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On board tests of the organism detection tools BallastCAM, FluidImaging, USA, Hach-PAM-fluorometer, USA, and Walz- Water-PAM-fluorometer, Germany. Results and findings

  • GoConsult, Independent Researcher
  • Dr. Matej David Consult
On board tests of the organism detection tools BallastCAM,
FluidImaging, USA, Hach-PAM-uorometer, USA, and Walz-
Water-PAM-uorometer. Results and ndings
Stephan Gollasch
Matej David
Dr. Matej David Consult s.p.
December 2012
Stephan Gollasch
Grosse Brunnenstrasse 61
22763 Hamburg
Matej David
Dr. Matej David Consult s.p.
Korte 13e
6310 Izola
Reporting Period 7, March 2012 to August 2012
This report should be quoted as follows
Gollasch S, David M, 2012. On board tests of the organism detection tools BallastCAM, FluidImaging,
USA, Hach-PAM-fluorometer, USA, and Walz-Water-PAM-fluorometer. Results and findings. Prepared for
Interreg IVB North Sea Ballast Water Opportunity project: 10 pp.
The contents and views contained in this report are those of the authors, and do not necessarily
represent those of the Interreg IVB North Sea Region Programme. Although careful consideration is
given to its content, we cannot be held responsible for its use.
1BACKGROUND ................................................................................................................... 2
1.1..ONBOARD USE OF THE SYSTEMS ........................................................................................ 3
2FINDINGS AND RECOMMENDATIONS ................................................................................ 4
2.1..BALLASTCAM .............................................................................................................. 4
2.2..ACTIVE FLUOROMETERS .................................................................................................. 5
2.3..INDICATIVE VS. IN-DEPTH SAMPLE ANALYSIS ......................................................................... 8
ACKNOWLEDGEMENTS .............................................................................................................. 9
REFERENCES ........................................................................................................................... 10
BWO on board test of selected organism detection technologies
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In Ballast Water Opportunity (BWO) one of the duties in WP4 are to check the applicability of potential
organism detection technologies onboard of commercial vessels. A screening of technologies was
completed and a comprehensive report was submitted in an earlier reporting period (Gollasch &
Stehouwer 2012) which benefitted from the authors work in another study (Gollasch & David 2010). This
report is an additional contribution of BWO Deliverables
D4-3 Identification of additional techniques for Compliance Enforcement and Monitoring based
on outcome WP2 and
D4-14 Technology assessment for compliance enforcement the relation between the BWMC
policies, enforceability and technology.
One of the challenges when biologically analyzing ballast water samples, e.g. for compliance control
purposes with the D-2 standard, is the prompt and accurate enumeration of viable organisms per size
class found in the samples.
From the candidate technologies listed in Gollasch, Stehouwer & David (2012), three methods were
selected for a closer consideration to analyse phytoplankton organisms in the size range below 50 and
above 10 micron in minimum dimension, i.e. for their applicability on board:
1. the BallastCAM of FluidImaging, USA,
2. the Hach-PAM1-fluorometer (AquaPen Version, USA, and
3. the Walz-Water-PAM-fluorometer.
FluidImaging developed the BallastCAM as an instrument to analyse ballast water samples with a focus
on phytoplankton organism less than 50 micrometres and greater than or equal to 10 micrometres in
minimum dimension. Its suitability for zooplankton organisms in this size class or above 50 micron in
minimum dimension needs to be tested. However, the system was never used and tested on board of a
commercial vessel, but this is likely the environment such analytical tools need to be operated in should
in the future port state control officers plan to check on the spot if the ballast water is in compliance with
the D-2 standard. It should be noted that the system does not distinguish between viable and dead
particles, but it delivers particle counts and photos of each particle counted so that a later analysis may
reveal numbers of viable organisms, although the accuracy level of such an approach is questionable. To
familiarize the authors with the use of the BallastCAM, Kevin Stewart of FluidImaging met the authors
and demonstrated the use of the BallastCAM in a hotel room in New York. For this purpose an ocean
water sample containing a natural plankton assemblage was used.
The Hach-PAM-fluorometer, which was provided by Bio-UV (see below), was developed as test kit for
compliance control with the D-2 standard and it works in a similar way as the Walz-Water-PAM-
fluorometer (Gollasch et al, 2012). The Hach system tested here is a pre-prototype unit which will be
further modified to better fit for purpose. This especially refers to increase its sensitivity to detect lower
algal cell concentrations (Harbridge (Hach) pers. comm.).
1 PAM = Pulse Amplitude Modulated (fluorometry)
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Both systems deliver a bulk measurement of viable algae, i.e. no organism count. However, the stronger
the measured viability signal (for the Walz-Water-PAM-fluorometer in combination with a biomass
measurement), the higher seems to be the number of viable cells in the sample.
The voyage during which all three systems were tested was undertaken on the container vessel MV
MARFRET SORMIOU between New York, USA and Monzanillo, Panama in July/August 2012. This voyage
was undertaken to challenge the Bio-UV (France) ballast water treatment system´s performance as a
consultancy work of GoConsult. Ballast water was taken up in the Port of Kingston, Jamaica and later
discharged in the Monzanillo port, Panama.
After all sampling and sample processing to document the performance of the Bio-UV ballast water
treatment system was completed remaining samples were analyzed with the FuidImaging BallastCAM
(Figure 1) and a Hach-PAM-fluorometer. The measurements of the Walz-Water-PAM-fluorometer were
measured as part of the standard sample processing and was considered here for comparison.
In addition a second voyage was undertaken in October 2012 with ballast water uptake in Rotterdam,
the Netherlands. Here the Hach and Walz Water PAM were used to analyse the samples.
On both voyages, untreated ballast water pumped onboard as well as treated ballast water, when
discharged from the vessel, was analyzed.
Figure 1: The BallastCAM of FluidImaging in on board use by Stephan Gollasch (top) and Matej David (bottom).
BWO on board test of selected organism detection technologies
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The FluidImaging BallastCAM is a compact instrument with a moderate weight of ca. 20 kg. Due to this
design it seem feasible that port state control officers, when planning to undertake ballast water
requirements compliance checks, may bring such an instrument on board a vessel to analyze for
organism numbers on the spot, i.e. during the onboard sampling event.
The BallastCAM is easy to use is and is designed to be operated by a “non-biologist” so that port state
control officers can use it after a short training session. However, we noted that the keyboard is not easy
to use and the software does not allow the copy/paste function for names when saving files or creating
folders. Further, it is difficult to replace the flow cell because this is obstructed and not at all conveniently
available. A flow cell replacement may be needed in cases when particles become stuck in the
observation field of view, which happened during our test. Possibly a rubber layer on the outer screw to
fix the flow cell could be helpful not to damage the cell by tightening the screw too much. The size
dimension of this flow cell (200 microns) further limits its use for organisms in size ranges > 200
The rapid sample processing time would promptly enable port state mitigation measures should the
ballast water samples indicate non-compliance with the D-2 standard. However, although the BallastCAM
rapidly counts objects and their sizes in a water sample, a weakness of the BallastCAM is that, in its
current design, it cannot distinguish between living and dead organisms/particles, which is a key feature
to proof compliance with the D-2 standard. The use of stains to overcome this situation may be
To proof that the D-2 standard was met, in addition to the viability of organisms, their size needs to be
documented in minimum dimension. During a zooplankton workshop (organisms above 50 micron in
minimum dimension), held at NIOZ, Texel, The Netherlands in Fall 2010, as an event of the Interreg IVB
Ballast Water Opportunity project, it was agreed that for the visual measurement of the minimum
dimension the smallest visible axis of an organism should be chosen and the widest point on this axis be
measured (see examples in Figure 2). If organisms are near the 50 um minimum dimension, efforts
should be made to measure the third (less visible) dimension.
For organisms in the smaller size class of the D-2 standard, targeted to be analyzed by the BallastCAM,
the same principle to measure the minimum dimension as outlined above should apply. The BallastCAM
has several features to analyze the size of the objects identified, but it remains unclear how close these
measurements get to identify the true minimum dimension. Tests may be undertaken by using
organisms of different shapes to identify how close the BallastCAM measurements are to identify the
minimum dimension in comparison to a visual observation by an expert.
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Figure 2: Minimum dimension measurements (red line) for selected organism types: A = mussel larvae, B and C = gastropod
larvae, D = foraminifera (phytoplankton) and E = copepod. (Photos A - D: Stephan Gollasch, H:
The Hach-PAM active fluorometer is of very compact design (pocket size). It is battery-powered and
therefore it can independently be used also at any sampling point. The simple use allows a non-biologist
to work out the samples and the results are available promptly.
Figure 3: The Hach-PAM active fluorometer operated by Stephan Gollasch.
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The Walz-Water-PAM-fluorometer is also of compact design, but it is bigger than pocket size and a laptop
computer is recommended to operate the system, hence it should be operated on a desk. The use is
simple so that a non-biologist can work out the samples and the results are available promptly. In
addition to the viability measurement, the biomass is measured and this gives additional information
regarding the concentration of cells in the sample.
A comparison of the performance of the two active fluorometers used is only enabled on a very limited
basis due the low number of samples analysed. However, when comparing the viability measurements of
the HACH and Walz instruments, in the first set of samples the HACH instrument measured lower values
compared to the Walz instrument. In the second set of samples the reverse situation was observed. In
the last test run the measured Fv/Fm values showed the highest difference (Hach 0,343 and Walz 0,466)
(Figure 3). In conclusion, both instruments show the same result pattern, but are different in sensitivity
and accuracy, especially in low value measurements.
It is recommended to repeat such comparative studies with a larger number of samples possibly also
containing different algae and sediment concentrations.
Figure 4: The Walz-Water-PAM-fluorometer operated by Matej David.
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Figure 5: Comparison of algae viability measurements (Fv/Fm) of the Hach and Walz Water PAM.
The HACH instrument cannot be adjusted to enable measurements with very low or very high load of
algae and it is unclear in which range of algal concentrations the instrument can be operated. We
observed in measurements of treated water at discharge that the instrument reads “low value”. In
contrast the Walz instrument can be adjusted by manipulating the gain according to the measurements,
so that also algae in very high and very low concentrations can be measured.
In Figure 4 results of performance comparison experiments of the Hach and Walz Water PAM are shown.
A very good correlation between the two tools is documented at higher concentrations of organisms
(Harbridge pers. comm.).
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Figure 4: Comparison of algae viability measurements (Fv/Fm) of the Hach and Walz Water PAM made by N. Welschmeyer
(Moss Landing Marine Laboratories). Measurements were made on 9 species of algal cultures reared to different levels of
nutrient stress to create a wide range of Fv/Fm values.
According to the IMO BWM Convention two sample processing approaches are available. One is an
indicative check of a sample, which likely delivers results promptly, but with a certain level of in-
accuracy. In contrast a detailed sample analysis may be carried out with the highest possible accuracy.
The currently constructed BallastCAM cannot analyze the viability of objects, and therefore a detailed
compliance control check with the D-2 standard is not enabled. The indicative sample analysis also needs
to show the presence/absenceof viable organisms in a ballast water sample which is not possible with the
current BallastCAM version. However, all objects identified by the BallastCAM are individually
photographed by a color camera. For an indicative check of the ballast water, the objects photographed
may be analyzed and those being undamaged and also containing chlorophyll may be assumed as viable.
The filters included in the BallastCAM operation software enable sorting of objects by color so that all
green, i.e. chlorophyll containing objects, can be counted per water volume. This result may be taken as
an indicative compliance check. The errors of such a method need to be carefully evaluated.
The active fluorometers tested here measure the chlorophyll content in living cells by triggering the
phytoplankton electron chain to respond. Such a response only exists in living cells thereby assessing
photosynthesis activity by utilizing the relationship of chlorophyll fluorescence and photosynthesis to
describe cell ‘health’. These instruments enable semi-quantitative cell density estimations with a greater
level of precision compared to standard fluorometers because responding cells are measured and not
only the chlorophyll content.
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Active fluorometers may have a similar ability as the BallastCAM for compliance checks. The weakness of
the active fluorometers is that they measure viability but do not deliver counts of cells, whereas the
BallastCAM can deliver counts, but it cannot measure viability. However, the active fluorometers deliver
additional information which seems essential for compliance checks, e.g. biomass, and some can also
measure very low cell concentrations. It seems reasonable to investigate the possible combination of
both instruments (active fluorometers and flow cameras) to enable counts of viable objects.
In cases where chlorophyll containing algal cells can be identified by the BallastCAM in the sample or the
active fluorometers do identify viability, it may indicatively be assumed that living algae are present in
the sample so that the ballast water treatment process was unsuccessful and the D-2 standard was met.
If a high biomass and a strong response of viable organisms is identified when measuring with active
fluorometers, this indicates gross exceedence of the D-2 standard. Similarly, the gross exceedence could
also be documented if the BallastCAM would identify a high number of chlorophyll containing objects.
We gratefully acknowledge the permission of the ballast water treatment systems vendor Bio-UV, France,
allowing us to operate the BallastCAM during the onboard performance tests of their ballast water
treatment system and especially Ronan Cadet who enabled the on board sample taking. We further
thank the crew of the test vessel MV MARFRET SORMIOU for their support during the ballast water
operations. This work was partly undertaken to support the Interreg IVB Ballast Water Opportunity
project where Gollasch is responsible for organism detection tools in ballast water.
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Gollasch S, David M 2010. Testing Sample Representativeness of a Ballast Water Discharge and
developing methods for Indicative Analysis. Tender N° EMSA/NEG/09/2010. Final Report prepared
for European Maritime Safety Agency, Lisbon, Portugal. 124 pp.
Gollasch S, Stehouwer PP, David M 2012. BWO Technical Outline and Requirements for Organism
Detection Systems for Establishing Compliance Enforcement. Final Report. Prepared for Interreg
IVB Project “Ballast Water Opportunity”. Submitted with 6th project reporting period. 88 pp.
... Many instruments and methods for the indicative and detailed ballast water sample analysis were evaluated in various studies (Schreiber et al., 1993;Veldhuis and Kraay, 2000;Shapiro, 2003;Buskey and Hyatt, 2006;Tang and Dobbs, 2007;Briski et al., 2011Briski et al., , 2013Cangelosi et al., 2011;Welschmeyer and Maurer, 2011;Gollasch and David, 2012;Gollasch et al., 2012aGollasch et al., ,b, 2013Zetsche and Meysman, 2012;Gollasch and Peperzak, 2013;Stehouwer et al., 2013;Drake et al., 2014;Wright et al., 2015). However, for the larger zooplankton and bacteria no reliable indicative sample analysis method was yet identified. ...
... PAM instruments were here tested in parallel with epifluorescence microscopy using fluorescein diacetate (FDA) stained samples which is one of the accepted methods for a detailed ballast water sample analysis (IMO, 2013a), and results were compared in the view of accuracy and for CME purposes. All PAMs selected for the tests were previously used on board of commercial vessels and it was shown that they can be operated in such an environment (Gollasch and David, 2012;Gollasch et al., 2012b). Other studies to compare the results of different PAMs were conducted recently, but had different objectives and did not consider as many PAMs as reported here (Drake et al., 2014;Wright et al., 2015) so that this study is to our knowledge the most comprehensive PAM comparison to date. ...
Different phytoplankton analysis methods (pulse-amplitude modulated fluorometry (PAM) and microscopy) were compared in preparation for compliance monitoring and enforcement with ballast water discharge standards. The key objective was to practically evaluate the performance of different new methods and tools to identify indicatively living phytoplankton cells of the size <50μm in minimum dimension and ≥10μm in minimum dimension as addressed by the Ballast Water Performance Standard (Regulation D-2, International Convention for the Control and Management of Ships' Ballast Water and Sediments, 2004). Four different PAM instruments were selected for the tests based upon knowledge and experience gained in different ballast water sample processing studies. The measurements of the PAM instruments were compared with epifluorescence microscope algae cell counts using fluorescein diacetate as viability stain. It was concluded that PAM fluorometry is a suitable method for indicative phytoplankton analysis of ballast water and the most accurate PAM instruments were identified.
Full-text available
This study investigates different UV doses (mJ/cm(2)) and the effect of dark incubation on the survival of the algae Tetraselmis suecica, to simulate ballast water treatment and subsequent transport. Samples were UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of ≥400mJ/cm(2) rendered inactivation after 1day as measured by all analytical methods, and are recommended for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100-200mJ/cm(2)) gave considerable differences of inactivation between experiments and analytical methods. Nevertheless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100mJ/cm(2) and ≤200mJ/cm(2) can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable results demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation when algae are treated with low UV doses.
Full-text available
A wealth of organism detection technologies are available. However, this report is meant to give an overview of methods and does not attempt to deliver a fully comprehensive summary. Most of the methods considered were not initially designed for the purpose of biological ballast water sample analysis. It further became clear that not a single method is available to identify all organism groups addressed in the Ballast Water Performance Standard (D-2 of the IMO BWM Convention). The practicality of selected methods was tested by using them on board of commercial vessels. A particularly challenging condition is that no laboratory facilities are available on such vessels for sample processing. Further, compliance tests with the D-2 standard requires a high detection limit, i.e. a high sensitivity of methods. As a result it becomes clear that a compromise is needed to select the most appropriate method. Aspects to consider include:  Accuracy / precision;  Reliability;  Time to a result;  Expertise;  Portability; and  Costs.