Acute Nitrite Toxicity and Methemoglobinemia in Milkfish

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Aquaculture, 61 (1987) 33-40 33 
Eisevier Science Publishers B.V., Amsterdam - Printed in The Netherlands 
Acute Nitrite Toxicity and Methemoglobinemia in 
Juvenile Milkfish (Chanos chanos Forsskal) 
J.M.E. ALMENDRAS 
Southeast Asian Fzkheries development Center, Tigbaum, Itoilo (The Philippines) 
SEAFDEC Contribution No.209 
(Accepted 30 July 1986 ) 
ABSTRACT 
Almendras, J.M.E., 1987. Acute nitrite toxicity and methemoglobinemia in juvenile milkfish 
(Chanos chanos Forsskal). Aquaculture, 61: 33-40. 
Nitrite was about 55 times more toxic to milkfish juveniles in fresh water than in 16%0 brackish 
water: the 48-h median lethal concentrations were 12 mg NO,-N/l (95% confidence limit = 7.4-19.6) 
and 675 mg NO,-N/l (35% confidence limit = 435.81,045.4) respectively. Methemoglobin levels 
were higher for a given eon~ntration of nitrite in milkfish kept in fresh water than in the brackish 
water. Methemoglobin decreased to a normal level within 24-26 hours of the removal of nitrite. 
INTRODUCTION 
Nitrite is an intermediate product in the biological oxidation of ammonia to 
nitrate. In intensive culture systems, harmful concentrations of nitrite can 
accumulate. The toxicity of nitrite is partly due to its ability to oxidize hemo- 
globin to methemoglobin with the consequent loss of respiratory efficiency, 
although another as yet unknown toxic action may be the cause of death (Smith 
and Williams, 1974; Crawford and Allen, 1977). 
Numerous studies have shown the protective effect of chloride on nitrite- 
induced mortality and methemoglobinemia in salmonids (Crawford and Allen, 
1977; Perrone and Meade, 1977; Russo and Thurston, 1977; Meade and Per- 
rone, 1980; Russo et al., 1981; Eddy et al., 1983) and in the channel catfish, 
Ictulurus punetutus ( Tomasso et al., 1979,198O; Bowser et al., 1983 ) . Calcium 
has been reported to reduce nitrite-induced mortality but not methemoglobi- 
nemia in chinook salmon, Oncorhynchus tshawytscha (Crawford and Allen, 
1971) . However, calcium had little effect on the tolerance to nitrite toxicity in 
channel catfish (Tomasso et al., 1980) in contrast with its large effect reported 
in steelhead trout ( W~emeyer and Yasutake, 1978). The bic~bonate ion from 
0 1987 Elsevier Science Publishers B.V. 
34 
TABLE 1 
Summary of parameters recorded during the toxicity experiments 
Salinity PH 
(so) range 
Temperature 
(“Cl 
Calcium (m&i) 
Average +- S.D 
Chloride f mg/l ) 
Average + SD. 
0 7.9-8.3 27.4-27.7 49.28 + 13.65 26.79 + 7.35 
16 8.0-8.5 27.4-27.8 252.80 + 38.12 8884.16 I!I 303.54 
sodium bicarbonate was less effective than chloride in preventing methemo- 
globinemia in channel catfish (Bowser et al., 1983). 
The present study was conducted to investigate the relative toxicity and the 
levels of methemoglobin in fresh-water and brackish-water-adapted milkfish 
juveniles after a 48-h exposure to nitrite. 
METHODOLOGY 
Milkfish juveniles ( 31 -t- 9.5 g) reared in brackish-water ponds were accli- 
mated to fresh water and brackish water 5 weeks prior to the experiments in 
separate l-ton fiberglass tanks. The freshwater acclimation tank was supplied 
with well water at a flow rate of 288 l/h. The brackish-water acclimation tank 
was supplied with fresh water at 240 l/h and sea water at 200-290 l/h. Both 
fresh and sea water were allowed to mix in a 10-l container before overflowing 
into the tank. The salinity of the resulting brackish water was 13-18%0. Fish 
were fed a commercial diet (40% protein) ad libitum every 24 h. Feeding was 
discontinued 48 h before stocking the fish into the experimental tanks. 
For the toxicity test, standard static bioassay procedures were followed 
(American Public Health Association et al., 1971). Sixteen 60-l tanks were 
filled with 40 1 aerated fresh water or 16%0 brackish water and stocked with 6 
.fish each, 24 h prior to the addition of the toxicant. The water in each tank 
was renewed at the start of the toxicity test. Twelve fish were exposed to each 
of the geometrically increasing doses (progression factor = 0.5) of nitrite added 
as reagent grade NaNQ,,. The water quality in each of the tanks was measured 
after the 48-h bioassay (Table 1) . After the experiment, measured nitrite lev- 
els were never 10% higher or lower than the theoretical levels. 
Mortalities were monitored every 12 h and totalled after 48 h. Blood samples 
were taken by severing the caudal peduncle of 4-5 fish picked randomly from 
the survivors. Methemoglobin ( MHb) was determined spectrophotometri- 
tally and expressed as a percentage of the total hemoglobin (Dubowski, 1960). 
The 48-h median lethal concentration f LC50) and the 95% confidence limits 
were determined according to the method of Litchfield and Wilcoxon (1949). 
Test water nitrite concentrations were determined by the method of Strick- 
0%. 48-h LCw= 12(7.~-19.6)mg/lNO2-N 
16%. C8-h LC50= 675(435.8- lOZ5.4)mg/t NO2-N 
3.5 
1.75 3.5 7.0 1c 28 56 112 22.4 hf.8 896 1792 
Nitrite Concentration (mg/l NO2-Nl 
Fig. 1. Mortality in juvenile milkfish exposed to various levels of nitrite for 48 h in fresh water and 
16%0 brackish water. 
36 
I 
0-t 
0 O.&O 0. k t.;s 25 710 1; 
Nitnte Concentration bqtt NC&-N i 
Fig. 2. Levels of mathem~~i~bi~ in juvenile milk&h after a 48-h exposure to different levels of 
nitrite in fresh water. 
fn freshwater-abated rn~~k~sh, the MHb level increased si~i~~~tly at 
0.875 mg NO,-N/l, and a slight but insignificant increase was also observed at 
0.440 mg N&-N/l (Fig. 2 1. A small but highly significant increase in the %M~ 
was found in brackish-water-adapted fish exposed to 14 mg NO,-N/I which 
was the lowest concentration tested (Fig. 3 ) . 
~orn~ari~g the MHb levels and the relative nitrite toxicity in both media, it 
can be seen that a certain level of MHb does not correspond to a definite level 
of rno~~ity~ For example, in brackish water with 448 mg NO,-N/l, 75.72% 
MHb was observed with only 33.33% mortality while in fresh water with 14 
mg NO,-N/l, 75.65% MHb was observed with 5~.33% mortality. 
A change in behaviour in the fish occurred about 6 h a&r the ad~tion of 
nitrite. They were observed to remain motionless near the tank bottom. 
In the recovery experiment, a 12-h exposure to 14 mg NC&-N/l in Trash water 
elicited a level of 68.73% MHb (Fig. 4 1. However, after 24 h in a toxicant-free 
medium, MHb values were lowered to a near-Norman level of 9.17. 
37 
0 14 28 56 112 224 448 696 
Nitrite Conc~ntra~~~ (mgll NOz-N) 
Fig. 3. Levels of methemoglobin in juvenile milkfish after a 48-h exposure to different levels of 
nitrite in 16% brackish water. 
DISCUSSION 
The results indicate that nitrite is about 55 times more toxic to milkfish 
juveniles in fresh water than in 16%0 brackish water. The chloride ion has been 
found by many authors to be primarily responsible for inhibiting nitrite tox- 
icity (Crawford and Allen, 1977; Perrone and Meade, 1977; Wedemeyer and 
Yasutake, 1978; Tomasso et al., 1979,198O). It has been hypothesized that the 
chloride ion protects fish by direct competition with the nitrite ion for trans- 
port across the gill membrane (Perrone and Meade, 1977). Plasma nitrite con- 
centrations in coho salmon have been shown to be significantly lower in fish 
kept in a medium with high chloride concentrations than in fish in a low chlo- 
ride concentration (Meade and Perrone, 1980). 
The presence of calcium ions in the medium has an ad~tiou~l protective 
effect against the nitrite toxicity. In coho salmon, nitrite-induced mortality 
but not methemoglobinemia is reduced when the calcium level is high (Craw- 
Hours 
Fig. 4. Levels of methemoglobin in juvenile milkfish after being exposed to 14 mg/l nitrite for 12 
h in fresh water and then being allowed to recover in a toxicant-free medium. 
fordand Allen, 1977). Although calcium is not as effective as chloride in pre- 
venting nitrite-induced toxicity and methemoglobinemia, its mode of protection 
could be due to its ability to reduce diffusion by altering membrane permea- 
bility (Potts and Fleming, 1970). 
The 48-h LC50 of nitrite for milkfish juveniles kept in 16%0 brackish-water 
is about 675 mg NO,-N/l, This value is inor~na~ly high compared with the 
average pond nitrite level of 0.04 mg NO,-N/l (Baticados, personal commu- 
nication, 1986)) thus ruling out the possible role of nitrite in mass milkfish 
kills in brackish-water ponds. However, the same cannot be said for milkfish 
in fresh water. This study suggests that a Cl- /NO, ratio of around 60 should 
prevent a significant increase in MHb after a 48-h exposure to nitrite, whereas 
a ratio lower than 15 would cause about 10% mortality. 
Although the oxidation of hemoglobin to MHb is a contributory factor in 
fish mortalities, it is not the primary mechanism of nitrite toxicity (Smith and 
Williams, 1974). In this study there was no correlation between mortality and 
%MHb. The mortality of milkfish with the same level of MHb f approx. 75%) 
was about twice as great in fresh water as in 16%0 brackish water. 
In the recovery experiment, MHb levels returned to normal 24 h after the 
39 
removal of nitrite. A similar pattern of recovery was shown by channel catfish 
(Ictuturus punctutus) exposed to 5 mg/l nitrite for 6 h and then transferred to 
nitrite-free water (Huey et al., 1980). Moreover, younger fish have a greater 
tolerance to nitrite than their adult counterparts, perhaps because of a more 
efficient MHb reductase system (Kiese, 1974; Perrone and Meade, 1977). 
ACKNOWLEDGEMENTS 
I am grateful to IDRC, Canada, for funding, to the SEAFDEC TRS Physi- 
ology Laboratory staff for technical assistance and to an anonymous reviewer 
for valuable comments and suggestions. 
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