Professional Documents
Culture Documents
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
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
ii
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
vi
vii
viii
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
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
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.
References
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13
14
15
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
0.73* 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
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
24
25
Williams, E.M., Eddy, F.B. 1986 Cloride Uptake In Freshwater Teleosts And Its
Relationship To Nitrite Uptake And Toxicity. J.Comp. Physiol.
B.156:867-872.
Willians, E.M., Glass, M.L., Heisler, N. 1993 Effects Of Nitrite-Induced
Methaemoglobinaemia On Oxygen Affinity Of Carp Blood. Enviorn.
Biol. Fishes.37:407-413.
Woo, N.Y.S. Chiu, S.F. 1997 Metabolic And Osmoregulatory Responses Of The
Sea Bass Lates Calcarifer To Nitrite Exposure. Environ. Toxicol. And
Water Qual., 12(3):257-264.
26
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
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
32
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)
(2)
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
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
41
42
44
45
46
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
51
52
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
12
8
4
0
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).
55
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
++
++
++++
++++
++
-
+++
+++
+++
+++
++
++++
++++
++++
+++
++
+
+/++
++
++
++
+++
+++
+++
++++
-
+++
++
+
++
++
++
+/-
++
+++
++
+++
+++
++
++++
+
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
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
60
61
62
63
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
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
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
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
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
200
400
600
800
1000
-1
plasma [ammonia] (mol l )
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
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
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
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
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).
120
Percentage Survival
100
80
***
60
***
40
20
0
control
T1
T2
Exposed concentrations
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
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
85
86
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
91
92
93
94
95
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
99
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
102
**
160
20
80
80
oo o
o o o
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
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)
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
107
108
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.
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
111
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
113
Cholestoral
300
Clarius
Idellus
250
200
150
100
50
0
Cotrol
96 hrs
15 days
30 days
Total Protein
10
8
6
4
2
0
Cotrol
96 hrs
15
days
Clarius
Idellus
30
days
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
1.0
0.8
0.6
0.4
0.2
0.0
Clarius
Idellus
Creatinine
Cotrol 96 hrs
15
days
30
days
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
Clarius
Potassium
12
Idellus
10
8
6
4
2
0
Cotrol
96 hrs
15 days
30 days
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
118
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
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
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
Inhibition (% of control)
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
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
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
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
131
132
133
134
Enzymatic activity
(mmol NAD+/min/g Hb
25000
Control
Cd
Meta
Deca
20000
15000
10000
5000
0
1
7
Time after administration (days)
135
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)
136
137
138
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.
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
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
143
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.
144
References
McLeay, D.J. and R.P. Scroggins. 2000. Biological test method: reference
method for determining acute lethality of effluents to rainbow trout.
Report EPS 1/RM/13 2nd Edition, Environment Canada 23 p.
McLeay, D.J. and J.B. Sprague. 1990. Biological test method: acute lethality test
using rainbow trout. Report EPS 1/RM/9, Environment Canada 51 p
McLeay, D.J. and J.B. Sprague. 1990. Biological test method: acute lethality test
using Daphnia magna. Report EPS 1/RM/11, Environment Canada 57
p
Miller, J.A., R.P. Scroggins and D.J. McLeay. 2000. Biological test method:
reference method for determining acute lethality of effluents to
Daphnia magna. Report EPS 1/RM/14 2nd Edition, Environment
Canada 21 p.
St. Laurent, D., G.L. Stephenson and K.E. Day. 1992. Biological test method:
reference method for determining acute lethality of effluents to rainbow
trout. Report EPS 1/RM/25, Environment Canada 41 p.
Sigma Resource Consultants. 1983. Summary of water quality criteria for
salmonid hatcheries. Dept Fisheries & Oceans report SECL 8067. 163
p.
Stockner, J.G. 1987. Lake fertilization: the enrichment cycle and lake sockeye
salmon production. Can Spec Pub Fish Aquat Sci 96: 198-215
145
146