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Jimnuil of Shellfish Research. Vol. 17, No. 3, 6X3-687. l'»8.
EFFECTS OF CHRONIC EXPOSURE OF GREENLIP ABALONE, HALIOTIS LAEVIGATA
DONOVAN, TO HIGH AMMONIA, NITRITE, AND LOW DISSOLVED OXYGEN
CONCENTRATIONS ON GILL AND KIDNEY STRUCTURE
JAMES O. HARRIS,' GREG B. MAGUIRE,' AND
JUDITH H. HANDLINGER'
'Department of Aiiiiaciilture
Uiiiversit}' of Tasmania
PO Box 1214
Launceston. Tasmania. Australia. 7250
'Department of Primary Industry and Fisheries
Fish Health Unit
PO Box 46
Kings Meadows. Tasmania. Australia. 7249
ABSTRACT After chronic, sublethal bioassays of juvenile greenlip abalone, Haliotis laevigata Donovan, to reagent ammonia, nitrite,
and low dissolved oxygen, tissue samples were dissected for histological analysis. Exposure to the highest ammonia treatment (0.188
mg of free ammonia-nitrogen ([FAN]L~')) resulted in little difference to the gills of these abalone, relative to the controls (0.006 mg
of FAN L"'), whereas at this concentration, the right kidney showed decreased tubule definition and enlarged tubule lumen. Exposure
to 7,8 mg of NO,-N L~', resulted in gill lamellar thickening and epithelial lifting along with aproliferation of mucous cells. The
proportion of kidney cell contents occupied by granules increased at this nitrite concentration. Associated with this change in kidney
structure was an increase in basally located eosinophilic cytoplasm. Gill mucous cells from abalone exposed to depressed dissolved
oxygen levels (55'7r oxygen saturation) exhibited more intense stainmg, mdicative of achange in mucous composition. Some necrosis
of gill epithelium was evident, either as a result of or in association with the occurrence of ciliates (Ancistrocomidae) between the gill
lamellae. Right kidney tissue did not exhibit any obvious changes in relation to exposure to low dissolved oxygen levels. Chronic
exposure to slight oxygen supersaturation {11 795-) caused no apparent effects on gill or kidney structure.
KEY WORDS: abalone, Haliotis laevigata, ammonia, nitnte, oxygen, histology
INTRODUCTION
The present expansion of abalone aquaculture (Hone and
Maguire 1996) brings with it the likelihood of encountering sub-
optimal water quality, especially where recirculating aquaculture
systems are used (Jirsa et al, 1997). The culture of animals in
nonoptimal environments may result in deaths, as a direct result of
one or more components of the environment or from infectious
diseases activated indirectly by suboptimal environments, or in
decreased productivity (Tomasso 1996).
The external environment can, if suboptimal, produce delete-
rious changes in aquatic animals. The overt signs of toxicity are
nearly alvsays preceded by biochemical, physiological, and/or
morphological changes in the organism (Meyers and Hendricks
1985). Often, the gills are among the organs most affected by
waterbome pollutants (Mallat 1985). because the respiratory sur-
face provides an extensive interface with the aquatic environment.
In many fish, the kidney often forins asite of histological changes
in response to toxicants (Russo 1985). Abalone are diotocardians,
possessing two kidneys of differing functions. The role of the right
kidney is believed to be in nitrogen excretion because of the pres-
ence of excretory vacuoles (Andrews 1985). Some resorption of
solutes, which is also the main function of the left kidney, is also
believed to occur, because of the presence of coated pits opening
between microvilli (Voltzow 1994), The role of the right kidney in
nitrogen excretion and protein turnover suggests apossible role
with the toxicants considered in this study.
The purpose of this study was to bring together histological
observations of juvenile greenlip abalone, Haliotis laevigata, from
three chronic, sublethal bioassay studies. Specifically, the effects
of sublethal exposure for 2-3 mo to high ammonia, nitrite, or low
dissolved oxygen on gill and right kidney histological structure
were investigated,
MATERIALS AND METHODS
Juvenile greenlip abalone were sampled after chronic sublethal
exposure for 58-82 days to ammonia (as NH4CI) (Harris et al.
1998). nitrite (as NaNO,) (Harris et al. 1997). or low dissolved
oxygen (Harris et al. in press). The experimental ranges were
0.006-0.188 mg, free ammonia-nitrogen (FAN), L"' (0.237-9.04
mg total ammonia-nitrogen L^'), 0.024—7.80 mg NO2-N L^' and
8.9^.2 mg of dissolved oxygen L"' (117-57% dissolved oxygen
saturation).
Five abalone were sampled from two of the triplicate bioassay
tanks for each treatment and bled, via an incision in the foot, for
2-3 min before being dissected to remove the posterior portion of
the viscera containing the gills and kidney. This tissue was fixed in
phosphate-buffered formalin at room temperature (15-18°C) and
then dehydrated through agraded ethanol series to xylene in a
Tissue-Tek II tissue processor. Dehydrated tissue samples were
embedded in paraffin resin on aShandon Histocentre 2and sec-
tioned on aMicrom MM 340 microtome at 4p.m. Routine Harris"
hemotoxylin and eosin staining was carried out on all tissues pro-
cessed with aShandon Linistain GLX automatic tissue stainer. All
sections were mounted in DPX and examined under alight mi-
croscope.
RESULTS
Exposure to the highest ammonia treatment (0.188 mg of FAN
L~') resulted in little difference to the gills of these abalone. rela-
683
684 Harris et al.
tive to the controls (0.006 mg of FAN L"') (Fig. I). At this treat-
ment level, however, the right kidney of all sampled abalone
showed less definition in the tubules, and the lumen of the tubules
appeared enlarged (Fig. 2). At 0.1 10 mg of FAN L"', 10% of the
sampled abalone showed both reduced right kidney definition and
an enlarged lumen, whereas 20Vf of abalone showed reduced right
kidney definition and 20% of abalone e.xhibited enlarged right
kidney lumen. Typical kidney structure for abalone not exposed to
elevated levels of ammonia or nitrite or to low dissolved oxygen is
shown in Figure 3.
From exposure to 7.8 mg of NO^-N L"', thickening of the
lamellae and epithelial lifting of gills were evident in all observed
abalone. along with aproliferation of mucous cells at the junction
of the lamellae and central gill axis (Fig. 4). Mucous cells, com-
mon at the distal tip of the lamellae, also extended further toward
the base of the lamellae than for other treatments and control
abalone. These mucous cells are evident both as complete and
apparently discharged cells with adhered mucous, often giving a
ragged appearance to the lamellae and contributuig to the poor
brush border definition observed here (Fig. 5). Abalone exposed to
concentrations less than 7.8 mg of NO^-N L^' showed typical gill
structure including the principal gill filament and lamellar junction
(Fig. 6) and lamellar tip (Fig. 7). At 4.15 mg of NO,-N L"', 20%
of sampled abalone showed thickened gill lamellae, 10% showed
lifting of gill epithelium, and 40% showed aproliferation of gill
mucous cells. At the highest nitrite concentration (7.8 mg of
NO,-N L"'), the overall height of the kidney tubule cells was
increased. This was due to an increase in both the amount of
pigment granules in the supranuclear region (toward the lumen
surface) and the amount of eosinophilic (protein rich) cytoplasm
located in the subnuclear region toward the base membrane (Fig. 8).
Gill mucous cells from abalone exposed to depressed dissolved
oxygen levels (55% oxygen saturation) exhibited more intense
staining, indicative of achange in the composition of mucous.
Necrosis of gill epithelium was evident in all sampled abalone at
this dissolved oxygen concentration, either as aresult of or in
association with the occurrence of ciliates (family Ancistrocomi-
dae; D. Lynn pers. comm.) between the gill lamellae (Fig. 9),
observed in 80% of sampled abalone at this concentration. At 63%
Figure 2. Right kidney of abalone exposed to (1. 18S m» of FAN L'.
Magnitlcation. x400. (A) Enlarged lumen of kidney tubule.
of oxygen saturation, necrosis of gill tissue was evident in 20% of
sampled abalone, whereas ciliates were observed in 40% of the
abalone. Right kidney tissue did not exhibit any obvious changes
in relation to exposure to low dissolved oxygen levels. One treat-
ment was maintained at 117% oxygen saturation, and no adverse
effects on gill or kidney structure were evident.
DISCUSSION
Some effects of ammonia on the histological structure of fish
have been documented (see Russo 1985), although data for inver-
tebrates are less common. FAN levels of 0.04-0.4 mg L~' have
been shown to induce inflammation and degeneration of gills and
kidneys for a variety of fish species (Russo and Thurston 1991).
The swollen, rounded secondary lamellae observed in rainbow
trout after long-term exposure to ammonia (Smart 1976) were not
observed in this study. However, among fish, the effects of am-
monia are varied, because not all authors found hyperplasia and/or
other degenerative changes to the gill structure. Sublethal ammo-
Figure 1. (iills of control ahalone Inim aiiiiiionia l)loassav. .Magnifi-
cation, xlUO. (A) principal lllanient: (lil gill lamella: (C) distal tip of
gill lamella.
I'iuiiic -V Ki^lil kiiliH'\ ol control al)aliiiK' lioni iiilrilc l)ioassav. Mag-
nillcalion, x4(M», (A) Kxterior perimeter of normal kidney tubule.
Ammonia, Nitritk. and Hypoxia Effects on Abalone 685
Figure 4. Distal tip of gill lamellae exposed to 7.80 nig of N(),-N L" .
Magnification, x4()0. lAl Lifting of epithelium: (B) proliferation of
mucous cells.
nia levels are also known to cause histological changes in the
kidneys of many fish (Colt and Armstrong 1981). The observed
differences in cell definition indicate that external ammonia had
some effect on kidney structure in greenlip abalone, even though
survival at this concentration was under 50% for aminimum of 58
days of exposure (Harris et al. 1998).
The long term effects of nitrite on histological structure have
not been well documented for aquatic invertebrates. Nitrite is
known to bioaccumulate in gill, liver, brain, and muscle tissue of
fish (Margiocco et al. 1983) and to increase susceptibility to dis-
eases (Hanson and Grizzle 1985). Michael et al. (1987) reported
gill hypertrophy and hyperplasia in Clarias lazera. with some
degree of gill epithelial lifting and necrosis. Gill degeneration,
observed in rainbow trout within 3wks of exposure to nitrite, was
noted to disappear with increasing exposure time (Wedemeyer and
Yasutake 1978). The observed changes in number and location of
mucous cells in greenlip abalone gills suggest ahypersecretion of
Figure 6. Junction of gill lamellae and principal filament of control
abalone from nitrite bioassay. Magnification, x400.
mucous as aresponse to chronic sublethal nitrite exposure at 7.8
mg of NOtN L"' for a minimum of 82 days of exposure. Al-
though these changes were more evident than for exposure to
aminonia, asurvival rate of 73% indicates that these changes may
not affect survival as much as growth rate (Hams et al. 1997). The
observed increase in right kidney pigment and granule deposition
may be areflection of increased kidney protein, and hence cell,
turnover. Arillo et al. (1984) hypothesized that tissue hypoxia, as
aresult of nitrite exposure, was the contributing factor to acute
toxicity for rainbow trout. The tissue hypoxia of fish is mediated
through production of methemoglobin from the respiratory pig-
ment hemoglobin. Because this pigment does not occur in abalone,
not surprisingly, the lesions seen with nitrite toxicity differ from
those seen with anoxia.
The effects of low dissolved oxygen on abalone in this study
have some parallel in the literature. Histopathological effects of
oxygen supersaturation to the red abalone, Haliotis riifescens (El-
ston 1983, Elston and Lockwood 1983) included the presence of
Figure 5. Junction of gill lainclluL' and principal I'lianRnt of abalone
exposed to 7.80 mg of NOj-N L"'. Magnification, x4(H), (A) Complete
mucous cell; (Bl discharged mucous cell with adhered mucous.
Figure 7. Distal tip of gill lamellae from control abalone from nitrite
bioassay. Magnification, x400. (A) Normal gill lamellar tip showing
mucous cells.
686 Harris et al.
Figure 8. Right kidney of abalone exposed to 7.80 mn ol N(),-N I, '.
Magnification. x400. (Al Pigment granules in supranuclear region of
kidney tubule cell; (B) eosinophilic cytoplasm in subnuclear region of
kidney tubule cell.
Figure '>. .lunction of gill lamellae and principal Tdament of abalone
exposed to 559r oxygen saturation. Magnification, x400. (Al Intensely
stained gill mucous cells; (B) necrotic tissue; (C) ciliate between gill
lamellae (family Ancistrocomidae).
gaseous emboli in epipodial. oral, and pedal tissues. Elston (1983)
reported the formation of these emboli at 150% oxygen saturation,
whereas at 111% oxygen saturation, no emboli were evident in the
abalone sampled in this study. Leitman (1992) reported increased
bacterial counts from effluent water of abalone tanks at 143%
oxygen saturation. The occurrence of ciliates at the 55% oxygen
saturation treatment suggests an increased susceptibility to disease
at depressed oxygen saturation. Walters and Plumb (1980) deter-
mined that low dissolved oxygen levels increase the susceptibility
of channel catfish to bacterial infection. Survival of abalone also
decreased to 59% of controls at this concentration (Harris et al.
in press).
Some of the responses seen to the toxicants in this study are
representative of the situation for many other aquatic animals.
Mallat (1985) reviewed the literature on structural changes, in fish
gills, induced by toxicants and determined that hislopathological
gill lesions are largely nonspecific in nature, with changes in gill
epithelium, bulbing or fusing of gill lamellae, hypersecretion and
proliferation of mucocytes, and changes in chloride cells and gill
vasculature common to many different exposure conditions. Mallat
(1985) also determined that the frequency of gill lesions is greater
from acute rather than sublethal exposure and in freshwater situ-
ations rather than marine. This may explain the lack of effect of
ammonia on the abalone gills and the subtle nature of changes has
occurred during sublethal exposure of greenlip abalone to ammo-
nia, nitrite, and low dissolved oxygen.
In this study, we have identified different histological changes
for each environmental stressor, but the effects with low dissolved
oxygen may have been dependent on ciliate infestation. Future
research will involve bioassays for combinations of stressors and
also abroadening of stress attributes to include biochemical
changes. The overall aim is to establish aset of stressor-specific
changes that can be used for diagnostic purposes.
ACKNOWLEDGMENTS
We thank the Fisheries Research and Development Corporation
for research funding, the Tasmania Research Council for scholar-
ship funding. Marine Shellfish Hatcheries for hosting the bioassay
work. Dr. Geoff Allan for use of rotameters, and Mr. Stephen
Hindrum and Mr. Deon Johns for technical assistance. We also
thank Dr. Stephen Edwards for critical assessment of the manu-
script and Prof. Denis Lynn for identification of the ciliates.
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