This article was downloaded by: [Florida State University]

On: 02 January 2015, At: 06:06

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office:
Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

The Progressive Fish-Culturist

Publication details, including instructions for authors and subscription
information:
http://www.tandfonline.com/loi/uzpf20

Effect of Nitrite on Methemoglobin and Total

Hemoglobin of Juvenile Rainbow Trout
a a
D. A. Brown & D. J. McLeay
a
British Columbia Research Council , 3650 Wesbrook Crescent, Vancouver 167,
Canada
Published online: 09 Jan 2011.

To cite this article: D. A. Brown & D. J. McLeay (1975) Effect of Nitrite on Methemoglobin and
Total Hemoglobin of Juvenile Rainbow Trout, The Progressive Fish-Culturist, 37:1, 36-38, DOI:
10.1577/1548-8659(1975)37[36:EONOMA]2.0.CO;2

To link to this article: http://dx.doi.org/10.1577/1548-8659(1975)37[36:EONOMA]2.0.CO;2

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”)
contained in the publications on our platform. However, Taylor & Francis, our agents, and our
licensors make no representations or warranties whatsoever as to the accuracy, completeness, or
suitability for any purpose of the Content. Any opinions and views expressed in this publication are
the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis.
The accuracy of the Content should not be relied upon and should be independently verified with
primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever
caused arising directly or indirectly in connection with, in relation to or arising out of the use of the
Content.

This article may be used for research, teaching, and private study purposes. Any substantial
or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or
distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can
be found at http://www.tandfonline.com/page/terms-and-conditions
 EFFECT OF NITRITE ON METHEMOGLOBIN
AND TOTAL HEMOGLOBIN OF JUVENILE
RAINBOW TROUT
D. A. BROWN and D. J. MCLEAY

British Columbia Research Council

3650 Wesbrook Crescent, Vancouver 167, Canada
Downloaded by [Florida State University] at 06:06 02 January 2015

NITRITE TOXICITY IS A PROBLEM in recycling were measured by a modification of the

fish hatchery water and efforts are being method of Evelyn and Malloy [5]. Turbidity
made to remove nitrite by biological oxidation was a problem in the blood-buffer solution and
[2,3,6]. This study measured the acute toxicity was removed by centrifugation at 2,000 rev-
of nitrite to rainbow trout (Salmo gairdneri) olutions per minute for 5 minutes. In order to
and the sublethal effects of nitrite upon their avoid the increase in methemoglobin that oc-
blood methemoglobin and hemoglobin levels. curs in standing blood-buffer solutions, all de-
terminations were completed within 5 min-
utes of removing the blood from the fish.
MATERIALS AND METHODS
RESULTS
Post-alevin rainbow trout were obtained
from a commercial trout farm and aeelimated Results in table 1 indicate that the test sys-
to water of pH 6.4-6.7, conductance 15-30 mi- tem effectively maintained consistent NO2-N
eromhos per centimeter, EDTA hardness 3-9 concentrations in the test compartments. The
milligrams of ealsium carbonate per liter, al- 96-hour LC50 value was 230 micrograms of
kalinity 2-8 mg CaCO3/liter and temperature nitrite nitrogen per liter.
12ø -+ 2øC. The fish used during the study were
9.3 -+ 1.1 centimeters in length and 9.1 _+ 3.0 Table 1.--Concentrations of NO2-N in
grams in weight. experimental streams initially and after
Groups of 10 fish were placed in compart- 96-hour exposureor at time of death of last
ments 190 by 12 by 15 centimeters containing fish
concentrations of sodium nitrite from 0 to 10
milligrams of nitrite nitrogen per liter. The Measured concentration •g NO2-N/liter
Treatment
solutions were recycled through the test com- •g NO2-N/liter 0 hour 96 hours or time of death
partments at a rate of 10 liters per minute. of last fish
Fresh solutions of nitrite were added continu-
0 (control) 1 7
ously to each test compartment at the rate of 2 15 17 16
liters of solution per gram of fish each day. The 25 26 25
concentration of nitrite was monitored 50 53 49
75 72 75
throughout the 96-hour exposure period using 100 104 105
the technique of Wood et al. [8]. The 96-hour 200 209 201
LC50 value of NO2-N in this system was calcu- 300 315 307
lated according to standard methods [•]. 500 506 505

Total hemoglobin and methemoglobin levels 1,000 1,010 1,010

10,000 10,040 10,200
in the blood of fish surviving 96-hour exposure
36 THE PROGRESSIVE FISH-CULTURIST
 Table 2.--Methemoglobin, total hemoglobin,and percentage hemoglobinafter 96-hour exposure to
varying nitrite concentrations

Total Percentage
Nitrite concentration No. of Methemoglobin hemoglobin methemoglobin
(/•g NOs-N/liter) fish (g/100ml) (g/100ml) of total hemoglobin
0 (control) 10 0.10 +- 0.10a 6.84 _+0.76 0.9 -+ 0.8
15 10 0.27 +- 0.17' 6.32 _+ 0.93 4.2 +_ 2.5*
25 10 0.22 _+ 0.18' 6.71 _+ 1.16 3.2 -+ 2.8*
50 10 1.01 +- 0.79* 6.52 _+ 0.74 16.0 _+ 13.4'
75 10 0.87 -+ 1.03' 5.95 -+ 1.54 18.0 _+ 24.4*
100 10 1.20 +_ 1.25' 5.73 _+ 0.86* 20.9 +- 21.7'
200 6 3.96 +- 0.59* 4.80 _+ 0.54* 82.3 _+ 6.2*
300 3 3.87 _+ 1.28' 4.86 _+ 1.01' 78.5 _+ 9.6*
amean ñ SD. *P < 0.05 Student's t-test.

Points represent mean values

Total hemoglobin and methemoglobin levels Bars represent 95% confidence interval
8-
for the fish which survived the 96-hour expo-
Downloaded by [Florida State University] at 06:06 02 January 2015

sure are presented in the figure. Total hemo- E

0 •
globin was unchanged at concentrations up to
100 /zg NO2-N/1; at this concentration there •6 I
was a significant reduction to 5.73 grams per
100 milliliters of blood from the control value
•O•
•4-
/ T•{:øta' hemøglø
•l ß
of 6.84 g/100 ml blood. At a concentration of 300 • •0 Methemoglobin
/zg NO2-N/1, total hemoglobin had fallen to 4.86
g/100 ml blood (table 2). Unlike total hemoglo-
bin, methemoglobin was changed significantly
at the lowest nitrite concentration, increasing o.,,c•c• I I I I I I
from 0.10 g/100 ml blood for control fish to 0.27 0 50 I00 150 200 250 :500
CONCENTRATION,
nitrite ion )JCjN/liter
g/100 ml blood for 15 /zg NO:-N/1. At higher
nitrite concentrations methemoglobin con- Effect of nitrite concentration .on total hemoglo-
tinued to increase, reaching a maximum of 3.9 bin and methemoglobin in blood of juvenile
g/100 ml blood for 200 and 300 /zg NO•-N/1 rainbow trout.
(table 2).
There was a small increase in the percent-
age of methemoglobin of total hemoglobin Sublethal effects must also be considered for
from the control value of 0.9 percent to about 4 effective hatchery operation. Both reduction
percent in fish exposed to nitrite concentra- of total hemoglobin and conversion of hemo-
tions of 15 to 25/zg NO:-N/1. Increases to about globin to methemoglobin in the fish blood will
20 percent occurred in fish exposed to 50 to 100 reduce oxygen transport capability inducing a
/zg NO2-N/1. The percentage ofmethemoglobin stress that may have detrimental effects on
in the 200 and 300/zg NO2-N/1 concentrations exposed fish. Mayo et al. [6] found severe
increased to 80 percent (table 2). pathological changes and an increase in bac-
terial gill disease in fish exposed to high levels
of nitrite and ammonia.
DISCUSSION
The 4 percent methemoglobin of total
High mortalities of rainbow trout occurred hemoglobin found in fish exposed to 15 to 25/zg
in recycling fish hatchery water when NO:-N NO:-N/1 may have little effect on oxygen
reached 200 /xg/1, according to Birkbeck [œ]. transport capability. However the increase to
This observation agrees with our estimate of 20 percent methemoglobin resulting from ex-
230 /zg NO2-N/1 for the 96-hour LC50 of posure to 50 to 100/zg NO:-N/1 could well affect
nitrite-exposed rainbow trout. For rainbow fish surviva'l and requires further evaluation.
trout to survive, nitrite levels must be kept Smith and Williams [7] found that in fish ex-
lower than 200/zg NO:-N/1. posed for 48 hours to 150/zg NO:-N/1, no mor-
VOL. 37, NO. 1, JANUARY 1975 37
 talities occurred but 70 percent of the hemo- REFERENCES
globin had been converted to methemoglobin. 1. AMERICAN PUBLIC HEALTH ASSOCIATION.
Our results are similar, i.e. after a 96-hour 1971. Standard methods for the examination of water
exposure to 200 and 300 t•g NO2-N/1, 80 percent and wastewater, 13th edition. New York. 874 p.
of the fish blood hemoglobin was in the 2. BIRKBECK, A. E.
methemoglobin form, and some mortalities 1973. Recycling of fish hatchery wastewater. Pre-
sented at the 40th Annual Convention of the Pacific
did occur. Therefore, severe sublethal effects Northwest Pollution Control Association.
can be expected to occur at nitrite concentra- 3. BURROWS,R. E., and B. D. COMB.
tions somewhere between 100 and 200 t•g 1968. Controlled environments for salmon propaga-
NO2-N/1. tion. Progressive Fish Culturist, vol. 30, no. 3, p.
123-136.
These results are based on only a 4-day ex-
4. CAMERON,J. M.
posure period and' thus do not take into ac-
1971. Methemoglobin in erythrocytes of rainbow
count any long term adaptation of the fish to trout. Comparative Biochemistry and Physiology,
nitrite. Such an adaptation may occur if fish vol. 40A, p. 743-749.
have a methemoglobin reduetase system simi- 5. EVELYN, H. A., and H. T. MALLOY.
lar to mammals. However Cameron [4] indi- 1938. Microdetermination of oxyhemoglobin,
methemoglobin, and sulfhemoglobin in a single
Downloaded by [Florida State University] at 06:06 02 January 2015

cates that no such system has yet been de-

sample of blood. Journal of Biological Chemistry,
scribed in fish. In the absence of such an adap- vol. 126, p. 655-662.
tive mechanism, poor growth and condition in 6. MAYO, R. D., P. L. LIA0, and W. G. WILLIAMS.
hatehew-reared fish may occur even at ex- 1972. A study for development of fish hatchery water
tremely low nitrite levels after chronic periods treatment systems. Prepared for Walla Walla Dis-
trict Corps of Engineers in cooperation with U.S.
of exposure.
Bureau of Sport Fisheries and Wildlife. Kramer,
Chin and Mayo, Seattle. 142 p.
ACKNOWLEDGMENTS 7. SMITH, C. E., and W. G. WILLIAMS.
1974. Experimental nitrite toxicity in rainbow trout
The authors gratefully thank Mr. A. E. and chinook salmon. Transactions of the American

Birkbeck for presenting us with background Fisheries Society, vol. 103, no. 2, p. 389-390.
8. WOOD,E. D., F. A. J. ARMSTRONG,and F. D. RICHARDS.
information for this study and Drs. T. E. How-
1967. Determination of nitrate in sea water by
ard and C. C. Walden for reviewing the manu- cadmium-copper reduction to nitrite. Journal of
script. Marine Biology, vol. 47, p. 23-31.

38 THE PROGRESSIVE FISH-CULTURIST