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Nitrate and Nitrite Toxicity to Salmonoid

Fishes
a
Deborah T. Westin
a
Marine Experiment Station University of Rhode Island , Kingston, Rhode
Island, 02881, USA
Published online: 09 Jan 2011.

To cite this article: Deborah T. Westin (1974) Nitrate and Nitrite Toxicity to Salmonoid Fishes, The Progressive
Fish-Culturist, 36:2, 86-89, DOI: 10.1577/1548-8659(1974)36[86:NANTTS]2.0.CO;2

To link to this article: http://dx.doi.org/10.1577/1548-8659(1974)36[86:NANTTS]2.0.CO;2

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 NITRATE AND NITRITE
TO SALMONOID
DEBORAH
Marine Experiment Station
University of Rhode Island, Kingston, Rhode Island 02881
ANALYSIS OF THE WATER IN 1,000-gallon recir-
culating biological filters at the University of
Rhode Island, Marine Experiment Station,
Jerusalem, R.I., revealed nitrate levels from
200 to 300 ppm, nitrite levels up to 0.4 ppm,
Downloaded by [UQ Library] at 02:04 16 November 2014
and ammonia levels varying from less than
0.1 to 1.0 ppm, indicating successful nitrifica-
tion but little or no denitrification activity with-
in these filters. These recirculation systems
with filters were supporting up to 100 pounds
of chinook salmon (Oncorhynchus tshawtscha)
at the time these measurements were made.
Fingerlings were obtained from eggs from Dr.
Lauren Donaldson's stock at the University of
Washington. As conventional flows through
fish hatcheries do not have nitrate or nitrite
build-ups other than those in their source
waters, the possible toxicity of these simple
nitrogen compounds to fish has received little
attention. Therefore, a' study was undertaken
to establish median tolerance limits for selected
salmonoids to nitrate and nitrite.
BACKGROUND
Wide ranges of nitrate levels have been re-
ported. These include 10.6 ppm in a Cortland,
New York, 47øF spring [14], 112 ppm in a
closed-circulating seawater aquariura in Japan
[10], and 1,370 ppm in a British aquarium [8]
that supported healthy fish. Experimentally
determined nitrate toxicity to fish species ap-
pears to span a much wider range. In 1917,
Powers [9], reporting on the relative toxicity
of five chloride and nitrate salts to goldfish,
found that the nitrates were less toxic in three
of these cases. In 1939, Jones [2] made a study
of the toxicity of nitrates of six alkali earth
and six earth metals to the stickleback (Gas-
86
TOXICITY
FISHES
T. WESTIN
terosteus aculeatus). These sticklebacks sur-
vived concentrations of 500 mg/1 sodium ni-
trate and 800 mg/1 calcium nitrate for 10 days.
In 1954, Trama [13] made a study of the toxi-
city of chloride, nitrate, and sulfate salts of
sodium, calcium, and potassium to bluegills.
Based on the experiments with the sodium salts,
Trama [13] found the relative toxicity of the
anions to be chloride less than nitrate, which
was less than sulfate. He reported the 96-hour
median tolerance limit (TLm) for bluegills at
20øC to be 12,000 mg/1 sodium nitrate and
3,000 mg/1 potassium nitrate.
Klinger [3], reporting on the toxicity of so-
dium nitrite to minnows (Phoxinus laevia),
observed that 50 mg/1 could be fatal in 14 days.
He found that 10 mg/1 was the threshold con-
centration above which toxic effects ensue, and
that 0.03 mg/1 was the minimum detectable
level for these minnows. McCoy [6] reported
on tests of NO.,-N levels found in lakes and
stream systems to 13 species of fish. She found
great differences in susceptibility of the fishes
to NO.,-N levels. Logperch (Percina caprodes)
were the most sensitive of those species tested,
dying in less than 3 hours at 5 ppm NOo.-N
(16.5 ppm NO.,). By contrast, carp (Cyprinus
carpio) and black bullheads (Ictalurus melas)
survived 40 ppm NO•-N (132 ppm NO2) be-
yond the 48 hours of the test exposure.
MATERIALS AND METHODS
Bioassay methods were followed as outlined
by APHA [1] using reconstituted deionized
water [4] for the freshwater experiments and
Instant Ocean (Aquarium Systems, Inc., East-
lake, Ohio) in deionized water for the 15ø/oo
experiments. The source of nitrate and nitrite
THE PROGRESSIVE FISH-CULTURIST
 toxicants was sodium nitrate and sodium nitrite
added to obtain calculated concentrations on the
basis of milligrams per liter (ppm) of nitrate
and nitrite per test container of 10 fish. Each
container was supplied to saturation with com-
pressed air through an airstone. Experimental
fish were taken from chinook fingerlings at the
Marine Experiment Station and rainbow trout
(Salmo gairdneri) fingerlings were supplied by
the Rhode Island State Trout Hatchery at Per-
ryville, where exposure to nitrate was measured
as 6 ppm or less.
The 96-hour and 7- and 10-day median toler-
ance limit (TLm) concentrations and their 95
percent confidence limits for all experiments
were calculated from the data collected using
the procedure outlined by Litchfield and Wil-
coxon [5]. A pairwise comparison of the TLm
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use was restricted to freshwater due to the
chloride interference with increasing salinity.
RESULTS AND DISCUSSION
The table summarizes the results and in-
cludes the ranges of weight and length for the
fish used in bioassays as well as the calculated
96-hour and 7- and 10-day median tolerance
limits and their 95 percent confidencelimits
[5].
Relative potency calculations revealed that
for both the chinooksand rainbow trout, ni-
trate at 15ø/oo was statistically (p = 0.05) more
active than in freshwater. This relative activity
of nitrate after a 96-hour exposureperiod for
the chinook salmon was between 1.24 and 1.38
times more toxic at 15ø/oo than in freshwater.
For the rainbow trout the relative activity at

15ø/oo was from 1.14 to 1.41 times that in

values showed that all lines fitted
ate significantly from parallelism and, there-
fore, the relative potency and its 95 percent
confidence limits were calculated.
Controls were run concurrently
toxicity experiment. Control fish showed no
stress, even when ammonia levels rose to 5.0
ppm. This concentration was not reached in
the nitrate and nitrite test solutions since they
were changed at the end of 96 hours. Monitor-
ing revealed that the highest nitrate concentra-
tion reachedin any of the control containers
was 5.58 ppm for the trout in freshwater. The
highest nitrite of any control was 0.0082 ppm
and the highest in the non-nitrite tests was
0.1013 ppm NO.• in the 4,200 NO• solution.
The nitrate and ammonia
made during this study were with the specific-
ion electrodes (Orion Research, Inc., Cam-
bridge, Mass.). The nitrate determinations
were based on the method outlined by Strick-
land and Parsons [12]. The nitrate electrode
did not devi.-
freshwater. Figure I showsthe effect of vary-
ing salinity on the rainbow trout after exposure
to nitrate concentrations for 7 days, when the
activity of the nitrate was 1.16 (1.01, 1.33)
with each times more toxic at 15ø/oo than in freshwater.
The effect of exposure time was also shown to
increase mortality among both chinooks and
trout in both the freshwater and the 15ø/oo ni-
trate tests and of the chinooks in the nitrite
tests. Figure 2 shows the •ffect of lengthened
exposure time on the mortality of chinook fin-
gerlings in freshwater to nitrite concentrations.
The relative activity of nitrite to chinook fin-
gerlings in freshwater was 2,000 (1,480, 2,700)
times that of nitrate in freshwater. Similar
analysis of Trama's [13] bluegill experiments
determinations (fish 1-9g) yielded a 96-hour TLm of 8,800
(8,446, 9,064) ppm nitrate. Using this calcu-
lation, the relative activity of nitrate to the
chinooks tested was determined to be about one
and one-half times more than that to Trama's
bluegills in slightly warmer water.

Summary o! bioassayresults
Size ranges Median tolerance limits (and 95 percent confidence
Average limits) ppm artion
Speciesand ,Temperature Weight Length number of
toxicant x (øC) range (g) (mm/FL) fish/conc. 2 96-hour 7-day
Nitrate:
Chinook (fw) ................ 14 -16.8 0.978- 9.66 52-95 21 18 5,800 (5,087, 6,612) 4,800 (4,528, 5,088)
Chinook (15ø/oo) ........13 -14 2.733- 9.89 67-100 23 23 4,400 (4,151, 4,664) 4,000 (3,810, 4,160)
Trout (fw) ...................... 13 -14.6 1.154- 5.15 52-80 20 24 6,000 (5,768, 6,240) 4,700 (4,433, 4,982)
Trout (15ø/oo) .............. 13 -14.4 2.00-21.0 76-115 16 12 4,650 (4,402, 4,901) 4,000 (3,448, 4,640)
Nitrite: 10-day
Chinook (fw) ................ 13.6-15.6 1.50-10.55 51-103 23 17 2.9 (2.0, 4.2) 2.4 (1.87, 3.07)

x fw=freshwater and toxicant; 15ø/oo=15ø/oo salinity and toxicant.

2 For 96-hour, 7-day or 10-day calculations.

VOL. 36, NO. 2, APRIL 1974 87

 ioo

IOO fresh water

e=15%o salinity

,•,

% 50-- ß . o 2 50
o

/- / 0:96 hours
/ / .ß:7days

_.//•o
I •I •,
--

I0-- IO
ß

OO I I
0 2500 4000 5000 6000 7000 o 1.0 3.0 5.0 IO

ppm NO3 ppmNO2

Figure 1.-•The effect of varying salinity on the Figure 2.--The effect of exposure to nitrite on
Downloaded by [UQ Library] at 02:04 16 November 2014

mortality of rainbow trout fingerlings after 7 the mortality of chinook salmonfingerllngsin

days exposure to nitrate. freshwater.

All of the trout subjected to nitrate concen-
trations showed acute signs after 2 days ex-
posure, while these signs were not observed in
the lower nitrate (i.e. 4,400 and less) concen-
trations of the chinook experiments until after
5 to 8 days. These signs included an inability
to swim upright, labored respiration, and
little movement altered
ming. Initial reactions were generally the
same for all fish and agreed with the observa-
tions recorded by Klingler [3]. The fish swam
about in the containers yawning, or gulping,
and exhibiting accelerated opercular move-
ments. Some broke the
"nose" as if trying to escape,while others swam
near the surface but did not break
control fish, once accustomed to the containers,
settled quietly and remained near the bottom
unless disturbed. The fish exposed to nitrite
were generally more sluggish in their behavior
than those exposed to nitrate. Death of fish
exposedto either of these toxicants usually fol-
lowed a period of lying at the bottom of the
container with opercular movements the only
sign of life. Some fish were observed to con-
vulse, but the majority died quietly. Many of
these behavior patterns were described by
Shepard [11] as indicative of oxygen depriva-
tion, although, the dissolved oxygen was at
least 7 ppm in each container.
Many of the fish showed changes in body
color from the normal silver-grey of chinooks
and the irridescence of the trout to almost
black. Klingler [3], however, stated that no
changein color occurred at any stage of nitrite
exposure to his minnows.
Some of the chinooks and trout exposed to
with erratic swim- nitrate were observedto have pale pink to dark
red-brown gill filaments, while many of the
chinooks exposed to nitrite had chocolate
colored gills. There were no abnormalities
present within the tissues examined histopath-
ologically from the fish exposedto any nitrate,
surface with their nitrite, or ammonia concentrations.
it. All of the
RECOMMENDATIONS
With the knowledge gained from these ex-
periments and observations of the fish in our
rearing facility mentionedearlier, it is possible
to estimate an application factor that will allow
prediction of a "safe" level of nitrate and ni-
trite for salmonolds. Since most rearing for
growth and/or release to the sea of these fish
involves increasing the salinity, perhaps a fair
application factor for nitrate concentrations
allowable in fish aquacultural practices would
be 1/10 of the 7-day lethal concentration to 10
percent (LC10) at 15%o. The choice of LC10

88 THE PROGRESSIVE FISH-CULTURIST

 is based mainly on the assumption that 10 per-
cent mortality in any hatchery operation is un-
satisfactory. This then would give a maximum
allowable concentration for chinooks of
ppm NO3 and 250 ppm for trout. But best re-
sults of growth and good health, based on ob-
servations of chinooks in recirculating
systems with varying nitrate concentrations
and fish health, would not exceed 1/100 of this
LC10 concentration, or 25 to 35 ppm. This is
10 to 20 ppm less than the recommended limit
for farmstead uses as reported by the National
Technical Advisory Committee [7]. Following
this reasoning, a maximum allowable level for
nitrate in freshwater (1/10 of 10-day LC10)
would be 0.12 ppm NO.,. Since nitrite is known
to be much more toxic to most animals, the
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1/100 factor (0.012 ppm NO._,), which is less
than Klinger's 0.03 ppm detectable level, is
probably safer still. Once the mode of action
of nitrate as a toxicant can be defined
clearly, probably through measurements of
blood nitrate and nitrite levels, and the sub-
lethal effects of nitrate and nitrite to fish can be
established, these application factors will be-
come more solidly defined.
ACKNOWLEDGMENTS
For support of this work, which was toward
a Master's degree, the author extends apprecia-
tion in part to Sea Grant and in part to the
State of Rhode Island. For the supply of the
salmonolds tested, thanks is extended to Dr.
Lauren Donaldson of the University of Wash-
ington and to the Rhode Island State Trout
Hatchery at Perryville. The author wishes to
thank Dr. R. E. Wolke, Sea Grant Pathology
Laboratory at the University of Rhode Island,
for his gross and histopathological observations
of the fish. The author also wishes to extend
her thanks to the staff of the Marine Experi-
ment Station for their help and encouragement.
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1965. Standard methods for examination
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1939. The relation between the electrolyte solu-
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89