<figure><img src="image_1.png" /></figure>

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HARMFUL AQUATIC ORGANISMS IN BALLAST

WATER Information on the development and
assessment of a new analytical method for enumerating
organisms in the 10 to 50 µm size class and in the
greater than 50 µm size class in treated ballast water

Julia Baudart, Charlène Cérésola

► To cite this version:

Julia Baudart, Charlène Cérésola. HARMFUL AQUATIC ORGANISMS IN BALLAST WATER Information on the development and assessment of a new analytical method for enumerating organisms in the 10 to 50 µm size class and in the greater than 50 µm size class in treated ballast water. International Maritime Organization. 2019. hal-05007713

HAL Id: hal-05007713

https://hal.science/hal-05007713v1

Submitted on 26 Mar 2025

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Public Domain
MARINE ENVIRONMENT PROTECTION
COMMITTEE
74th session
Agenda item 4

MEPC 74/INF.17

7 March 2019

ENGLISH ONLY

# HARMFUL AQUATIC ORGANISMS IN BALLAST WATER

## Information on the development and assessment of a new analytical method for enumerating organisms in the 10 to 50 µm size class and in the greater than 50 µm size class in treated ballast water

Submitted by France

## SUMMARY

**Executive summary:** This document summarizes the recent research about a new application of the CV6 dye for the control of the viable organisms in ballast water, to detect viable phytoplankton and zooplankton belonging to both size classes (> 50 µm and 10 to 50 µm) defined in the regulation D-2 of the BWM Convention. It provides information on the analytical method used in a device under development, combining membrane filtration and the CV6 vital stain, and presents preliminary results for the scientific assessment of the method compared to currently accepted methods for ballast water analysis.

**Strategic direction, if applicable:**

**Output:** 1.14

**Action to be taken:** Paragraph 18

**Related documents:** BWM.2/Circ.42/Rev.1 and MEPC 74/4/10

## Introduction

1. Verification of compliant discharge of ballast water, according to the D-2 ballast water performance standard of the BWM Convention stipulating a maximum allowable number of viable organisms in treated ballast water, has led to a need for rapid, easy and accurate assessment tools and protocols.

2. BIOSEA2 research project was led, from 2015 to 2017, by the Laboratory of Microbial Biodiversity and Biotechnology (LBBM) to assess and validate a combination of approaches and methods, as the first step to develop a new device able to perform automatic counting of

<figure><img src="image_2.png" /></figure>
viable phytoplankton and zooplankton in both size classes (> 50 µm and 10 to 50 µm in minimum dimension) determined by regulation D-2 of the BWM Convention.

3 LBBM is a Joint Research and Service Unit (USR3579) of the Sorbonne Universities (Univ. Pierre and Marie Curie) and of the French National Centre for Scientific Research (CNRS); it is located in Banyuls-sur-Mer (66), France.

4 This document summarizes the project main activities and preliminary results about a new application of a commercialized vital stain (ChemChrome V6) for fluorescent marking of the viable organisms, used in combination with a solid phase analysis with a specifically developed device (here named the FluoCount), designed to perform an automatic counting of the two size classes of organisms after membrane filtration of sample.

Scientific assessments of the CV6 for staining a large diversity of phytoplankton and zooplankton taxa

5 The ChemChrome V6 (CV6) is a commercialized fluorescent viability indicator; this dye is a fluorogenic ester that is converted into free fluorescein in presence of active esterase enzymes, which are commonly associated to cell viability. The CV6 was already assessed to measure the esterase activity of prokaryotes (bacteria; Parthuisot et al., 2000) and microeukaryote (Naegleria lovaniensis amoebae; Johnson et al., 2007). Both categories of microorganisms are easily detected by flow cytometry, solid-phase cytometry and fountain flow cytometer, devices able to detect produced fluorescein at the organism scale.

6 In order to determine the capability of the CV6 for labelling a large diversity of organisms belonging to the phytoplankton and zooplankton, the CV6 was tested for a panel of phytoplankton strains provided from collections (table 1) and for larvae of *Artemia salina* at different stages of their developments and after visualization under epifluorescence microscopy.

<table><caption>Table 1: List of phytoplankton strains tested in the study</caption><thead><tr><th>Taxons</th><th>taxonomic class</th><th>Species/strains</th><th>CV6 signal</th><th>Estimated cell length (µm)</th><th>Origine of collection</th></tr></thead><tbody><tr><td>Diatoms</td><td>Baccillariophyceae</td><td>Achnanthes spp (strain 2B Texas)</td><td>+</td><td>30</td><td>LECOB</td></tr><tr><td>Diatoms</td><td>Baccillariophyceae</td><td>Achnanthes spp (strain 1A)</td><td>+</td><td>25-30</td><td>LECOB</td></tr><tr><td>Green algae</td><td>Chlorophyceae</td><td>Dunaliella tertiolecta (strain 1)</td><td>+</td><td>5-18</td><td>LECOB</td></tr><tr><td>Green algae</td><td>Chlorophyceae</td><td>Dunaliella tertiolecta (strain 2)</td><td>+</td><td>10</td><td>LECOB</td></tr><tr><td>Haptophytes</td><td>Prymnesiophyceae</td><td>Prymnesium parvum</td><td>+</td><td>9</td><td>LECOB</td></tr><tr><td>Diatoms</td><td>Mediophyceae</td><td>Thalassiosira weissflogi (strain</td><td>+</td><td>10</td><td>RCC</td></tr><tr><td>Diatoms</td><td>Mediophyceae</td><td>Thalassiosira pseudonana (strain</td><td>+</td><td>4</td><td>RCC</td></tr><tr><td>Diatoms</td><td>Mediophyceae</td><td>Chaetoceros spp (strain RCC4495)</td><td>+</td><td>chain</td><td>RCC</td></tr><tr><td>Cryptomonads</td><td>Cryptophyceae</td><td>Cryptomonas sp.</td><td>+</td><td>15</td><td>LECOB</td></tr><tr><td>Cryptomonads</td><td>Cryptophyceae</td><td>Rhodomonas sp. (strain 1)</td><td>+</td><td>10</td><td>LECOB</td></tr><tr><td>Cryptomonads</td><td>Cryptophyceae</td><td>Rhodomonas sp. (strain 2)</td><td>+</td><td>10</td><td>LECOB</td></tr><tr><td>Diatoms</td><td>Baccillariophyceae</td><td>Navicula incerta</td><td>+</td><td>19</td><td>LECOB</td></tr><tr><td>Diatoms</td><td>Baccillariophyceae</td><td>Phaeodactylum tricomutum</td><td>+</td><td>20</td><td>LECOB</td></tr><tr><td>Diatoms</td><td>Baccillariophyceae</td><td>Nitzschia sp</td><td>+</td><td>42</td><td>LECOB</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Lingulodinium polyedrum (strain ME</td><td>+</td><td>42-54</td><td>ME</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Alexandrium ostenfeldi (strain ME Ao)</td><td>+</td><td>39-54</td><td>ME</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Alexandrium minutum (strain ME Am)</td><td>+</td><td>17-25</td><td>ME</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Alexandrium catenella (strain ME Ac)</td><td>+</td><td>20-50</td><td>ME</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Alexandrium tamarense (strain ME At)</td><td>+</td><td>25-46</td><td>ME</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Karenia brevis (strain ME Kb)</td><td>+</td><td>20-40</td><td>ME</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Heterocapsa triquetra (strain ME Ht)</td><td>+</td><td>20-30</td><td>ME</td></tr><tr><td>Green algae</td><td>Prasinophyceae</td><td>Tetraselmis sp</td><td>+</td><td>10</td><td>LECOB</td></tr><tr><td>Haptophytes</td><td>Prymnesiophyceae</td><td>Hymenomonas coronata</td><td>+</td><td>10</td><td>LECOB</td></tr><tr><td>Eukaryote</td><td>Raphidophyceae</td><td>Chattonella subsalsa (strain CS0004)</td><td>+</td><td>30-50</td><td>ME</td></tr><tr><td>Eukaryote</td><td>Raphidophyceae</td><td>Chattonella subsalsa (strain CS0507)</td><td>+</td><td>30-50</td><td>ME</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Prorocentrum lima (strain RCC1520)</td><td>+</td><td>40</td><td>RCC</td></tr><tr><td>Dinoflagellates</td><td>Dinophyceae</td><td>Prorocentrum micans (strain ME Pm)</td><td>+</td><td>35-70</td><td>ME</td></tr><tr><td>Green algae</td><td>Chlorophyceae</td><td>Chlamydomonas spp (strain RCC443)</td><td>+</td><td>8</td><td>RCC</td></tr><tr><td>Cryptomonads</td><td>Prymnesiophyceae</td><td>Isochrysis galbana Parke</td><td>+</td><td>6</td><td>LECOB</td></tr><tr><td>Cryptomonads</td><td>Prymnesiophyceae</td><td>Pavlova lutheri</td><td>+</td><td>6</td><td>LECOB</td></tr><tr><td>Cryptomonads</td><td>Prymnesiophyceae</td><td>Hymenomonas coronata</td><td>+</td><td>10</td><td>LECOB</td></tr></tbody></table>

7 The labelling is rapid (10 minutes) and performed directly in the sample at room temperature. The fluorescent organisms can be observed into a Sedgewick-Rafter counting
chamber etched with 1 mm² grids for microscopic observations. The autofluorescence without CV6 dye of algae populations was tested in parallel. As shown in figures 1 and 2, tested phytoplankton strains and larvae of *Artemia salina* were positive for CV6 and no autofluorescence was measured under blue light without the CV6.

<figure><img src="image_3.png" /><figcaption>Figure 1: Photographs collected with an epifluorescence microscope under blue light excitation of CV6 labelled phytoplankton strains</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Figure 2: Photographs collected with an epifluorescence microscope under blue light excitation after CV6 labelling of (A) just hatched-larvae of *Artemia salina* and (B) 1 day-larvae</figcaption></figure>

8 In order to determine the capability of the CV6 for labelling planktonic organisms in a natural assemblage, the CV6 was tested from several natural samples collected at different seasons. The plankton was collected in the bay of Banyuls-sur-mer (France), a northwestern Mediterranean Sea coastal area, by using a plankton net (10 µm of mesh size for the phytoplankton and 50 µm of mesh size for the zooplankton) and a vessel. As shown in figure 3, a large diversity of taxa was positive for CV6 and no autofluorescence (samples without CV6) was observed.
<figure><img src="image_5.png" /><figcaption>**Figure 3:** Examples of photographs collected with an epifluorescence microscope under blue light excitation after CV6 labelling (left) and under white light (right) of natural assemblage collected with (A) a plankton net with mesh size 10 µm and (B) a plankton net with mesh size 50 µm)</figcaption></figure>

Scientific assessments of the CV6 for detection of viable phytoplankton

9. The CV6 dye was assessed on cultured phytoplankton exposed or not to a lethal treatment (heat treatment, chemical fixative agent and UV-C) to determine the capability of the dye to detect living organisms. The analyses were performed with a flow cytometry FACSCanto II (Becton Dickinson) to compare the fluorescence of dead and living cells to fluorescent particle standards.

10. Cultured phytoplankton strains with a size ranging from 10-70 µm (table 1) were firstly analysed with and without CV6 to test their autofluorescence and to measure the green fluorescence intensity of labelled cells. No autofluorescent signal was measured on the FL1 channel for unlabelled cells and the green fluorescence intensity of labelled cells ranged between 11.1% to 184.0% of the fluorescent particle standards.

11. The CV6 was tested during the growth phases of the diatom *Thalassiosira weissflogii* RCC76. The growth phases were monitored at 22, 45, 59 and 93 hours after the start of the culture. The total cells, with a red fluorescence excited by a 488-nm laser and indicating cellular chlorophyll contents and the viable cells (positive for CV6) detected on the FL1 channel, were monitored for each time series. As shown in figure 4, 93% to 97.7% of the cells positive for chlorophyll were also positive for CV6 during the exponential phase and the relative fluorescence was 50% of the fluorescent standard used in the analysis. These results confirm the efficiency of the CV6 to detect the viable cells during the growth of algae.
<figure><img src="image_6.png" /><figcaption>Figure 4: Concentrations of total cells (chlorophyll +) and viable cells (CV6 +) during the growth phases of *Thalassiosira weissflogii* RCC76</figcaption></figure>

In order to assess the CV6 as a vital stain, we analysed the fluorescent signal of *Thalassiosira weissflogii* RCC76 after CV6 labelling treated and not treated with a lethal process (heat treatment, 5 min at 80°C, or addition of a lethal fixative agent-glutaraldehyde at 1% final) and by using a flow cytometer. As shown in figures 5 and 6, the fluorescence signals of living cells were easily distinguished from the dead cells for both lethal treatments. The relative fluorescence of dead populations was compared to the standard of fluorescence and were 0.1% when killed by heating and 1-2 % when killed by the chemical fixative agent, 50 fold to 500-fold lower than those measured from the living cells.

<figure><img src="image_7.png" /><figcaption>Figure 5: Flow cytometry dot plot for *Thalassiosira weissflogii* not treated (A) and heat treated (B) after CV6 staining</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Figure 6: Distribution of green intensities of *T. weissflogii* measured on FL1 channel (expressed in log) after CV6 labelling of cells killed with glutaraldehyde</figcaption></figure>
13 The capability to use the CV6 dye to measure an effect of a UVC-Low pressure (UVC-LP) disinfection treatment was performed on cultured phytoplankton cells and flow cytometry analysis. A treatment #1 consisted of two UVC-LP exposures at 120 mJ/cm² separated in time of 3 hours and the treatment #2 consisted of the same procedure but separated in time by 24 hours. Experiments were done in a glass petri dish (45 mm) inoculated with 18 ml of cultured cells in the exponential growth phase. Between both UVC exposures, cells were kept in static conditions and in the dark to simulate the ballast environment. Cells were analysed before and after both exposures and compared to untreated cells over time. Results presented in figure 7 showed a difference in sensitivity to UVC treatments of the dinoflagellate, *Alexandrium minutum* strain ME Am, and the diatom *Thalassiosira weissflogi* strain RCC76. These differences could be explained by the resistance of the members of *Thalassiosira* genus to UVC (LP) treatment as reported in Liebich et al., 2012. The results showed that the CV6 can be used to determine the efficiency of a UVC disinfection treatment in combination with the flow cytometer device.

<figure><img src="image_9.png" /><figcaption>Figure 7: Percentage of viable cells (i.e positive for CV6) in response of two UVC treatments and without treatment</figcaption></figure>

Scientific assessments of the FluoCount device

14 A specific device, able to automatically detect green fluorescence on a membrane filter and to count fluorescent organisms, was developed during the project. This "FluoCount" is a low-cost device specifically designed to perform the automatic detection and to count the green fluorescent organisms belonging to both size classes after membrane filtration of sample. The system includes a light source (LED), a dichroic mirror, a camera and a holder to analyse a membrane sample. The analysis is performed after filtration of sample through a membrane filter (25 mm diameter). The pore-size of membranes was adapted to the targeted size class: for the size class 10 to 50 µm, a 12 µm pore size membrane is used; for the size class > 50 µm, a 30 µm pore size membrane is used. The software processes the image to measure the intensity of green signal and to count automatically the viable organisms. (figure 8)
<figure><img src="image_10.png" /><figcaption>Figure 8: Schematic diagram for the automatic detection and counting of viable phytoplankton and zooplankton with the FluoCount device</figcaption></figure>

15 The analytical performances of the FluoCount device and the software were firstly determined on fluorescent particle standards (microspheres) that are usually used to assess the performance of the measuring instruments such as flow cytometers and microscopes. The standards had green fluorescence intensities close to those of stained CV6 organisms. Two standards, Microspheres "6-10" and Microspheres "10" were selected to calibrate the FluoCount and were used to simulate phytoplankton and zooplankton green fluorescence after CV6 labelling, respectively. The number of particules measured by the Fluocount were compared with those measured with a solid phase cytometer (ChemScan®). The ChemScan® is a referenced device to automatically detect and count fluorescent organisms with low abundance onto a membrane. The analyses with the ChemScan® were made by using the same membrane-sample as those analysed with FluoCount device. The capability of the FluoCount device to count with the same performances than the ChemScan®, was confirmed as shown in figure 9. The correlation coefficient (R²) values were 0.980 and 0.996, for standards used for phytoplankton and zooplankton, respectively, and the relative standard deviation (RSD) averages were 23.9% and 21.8% for FluoCount and ChemScan® devices, and confirmed the reproducibility of the measures with both devices.
<figure><img src="image_11.png" /><figcaption>Figure 9: Relationships between the FluoCount and ChemScan® count for (A) Microspheres used as standard for phytoplankton and (B) Microspheres used as standard of fluorescence for zooplankton</figcaption></figure>

Scientific assessments of the FluoCount device in conjunction with the CV6 dye to count viable organisms greater than 50 µm

16 The analytical performance of the FluoCount device and the software (zooplankton application) were determined after CV6 labelling of larvae of *Artemia salina*, used as a zooplankton model. As shown in figure 10, the correlation coefficient (R²) value was 0.994 when FluoCount are compared to the number of organisms added. The RSD average was 6.3% for FluoCount. The detection limit of the assay was determined as 1 larvae detected per membrane with accuracy.

<figure><img src="image_12.png" /><figcaption>Figure 10: Relationship between the FluoCount after CV6 labelling and the number of nauplii of Artemia salina in culture (organisms > 50 µm)</figcaption></figure>

17 As recommended by BWM.2/Circ.42/Rev.1, the organisms can be counted through microscopic observations. As shown in figure 11, the correlation coefficient (R²) value was 0.999 between FluoCount and manual count with microscope, the reproducibility was
similar for both devices (5%) and results confirm the capability of the FluoCount to perform an
automatic count with a high accuracy.

<figure><img src="image_13.png" /><figcaption>Figure 11: Results with the FluoCount method and the manual counts (under microscope) of *Artemia salina nauplii* in culture and labelled with CV6</figcaption></figure>

**Action requested of the Committee**

18 The Committee is invited to take note of the information provided.

***
<figure><img src="image_14.png" /></figure>
# ANNEX

## References

Parthuisot N, Catala P, Lemarchand K, Baudart J, Lebaron P. 2000. *Evaluation of ChemChrome V6 for bacterial viability assessment in waters*. J Appl Microbiol. 89(2):370-80.

Johnson PE, Deromedi AJ, Lebaron P, Catala P, Havens C, Pougnard C. 2007
*High throughput, real-time detection of Naegleria lovaniensis in natural river water using LED-illuminated Fountain Flow Cytometry*. J Appl Microbiol. 103(3):700-10.

Liebich V, Stehouwer PP, Veldhuis M. 2012. *Re-growth of potential invasive phytoplankton following UV-based ballast water treatment*. Aquatic Invasions. 7(1): 29-36.