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Aquatic Toxicology:

Mechanisms and
Consequences
SYMPOSIUM PROCEEDINGS

Chris Kennedy
Alan Kolok
Don MacKinlay
International Congress on the Biology of Fish
University of British Columbia, Vancouver. CANADA

Aquatic Toxicology:
Mechanisms and
Consequences
SYMPOSIUM PROCEEDINGS

Chris Kennedy
Alan Kolok
Don MacKinlay

International Congress on the Biology of Fish


University of British Columbia, Vancouver. CANADA

Copyright 2002
Physiology Section,
American Fisheries Society
All rights reserved
International Standard Book Number (ISBN) 1-894337-22-0

Notice
This publication is made up of a combination of extended abstracts and full
papers, submitted by the authors without peer review. The papers in this
volume should not be cited as primary literature. The Physiology Section of
the American Fisheries Society offers this compilation of papers in the
interests of information exchange only, and makes no claim as to the
validity of the conclusions or recommendations presented in the papers.
For copies of these Symposium Proceedings, or the other 50 Proceedings in the
Congress series, contact:
Don MacKinlay, SEP DFO, 555 West Hastings St.,
Vancouver BC V6B 5G3 Canada
Phone: 604-666-3520
Fax 604-666-6894
E-mail: mackinlayd@pac.dfo-mpo.gc.ca

Website: www.fishbiologycongress.org

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PREFACE
This symposium provides a forum for the presentation and discussion of
relevant toxicological investigations involving fish. Like the previous 2
symposia on piscine toxicology in the Fish Biology Congress series, this one
was held because there is an immediate and dire need to understand the various
direct and indirect impacts of both natural and anthropogenic contaminants on
fish and fisheries. The talks in this series range from the molecular level,
through biochemistry and physiology, to effects on populations. These papers
highlight the multidisciplinary nature of aquatic toxicology, and the diversity of
approaches used to understand the mechanisms of toxicity and the relevance of
those toxicities to individuals, populations and ecologies. In this series, papers
discuss toxic mechanisms, comparative approaches to toxicology, various
toxicants including organics and metals, effects on different biochemical,
physiological and organ systems, as well as endeavors with applicability to
population-level impacts. The cumulative knowledge communicated in this
symposium will enhance our understanding of piscine toxicology, an area often
underrepresented in the general world of basic and applied fish biology.
Symposium Organizers:
Chris Kennedy, Simon Fraser University, Burnaby B.C., Canada
Alan Kolok, University of Nebraska, Omaha NE, USA
Don MacKinlay, Fisheries & Oceans Canada, Vancouver, B.C., Canada

iii

CONGRESS ACKNOWLEDGEMENTS
This Symposium is part of the International Congress on the Biology of Fish,
held at the University of British Columbia in Vancouver B.C., Canada on July
22-25, 2002.
The sponsors included:
- Fisheries and Oceans Canada (DFO)
- US Department of Agriculture
- US Geological Service
- University of British Columbia Fisheries Centre
- National Research Council Institute for Marine Biosciences
- Vancouver Aquarium Marine Science Centre
The main organizers of the Congress, on behalf of the Physiology Section of the
American Fisheries Society, were Don MacKinlay of DFO (overall chair, local
arrangements, program and proceedings) and Rosemary Pura of UBC
Conferences and Accommodation (facility arrangements, registration and
housing). Thanks to Karin Howard for assistance with Proceedings editing and
word-processing; to Anne Martin for assistance with the web pages; and to
Cammi MacKinlay for assistance with social events.
I would like to extend a sincere thank you to the many organizers and
contributors who took the time to prepare a written submission for these
proceedings. Your efforts are very much appreciated.
Don MacKinlay
Congress Chair

iv

TABLE OF CONTENTS
The impact of metal ions on some components of glutathione cycle in carp gill
cells
Arabi, M. and M.S. Heydarnejad........................................................................................1
Biochemical effects of environmental nitrite in matrinx
Moraes, Gilberto, et al......................................................................................................15
DNA synthesis after exposure to heavy metals in the testis of the spiny dogfish
Redding, Michael..............................................................................................................27
Quantitative PCR of Atlantic salmon CYP1A in gills
Rees, C, S McCormick, and W Li......................................................................................31
Expression of multi-drug resistance (MDR) p-glycoprotein and associated
proteins (MRPs) in the flounder; effects of exposure to the
polyaromatic hydrocarbon, benzo(a)pyrene
Cramb,G, Cutler,et al. .. ...................................................................................................33
Sediment-associated pollutants in the marine environment: a multi-biomarker
approach for assessing sediment toxicity in turbot
Kilemade, M., Hartl, M. et, al. .........................................................................................39
Responses of Atlantic salmon and bivalve molluscs to paralytic shellfish
Eddy, FB, et al. .. ..............................................................................................................43
The effect of environmental levels of freshwater contaminants on juvenile
Atlantic salmon: Implications for marine survival
Lower, Nicola and Andy Moore........................................................................................47
Chloride cell changes induced by nitrite exposure in an amazonian fish species
Da Costa O.T.F. and M.N. Fernandes..............................................................................51
Behavioral and Neurophysiological Effects of Carbamate Pesticides on
Olfactory Capabilities in Pacific Salmon
Jarrard, H and C Kennedy.. .............................................................................................63

Effects of exposure to sub-lethal concentrations of ammonia and hypoxia on the


swimming performance of brown trout
Shingles, A. et al. .. ...........................................................................................................69
Effects of chlorpyrifos (Lorsban)on reproductive performance of guppy
De Silva, M.. .....................................................................................................................75
Maternal transfer of copper resistance in fathead minnows
Peake, EB, et al. ...............................................................................................................87
Reduced gonadal somatic index and external coloration following exposure to
P,P-dde in adult male Fundulus heteroclitus
Monosson, Emily et al. .....................................................................................................93
Blood cell responses of the tropical fish Prochilodus scrofa to acute copper
exposure and subsequent recovery
Cerqueira, Carla and M.N. Fernandes.............................................................................99
Toxicity of cadmium: a comparative study in the airbreathing fish, Clarias
batrachus and in non-airbreathing Ctenopharyngodon idellus
Joshi, P.K. & M. Bose.....................................................................................................109
Effects of vanadate oligomers on lipid peroxidation and antioxidant enzymes in
the Lusitanian toadfish kidney and liver: short term-exposure
Figueiredo, Ins, et al. ...................................................................................................119
Incubation and pH-dependent effects of vanadate oligomers and cadmium with
Halobactrachus didactylus sarcoplasmic reticulum calcium pump
Leonardo, Llia, et al. .. ..................................................................................................123
Histological analysis of vanadate oligomers effects on heart, kidney and liver of
the Lusitanian toadfish: an acute exposure study
Borges, Gisela, et al. ......................................................................................................129
Cadmium and vanadate oligomers comparative effects on the toadfish
erythrocyte
Soares, Sandra, et al. .....................................................................................................133

vi

Toxicity of Lake Enrichment nutrients to aquatic life


MacKinlay, Don and Craig Buday .................................................................................139

vii

viii

THE IMPACT OF METAL IONS ON SOME COMPONENTS OF


GLUTATHIONE CYCLE IN CARP GILL CELLS
Mehran Arabi
and Mohammad Saeed Heydarnejad
Department of Biology, Shahrekord University, Shahrekord- 88186,
POB 115 , Iran .
mehranarabi@yahoo.com
Abstract
The extent of cellular damage was investigated after in vitro addition of two
metal ion componds Viz. CuSo4 and HgCl2 in various concentrations 300, 500,
700, 1000 and 3000 mM to gill cells preparation of freshwater Fish Carp
(Cyprinus carpio L.). The objectives of current study were to determine the
influence of these metal ions on the levels of TBARS/Lipid peroxidation (LPO)
and GSH, also to evaluate the role of BSA (0.5 & 1.0%) and DMSO (0.5%) as
ROS scavangers to encounter the relative processes.
The outcomes of this report are: (a) Copper and mercury increased the rate of
LPO dose-dependently ( r = + 0.995 and r = + 0.993 respectively, P< 0.001) but
the GSH Content was only marginally affected ( r = -0.787 and r = -0.844
respectively, P <0.05). (b) CuSO4 played a more potent role in oxidative damage
to fatty acid chains than HgCl2 in gill cells preparation. (c) Depleting of GSH
molecules by copper had a wider range than mercury. (d) In the highest
concentration of metal ions (3000 mM) both DMSO and 1.0% BSA showed a
pro-oxidative potential to elevate the levels of TBARS (P<0.001) but for other
concentrations when supplemented with three scavanges , it was found a fall in
the levels of the latter. (e) Addition of 1.0% BSA to medium containing 3000
mM of metal ions caused a significant decline in GSH content (P<0.01).
Collectively, these findings indicate that copper and mercury have deleterious
influence on membrane integrity and GSH content as well in a relative dosedependent manner.
The complexes of metal ions and thiol residues of cell proteins could also act as
a more potential cell toxicant leading to disturbances in cell functions towards
cell death.

Introduction
Common Carp (Cyprinus carpio L.) is an important commercial species around
the world to feed populations and is as an economic rather than an ornamental
fish. As a group, carp provide 4 million metric tonnes of fish annually - over a
quarter of all fish culture worldwide. Reducing the number of these precious
animals for water contamination (e.g. metal ion toxicity) is as a paradox to
heavy demands. Teleosts, functionally, have four pairs of gill arches furnished
with tiny structures called gill lamellae. The latter, are rich in capillary
networks and covered with a simple squamous epithelial cells which are
responsible for gas exchanges in aquatic media. Due to direct exposure of gills
in the water medium, it has been dominantly accepted that they are the main site
to water contamination and toxicity (Karan et al. 1998; Dalzell and Macfarlane
1999). Coping with to fish is very crucial. The water companies are routinely
consuming metal ions in aquatic media e.g. Iron sultate is used to combat algal
blooms in reservoirs (Hayes et al. 1984); Copper sulfate is one of the most
widely used algicides for the control of phytoplanktons in lakes, reserviors, and
ponds, it is also used for aquatic weed control (Karan et al. 1998); Mercuric
chloride is also mainly used to control the mass of other fish partners as
mollusicide (Radhakrishnaiah et al. 1993). It has been shown that some metal
ions in fish express deleterious impacts on some organs such as liver, gill,
gonads and components of blood as well (Radi and Matkovics 1998; Mukherjee
et al. 1994; Karan et al. 1998 and Akahori et al. 1999).
A number of researchers have observed that the reactive oxygen species (ROS)
such as superoxide anion (.O2-) and hydroxyl radical (.OH) through their own
generating systems can stimulate the peroxidation of polyunsaturated fatty acids
(PUFAs) of the biological membranes which is called lipid peroxidation, LPO
(Halliwell and Gutteridge 1985; Radi and Matkovics 1988).
Continued fragmentation of fatty acid side chains to produce further more
aldehyde and hydrocarbons will eventually leads to LPO propagation and
complete loss of membrane integrity and cell functions. Therefore, LPO is an
useful index to measure the membrane integrity and relative lesions. To
overcome this degenerative process cells are well-equipped with a powerfull
battery of defence systems (Antioxidants) to mop up and then neutralize the
ROS (Sharma and Agarwal 1996).
The ubiquitous tripeptide, reduced glutathione (L-gamma-glutamyl-LCysteinylglycine, GSH), most prevalent intracellular thiol ( - SH) functions as a
2

quick and vibrant antioxidant in cells by donating one hydrogen atom from two
molecules to a toxic substance (Meister and Anderson 1983). Glutathione is
present in the oxidized (GSSG) form, which is readily converted to the GSH
form by the enzyme glutathione reductase ( GRD). It has been reported that
glutathione is present mainly in its reduced form in biological tissues, at
concentrations as high as 2180 mg/g of tissue and in form of GSSG to be present
in much smaller concentrations, ranging from 0 to 288 mg/g of tissue (Hissin
and Hilf 1976). A shift to a more oxidative state or any imbalance between
production and degradation of ROS in animal tissues may causes LPO, plasma
membrane alternations, inactivation of enzymes, ect. (Radi and Matkovics 1988;
Matta et al. 1999; Akahori et al. 1999; Anand et al. 2000).
The current study aimed to investigate the influence of different concentrations
of two metal ion compounds viz. copper sulfate (CuSo4) and mercuric chloride
(HgCl2) on some properties of carp gill cells; membrane integrity and GSH
content. We also estimated the scavanging activity of 0.5 & 1.0% small
molecule protein, bovine serum albumin (BSA) and 0.5% dimethylsulfoxide
(DMSO) to possibly generated ROS in different media.
Material and Methods
Reagents
Thiobarbituric acid (TBA), and DTNB (5,5'-dithio-bis [ 2-nitrobenzoic acid])
were obtained from Merk, Darmstadt ( Germany). All other chemicals were
from Himedia company (India) and were of analytical grade. All solutions were
made with triple-distilled water.
Collection of Samples
Six fresh carps were collected from local fish farm. As soon as possible,
branchial arches were gently taken out and kept in chillled sorenson's buffer (pH
7.4) and transfered to lab and kept at 4C for a short period of time till final
processing.
Protocol for the estimation of Lipid peroxidation and GSH Content
a) Preparation of tissue homogenate
After careful removal of the cartilageous portions supporting gill arches, the gill
filaments gently cleared of other parts and washed several times with chilled
0.154 M NaCl solution (0.308 Osmolar) to remove blood. After that, the bulk of
gill fillaments grinded resulting in bared ones and free bands of lamellae. The
latter, separated from bony parts by means of ordinary tea-strainer.
3

Consequently, the resulting filtrate homogenized in 0.05 M phosphate buffer


(pH 7.4) using polytron homogenizer at a speed of 13000 rpm in ice bath. The
homogenate was then centrifuged (3000 rpm, 10 min). The supernatant was
used for assays throughout the work. All the above steps commencing from
dissection of the tissue till the preparation of homogenate were performed at 04C.
b) Estimation of Proteins
Cell protein content was estimated by the modified sodium dodecyl sulphate Lowry method of Lees and Paxman (1972) using BSA as standards. Activities
of measured parameters were calculated and expressed per mg protein.
c) Assay of Lipid Peroxidation by Spectrophotometric method
The LPO was estimated in terms of Thiobarbituric acid reactive substances
(TBARS), particularly Malondialdehyde (MDA) by the method of Ohkawa et al.
(1979) with slight modifications. The reaction mixture (3ml) contained 0.1 ml of
homogenate supernatant, 0.2 ml of 8.1% sodium dodecyl sulphate, 1.25 ml of
20% glacial acetic acid (pH 3.5), 1.25 ml of 1.2% aqueous solution of TBA and
0.2 ml distilled water in control group instead of adding 0.1 ml of metal ions or
antioxidants in treated one. Finally, after heating, adding 3 ml of n-butanolpyridine mixture and centrifuging at 2200 xg, the amount of MDA formed was
measured by the absorbance of the upper organic layer at 532 nm which is the
max of MDA (Extinction Coeff. of 1.56 x 105 / M/cm) using appropriate
controls.
d) GSH content measurement
Reduced glutathione residue after treatments as well as in control group was
assayed according to the method of Sedlak and Lindsay (1968) using DTNB
with minor modification.
Statistical Analysis
Data reported in the paper are means of two or more assays. All measurements
were performed in triplicate. The data are given as means + standard deviation
(S.D.). The comparison of the control and treated series was statistically
analysed by Student's t-test and validity of investigation was expressed as
probablity (p) values. Values of P>0.05 and P<0.05 were considered to be nonsignificant and almost significant; values of P<0.01 and P<0.001 were
considered significant and very significant respectively. Correlations were

evaluated using linear regression (LR) analysis between parameters assayed in


the present study.
Results
Influence on TBARS levels
As showed in table 1 production of MDA/TBARS in carp gill cells preparation
was induced by the presence of various concentration of copper in a dosedependent and linear manner (p<0.001, t-test). There was a positive and strong
correlation (r = +0.995, see fig.1A) between the elevated Cuso4 concentrations
and TBARS levels. When ROS scavangers (DMSO & BSA) supplemented to
the media a sharp significant decrease was observed in TBARS levels (P<0.001)
for all concentration of Cuso4 as compared to treated ones with same
concentration of metal ion (alone), but here at the concentration of 3000 M for
DMSO and 1.0% BSA instead of decreasing there was a dramatic increase in
TBARS production (P<0.001) which is due to their pro-oxidant activities. Also
all three scavangers exerted a significant fall in TBARS levels as compared to
control group (no metal ion treatment and supplementation) which expressed a
spontaneously LPO production in medium, Table 1 (P<0.01 & P<0.001,
cf.different data).
The data demonstrated in Table 2 show that the addition of upgrade
concentrations of HgCl2 caused highly significant elevation in TBRAS
production (P<0.001). The strength of associatioin between metal ion effect and
LPO generation was found positive and also considerable (r=+0.993, see fig.
1B). As soon as supplementation it was clearly observed that BSA (0.5 &
1.0%) can inhibit the extent of MDA/TBARS production (P<0.001) when
compared to the same samples treated with metal ions (alone) but DMSO
affected the LPO rate only marginally (P<0.05 & P <0.01, cf. data). Here again
we found out that when 0.5% DMSO and 1.0% BSA added to media with 3000
M metal ion a drastic increase in TBARS levels was occured (P<0.01 &
P<0.001, respectively). Addition of the all three scavangers to control group
resulted in a fall in TBARS levels (P<0.01 & P<0.001, see Table 2). The
protective effect of 0.5% BSA for holding the membrane integrity was found
much more better than DMSO & 1.0% BSA (P<0.001).
Influence on reduced glutathione (GSH) content
Table 3 summarizes the effect of CuSO4 concentrations on the GSH Pool in
Carp gill cells preparation. This evaluation showed only 3 concentration of
5

copper (700-3000 mM ) could change the GSH content which was a slightly
significant decrease (P<0.05). A negative correlation represented between metal
ion influeuce and GSH levels in preparation (r = -0.787, see fig. 1A). No
significant change was observed in the levels of GSH when DMSO & 0.5%
BSA were added (P>0.05). There was an interesting finding when 1.0% BSA
added to assay mixture that was a significant diminution for GSH content as
compared to control value (P<0.05 & P<0.01, cf. different data), Moreover,
supplementation of the ROS scavanger 0.5% BSA to control group (alone) led
to an elevation in GSH levels (P<0.05) and no significant alteration following
addition of two others (P>0.05).
The data mentioned in Table 4 reveal that addition of various concentration of
mercury (HgCl2) to gill cell preparations of carp could substract the levels of
GSH dose-dependently but slightly significant (P<0.05) for two higher
concentrations namely 1000 & 3000 M. A negative significant correlation
existed between this parameter and metal ion concentrations which is given in
fig. 1B (r = -0.844). Supplementation of DMSO to assay mixture containing
metal ion resulted in a slight diminution but non-significant (P>0.05) in GSH
content. 0.5% BSA caused an almost significant elevation (P<0.05) to GSH
content when compared to respective samples treated with same metal ion
concentration (alone) (1000 & 3000 M). Addition of 1.0% BSA could not
change the GSH levels significantly in series treated with mercury except at the
highest concentration which led to a sharp significant (p<0.01) diminution as
compared to control group (cf. Table 4). Among the ROS scavengers used in
this experiment only 0.5% BSA could change and elevate the levels of GSH
when compared to the control data (P<0.05).
Discussion
As cited in the introduction there is a growing body of evidence to indicate that
metal ions can exert some deleterious impacts on a number of tissues of fish
Carp (Cyprinus Carpio L.). For instances, mercury concentration effect in vivo
model is believed to be associated with lowered succinate dehydrogenase
activity and O2 consumption (Radhakrishnaiah et al 1993) and also an induction
of severe proteolysis in carp gill cell metabolism (Suresh et al 1991). Radi et al
(1988) reported a sharp fall in antioxidant activity of some enzymes e.g.
glutathione peroxidase (GPx) and an elevation in the rate of LPO and also
protein contents after mercury treatments in liver, white muscles and gill of carp.
On the other hand, copper increases the activity of some key metabolism
enzymes such as alkaline phosphotase, alanine and aspartate aminoteransferases
6

and showed induction of some lesions in carp gill e.g. epithelial hyperplasia and
chloride cells dysfunction (Karan et al 1998). In addition Kurant et al (1997)
reported that copper treatment in carp liver cells induces a diminution in the
content of low molecular weight protein thiols.
The reports on the effect of metal ions toxicity as an in vitro model are not too
many, this kind of work is able to open new lines of intracellular compartments
when exposed to pollutants. Owing to the role of metal ions as important
catalysts in living organisms finding the knowledge regarding to other facets of
these compounds sounds much vital.
Under the present experimental conditions, the results presented here have
clearly demonstrated that the elevated metal ion concentrations for copper and
mercury is associated with high production of the TBARS (p < 0.001, Table 1 &
2). We observed a strong link between concentration effect of metal ions used
and extent of TBARS. The correlation coefficients for copper and mercury
amounted to + 0.995 and + 0.993 at 370C respectively (p < 0.001, Fig. 1. A &
B). Here, copper and mercury acted as potent oxidants to biological
membranes, both plasma and intracellular membranes. Therefore, these results
indicate that in spite of physiological role of metal ions in maintenance of cell
functions, could also function as toxic agents when present in excess.
The metal ions as transition metals cause cellular damages via formation of
highly reactive oxygen free radical viz .OH. LPO initiation phase results in the
formation of lipid hydroperoxides (Lipid OOH) in the presence of .OH
(Sharma and Agarwal 1996) which is derived from .O2- and hydrogen peroxide
(H2O2), generally metabolically generated in medium. Neither .O2- nor H2O2 is
energetic enough to initiate LPO directly, but in presence of catalytic amounts of
metal ions, they can react and form .OH radicals under a net equation, Harber
Weiss reaction (Halliwel and Gutteridge 1984):
metal ion
.O2- + H2O2
.
O2 + .OH + OHcatalyst
Here, the reduction of membrane integrity and fluidity is as a consequence of
enhancement of LPO process. On the other hand, with commencing LPO
cascade, the release of significant quantities of lipid OOH in medium will be
occurred, which is presumably a consequence of the activity of membraneous
enzyme phospholipase A2 (PLA2) by cleaving oxidized PUFAs from the 2position of phospholipid glycerol backbone so that they can then be
7

metabolized by GPx to the corresponding alcohols (Halliwell and Gutteridge


1985; Alvarez and storey 1995). It has been determined that metal ions like
mercury can alleviate the rate of GPx activity in carp gill and liver resulting in
accumulation of lipid OOH in cell and LPO propagation throughout the cell
membranes which injures the membrane integrity (Radi et al 1988).
Furthermore, as Huang et al (1993) and Halliwel (1995) suggested it is possible
that lipid-OOH formed in the biological membrane systems by the effect of
metal ions convert to the form of peroxyl radical (Lipid O2.) which is able to
reattack the membrane.
It is of great interest to know that MDA formed during LPO could conceivably
reacts as bifunctional reagent to cross-link through schiffs base formation
between some classes of phospholipids (Eichenberger et al 1982). Lack of the
uniformity to these cross-links in membranes will be led to physical forces
which may disturb the membrane lipid distributions (Yuli et al 1981).
We found that GSH was marginally affected by the elevated metal ion
concentration effect (p < 0.05, Table 3 & 4 and fig. 1 A & B) to a lesser content.
GSH can react with peroxyl radicals to achieve a steady state for themselves and
itself converts to thiyl radical (GS.) which is not rapid as oxidative attack at
polyunsaturated membrane lipids:
GSH + Lipid O2.
lipid OOH + GS.
It seems that the metal ion extrusion from cells presumably involves movement
of diffusible complexes such as Hg GSH, it also gives rise to alleviating in the
level of thiol groups pool including GSH in medium.
Due to more reactivity of copper with GSH molecules than mercury, it can be
assumed that copper could present more oxidative stress on living organism
making its cells being more susceptible to damages. Despite the extensive
evidence implicating the depletion and / or oxidation of GSH in a wide variety
of experimental toxicities (Smith et al 1996), here it has been shown that GSH
content was not much altered under the influence of metal ion stress (p < 0.05).
An explanation here is that relative inhibition following metal ion impact on
some enzymatic defence systems such as GPx (Radi et al 1988), converts lipid
OOH to respective alcohol through oxidation of GSH to GSSG, causes no
remarkable alteration in GSH content due to less GSH consumption by inhibited
enzyme.
Supplementations were carried out to check the protective role of BSA (0.5 &
1.0%) and DMSO (0.5%) in media. DMSO as a lipophilic OH. scavanger
8

normally is used to mop up ROS and protect biological membranes against


oxidation. On the other hand, BSA also as a OH. scavanger might play a far
more important role in protection of plasma membrane from peroxidative
injuries by binding to and neutralizing the lipid hydroperoxides liberated by
PLA2 (Alvarez and storey 1995). BSA can impair the LPO cascade either by
acting as a sacrificial antioxidant or as a chelator of the metal ions that promote
this process (Halliwel and Gutteridge 1989). Another intriguing possibility from
cha and Kim (1996) is that BSA is able to exhibit peroxidase activity, providing
that thiol-reducing equivallents are available.
Addition of 1.0% BSA and DMSO caused a significant fall in the level of
TBARS upto concentration of 1000 M of metal ions and after that led an
increase in MDA production (Table 1 & 2), this is due to of the fact that thiol
groups which exist in these antioxidants could play a multifaceted role as prooxidants by autoxidizing to produce .O2- in the presence of metal ions (Tien et al
1982).
R SH + metal ion (oxidized)
RS + metal ion (Reduced).
Metal ion (Reduced) + O2
metal ion (oxidized) + .O2Collectively, our results showed that 0.5% BSA exerts a positive influence in
protection against fatty acid peroxidation than 1.0% BSA and DMSO as well
(Table 1 & 2) in control and treated ones.
The intracellular compartmentations of GSH molecules including the nucleus,
mitochondrial matrix and endoplasmic reticulum have many important
implications for cells that are exposed to toxic compounds or to other stresses
(Smith et al 1996). It is noteworthy that there is a kinetically distinct pool of
GSH in the nucleus, estimated to be 5 to 10% of the total GSH and may be
concentrated at the nuclear membrane surface (Loh et al 1990; Britten et al
1991) when an adequate stimulation is made, it might be released and added to
cytoplasmic pool.
We found that among the antioxidants tested only 0.5% BSA yielded an
alteration in the GSH content of the control group of preparations (p < 0.05, see
Table 3 & 4). It seems that BSA acted as a stimulation either for releasing the
stored GSH molecules from their cellular compartments or to detach the masked
GSH molecules from trapping points in the preparation. However the exact
mechanism of this phenomenon remains to be elucidated.
As mentioned earlier, thiol groups in cases of DMSO and 1.0% BSA could
imposed a severe peroxidation on cell membranes, however 1.0% BSA has been
9

found to be more toxic than DMSO to substract the GSH content when
accompanied with metal ions particularly in higher concentrations. More
analysis needs to be conducted.
Taken together, these findings revealed that used metal ions namely copper and
mercury are very harmful to gill cells of fish carp when are expressed in excess.
These metal ions not only act as anti fluidity agents for membrane lipids but
also in making a complex with cell protein thiols will be able to express more
toxic effects to push the cell towards dysfunction.
Acknowledgement
We are extremely grateful to Dr. (Mrs.) Noha Eftekhari for conducting
supplemental statistical analyses to check those data reported here.
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13

14

BIOCHEMICAL EFFECTS OF ENVIRONMENTAL NITRITE


IN MATRINX (Brycon cephalus)
Gilberto Moraes
Department of Genetics and Evolution, Federal University of So Carlos. Rod.
Washington Luiz, Km 235.
So Carlos, SP, Brazil. CEP 13565-905.
E-mail <gil@power.ufscar.br>
Ive Marchioni Avilez, Alexandre Eneas Altran
and Lucia Helena de Aguiar
Department of Genetics and Evolution Federal University of So Carlos.Rod.
Washington Luiz, Km 235.
So Carlos, SP, Brazil. CEP 13565-905.
Introduction
Nitrite usually occurs in aquatic environments as a product of bacterial activity.
It normally comes from oxidation of ammonia and this (nitrification) depends on
the water aeration. Another source of water nitrite is the industrial wastes
(Nikinmaa, 1992; Heckman et al., 1997). The fish-culture systems also increase
ammonia and nitrite, followed by undesirable consequences (Hargreaves, 1998;
Hagopian & Riley, 1998). The organismal disorder arisen from such
environmental disturbances are particularly observed in fishes.
The prompt nitrite effect in fishes is observed at the blood level. Plasma can
accumulate it (Shechter, 1972), working as a vehicle to spread it over the tissues.
Within the red blood cells it oxidizes the hemoglobin-Fe2+ to Fe3+ yielding
methemoglobin, unable to transport oxygen. This effect is supposed to result in
tissue hypoxia (Cameron, 1971; Bath & Eddy, 1980; Doblander, et al., 1996,
Vedel, et al., 1998) even in the presence of oxygen (functional hypoxia). The
intensity of methemoglobin formation is dependent on the non-oxygenated level
of hemoglobin (Jensen, 1990; Jensen, 1992; Willians, et al., 1993).
Methemoglobin content varies among the species and depends of the nitrite
levels and the exposure time. The increase of nitrite concentration increases lead
to raise methemoglobin concentration (Schoore, et al., 1995). Usually, the fish
plasma concentration of nitrite is higher than environmental one.

15

Environmental nitrite can cause physiological disturbs as decreases in total


hemoglobin, hematocrit and RBC. This phenomena can be explained by
erythrocyte hemolysis (Kundsen & Jensen, 1997).
In freshwater teleosts, nitrite exposure is followed by several osmoregulation
responses as hyponatremia, hypochloremia (Jensen et al, 1987), branchial
chloride cell failure (Gaino et al, 1984), and inhibition of chloride uptake
(Willians & Eddy, 1986).
Nitrite exposed fish is completely recovered in clean water (Huey & Beitinger,
1981), and a couple of mechanisms involved in such process are proposed. The
first is NADH-Methemoglobin reductase system (Diaforase I) reduces
hemoglobin-Fe3+ to Fe2+ and it role in nitrite detoxification has been studied in
fish (Scott and Harrington, 1985; Woo & Chiu, 1997). The nitrate synthesis, the
more oxidized form of nitrite, is a second way of detoxification (Doblander &
Lackner, 1997) and catalase and citocrome oxidase-aa3 is proposed to take a
share in such process (Doblander & Lackner, 1996). However, both mechanisms
are still unclear and further studies should establish the role of those enzymes in
fish detoxification of nitrite.
In this study the environmental nitrite effects hematological and osmoregulator
response, the methemoglobin formation and NADH-methemoglobin reductase
system will be investigated in the neotropical teleost Brycon cephalus
(matrinx).
Material and methods
Juveniles of B. cephalus (matrinx) ranging 90 5g (means SD) were
obtained from the fish farm Aguas Claras, Mococa, SP, Brazil. The fish were
brought to the aquaculture facilities of the Comparative Biochemistry
Laboratory. Before the experiments, fish were equally divided in four tanks of
250L, covered with black plastic sheets, provided with well-aerated water.
Quality of water was measure by APHA (1980) (pO2 = 7.5 ppm, pH = 6.8 0.2,
temperature = 23 1oC, conductivity = 74.3 4.8 S. cm-3, alkalinity = 37 mg/L
of CO3-, hardness = 28 mg/L of CO3-, ammonia concentration = 0,01 p.p.m,
nitrite concentration = 0 ppm). The indoor-experimental tests were performed in
August.

16

Experimental design
Kept starved for 1 day, one tank nitrite free was the control. In a semi-static
system (with water replace each twenty four hours), six fish per tank were
exposed to 1, 2, and 3 p.p.m of NO2 by 48 h. After this, the fish were collected
and anesthetized with MS 222. A blood sample was drawn from the caudal vein
and divided into aliquots for further analysis.
Blood analysis
Microhematocrit was done with blood samples centrifuged at 12.000 g for 3 min
in capillary tubes. Total hemoglobin was colorimetricaly determined at 540 nm
in samples containing 10 L of blood into 2.0 mL of Drabkin solution.
Methemoglobin was optically quantified at 563 nm as Matsuoka (1997). Red
blood cells were counted under light microscope with a Neubauer chamber and
the mean corpuscular volume (MCV), the mean corpuscular hemoglobin (MCH)
and the mean corpuscular hemoglobin concentration (MCHC) were calculated.
One blood aliquot was centrifuged at 12.000 g for 3 min and the plasma was
used to flame photometric determinations of Na+ and K+, and optical
determination of Cl at 480 nm (APHA, 1980) and NO2 at 520 nm (Shechter, et
al., 1972).
NADH-Methemoglobin reductase system
One aliquot of blood was re-suspended into 0.9% saline solution and centrifuged
at 1.000 g for 10 min. This procedure was repeated thrice and the cells were resuspended into 0,04 mL of mercaptoethanol-EDTA solution and the
erythrocytes were lysed by termal shock (with liquid nitrogen). This hemolysis
was used as enzyme source. The enzyme assay was performed into a buffer
solution 0.2M Tris-HCl pH 7.5, 1,2 mM 2.6-dichlorophenol indophenol, 6 mM
NADH, and a suitable enzyme aliquot. The substrate consume was optically
followed at 600nm and one unit equals a decrease in absorbancy of 1.0 per
minute, in 25C as Beutler (1984). The specific activity is expressed U (units)
per mg of hemoglobin (U/mg total Hb).
Statistics
For comparison of the dates was used STATISTICA 5.5 software. Normality of
the data set was evaluated by the SHAPIO-WILL W test with 95% of
17

confidence limit. The parametric test ANOVA was used to compare the groups
and the Post-Test of multiple comparisons DUNCAN was applied considering p
< 0.05.
Results
Increasing concentrations of environmental nitrite affected the blood parameters
of matrinx (Table I). The methemoglobin and the plasma nitrite increased very
sharply keeping high values (figure 1).
Hematocrit decreased in the all the nitrite exposures, however total hemoglobin
and the red blood cell number did not change. The MCV, MCH and MCHC did
not change too.
The NADH- methemoglobin reductase enzyme system was detected in the red
blood cells of B. cephalus. . That enzyme activity was found in all the fish and it
was not affected by nitrite exposure (Table1). The figure 1 shows the trend of
this enzyme during the nitrite exposure for 48 hours.
No significant effects were found in plasma protein, K+, Na+ and Cl under
nitrite exposure.

18

Table 1.Hematological parameters of Brycon cephalus exposed to environmental nitrite for 48h.
NITRITE (ppm)

PARAMETER
Total blood
MetHb
Ht
Total Hb
RBC
MCH
MCV
CMCH
NADH-MetHb reductase
Plasma
NO2Na+
K+
ClProtein

0
0.17 0
35.8 2
9.06 2
2.40 0.2
37.96 4.9
133.6 13
2.850.39
0.330.14

1
56.1* 6
26.0* 3
8.50 2
1.86 0.5
47.30 4.8
145.6 14
3.220.40
0.520.20

2
84.3* 7
24.0* 4
7.35 1
2.00 0.6
39.02 2.6
127.4 12
3.050.41
0.470.06

3
78.4* 8
23.9* 2
8.10 1
1.98 0.3
41.72 4.1
122.4 6
3.410.42
0.430.12

0.01 0.00
144.7 12
3.1 0.5
130.4 6
0.45 0.04

0.23* 0.02 0.34* 0.04


130.5 10
128.5 12
2.7 0.3
3.4 0.3
134.2 7
123.1 12
0.49 0.05 0.43 0.04

0.73* 0.05
103.0 9
3.2 0.4
110.6 12
0.41 0.01

The values are expressed as: Ht (%), Total Hb (g.dL1), Red Blood Cells-RBC (106.mm3), Mean Corpuscular
Hemoglobin-MCH (pg.cell1), Mean Corpuscular Volume-MCV (mm3), mean corpuscular hemoglobin
concentration MCHC (%), NADH-methemoglobin reductase (U.mg total Hb-1) Methemoglobin-MetHb
(%), Na+ and K+ (mEq.L1), Cl (nmol.mL1 ), Protein (mg.mL1), NO2 (nmol.ml1). The mark (*) means
significantly different (p< 0.05) as compared to the control.
19

MetaHb Reductase %
MetHb %

100
75
50
25
0
100
75
50
25

Plasma NO %

0
100
75
50
25
0
0

1
2
Nitrite ppm

Figure 1. Relative concentration (%) of blood methemoglobin reductase,


methemoglobin and plasma nitrate of Brycon cephalus (matrinx) exposed to
NO2- for 48 h, considered as 100% the maximum value of the parameter.

20

Discussion
Nitrite exposure in fish is supposed to result in tissue hypoxia that usually
causes significant stress (Huey et al, 1980, Arillo et al, 1984, Hilmy et al, 1987).
As a common classical response to stress from hypoxia, the increase of
hematocrit should be expected. This strategy increases the number of red cells
and the content of hemoglobin to keep the oxygen availability (Swift, 1981;
Peterson, 1990). However, these responses were not detected in several fishes
(Eddy, et al., 1983; Hilmy, at al 1987; Tucker, at al 1989; Jensen, 1990;
Knudsen & Jensen, 1997; Woo & Chiu, 1997), or are thinly discussed. In this
particular, decrease of the hematocrit of matrinx can be attributed to the blood
cell lyses (Jensen, 1990; Knudsen & Jensen, 1997), for the reduction of number
of cells without changes of MCV, MCH and MCHC. This response should
remove the blood methemoglobin but it will reduce the hemoglobin availability.
Decrease of hematocrit in matrinx without changes of the red blood cell
number and total hemoglobin is suggestive of hemolytic anaemia, which is a
posterior response to functional anemia (Scarano & Saraglia, 1984). Those
authors propose the increase of methemoglobin as an early functional anaemia.
The NADH-methemoglobin reductase system has been detected in the most
animals in nature, as well as in .B. cephalus. This enzyme recovers the
hemoglobin from methemoglobin keeping the equilibrium between both forms.
Among the fishes, it was reported in the channel catfish Ictalurus punctatus
(Huey & Beitinger, 1981), the rainbow trout Salmo gairdneri, Oncorhynchus
kisutch, Oncorhynchus nerka, Salmo malma (Scott and Harrington, 1985) and
Lates calcarifer (Woo and Chiu, 1997) and others.
Several studies attribute to the nitrite exposure the increase of methemoglobin
concentration. Also, the recovery of the methemoglobin levels to normal values
is observed as the fish return to nitrite free water. Schoore and col (1995)
attribute this fact to the activity of NADH-methemoglobin reductase system, in
spite of it was not assayed. One study on enzyme changes in fish exposed to
nitrite is reported by Woo and Chiu, (1997) but no significant change was
observed. The same occurred in matrinx exposed to nitrite, as the level of
NADH-methemoglobin reductase system did not change under any level of
environmental nitrite. However, the presence of that system is very important
for the species because it prevents the hemoglobin oxidation. The fact of
NADH-methemoglobin reductase system be unchanged in matrinx, does not
21

mean that this enzyme is not working, but more likely it was not induced as
proposed to Lates calcarifer (Woo & Chiu, 1997).
As well as the nitrite attacks the heme group of hemoglobin it is possible that
other heme proteins are affected, like the NADH-methemoglobin reductase
system. This enzyme also presents a heme group in the molecular structure
(citocromo b5). This attack could camouflage an increase of this enzyme
production. In rainbow trout, Arillo and col. (1984) suggest that nitrite could
attack hemoproteins as citocrome P450 in liver.
The main characteristic of fish nitrite exposure is the increase of methemoglobin
and plasma nitrite concentration (Cameron, 1971; Shechter et al, 1972). These
levels are usually very low and the increase depicts different trends. The
increase of plasma nitrite concentration reveals an exponential tendency and the
methemoglobin concentration reached a plateau. This characteristic suggests an
equilibrium of methemoglobin formation by the reductase system activity (Huey
et al, 1980).
The exposure of matrinx to nitrite did not change the ion concentrations. In the
marine teleost Lates calcarifer the enhancement of plasma sodium and chloride
has been reported (Woo and Chui, 1997) and such fact should be associated to
environmental seawater. Plasma potassium concentration is proposed to be
associated to nitrite uptake. The increase of plasma K+ in Cyprinus carpio
exposed to NO2- is reported, and the direct correlation for both ions leads to such
assumption (Jensen, 1990). In matrinx, the plasma concentration of pattern
nitrite followed the environmental one but the plasma level of potassium
remained constant indicating that hipercalemia did not occur in matrinx. The
Cl- concentration did not change in the most of the freshwater teleosts fish.
However, nitrite uptake by chloride cell in gills occurs by competitive
interaction between Cl- and nitrite with the uptake sites. Gaino and col. (1984)
suggest that there is no decrease of Cl- concentration because the hipertrophia of
some gill chloride cells. Other exchange mechanism may be an hyperactive
response working jointly to chloride cells to maintain the physiological Cllevels, despite the nitrite competition or decrease of HCO3-production. This
process could result in degeneration in these cells. That author showed that
nitrite inhibits the carbonic anhydrase of the gills (Cl- exchange with HCO3- in
gills) in vitro.
The present data call attention to the fact that the anti-oxidative mechanism to
prevent the hemoglobin conversion to methemoglobin in the freshwater teleost
22

matrinx exposed to nitrite is not enough efficient. No other mechanism to


prevent nitrite deleterious effects seems to work in matrinx, since the external
and the plasma concentration of nitrite was practically the same. Those fact plus
the osmotic disturbs in matrinx, are cumulative and certainly responsible for
the great sensibility of the species to the nitrite poisoning.
References
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And Ultrastructural Effects Of Nitrite In Rainbow Trout: Liver
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Doblander, C., Lackner, R. 1997 Oxidation Of Nitrite To Nitrate In Isolated
Erytrocytes: A Possible Mechanism For Adaptation To Environmental
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Eddy, F.B., Kunzilik, P.A., Bath, R.N. 1983 Uptake And Loss Of Nitrite From
The Blood Of Rainbow Trout, Salmo Gairneri Richardson, And
Atlantic Salmon, Salmo Salar L. In Fresh Water And Dilute Sea Water.
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Gaino, E., Arillo, A., Mensi, P. 1984 Involvement Of The Gill Chloride Cells Of
Trout Under Acute Nitrite Intoxication. Comp, Biochem. Physiol. A.,
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Hagopian, D.S., Riley, J.G. 1998 A Closer Look At The Bacteriology Of


Nitrification. Aquacultural Engineering, 18:223-244.
Hargreaves, J. A. 1998 Nitrogen Biogeochemistry Of Aquaculture Ponds.
Aquaculture, 166:181-212.
Heckman, C.W., Campos, J.L.E., Hardoim, E.L. 1997 Nitrite Concentration In
Well Water From Pocon, Mato Grosso, And Its Relationship To
Public Health In Rural Brazil. Bull. Environ. Contam. Toxicol., 58:815.
Hilmy, A.M., El-Domiaty, N.A., Weshana, K. 1987 Acute And Chronic
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Channel Catfish Ictalurus Punctatus. Can. J. Zool., 60:1511-1513.
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Blood Respiratory Proprieties, Acid-Base And Electrolyte Regulation
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Jensen, F.B. 1990 Nitrite And Red Cell Function I Carp: Control Factors For
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Jensen, F.B. 1992 Influence Of Haemoglobin Conformation, Nitrite And
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24

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25

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26

DNA SYNTHESIS AFTER EXPOSURE TO HEAVY METALS IN THE


TESTIS OF THE SPINY DOGFISH (SQUALUS ACANTHIAS)
J. Michael Redding
Department of Biology, Tennessee Technological University
Cookeville, TN 38501 USA
931-372-3135/931-372-6257/mredding@tntech.edu
EXTENDED ABSTRACT ONLY DO NOT CITE
Contamination of aquatic environments with metal compounds poses a serious
risk to the health of aquatic species and terrestial species that rely on food from
aquatic environments. Widespread metal contamination of both marine and
freshwater systems has been reported, and a large body of literature documents
deleterious effects of such pollution on various species. Of particular concern is
the tendency of animals, especially carnivores, to accumulate metals from
dietary sources, thereby increasing their risk for dose-dependent toxic effects.
The spiny dogfish, Squalus acanthias, and other elasmobranchs are especially
susceptible to such cumulative effects because they are long-lived, carnivorous
animals whose home range includes coastal marine habitats which tend to have
the highest concentrations of metals. Several studies have documented high
metal concentrations in tissues of the spiny dogfish and other members of the
genus Squalus (e.g., Taguchi, 1979). Metal intoxication may be directly
detrimental to the health of the dogfish populations by decreasing survival or
reproductive fitness.
The known toxic effects of metals are diverse. Effects are thought to be exerted
via the formation of stable complexes with many different biological molecules
including proteins, DNA, RNA, and phosphorylated compounds. Specific
mechanisms of metal toxicity have been characterized extensively for
mammalian systems, but much less is known about non-mammalian vertebrates.
Information on the specific cellular effects of metals on male reproductive
systems in vertebrates is sparse but suggestive of profound disturbances (e.g.,
Clarkson, et al., 1983).
The dogfish testis has proven to be an excellent model for studying the
regulation of vertebrate spermatogenesis (DuBois and Callard, 1991). Distinct
developmental stages of spermatocysts (germ cell:Sertoli cell units) can be

27

isolated and cultured in vitro for at least two weeks. Moreover, mitotic activity,
as indicated by DNA synthesis, is maintained quantitatively during this period
and is responsive to stimulatory and inhibitory factors (Piferrer, et al., 1993).
Thus, this model system would seem suitable for toxicological studies of
vertebrate spermatogenesis. The purpose of this study was to determine the
effects of various metals on DNA synthesis in the testis of the spiny dogfish.
For each experiment, spermatocysts were isolated from zone I tissue from testes
of 2-4 sharks and maintained in culture with Leibovitz L-15 medium, modified
for use with elasmobranch tissue (DuBois and Callard, 1991). After various
periods of treatment with metals, 5.0 uCi/ml of 3H-thymidine was added to the
cyst cultures for 6-24 hr before harvesting the cysts. Harvested cysts were
washed twice with saline solution augmented with excess unlabelled thymidine.
Then, cysts were treated with ice-cold 10% trichloroacetic acid for 1-24 hr.
Cysts were then washed again before solubilizing overnight in 0.2 M NaOH.
Aliquots of the solubilized cysts were analyzed for radioactivity (cpm). Data
were not standardized by sample protein concentration as previously reported;
in these experiments such standardization would not significantly change the
results. Results from cysts treated with metals were standardized as a
percentage of the mean of untreated controls. The standardized means of
treatment groups were compared to that of untreated control groups by an
unpaired t-test with a pooled variance estimate.
Preliminary experiments showed that mercuric chloride (HgCl2) at
concentrations greater than 100 uM inhibited synthesis of DNA. Subsequently,
the effects of equivalent concentrations (100, 500, 1000 uM) of HgCl2, the
organic mercurial parachloro-mercuric-phenol-sulfonic acid (PCMBS), sodium
vanadate (NaVO3), zinc chloride (ZnCl2), and cadmium chloride (CdCl2) were
evaluated. Effects of these metals were compared to untreated controls, positive
controls treated with bovine insulin (10 ug/ml), and negative controls treated
with isobutyl-methylxanthine (1 mM, IBMX). Results of this experiment are
shown in Table 1.
Mercurial compounds showed dose-dependent inhibition of DNA synthesis. Of
these HgCl2 was most potent, virtually negating DNA synthesis between 100
and 500 uM. Cadmium stimulated DNA synthesis at 100 uM but markedly
inhibited it at 500 and 1000 uM. Vanadate was relatively ineffective, reducing
DNA synthesis only to 71% of controls at 1000 uM. In contrast, zinc slightly
stimulated DNA synthesis at all concentrations. Beyond simply identifying
their effective concentrations, these results demonstrate the specificity of
28

various metals with respect to their effects on DNA synthesis in shark testis.
This specificity may reflect differences in the capacity and affinity of metal
binding proteins, such as metallothionine, that effectively sequester metals and
prevent them from affecting critical cellular processes. It is evident from these
results that DNA synthesis in the Squalus testis is sensitive to metal
intoxication, generally supporting previous results from mammalian models
(Clarkson, et al., 1983). These results support the use of Squalus testis as a
model system for toxicological studies of vertebrate spermatogenesis.
[This research was supported by a fellowship from the Lucille P. Markey
Charitable Trust via the Mount Desert Island Biological Laboratory. I thank
G.V. Callard, D.S. Miller, D. Barnes and A. Kleinzeller for valuable advice and
material support.]
Table 1. DNA synthesis rates of Squalus zone I spermatocysts cultured in vitro
with metals, insulin (10 ug/ml, positive control), or IBMX (1 mM, negative
control) for 24 hr and exposed for the last 12 hr to radiolabelled thymidine.
Results are shown as a mean (SE) percentage of control cultures. Sample size
was four for each treatment and eight for the untreated control. All means
except those noted by "ns" were significantly (P< 0.01) different from the
control.
Treatment
Control
Insulin
IBMX
HgCl2
PCMBS
CdCl2
NaVO3
ZnCl2

Metal Concentration (uM)


0
100
100 (2)
--192 (7)
--34 (3)
----84 (2)
--95 (3)ns
--128 (6)
--98 (3)ns
--117 (6)

29

500
------3
62
69
--117

(1)
(3)
(2)
(6)

1000
------0 (0)
8 (3)
21 (3)
71 (4)
127(12)

References
Clarkson, T.W., Nordberg, G.F., and Sager, P.R. 1983. Reproductive and
developmental toxicity of metals, Plenum Press, New York.
DuBois, W. and Callard, G.V. 1991. Culture of intact Sertoli/germ cell units
and isolated Sertoli cells from Squalus testis: I Evidence of stagerelate functions in vitro. J. Exp. Zool. 258:359-372.
Piferrer, F., Redding, M., DuBois, W., and Callard, G. 1993. Stimulatory and
inhibitory regulation of DNA synthesis during spermatogenesis:
studies in Squalus acanthias.
Taguchi, M., Yasuda, K., Toda, S., and Shimizu, M. 1979. Study of metal
contents of elasmobranch fishes: Part I Metal concentration in the
muscle tissues of a dogfish, Squalus mitsukurii. Mar. Environ. Res. 2:
239-249.

30

QUANTITATIVE PCR ANALYSIS OF ATLANTIC SALMON


CYP1A IN GILLS
Christopher B. Rees
Department of Fisheries and Wildlife
Michigan State University
13 Natural Resources Building
East Lansing, MI 48823
Phone: 517-432-1141
Fax: 517-432-1699
Email: reeschri@msu.edu
Stephen D. McCormick
USGS, Leetown Science Center, Conte Anadromous Fish Research
Center
One Migratory Way, PO Box 796
Turners Falls, MA 01376
Phone: 413 863-3804
Fax: 413 863-9810
Email: mccormick@umext.umass.edu
Weiming Li
Department of Fisheries and Wildlife
Michigan State University
13 Natural Resources Building
East Lansing, MI 48823
Phone: 517-432-1141
Fax: 517-432-1699
Email: liweim@msu.edu
EXTENDED ABSTRACT ONLY - DO NOT CITE
Environmental pollutants are hypothesized to be one of the causes of
recent declines in wild populations of Atlantic salmon (Salmo salar)
across Eastern Canada and the United States. Some of these pollutants,
such as polychlorinated biphenyls and dioxins, are known to induce
expression of the CYP1A subfamily of genes. We applied a highly
sensitive technique, quantitative reverse transcription-polymerase chain
reaction (Q-RT-PCR), to measure CYP1A induction and expression
patterns in Atlantic salmon gills. This assay was used to detect patterns
31

of CYP1A mRNA levels, a direct measure of CYP1A expression, in


Atlantic salmon exposed to pollutants under both laboratory and field
conditions. Two groups of Atlantic salmon juveniles (48-76 g)
received an intraperitoneal injection of 50 g g-1 -naphthoflavone
(BNF) or vehicle only (n=10 for both groups). Non-lethal gill biopsies
were taken for each treatment group prior to injection and 1,2, and 7
days post-injection. The same fish were used at each sampling point.
After RNA extraction and Q-RT-PCR analysis, control fish showed
static levels of CYP1A over the course of sampling. Induced salmon
demonstrated similar levels of CYP1A to control fish at time zero and a
significant induction over the course of each additional sampling
period. The quantitative RT-PCR was also applied to salmon sampled
from two streams (n=10 for each stream) in Massachusetts, USA.
Salmon gill biopsies sampled from Millers River (South Royalston,
Worcester County), known to contain polychlorinated biphenyls
(PCBs), showed a significant induction over those collected from a
stream with no known contamination (Fourmile Brook, Northfield,
Franklin County). Gill biopsies coupled with Q-RT-PCR analysis is a
novel, sensitive, and accurate method to estimate CYP1A expression
dynamics in gill tissues of Atlantic salmon. This method has the
potential to be a valuable tool in environmental assessment of not only
wild Atlantic salmon populations, but many other populations of
salmonids as well.
Acknowledgements
This work is supported by the National Marine Fisheries Service and
the Great Lakes Fishery Commission.

32

EXPRESSION OF MULTI-DRUG RESISTANCE (MDR)


P-GLYCOPROTEIN AND ASSOCIATED PROTEINS (MRPs) IN
THE FLOUNDER; EFFECTS OF EXPOSURE TO THE
POLYAROMATIC HYDROCARBON, BENZO(a)PYRENE.

Gordon Cramb
School of Biology, University of St Andrews, Bute Medical Buildings,
St Andrews, Fife, Scotland, KY16 9TS.
Email gc@st-and.ac.uk; Tel. 044/0 1334 3530; Fax 044/0 1334 3600.
(1)

Christopher P. Cutler, (1)Jean-H. Lignot, (1)Anne-Sophie Martinez,


Belinda S. Chesman, (2)Susan C. Frankling and (2)J.Anne Brown,
(1)
School of Biology, University of St Andrews,
Bute Medical Buildings, St Andrews, Fife, Scotland, KY16 9TS
(2)
School of Biological Sciences, University of Exeter,
Hatherley Laboratories, Exeter, EX4 4PS

(2)

EXTENDED ABSTRACT ONLY - DO NOT CITE


Introduction
Aquatic organisms are frequently faced with periods of exposure to various
environmental pollutants, often as the result of the release of chemicals from
agricultural and/or industrial activities. At present, a number of potential
biomarkers have been characterised in terms of their sensitivity to a wide
range of natural and anthropogenic xenobiotics. In addition to these classic
biomarkers, there is increasing evidence to suggest that certain members of
the ATP Binding Cassette (ABC) transporter superfamily, including the
multi-drug resistance (MDR)/p-glycoprotein and multi-drug resistance
associated protein (MRP) sub-gene families, are also involved in the
survival of marine organisms exposed to various environmental pollutants.
The products of these genes are active transporters that couple the
hydrolysis of ATP with the epithelial transport of organic cations and
anions. The normal physiological role for these proteins is in the transport
of bile salts and organic ions within the liver and intestinal parenchyma
33

(Meier and Stieger, 2002), however as a result of their wide substrate


specificity these complex membrane proteins can also transport, and
therefore aid in the excretion of, a wide range of environmental toxins. As
a result, the products of these gene families have been implicated in the
development of cellular resistance to various environmental xenobiotics.
To date, very little information is available about the regulation of
expression and function of the MDR/p-glycoprotein/MRP families in
marine teleosts and how these genes may confer protective resistance
following exposure to various environmental pollutants. In this study we
report the cloning, sequencing and expression of several members of the
MDR and MRP gene families from the European flounder (Pleuronectes
flesus). The levels of expression of these gene products were monitored
after exposure of fish to the polyaromatic hydrocarbon, benzo(a)pyrene
(B(a)P).
Materials and methods
Juvenile flounder were maintained in normal seawater or in seawater
containing 0.5ppm B(a)P for up to 6 days before removal of tissues for
RNA extraction (Cutler et al., 2000), plasma membrane preparation
(McCartney and Cramb, 1993) and sampling of bile for analysis of B(a)P
metabolites by fluorescence spectroscopy. Cloning of the MDR and MRP
cDNAs was conducted using Invitrogen's TA Cloning Kit (Leek, The
Netherlands) following amplification of fragments using degenerate primers
in RT-PCR and then 3'- and 5'-RACE specific primers with Marathon
cDNA (Clontech, Basingstoke, UK). Fragments were sequenced using a
Big Dye Terminator Cycle Sequencing kit (Perkin Elmer Biosystems,
Warrington, UK) and radiolabelled (Amersham Pharmacia Biotech,
Megaprime labelling kit; Bucks, UK) for use in Northern blots. Purified
plasma membrane fractions were run on denaturing SDS PAGE gels, blotted
onto PVDF membranes and processed for Western blotting using specific
antisera raised to two isoforms of flounder MDR. Liver and gut tissues
were
fixed
in
4%
paraformaldehyde
and
processed
for
immunohistochemistry. Sections were probed with MDR isoform-specific
antisera and then incubated with FITC-conjugated donkey anti-rabbit IgG
before visualisation on a fluoresecent microscope.
Results and Discussion
RT-PCR and Marathon RACE techniques amplified full-length cDNA
34

clones for two members of the MDR family (termed MDR-A and MDR-B)
from the European flounder. Comparison of sequence homologies between
the teleost MDRs and the genebank data bases indicated that MDR-A is a
member of the bile transporter sub-family that is known as sister of pglycoprotein or abbreviated to sp-gp. MDR-B amino acid sequences are
more consistent with the multi-drug resistance/p-glycoprotein family,
however due to heterogeneity within the sequences it is not possible to
assign MDR-B as the homologue of any one member of the mammalian
MDR family. In addition to these two members of the flounder MDR gene
family, cDNA fragments of five members of the related multi-drug
resistance associated protein (MRP) family were amplified and cloned.
These cDNA fragments have subsequently been characterised, based on
sequence comparisons) as homologues of mammalian MPRs 1 to 5.
Although expressed in most tissues at low levels MDR and MRP isoforms
exhibited differential tissue expression with the liver being the major site of
expression of MDR-A, the intestine and brain for MDR-B and mixed
expression profiles for the MRP isoforms (Fig. 1). Expression profiles
were not affected to any major degree by exposure of fish to B(a)P. Highly
purified plasma membrane fractions were isolated from both the liver and
the intestine and samples run on denaturing SDS-PAGE for Western
blotting. Multiple immunoreactive bands ranging in size from 100kDa to
>250 kDa were apparent in both tissues and with antisera raised to both
MDR-A and MDR-B with no marked differences between control and
B(a)P-treated fish (see Fig 2). Immunohistochemistry revealed that, as
expected, the bile canniculae were the main site of expression of both MDR-

35

36

A and MDR-B isoforms in the liver. In the intestine both proteins were
expressed within in the apical membrane of the intestinal epithelia although
MDR-A was also found in duodenal gland-like structures which lie under
the absorptive cell layer.

Acknowledgements
This study was funded by a project grant from the Natural Environment
Research Council - GR3/12467.

37

References
Cutler, C.P., Brezillon, S., Bekir, S., Hazon, N. and Cramb, G 2000.
Expression of a duplicate Na, K-ATPase 1 isoform in the
European eel (Anguilla anguilla) Am.J.Physiol. 279: R222-R229.
McCartney, S. and Cramb, G. 1993. Effects of a high salt diet on hepatic
atrial natriuretic peptide receptor expression in Dahl salt-sensitive
and salt-resistant rats. J.Hypertens. 11: 253-262.
Meier, P.J. and Stieger, B 2002. Bile salt transporters. Ann. Rev. Physiol.
64: 635-661

38

SEDIMENT-ASSOCIATED POLLUTANTS IN THE MARINE


ENVIRONMENT: A MULTI-BIOMARKER APPROACH FOR
ASSESSING SEDIMENT TOXICITY IN TURBOT
Kilemade, M., Hartl, M. G. J., OHalloran, J.
Department of Zoology and Animal Ecology
University College Cork, Ireland
Telephone: +353214904594, fax: +353214904593; e-mail: m.hartl@ucc.ie
Sheehan, D.
Department of Biochemistry
University College Cork, Ireland
van Pelt, F. N. A. M.
Department of Pharmacology and Therapeutics
University College Cork
OBrien, N. M.
Department of Food Science, Food Technology and Nutrition
University College Cork, Ireland
EXTENDED ABSTRACT ONLY- DO NOT CITE
Sediments in the aquatic environment have become an area of concern due to
their potential for accumulating toxic compounds and acting as a secondary
pollutant source to benthic fauna. Owing to their predominantly benthic life
style, fish of the order Pleuronectiformes (flatfish) are particularly vulnerable to
sediment-associated pollutants. This and the relative ease of obtaining
specimens, from either commercial hatcheries or local estuaries, makes them the
preferred choice for studying sediment-water-organism interactions in benthic
fish (Courtney et al., 1980; Hartl et al., 2001).
Here we report on the first phase of an ongoing project applying a multibiomarker approach to the toxicity of field-collected sediments from a polluted
estuary to juvenile turbot (Scrophthalmus maximus, L.). Laboratory experiments
using fish exposed to spiked sediments have been instrumental in establishing

39

biomarkers for single compounds. The aim of the present study was to
determine a suite of biomarkers, in combination with chemical and statistical
analysis, capable of establishing cause and effect relationships of exposure to
sediments containing complex mixtures of pollutants.
Sediments were sampled at low tide from two sites, Whitegate and Aghada, in
Cork Harbour, Co. Cork, Ireland, where sediments have previously been shown
to contain elevated levels of organic pollutants, particularly polychlorinated
biphenyls and organotin compounds (Boelans et al., 1999) and from a clean
reference site at Ballymacoda, Co. Cork, Ireland (Byrne and O'Halloran., 2000).
The top layer of the sediments were collected, thoroughly mixed and stored at
4C over night. Subsamples were frozen (-20C) prior to chemical analysis. A
layer (approx. 5 cm) of sediment was applied to 500 litre aquaculture tanks filled
with aerated seawater (S = 35; 15C).
Following 7 days acclimation, 60 turbot (30g 5) were added to each tank and
exposed for 21 days. Individuals were sampled at 0, 7, 14 and 21 days and
sacrificed. Blood samples were taken from the caudal vein for the analysis of
blood osmolality, haematocrit, differential cell counts, serum protein and DNA
single strand breaks. A Comet assay, for the analysis of DNA single strand
breaks, was also performed on skin, gill, spleen and head kidney cell
preparations. The liver and two gill arches from both the upper and the lower
gill pouch were removed, shock-frozen in liquid nitrogen and stored at 70C
until further analysis of P450 induction (measured as EROD activity in hepatic
S9 post-mitochondrial fractions) and membrane-bound Na+/K+-ATPase activity,
respectively.
Results from preliminary experiments showed that turbot exposed to
contaminated sediments displayed an increase in DNA single strand breaks in
gill cells, haemocytes and haemopoietic tissues when compared to those
exposed to sediments from the clean site. There was also an increase in blood
osmolality in fish exposed to the polluted sediments, indicating an increase in
membrane permeability, due to the possible interaction of lipophillic organic
compounds with gill epithelia, and the resulting osmotic loss of water across the
membrane. The blood parameters, haematocrit, differential cell counts and
serum protein, remained unchanged during the exposure period in all treatments.
Although sediments spiked with a single compound have aided the
understanding of toxicological mechanisms, they generally have limited
environmental relevance, in particular by disregarding potential synergistic

40

and/or antagonistic pollutant effects. By using an array of relevant biomarkers


combined with chemical and statistical (e.g. Principal Component Analysis)
analysis, we are currently assessing the toxicological effects of field-collected
sediments from Cork Harbour and the principle pollutants involved.
References
Boelans, R. G. V., Maloney, D. M., Parsons, A. P. & Walsh, A. R. (1999).
Ireland's Marine and Coastal Areas and Adjacent Seas - An
Environmental Assessment Marine Istitute, Dublin: 388.
Byrne, P. A. & O'Halloran, J. (2000). Acute and sublethal toxicity of estuarine
sediments to the manila clam, Tapes semidecussatus. Environ. Toxcol.
15: 456-468.
Courtney, W. A. M. & Langston, W. J. (1980). Accumulation of polychlorinated
biphenyls in turbot (Scrophthalmus maximus) from sea water sediments
and food. Helgol. Meeresunters. 33: 333-339.
Hartl, M. G. J., Hutchinson, S. & Hawkins, L. E. (2001). Sediment-associated
tri-n-butyltin chloride and its effects on osmoregulation of freshwateradapted 0-group European flounder, Platichthys flesus (L.). Aquat.
Toxicol. 55: 125-136.

41

42

RESPONSES OF ATLANTIC SALMON


AND BIVALVE MOLLUSCS TO
PARALYTIC SHELLFISH
F B Eddy
Department of Biological Sciences
University of Dundee, Dundee, DD1 4HN, Scotland, UK.
f.b.eddy@dundee.ac.uk
Matt J Gubbins and Susan Gallacher
FRS Marine Laboratory, PO Box 101, Victoria Road
Aberdeen AB11 9DB, Scotland, UK
EXTENDED ABSTRACT ONLY DO NOT CITE
Paralytic shellfish poisoning (PSP) toxins are a group of potent neurotoxins
produced by certain strains of marine dinoflagellates. Blooms of these algal
species can result in the passage of PSPs through marine food webs, with
detrimental effects on the marine environment and human health. These toxins
have been implicated as the causative agent in some of the many fish kills that
have occurred during blooms of PSP producing dinoflagellates. As such, PSPs
represent a potential threat to fisheries resources and aquaculture. Little is
known of the fate of these compounds in fish, but they have been shown to
accumulate in the liver of mackerel sampled after bloom events. Analysis of fish
tissues also suggests that transformation of these toxins does occur following
absorption since the PSP analogues differ from those to which the fish were
exposed. The induction of xenobiotic metabolising enzyme activities has also
been noted in Atlantic salmon exposed to PSPs and may represent a
detoxification mechanism.
Better understood is the fate of PSPs in shellfish. Bivalve molluscs accumulate
significant of PSPs during toxic blooms and pose a serious heath risk to
vertebrate consumers. Many species of marine invertebrates are reported to be
resistant to the effects of neurotoxins, a trait that is innate in some species and
acquired in others. There are reports of PSP biotransformations in shellfish,
43

namely epimerisations, decarbamoylations and reductive eliminations that alter


the profile of the toxins as they are transferred from causative dinoflagellate to
shellfish tissue. There are also reports of protiens, inducible on exposure to PSPs
that can bind to saxitoxin. Such a mechanism may be responsible for
inactivating PSPS and could confer resistance to invertebrates that express these
proteins.
Little is known about the sub-lethal effects of exposure from this group of toxins
on marine organisms. Laboratory based exposure experiments on Atlantic
salmon (Salmo salar) indicate that intra-peritoneal exposure to low doses (2-4
g/kg) of saxitoxin causes an induction of hepatic glutathione S-transferase
(GST) activity within four days. Doses approximating the LD50 for this
compound (4 g/kg) had little effect on blood plasma ionic concentration of
surviving fish.
Mussels (Mytilus edulis), like other invertebrates, appear insensitive to the
paralytic effects of PSTs. Exposure to high doses (intra-muscular, >100 g/100g
soft tissue) of saxitoxin, however, causes an induction of digestive gland GST
activity. This is in contrast to scallops (Pecten maximus) which showed no
induction of GST activity after acquiring high digestive gland toxicities from
feeding on cultures of toxic dinoflagellates. After toxic events, scallops retain
PSTs considerably longer than mussels. It is suggested that the induction
response of GST in mussels may be partly responsible for this discrepancy in
toxicokinetics between the two species.
In conclusion further work is required to define the metabolic pathways leading
to the detoxification and excretion of saxitoxins and related compounds.
References
MJ Gubbins, FB Eddy, S Gallacher and RM Stagg (2000). Paralytic shellfish
poisoning toxins induce xenobiotic metabolising enzymes in Atlantic
salmon (Salmo salar). Marine Environmental Research 50, 469-483.
M J Gubbins, EA Guezennec, F B Eddy, S Gallacher & RM Stagg,. (2001).
Paralytic shellfish toxins and glutathione S-transferases in artificially
intoxicated marine organisms. GM Hallegraeff, SI Blackburn, CJ Bolch
& RJ Lewis (eds), Harmful Algal Blooms 2000. Intergovernmental
Oceanographic Commission of UNESCO. Paris, 2001. 387 - 390

44

Fig 1. Hepatic glutathione S-transferase (GST) activities of Atlantic salmon


administered intra-peritoneal injections of physiological saline,
saxitoxin and extraxts of a toxic (Alexandrium fundyense) and a nontoxic (Scrippsiella trochoidea) strain of dinoflagellate over 21 days.
Error bars represent + or SEM, n=10 (except for saxitoxin group
where n=9). Ab, groups with different notation are significantly
different (P<0.05)

45

46

THE EFFECT OF ENVIRONMENTAL LEVELS


OF FRESHWATER CONTAMINANTS
ON JUVENILE ATLANTIC SALMON (Salmo salar L.):
IMPLICATIONS FOR MARINE SURVIVAL
Nicola Lower
Centre for Environment, Fisheries and Aquaculture Science,
Lowestoft Laboratory, Pakefield Road, Suffolk, NR33 0HT, UK
Tel: +44 (0) 1502 562244 Fax: +44 (0) 1502 513865
E-mail: N.J.Lower@cefas.co.uk
Andy Moore
Centre for Environment, Fisheries and Aquaculture Science,
Lowestoft Laboratory, Pakefield,Road, Suffolk, NR33 0HT, UK
Tel: +44 (0) 1502 562244 Fax: +44 (0) 1502 513865
E-mail: A.Moore@cefas.co.uk
EXTENDED ABSTRACT ONLY DO NOT CITE
There is increasing concern over the continuing decline of wild stocks of
Atlantic salmon, Salmo salar, throughout the North Atlantic, and the impact
on commercial and recreational fisheries. Recent research has demonstrated
that the freshwater and marine environments cannot be considered in
isolation and that conditions within the freshwater zone experienced by
Atlantic salmon may be critical to their subsequent survival in the sea. In
particular, exposure of juvenile salmon to a range of sub-lethal
concentrations of freshwater contaminants, such as pesticides, may operate
to reduce survival in fish once they have migrated to sea.
Environmental levels of a wide range of pesticides have previously been
shown to deleteriously effect Atlantic salmon reproduction and fecundity,
by disrupting pheromone-mediated spawning and reducing fertilisation rates
(Moore and Lower, 2001; Moore and Waring, 2001). However, the effects
of such exposure on other critical life history stages, for example
developing embryos and the parr-smolt transformation, and the subsequent
survival of salmon in the sea is less clear. The aim of the present studies
was therefore to determine the impacts of freshwater contaminants on smolt
physiology, behaviour and marine survival and to further investigate the
sub-lethal effects of contaminants on reproduction and the emergence of
juvenile salmonids.
47

Two types of persistent freshwater contaminants were studied: flame


retardants and pesticides derived from intensive agricultural. Flame
retardants are substances used in the manufacture of a wide range of
materials such as plastics and textiles. These chemicals enter the aquatic
environment as point sources either through leaching from landfills or from
effluents derived from the manufacturing process. The majority of flame
retardants contain brominated organic compounds, making them persistent
and lipophilic with the ability to bioaccumulate. Pentabromodiphenyl ether
(PeBDE), tetrabromobisphenyl-A (TBBPA) and hexabromocyclododecane
(HBCD) are the most frequently used brominated flame retardants, and they
are all found at low levels in many European rivers (de Wit, 2002). The
widespread usage of pesticides in agriculture has resulted in the extensive
contamination of many rivers and tributaries supporting salmon populations.
Three such pesticides are cypermethrin, a synthetic pyrethroid insecticide
primarily used in crop sprays and in sheep dips; diazinon, an
organophosphate insecticide; and atrazine, a pre- and post-emergence
herbicide used in the control of annual and perennial grass and broad-leaved
weeds.
In a series of separate studies, pre-smolts (Salmo salar) were continuously
exposed to either environmental levels of flame retardants (PeBDE, TBBPA
and HBCD) or the pesticides (cypermethrin, diazinon and atrazine). Fish
were maintained in a freshwater flow-through system and contaminants
were introduced using a peristaltic pump. Dosing periods ranged from 10 to
14 days, or in the case of the atrazine study, a period of 2 months. A
number of fish from each group were then sampled for some of the
physiological parameters associated with the parr-smolt transformation
(plasma ion levels [Na+, K+, Cl-], gill Na+ K+ ATPase activity and plasma
thyroid levels [triiodothyronine, T3; thyroxine, T4]. The remaining fish in
each group were subsequently exposed to a seawater challenge test (SWC)
for 24 hours to assess osmotic capabilities and adaptation of the fish to
saltwater. At the end of this test, all fish were again sampled and the above
physiological parameters measured. In the atrazine study, fish were first
PIT-tagged, exposed to the pesticide and subsequently released into an
experimental stream channel in order to determine any effect of exposure on
downstream migratory behaviour.
Exposure of the smolts to the flame retardants (0.5, 5, 10, 50, 100ngl 1) had
little or no effect on gill Na+ K+ ATPase activity or plasma Na+, K+, or Cllevels. Thyroid hormone levels significantly increased in the control group
following exposure to seawater, but exposure to 0.5ngl-1 PeBDE abolished
this increase and both T3 and T4 plasma levels were significantly lower
compared to the SWC control. All fish in the groups dosed with higher
48

levels of PeBDE died during the SWC. Exposure to atrazine (0.5, 5gl-1)
also had no effect on the plasma ion levels in smolts, but the gill Na+ K+
ATPase activity was significantly lower in the fish dosed with 0.5gl-1
atrazine compared to the SWC control.
Current research is focused on modelling the impacts of environmental
levels of both individual pesticides and mixtures on smolts, reproductively
mature parr, and developing embryos to determine possible synergistic or
additive effects. In one study, salmon eggs and milt were briefly exposed
during fertilisation to pesticide-dosed water, before being incubated in
artificial redds. Preliminary results indicate that exposure even at this stage
of the salmonid life cycle can have implications for the production of
quality juveniles, as both timing of emergence and mortality of fry were
effected.
In conclusion, exposure of environmental levels of waterborne
contaminants within freshwater has been shown to disrupt a number of
sensitive stages in the life cycle of the salmon. This has implications for the
number of juvenile salmon recruited into the population, their subsequent
survival in the marine environment and the numbers of returning adults.
Acknowledgements
This work was funded by DEFRA. The authors wish to thank Lorraine
Greenwood for assistance with the gill ATPase assay.
References
De Wit, C.A. 2002. An overview of brominated flame retardants in the
environment. Chemosphere. 46: 583-624.
Moore, A. and Lower, N. 2001. The impact of two pesticides on olfactorymediated endocrine function in mature male Atlantic salmon
(Salmo salar L.) parr. Comparative Biochemistry and Physiology
Part B. 29:269-276.
Moore, A. and Waring, C.P. 2001. The effects of a synthetic pyrethroid
pesticide on some aspects of reproduction in Atlantic salmon
(Salmo salar L.). Aquatic Toxicology. 52 (1):1-12.

49

50

CHLORIDE CELL CHANGES INDUCED BY NITRITE EXPOSURE


IN AN AMAZONIAN FISH SPECIES
Oscar Tadeu Ferreira da Costa
Federal University of So Carlos
Post-Graduate Program in Ecology and Natural Resources
C. Postal 676, Phone: 55 16 260-8314
Department of Morphology
University of Amazonas
Marisa N. Fernandes
Federal University of So Carlos
Department of Physiological Sciences
C. Postal 676, Phone: 55 16 260-8314
email: dmnf@power.ufscar.br
Abstract
The gill chloride cells of the juvenile Amazonian fish Colossoma macropomum
were analyzed using light and scanning and transmission electron microscopy
after 96h exposure to 0.04 and 0.2 mM nitrite (NO2-). Although the number of
chloride cells decreased significantly in the lamellar epithelium, no decrease was
found in the interlamellar region of the gill filament. A positive dose-effect was
evidenced by ultrastructural changes in chloride cells. NO2- exposure caused
significant reduction on the apical surface area of individual chloride cells (p <
0.05), with a resulting reduction of the fractional area of these cells in both the
lamellar and filament epithelium. Swelling of endoplasmic reticulum cisternae,
nuclear envelope and mitochondria were the main changes found in the chloride
cells. Cristae lysis and matrix vacuolization characterized the mitochondrial
changes. The overall ultrastructural changes in the chloride cell indicated
cellular functional disruption caused by exposure to nitrite.
Introduction
Nitrite (NO2-) is toxic to aquatic organisms. High NO2- levels may develop as a
result of an imbalance between the bacterial nitrification and denitrification
processes of ammonia (Lewis and Morris, 1986). Some environmental
conditions such as temperature and high levels of organic matter favor the

51

occasional development of NO2- in the Amazonian environment and in fish


culture systems. The active mechanism for NO2- uptake by the gill chloride cell
and its possible toxic effect on these cells were discussed by Williams and Eddy
(1986). However, most studies have investigated the toxic effects of NO2- on
hemoglobin, the P450 enzymatic system and mitochondrial cytochrome oxidase.
Few studies have focused on the ultrastructural changes in gill chloride cells.
The main purpose of the study reported on herein was to evaluate the effect of
NO2- on the number and ultrastructure of the chloride cells of an Amazonian
serrasalmid species, Colossoma macropomum (Cuvier, 1818), popularly known
as tambaqui.
Materials and Methods
Experimental animals
Juvenile specimens of tambaqui, Colossoma macropomum, weighing 66 3 g,
were obtained from the Aquaculture Research and Training Center
CEPTA/IBAMA (Pirassununga, SP). The fish were kept for at least 4 weeks in
1000-L holding tanks supplied with running aerated water (at a temperature of
21oC; pH 7.4; water PO2 > 130 mmHg; Ca++, 1.71 mg/L, Na+, 0.78 mg/L, Cl-,
0.5 mg/L, NO2- < 0.004 mM). The animals were fed daily to satiation with
commercial fish food pellets. Feeding was suspended two days before the
experiments. The photoperiod adopted was 12 h daylight and 12 h night during
acclimation and throughout the experimental period.
Nitrite exposure
Six fish per treatment were placed in an experimental aquarium (63 L) supplied
with continuous aerated water from the same source as the holding water, and
acclimatized for 24 h prior to adding reagent grade sodium nitrite to provide the
selected concentrations of 0.04 and 0.2 mM NO2- (respectively 31% and 154%
of 96 h LC50 NO2- estimated for tambaqui (96h LC50 NO2- = 0.13 mM; Costa
and Fernandes, 2000). The flow through the experimental aquarium was
adjusted to 1.0 L/min and the NO2- exposure was maintained for 96 hours. The
entire volume of water in each tank was completely renewed twice a day. The
nitrite concentration was measured and adjusted according to Strickland and
Parsons (1972). Similar tests, although without the addition of NO2-, were used
for control purposes. Following the 96 h exposure to NO2-, the fish were
removed from the test aquarium, anesthetized (benzocaine, 1:10,000), and
sacrificed with a blow to the head.

52

Sampling and tissue processing


The second gill arch on the right-hand side was immediately excised and washed
in a 0.9% saline-saccharose solution. For light microscopy (LM), filaments (n =
6) still attached to the septum of the arch were immersed in 4%
paraformaldehyde, 1% glutaraldehyde in 0.1 M of sodium phosphate-buffer (pH
7.2) fixative solution for 24 h. The samples were dehydrated and embedded in
Historesin LKB (Reichert-Jung). Serial sections (1 m thick) from the trailing to
the leading edge of the filament (longitudinal sections perpendicular to the surface
of the secondary lamella) were stained with 0.4% toluidine blue and subsequent
PAS (periodic acid-Schiff reaction, Kiernan, 1990) and analyzed under an Olympus
CBA-K compound microscope equipped with a JVC video camera.
For transmission electron microscopy, small pieces of gills (n = 4) were fixed in
2.5% glutaraldehyde solution buffered with 0.1 M sodium phosphate at pH 7.3
for 2 h. The samples were post-fixed in a sodium phosphate-buffered 1% OsO4
solution, dehydrated with an acetone series, and embedded in Araldite 6005
(Ladd Research). Ultra thin sections (60-70 nm) were contrasted with uranyl
acetate and lead citrate and examined under a Philips CM 120 electron
microscope at 100-kV accelerating voltage.
For the analysis by scanning electron microscopy, pairs of filaments attached to
the septum (n = 4) were fixed in 2.5% glutaraldehyde solution buffered with 0.1
M sodium phosphate at pH 7.3 for 2 h. The samples were dehydrated with a
graded ethanol series up to pure ethanol, followed by ethanol/acetone up to pure
acetone and CO2 critical point dried. Filament pairs were glued with silver paint
onto the specimen stub, coated with gold in a vacuum sputter and examined
under a DSM 940 ZEISS Scanning Microscope at 25 kV.
Morphometry and histopathological analyses
Morphometric analyses were performed on 1 m serial sections stained with
toluidine blue-PAS. Digitized images from each section were analyzed using
SigmaScan 3.0 Image Analyser software (Jandel Scientific) and a Merz grid
(Merz, 1967) superimposed over the video monitor to ensure random orientation
of the measurements on each section with 1000 times magnification. Chloride
cells were counted in five consecutive lamella and interlamellar regions
(filament) of three different filaments per fish (30 measurements per fish). Each
cell count and measurement was made using a randomized blind method in
53

which the counter did not know whether the tissue sections were from nitritetreated experimental or untreated control animals. A semi-quantitative analysis
of the ultrastructural components of chloride cells (10,000x) and mitochondria
(35,000x) was made with high magnification.
Statistics
All data were expressed as mean standard error of the mean. Statistical
significance between data sets was determined by one-way ANOVA, followed
by Bonferroni's multiple comparison tests. Regression analysis and correlation
coefficients between the variables were also calculated. A statistical significance
of p < 0.05 was adopted.
Results
The secondary lamellae of C. macropomum were regularly spaced on both sides
of gill filament and consisted of two epithelial layers of cells kept apart by rows
of pillar cells interspersed with blood vessels. The control fish of this species
presented numerous chloride cells on the secondary lamella.
NO2- exposure produced drastic morphological changes in the gill. There was a
gradual increase in the interlamellar distance from 18.42 0.85 m in control
conditions to 21.94 0.95 m after exposure to 0.2 mM NO2-. The several layers
of undifferentiated filament cells were significantly reduced after exposure to
NO2-.
Nitrite exposure reduced the chloride cell number in the lamellar epithelium by
51-57% (p < 0.05), but no significant changes were found in the number of these
cells in the filament (interlamellar region) (Fig. 1). The total number of chloride
cells (lamellar plus filament) was reduced by 49% and 45% (to 0.04 and 0.2 mM
NO2-, respectively) (Fig. 1).

54

Number of cells per five interlamellar spaces


16

Chloride cells from lamella

Chloride cells from filament 9 Total chloride cells

12
8
4
0

Control 0.04 mM 0.2 mM

Control 0.04 mM 0.2 mM

Control 0.04 mM 0.2 mM

Figure 1. Chloride cell count at the lamellae (A), filament (B) and total number of
chloride cells (lamellae plus filament). The number of chloride cells was only reduced in
the lamellae. * Significantly different (p < 0.05) from control. Data presented as mean
standard error of the mean (n = 6).

Table 1 summarizes the cytological analysis of chloride cells of control C,


macropomum and fish exposed to NO2- for 96 h. The chloride cells were rich in
mitochondria and presented a well-developed tubular network system similar to
other freshwater teleosts (Laurent and Dunel, 1980). The lamella in control fish
was lined with chloride cells (Fig. 2A). Chloride cells of the control group were
round, with a basally located nucleus, which normally presented
heterochromatin in the internal membrane of the nuclear envelope (Fig. 2A-C).
Ellipsoid-shaped mitochondria were arranged predominantly near the apical
region. Some cisternae of the tubular network were visible close to mitochondria
(Fig. 2C). The smooth endoplasmic reticulum (SER) was extensive, through not
dilated. Spherical vesicles were dispersed in the cytoplasm. The apical surface
of chloride cells, in some cases, displayed a few short microvilli and, in others,
was buried by pavement cells.
The exposure to 0.04 mM NO2- for 96 h of NO2- caused a decrease in the number
of chloride cells, resulting in the increased distance of the interlamellar spaces
(Fig. 3A). Hypertrophied chloride cells were visible in the lamellae. Chloride
cells exhibiting signs of degeneration in cellular compartments and nuclei
displaying a slight swelling of the perinuclear space were common (Fig. 3C).

55

Table 1. Ultrastructural changes in the gill chloride cells of tambaqui, C.


macropomum, after 96 h exposure to NO2-. The data are the mean
values of semi-quantitative evaluations from four specimens per group.
[NO2-] (mM)

Cytological Parameters
0.00
Nuclei
Deformation of nuclear envelope
Dilation of nuclear envelope cisternae
Smooth endoplasmic reticulum (SER)
Overall amount
Dilation of cisternae
Branching aspect
Fragmentation of cisternae
Formation of SER whorls
Lysosomal elements
Overall amount
Myelinated bodies
Cytoplasmic vacuoles
Mitochondria
Overall amount
Morphological heterogeneity
Formation of mitochondria clusters
Formation of myelin-like whorls
Vacuolization
Outer membrane rupture
Association with SER cisternae
Association with lysosomal elements

0.04

0.20

++
++

++++
++++

++
-

+++
+++
+++
+++
++

++++
++++
++++
+++
++

+
+/++

++
++
++

+++
+++
+++

++++
-

+++
++
+
++
++
++
+/-

++
+++
++
+++
+++
++
++++
+

- absent; +/- very little developed; + little developed; ++ moderately developed;


+++ strongly developed; ++++ very strongly developed.
The mitochondria showed great morphological heterogeneity. These organelles
were partly associated with myelin-like membrane whorls, displaying
vacuolization in the matrix region and showing a tendency to

56

A
A

BC

PVC

25 m

CC

0.3 m

1.4 m

Figure 2. Control fish. A. Gill filament showing lamellae (L) and chloride cells
(arrowheads); B. Chloride cell cytoplasm showing mitochondria (M),
tubular system (T) and nucleus (N); C. Photomicrograph showing
chloride cells (CC) and short microvilli (arrowhead) on the apical
surface. PVC, pavement cell.
aggregate in small mitochondrial clusters. The total amount of SER increased
and the lysosomal elements, particularly small myelinated bodies, proliferated in
the cytoplasm. Furthermore, chloride cells showed a large number of swollen
mitochondrial profiles, which were often intimately associated with dilated SER
cisternae and lysosomal elements. After exposure to 0.2 mM NO2, the lamellae
were thinner, with few chloride cells on their epithelium (Fig. 3A). The chloride
cells displayed a proliferation of SER (Fig. 3A-C). In addition, the SER
cisternae showed signs of fragmentation and formation of SER whorls.
Exposure to this concentration of NO2- resulted in a high density of cytoplasmic
vacuoles, which were very similar to lysosomal elements (Fig. 3C). The
mitochondria displayed pronounced matrix vacuolization, partial lysis of cristae,
and destruction of mitochondrial membranes (Fig. 3C). At this stage, vacuolated
chloride cells were overlapped by epithelial cell extensions and had lost contact
with the environments water. Despite this generalized pathological condition, a
few chloride cells were hypertrophied, with their apical surface in contact with
the environments water.

57

A
A

EP
M
CC

M
SER

CC
M

MW

L
SER
M

10 m

SER
2 m

CD

PVC
CC1

W
SER

CC2
V
2,5 m

Figure 3. C. macropomum exposed to 0.04 (A-B) and 0.2 (C-D) mM NO2-. A.


Cross section of lamellae (L) showing the chloride cells (arrows) on
lamellar epithelium; B. Chloride cell (CC) partially covered by
pavement cell (arrowhead). Note the heterogeneity of mitochondria
(M); C. A few chloride cells (arrow) on lamellar epithelium; D.
Chloride cell (CC1) presenting apical pit (arrowhead) and chloride cell
(CC2) buried in lamellar epithelium. PVC: pavement cell; SER:
Smooth endoplasmic reticulum; V: vacuole
Discussion
The morphology of tambaqui, Colossoma macropomum, gills is similar to that
described for other teleost species (Laurent and Dunel, 1980). The
Importance of the role of the gills on gas exchange (Hughes, 1972), ion- and
acid-base equilibrium (Evans, 1980), and nitrogen excretion (Haswell et al.,
1980) is proportional to their susceptibility to changes in the environment. The

58

gills are the main sites for nitrite uptake; hence, the environmental Clconcentration is a determining factor influencing NO2- toxicity (Eddy et al.,
1983). High ambient Cl- concentrations inhibit the uptake of NO2- and protect
against its toxic effects (Perrone and Meade, 1977; Bath and Eddy, 1980), for
Cl- is a competitive inhibitor of NO2- uptake (Bath and Eddy, 1980).
The high density of chloride cells in the lamellar epithelium of C. macropomum
may be due to the low ion concentration in the environmental water, which
characterizes Brazils continental waters. Several studies have shown high
chloride cell proliferation in fish living in ion-poor water (Perry, 1997), although
Moron (2000) and Fernandes and Perna-Martins (2002) noted the significantly
variable number of chloride cells in Brazils freshwater fish.
Nitrite exposure caused a sharp reduction of chloride cells in lamellar
epithelium, which may be related to increased cell death and low cellular
differentiation in this epithelium. Indeed, most of the ultrastructural changes in
chloride cells displayed a morphological pattern of necrosis. Previous studies
have suggested that nitrite increases chloride cell activity to maintain ion
homeostasis (Gaino et al., 1984), which, incombination with the direct toxic
effects of nitrite, contributes to reduce the cells cycle.
Our results are, in general, congruent with those found in the ultrastructure of
chloride cells of Oncorhynchus mykiss exposed to 450 gN-NO2/L (0.03 mM
NO2-) for 72h (Gaino et al., 1984). Furthermore, we also found signs of severe
damage in chloride cell mitochondria after NO2- exposure, evidencing clear
irreversible cellular damage. NO2- binds to heme moieties of mitochondrial
cytochrome components of the respiratory chain, inhibiting respiration, and
stopping or reducing ATP production (Cotran et al., 1994). It may cause
mitochondrial swelling and rupture of mitochondrial cristae. Mitochondrial
swelling has been related to the toxic action of compounds deriving from nitrite
in rat liver hepathocytes (Rusu et al., 1980).
In conclusion, the changes observed in the gill chloride cells investigated in this
study indicate that the cellular structures involved in the process of energy
production become severely damaged by exposure to nitrite.
Acknowledgments
This research work was supported by Fundao de Amparo Pesquisa do
Estado de So Paulo (FAPESP) and Conselho Nacional de Desenvolvimento

59

Cientfico e Tecnolgico (CNPq), Brazil. O.T.F.Costa acknowledges CAPES for


the award of a scholarship.
References
Bath RN and Eddy FB 1980 Transport of nitrite across fish gills. J. Exp. Zool. 214:
119-121.
Costa OTF, Ferreira DJS, Mendona FLP and Fernandes MN 2000 Acute toxicity
of nitrite to freshwater teleost tambaqui, Colossoma macropomum
(Teleostei, Serrasalmidae). Comp. Biochem. Physiol.(Suppl) 126B: 26.
Cotran RS, Kumar V and Robbins SL 1994 Robbins Pathologic Basis of Disease.
(Schoen, FJ, ed.). W. B. Saunders Company: Philadelphia.
Evans DH 1980 Kinetic studies of ion transport by gill epithelium. Am. J. Physiol.
238: R224-230.
Eddy FB, Kunzlik PA and Bath RN 1983 Uptake and loss of nitrite from the
blood of rainbow trout, Salmo gairdneri Richardson, and Atlantic
salmon, Salmo salar L. in freshwater and in dilute sea water. J. Fish
Biol. 23: 105-116.
Fernandes MN and Perna-Martins SA 2002 Chloride cell responses to long-term
exposure to distilled and hard water in the gill of the armored catfish,
Hypostomus tietensis (Loricariidae). Acta Zool (in press).
Gaino E, Arillo A and Mensi P 1984 Involvement of the gill chloride cells of trout
under acute nitrite intoxication. Comp. Biochem. Physiol. 77A: 611-617.
Haswell MS, Randall DJ and Perry SF 1980 Fish gill carbonic anhydrase: acidbase regulation or salt transport. Am. J. Physiol. 238: R240-245.
Hughes GM 1972 Morphometrics of fish gills. Respir. Physiol. 14: 125.
Kiernan JA 1990 Histological and histochemical methods: theory and practice.
Pergamon Press, London

60

Laurent P and Dunel S 1980. Morphology of gill epithelia in fish. Am. J.


Physiol. 238: R147R159.
Lewis WM and Morris DP 1986 Toxicity of nitrite to fish: A review. Trans. Am.
Fish. Soc. 115: 183-195.
Merz WA 1967 Die Streckenmessungen an gerichteten Strukturen im
Mikroskop und ihr Anwendung zur Bestimmung von OberflachenVolumen-Relationen im Knochengewebe. Mikroskopie 22: 132142.
Moron SE 2000 Efeito das concentraes de ons Na+, Ca2+ e Cl- na morfologia
branquial e nos parmetros fisiolgicos de Hoplias malabaricus e
Hoplerythrinus unitaeniatus (Teleostei Erythrinidae). Ph D thesis.
Universidade Federal de So Carlos, 81 p.
Perrone SJ and Meade TL 1977 Protective effects of chloride on nitrite toxicity
to coho salmon (Oncorhynchus kisutch). J. Fish. Res. Board Can. 34:
486-492.
Perry SF 1997 The chloride cell: structure and function in the gills of freshwater
fishes. Ann. Rev. Physiol. 59: 325347.
Rusu MA, Preda N, Graciun C, Gadaleanu V, and Bucur N 1980
Histoenzymological and ultrastructural changes in rat following the
administration of aminopyrine and nitrite (nitrosoaminopyrine). In: Nnitroso-compounds: Analysis formation and occurrence (Walker EA,
Castegnaro M, Gricinte L, Borzsonyi M, eds.), Lyon IARC Scientific
Publ. no. 31.
Strickland JDH and Parsons TR 1972 A Practical Handbook of Seawater Analysis.
Fish. Res. Bd. Canada Bull 167.
Williams EM and Eddy FB 1986 Chloride uptake in freshwater teleosts and its
relationship to nitrite uptake and toxicity. J. Comp. Physiol. 156B: 867872

61

62

BEHAVIORAL AND NEUROPHYSIOLOGICAL EFFECTS OF


CARBAMATE PESTICIDES ON OLFACTORY CAPABILITIES
IN PACIFIC SALMON
Hugh E. Jarrard
Department of Biological Sciences, Simon Fraser University
Burnaby, BC V5A1S6
Tel (604) 291-5634, Fax: (604) 291-3496, Email: hej@sfu.ca
and
C.K. Kennedy
Department of Biological Sciences, Simon Fraser University
Email: ckennedy@sfu.ca
EXTENDED ABSTRACT ONLY DO NOT CITE
Introduction
The presence of pesticides within aquatic environments can induce
physiological and behavioral changes in teleosts that, although sublethal, impair
the survivability and ecological fitness of the organism. We are interested in
carbamate pesticide effects on olfactory behaviors and physiology in the Pacific
salmon, a teleost that relies heavily on olfaction for successful completion of its
life history. Specific impairment of olfactory-based behaviors by pesticides has
been little studied in teleosts. Among salmonids, sublethal exposure to diazinon
(organophosphate) reduces the ability of Chinook (O. tshawytscha) to react
appropriately to alarm pheromones and to home [Scholz, 2000 #109]. An
important issue is the locus and mechanism by which pesticide exposure may
impair olfactory behaviors. Work at the neurophysiological level in Atlantic
salmon (Salmo salar) suggests that pesticides can act on olfactory receptor
neurons (ORNs), impairing their ability to respond to odorants of biological
importance (e.g. Moore 2001).

63

We have begun to examine more thoroughly the mechanisms involved in


pesticide impairment of salmonid olfaction by establishing a baseline of
carbamate effects on Coho (O. kisutch) at behavioral and neurophysiological
levels utilizing Y-maze avoidance-behavior assays and electro-olfactrograms
(EOG), a sensitive measure of multi-unit voltage potentials created by ORNs in
response to odorant stimulation (e.g. Hara 1973). The carbamates chosen are
compounds of current concern with moderate to high aquatic toxicity and a
limited refereed database regarding their olfactory toxicity.
Methods
Experimental Animals
Juvenile Coho salmon were obtained locally at ages 35 months for Y-Maze
experiments (mean weight= 0.97 g), and 10-15 months (mean weight= 16.0 g)
for EOG experiments. Fish were maintained at natural photoperiod/temperature
in flow-through tanks supplied with dechlorinated `background water (BKD;
pH 6.26.8, hardness 3.49 -6.19 mg/L CaCO3).
Pesticide Exposures
For Y-maze experiments, fish were tank exposed for 21 days prior to behavioral
testing to either carbofuran (0.075 ppm), 3-iodopropynylbutylcarbamate (IPBC,
0.048 ppm), mancozeb (1.1 ppm), or control condition in chilled (10-11C)
static 18L aquaria. Fish were fed 2% b.w. daily, and tanks were cleaned,
refilled, and exposures refreshed every other day. For EOG experiments,
exposures were acute (30 mins) and applied locally to the olfactory epithelium
(OE). All pesticides dissolved in BKD immediately prior to use (exception:
IPBC dissolved in polyethyelene glycol at 10 mg/ml; same vehicle also given to
controls).
Behavioral Testing
Effects of sublethal pesticide exposure on olfactory behaviors were examined
using salmonid avoidance of L-Serine (10-8 M; SER) in a two choice Y-trough
maze (Fig. 1A; after Rehnberg et al. [1985]). Behavioral assays began with a
single nave fish in the start box with all gates closed. After 5 min. acclimation,
SER was introduced into one arm of Y-maze for 5 min., then all gates were
opened simultaneously. Fish were given 10 min. in maze to explore and choose
an arm, after which all gates were dropped and the choice noted. The arm
receiving SER was changed every second run, and the maze cleaned, in order to
eliminate any bias.

64

Neurophysiological testing
EOGs were recorded from coho parr after Evans and Hara (1985). Individual
fish were anaesthetized (2-phenoxyethanol, 0.4 ml/L), and paralyzed with intramuscular injections of Flaxedil (2.4 mg/kg b.wt.). Fish were then wrapped in
gauze, secured in a Plexiglas trough, and maintained under anesthesia by gill
perfusion. After removal of skin overlying the OE, a gravity-fed stream of BKD
was passed over the exposed OE into which brief pulses of odorant (10-5 M
SER) were introduced. EOG responses to SER delivery (Fig. 2A) were recorded
from the OE using an Ag/Ag-Cl electrode, then filtered, amplified, and
displayed on a computer. Experimental trials consisted of the collection of preexposure EOG responses (PRE; n=5), followed by acute OE pesticide exposure,
then post-exposure EOG responses (POST; n=5).
Results
Y-Maze Behavior
While statistical analysis has not yet been conducted on this data, it appears:
Non-exposed control fish consistently avoid SER-scented arms
In contrast, carbofuran- and mancozeb-exposed groups choose
scented/unscented arms at a 50:50 ratio (suggesting anosmia)
IPBC-exposed fish avoid the SER-scented arm more frequently than
expected based on chance alone.
A

12

Scent ed
10

Unscent ed
No Choice

8
6
4
2

gate
inflow
drain
pump
SER

0
Contr ol

Car bof ur an

IPBC

Mancozeb

Exposur e Gr oup

Figure 1. Y-Maze Behavioral Assay. A. Flow-through Y-maze apparatus.


Flow: 6.4 L/min., maze length: 2.3 m. B. Effects of Pesticide Exposure on
SER Avoidance. Plot displays number of fish choosing each arm per exposure
group (carbofuran 0.075ppm, IPBC 0.048ppm, mancozeb 1.1ppm).

65

EOG Results
In preliminary results, significant reduction in EOG amplitude after pesticide
exposure (in comparison with H2O-exposed controls, one-tailed t test, p < 0.05)
occurred after acute 30 min. exposure:
for carbofuran at 0.0001, 0.002, 0.01, and 2ppm
for IPBC at 0.00048, 0.0048, and 0.048 ppm
and, for mancozeb at 0.022, 0.22, and 2.2 ppm.

10-5 SER

peak amplitude
0.2 mV
5 secs

Fig. 2. Pesticide Effects on


EOG A. EOG response to
SER delivery. B. Dosedependant carbamate effects
on EOG amplitude. Data as
POST EOG amplitude as % of
PRE EOG amplitude.

66

Conclusions
Carbamate pesticides interfere with salmon olfactory capabilities at
both behavioral and neurophysiological levels
This interference appears as loss of ability to avoid SER at low concentrations in Y-maze tests and as a significant decrease in the population
of ORNs responding to SER in EOG responses.
These effects appear at 50% of the 96 hr. LC50 for these compounds in
behavioral tests (LC50s: carbofuran=200ug/l, IPBC=100ug/l,
mancozeb=2.2mg/L), and at several orders of magnitude below that in
neurophysiological studies.
Ongoing work is focused on establishing both a clear correlation
between these different classes of effects and the mechanism by which
carbamates exert their effects in the olfactory periphery.
Acknowledgements
We wish to acknowledge the generous assistance of Dr. Kerry Delaney in
establishing a functional EOG apparatus, the Capilano Hatchery for generous
salmon supply, and Doug Wilson for equipment loans. HEJ supported by NIH
NRSA Postdoctoral Training Fellowship F32-NS10973.
References
Evans, R. E. and T.J. Hara (1985). The characteristics of the electro-olfactogram
(EOG): its loss and recovery following olfactory nerve section in
rainbow trout (Salmo gairdneri). Brain Res 330(1): 65-75.
Hara, T. J. (1973). Olfactory responses to amino acids in rainbow trout, Salmo
gairdneri. Comp Biochem Physiol A 44(2): 407-16.
Moore, A. and C. Waring (2001). The impact of two pesticides on olfactorymediated endocrine function in mature male Atlantic salmon (Salmo
salar L.) parr. Comp Biochem Physiol B Biochem Mol Biol 129(2-3):
269-76.
Rehnberg, BG and CB Schreck. (1987) Chemosensory detection of predators by
coho salmon (Oncorhynchus kisutch): behavioral reaction and
physiological stress response. Can. J. Zool. 65:481-485.

67

Scholz, N. (2000). Diazinon dirsupts predator. Can J of Fish Aquat Sci 57:
1911-1918.

68

EFFECTS OF EXPOSURE TO SUB-LETHAL CONCENTRATIONS OF


AMMONIA AND HYPOXIA ON THE SWIMMING PERFORMANCE
OF BROWN TROUT (SALMO TRUTTA)

A. Shingles,
School of Biosciences, University of Birmingham,
Birmingham, B15 2TT, United Kingdom.
Tel +44-(0)121-4145472; Fax +44-(0)121-4145925; a.shingles@bham.ac.uk
D.J. McKenzie1, S. Ceradini2, A.Z. Dalla Valle3, A. Moretti2 & E.W. Taylor1
1
Biosciences, University of Birmingham, Birmingham, UK;
2
Environment Unit, CESI SpA, Milan, Italy.
3
Pharmacological Sciences, University of Milan, Milan, Italy.
EXTENDED ABSTRACT ONLY DO NOT CITE
Introduction
Ammonia is toxic to all vertebrates. It has become a pervasive pollutant of
aquatic habitats but is also, for the majority of aquatic organisms, an endproduct of protein metabolism. In teleost fish ammonia can, therefore,
accumulate to toxic levels either as a consequence of exposure to elevated water
ammonia concentrations or as a consequence of impaired excretion of the
endogenous metabolite.
Ammonia accumulates in fish during exposure to sub-lethal concentrations of
heavy metals such as copper and, in brown trout, this has been linked
statistically to a decline in the ability to perform exercise (Beaumont et al.,
1995). Beaumont et al. (1995) found a negative linear relationship between
plasma ammonia concentrations and maximum sustainable swimming speed
(Ucrit) in brown trout exposed to a sublethal combination of copper and acidic
water, and went on to demonstrate that the ammonia accumulation caused a
partial depolarisation of muscle membrane potential (Beaumont et al., 2000).
Shingles et al. (2001) demonstrated that exposure to elevated water ammonia
alone was sufficient to reduce Ucrit in rainbow trout (Oncorhynchus mykiss),
with evidence that this was linked to a partial depolarisation of muscle.
69

Objectives of the current study included to investigate whether a linear


relationship between plasma ammonia concentration and Ucrit could be elicited
in brown trout by ammonia exposure alone, and to investigate the mechanisms
for impaired performance.
Hypoxia is a growing problem in many aquatic habitats and, therefore, can be
expected to occur concurrently to elevated ammonia. Hypoxia impairs
swimming performance in salmonids by limiting aerobic scope (Bushnell et al.,
1984). Thus, a further objective of the current study was to determine how
concurrent exposure to ammonia and hypoxia influenced exercise performance
in brown trout.
Methods
To investigate the relationship between plasma ammonia concentration and
swimming performance, adult brown trout (mean mass approx. 500g) were
exposed to two different sub-lethal concentrations of NH4Cl in the water,
nominally 100 mol l-1 and 200 mol l-1, for 24h in hard water at 15 C and pH
8.2. Their swimming performance, and associated respirometry, were then
investigated in a Brett-type swimming respirometer (Shingles et al., 2001), in
comparison to controls in normal water. In parallel, the effects of the ammonia
exposure regimes on plasma ammonia and on the membrane potentials (EM) of
red muscle, white muscle, heart and brain were investigated on trout with an
indwelling catheter in the dorsal aorta.
To investigate the effects of ammonia and hypoxia, the experimental series were
repeated, but trout were exposed to mild hypoxia at a nominal water O2 partial
pressure of 80 mmHg for 1h prior to, and then during, the various
measurements.
Results
Exposure to either 100 mol l-1 or 200 mol l-1 NH4Cl caused an increase in
plasma total ammonia to 38642 mol l-1 or 77192 mol l-1, respectively,
compared with 13329 mol l-1 in control fish (mean SE, n = 6). This
ammonia accumulation was associated with a significant decline in Ucrit from
2.240.15 bodylengths s-1 (BL s-1) in control trout to 1.460.09 BL s-1 or
1.080.16 BL s-1 in trout exposed to 100 mol l-1 or 200 mol l-1 NH4Cl,
revealing a direct negative relationship between plasma ammonia concentration
and Ucrit (Figure 1). The linear relationship was surprisingly similar to that
70

observed by Beaumont et al. (1995) in brown trout exposed to sublethal copper


at low pH, which is shown on the figure for comparison.

3.0

Ucrit (BL s-1)

2.5
2.0
1.5
1.0
0.5
0.0
0

200
400
600
800
1000
-1
plasma [ammonia] (mol l )

Figure 1. The least squares linear regression relationship between plasma


ammonia concentration and maximum sustainable swimming speed
(Ucrit) in brown trout exposed to three water concentrations of ammonia
(this study, solid symbols, solid line); as reported for brown trout that
accumulate ammonia following exposure to a sub-lethal combination of
copper and low pH (Beaumont et al., 1995, dashed line), and in
rainbow trout exposed to two water concentrations of ammonia (plotted
from data reported in Shingles et al. 2001, open symbols, dotted line).
The error bars on the symbols are 1 SEM of the mean values for
plasma ammonia and Ucrit, n = 6 or 7. For brown trout exposed to
ammonia (this study), the linear regression was described by the
equation: mean Ucrit = -0.0018(mean[ammonia]) + 2.347 (R2 = 0.903, n
= 3). Beaumont et al. (1995) reported an equation based on individual
values rather than means: Ucrit = -0.0020[ammonia] + 2.089 (R2 =
0.670, n = 30). For rainbow trout (Shingles et al., 2001), the equation
was: mean Ucrit = -0.0024(mean[ammonia] + 2.674 (n = 2).
71

Also, the figure shows the relationship between plasma ammonia and Ucrit in
rainbow trout, replotted from the data reported in Shingles et al. (2001). The
relationship between the plasma ammonia concentration and Ucrit was very
similar in the two species. Interestingly, however, a significantly higher water
ammonia concentration was required in rainbow trout to elicit the same plasma
ammonia accumulation (Shingles et al., 2001).
The respirometry measurements revealed that the impaired performance in the
brown trout exposed to ammonia was associated with reduced swimming
efficiency, and with a partial depolarisation of EM in white muscle and the brain,
although there were no significant effects on EM of the red muscle and heart.
Exposure to hypoxia caused a 45% decline in Ucrit in control animals, down to
1.230.09 BL s-1, as a consequence of the expected limitation to aerobic scope.
Hypoxia did not, however, cause the same proportional decline in performance
in the ammonia-exposed fish; both groups had a Ucrit of approximately 1 BL s-1.
Therefore, hypoxia had no further effect on the reduced performance of animals
exposed to 200 mol-1 NH4Cl.
Conclusions
The results provide further evidence that the impaired swimming performance of
trout following exposure to sub-lethal concentrations of copper in acid water
(Beaumont et al., 1995) can be attributed to the accumulation of ammonia.
Ammonia accumulation has similar effects on performance in both rainbow and
brown trout, but rainbow trout appear better able to limit plasma ammonia
accumulation during exposure to elevated water ammonia. The fact that
hypoxia did not elicit any further decline in Ucrit in trout exposed to NH4Cl
indicates that ammonia impairs performance by a mechanism unrelated to
oxygen supply in brown trout, perhaps through effects on nerve and white
muscle function.
References
Beaumont, M.W., P.J. Butler and E.W. Taylor. 1995. Plasma ammonia
concentration in brown trout (Salmo trutta) exposed to acidic water and
sublethal copper concentrations and its relationship to decreased
swimming performance. J. Exp. Biol. 198: 2213-2220.

72

Beaumont, M.W., E.W. Taylor and P.J. Butler. 2000. The resting membrane
potential of white muscle from brown trout (Salmo trutta) exposed to
copper in soft, acidic water. J. Exp. Biol. 203: 2229-2236.
Bushnell, P.G., J.F. Steffensen and K. Johansen. (1984). Oxygen consumption
and swimming performance in hypoxia-acclimated rainbow trout Salmo
gairdneri. J. Exp. Biol. 113: 225-235.
Shingles A., D.J. McKenzie, E.W. Taylor, A. Moretti, P.J. Butler and S.
Ceradini. (2001). Effects of sub-lethal ammonia exposure on swimming
performance in rainbow trout (Oncorhynchus mykiss). J. Exp. Biol.
204: 2699-2707.

73

74

EFFECTS OF CHLORPYRIFOS (LORSBAN) ON REPRODUCTIVE


PERFORMANCES OF GUPPY (POECILIA RETICULATA)
De Silva, P. M C. S
Department of Zoology, University of Ruhuna,
Matara, Sri Lanka.
Telephone: 0094 41 27025
Fax: 0094 4122683
Email: chathura@zoo.ruh.ac.lk
Samayawardhena, L. A,
Department of Zoology, University of Ruhuna,
Matara, Sri Lanka.
Telephone; 0094 41 27025
Fax: 0094 4122683
Email: samaya@zoo.ruh.ac.lk
Abstract
Toxicity of Chlorpyrifos on reproductive disorders including altered fertility,
reduced viability of offspring, impaired hormone secretion and modified
reproductive anatomy were little concerned. In the present study, we selected
Guppy (Poecilia reticulata) to investigate the reproductive effects of Lorsban, a
common insecticide in Sri Lanka. Male and female guppy were selected with
proven fertility from our own colony and the groups of fish (n=12x6/group)
were exposed to pre-determined 2g / l, 0.002 g /l Chlorpyrifos based on the
96 hrs LC50 for guppy. Mating behavior of pairs was recorded on the 2nd day of
exposure. Offspring were counted and survival recorded on the 14th day.
Gonopodial thrusts (4 /15 min, in 2 g/l and 8/15 min in 0.002 g/l) were
significantly different from the control (11/15min, in the control). Similarly, live
birth reduced significantly to 8/female in 2 g/l compared to 27/female in the
control group. Survival of offspring at the 14 days was reduced to 66% in 2 g/l
group. Our findings show that low soluble concentrations of Chlorpyrifos can
impair reproductive behavior and capabilities of Guppy to a significant extent.
F1 generation of treated fish showed reduced survival suggested importance of
mating behavior. Pesticide exposure throughout embryonic development could
result weak offspring and lesser their survival. Our study confirmed

75

Chlorpyrifos could potentially alter mating behavior, live birth and F1 survival
of Guppy.
Introduction
Chlorpyrifos (O, OdiethylO (3,5,6trichlor2pyridyl) phosphorothioate) is a
broad-spectrum organophosphate insecticide with growing concern due to its
aquatic toxicity (Foe, 1998; Bailey et al 1997). The toxicity effects may include
neurological, behavioral and possibly reproductive effects (Mueller-Beilschmidt,
1990; Hill, 1995). Recent studies have revealed that Chloropyrifos together with
Diazinon are responsible for most of the toxicity to aquatic organisms (de
Vlaming et al., 1993; Moor et al., 1998; Foe et al., 1998). Especially it is very
highly toxic to fresh water fish and aquatic invertebrates. The agricultural,
residential and commercial use of Chloropyrifos on pest control leads to
presence of Chloropyrifos in sufficient concentrations in agricultural runoff and
as well as in urban storm water runoff resulting high toxic effects on
Ceridodaphnia and Mysidopis, two zooplankton species (Corner et al., 1998;
Larson et al., 1998). Similar toxicity assessment subjected by Lee and JonesLee (1997).
Toxicity of Chloropyrifos on zooplankton is well documented, but less work
carried on fish. Johnson and Finley (1980), Odenkirchen et al, (1988)
documented its acute toxicity. Reproductive disorders including altered fertility,
reduced viability of offspring, impaired hormone secretion and modified
reproductive anatomy were little concerned. Few early studies give some viable
information on this aspect. Dursban exposure on fathead minnows for 200 days
resulted a decline of offspring in first generation survival (USPHS, 1989).
Growth of early life stage of California grunion (Leuresthes tenuis) was reduced
20% and 26% in two low concentrations of Chloropyrifos (Odenkirchen et al.,
1988). But the validity of reproduction as a key parameter to evaluate the
impacts of chemical pollutants Kime (1999) tends us to study the Chloropyrifos
toxicity with some aspects of reproduction.
Guppy (Poecilia reticulata, Peters) is selected as the model organism, a
livebearer fish species that was introduced to Sri Lanka as a biological tool in
mosquito control. The experimental organism Guppy is a viviparous fish with a
short reproductive period (Houde, 1997). Male guppies approach two methods
of mating behaviour, sigmoid displays and gonopodial thrusts (Evens et al.,

76

1999). They perform a sigmoid display in which the body is held in S- shape
while fins are extended and quivered.
Alternatively they may attempt sneaky mating, in which the female is
approached sideways or from behind and the modified anal fin the gonopodium
is thrust toward the genital pore. Successful mating produced a litter size range
from 12 46 in monthly intervals depending on circumstances (Hutchins, 1996).
In this study we observed their mating behaviour representing an organism level
parameter, brood size and the survival of the offspring as a population level
parameter with two more or less similar concentrations of Chloropyrifos.
2 Materials and Methods
2.1 Study population and their maintenance
We collected wild guppies (Poecilia reticulata) from the urban cannel systems
around the Nilwala river basin in the southern region of Sri Lanka. Fishes were
returned to the laboratory and stocked in (200 l) tanks. This stock aquarium
received fully aerated water from a header tank. The water temperature was kept
at 26 2 C0.These fishes were fed with special aquaria food purchased through
local market. By maintaining this colony we select female guppy that belongs to
the highest length class (3.51.0 cm) was selected as the test female animals
assuming that they were well suited to give birth to offspring. They were
separated into (18 l) tanks and monitored for 4 6 weeks, until they had given
birth to offspring due to early fertilization in stock aquaria. Adult males
representing the length class (2.0 1.0 cm) were selected based on their early
sexual behaviour.
2.2 Stress exposure
Pre determined 2 g / l, 0.002 g / l were used as the exposure concentrations
based on the 96 Hrs LC 50 for guppy (our own study). Each pair of guppy was
transferred to the tanks (23x23x35cm), that contained 10 l of Lorsban EC 40%
(Chloropyrifos) solutions for consecutive three days together with controls.
Chlorinated free tap water was used in the process of dilution of the pesticide.
Test solutions were changed every 24 hours followed by the addition of fresh
Chloropyrifos solution. After three day exposure time male guppies were
removed and the females were kept in the test solutions until they produced their
brood.

77

2.3 Mating behaviour


Sigmoid displays and gonopodial thrusts characterize male mating behaviour of
guppy. In sigmoid displays the cooperation of the female is necessary hence we
observed the number of gonopodial thrusts as our observation because it is an
alternative mating tactic, which does not require female reception. When the
modified anal fin, gonopodium made contact with the female genital region it
was referred as a successful attempt of mating. A day after exposure to the
concentrations the male mating behaviour was observed for consecutive two
days. The number of gonopodial thrusts performed by each male in the tanks
was calculated over 15 minutes using a counting deviser.
2.4 Reproductive rate and Offspring survival
The reproductive capabilities of the pair were estimated by counting the total
number of offspring born to the female. The reproductive rate was calculated as
the average number of offspring born per female for each concentration and the
control. The number of offspring born to each pair was separated into glass
tanks (23 x 23 x 35 mm) and monitored for consecutive two weeks. The
mortality in each set of offspring was observed in every 24 hrs and the total
number of dead offspring was determined after a period of two weeks.
Statistical analysis
The 96 hrs LC50 was determined through probit analysis, using the software
SPSS for windows 98.The relationship between the observations in exposed
concentrations and control experiments were investigated by ANOVA modeling
and comparisons by Student-Newman-Keuls test. All analysis was subjected at
< 0.001 probability level.
3.Results
During the overall study period General appearance was prime in condition and
no pathological effects were observed. Also there were no changes in feed
intake in both control and treatment groups. The 96 hrs LC 50 was 7.17 g / l.
Mating behavior observed as number of gonopodial thrusts performed by male
in 8.00 hr, 12.00 hr, and 6.00 hr respectively, with control and two treatments as
given in Figure 1.

78

14

12

10

control

T1

T2

0
8. 00am

1 2. 00pm

6. 00pm

T i me

Fi gu r e 1 : M e a n n um be r of g on op od i a l t h r ust s p e r f o r m e d b y m a l e i n
di f f e r e nt p e r i od s of t h e d a y .

It is quite evident that the time and the number of attempts were not significantly
different. But number of gonopodial thrusts by male was considerably reducing
with increased concentration of LORSBAN. In control mean number of
gonopodial thrusts were recorded as 11 while in males in 2 g / l (T 1) it was 4
and 8 in the lowest concentration 0.002 g / l (T 2) (Figure 2).
14

12

10

0
con t r ol

T1

T2

E xposed concent r at i on

Fi gur e 2 : M e a n numbe r of gonopodi a l t hr us t s pe r f or me d by ma l e i n


e x pos e d c onc e nt r a t i ons ( * * * P < 0 . 0 0 1 ) .

Each of these values were significantly different at P<0.001. The litter size
calculated as number of offspring per female varies to minimum number of 18
to maximum of 36 in the control, which made it to average of 27 offspring per
female. Female guppy in exposed concentration 2 g / l recorded least number
of offspring ranging 5 11 and mean of 8 offspring per female. But in the
lowest concentration the mean number of offspring per female has risen to 24.

79

Although the mean number of attempts were reduced at 0.002g / l, still


exposed females were able to produce a closer number of offspring as the
control (Figure 3).

40

35

Number of offspring

30

Max

25

20

Min

15

Mean

10

0
control

T1

T2

Exposed concentration

Figure 3: Mean,maximum,and minimum number of offspring per female w ith different concentration (Mean,***P<
0.001).

Produced offspring in control for additional 14 days were recorded a highest


percentage survival, over 86% while in the concentration of 2g / l it was less
than 66. Quite contrast to the observations related to number of offspring in the
control and exposed concentration, 0.002g / l, the percentage survival of this
concentration significantly lesser than control (Figure 4).

120

Percentage Survival

100

80

***

60

***
40

20

0
control

T1

T2

Exposed concentrations

Figure 4: Recorded maximum,minimum and mean percentage survival with exposed


concentrations( ***P< 0.001)

80

Max
Min
Mean

4.Discussion
Fish exposed to xenobiotic pollutants have manifested a range of reproductive
defects including behavioral, anatomical, physiological levels from reduced
fertility to alternation of sexual behaviour and one of the key parameters to
evaluate the impact of xenobiotics (Jones et al, 1997; Kime, 1999).
Reproductive behavior it self is a key phase of the reproduction cycle of Guppy,
which ensures the successful mating. Endler (1987) and Houde (1997) have
illustrated the two approaches as sigmoid displays and alternative tactic
gonopodial thrusts difference in change of temperature, in presence of predators.
But the effects of pesticides the sexual behaviour was either poorly highlighted
or neglected. Although the degradation of Chloropyrifos is rapid still it can
cause serious consequences on fish as it coincides with this key phase of their
reproduction cycle, mating behaviour. Our study suggests that even low
concentrations of Chloropyrifos well below LC50 value heavily ceased male
mating behaviour. Similarly Matthiessen and Logan (1984) suggested that 0.002
0.0015 mg/l exposure of Endosulfan inhibited male reproductive behavior of
Sarotherodon mossambicus.
Moore et al., (1997) focused on interesting phenomenon stating as successful
mating behavior sometimes depend on the secretion of chemicals by female fish
to elicit a response that both triggers production of sperm and male mating
behaviour. Pesticides such as carbofuran and diazinon both could disrupt male
fish to detect such chemicals. Similar mechanism could be suggested for
Chloropyrifos since its effects are more similar to diazinon. Further research on
this aspect might be able to confirm this mechanism. In our early study using
guppy juveniles and Chloropyrifos suggested that exposed animals had signs of
paralysis even in low concentrations of this pesticide. These signs of paralysis
which caused them, incapable of moving might lead to the reduction of their
mating behaviour. Male guppy reduces their mating tactics in the presence of
predators (Endler, 1987). In natural environment impacts of Chloropyrifos alone
with predators might cause the situation worst.
Guppy, which can produce the brood to the external as fries and its short
reproductive cycle, provides an excellent model to examine effects of
Chloropyrifos on female fertility. The sperm storage of female guppy ensures
that female could fertilized new embryos even if she was unable to remate
(Constantz, 1989). Since we used females, which confirmed as no early mating,
prior to exposure of Chloropyrifos we strongly suggest that reduction of average
81

number of offspring per female associated with pesticide exposure. It is further


highlighted as the highest concentration was used have been recorded the least
number of offspring.
It is true that male vertebrates produce more sperm than eggs. But studies on
fish and mammals confirmed that small change of sperm quality and quantity
could reduce female fertility which might resulted fewer offspring (Kime, 1999).
Change of mating behaviour in exposed males could lead to change of sperm
quality and quantity resulting few offspring in exposed groups. The validity of
this observation needs further research on Chloropyrifos effects on sperm
quality and quantity. Decrease production of yolk protein resulting from
inhibition of ovarian or liver function may lead to small number of eggs (Tyler
et al, 1990). In this study we did not focus on this aspect but this relationship
should be revealed with this experiment as well. Several authors suggested
decreasing number of offspring with various pesticides. Yasuno (1980) used the
same species Guppy as model organism to evaluate the effects of Fenitrothion
and he suggested that reduced number of juveniles in pesticide contaminated
female population.
Exposure of Dursban on Fathead minnows for 200 days resulted the reduction of
first generation of offspring. Hose et al, (1989) and Thomas et al, (1989)
suggested similar results on fish as the test species. Another aspect that could
have a possible relationship was percentage survival of the juveniles born.
Although females in the lowest concentration were able to produce quite similar
number of offspring with control their survival was significantly lesser than
control. Xenobiotics could pass on from mother to offspring when it was
developing. Pesticide exposure throughout this embryonic development could
result week offspring that makes them struggle to survival. Less than 66 % of
survival in exposed concentrations of this study revealed that fries affected by
Chloropyrifos, although they were not entirely exposed. Contaminated yolk
passed on from mothers who have accumulated high pesticide burden and liver
alterations itself might lead to the nutrition content and quality of the eggs,
which were the possible causes for weak offspring production. Also we suggest
that Chloropyrifos could decrease the responses to stresses and further decrease
in growth and metabolism could possibly affect these juveniles, ability to
survival. In wild they might highly struggling to survival since they are
vulnerable to combination of stress conditions including effects of pesticides.
There is increasing evidence that some of the problems found in fish now being
applied to human population as well (Colborn, et al 1996). So suggested effects
82

of chlorpyrifos on guppy might insight into a much more general view as well.
We can conclude that LORSBAN (Chlorpyrifos) could potentially impair
mating behavior and the effects could be extended to survival of F1 up to 14
days after birth.
References
Bailey, H. C., Miller, J.L., Miller, M.J., Wiborg, L.C., Deanovic, L., Shed, T.
1997. Joint acute toxicity of diazinon and chlorpyrifos to Ceriodaphnia
dubia. Environmental Toxicology and Chemistry.16 (11). 2304-2308.
Colborn.T, Dumasanoski and Myres, J.P. 1996. Our Stolen future. Little brown.
London.
Connor, V., 1995. Status of urban storm runoff projects. Central Valley
Regional Water Quality Control Board, Sacramento, CA.
Constanz, G.D. 1989. Reproductive biology of poeciliid fishes. Ecology and
evolution of livebearing fishes (Poeciliiadae)(Ed.by G.Kmeffe & F.F
Snelson, Jr) pp33-50. EnglewoodCliffs, New Jersy, Prentice Hall.
deVlaming, V. DiGiorgio, C. and Deonovic, L., "Insecticide-Caused Toxicity in
the Alamo River," Presented at the NorCal SETAC annual meeting,
Reno., NV, June. (1998).
Endler, J. A. 1987. Predation, light intensity and courtship behaviour in Poecilia
reticulata (Pisces: Poeciliidae). Anim.Behav.35. 1376-1385.
Evens.J.P, Magurran.A.E. 1999. Geographic variation in sperm production by
Trinidadian guppies. Proc.R.Soc.Lond. B266, 2083-2087.
Foe, C., Deanovic, L., Hinton, D. 1998. Toxicity Identification Evaluations of
Orchard Dormant Spray Storm Runoff. California Regional Water
Quality Control Board, Central Valley Region, Sacramento, CA.
Hill, E.F. 1995. Organophosphorus and Carbamate Pesticides. Pp. 243-274 in D.
J. Hoffman, B.A. Rattner, G. A. Burton, Jr., and J. Cairns, Jr. (eds.).
Handbook of Ecotoxicology. Lewis Publishers, Boca Raton, FL.

83

Hose, J.E., Cross., Smith, S.G ., Diehl.D.1989. Reproductive impairment in a


fish inhabiting a contaminated coastal environment off southern
California. Env. Poll. 57, 139-148.
Houde, A. E. 1997.Sex, colour, and mate choice in Guppies. Princeton
University Press. New Jercy.USA.
Hutchins,L.1996.Freshwaterresources.OnlinePublication.http://www.Aqualink.c
om/Fresh/Z1-Guppy.
Jones, J .C., Reynolds J.D. 1997.Effects of pollution on reproductive behaviour
of fishes. Rev. Fish.Biol. & Fisheries 7. 463-491.
Johnson, W.W., Finley, M.T. 1980. Handbook of acute toxicity of chemicals to
fish and aquatic invertebrates. U.S.Fish.Wild.Serv.Resour.Pub.137, 9899.
Kime.D.E. 1999. Endocrine disrupting chemicals.,ed R.F Hester and R.M
Harrison, Issues in Environ.Sci.and Tech.12. 27-48.
Landis, W.G., Yu, M.H. 1995. Introduction to Environmental Toxicology:
Impacts of Chemicals Upon Ecological Systems. Lewis Publishers,
Boca Raton, FL.
Larsen, K. L, Connor, V.M and Hinton, D.E."Sacramento River watershed
Program Toxicity Monitoring Results 1996 - 1997,SETEC annual
meeting Reno, NV.June.(1998).
Lee, G.F., Jones-Lee.1998. A Development of a regulatory approach for OP
pesticide toxicity to aquatic life in receiving waters for urban storm
water runoff. SETAC Meeting, Reno, NV.
Matthiessen, P.Logazn, J.W.M.1984.The effects of low concentrations of
endosulfan insecticide on reproductive behavior in the tropical cichild
fish, Sarotherdon mossambicus. Bull. Contam. Toxicol.33.575 - 583.
Mueller-Beilschmidt, D. 1990. Toxicology and environmental fate of synthetic
pyrethroids. J. Pest. Reform .10(3). 33-34.
Moore, M. T., Huggett, D.B., Gillespie, W.B., Rodgers, J.H.J., Cooper, C.M.
1998. Comparative toxicity of chlordane, chlorpyrifos, and aldicarb to
four aquatic testing organisms. Archives Environmental Contamination
and Toxicology. 34. 152-157.
84

Odenkirchen, E.W., Eisler, R. 1988.Chlorpyrifos hazards to fish, wild life, and


invertebrates synoptic review. U.S fish wild. Serv Biol. Rep.85, 9-11.
Thomas, P. 1990.Teleost model for studying the effects of chemicals on female
reproductive endocrine function. J. Exp. Zool.Suppl.4. 126-128.
Tyler.C.R, Sumpter.J.P.1996.Oocyte growth and development in teleosts.
Rev.Fish.Biol.Fisheries.6, 287.
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Review) for the Registration of Pesticide Products Containing
Chlorpyrifos. Washington, DC, 1989.5-44.
Yasuno.M, Hatakeyama.S, Miyashita.M., 1990. Effects on reproduction in the
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Acknowledgements
Authors acknowledge University of Ruhuna research grant (RU/SF/RP/99/05)
for providing financial assistance.

85

86

MATERNAL TRANSFER OF COPPER RESISTANCE IN FATHEAD


MINNOWS
Elizabeth B. Peake
Biology Department, University of Nebraska at Omaha
6001 Dodge Street, Omaha, Nebraska 68182-0040, USA
phone (402) 221-4474, fax (402) 221-4886
e-mail: peake@cox.net
Laura L. Tierney, Jessica C. Locke, and Alan S. Kolok
Biology Department, University of Nebraska at Omaha
EXTENDED ABSTRACT ONLY DO NOT CITE
The overall objective of this research was to determine if differential resistance
to copper (Cu) could be transmitted from adult fish to larval offspring.
Differential resistance can be transferred from adults to offspring in two
different ways. The first is genetic inheritance. This is supported in the literature
by several studies that found that differences in Cu resistance were significantly
correlated with differences in allozyme genotypes (e.g. Schlueter et al., 1995).
The second way is by maternal transfer of non-genetic material (e.g. Lin et al.,
2000). We conducted two experiments with fathead minnows (Pimephales
promelas) to investigate these two potential methods for transfer of Cu
resistance from parents to larval offspring.
In our first experiment, we tested the hypothesis that genetic differences in
adults were a major influence in determining the resistance of larvae. We used
the same methods as detailed in Kolok (1998) to classify 48 male and 48 female
adult minnows as being either Cu-susceptible or Cu-resistant. Critical swimming
speeds (Ucrits) were measured for the 96 minnows before and after an 8-9 d
exposure to 150 g/L Cu. Cu resistance or susceptibility was based on the
percent decrease in Ucrit after Cu exposure. After the minnows were classified,
the 12 most Cu-susceptible pairs and 10 most Cu-resistant pairs were bred
together.
A 7-d survival test (U.S. EPA, 1994) was conducted on the larvae produced by
Cu-resistant, Cu-susceptible, and nave (previously unexposed) parents.
Eighteen 600-ml beakers, each with 250 ml of Cu solution, served as test
chambers. Larvae <24 h old from five to seven breeding pairs were distributed

87

evenly among the test chambers, with about 15 larvae per test chamber. There
were triplicates at each of six Cu concentrations (0, 100, 200, 400, 800, and
1600 g/L), with static renewal of 80% daily with freshly made test solutions.
Actual Cu concentrations were analyzed by flame atomic absorption
spectrometry. Mortality was recorded daily. Figure 1 shows mortality for the
four groups, with replicates pooled.

CONTROL 1

RESISTANT

CONTROL 2

SUSCEPTIBLE

MORTALITY

1
0.8
0.6
0.4
0.2
0

500
1000
Cu CONCENTRATION (ug/L)

1500

Figure 1. Mortality after 7 days for four larval groups at six actual Cu
concentrations, 0-1400 g/L Cu.
The LC50s of the larvae from Cu-susceptible and Cu-resistant parents were
surprisingly similar (922 and 924 g/L, respectively) and significantly greater (ttest, p=0.02) than those of controls (412 and 210 g/L). Cu-susceptible parents
did not produce Cu-susceptible larvae; therefore the data did not support the
hypothesis that the relative Cu tolerance of the larvae would be similar to that of
their parents.

88

In the second experiment, we tested the hypothesis that larvae would be more
Cu resistant if their female parent had been previously exposed to Cu 1 week
prior to breeding. We bred 12 pairs of minnows and conducted 96-h time-todeath tests on their larvae. About 20 larvae <24 h old from each breeding pair
were placed in each of three 600-ml beakers. Each of these test chambers had
350 ml of Cu solution at 0, 400, or 800 g/L Cu, with daily static renewal of
80% with freshly made test solutions. Mortality was recorded 3, 6, 12, 24, 48,
and 96 h after test initiation. Six females were exposed to 100 g/L Cu for 5 d,
while the remaining females were sham-exposed. Females were then bred with
their original partners, and 96-h larval time-to-death tests were again conducted
as above. Figure 2 shows cumulative survival for the larval groups.

1.1
PRE-EXPOSURE
POST-EXPOSURE

CUMULATIVE SURVIVAL

1
0.9
0.8

0.7
0.6
1

24

48

24

48

72
96
PRE-EXPOSURE
POST-EXPOSURE

0.9
0.8
0.7

B
0

72

96

HOURS
Figure 2. Survival for pre- and post-exposure groups of larvae from female
parents exposed to (A) 100 g/L Cu and (B) 0 g/L Cu.

89

For both groups, the effects of each Cu test solution concentration was
significantly related to time-to-death, and their inclusion significantly improved
the fit of the model to the data. The pre-exposure and post-exposure groups of
larvae from sham-exposed females were not significantly different in
survivorship. For larvae from Cu-exposed females, however, post-exposure
survivorship was significantly higher than pre-exposure survivorship; indicative
of maternal transfer.
We were surprised to find that Cu susceptibility in the adults was not directly
correlated with Cu susceptibility in their larvae. One reason may be that the
mechanism for Cu susceptibility may be different between these two life history
stages. Upon closer examination (our second experiment), it became apparent
that maternal transfer had a much more important influence on the relative Cu
resistance of larvae than we initially anticipated.
Acknowledgements
We thank the Biology Department and the University Committee on Research,
University of Nebraska at Omaha (UNO) (Omaha, NE, USA) for funding. The
University of Mississippi Environmental Toxicology Research Laboratory
(Oxford, MS, USA) and the University of Nebraska-Lincoln Groundwater
Chemistry Laboratory (Lincoln, NE, USA) assisted in the analysis of water
samples for Cu. We also thank laboratory workers Koryn Boss, Sarah
Cederstrand, Randy Johnson, Darcy L'Etoile-Lopes, and Tony Mertz as well as
the Animal Care Services staff at UNO.
References
Kolok, A.S., E.P. Plaisance and A. Abdelghani. 1998. Individual variation in the
swimming performance of fishes: An overlooked source of variation in
toxicity studies. Environ. Toxicol. Chem. 17:282-285.
Lin, H.C., S.C. Hsu and P.P. Hwang. 2000. Maternal transfer of cadmium
tolerance in larval Oreochromis mossambicus. J. Fish Biol. 57:239-249.
Schlueter, M.A., S.I. Guttman, J.T. Oris and A.J. Bailer. 1995. Survival of
copper-exposed juvenile fathead minnows (Pimephales promelas)
differs among allozyme genotypes. Environ. Toxicol. Chem. 14:17271734.

90

U.S. Environmental Protection Agency. 1994. Short-term methods for


estimating the chronic toxicity of effluents and receiving waters to
freshwater organisms. 3rd Ed. EPA/600/4-91/002. Environmental
Monitoring Systems Laboratory, Cincinnati, OH, USA.

91

92

REDUCED GONADAL SOMATIC INDEX AND EXTERNAL


COLORATION FOLLOWING EXPOSURE
TO P,P-DDE IN ADULT MALE FUNDULUS HETEROCLITUS
Emily Monosson,
Box 329/15 North Street Montague MA 01351
phone/FAX 413-367-0052emonosson@forwild.umass.edu
Stephen D. McCormick, Michael F. ODea, USGS, Leetown Science Center,
Conte Anadromous Fish Research Center, Turners Falls, MA
Gregory M. Weber, USDA/ARS National Center for Cool and Cold Water
Aquaculture, 11876 Leetown Road, Kearneysville, WV 25430
EXTENDED ABSTRACT ONLY DO NOT CITE
The persistent organochlorine, p,p-DDE (a lipophilic metabolite of DDT) is a
potent antiandrogen that binds the androgen receptor (AR) in rats (Kelce et al.
1995). p,p-DDE also binds ARs in fish in vitro (Thomas 2000) although in
vivo studies investigating the antiandrogenic activity of p,p-DDE in fish have
produced conflicting results. Carlson et al. (2000) reported a lack of effects by
either p,pDDE or o,pDDT on sexual development in rainbow trout embryos,
whereas Baatrup and Junge (2001) recently demonstrated that p,pDDE can act
as an antiandrogen in male guppys. We evaluated the effects of p,pDDE
exposure on gonadal growth, coloration (male Fudulus develop yellow
coloration when mature), and blood plasma concentrations of testosterone (T)
and 11-ketotestosterone (KT).
Two similar studies were conducted in 1998 and 1999. Fundulus collected from
Stony Brook, NY in the fall of 1997 were held approximately six months prior
to the 1998 study (10 ppt salinity, 10oC) and for 1.5 years prior to the 1999
study. In March 1998, 56 Fundulus were allocated to tanks (12 fish per tank,
with two tanks per dose), acclimated for five days and injected with either 0, 10
or 100 ppm p,pDDE dissolved in olive oil. Temperature was then increased on
the day of injection over a period of 4 days to 20oC to induce maturation. Fish
were sampled 0, 2 and 4 weeks after injection, visually inspected for color
development, and condition factor (CF), gonadal somatic index (GSI) and
hepatic somatic index (HSI) were recorded. Na+,K+-ATPase activity was

93

measured in gill tissue as a general indicator osmoregulatory status. In 1999 we


again rated coloration on a scale from 1 (little or no yellow) to 3 (bright yellow),
since ratings were qualitative they were confirmed blind by others in the lab.
Blood plasma concentrations of T and KT were also measured using previously
established radioimmunoassays. Statistical results were calculated using
ANOVA, with HSD for unequal cell numbers. A nonparametric analysis
employing a Kruskal-Wallis ANOVA by ranks post-hoc test in STATISTICA
was used for hormone analysis since a large number of samples were below
detection limits.
Results from both 1998 and 1999 were fairly consistent. In general pp-DDE
did not affect general indicators of health including weight, HSI, or Na+,K+ATPase activity (range from 6.2 to 7.0 mole/mg protein/min). There was,
however, a decrease in CF in 1998 in fish dosed with 100 ppm p,p-DDE (from
1.12 to 1.05), although we did not observe a reduced CF in 1999. Exposure to
100 ppm p,p-DDE caused a 28% decline in GSI in the maturing male fish in
1999 and a 22% decline (which was not significant) in

Fig. 1. Gonadal (testicular) somatic index (GSI) following four weeks of


exposure to p,p-DDE in Fundulus heteroclitus shown as a percent of mean
control GSI (+/- STDEV). Data for studies conducted in 1998 and 1999.

94

1998 (Figure 1). Additionally we observed a decrease in yellow coloration of


the males in 1999 (Table 1). Differences in coloration, though not quantified,
were noted in 1998 as well. Development of coloration is an androgen
dependent process in most male fish often involving KT (reviewed in Borg
1994). The results of this work are consistent with effects of p,p-DDE in the
guppy (Baatrup and Junge 2001) where high doses of p,p-DDE caused reduced
coloration, sperm count, gonad size and courtship behavior (1 ppm of p,p-DDE
in food was estimated to result in a daily dose of 15 g/g fish [Baatrup and
Junge 2001] or a maximum of 450 ppm over 30 days.) Our exposure levels are
closer to the lower dose used by Baatrup and Junge (2001) although our route of
exposure was quite different (a single injection at the initiation of the study
verses a daily exposure throughout the course of the study). Additionally we
measured plasma concentrations of T and KT. Unfortunately too many values
for T were below the detection limit of our assay to conduct statistical analysis.
KT concentrations did not appear to be altered five weeks after p,p-DDE
exposure (Table 1). Since a large number of samples were below detection
limits for T and KT it is difficult to draw conclusions regarding the effects of
p,p-DDE exposure on these androgens. Although by taking the conservative
approach of assigning the detection limit to those samples below that limit, our
data suggests that exposure to p,p-DDE did not alter concentrations of KT. Nor
did concentrations of KT appear to differ among groups when calculated as
ng/ml steroid/g gonad (data not shown). The results of this study suggest that
p,p-DDE exposure in adult fish interferes with gonad growth and development
of nuptial coloration in Fundulus, possibly by acting as an antiandrogen.

95

Table 1. Effects of p,p-DDE on morphometric and steroidal endpoints in


Fundulus heteroclitus following two or four weeks of exposure data are
expressed as mean(stdev).
tmt
N
Wta
L
HSI
mg/
g
cm
%
kg
Results 1998 2 weeks after initial exposure
7.7
1.0
0
8
5.2
(1.0)A
(0.6)B
(2.2)A
8.1
1.3
10
8
5.9
(0.5)A
(0.3)B
(1.3)A
8.1
1.5
100
8
4.6
(0.8)A
(0.4)B
(0.9)A
Results 1999 4 weeks after initial exposure
7.4
0.9
0
5
4.6
(0.8)A
(0.2)B
(1.3)A
7.5
0.9
10
7
5.1
(1.0)A
(0.3)B
(2.0)A
7.6
0.8
100
8
4.6
(0.5)A
(0.2)B
(0.9)A
Results 1999 4 weeks after initial exposure
0
12
10.9
9.5
1.7
(2.0)A
(0.5)A
(0.3)B

GSI
%

CF

KTb
ng/ml

Tc
ng/
ml

Color
Score

4.1
(1.1)B
3.7
(0.8)B
4.5
(0.9)B

1.10
(0.04)A
1.08
(0.04)A
1.19
(0.04)A

3.7
(0.2)B
3.2
(0.7)B
2.9
(0.7)B

1.12
(0.03)A
1.15
(0.03)A
1.05
(0.02)B

2.5
(0.5)A

1.25
(0.04)A

0.46
(0.08)B

10

11

11.0
(3.1)A

9.5
(0.8)A

1.7
(0.3)B

2.8
(0.5)A

1.26
(0.04)A

0.67
(0.30)B

100

12

12.9
(3.2)A

9.9
(0.7)A

1.8
(0.4)B

1.8
(0.5)B

1.31
(0.31)A

0.48
(0.17)B

0.14
(0.0
2)
A
0.21
(0.1
1)
A
0.16
(0.0
5)
A

1.9
(0.7)
B
1.9
(0.7)
B
1.2
(0.5)
A

Different letters are significantly different (p<0.05) using ANOVA and HSD
for an unequal N post-hoc test.b KT measurements are for individual fish.
Sample detection limit was 0.40 ng/ml to be conservative we used 0.40 ng/ml
for samples that were below detection limit. There were several samples below
detection limit (0, 5, 4 and 8 in the Time 0, 0 ppm, 10 ppm and 100 ppm groups
respectively). Because of the large number of samples below detection we used
a nonparametric posthoc test for KT analysis (Kruskal-Wallis ANOVA by
ranks) cSamples from 3-4 individuals were pooled for analysis of T, so N=4 per

96

treatment except Time 0 where N=3. Sample detection limit was 0.13 ng/ml, to
be conservative we used 0.13 ng/ml for samples that were below detection limit.
There were 3, 1 and 3 values below detection in the 0, 10 and 100 ppm groups
respectively, so no statistical analysis was conducted. d n=not measured in this
study.
References
Baatrup, E, M Junge. 2001. Antiandrogenic Pesticides Disrupt Sexual
Characteristics in the Adult Male Guppy (Poecilia reticulata). Env
Health Perspec 109:1063-1070
Carlson DB, Curtis LR, Williams DE. 2000. Salmonid sexual development is
not consistently altered by embryonic exposure to endocrine-active
chemicals. Env Health Perspec 108:249-255
Kelce WR, Stone CR, Laws SC, Gray LE, Kemppainen JA, Wilson EM. 1995.
Persistent DDT metabolite p,p-DDE is a potent androgen receptor
agonist. Nature 375:581-585
Kodric-Brown A. 1998. Sexual dichromatism and temporary color changes in
the reproduction of fishes. Am Zool 38:70-81
Thomas P. 2000. Chemical interference with genomic and nongenomic actions
of steroids in fishes: role of receptor binding. Mar Env Res 50:127-134

97

98

BLOOD CELL RESPONSES


OF THE TROPICAL FISH PROCHILODUS SCROFA
TO ACUTE COPPER EXPOSURE AND SUBSEQUENT RECOVERY
Carla C. C. Cerqueira
Federal University of So Carlos
Post-Graduate Program in Ecology and Natural Resources
C. Postal 676, Phone: 55 16 260-8314
Marisa N. Fernandes
Federal University of So Carlos
Department of Physiological Sciences
C. Postal 676, Phone: 55 16 260-8314
email: dmnf@power.ufscar.br
Abstract
Changes in Prochilodus scrofa blood cells were investigated after 96-h of
exposure to copper and following transference to clean water. Hematocrit, red
blood cells and hemoglobin concentrations showed a significant increase after
copper exposure, remaining high until the 7th day after transference to clean
water. The immature blood cells (erythroblasts) also increased significantly, but
did not differ from the controls on the 7th day in clean water. The changes in
leukocytes occurred only in the percentage of lymphocytes, which was
significantly reduced after 96-h copper exposure, remaining lower on the first
and second day in copper-free water. Thrombocytes increased significantly in
fish exposed to copper and remained high on the 7th day in clean water. The
changes in the blood cells of P. scrofa reflect the animals responses to stress
caused by copper; however, after their transfer to clean water, most of the
changes involved a compensatory physiological mechanism that allowed the fish
to recover from copper-related damage.
Introduction
Blood cell responses are important indicators of changes in the internal and/or
external environment of animals. In fish, exposure to chemical pollutants can
induce either increases or decreases in hematological levels. The growing use of

99

copper in the metallurgic industry has resulted in an increase of copper ions in


the natural waters of southeastern Brazil. Previous studies of the neotropical fish
Prochilodus scrofa exposed to copper revealed drastic changes in red and white
blood cells as well as in the thrombocytes (Cerqueira, 2000; Mazon et al., 2003).
Few studies, however, have focused on the process of recovery of these cells
after copper was removed from water. Hence, the main purpose of this study
was to evaluate the effect of acute copper exposure on P. scrofa blood cells and
to discover how long the fish took to recover after returning to an environment
of improved water quality.
Materials and Methods
Three to five-month-old juvenile Prochilodus scrofa (W = 15-75 g) were
provided by the Hydrobiology and Aquaculture Station of Furnas Hydroelectric
Power Plant, Furnas, MG, Brazil, and kept in tanks at 25 1oC (1000 L) with a
continuous flow of dechlorinated tap water (water composition: pH = 7.3 0.2;
alkalinity = 23.7 1.9 mg L-1 as CaCO3; conductivity = 8.3 0.3 S and
hardness = 24.5 0.2 mg L-1 as CaCO3) and aeration (100% O2 saturation) for at
least one month prior to the experiments. The laboratory photoperiod was
12D:12L. The fish were fed with balanced fish food suitable for this species
provided by the Aquaculture Research and Training Center - CEPTA/IBAMA.
Water temperature, pH, hardness and alkalinity were the same as the mean
values found in P. scrofas natural habitat (CETESB 1992-2000).
After acclimation to laboratorial conditions, the fish were randomly divided into
two groups and each group transferred to static test aquariums not exceeding 1g
fish L-1. Group 1 (the control group) was kept in copper-free water while group
2 (the group exposed to copper) was exposed to 29 gCu L-1 (96-h LC50 for P.
scrofa; Mazon and Fernandes, 1999). After 96 h, each group was transferred to
an aquarium with clean flowing water (recovery period). The physicochemical
characteristics of the water in the aquariums of both groups were maintained
constant throughout the experimental period and were the same as those
prevailing during the acclimation period (except for the copper concentration in
the aquarium of group 2). The copper agent used was CuSO4.5H2O and the
copper concentration in the water was measured using Atomic Absorption
Spectrophotometry. No copper was detected in the water of the control and
recovery aquariums.

100

To evaluate the changes in blood cells after exposure to copper and their
reversibility following transference to copper-free water, random fish samples (n
= 8) from each group, i.e., the control group and the one exposed to copper,
were taken after the fish had been held for 96 h in a static system and 1, 2, 7, 15,
30 and 45 days after their transference to clean water. The fish were
anaesthetized with 0.01% benzocaine and blood samples were withdrawn from
the caudal vein into heparinized plastic tubes.
Hematocrit (Hct), red blood cell count (RBC) and hemoglobin concentration
([Hb]) were conducted immediately. Hct was determined by spinning the blood
sample contained in heparinized capillary tubes in a microhematocrit centrifuge.
The RBC count was carried out in a modified Neubauer chamber after saline
dilution of the blood, while the [Hb] was determined by the
cyanomethaemoglobin method. The mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin
concentration (MCHC) were calculated from previous blood measurements.
Blood smears were fixed with methanol and stained with Leishman solution for
counts of immature red blood cell, thrombocytes and leukocytes by 5000 cell
count, according to the method described by McKnight (1966). To prevent
errors arising from uneven cell distribution, the slides were divided into four
segments and cells were counted in fields contained in parallel rows
commencing from outside edge of the slide toward the inside. Differential
leukocyte counts were made by identifying 200 leukocytes in each slide (Dick
and Dixon, 1985). The leukocytes were classified according to their general
shape and affinity to the dye (Takashima and Hibiya, 1995).
The data are presented as mean SEM. The control group data are given all
together, since no significant changes were found among them. After the
uniformity of the groups data was verified using the Bartlett test, the parametric
analysis of variance (ANOVA) was applied to determine differences in the level
of significance among the groups. Tukeys test with a 95% confidence limit was
applied to compare the mean values whenever a level of significance occurred
(GraphPad InStat Software, San Diego, CA).
Results
No fish from the control group died during the experiment; however, 48% of the
fish from the group exposed to copper died during its 96-h exposure. After its

101

transfer to aquariums with clean flowing water, no further mortality occurred in


this group.
Figures 1 and 2 and Table 1 show the changes in the blood cells after 96 h of
copper exposure and following transference to clean water. Hct, RBC and [Hb]
were significantly higher in fish exposed to copper, particularly on the 1st and
2nd days following transference to clean water (Fig 1). On the 7th day in clean
water, RBC and [Hb] were still significantly higher than in the control fish, but
significantly lower than they had been immediately following 96 h of exposure
to copper. VCM and CHCM increased after 96 h of exposure to copper but were
similar to the values of the controls following transference to clean water. The
percentage
of
immature
erythrocytes
(polychromatophilic
and
orthochromatophilic erythroblasts) increased after copper exposure, remaining
high on the 1st and 2nd days after the transference to clean water (Table 1).
Table 1. Mean values SEM of polychromatophilic erytroblasts (PCE) and
orthochromatophilic erytroblasts (OCE) of P. scrofa after 96h exposure to
copper and subsequent recovery
n
PCE
OCE
(%)
(%)
Control
56
2.7 0.05
1.3 0.08
96h LC50 copper exposure
8
4.5 0.12*
3.8 0.07*
Recovery (days)
1
8
3.8 0.06*o
2.1 0.07*o
2
8
3.5 0.08*o
1.9 0.10*o
o
7
8
2.6 0.13
1.4 0.07o
o
15
8
2.5 0.13
1.4 0.07o
o
30
8
2.6 0.09
1.3 0.10o
o
45
8
2.7 0.09
1.4 0.08o
* indicates significant difference from the controls (p<0.05) and o indicates
significant difference from 96h LC50 copper exposure (p<0.05)

102

**

160

20

80

80

oo o

o o o

MCH (pg. cell-1 )

800

RBC x 10 4 (mm 3 )

* ** *

40

80

o o o

***

oo o

MC H C (%)

Hemoglobin (%)

20

Cont 96h 1 2 7 15 30 45
Recovery (days)

*
o o

40

10

40

400

o o

120

MCV (m3)

Hematocrit (%)

40

o
o

Cont 96h 1 2 7 15 30 45
Recovery (days)

Figure 1. Changes in hematocrit (Hct), red blood cells (RBC), whole blood
hemoglobin concentration ([Hb]), mean cell volume (MCV), mean
corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin
content (MCHC) of P. scrofa after 96h of copper exposure and
subsequent recovery in clean water. The bars represent the mean values
( SEM). Control fish (n = 56; open bars); 96h copper exposed fish (n
= 8; black bars) and recovery (n = 8 each time; stippled bars). *
indicates significant difference from the controls (p < 0.05); o indicates
significant difference from 96h copper exposed fish (p < 0.05)

103

Differential leukocyte counts (Fig. 2A) showed that lymphocytes were


the most frequent white blood cells in the control P. scrofa (63 1 %) and
the proportion of these cells was reduced to 50% in fish exposed to 29
gCu L-1. A further significant reduction was found during the first two
days of the recovery period and, on the 7th day, the percentage of
lymphocytes was similar to that of the controls. The percentage of
neutrophils was low compared to monocytes in the control fish. Neither of
the cell types showed significant changes after exposure to copper.
Basophils were not found in the prepared smears and eosinophils were very
rare (less than 0,20 %).
The percentage of thrombocytes increased in fish exposed to copper and
was found to be similar to the control fish on the 15th day in clean water
(Fig. 2B).
A

B
8
o

o
*

o
*

o
*

40

Cont 96h 1

15

Recovery

Thrombocytes (%)

Leukocytes (%)

80

45
(days)

o
*

o
*
o
*

4
o

30

Cont 96h 1

2 7 15 30 45
Recovery (days)

Figure 2. A. Lymphocyte (open bars), monocyte (black bars) and


neutrophil (stripped bars) percentages of P. scrofa leukocytes
after 96h copper exposure and subsequent recovery in clean
water. B. Thrombocyte percentage of P. scrofa leukocytes after
96h copper exposure (black bar) and subsequent recovery in clean
water (stripped bars); control fish (open bar). The bars represent
the mean values ( SEM). Control fish (Cont, n = 56); 96h copper
exposed fish (n = 8) and recovery (n = 8 each time). * indicates
significant difference from the controls (p < 0.05); o indicates
significant difference from 96h copper exposed fish (p < 0.05)

104

Discussion
The direct effects of copper on circulating blood cells were usually associated
with an increased disintegration of erythrocytes or, in the case of more sensitive
species, to damage of the hemopoietic system (Svobodov et al., 1994).
However, some contradictory responses have been found (Wilson and Taylor,
1993; Heath, 1995; Nussey et al., 1995a,b) even in the same species. In P.
scrofa, the increase of Hct, RBC and [Hb] with significant changes in the MVC
and MCHC blood indices suggests a possible hemoconcentration after copper
exposure for 96h. Similar increases of Hct, RBC and [Hb] were also reported by
Mazon et al. (2003) in P. scrofa exposed to copper, but the changes were not
coupled to changes in the blood indices. During the 7 days of the recovery
period, the changes in the red blood cells (increase in the RBC and Hb
concentration with no significant changes in the MCH and MCHC blood indices
and cell size) suggest a compensatory response of this species to heighten the
bloods O2 carrying capacity.
The reduction of the lymphocyte percentage seems to be a general response to
metal exposure (Mishra and Srivastava, 1980; Dick and Dixon, 1985;
Svobodov et al., 1994) caused by increasing corticosteroid levels in the blood.
Neutrophil and monocyte percentages in blood are expected to decrease during
acute copper exposure (Svobodov et al., 1994), since these blood cells are vital
to protect the body against bacterial infection in damaged tissue. However, no
changes were found in P. scrofa either following acute copper exposure in
which cell degeneration, rupture and peeling of lamellar epithelial are known to
be intense (Mazon et al., 2002), nor during the recovery period.
Because thrombocytes are the blood cells involved with blood clotting, their
increased percentage in P. scrofa exposed to copper may evidence a
compensatory response to reduce bleeding from the damaged branchial vascular
tissue. The return of thrombocyte percentages to the levels of the control fish in
clean water coincided with the main changes observed in the restoration of gill
tissue (Cerqueira et al., 2002).
In conclusion, the changes in the blood cells reflect the responses to the effects
of stress caused by copper and, after transference to clean water, most of the
changes are evidence of compensatory responses that enable fish to recover
from copper-related damage.

105

Acknowledgments
This research was supported by Fundao de Amparo a Pesquisa do Estado de
So Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientfico e
Tecnolgico (CNPq), Brazil. C.C.C. Cerqueira acknowledges CAPES for the
award of a scholarship.
References
Cerqueira, CCC
2000 Recuperao do tecido branquial, parmetros
hematolgicos e inicos de curimbat, Prochilodus scrofa
Steindachner, 1881 (Characiformes, Prochiloontidae) aps exposio
ao cobre. Ms thesis. Universidade Federal de So Carlos, So Carlos,
SP, Brazil, 86 p.
Cerqueira, CCC and Fernandes, MN 2002 Gill tissue recovery after copper
exposure and blood parameter responses in the tropical fish
Prochilodus scrofa. Ecotoxicol. Environm. Safety 52, in press.
CETESB 1992-2000 Relatrio de Qualidade das guas Interiores do Estado de
So Paulo. So Paulo, Brasil
Dick, PT and Dixon, DG 1985 Changes in circulating blood cells levels of rainbow
trout, Salmo gairdneri Richardson, following acute and chronic exposure
to copper. J. Fish Biol. 26: 475-484.
Heath, AG 1995 Water Pollution and Fish Physiology. CRC Press, Boca Raton,
Fl.
Mazon, AF and Fernandes, MN 1999 Toxicity and differential tissue
accumulation of copper in the tropical freshwater fish, Prochilodus
scrofa (Prochilodontidae). Bull. Environ. Contam. Toxicol. 63: 797804.
Mazon AF, Cerqueira, CCC and Fernandes, MN 2002 Gill cellular changes
induced by copper exposure in the South American tropical freshwater
fish Prochilodus scrofa. Environ. Res. 88: 52-63.
Mazon, AF, Monteiro, EAS, Pinheiro, GHD and Fernandes, MN 2003
Hematological and physiological changes induced by short-term

106

exposure to copper in the freshwater fish, Prochilodus scrofa. Braz. J.


Biol. 63: in press.
McKnight, IM 1966 A hematological study on the mountain whitefish,
Prosopium williamsoni. J. Fish. Res. B. Can. 23: 45-64.
Mishra, S and Srivastava, AK 1980 The acute toxic effects of copper on the blood
of a teleost. Ecotoxicol. Environ. Safety 4: 191-194.
Nussey, G, Van Vuren, JHJ and Du Preez, HH 1995a Effect of copper on
haematology and osmoregulation of the Mozambique tilapia,
Oreochromis mossambicus (Cichlidae). Comp. Biochem. Physiol.
111(C): 369-380.
Nussey, G, Van Vuren, JHJ and Du Preez, HH 1995b Effect of copper on the
differential white blood cell counts of the Mozambique tilapia
(Oreochromis mossambicus). Comp. Biochem. Physiol. 111(C): 381388.
Svobodov, Z, Vykusov, B and Mchov, J 1994 The effects of pollutants on
selected haematological and biochemical parameters in fish. In
Sublethal And Chronic Effects Of Pollutants On Freshwater Fish
(Mller, R and Lloyd, R, eds). Fishing New Books, London.
Takashima, F and Hibiya, T 1995 An Atlas Of Fish Histology. Normal And
Pathological Features. 2nd. Ed. Kodansha, Tokyo.
Wilson, R and Taylor, EW 1993 The physiological responses of freshwater
rainbow trout, Oncorhynchus mykiss, during acutely lethal copper
exposure. J. Comp. Physiol. 163(B): 38-47.

107

108

TOXICITY OF CADMIUM: A COMPARATIVE STUDY IN THE


AIR BREATHING FISH, CLARIAS BATRACHUS AND IN
NON-AIR BREATHING ONE, CTENOPHARYNGODON IDELLUS
P.K. Joshi
Head of the Department, Dept. of Zoology
Govt. P. G. College. MHOW
Dist.Indore (M.P.) INDIA
Phone-91-0731-478204
E-mail PRAMOD772000@yahoo.com
Manjushree Bose
Ram Rahim Colony RAU
Dist.Indore (M.P.) INDIA
Phone-91-0731-856219
E-mail - manjushree_bose@rediffmail.com
Abstract
Heavy metals and their salts constitute a very important group of
environmental pollutants since they are potent metabolic inhibitors. The
inherent toxicity of a metal depends upon its capacity to disturb the dynamic
life processes in biological system by combining with cell organelles,
macromolecules and metabolites. Cadmium is considered as non-essential
element. This study was performed to begin an assessment of effect of the
heavy metal on biochemistry of blood serum because blood is a good patho
physiological indicator. Test animals used for the study were Clarias
batrachus and Ctenopharyngodon idellus. Both the fishes responded
differently to the same toxicant and for same duration of time. In Clarias
there was a decrease in glucose, cholesterol, total protein, urea and
creatinine value. While in Ctenopharyngodon only glucose and sodium
showed a decrease but all the other parameters showed elevation in values.
After studying the result of present work it is clear that Cadmium very much
affects the energy metabolism, which in long term cause the death of the
individual organism and affects the whole community.
Introduction
It is now well realized that environmental problems have increased
exponentially in recent decades mainly because of rapid growth in human
population and increased demand for several household materials. While on
one hand technological development has improved the quality of life, on the
109

other hand it has created a number of health hazards. The toxic chemicals
discharged into air, water and soil get into food chain from the environment.
By entering into the biological system they disturb the biochemical
processes leading to health abnormalities, in some cases to fetal
consequences (Pratima Gupta, 1998). In 1975 U. S. Environmental
Protection Agency (U S E P A), Occupational Safety and Health
Administration (O S H A), Consumer Product Safety Commission (C P S C)
listed 24 extremely hazardous substances. These include heavy metals also.
One such important heavy metal is cadmium (Cd). Cadmium is a wellknown cumulative poison in animals that belongs to group b of the
periodic table. Cadmium enters surface water with the discharge of
industrial wastes or by leaching of soil, to which sewage sludge is added. It
is biologically very reactive and therefore gives rise to both acute and
chronic poisoning. Nariagu (1983) emphasized elaborately on effects of
cadmium on aquatic organisms. Many reports are available on the effect of
Cd on fish blood. Blood is a good bio indicator or a diagnostic tool to study
the problem in organ function. The measurement of biochemical changes in
blood of fish under exposure to any toxicant may be used to predict effects
upon chronic exposure. Present work was a comparative study with a
siluroid air-breathing catfish Clarias batrachus and a cyprinoids non airbreathing fish Ctenopharyngodon idellus under sub lethal Cd intoxication.
Effects were studied on some serum biochemical parameters.
Materials and Methods
Live and healthy Clarias batrachus were purchased from local fish market
and Ctenopharyngodon idellus were collected from the pond of village
Santer near MHOW. Fishes were checked for injury and disease, and then
washed in .1% KMnO4 solution for 5 minutes. After acclimation of 15 days,
42 fishes of each category were selected for experiment, irrespective of their
sex. The average length and weight of Clarias batrachus was 17+- cm. and
100+_ gm. The average length of Ctenopharyngodon idellus was 14+_ cm.
and weight 100+_ gm. Prior to experiment toxicity tests were conducted to
determine the LC50 and safe concentration values of CdCl2 for 96 hours. The
physico-chemical analysis of water was done according to Standard
Methods published by A.P.H.A. (1992). Both the fishes were divided into 4
equal groups of 6 fishes each. First 3 groups of both the fishes were
maintained in sub lethal concentration of CdCl2 separately for 96 hours, 15
days and 30 days. Sub lethal concentration of CdCl2 for Clarias batrachus
was 2.5 ppm. and for Ctenopharyngodon idellus was 2.0 ppm. Fourth
groups were served as control for respective groups. All control and treated
fishes were fed once daily during the tenure of experiment. Both control and
treated fishes were sacrificed at time intervals and blood was collected by
serving the caudal peduncle using a sharp knife. Serum was separated from

110

the formed elements through the centrifugation at 3000 rpm. for 15 minutes.
Seven biochemical parameters were analyzed in serum, are:
1.
2.
3.
4.
5.
6.
7.

Glucose (mg/100 ml) - GOD POD method of Trinder.


Cholesterol (mgm/dl) - CHOD PAD method of R. Kettermann.
Urea (mg%)
- DAM method of D. R. Wybenga.
Total Protein (gm%) - Biuret method of T. E. Welchelbaum.
Creatinine (mg/dl) - Jaffs method of H. P. Seilig and H. West
Sodium (mMol/li)
- Flame Photometry method of John D. Baur.
Potassium (mMol/li) - Flame Photometry method of John D. Baur.

Results
During the course of experiments no mortality were recorded in both the
types of fishes exposed to sub lethal concentration of Cadmium chloride.
Certain changes were observed in the coloration, feeding behavior and
activeness of the fishes. Both the types of fishes initially became more
active but later their activity ceases. In both the types of fishes coloration
fades a little, fluctuating responses were observed in feeding behavior. Table
1. shows the biochemical indices recorded from exposing Clarias batrachus
to 2.5 ppm Cadmium chloride for 96 hours, 15 days and 30 days.
Differences were measured against the control values determined under
controlled laboratory conditions. The value of glucose shows a gradual fall
of 54%, cholesterol, total protein, creatinine, urea and potassium values
show a regular increase while sodium levels show an initial increase of 11%
but at the end of experiment it lowers to .7%.
Table 1. Serum biochemical data of the Clarias batrachus exposed to
Cadmium chloride (2.5 ppm).
SNO

PARAMETER

1Glucose
2Cholesterol
3Total protein
4Urea
5Creatinine
6Sodium
7Potassium

Control

47.0
170.0
4.0
16.300
0.200
134.000
6.000

96 hrs

15 days

30 days

32.3
185.0
4.2
16.500
0.300
149.000
7.100

27.4
192.0
4.2
17.100
0.400
138.000
8.100

21.4
209.0
5.4
18.400
0.500
133.000
8.500

Ctenopharyngodon idellus responded slightly different from Clarias


batrachus. (Table 2.) In this fish all the parameters gave fluctuating results.
Glucose level initially increases up to 29% but later it decreases lower than
normal level. Cholesterol, urea and creatinine had a regular decrease up to
66%. Total protein value increases 14% up to 15 days then it lowers to

111

normal in 30 days, while sodium and potassium levels gradually elevated


during the total period of experiment.
Table 2. Serum biochemical data of the Ctenopharyngodon idellus
exposed to Cadmium chloride (2.0 ppm).
SNO

PARAMETER

1Glucose
2Cholesterol
3Total protein
4Urea
5Creatinine
6Sodium
7Potassium

Control

96 hrs

15 days

30 days

66.6
266.6
7.0
20.600
0.900
112.600
9.200

86.0
210.0
7.5
20.500
0.600
120.000
9.800

72.0
170.0
8.0
18.300
0.500
124.000
10.200

60.0
104.7
7.0
15.200
0.300
129.000
11.200

112

Discussion
Heavy metals are widely distributed in free water sources and are harmful to
aquatic fauna. Biochemical parameters are the best indicators of stress
situations caused by heavy metals. In Clarias there was a decrease in
glucose value. Cadmium like heavy metals have affinity for ligands like
phosphate, cystenyle and histidyl side chains of proteins, can bind with
carrier protein molecules resulting in inhibition of sugar and amino acid
transport (Alvarado, 1966). According to Passov et al,. (1966) metal ions
block the active absorption of glucose by the intestinal epithelial cells. Many
other workers reported hypoglycemic condition in air breathing fishes due to
contaminants (Kurde1990, Sastry 1984). This may be to cope with highenergy demand in stress situations. Clarias
is
more
active
than
Ctenopharyngodon, toxicity tests showed that cadmium is more toxic to
non-air breathing fishes. (Figure 1) In glucose levels of Ctenopharyngodon,
showed initial increase and then a decrease. It may be due to liver
impairment to utilize glucose for glycogenolysis (Shastry and Sunita, 1982).
Such a situation may be attributed to higher activities of enzymes
participating in gluconeogenetic mechanisms, since enzymes of
gluconeogenesis are reported to be induced by various toxicants (Shaikh and
Hiradhar, 1985).

Glucose

100

Clarius
Idellus

80
60
40
20
0
Cotrol

96 hrs

15 days

30 days

Figure 1. Alterations in blood glucose levels in both the fishes.


Clarias batrachus showed increase in cholesterol value while a slow
decrease was observed in Ctenopharyngodon. (Figure 2) According to
(Kurde, 1990) 60 80 % of total serum cholesterol is in esterified form &
esterification occurs mainly in liver. Cadmium damages the liver, proportion
of esterified cholesterol decreases. Hyper cholestrolemia observed in
Clarias it may be due to impairment of liver and inhibition of enzymes,
which converts cholesterol into bile acid (Murrey 1990). Reduced
lipoprotein lipase activity plays a role in the increment of plasma lipid (Asha
Agrawal and Poonam Sharma, 1999).

113

Cholestoral

300

Clarius
Idellus

250
200
150
100
50
0
Cotrol

96 hrs

15 days

30 days

Figure 2. Changes in serum cholesterol level.


Proteins play a vital role in physiology of living organisms. All biological
activities are regulated by enzymes and hormones, which are also proteins.
Assessment of protein content can be considered as a diagnostic tool to
determine the physiological phases of the cells. (Kapila Manoj 1999)
Cadmium competes with Zn for the same sulphahydral group and binds
more firmly. Proteins are too sensitive and early indicators of heavy metal
poisoning. Kapila Manoj, 1999 & Kurde, 1990 observed elevation in protein
content of rat serum due to textile mill effluents. B.Rajanna et al,. (1981)
reported enhancement in protein content due to cadmium. (Figure 3) The
increase in protein content was due to enhancement of microsomal protein
synthesis suggested by many workers. Kidney is the target organ of
cadmium poisoning.

Total Protein

10
8
6
4
2
0
Cotrol

96 hrs

15
days

Clarius
Idellus

30
days

Figure 3.Changes in serum total protein level.


Teleost fishes are primarily amminotelic but their blood contains significant
amount of urea and indeed in some teleosts it may account for 20 % or more
of the total nitrogen excreted. Occurrence of uremia was reported by many
workers (Gupta and Bhargava 1985, Kurde 1990). Renal disorders also

114

elevate serum urea values. The level of urea was influenced by protein
content of diet.

Clarius
Idellus

Urea

25
20
15
10
5
0
Cotrol 96 hrs

15
days

30
days

Figure 4. Changes in serum urea levels.


A high protein diet raises the serum urea and low protein diet lowers it.
(Figure 4) Clarias is an active fish in stress condition it shows higher
activity while Ctenopharyngodon become sluggish and doesnt show more
activity, this type of expression in serum urea level was may be due to their
feeding habits. Creatinine is another nitrogenous waste product that is
eliminated by the kidneys, when excretion is suppressed in renal
insufficiency. The value is unaffected by protein intake. According to Lall
et.al,. (1997) rise in creatinine value is an indication of renal tubular damage
due to cadmium-induced naphrotoxicity (Kazuo et al,. 1980) Figure 5.
Shows the regular increase in serum creatinine value in Ctenopharyngodon
it proves that Cadmium is more nephrotoxic to non-air breathing fishes.

1.0
0.8
0.6
0.4
0.2
0.0

Clarius
Idellus

Creatinine

Cotrol 96 hrs

15
days

30
days

Figure 5. Alterations in serum creatinine value.


Minerals are mainly responsible for the maintenance of osmotic pressure in
blood and proper function of all types of tissues. (Mohanty & Mishra 1983).
The presences of activator ions of alkali metal series (Na+, K+) are essential

115

for activity of many enzymes. Toxic metals can alter the concentration of
electrolytes in blood. ATP and its related systems have been documented
well to participate in several metabolic processes. Na+ -K+ ATPase, located
in the cell membrane, has been implicated in the active transport of Na+ and
K+ across the cell membrane (B.Rajanna et al., 1981); changes in the levels
of plasma ions in present study were due to gill damage and inhibition of
Clarius

Sodium

160
140
120
100
80
60
40
20
0

Idellus

Cotrol

96 hrs

15 days

30 days

Figure 6. Changes in blood sodium level.

Clarius

Potassium

12

Idellus

10
8
6
4
2
0
Cotrol

96 hrs

15 days

30 days

Figure 7. Changes in blood potassium level.


enzyme activity. Woodling (1999) suggested that plasma ion decrease or
increase is due to kidney damage and altered enzyme activity and it has been
an indicator of impending death. Changes in the value of sodium in both the
fishes was may be due to the structural difference in gills and tolerance to
toxicant. Through reviewing the available literature it may be due to lateral
line imbalance and hormonal disorder by affected endocrine organs through
heavy metals.

116

Conclusion
After the above discussion it had been concluded that heavy metals causes
deleterious effects on fishes and very much altars the biochemical
characteristic of blood. In sub lethal concentration it may not be fetal for an
individual organism but it does affect the growth rate and reproduction
resulting in disturbance to whole community and tropic levels of food
chains, ultimately the ecosystem.
References
Alvarado, F.1966.Transport of sugars and amino acids in the intestine.
evidences for a common carrier. Science.151: 1011-1012.
A.P.H.A., A.W.W.A.and W.P.C.F. 1970. Standard methods for the
examination of water and wastewater. 18th Ed. American Public
Health Association. Washington.
Asha Agrawal and Poonam Sharma. 1999. Effect of sulphur dioxide on total
lipid and cholesterol level in the blood of albino rats. Journal of
Environ.Biol.20 (4). 335-338.
Gupta, R.C. and S. Bhargava. 1985. Practical Biochemistry. CBS Publishers
And Distributors, Delhi (India).
Gupta Pratima. 1998. Cadmium toxicity and thyroid function with special
reference to 5-monodeiodinase enzyme activity a comparative
study in birds and mammal. Ph.D. Thesis.
Kapila Manoj and G.Raghothaman. 1999. Mercury, copper and cadmium
induced changes in the total protein level muscle tissue of an edible
estuarine fish Boleophthalmus dessumieri. Cuv.J.Envi. Biol.20 (3),
231-234.
Kazuo T.Suzuki, Mitsuru Yamamura, Yasuko K. Yamada and Fujio
Shimizu. 1980. Decreased copper content in rat kidney
Metalothionine and its relation to acute cadmium toxicity.
Toxicology Letters, 7.137-142.
Kurde Sushama.1990. Effect of textile mill effluents and dyes on the
heamatological parameters in albino rats. Ph.D.Thesis.
Lall, S.B., N.Das, R.Rama, S.S.Peshin, S.Khatter, K.Gulati and S.D.Seth.
1997. Cadmium induced nephrotoxicity in rats. Indian. J. Exp.
Biol. Vol 35, pp 151-154.
117

Mohanty, B.K. and B.N.Mishra. 1983. Effect of mercurial drug (Kajyoli) on


Albino rat blood.J.Envi.Biol. 4 (4) 201-206.
Murray,R.K. 1991.Harpers Biochemistry 22 nd edition Prentice Hall
International Inc. pp-678.
Nariagu, O.Jerome and John.B.Sprangu. 1983. Cadmium on the aquatic
environment. Vol.9, A. Wietly Interscience Publication.
Rajanna, B., K.D.Chapatwala, D.D.Vaishnav and D.Desaiah.1981.Changes
in ATPase activity in tissues of rat fed on cadmium. J. Envi. Biol.
2(1) 1-9.
Sastry, K.V. and Sunita. 1983. Alterations in the intestinal absorption of
Xylose induced by heavy metals in fresh water teleost fish Channa
punctatus. Poll.Res.Vol. 2(2): 45-48.
Sastry and Sunita. 1982. Effect of cadmium chromium on the intestinal
absorption of glucose in snake head fish Channa
punctatus.Toxicology Letters,10.293-296.
Shaikh, Y.A. and P.K.Hiradher. 1985. Fluoride induced changes in blood
Glucose, tissue glycogen and succinate dehydrogenase (SDH)
Activity in the mudskipper Beleophthalmus dessumeiri. Proc.
Symp. Assess. Envir. Pollu, 93-99.
Woodling, John. D. 1999.Physiological and weight changes of wild brown
Trout inhabiting water with acutely toxic cadmium and zinc
concentration : An in situ study. Proc.of World Fish Convention.
Acknowledgement
Authors wish to express their humble gratitude to Dr. Lokesh K. Agrawal
for providing facilities for present work.

118

EFFECTS OF VANADATE OLIGOMERS


ON LIPID PEROXIDATION AND ANTIOXIDANT ENZYMES
IN THE LUSITANIAN TOADFISH KIDNEY AND LIVER:
SHORT-TERM EXPOSURE
Ins Figueiredo 1
Group of Comparative Cardiovascular , CCMar, Faculty of Marine and
Environmental Sciences, University of Algarve, Campus de Gambelas,
8000-810 Faro, PORTUGAL
Phone: 351 89 800 900; Fax: 351 89 818353
E-mail: inesu2@hotmail.com

Sandra Soares1; Gisela Borges1; Natrcia Joaquim1;


Manuel Aureliano2; Josefina Coucelo1
2
CMQA, Chemistry Dept., FCT, University of Algarve
EXTENDED ABSTRACT ONLY - DO NOT CITE
Introduction
Vanadium is a transitional metal to which a special attention is given in
questions of environmental management and health (Nriagu, 1998). Several
animal studies associate vanadium with oxidative stress and pointout liver and
kidney as major targets of metal toxicity (Stohs and Bagchi, 1995). As well as
other toxic metals, vanadium is known to exhibit the ability to produce reactive
oxygen species, resulting in lipid peroxidation and antioxidant enzymes
alterations, namely superoxide dismutase, catalase and glutathione peroxidase
(Byczkowski and Kulkarni, 1998). However, the contribution of vanadate
oligomers, in this case meta and decavanadate, for vanadium toxicity in
these tissues is not clarified. Thus, the objective of this work was to evaluate
antioxidant defence system responses induced by an acute exposure to a sublethal concentration (5mM) of meta and decavanadate, on the kidney and
liver of Halobatrachus didactylus (Lusitanian toadfish).

119

Methodology
The H. didactylus individuals were collected from Ria Formosa (South coast of
Portugal) and divided in three groups: Control (CTRL), injected
intraperitoneously (i.p.) with 0.9% NaCl; Metavanadate (Meta V), injected i.p.
with 1 ml/Kg of metavanadate (5mM); Decavanadate group (Deca V),
injected i.p. with 1 ml/Kg of decavanadate (5mM) Subgroups of 3 individuals
were sacrificed 0, 1 and 8 days after intoxication.
Liver and kidney were collected after sacrifice and cytosolic and mitochondrial
fractions were prepared for determination of catalase (CAT), superoxide
dismutase (SOD) and glutathione peroxidase (Total GPx and Se-GPx) activities.
Lipid peroxidation products were determined in homogenates using TBA
method.
The results are shown in percentual variation of group averages, compared with
CTRL group.
Results and Discussion
Different effects for both vanadate solutions in liver and kidney, were observed.
In the kidney (Table 1), antioxidant enzymes activities and lipid peroxidation
increased both in Meta V and Deca V groups. Major alterations occured in Deca
V CAT cytosolic, after 8 days, SOD mitochondrial, after 24 hours and Se-GPx
activities (Table 1). Also, there was a significant increase in lipid peroxidation
on Deca V group, which indicates an ineffective response of the cellular defence
mechanisms against oxidative stress caused by this metal. The same study
applied to the cardiac muscle (Aureliano et al., 2002) also revealed significant
changes in antioxidant enzymes activities and lipid peroxidation, indicating that
decameric vanadate species induce stronger toxic effects than other vanadate
species.

120

Table 1. Percentual variation of kidney antioxidant enzymes activity and lipid


peroxidation, after 24 hours and 8 days of meta or decavanadate
exposure.
Parameter
CAT
SOD
GPx
TBARS

Fraction
Cytosolic
Mitochondrial
Cytosolic
Mitochondrial
Total
Se-GPx
Total

% of Variation
24 Hours
8 Days
Meta V Deca V Meta V Deca V
70.9
39.2
-57.8
295.0
23.0
0.5
-42.6
-53.2
-29,6
-48.1
-18.3
33.1
24.1
139.2
65.2
85.0
9.1
43.1
4.2
64.7
11.6
115.3
70.0
137.0
61.4
8.8
34.7
257.3

In the liver (Table 2), CAT and SOD activities were in general stimulated in
both groups. Deca V group, after 24 hours, has shown the highest difference in
comparison to CTRL group (139.4%). There were no significant alterations in
lipid degradation products. These results indicate that, in the liver, the
antioxidant enzymes play an important role against oxidative stress.
Table 2. Percentual variation of liver antioxidant enzymes activity and lipid
peroxidation, after 24 hours and 8 days of meta or decavanadate
exposure
Parameter
CAT
SOD
GPx
TBARS

Fraction
Cytosolic
Mitochondrial
Cytosolic
Mitochondrial
Total
Se-GPx
Total

% of Variation
24 Hours
8 Days
Meta V Deca V Meta V Deca V
7.9
23.7
55.3
19.9
14.1
139.4
-44.9
11.0
66.8
13.0
21.9
6.8
18.8
10.6
6.3
9.5
-34.1
-16.1
-36.7
58.0
-8.2
-19.9
-20.4
-33.5
-42.1
-30.1
-49.5
-33.6

121

A similar study with cadmium in the heart, kidney and liver (Coucelo et al.,
2000) also report an increase of CAT and SOD activities in the liver. The
antioxidant enzymes activities in kidney had the same pattern, except in CAT
activity, that decreases after 24 hours and an increase after 7 days.
All oligomeric species of vanadate studied induced oxidative stress in both
tissues, but have also shown to affect differently antioxidant enzymes activities
and lipid peroxidation. Apparently, decavanadate induces stronger antioxidant
responses than metavanadate and stronger effects, as well as lipid
peroxidation, in the kidney.
References
Aureliano, M., N. Joaquim, A. Sousa, H. Martins and J.M. Coucelo. 2002.
Oxidative stress in toadfish (Halobactrachus didactylus) cardiac muscle:
acute exposure to vanadate oligomers. J. Inorg. Biochem. 90: 159-165
Byczkowski, J.Z. and A.P. Kulkarni. 1998. Oxidative stress and pro-oxidant
biological effects of vanadium. In "Vanadium in the environment. Part 2:
Health Effects. John Willey & Sons, Inc. N.Y. pp. 235-264
Coucelo, J. M., N. Joaquim, V. Correia, M.J. Bebianno and J.A. Coucelo. 2000.
Cellular responses to cadmium toxicity in the heart, kidney and liver of
Halobatrachus didactylus. Ecotox. Environ. Rest. 3: 29-35
Nriagu, J. O. (1998). History, occurence and uses of vanadium In "Vanadium in
the environment. Part 1: Chemistry and Biochemistry. Willey & Sons,
Inc. , N.Y. pp. 1-22
Stohs, S. J. and D. Bagchi. 1995. Oxidative mechanisms in the toxicity of metal
ions. Free Rad. Biol. Med. 18: 321-336

122

INCUBATION AND PH-DEPENDENT EFFECTS


OF VANADATE OLIGOMERS AND CADMIUM
WITH Halobatrachus didactylus SARCOPLASMATIC RETICULUM
CALCIUM PUMP
Llia Leonardo
Biochemistry Lab., CMQA, Chemistry Dept., FCT
University of Algarve, Campus de Gambelas, 8000-810 Faro, PORTUGAL
Phone: +351 89 800 900; Fax: +351 89 818 353
E-mail: lilialeonardo@hotmail.com
S.S. Soares1, N. Joaquim1, J.M. Coucelo. 1 and M. Aureliano2
Group of Comparative Cardiovascular , CCMar, Faculty of Marine and
Environmental Sciences
2
Biochemistry Lab., CMQA, Chemistry Dept., FCT
University of Algarve, Campus de Gambelas, 8000-810 Faro, PORTUGAL
1

EXTENDED ABSTRACT ONLY DO NOT CITE


Introduction
The Ca2+-ATPase of sarcoplasmatic reticulum (SR) membrane is a
transmembranar protein that catalyses both the hydrolysis and synthesis of ATP
(Chini et al; 1993) playing an essential role in relaxation and contraction of the
muscle. The activity of Ca2+-ATPase is normally described by a cycle
postulating the existence of two protein conformations, E1 and E2, with high
and low affinity for Ca2+ and ATP, respectively (Inesi, 1985). The alternation
between the two distinct conformations of Ca2+-ATPase during the transport
cycle seems affect the interactions with different oligomeric species of vanadate
(e,g., decameric and tetrameric) that affects the activity of the calcium pump
(Aureliano and Madeira, 1998). Such as, vanadium, cadmium is well known by
its toxic effects in live organisms, being an inhibitor of the calcium pump even
at very low concentrations.

123

In this work we compare the effects of cadmium and vanadium on the ATP
hydrolysis by the calcium pump at different pH values and incubation times
from skeletal muscle of Halobatrachus didactylus (Schneider, 1801).
Material and Methods
Ca2+-ATPase isolation and characterization
SR vesicles derived from H. didactylus Lusitanian toadfish skeletal muscle
were prepared as described elsewhere (Aureliano and Madeira, 1994).
Metal stock solutions and vanadate stability
Cadmium stock solution (50 mM) was prepared from cadmium chloride.
Vanadate stock solutions (50 mM) metavanadate and decavanadate were
prepared from ammonium metavanadate, according to described elsewhere
(Aureliano and Madeira, 1994). The stability of vanadate solutions at the
different experimental conditions was analysed by measuring the absorbance at
400 nm.
Hydrolysis of ATP by calcium pump
ATP hydrolysis was measured by colorimetry, through inorganic phosphate
analysis. Experiments were proceeded at 25C in a reaction medium containing
0.1 M KCl, 25 mM HEPES, 5 mM MgCl2, 50 mM CaCl2, pH 6.0, 7.0 or 8.0, in
the absence and presence of 2 M of cadmium or 2 mM (total vanadium) of
metavanadate or decavanadate, with or without 1 hour incubation with
0.285 mg/ml protein. The reaction was started by the addition of 420 mM MgATP.
Results
pH effects on the vanadate solutions stability
The decavanadate solutions stability decreases with pH (pH 6.0>7.0>8.0)
whereas metavanadate solutions are stable.

124

pH effects on Ca2+-ATPase activity in vanadium or cadmium inhibition

Inhibition (% of control)

The ATP hydrolysis by the SR Ca2+-ATPase is more strongly affected by


decavanadate than metavanadate and it depends on the pH. The relative
inhibitory effects for metavanadate and decavanadate regarding pH was: pH
6.0>8.0>7.0, whereas for cadmium the ATP hydrolysis was more inhibit at pH
8.0 (pH 8.0>7.06.0) (Figure 1).

100
80

meta 2 mM

60

deca 2 mM

cadmium
2 M
cdmio 2 mM

40
20
0
pH 6.0

pH 7.0

pH 8.0

Figure 1. Effects of pH on the inhibition of calcium pump ATP


hydrolysis by metavanadate, decavanadate 2 mM or
cadmium 2 M (n=4).

Effects of Ca2+-ATPase incubation with vanadium or cadmium


After 60 minutes of incubation decavanadate inhibition on ATP hydrolysis is
always higher than metavanadate. For both vanadate solutions the relative
order of inhibition upon pH is: pH 6.0>8.0>7.0. In fact, the lowest inhibition is
observed at pH 7.0 where metavanadate and decavanadate affect by 49%
and 80%, respectively the ATPase activity. Unlike vanadium, the protein
incubation with cadmium increases the inhibition of ATP hydrolysis within pH
increase from 3% to 24% (Figure 2).

125

Inhibition (% of control)

meta 2 mM
deca 2 mM

100

cadmium
M
cdmio 2 2mM

80
60
40
20
0

pH 6.0

pH 7.0

pH 8.0

Figure 2. Effects of pH on the inhibition of calcium pump ATP


hydrolysis by metavanadate, decavanadate 2 mM or
cadmium 2 M after 1 hour of incubation (n=4).

Conclusions
It is concluded that, at pHs 6.0, 7.0 and 8.0, cadmium inhibits strongly calcium
pump ATP hydrolysis and the cadmium incubation with the protein favours
enzymatic inhibition. Decavanadate affects more strongly the ATP hydrolysis
by Ca2+-ATPase than metavanadate, being the relative order of inhibition
affected by pH as followed: pH 6.0>8.0>7.0. It is suggested that oligomeric
species of vanadate inhibition of ATP hydrolysis is favoured by the E2
conformation (pH 6.0). Further studies are in course to clarify contribution of
vanadate species to vanadium interaction with the calcium pump of
Halobatrachus didactylus.
Acknowledgements
Sandra Soares thanks to Portuguese Foundation for Science and Technology,
FCT and by POCTI program financed through FEDER. Research project
38191/QUI/2001 and University of Algarve.

126

References
Aureliano, M. and V.M.C. Madeira. (1994). Interactions of vanadate oligomers
with sarcoplasmic reticulum Ca2+-ATPase. Biochim. Biophys. Acta
1221: 259-271
Chini, E.N., F.G.S. de Toledo, M.C. Albuquerque and L. de Meis (1993). The
Ca2+-transporting ATPases of rabbit and trout exhibit different pH- and
temperature-dependences. Biochem. J. 293: 469-473
Inesi, G. (1985). Mechanism of calcium transport. Annu. Rev. Physiol. 47: 573604
M. Aureliano and V. M. C. Madeira (1998) Vanadium in the Environment. Part
1: Chemistry and Biochemistry, Wiley & Sons, Inc., New York, pp.
333357

127

128

HISTOLOGICAL ANALYSIS OF VANADATE OLIGOMERS


EFFECTS ON HEART, KIDNEY AND LIVER
OF THE LUSITANIAN TOADFISH:
AN ACUTE EXPOSURE STUDY
Gisela Borges1
Group of Comparative Cardiovascular , CCMar, Faculty of Marine and
Environmental Sciences, University of Algarve, Campus de Gambelas,
8000-810 Faro, PORTUGAL
Phone: +351 89 800 900; Fax: +351 89 818353
E-mail: gisela_borges@megamail.pt

Paula Mendona1; Natrcia Joaquim1; Manuel Aureliano2; Josefina


Coucelo1
2
CMQA, Chemistry Dept., FCT, University of Algarve
EXTENDED ABSTRACT ONLY - DO NOT CITE
Introduction
The transitional element vanadium is an anthropogenic toxic metal produced
by the burning of fossil fuels (Barceloux, 1999). In trace concentrations
vanadium may exert benefit effects (1-10 nM), although in higher
concentrations it becomes toxic (>100 mM) (Stohs and Bagchi, 1995; Crans
et al, 1998).Vanadium toxicity has been associated mainly to oxidative
stress in kidney and liver (Stohs and Bagchi, 1995), but several effects on
cardiovascular system were also reported. However, histopathological
effects of vanadium on these tissues are not clarified. Therefore, the
objective of this work is to study the effects of an acute exposure to a sublethal concentration (5 mM) of two different vanadate solutions in cardiac,
renal and hepatic tissues of the Lusitanian toadfish Halobatrachus
didactylus (Schneider, 1801). It is also our objective to verify if these
histopathological effects depend on the vanadate oligomers administered:
metavanadate or decavanadate.
Material and Methods
Specimens of H. didactylus were divided in three groups: Control, Meta and
Deca groups, consisting of 10 individuals each. The Meta and Deca groups
were injected with 1ml/Kg of a metavanadate solution (5 mM), containing
129

mainly metameric species and a decavanadate solution (5 mM) containing


mainly decameric species, respectively. Five specimens of each group were
sacrificed 1 and 7 days after the exposure.
The organs were collected and the hearts were weighted in order to calculate
the relative ventricular mass (RVM). Sections of ventricular, renal and
hepatic tissues were subjected to histological procedures, stained with
Haematoxylin-eosin and examined through light microscopy in order to
evaluate tissue alterations induced by vanadium. Ventricular sections were
also stained with Picrosirius and observed through bipolarising microscopy
to calculate the ventricular wall structural elements area fraction
(percentage): collagen type I (coll. I), collagen III (coll. III) and muscular
tissue, as described previously (Coucelo et al., 2000).
Mann-Whitney test, a non-parametric variance test with a significance level
of 0.05 was applied to compare contaminated groups with control.
Results and Discussion
In the heart, 1 and 7 days after exposure to metavanadate the RVM was
not affected (p>0.05), while decavanadate, induced a significant increase
at day 7 (p<0.05), relatively to the Control group.
Although no evident cardiac tissue lesions were observed in the
contaminated groups, the study of the cardiac tissue components area
fractions revealed significant alterations (p<0.05) in the myocardial tissue
organization. Metavanadate induced a decrease of coll. III at day 1 and a
decrease of coll. I after 7 days, while decavanadate decreased coll. I and
III, just after 1 day. The muscle tissue area fraction was increased at day 1
by metavanadate, while decavanadate augmented this component after
1 and 7 days of exposure (Figure 1).
The observed results indicate that, in general, there was a collagen decrease
and a muscle tissue increase. Studies indicate that a collagenase activation
and fibrillar collagen breakdown are responsible for the dilation, the change
in shape and the increase in distensibility of the cardiomyopathic left
ventricle (Caulfield and Janicki, 1997), which is in concordance with our
results. However, metavanadate contrarily to decavanadate did not
induce RVM increase, although a collagen decrease was registered.

130

0.70 0.23
*

4
Area (%)

1.68 2.50
*

2.49 1.59

1.70 0.75

2
1
0
1

a)

Days

Area (%)

0.33 1.12

0.33 0.14
0.23 0.19
* 0.22 0.08
*

0.21 0.16
*

0
1

50.84 0.945
*

Area (%)

60

7
Days

51.1 2.8
*

46.2 1.7

53.7 4.4
*
50.8 5.4

b)

b)
a)

40
20
0

50.84 0.945
*

c)

Days

Control

Meta

Deca

Figure 1. Cardiac tissue components area fraction (%) in the images


containing the myocardial region: a) collagen type I; b) collagen type
III; c) muscle tissue; * p<0.05.
In the kidney of contaminated individuals, the renal tubules presented no
lumen and showed complete disorganization and different necrosis states of
epithelial cells (picnosis and karyolysis). The interstitial tissue was

131

hypercromatic. At day 7, these features were more severe than at day 1,


although metavanadate and decavanadate produced similar effects.
The hepatic tissue alterations included hypertrophied nuclei and
hepatocytes, necrotic hepatocytes and diminished cytoplasmic content.
Metavanadate and decavanadate induced more severe effects at day 7,
and the effects due to decavanadate were also more severe.
Conclusion
The studied oligomeric species of vanadate promoted evident tissue lesions
in the kidney and liver but not in cardiac tissue. However, decavanadate
induced a dilatation of the ventricle due to a decrease in the myocardial
collagen fibers percentage area, which may be related to ventricular
dysfunction. In general, decavanadate induced stronger histopathological
changes than metavanadate.
Acknowledgements
Gisela Borges was supported by a research grant from UIC (Unidade de
Interveno Cardiovascular , Hospital Particular do Algarve).
References
Barceloux, D. 1999. Vanadium. Clin.Toxicol. 37 (2): 265-278
Coucelo, J.M., N. Joaquim, V. Correia, M.J. Bebianno and J.A. Coucelo.
2000. Cellular responses to cadmium toxicity in the heart, kidney
and liver of Halobatrachus didactylus. Ecotox. Environ. Rest. 3
(1): 29-35
Crans, D., S. Amin and A. Keramidas 1998. Chemistry of relevance to
vanadium in the environment in Nriagu, J. (Ed.). Vanadium in the
environment. Part 1: Chemistry and biochemistry. John Wiley &
Sons, Inc. New York. pp. 73-95
Caulfield, J. B. and J. S. Janicki. 1997. Structure and function of myocardial
fibrillar collagen. Technol. Health Care. 5: 95-113
Stohs, S.; Bagchi, D. (1995). Oxidative mechanisms in the toxicity of metal
ions. Free Rad. Biol. Med. 18: 321-336

132

CADMIUM AND VANADATE OLIGOMERS COMPARATIVE


EFFECTS ON THE TOADFISH ERYTHROCYTE
S.S. Soares1
Group of Comparative Cardiovascular , CCMar, Faculty of Marine and
Environmental Sciences, University of Algarve, Campus de Gambelas, 8000810 Faro, PORTUGAL
Phone: +351 89 800 900; Fax: +351 89 818 353
E-mail: soares_ss@hotmail.com
1

M. Aureliano2, N. Joaquim1, J. M. Coucelo1


CMQA, Chemistry Dept., FCT, University of Algarve

EXTENDED ABSTRAT ONLY DO NOT CITE


Introduction
The increasing levels of toxic metals in the environment, specially in marine
environments, due to anthropogenic activities over the last years, make it
important to study its toxicity mechanisms. Heavy metals, such as cadmium and
vanadium, are known to cause extremely harm on biological systems. Cadmium
presents a high potential as a toxic substance, even in low concentrations (Hu,
2000). On contrary, vanadium is considered benefit to live organisms in
vestigial concentrations (1-10 nM), although it becomes toxic in higher
concentrations (>100 M) (Harland and Harden-Williams, 1994). Several
haematological changes, compromising oxygen transport efficacy (cell
destruction, hemolytic anemia and methaemoglobinemia), have been described
upon metal intoxication. In teleost species this is specially important since 10%
of haemoglobin is in the form of methaemoglobin (Lewis and Morris, 1986). In
the present study, biochemical and morphological in vivo toxic effects of
cadmium and vanadium were evaluated in red blood cells from Halobatrachus
didactylus (Schneider, 1801), in order to: 1) compare cadmium and vanadium
effects in methaemoglobin reductase activity; 2) evaluate the contribution of
different vanadate oligomeric species to vanadium toxicity; 3) study cadmium
and vanadium interaction with haemoglobin and 4) determine morphological
changes induced in red blood cells by cadmium and vanadium.

133

Material and Methods


H. didactylus Lusitanian toadfish individuals were collected in Ria Formosa
lagoon (Portuguese south coast) and divided into five groups: Control 1, non
injected; Control 2, injected intraperitoneously (i.p.) with 0.9% NaCl (placebo);
Cd, injected i.p. with 5 mM of Cd (CdCl2); Meta, injected i.p. with a
metavanadate solution containing 5 mM of total vanadium; and Deca, injected
i.p. with decavanadate containing 5 mM of total vanadium. Metal solutions
were diluted into final concentration (5 mM) in 0.9% NaCl. Metavanadate and
decavanadate solutions were prepared from ammonium metavanadate,
according to described elsewhere (Aureliano and Madeira, 1994). All solutions
were administrated in a dosage of 1 mL solution/Kg of body weight. Subgroups
of 4 individuals of each group were sacrificed after 1 and 7 days. Blood samples
were collected using heparin as an anticoagulant.
Methaemoglobin reductase activity was determined by Board (1981) method.
Cadmium and vanadium interactions with haemoglobin were studied in red
blood cells haemolysates, from a group of 6 non-injected fishes. The obtained
supernatant was incubated with metal concentration ranging from 0.5 to 5 mM,
at 25 C, up to 90 minutes and haemoglobin spectrum changes were analyzed by
spectroscopy.
Erythrocyte number, haemoglobin level and haematocrit were determined, in
blood samples, by routine methods. Morphological changes, induced by
cadmium and vanadium on red blood cells were studied on haematological
preparations with Giemsa stain.
The Mann-Whitney test was applied to test differences between groups, on all
the parameters analysed. The significant level used was p <0.05. Control 1 and
Control 2 showed no significant differences and for result analysed were
considered together as Control.
Results
Cadmium and vanadium effects on methaemoglobin reductase activity
It was observed a 20% decrease, relatively to Control, in methaemoglobin
reductase activity 1 day after cadmium injection, while metavanadate induced

134

Enzymatic activity
(mmol NAD+/min/g Hb

a 15 to 67% increase. Decavanadate had no significant effects on


methaemoglobin reductase activity (Figure 1).

25000

Control

Cd

Meta

Deca

20000
15000
10000
5000
0
1
7
Time after administration (days)

Figure 1. H. didactylus methaemoglobin reductase activity variation


1 and 7 days after 5 mM cadmium or vanadium (metavanadate or
decavanadate) in vivo administration (n=4). Standard deviation
ranging from 2 to 25 mol NAD+/min/g Hb.

Cadmium and vanadium interactions with haemoglobin


A 0.5 mM decavanadate solution (which contains only 50 M of decameric
species) induced haemoglobin oxidation to methaemoglobin after 30 minutes

135

incubation, whereas a slight spectrum change was detected for 5 mM of


metavanadate and 5 mM of cadmium does not affect haemoglobin spectrum
(Figure 2).

Absorbance

A
1,5

1
0,5

0
500

520

540

560

580

600

620

640

660

Wavelenght (nm)

Absorbance

B
2,5
2
1,5
1
0,5
0

c
a
500

520

540

560

580

600

620

640

660

Wavelength (nm)

Figure 2. A: a) Haemoglobin and b) methaemoglobin spectra; B:


Haemoglobin spectra after 90 minutes of incubation with a) Cd 5
mM, b) Meta 5 mM and c) Deca 50 M (0.5 mM total vanadium), at
25 C in phosphate buffer 0.5 M pH 7.2.

136

Cadmium and vanadium effects on haematological parameters


After 7 days of metals administration distinct changes were found in the
haematological parameters. Only decavanadate solution induced a decrease in
erythrocytes count and haematocrit relatively to Control 14% and 31%,
respectively, whereas the haemoglobin concentration decreased 29, 22 and 36%
in Cd, Meta and Deca groups, respectively, as it was observed an increase of
erythrocytes cellular volume. In Cd group, the cell hypertrophy was followed by
a nuclear volume increase.
Conclusions
It is concluded that different vanadate oligomers contributed differently to
vanadium toxicity in H. didactylus. Stronger effects were observed for
decameric species in causing haematological changes in the toadfish
erythrocyte. Upon administration, apparently cadmium induced an enzymatic
activity decrease of the methaemoglobin reductase in vivo, while
metavanadate solution promoted its stimulation and decavanadate had no
effect. Vanadate species seems to be more effective causing haemoglobin
oxidation in vitro than cadmium and, among vanadate species, decavanadate
promoted a stronger effect in this process. Moreover, cadmium and vanadate
oligomers induce a reduction of haemoglobin concentration, as well as the
increase of erythrocytes cellular volume. However, decameric species of
vanadate exhibit a more deleterious effect, promoting the reduction of
erythrocytes count and hematocrit.
Acknowledgements
S.S. Soares thanks to Portuguese Foundation for Science and Technology, FCT
and by POCTI program financed through FEDER. Research project
38191/QUI/2001 and University of Algarve.
References
Aureliano, M. and V.M.C. Madeira. (1994). Interactions of vanadate oligomers
with sarcoplasmic reticulum Ca2+-ATPase. Biochim. Biophys. Acta 1221:
259-271
Board, P.G. 1981. NADH-ferricyanide reductase, a convenient approach to the
evaluation of NADH-methaemoglobin reductase in human erythrocytes.
Clin. Chim. Acta 109: 233-237

137

Harland, B. and B. Harden-Williams. 1994. Is vanadium of human importance


yet? J. Am. Diet. Assoc. 94(8): 891-894
Hu, H. 2000. Exposure to metals. Occup. Environ. Med. 27(4): 983-996
Lewis, Jr W.M. and D.P. Morris. 1986. Toxicity of nitrite to fish: A review.
Trans. Amer. Fish. Soc. 115: 183-195
Winterbourn, C. 1985. Free-radical production and oxidative reactions of
hemoglobin. Environ. Health Perspect. 64: 321-330

138

TOXICITY OF LAKE ENRICHMENT NUTRIENTS


TO AQUATIC LIFE
Don MacKinlay,
Fisheries & Oceans Canada
555 West Hastings St. Vancouver V6B 5G3 Canada
E-mail: mackinlayd@pac.dfo-mpo.gc.ca
Craig Buday,
Pacific Environment Laboratory, Environment Canada
Introduction
The Lake Enrichment Program adds nutrients to lakes in British Columbia to
increase their productivity as nurseries for sockeye salmon fry (Stockner, 1987).
Since 1985, the nutrients used have been a mixture of urea ammonium nitrate
(32-0-0 or 28-0-0) and ammonium polyphosphate (10-34-0), both of which are
supplied in concentrated liquid form. Before 1997, these nutrients were added to
the lakes by spraying a fine mist onto a designated 'application zone' from
aircraft (twin engine airplanes or helicopters) fitted for crop dusting. Currently,
nutrients are added by introducing the liquid nutrient mix directly into the
propeller wash from a boat cruising on the lake.
The goals of this study were to:
1. Determine the toxicity of the concentrated nutrient solutions to aquatic life,
2. Determine which chemical species is the toxic fraction, and
3. Compare the toxicity levels to the concentrations that would be found
during an application of nutrients to the lake.
Methods
Toxicity Tests
Toxicity testing was carried out at the Pacific Environmental Science Centre in
North Vancouver, Canada in 2001. 96-hr acute lethality tests were performed on

139

rainbow trout (Oncorhynchus mykiss) for both the 28-0-0 and the 10-34-0
solutions. A 48-hr a acute lethality test was performed on the freshwater
microcrustacean, Daphnia magna (Figure 1). The 96 hour LC50 is the
concentration of sample that is calculated to be lethal to 50% of the test fish over
an exposure period of 96 hours.A 72-hr IC50/IC25 growth inhibition test was
performed on the green alga, Selenastrum capricornutum using the 10-34-0
solution.

Figure 1. Daphnia magna, a test organism for freshwater aquatic life


toxicity studies.
The rainbow trout LC50 tests followed protocols outlined in McLeay (1990,
2001) and the Dapnia LC50 test followed protocols outlined in (Miller et al.
2000). Both tests were conducted using well water as the contol and the diluent
for the test concentrations. Five test concentrations were used: 5600, 3200,
1800, 1000, 560 and 320 mg/L and a control. Each concentration had one
replicate with 10 test organisms per replicate in 30 kg of test solution for the
Rainbow trout and 200 mL for the Daphnia. Rainbow trout mortality was
recorded after 5, 10, 20, 40, 80 minutes and 24, 48, 72, 96 hours of exposure at
15+1oC. Cumulative Daphnia mortality was recorded after 24 and 48 hours of
exposure at 20+2oC. The 96-hr and 48-hr mortality data, respectively for
rainbow trout and Daphnia, were analyzed using the Stephan Program to
calculate the LC50 and 95% confidence intervals. A phenol reference test was
conducted for the rainbow trout, and a sodium chloride reference test was

140

conducted for the Daphnia, to ensure that the organism sensitivity was within
acceptable quality control warning chart limits.
The Selenastrum growth inhibition test followed protocols outlined in (McLeay,
2000). Tests were conducted using de-ionized water as the control and the
diluent for the test concentrations, which were: 10000, 5000, 2500, 1250, 625,
312.5, 156.3, 78.1, 39.1, 19.5 mg/L and a control. Each concentration had five
replicates with an initial approximate concentration of 10,000 organisms per
replicate in 220 L of test solution. Algal cell yield was recorded using a
Coulter (particle) Counter after 72 hours at 23+1oC. Algal cell yield data was
analyzed using ToxStat V3.5 to calculate the IC50, IC25 and 95% confidence
intervals. The 72 hour IC50 and IC25 is the concentration of sample estimated to
cause a 50% and 25% inhibition in growth of the algae over an exposure period
of 72 hours. A copper reference toxicant test was conducted to ensure that the
organism sensitivity was within acceptable quality control warning chart limits.
Chemical Analyses
Nutrient solution chemistry samples from both solutions were prepared at the
LC50 or IC50 concentrations following the toxicity tests. The chemistry samples
were analyzed for total metals, nitrogen ammonia, nitrogen nitrate and nitrite,
and total nitrogen using an ICAP spectrophotometer and standard methods,
respectively.
Application Dilution
The nutrient solution is added to the lake by filling tanks or the hold on a boat
with the solution and cruising down the middle of the lake while pumping the
nutrients into the propeller wash behind the boat (Figure 2). The propeller wash
is an extremely turbulent zone of water that spreads out behind the boat in three
dimensions.
Application concentrations were calculated from conditions found in lake
enrichment projects in British Columbia. The diluted concentration of nutrients
when it first enters the water can be calculated from the total amount of nutrient
solution added per trip or from the instantaneous addition rate:

141

CN =

NT
DT * AXS

CN =

FA * * 60
VB * AXS

where: CN is the final concentration of the nutrient solution (in mg/L), NT is the
total amount of nutrients added (in kg), DT is the total distance travelled during
the application (in km), AXS is the cross sectional area influenced by the
propeller wash (in m2), FA is the flow of nutrients into the lake during
application (in L/min), is the specific gravity of the nutrient solution (about
1.4, dimensionless), VB is the velocity of the boat (in km/hr), and 60 is a factor
that converts hours to minutes.
The concentration of the nutrient elements (N and P) would be proportionately
less, according to which source nutrient was used. The 28-0-0 contains 28% N
as a combination of ammonia, nitrate and urea. The 10-34-0 contains 10%
nitrogen and 34% phosphorus as P2O5, which translates to about 14.6%
elemental P.

Figure 2. Application vessel for the Adams Lake Enrichment Project, 1997.

142

Results and Discussion


Toxicity Tests
The test results indicate that the N-rich 20-0-0 is about twice as toxic to trout as
the P-rich 10-34-0 (Table 1).
Table 1. Toxicity levels of fertilizers used in Lake Enrichment Program
Chemical
28-0-0
10-34-0
10-34-0
10-34-0

Test
Organism
Trout.
Trout.
Daphnia
Selenastrum

Type of
Test
LC50
LC50
LC50
IC50

Length
of Test
96 hrs
96 hrs
48 hrs
72 hrs

Effective
Concentration
585.1 mg/L
1341.6 mg/L
1229.4 mg/L
1745.0 mg/L

95%
C.I.
(mg/L)
454-745
1000-1800
1000-1800
1674-1818

Chemical Composition of Test Solutions


Heavy metals were all below detection limits (0.03 mg/L or 0.005 mg/L,
depending on the element) for all test solutions (including the toxic heavy
metals: aluminum, arsenic, cadmium, chromium, cobalt, copper, iron, lead,
manganese, tin and zinc). The total nitrogen concentration of the lethal
solutions is very similar for rainbow trout and Daphnia at approximately 130150 mg/L (Table 2). This indicates that it is probably nitrogen, in some form,
that causes the lethality. The nitrogen content of the 28-0-0 is approximately 1/3
ammonia and 1/3 nitrate. The other major component is urea, which was not
analysed from these samples. It is possible that the urea degraded into ammonia,
which is known to be toxic (Sigma, 1983), over the duration of the test.

143

Table 2. Concentrations of nitrogen compounds in the test fertilizers at the lethal


concentrations.
Organism
Fertilizer
Test Solution
Concentration
Ammonia
Nitrite
Nitrate
Total N

Rainbow
Trout
28-0-0
585 mg/L

Rainbow
Trout
10-34-0
1341 mg/L

Daphnia

Selenastrum

10-34-0
1229 mg/L

10-34-0
1745 mg/L

42 mg/L
<0.002 mg/L
42.2 mg/L
130 mg/L

115 mg/L
<0.003 mg/L
8.3 mg/L
149 mg/L

112 mg/L
<0.003 mg/L
11.6 mg/L
139 mg/L

160 mg/L
<0.003 mg/L
<0.2 mg/L
209 mg/L

In-Lake Dilution
At the Adams Lake Enrichment Project, the boat travels approximately 20 km in
the application zone, discharging 7000 kg of nutrient solution. The boat travels
at 10 km/hr and discharges the nutrient solution at about 42 L/min. I estimate
that the propeller wash of the vessel shown in Figure 2, fully loaded with
nutrient solution, causes a turbulent zone approximately 4 m wide by 3 m deep.
The smaller vessel used on the Great Central Lake Enrichment Project causes a
smaller turbulent zone (~3m wide by 2 m deep), but it discharges nutrients at
less than half the rate of the Adams Lake project.
Inserting the Adams Lake conditions into the formulae shows that the nutrient
solution is diluted to about 29 mg/L within the propeller wash. This value is
about 5% of the LC50 value for the most toxic of the nutrient solutions on the
most sensitive species. However, this is the highest concentration of the nutrient
entering the lake water. The nutrient solutions are extremely soluble and
continue to disperse and dilute so rapidly that water sampling a day or two later
cannot find a gradient in nutrient concentrations in a lake being enriched.
Conclusions
The inorganic fertilizers that are used to enrich lakes can be toxic to aquatic life
in high concentrations. However, such high concentrations are not likely to be
encountered during standard techniques of adding nutrients to lakes because of
the turbulence of the immediate application zone and the rapid dilution with
surrounding lake water.

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References
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