Salinity Tolerance
Non-lethal endpoints indicative of a fish approaching a moribund state
are necessary to avoid unnecessary physical distress to animals and for
the collection of useable tissue samples. Aberrant swimming and
movements are an indicator of this approaching state, and it was
initially assumed based on previous experiments determining the maximum
critical salinity (CSMAX) that the appropriate endpoint
was the inability to maintain upright position, or loss of equilibrium
(LOE). Preliminary experiments conducted with six individuals acclimated
using a continuous increase of 6 g/kg indicated that this behavior did
not precede more severe impacts including pronounced lethargy and
cessation of feeding, and that a different endpoint was necessary. Fish
approaching a moribund state exhibited several behaviors including:
constant fin movement, inability to maintain position in the water
column, sustained darkened coloration, and highly reduced feeding.
Unfortunately, none of these were consistently observed in all fish. The
most consistent behavior was found to be non-reactivity to a potential
threat, specifically a dip-net. Normally functioning fish will evade
capture in a net, and though fish appear to move less frequently under
salinity stress, they still will approach food pellets and avoid nets.
When a fish no longer evades the net, this appears to be the most
consistent response and indicator that they will become moribund before
the following monitoring timepoint, and for this reason we have used the
term “morbidity point” (MP). This behavioral response is presented as
an alternative endpoint when other responses such as LOE are not
applicable.
Two fish in the preliminary experiments which had reached MP were
rescued by reducing salinity, suggesting that this state not terminal if
the salinity stress is ameliorated. In trials with an MP endpoint,
constant fish monitoring allowed for capture of fish before death in
most cases as this was necessary for acquiring usable serum and tissues
for proteomics. Nonetheless, some individuals were found dead and MP
estimated to the closest hour since the previous monitoring timepoint
for determining maximum salinity tolerance. Experimental mortalities
were not used for proteomic analysis. No premature deaths were recorded
in the 14-day acclimation trials to target salinity.
In experiments with continuous gradual salinity increase at variable
rates, the mean salinity at MP were as follows: 32.0g/kg at 32g/kg/day,
69.0g/kg at 24g/kg/day, 106.8g/kg at 12g/kg/day, 117.0g/kg at 8g/kg/day,
and 114.6g/kg at 6g/kg/day (Figure 1A). Rate of salinity increase was
clearly important in determining CSMAX, but lower rates
avoid acute affects and resulted in relatively consistent
CSMAX. Finding the mean of the CSMAXfor the 8 g/kg/day and 6 g/kg/day rates indicates an acclimatory
CSMAX of approximately 115g/kg for this population and
experimental set-up. This result is not meant to represent the
CSMAX for the species generally, but rather to define
the pessimum salinity range for this population so that appropriate
samples representative of stages of upper range salinity tolerance could
be chosen for later proteomic analysis. For extended exposure to
constant salinity experiments, decreasing increments of 10g/kg lower
than the CSMAX (115g/kg) were used until a salinity was
found in which fish were able to survive indefinitely to determine the
critical salinity threshold. The mean times until MP at these salinity
levels were as follows: 263 hours at 105g/kg, 326 hours at 95g/kg, and
559 hours at 85g/kg (Figure 1B). At 75g/kg, 79% of individuals survived
for 10 weeks, which was the endpoint the experiment.