revised guidance on ballast water sampling and … · 2020-02-17 · i:\ppr\07\ppr 7-inf.5.docx e...

$: REVISED GUIDANCE ON BALLAST WATER SAMPLING AND … · 2020-02-17 · I:\PPR\07\PPR 7-INF.5.docx E SUB-COMMITTEE ON POLLUTION PREVENTION AND RESPONSE 7th session Agenda item 4 PPR$
I:\PPR\07\PPR 7-INF.5.docx

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SUB-COMMITTEE ON POLLUTION PREVENTION AND RESPONSE 7th session Agenda item 4

PPR 7/INF.5

11 December 2019 ENGLISH ONLY

Pre-session public release: ☒

REVISED GUIDANCE ON BALLAST WATER SAMPLING AND ANALYSIS

Additional data for validation of CV6 stain combined with the FluoCount as indicative method for enumerating organisms in the 10 to 50 μm size class and in the greater

than 50 µm size class in ballast water

Submitted by France

SUMMARY

Executive summary: This document summarizes the additional 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 10-50 µm) defined in regulation D-2 of the BWM Convention. It provides additional information on the analytical method used in a device under development, combining membrane filtration and the CV6 vital stain, and presents results for the scientific assessment of the method compared to currently accepted methods for ballast water analysis.

Strategic direction, if applicable:

1

Output: 1.14

Action to be taken: Paragraph 32

Related documents: BWM.2/Circ.42/Rev.1; MEPC 74/4/10, MEPC 74/INF.17 and PPR 7/4/1

Introduction

1 In document MEPC 74/INF.17 (France), the preliminary results of the development and assessment of a new analytical method that combines CV6 vital stain, membrane filtration and fluorescence detection in solid phase were presented, for enumerating organisms in the 10 to 50 μm size class and in the greater than 50 μm size class in treated ballast water.

2 This document brings additional data on the application of the commercialized dye ChemChrome V6 (CV6) for fluorescent vital staining of organisms, used in combination with a solid phase analysis by a specifically developed device (FluoCount) for enumerating organisms in the 10 to 50 μm size class and in the greater than 50 µm size class in natural waters.

PPR 7/INF.5 Page 2


3 Two field studies were performed in 2016 and 2019. Their main objective was to

evaluate the performance of the proposed indicative method for rapid count of organisms in

different qualities of waters in comparison with the approved detailed methods contained in

BWM.2/Circ.61 (Guidance on methodologies that may be used for enumerating viable

organisms for type approval of ballast water management systems).

4 This document details the results of the two tests series performed, one with natural

marine communities in water treated with UV exposure only (bench-scale test, in June 2016),

and the other with multispecies assemblies relevant to ballast water treated with filtration and

UV exposures (land-based test) in August 2019. Experimental test #1: benchscale evaluation for enumeration of living organisms after treatment with UV on natural biological communities

5 The test was conducted between 16 and 19 June 2016 at the Oceanological

Observatory of Banyuls sur mer, a French National Institute in Marine Biology, before and after

a disinfection of seawater performed with the UV reactor of a type approved ballast water

management system (BWMS). The objective was to validate the sensitivity of the method to

different levels of treatment on natural communities at bench scale.

6 The UV reactor applied medium pressure UV-C treatment and was composed of one medium pressure UV lamp of 6000 W and one UVI sensor. The flow rate operated during the test was 30 m3/h representing 100% capacity of the UV unit. Tests were performed on seawater collected in harbour with a motopump to provide the 30 m3/h flow rate to the UV reactor. Three conditions of UV exposure were tested during the assay: before treatment (0 W) and after treatment at 2000 W (equivalent to one exposure) and 4000 W (equivalent to two exposures). 7 For zooplankton sampling, 500 L of seawater were sampled immediately after UV exposures. The organisms were concentrated by using the Ballast water kit (HydroBios) described in Gollasch (2006) and the volume of concentrate was adjusted to 250 mL by adding dialyzed seawater (Hemoflow dialysis hollow fiber filter, HF80S). The flow rate applied during the collection of organisms was 60 L/min to reduce damage of organisms during filtration (as preconized by the manufacturer). The organisms positive for CV6 were analysed with replicate (n = 3) after filtration (polycarbonate membrane pore size 30 µm) of 1 mL subsampled from the plankton net concentrate and CV6 staining (the complete procedure is described in document MEPC 74/INF.17). The green fluorescent organisms positive for CV6 were immediately counted with the FluoCount by mean the ZOOCount application. The total procedure of analysis was done within 15 minutes. 8 For phytoplankton sampling, 1 L of water filtered through the 50 microns mesh size plankton net of the ballast water kit was collected in sterile bottle immediately after UV exposures. The phytoplankton cells positive for CV6 were analysed with replicate (n = 3) after filtration (polycarbonate membrane pore size 12 µm) of 1 mL subsample and CV6 staining (complete procedure described in document MEPC74/INF.17). The green fluorescent organisms positive for CV6 were immediately counted with the FluoCount by mean the PHYTOCount application. The total procedure of analysis was done within 15 minutes. 9 As shown in figure 1, the counts measured with the FluoCount for both classes size of organisms (> 50 µm and 10-50 µm in size) decreased gradually depending on the UV exposures (0 W, 2000 W and 4000 W). The repeatability of the results calculated as the average relative standard deviation (RSD) expressed in unit of %, is summarized in table 1. These results highlighted the capability of CV6/FluoCount method to estimate the number of active organisms for both classes after UV-C treatment from natural marine communities.

PPR 7/INF.5 Page 3


Figure 1: Counts of organisms positive for CV6 measured with FluoCount depending

on the UV-C treatment.

Table 1: Average count (Avg.), standard deviation (Std.) of CV6/FluoCount (n=3) and relative standard deviation (RSD) expressed in unit of %.

Experimental test #2: evaluation for enumeration of living organisms after full treatment on multispecies assemblies during land-based testing of ballast water management system 10 The study was performed during land-based testing of a UV-based ballast water management system (BWMS) in August 2019 at NIVA's test facility in Norway, according to the requirements of IMO and the United States Coast Guard (USCG). The objective was to compare the results given by the indicative method to the results given by the approved detailed methods. 11 BWMS applied filtration and UV treatment during ballasting operation, and UV treatment only during discharge operation. UV treatment dose was chosen for inactivation of organisms (compliance determined by the MPN method for the 10-50 µm category) but counts were also performed according to the USCG/EPA standard, using CMFDA/FDA. The retention time between ballasting and discharge operations was 24 hours approximately. BWMS was operated with flow rate regulation according to UV-Intensity (UVI) measurements. 12 Between 5 and 13 August 2019, four tests were carried out from three qualities of water: brackish water (one cycle BW), freshwater (one cycle FW) and seawater (two cycles SW). Each cycle required analysis of challenge water (untreated water), treated ballasted water (challenge water treated with filtration and UV exposure) and treated deballasted water (full treatment applied with retention).

0

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0 W 2000 W 4000 W

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LOFLO-UV treatment

0

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0 W 2000 W 4000 W

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etw

een 1

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LOFLO-UV treatment

Avg. Std. RDS Avg. Std. RDS

0W 39,7 1,15 2,9 1090,3 27,5 2,5

2000W 22,7 4,7 20,8 439,7 21,3 7,1

4000W 2,3 1,1 49,5 284,3 6,5 2,3

Organisms > 50µm/mL of

concentrateOrganisms 10-50µm/mL

UV treatment

PPR 7/INF.5 Page 4


13 Water sampling was performed by NIVA as follows:

- for untreated challenge water, a total of 1 m3 time-integrated sample of water was collected into a 1 m3 clean plastic tank during the entire pumping operation. For ≥ 50 µm organisms, 3x 20 L subsamples from this 1 m3 of time-integrated sample were collected into a clean plastic bucket and sieved through a plankton net with diagonal dimension of 50 µm. Each sieved sample was collected in 100 mL clean glass bottle. For ≥ 10-50 µm organisms, 1x1 L subsamples from this 1 m3 of time-integrated sample was collected in clean plastic bottle.

- for treated (ballasted and deballasted) waters, a total of at least three consecutive

1 m3 samples were collected and sieved successively during the entire pumping operation for ≥50 µm organism analysis into respective 1 m3 clean plastic tanks. Each sieved 1m3 sample was collected into one 100 mL clean glass bottle. For organisms 10-50 µm, a total of up to 20 L time integrated (non-sieved) sample was collected continuously during the entire pumping operation into a clean plastic jerry can. After homogenization by gentle shaking of the can, 1 x 1 L subsamples were collected in clean plastic bottle for ≥ 10-50 µm organism analysis.

14 Detailed analysis method for enumerating organisms > 50 µm was performed by NIVA from the 3 x 20 L or the 3 x 1 m3 sieved subsamples. The detailed method consisted in dissecting microscope with 10-40x magnification (NIVA method 17113) and results were expressed as living organisms > 50 µm in size/m3. 15 Detailed analysis methods for enumerating organisms 10-50 µm was performed by NIVA from the 1L subsampled. The densities of living organisms 10-50 µm in size were determined by two standard detailed analysis methods. The first one was the MPN culture method (Agilent Technologies-Cary Eclipse Fluorescence Spectrometer; NIVA method 17110). The second one was a staining method; FDA/CMFDA stains combined with epifluorescence microscope (excitation filter 485 nm; emission filter 530 nm) at 100-250 times magnification (NIVA method 17111). The results were expressed as living organisms of 10-50 µm in size/mL. 16 The requirements of BWMS Code (resolution MEPC.300(72)) regarding organisms density measured by approved methods in tested waters were fulfilled in all test cycles in inlets and controls (ballasting and discharge days) (table 2).

PPR 7/INF.5 Page 5


Table 2: Living organisms in the inlets and control waters (ballasting and discharge days) measured by the approved detailed methods (Avg., average; Std., standard deviation; MPN, Most probable number; Ci. Confidential interval). Source: NIVA.

17 Organisms > 50 µm were analysed by the CV6/FluoCount method from 1 x 20L sieved sample for challenge water and 1x1 m3 sieved sample for treated ballasted and deballasted water samples (see paragraph 13 for more details on the sampling process). Staining was performed after gentle filtration (polycarbonate membrane pore size 30 µm) of 1 or 10 mL of subsamples from the plankton net concentrate. After filtration, organisms were stained with the CV6 dye (complete procedure described in document MEPC 74/INF.17) for 10 minutes at dark and the green fluorescent organisms positive for CV6 were immediately counted with the FluoCount by mean the ZOOCount application. The average counts were determined from 3 X 1 mL of concentrate for challenge and ballasted water and from 3 x 10 mL of concentrate for deballasted water. The total procedure of analysis was done within 15 minutes. The counts were expressed as organisms/m3 by taking into account the total volume of the concentrate. The comparison between indicative method (CV6/FluoCount) and detailed methods (microscope observation) was performed from the same water sample. 18 The counts for the > 50 µm group of organisms measured with the CV6/FluoCount are presented in figure 2. They decreased gradually from challenge to deballasted waters. The estimations in deballasted water were close (i) to the counts obtained with the recommended method using microscope and (ii) close to the D-2 threshold value for compliance (< 10 org./m3). All deballasted samples were compliant with the D-2 discharge standard when organisms were counted by the microscope method (tables 3 and 4).

PPR 7/INF.5 Page 6


Figure 2: Count of organisms > 50 µm measured with CV6/FluoCount during the IMO

land-based tests conducted at NIVA’s test facility in August 2019. Table 3: Average counts (org./m3), standard deviation (Std.) and RDS expressed in unit of % of living organisms > 50 μm obtained by the microscope method. Source: NIVA.

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Deballasted water

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Water sample

Seawater_ test 2

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Freshwater_ test 1

<284

Avg. Std. RDS Avg. Std. RDS Avg. Std. RDS

BW-test 1 256792 12488 5 344 22 6 0.6 0.6 100

FW-test 1 109358 2403 2 229 82 36 <1 - -

SW-test 1 161304 17552 11 616 143 23 <1 - -

SW-test 2 150708 9191 6 760 172 23 <1 - -

Ballasting day Discharge day

Water quality

and test cycle N°Before treatment After treatment After treatment

PPR 7/INF.5 Page 7


Table 4: Average counts (org./m3), standard deviation (Std.) and RDS expressed in unit of % of living organisms > 50 μm obtained by the CV6/FluoCount.

19 The repeatability of results for organisms > 50 µm estimated with the RDS calculation ranged between 16 and 43% for CV6/FluoCount in challenge water and between 44 and 161% in deballasted water. For comparison, the RDS for the microscope results in challenge water, ranged between 2 and 11% (tables 3 and 4). 20 The CV6/FluoCount was significantly and positively correlated with the microscope

counts (Pearson test, r = 1, p < 0,0001 for = 0,05). The untransformed data obtained by both methods were compared with a paired t test. Both counts were not significantly different

(P=0,1201, for = 0,05) and the pairing was significantly effective (P value < 0.0001). The capability of the CV6 stain combined with the FluoCount device to count organisms > 50 µm in the same performance as the standard microscope method was confirmed as shown in figure 3.

Figure 3: Relationships between mean counts of organism > 50 µm measured with the microscope and the CV6/FluoCount during land-based testing of UV-based BWMS.

Challenge, ballasted and deballasted water samples are represented with white circles, crosses and black circles, respectively. The microscope analyses with a result under the limit of detection of the method were considered as negative and counted as x =0.

21 These results confirmed the capability of CV6/FluoCount method to estimate the number of active organisms > 50 µm in size before and after BWMS treatment combining filtration and UV treatments. The presented method offers the main advantage to be easy to perform and faster (< 15 minutes) 22 The organisms 10-50 µm counted by CV6/FluoCount were collected as described in paragraph 13. Nevertheless, at the stage of development of the PHYTOCount application, it is impossible to discriminate the organisms according to their size. To solve the problem, the 1L


BW-test 1 489000 122474 25 3657 344 9 12,6 5,5 44

FW-test 1 60200 11363 19 <284 - - 12,2 19,7 161

SW-test 1 332500 52034 16 4500 756 17 5 5 100

SW-test 2 485333 208838 43 1867 1048 56 29,2 13,8 47

After treatment After treatmentWater quality

and test cycle N°


Before treatment

y = 2.0459xR² = 0.85784

0

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100 000

1 000 000

0 1 10 100 1 000 10 000 100 000 1 000 000

CV

6/F

luo

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t (x+

1)/

m3

Microscope count (x+1)/m3

n = 11

PPR 7/INF.5 Page 8


of samples was filtered with a nylon filter 50 µm mesh size to remove organisms > 50 µm before analysis. Comparatively, no 50 µm prefiltration step was performed for the FDA/CMFDA analyses. The CV6/FluoCount analysis was performed from water samples as following: after gentle filtration (polycarbonate membrane pore size 12 µm) of 1 or 10 mL of prefiltered subsamples, organisms were stained with the CV6 dye (complete procedure described in document MEPC 74/INF.17) for 10 minutes at dark and the green fluorescent organisms positive for CV6 were immediately counted with the FluoCount by using the PHYTOCount application. The total procedure of analysis was done within 15 minutes. The average counts were determined from 3 X 1 mL for challenge and ballasted water and from 3 x 10 mL for deballasted water. The counts were expressed as organisms/mL. The comparison between indicative method and both detailed methods (MPN and FDA/CMFDA) was performed from the same water sample. 23 The counts for the 10-50 µm group of organisms measured with the CV6/FluoCount method are presented in the figure 4. As shown, counts decreased gradually from challenge

water to deballasted water.

Figure 4: Mean counts of organisms 10-50 µm measured with CV6/FluoCount during the IMO tests conducted at NIVA's test facility in August 2019. 24 For the 10-50 µm group of organisms in deballasted water, the counts were in compliance with the D-2 standard (< 10 org./mL) only for the BW test when analyzed with the CV6/FluoCount and only for the FW test when analyzed with the FDA/CMFDA method (tables 5 and 6). No compliance was given for the two seawater tests with both staining based approaches. Comparatively, all waters tested with the MPN method were in compliance with the D-2-threshold value (table 7).

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PPR 7/INF.5 Page 9


Table 5: Average counts (org./mL), standard deviation (Std.) and RDS expressed in unit of % of organisms 10-50 µm obtained by the FDA/CMFDA method. Source: NIVA.

Table 6: Average counts (org./mL), standard deviation (Std.) and RDS expressed in unit of % of organisms 10-50 µm obtained by the CV6/FluoCount method.

Table 7: Most Probable Number (MPN) (org./mL) and confidential interval (CI) of living

organisms 10-50 µm. Source: NIVA.

25 The repeatability of CV6/FluoCount results for counting organisms of 10-50 µm estimated with the RDS calculation ranged between 2 and 24% in challenge water and between 7 and 39% in deballasted water, respectively. For comparison, the RDS for FDA/CMFDA were in the same range of values (1-6% in challenge water and 9-40% in deballasted water) (tables 5 and 6). 26 The relationships between counts measured with both staining methods is presented in figure 5. The CV6/FluoCount was significantly and positively correlated with the

FDA/CMFDA counts (Pearson test, r = 0.8502, p= 0,0005, for = 0,05). The untransformed data obtained by both methods were compared with a paired t test. Both counts were not

significantly different (P=0,6039, for = 0,05) and the pairing was significantly effective (P value < 0.0001) The capability of the CV6 stain combined with the FluoCount device to count organisms 10-50 µm in a similar linear range of values than FDA/CMFDA was confirmed as shown in figure 5.

CMFDA/FDA >10-50µm (org./ml)


BW-test 1 1029 58 6 287 21 7 27 7 26

FW-test 1 1146 26 2 94 11 12 2.5 1 40

SW-test 1 1400 50 4 197 42 21 38 5 13

SW-test 2 1504 19 1 205 33 16 43 4 9

After treatmentWater quality

and test cycle N°


Before treatment After treatment


BW-test 1 332 14 4 74 16 22 7.9 3.1 39

FW-test 1 925 220 24 329 140 43 42.4 2.8 7

SW-test 1 690 17 2 456 98 21 61 9 15

SW-test 2 964 109 11 409 29 7 58.8 16.8 29

Water quality and

test cycle N°


Before treatment After treatment After treatment

MPN >10-50µm (org./ml)

MPN C.i. MPN C.i. MPN C.i.

BW-test 1 1100 570-2100 31 17-55 0.13 0.03-0.51

FW-test 1 1600 860-3000 8 2.7-23 <0.06 0.01-0.47

SW-test 1 1600 860-3000 40 16-98 0.13 0.03-0.51

SW-test 2 2000 1100-3800 30 14-68 0.06 0.01-0.44

Water quality

and test cycle N°


Before treatment After treatment After treatment

PPR 7/INF.5 Page 10


Figure 5: Relationships between mean counts of organism 10-50 µm measured with

CV6/FluoCount and FDA/CMFDA during the land-based test of UV-based BWMS. Challenge, ballasting and deballasting water samples are represented with white

circles, crosses and black circles, respectively.

27 The CV6/FluoCount shows the same precision than the standard FDA/CMFDA for counting organisms in the 10-50 µm category and the analysis is faster (< 15 minutes). 28 The comparison of the results obtained from MPN and staining based methods can be done by calculating the Log10 ratio between counts, as proposed by Blatchley et al. (2018) and graphical comparison is presented in figure 6. For the challenge water samples, results from MPN and FDA/CMFDA were similar (value close to 0) and same similarity was found from MPN and CV6-FluoCount. Both staining-based methods show the same abilities to quantify viable phytoplankton in multispecies assemblies relevant to untreated ballast water. 29 For the deballasted water samples, the ratio measured from the two staining-based methods were similar and ranged between -2,99 to -1,78 and -2,85 to -1,69 when CV6/FluoCount and FDA/CMFDA are compared with the MPN, respectively. Results suggested that both staining methods measure higher counts - in the same range - than the regrowth assay. The observed discrepancy between staining methods and regrowth assay from UV treatment system were already reported by Blatchley et al. (2018).

Figure 6: Log10-transformed ratio of MPN: stain counts for challenge water and deballasting water samples. Log10 (MPN : stain) = 0 implies no difference between assays.

n = 12

y = 0.5979xR² = 0.65333

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Lo

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/CV

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t)

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-1

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Lo

g (M

NP

/FD

A-C

MF

DA

)

PPR 7/INF.5 Page 11


30 The apparent treatment efficiency calculated as proposed by Blatchley et al. (2018)

as 𝐿𝑜𝑔10 (𝑖𝑛𝑡𝑎𝑘𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛

𝑑𝑖𝑠𝑐ℎ𝑎𝑟𝑔𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛) were compared between methods and were in a same range

of variation for all methods and for both organisms classes (table 8).

Table 8: Apparent treatment efficiencies obtained with the different methods in deballasted waters (discharge)

Water quality and test cycle N°

Counting method BW-test1 FW-test1 SW-test1 SW-test2

Organisms >50µm

CV6/FluoCount 4.59 3.69 4.82 4.22

Microscope+motility 5.63 5.04 5.21 5.18

Organisms 10-50µm

CV6/FluoCount 1.58 2.66 1.57 1.54

FDA/CMFDA 1.62 1.34 1.05 1.21

MPN 3.93 4.42 4.09 4.52

31 The additional data obtained from both tests series performed in 2016 and 2019 confirmed that the CV6/FluoCount method is a promising fast method for estimating the density of organisms in the 10 to 50 μm size class and in the greater than 50 μm size class in untreated and UV-treated ballast water. Action requested of the Sub-Committee 32 The Sub-Committee is invited to take note of the information provided.

***

PPR 7/INF.5 Annex, page 1


ANNEX References Blatchley ER, Cullen JJ, Petri B, Bircher K, Welschmeyer (2018) The biological basis for ballast water performance standards : « viable/non-viable » or « live/dead » ? Environmental Science & Technology. 52 : 8075-8086. Gollasch S (2006) A new ballast water sampling device for sampling organisms above 50µm. Aquatic Invasions. 1 : 46-50.

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