2.3.1 Methods to measure the organisms in this
category of the D-2 standard can be divided into two categories as
follows:
2.3.2 The presence of nucleic acid or ATP in a
sample may be taken as an indication of life, but it should be noted
that this nucleic acid or ATP could come from any living organism
of any size within the sample. There are no definitive methods available
to correlate the amount of nucleic acid or ATP with the amount, or
viability of organisms in the sample and, therefore, the presence
of these chemicals are limited as an indicative analysis methodology.
However, zero measurements of these chemicals may indicate that no
organisms are in the sample, i.e. the treatment process was successful
and in the D-2 standard is being met. Additionally, if nested filters
are used to isolate specific size groups, then ATP, which degrades
relatively quickly, can provide an indication of the potential presence
of a large concentration of organisms in one size class. If linked
to thresholds of ATP concentrations, this can be used to indicate
samples which are highly likely to be above the standard.
2.3.3 The same problems occur when using other
bio-chemical indicators to monitor the number of organisms in this
category. As many of the organisms in this size range are likely to
be phytoplankton, an obvious step would be to measure the level of chlorophyll a, a photosynthetic pigment which is essential
for photosynthesis in the sample. Zero concentrations may indicate
that there is no phytoplankton in the sample and chlorophyll a may
also be a good indicator as to whether a BWMS using an oxidizing process
was working to design dosages, as it might be expected to bleach such
pigments. However, caution has to be exercised as:
-
.1
chlorophyll a can persist in seawater
outside of a cell, therefore sampling should only be limited to the
particulate phase. However, nucleic acid and ATP can exist in dead
organisms, detrital material, senescent or dead cells, decomposing
macroalgae, plant detritus from terrestrial ecosystems and other non-living
particles, etc.;
-
.2 there may be zooplankton in the sample being
analysed;
-
.3 no cell count can be directly measured from
a chlorophyll a measurement, as many small cells may
provide a similar signal strength to that of fewer bigger cells; and
-
.4 no size distinction can be made and the chlorophyll
a could derive from phytoplankton in the larger size category
of the D-2 standard.
As a consequence, direct concentration measurements of this
chemical would be difficult to use in indicative analysis. A wealth
of portable tools exists to document the chlorophyll a
content in seawater.
2.3.4 One potential exception is the Pulse-Amplitude
Modulated Fluorometer (PAM) which measures the chlorophyll a fluorescence in
living cells by exciting chlorophyll a molecules and registering the subsequent
fluorescent signal. Such a response is only available in living cells and it should be
noted that this method only provides an indirect measurement of those phytoplankton that
use chlorophyll a in the sample, in both size categories of the D-2 standard.
Testing this methodology on ballast water discharges suggests that there is a
correlation between the ratio of variable and maximum fluorescence and the number of
phytoplankton in this size category. However, the relationship between fluorescence
signals and mixed assemblages of phytoplankton from different locations needs to be
validated.
2.3.5 For analysis of organisms above 10 microns
in minimum dimension, a flow cytometer may also be used. A common
element of these systems is that they automatically count objects,
including organisms, per size class in a fluid. The more simplified
systems cannot separate organisms from sediment and detritus, or living
from dead organisms. More sophisticated systems can also assess organism
viability for phytoplankton by using organism stains together with
flow cytometry. The separation of living phytoplankton from detrital
material and zooplankton is based on the presence of auto chlorophyll
fluorescence of phytoplankton cells. It should be noted, however,
that using chlorophyll a fluorescence as an indicator
of living organisms may result in over counting, as the molecule can
remain intact for a significant amount of time as has been proved
in preparing fixed (dead) samples. The practicability to use such
devices on board a ship should be carefully assessed before use. To
make a stable stream to produce adequate size of water particles,
the device should be set in perfectly horizontal. Also any vibration
should be isolated for accurate measurement.
2.3.6 Systems using flow cytometry deliver automated
results promptly and may be used to assess the number of living phytoplankton
in a sample after treatment with a viability stain. However, readings
provided by the flow cytometer should also be examined manually to
verify the automated readings. Concerns have been raised by users
that the viability of smaller algae may not always be categorized
correctly in these systems, as the viability signal may be too low
for detection. Other concerns include the efficiency of portable versions
and the limited ability of some of them to monitor organisms greater
than or equal to 50 micrometres in minimum dimension. Although these
systems may become a major tool in the future, there are elements,
such as the reliability of portable versions of the systems that limit
their use at the present time, which is especially the case for organisms
greater than or equal to 50 micrometres in minimum dimension. Also,
it is not clear if the time to analyse a sample is greater than can
be allotted in compliance testing. These can be overcome by taking
the sample off the ship and using a fixed or mobile system near to
the ship or the port.
2.3.7 Visual inspection could be another method
of indicative analysis that is a quick and simple way to justify the
need for detailed analysis. Taking an appropriate sample, concentrating
it if necessary, and visually inspecting it against the light may
show living organisms in the sample, but it should be noted that without
magnification a visual inspection is likely to result in only organisms
greater than or equal to 1,000 micrometres in minimum dimension being
detected, unless chains or clumps are formed by colony forming organisms
or the density of organisms is sufficiently large to colour the water.
An assessment of the viability in such an inspection is limited to
complete body movements of the organisms as organ activity and antennae
or flagella movements may not be seen. As samples from BWMS that are
not compliant are likely to contain organism levels that are orders
of magnitude above the D-2 performance standard, visual inspections
could be used in indicative analysis. However, it is assumed that
only organisms bigger than 1,000 micrometres in minimum dimension
may be determined in such way, therefore its use for this size category
is limited.
2.3.8 Visual inspection can also be undertaken
using a field stereomicroscope with a low magnification (e.g. x 10).
However, this methodology may require concentration of the sample
and may need analysis by a trained operator to detect viable organisms.
It should be also be noted that this methodology would be more efficient
and practicable for organisms greater than or equal to 50 micrometres
in minimum dimension.