3.1 Once detailed analysis has been instigated by the port State, they
should be prepared to undertake full analysis of the sample at an appropriate
laboratory.
3.2 Bacteria
3.2.1 There are already international standards in place to
analyse for the bacteriological indicators contained within the D-2 standard.
3.2.2 For Enterococci, ISO 7899-1 or 7899-2; or Standard
Method 9230 (in the United States) should be used, and ISO 9308-3, ISO 9308-1 or
Standard Method 9213D (in the United States) are appropriate for Escherichia coli. The
methods used should be quantitative and based on a 95-percentile statistical evaluation.
The number of laboratory samples should be sufficient to define the mean and standard
deviation of Log 10 bacterial enumerations.
3.2.3 For Vibrio cholerae ISO/TS 21872-1/13 is
appropriate. 100 ml of ballast water should be filtered and incubated according to
ISO/TS 21872-1. Analysis needs to be undertaken in a specialist laboratory.
3.3 Organisms of less than 50 micrometres and greater than or equal to 10 micrometres
in minimum dimension
3.3.1 Many of the analysis methods used to ascertain the numbers of organisms within
this category have already been discussed in section 2. However, section 2 focuses on
indicative analysis, rather than the more detailed analysis. Therefore, the following
sections examine these methodologies in more detail. Some of these methodologies
discussed here also relate to organisms greater than or equal to 50 micrometres in
minimum dimension.
3.3.2 Simple upright and inverted microscopes are very useful for the enumeration of
morphologically healthy organisms and motile organisms, as well as for measuring the
size of organisms. Using this technology needs some skill and experience to evaluate the
health of the individual organisms in the sample. However, this technology and
experience should be available globally.
3.3.3 Fluorescence generated from photosynthetic pigments can be used for more detailed
analysis of the morphological health of organisms and for the evaluation of stained
organisms and a microscope with fluorescence capabilities is needed. However, this
methodology only identifies phytoplankton (both living and dead) in the sample and makes
no size differentiation. Zooplankton should be analysed through the methods highlighted
in section 3.4.
3.3.4 Fluorescein di-acetate (FDA), chloromethylfluorescein diacetate (CMFDA) and
Calcein-AM vital stains have both been used to determine viability. When non-specific
esterases (enzymes found in live cells) are present, they cleave the acetate groups from
the stains, and the resultant fluorescein molecules fluoresce green when illuminated
with a blue light from an epi-fluorescence microscope. This method works best with live
samples. Microscopes with a fluorescence capability and operators with skills and
experience of analysis should be available at universities and research laboratories
worldwide. However, it should be noted that these stains do not always work on all
species or at all salinities and further research to validate this approach may be
needed to support the use of these stains for this type of analysis.
3.3.5 Flow cytometers are advanced technologies which can be used in a laboratory to
determine size, and viability of organisms in ballast water when a reliable vital
stain(s) is (are) used to indicate organism viability. Cytometer detected particles,
including organisms, can be processed visually or by a computer to quantify viable
organisms in that sample. These systems reduce manual labour but require specific
knowledge to operate them. High particle loads in ballast water may reduce the detection
limits of these methodologies and the volume of samples analysed. At present, portable
versions of these technologies have not fully been proven for use on ballast water
discharges, however, samples could be taken off the ship and analysed using a fixed or
mobile system near to the ship or the port.
3.3.6 Regrowth experiments, in which the visual appearance of photosynthetic organisms
in a sample is followed by a specific period in order to quantify the most probable
number (MPN), are methods to evaluate the number of organisms in a sample. However,
these are slow and are work intensive. In addition, a major drawback of this methodology
may be that specific growth factors during the incubation may not be fulfilled, giving a
risk of bias. Regrowth and reproduction may be seasonably variable, giving different
results at different times. Further, a viable organism may be in good health and
reproducing rapidly, or in poor health, not reproducing until health has improved.
Finally, this is likely to be time-consuming.
3.3.7 Bulk parameter measurements, such as photosynthetic activity, are also not
suitable for detailed analysis (please see paragraphs 2.3.2 and 2.3.3), but can be used
as supporting data for other methods used to determine the number of viable organisms in
the ballast water samples.
3.3.8 Planktonic organisms may be fragile and samples may need to be concentrated
further to aid the accurate quantification of organisms. There are many methods to
achieve this, however, care has to be taken to reduce physical stress as this may result
in reduced viability levels. A simple, rapid, flexible and cautious method for
concentrating plankton cells is the use of transparent membrane filters. If the sample
analysis is performed on board the sample can be filtered directly on to this membrane,
which can subsequently be placed directly under a microscope for examination. The sample
volume to be analysed would need to be adjusted depending on the cell density, however,
live, vital stained and fixed organisms within this size category can be evaluated on
these filters. If the representative analysis is performed at a laboratory, this process
for concentration should be performed at the laboratory just before starting the
staining process to avoid under-estimate of viable organisms. Importantly, the loss (if
any) of organisms (i.e. those cells passing through the filter and recovered in the
filtrate) would need to be determined. Alternatively, a filter mesh may be used to
concentrate the sample and the concentrated organisms may, after filtration, be
transferred into an observation chamber. Again, the loss of organisms through damage
must be quantified.
3.4 Organisms greater than or equal to 50 micrometres in minimum dimension in the D-2
standard
3.4.1 Paragraphs 3.3.2 to 3.3.8 are also applicable to the analysis of organisms in this
size category.
3.4.2 In addition, the following issues need to be considered when developing a
methodology for analysing organism numbers in this size category:
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.1 testing the sample for movement and response to
different stimuli are simple techniques for the examination of viable/dead
zooplankton under a stereomicroscope. The observation for organ activity, such as
heartbeats, may also contribute to the viability assessment. The use of a
filtering mesh (e.g. 50 microns in diagonal dimension) under the Petri dish of the
stereomicroscope, or the addition of 50 micron micro beads to the sample, may help
with size calculations and vital stains may also add value to these methodologies.
Separate guidelines on this issue are being developed through the land-based
facilities and the ETV protocol in the United States;
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.2 methods using a combination of flow cytometry and
microscopy have the disadvantage of high complexity, high price and small sample
sizes, which means the ballast water samples would have to be concentrated
further; and
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.3 the storage condition and time before analysis is
likely to be critical to reduce mortality in the sample.
3.4.3 It is therefore recommended that simple microscopic examination of organisms in
this size category is used for compliance monitoring. The microscopic examination of
organisms is a robust, simple and cheap methodology which can be completed in
laboratories worldwide.