pre-incubation time

320 A. Zitova et al.

3.8e + 4 µg/L, 6.2e + 4 µg/L, 9.1e + 3 µgL, respectively (Table 24.1). Increasing the

incubation time to 24 h resulted in Artemia being the most sensitive followed by

Jurkat cells and E.coli with EC

−24 h values of 3.6e + 3 µg/L , 8.6e + 3 µg/L and

3.0e + 4 µg/L respectively. Table 24.1 shows that a similar pattern is observed for

, with Artemia being the least sensitive at 0 h incubation and the most sensitive

at 24 h incubation periods.

24.3.2 Analysis of Industrial Wastewater Samples

The model organisms were also used to analyse a set of industrial wastewater sam-

ples for their residual biological toxicity. All these samples underwent treatment at

WWTP and were discharged into the environment. An initial screening of the sam-

ples (diluted 1:10 in assay medium) using Jurkat cells and 0 h pre-incubation time

revealed that only sample 4 caused a marked reduction in respiration, while the

others had practically no effect. Sample 4, which produced a respiration profile

similar to that of the negative control (i.e., almost complete inhibition, see Fig. 24.3a).

These samples underwent subsequent investigation to determine their dose and

time dependant effects on the respiration of Jurkat cells, E.coli and Artemia.

Sample 4 displayed the greatest level of toxicity with T values of 78.0615 for

(Jurkats), 12.4033 for (E.coli) and 9.6363 for (Artemia) at 0 h incubation. A dilu-

tion factor of 79 was required for sample 4 to achieve 50% inhibition of respira-

tion (Table 24.2). Of the three model organisms tested, Jurkats were shown to be

the most sensitive when an incubation period of 0 h was used followed by E.coli

and Artemia with 50% of control respiration achieved from dilution factors of

79, 67.1 and 12 respectively. Using the 24 h incubation period, Jurkat cells were

still the most sensitive however Artemia were shown have developed a greater

sensitivity then E.coli with dilution factors of 27.5, 13.3 and 3.8 respectively.

Samples 1, 2 and 3 showed no measurable effect on the respiration, whilst sam-

ples 5 and 6 showed some effects, but no clear pattern of toxicity at both 0 and

24 h incubation times.

Table 24.1 Heavy metals toxicity on various model organisms measured by oxygen

respirometry

Heavy metal

[µg/L] Cu

[µg/L]

− 0 h Artemia 6.5e + 5 6.2e + 4

Jurkat 2.1e + 4 9.1e + 3

E.coli 1.0e + 4 3.8e + 4

− 24 h Artemia 4.9e + 2 3.6e + 3

Jurkat 7.7e + 3 8.6e + 3

E.coli 9.5e + 3 3.0e + 4

Table 24.2 Comparison of wastewaters samples toxicity effect (EC

) on various model

organisms

Environmental sample [dilution factor

causing the effect]

n.1 n.2 n.3 n.4 n.5 n.6

− 0 h Artemia < 1 < 1 < 1 12.0 < 1 < 1

Jurkat < 1 < 1 < 1 79.0 < 1 < 1

E.coli < 1 < 1 < 1 67.1 5.2 < 1

− 24 h Artemia < 1 < 1 < 1 13.3 1.5 4.1

Jurkat < 1 < 1 < 1 27.5 < 1 < 1

E.coli < 1 < 1 < 1 3.8 < 1 3.9

Fig. 24.3 Effects on Jurkat cells respiration of six wastewater samples (top). Effect of increasing

concentrations of sample 4 (bottom). Incubation time 0 h, Jurkat cells 5 · 10

cells/ml

322 A. Zitova et al.

24.4 Discussion

The above results show the suitability and effectiveness of oxygen respirometry and

RST for the analysis of chemicals and environmental samples for their biological

hazard and toxicity. The ability to monitor the respiration of a panel of model

organisms and study changes in their metabolism induced by external toxicants

facilitated the assessment and ranking of heavy metals ions for their toxicity. Zn

was identified by each of the three model systems as being of greater toxicity then

, at both short (0 h) and long (24 h) exposure times a result which agrees with

similar assays conducted on Microtox system (Sillanpaa and Oikari 1996). In com-

parison to the same Microtox study the sensitivities of the various organism respiro-

metric-based assays described in this study were less sensitive at both incubation

times, however as different organisms were used for RST and Microtox a direct com-

parison is not possible. For Zn

and Cu

respectively the Microtox gave EC

values

of 2.6e + 2 µg/L and 1.02e + 3 µg/L respectively whereas the RST assays at 0 h incuba-

tion gave 1.0e + 4 µg/L (E.coli), 2.1e + 4 µg/L (Jurkats) and 6.5e + 5 µg/L (Artemia)

for Zinc and 3.8e + 4 µg/L (E.coli), 9.1e + 3 µg/L (Jurkats), 6.2e + 4 µg/L (Artemia) for

Copper. Increased incubation time was seen to increase the toxicological impact,

especially for the multi-cellular organism Artemia, where the EC

values dropped

from 6.5e + 5 µg/L to 4.9e + 2 µg/L for Zn

and 6.2e + 4 µg/L to 3.5e + 3 µg/L for

, respectively. The lack of a similar large increase in the cell-based assays is

assumed to be due to the more simple organization and physiology of the cultured

cells compared to multi-cellular organisms. The ability of longer exposure time to

increase sensitivity of Artemia but decrease it for the cell-based assays was also

observed when the environmental samples were tested.

The screening of environmental samples highlighted the power of the RST

approach for its ability to assay high numbers of samples simultaneously. The

microtitter plate format of the assay, is only limited by the number of wells in the

plate and the degree of automation. Even for manual liquid handling used in our

case, several hundred samples or assay points can be run each day. Qualitative and

objective (instrumental) toxicological assessment allowed samples to be rapidly

(1–2 h) screened, reliably identifying toxic samples, ranking them and determining

values. The ability of the platform to quantify the toxicity of real environmen-

tal samples such as wastewater has been demonstrated. The tracking of the toxicity

of a sample over time by comparing its effect on the respiration of the chosen test

organism and exposure time was shown. The versatility of the RST platform allows

the user to choose test organisms and customise the assay based on such factors as

the availability of culturing facilities, measurement instrumentation and trained

personnel. With this in mind the brine shrimp Artemia salina is an attractive model

for primary screening of samples for potential biological hazard and toxicity by

RST. Its ease of culture, high fecundity, low cost, facilitate the growth of sufficient

numbers of test organisms in laboratories with little knowledge of animal handling,

without the legal or ethical issues that surround the use of high animal models such

as mice. From the cell-based RST assays, test organisms such as E. coli look attrac-

tive, as they are also robust, easy to culture and use.

24 Biological Toxicity Testing of Heavy Metals 323

24.5 Conclusions

EU initiatives such as REACH and the Water Framework Directive have high-

lighted the need for high throughput screening for the analysis of toxic chemicals

and environmental samples. RST and simple respirometric in vitro assays with a

panel of model organisms have been shown to provide relevant and information-

rich toxicological data and to meet the requirements of such initiatives whilst avoid-

ing the legal and ethical issues of using conventional animal-based testing methods.

The flexibility and cost-effectiveness of the assays allows them to be adapted for

various test organisms such as bacteria, mammalian cell culture and small marine

animals. The microtitter plate platform (96- and 384-well) on which the assay are

conducted means that the number of samples amenable for testing is greatly

increased and only limited by the level assay of automation. These factors com-

bined with the rapid and sensitive nature of the assay clearly highlight the potential

for RST use in these increasingly important areas of environmental research, bio-

chemical and molecular toxicology.

Acknowledgements Financial support of this work by the Marine Institute and Marine RTDI

Measure, Productive Sector Operational Programme, National Development Plan 2000–2006

(Grant-aid agreement No AT-04-01-01), is gratefully acknowledged.

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