Recommendations for Representative Ballast Water Sampling. Final report of research study of the Bundesamt für Seeschifffahrt und Hydrographie (BSH), Hamburg, Germany. Order Number 4500025702

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Bundesamt für Seeschifffahrt und Hydrographie
Hamburg, Germany
RESEARCH STUDY
Recommendations for Representative Ballast Water
Sampling
(Order Number 4500025702)
FINAL REPORT

I
Recommendations for Representative Ballast Water Sampling
Title:
(Order Number 4500025702)
Bundesamt für Seeschifffahrt und Hydrographie (BSH)
Hamburg, Germany
Dr. Stephan Gollasch,
Researchers:
GoConsult, Hamburg, Germany
Prof. Dr. Matej David,
David Consult, Korte, Slovenia
Hamburg, Korte, January, 2013

II
Citation and disclaimer
This report should be quoted as follows:
Gollasch S. & David, M. 2013. Recommendations for Representative Ballast Water
Sampling. Final report of research study of the Bundesamt für Seeschifffahrt und
Hydrographie (BSH), Hamburg, Germany. Order Number 4500025702. 28 pp.
The contents and views contained in this report are those of the authors, and do not
necessarily represent those of the Bundesamt für Seeschifffahrt und Hydrographie.

Gollasch S., David M., BSH Project: Recommendations for Representative Ballast Water Sampling, Final report
III
TABLE OF CONTENTS
EXECUTIVE SUMMARY ................................................................................................. 1
1 INTRODUCTION ....................................................................................................... 3
2 MATERIALS AND METHODS ............................................................................... 4
2.1 TANKS AND BALLAST WATER................................................................................ 4
2.2 SAMPLING ARRANGEMENTS ................................................................................. 5
2.2.1 Sampling process ........................................................................................ 7
2.3 SAMPLES PROCESSING AND ANALYSES ............................................................... 13
3 RESULTS OF THE SHIPBOARD SAMPLING TEST ........................................ 15
3.1 ZOOPLANKTON (ABOVE 50 µM IN MINIMUM DIMENSION) .................................. 18
3.2 PHYTOPLANKTON (BELOW 50 µM AND ABOVE 10 µM IN MINIMUM DIMENSION) 20
4 HOW TO TAKE A REPRESENTATIVE SAMPLE? .......................................... 22
4.1 ZOOPLANKTON (ABOVE 50 µM IN MINIMUM DIMENSION) .................................. 22
4.2 PHYTOPLANKTON (BELOW 50 µM AND ABOVE 10 µM IN MINIMUM DIMENSION) 22
4.3 SAMPLING RECOMMENDATIONS ......................................................................... 22
4.3.1 Organisms 50 µm and above .................................................................... 23
4.3.2 Organisms below 50 and above 10 µm .................................................... 23
5 ACKNOWLEDGEMENTS ..................................................................................... 24
LIST OF FIGURES ........................................................................................................ 25
LIST OF TABLES ......................................................................................................... 26
REFERENCES .............................................................................................................. 27

Gollasch S., David M., BSH Project: Recommendations for Representative Ballast Water Sampling, Final report
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EXECUTIVE SUMMARY
The objectives of this project included to contribute to the ongoing discussions at IMO
regarding procedures and methods how to take representative biological samples of
discharged ballast water.
One vessel voyage was undertaken in November 2012 during which four ballast sampling
tests were conducted to meet the study objectives. These tests were conducted on the
container vessel COSCO Guangzhou with the support of Environmental Protection
Engineering S.A. (Perama, Greece), the developer of the ERMA FIRST ballast water
treatment system.
The samples were taken from the ship’s ballast water line during ballast water discharge. The
water flow was split in two equal parts to enable a parallel comparison of samples taken over
the entire time (OET) of the deballasting operation with samples taken sequentially. The
sequential sampling was arranged so that no time gap occurred between the individual
sequences, i.e. the sequential samples were 8-10 minutes in length, taken consecutively, so
that they covered the entire discharge period in 7-8 samples.
The study conducted focussed on organisms above 10 micrometers in minimum dimension.
All samples were analysed for living organisms greater than or equal to 50 micrometers in
minimum dimension (mainly zooplankton) and for organisms less than 50 micrometers and
greater than or equal to 10 micrometers in minimum dimension (mainly phytoplankton).
The sequential trials showed different organism numbers in each sequence of one test
indicating the patchy organism distribution inside the ballast tank. This was observed during
all sampling events and for both organism groups studied.
For organisms 50 micron and above in minimum dimension it was observed that, with the
exception of test 3, in all tests the organism concentration of the very first and last sequences
are above the mean organism concentration. In test 3 the highest organism concentration was
found in the end sequence and in the middle sequences.
For the phytoplankton samples below 50 and above 10 micron in minimum dimension in test
2 the first, fourth and end sequences delivered higher organism counts then the mean value
determined. In test 3 the first four sequences are above the mean organism concentration and
in test 4 the first, second and fifth sequence delivered higher organisms compared to the mean
value.
For the group of organisms 50 µm and above the samples taken over the entire time
contained fewer organisms compared to the sequential samples. We assume that the
sequential samples deliver more representative results.
Our study has shown that sequential samples taken in the very beginning and end during a
ballast tank is emptied are unlikely to give representative results of the organism
concentration because in these samples the organism count showed high variations, which
may result in under- or oversampling the organism concentration. These findings are in line
with an earlier study (Gollasch & David 2010a).

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Assuming the mean organism count from all sequences of one test shows the true organism
concentration in the tank, we recommend to avoid taking a sample during the very beginning
and end of the discharge time of a tank or tanks. By avoiding these time windows the samples
taken are nearest to the mean organism count of all sequences.
Although the best sampling time may be in the middle of a discharge, the organism
concentration still seems to be patchy so that we recommend taking at least two samples in
this time window. The mean value of the organism concentration in these two samples may be
taken to assume the real organism concentration.
For the group of organisms below 50 and above 10 µm, in contrast to the larger organisms,
the continuous drip over the entire time samples contained higher organism counts for the
smaller organisms compared to the mean of sequential samples.
The comparison of the concentration of the smaller organisms between the different
sequences of all tests showed that no clear trend can be identified during which time window
a more representative sample will be taken. Therefore we recommend taking at least two
samples during the discharge of a ballast water tank with avoiding taking a sample during the
very beginning and end of the discharge time of a tank or tanks. The mean organism count in
these two or more samples may be seen as the real organism concentration in the ballast water.
This study has shown that different approaches (i.e., short/long sampling times) in the
sampling process result in different organism concentrations. Therefore the selection of an
inappropriate sampling approach will influence the compliance control result. As a
consequence the organism concentrations in the ballast water discharge may therefore be
underestimated, so that an inefficient ballast water treatment system (BWTS) could be
recognised as compliant. In contrast, organism concentrations may be also overestimated, and
a BWTS complying with the D-2 Standard may fail in compliance tests.

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1 INTRODUCTION
This study was undertaken to contribute to the ongoing discussions regarding procedures and
methods to take samples which result in representative numbers of organisms in discharged
ballast water. Representative samples need to be taken according to the organism groups
addressed in the Ballast Water Performance Standard as outlined in Regulation D-2 of the
Ballast Water Management Convention.
Regulation D-2 of the Ballast Water Management Convention stipulates that ships meeting
the requirements of the Convention shall discharge:
• less than 10 viable organisms per cubic meter greater than or equal to 50 micrometers in
minimum dimension, and
• less than 10 viable organisms per millilitre less than 50 micrometers in minimum
dimension and greater than or equal to 10 micrometers in minimum dimension, and
• less than the following concentrations of indicator microbes, as a human health standard:
• Toxigenic Vibrio cholerae (serotypes O1 and O139) with less than 1 Colony Forming
Unit (cfu) per 100 millilitres or less than 1 cfu per 1 gramme (wet weight) of
zooplankton samples,
• Escherichia coli less than 250 cfu per 100 millilitres, and
• Intestinal Enterococci less than 100 cfu per 100 millilitres.
There are still uncertainties to assess compliance with the D-2 Standard due to the lack of
knowledge how to take representative ballast water samples. The need for a representative
sample, and the difficulties involved in obtaining such a representative sample, cannot be
overstressed. Consequently, Germany funded an on board scientific ballast water sampling
study comparing different sampling scenarios with the aim to evaluate how representative
samples for compliance control with the D-2 Standards may be taken.
This study was undertaken in November 2012 on the container vessel COSCO Guangzhou
with the support of Environmental Protection Engineering S.A. (Perama, Greece), the
developer of the ERMA FIRST ballast water treatment system. The tests were independent
from any possible onboard performance tests of the ERMA FIRST ballast water treatment
system. However, as ships currently are lacking in-line sampling points, this vessel was
selected as such sampling points were installed on this vessel for previously completed tests
of the performance of the treatment system.
The on board sampling team consisted of Stephan Gollasch and Matej David. Both are
leading scientists in Europe for ballast water sampling as they have shown in earlier projects
(Gollasch 1996, Gollasch et al. 2000a-c, David & Perkovic 2004, David et al. 2007, Gollasch
& David 2009, Gollasch & David 2010a,b, David & Gollasch 2011, Gollasch & David 2011).

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2 MATERIALS AND METHODS
2.1 TANKS AND BALLAST WATER
The study was undertaken on the container vessel COSCO Guangzhou (IMO Number
9305570). The vessel particulars are DWT 107,526 t, cargo capacity 9500 TEU, maximum
ballast water capacity 30,115 m³ in 35 tanks. The vessel voyage took place in November 2012
between ports along the Canadian west coast.
Table 1 - Main dimensions of the vessel and tank details.
Details
COSCO Guangzhou
Vessel type
Container
Length overall
350.57 m
Dead Weight Tonnage (DWT)
107,526 t
Container capacity
9,500 TEU
Total ballast water capacity
30,115 m³
Number of ballast tanks
35
Number of ballast pumps
2
Capacity of each ballast pump
500 m³/h
Number of BWTS installed
1
Capacity of BWTS
500 m³/h
Four ballast water tanks, Nr. 3 SBWT (side ballast water tank) and 4 SBWT port (P) and
starboard (S) were dedicated for the tests. All tanks were side tanks. Each of the tanks Nr. 3
SBWT were of a capacity of 637.4 m
3
, and Nr. 4 SBWT of 476.6 m
3
. Tank Nr. 4 SBWT (S)
was full when the sampling team arrived on board, it was ballasted during the voyage across
the Pacific Ocean (approximate location 50
ᵒ
45’N 166
ᵒ
51’E) on 28
th
Oct, 18-19h UTC (09-10h
Canada west coast local time). The remaining three tanks, Nr. 3 SBWT (P) and (S), and Nr. 4
SBWT (P), were completely empty, and were ballasted in the Port of Prince Rupert (Canada)
on 03
rd
November, 18.30-20.30h (local time).
The voyage was undertaken in November 2012 between Port of Prince Rupert and Vancouver
(Canada). The ballast water sampling programme is shown in Table 2.

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Table 2 – Ballast water sampling events and tank details.
Discharge test
date
Tank
Uptake date
Uptake location
Volume (m
3
)
04.11.12
4 SBWT (S)
28.11.12
Pacific Ocean
476.6
05.11.12
4 SBWT (P)
03.11.12
Prince Rupert
476.6
06.11.12
3 SBWT (P)
03.11.12
Prince Rupert
637.4
07.11.12
3 SBWT (S)
03.11.12
Prince Rupert
637.4
2.2 SAMPLING ARRANGEMENTS
The samples were taken from the ship’s ballast water line during ballast water discharge. The
in-line sampling point was located in a straight section of the ballast water pipe and its design
is shown in Figure 1.
Figure 1 – Design of the in-line ballast water sampling point.
The water flow was split in two equal parts (see Figure 2) to enable a parallel comparison of
samples taken over the entire time (OET) of the deballasting operation with samples taken
sequentially.

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Figure 2 - Flow splitter to generate two equal splits of ballast water enable a parallel
comparison of samples.
Three sampling bins with submersible pumps for overboard discharge of sampled water were
arranged (see Figure 3).
Figure 3 – Sampling point set-up with three sampling bins with submersible pumps.

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2.2.1 SAMPLING PROCESS
One split was used for sampling over the entire discharge time (OET) of the ballast water tank
and the second split was used for sampling in sequences (see Figure 4).
Figure 4 – Zooplankton sampling nets, left the sample taken over the entire time, right the two
nets used for the sequential samples.

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Page - 8 -
For zooplankton samples plankton nets with a removable filtering cod-end with a mesh size of
50 µm in diagonal dimension were used (see Figure 5).
Figure 5 - Unscrewing the removable cod-end form the zooplankton sampling net.
During all the period of sampling the nets were sitting in the water as deep as practicable to
avoid as much as possible negative impacts on organisms during the sampling period (see
Figure 6 ).
Figure 6 - Position of zooplankton net with a high water level in the water collecting bin.

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For organisms less than 50 micrometers in minimum dimension and greater than or equal to
10 micrometers in minimum dimension, an integrated “continuous drip” sample was provided
by taking samples every 100 litres in the OET sample, and every 50 litres in sequential
samples (see Figure 7 ).
Figure 7 - Taking a “continuous drip sample” with the jar to collect the sample in the bucket
in front of the yellow bin.
Overall approximately 6 litre samples were collected per each “continuous drip” sample (see
Figure 8).

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Figure 8 - Water buckets of one test run. In front of each bucket are the phytoplankton
samples which were taken in duplicate, one sample with living cells and another Lugol-
preserved.
The sequential sampling was arranged so that no time gap occurred between the individual
sequences, i.e. the sequential samples were 8-10 minutes in length, taken consecutively, so
that they covered the entire discharge period in around 7-8 samples. In each test the total
volume sampled for the OET sample and the total volume of all sequences were identical. The
sample volume of all individual sequences was the same during each test run. In total 4 ballast
water discharge sampling events were undertaken which resulted in 35 samples. One
sampling event was undertaken per day during 4-7 November 2012 (see Table 3).

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Table 3 – List of the samples taken also showing the start and end time of each sampling
event with duration and the water quantity sampled. Quantity sampled in litres, flowrate in
litres per minute.
Test
No.
Date Sample type
Start time
[h:min]
End time
[h:min]
Sampling
time
[h:min]
Time
between
sequences
[h:min]
Quantity
sampled
Sampling
flowrate
04.11.12 Discharge sequence 1 9:25 9:33 0:08 370 46
04.11.12 Discharge sequence 2 9:33 9:41 0:08 0:00 370 46
04.11.12 Discharge sequence 3 9:41 9:49 0:08 0:00 370 46
04.11.12 Discharge sequence 4 9:49 9:57 0:08 0:00 370 46
04.11.12 Discharge sequence 5 9:57 10:05 0:08 0:00 370 46
04.11.12 Discharge sequence 6 10:05 10:13 0:08 0:00 370 46
04.11.12 Discharge sequence 7 10:13 10:18 0:05 0:00 224 45
04.11.12 Discharge OET 9:25 10:18 0:53 2443 46
05.11.12 Discharge sequence 1 9:31 9:38 0:07 350 50
05.11.12 Discharge sequence 2 9:38 9:45 0:07 0:00 350 50
05.11.12 Discharge sequence 3 9:45 9:52 0:07 0:00 350 50
05.11.12 Discharge sequence 4 9:52 9:59 0:07 0:00 350 50
05.11.12 Discharge sequence 5 9:59 10:06 0:07 0:00 350 50
05.11.12 Discharge sequence 6 10:06 10:13 0:07 0:00 350 50
05.11.12 Discharge sequence 7 10:13 10:20 0:07 0:00 350 50
05.11.12 Discharge sequence 8 10:20 10:27 0:07 0:00 350 50
05.11.12 Discharge OET 9:31 10:27 0:56 2800 50
06.11.12 Discharge sequence 1 9:24 9:33 0:09 430 45
06.11.12 Discharge sequence 2 9:33 9:43 0:09 0:00 430 45
06.11.12 Discharge sequence 3 9:43 9:52 0:09 0:00 430 45
06.11.12 Discharge sequence 4 9:52 10:02 0:09 0:00 430 45
06.11.12 Discharge sequence 5 10:02 10:11 0:09 0:00 430 45
06.11.12 Discharge sequence 6 10:11 10:21 0:09 0:00 430 45
06.11.12 Discharge sequence 7 10:21 10:30 0:09 0:00 430 45
06.11.12 Discharge sequence 8 10:30 10:40 0:09 0:00 430 45
06.11.12 Discharge OET 9:24 10:40 1:16 3440 45
07.11.12 Discharge sequence 1 10:41 10:51 0:10 460 46
07.11.12 Discharge sequence 2 10:51 11:01 0:10 0:00 460 46
07.11.12 Discharge sequence 3 11:01 11:11 0:10 0:00 460 46
07.11.12 Discharge sequence 4 11:11 11:21 0:10 0:00 460 46
07.11.12 Discharge sequence 5 11:21 11:31 0:10 0:00 460 46
07.11.12 Discharge sequence 6 11:31 11:41 0:10 0:00 460 46
07.11.12 Discharge sequence 7 11:41 11:51 0:10 0:00 460 46
07.11.12 Discharge sequence 8 11:51 11:58 0:07 0:00 335 46
07.11.12 Discharge OET 10:41 11:58 1:17 3555 46
1
2
3
4

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Table 4 - Sampling event duration (sequential samples in blue shading, the samples taken over
the entire time (OET) in grey shading).

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2.3 SAMPLES PROCESSING AND ANALYSES
The study conducted focussed on organisms above 10 micrometers in minimum dimension.
All samples were analysed for living organisms greater than or equal to 50 micrometers in
minimum dimension (mainly zooplankton) and for organisms less than 50 micrometers in
minimum dimension and greater than or equal to 10 micrometers in minimum dimension
(mainly phytoplankton).
For phytoplankton analyses the samples were transported to the expert team of the Royal
Netherlands Institute for Sea Research (NIOZ), Texel, the Netherlands and two times 100
millilitres of sample were separated after mixing, and one of these was preserved with Lugol
Solution, the other was kept unpreserved (see Figure 8).
The zooplankton samples were processed directly after sampling and living organisms
counted on board the vessel (see Figure 9).
Figure 9 - Counting of zooplankton organisms on board using a stereomicroscope.
The viability of the phytoplankton organisms was measured on board by using a Pulse-
Amplitude Modulation (PAM) instrument which gives a bulk phytoplankton viability result
(see Figure 10). The details of this method are described elsewhere (Gollasch 2010, Gollasch
& David 2010, Gollasch et al. 2012).

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Figure 10 - The Pulse-Amplitude Modulation (PAM) instrument to measure phytoplankton
viability.
The phytoplankton samples were kept in a dark and cool environment during storage and
shipment to NIOZ. After arrival at NIOZ the samples were processed by using a cell counting
machine.

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3 RESULTS OF THE SHIPBOARD SAMPLING TEST
The data were sorted per organism size group (Table 5). For phytoplankton the number of
living cells less than 50 micrometers in minimum dimension and greater than or equal to 10
micrometers in minimum dimension and for zooplankton the number of living organisms
greater than or equal to 50 micrometers in minimum dimension is given. During the first test
run the viability measurement of the phytoplankton indicated that all algae were dead or dying
so that the data from this test were excluded from further analysis.
Table 5 - Number of living organisms in the samples (OET = sample taken over the entire
discharge time of the tank; S= sequence; ml= millilitre; m³ = cubicmeter; µm = micrometre).

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It was observed that the organism concentrations in the sequential samples of each test were
always different which indicated the patchy distribution of organisms in the tank.
Figure 11 – Numbers of living organisms 50 µm and above in minimum dimension/m3 of
ballast water. The blue columns show numbers of living organisms in different sequential
samples, the red columns in OET = sample taken over the entire discharge time of the tank,
and the orange columns show mean numbers of living organisms in sequential samples per
test.
0
200
400
600
800
1.000
1.200
1.400
1.600
1.800
2.000
S1
S2
S3
S4
S5
S6
S7
OET
SeqMean
S1
S2
S3
S4
S5
S6
S7
S8
OET
SeqMean
S1
S2
S3
S4
S5
S6
S7
S8
OET
SeqMean
S1
S2
S3
S4
S5
S6
S7
S8
OET
SeqMean
Test 1
Test 2
Test 3
Test 4
Sequences
number of
organisms
50 µm and
above/ m3
OET
number of
organisms
50 µm and
above/m3
Sequences
mean
number
50 µm and
above/m3

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Figure 12 - Numbers of living organisms below 50 and above 10 micron in minimum
dimension/m3 of ballast water. The blue columns show numbers of living organisms in
different sequential samples, the red columns in OET = sample taken over the entire discharge
time of the tank, and the orange columns show mean numbers of living organisms in
sequential samples per test.
Figure 13 shows the content in the concentrated samples for the living organisms 50 micron
and above. It is obvious that the sample taken over the entire time (OET) contains a lot more
material compared to the sequences. Considering the sequences, the beginning sequence has a
high material content, the lowest content was found in sequence 2 from which an increase was
observed to the end sequence.
0
5
10
15
20
25
30
35
40
45
R1
R2
R3
R4
R5
R6
R7
R8
OET
SeqMean
R9
R10
R11
R12
R13
R14
R15
R16
OET
SeqMean
R17
R18
R19
R20
R21
R22
R23
R24
OET
SeqMean
Test 2
Test 3
Test 4
Sequences
number of
organisms
10-50
µm/m3
OET
number of
organisms
10-50
µm/m3
Sequences
mean
number of
organisms
10-50
µm/m3

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Page - 18 -
Figure 13 – Concentrated samples of organisms 50 micron and above of test 3. Top centered
is the OET sample, below the OET sample the 8 sequential samples are shown.
3.1 ZOOPLANKTON (ABOVE 50 µM IN MINIMUM DIMENSION)
For organisms above 50 micron in minimum dimension Figure 14 shows that, with the
exception of test 3, in all tests the organism concentration of the very first and last sequences
are above the mean organism concentration. In test 3 the highest organism concentration was
found in the end sequence and in the middle sequences.

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Figure 14 – Blue columns show numbers of living organisms with 50 micron in minimum
dimension and above of the sequential samples, the red line shows the mean number of living
organisms in all sequential samples per test.
In three tests the highest organism count was made in the very last sequence (tests 1, 3 and 4),
whereas in test 2 this was found for the very first sequence. The lowest organism count was
observed in sequence 3 (test 1), in sequence 6 (test 2), in sequence 2 (test 3) and in sequence 5
(test 4).
The sequences which show an organism count nearest to the mean value were in test 1 in
sequence 5, in test 2 in sequence 2, in test 3 in sequence 5 and in test 4 in sequences 2 and 4.
The highest organism count of all sequences from all tests was observed in test 2 where the
holding time of the ballast water inside the tank before discharge was approximately 1 day.
Considering the mean organism counts of all sequences per test the organism number
decreases with increasing holding time.
For the larger organisms, the samples taken over the entire time contained much lower living
organism concentrations compared to sequences (in average 30-62% lower). The same was
observed in previous studies we conducted.
0
500
1000
1500
2000
2500
Seq 1
Seq 2
Seq 3
Seq 4
Seq 5
Seq 6
Seq 7
Te s t 1
Orgs
Mean
0
500
1000
1500
2000
2500
Seq 1
Seq 2
Seq 3
Seq 4
Seq 5
Seq 6
Seq 7
Seq 8
Te s t 2
Orgs
Mean
0
500
1000
1500
2000
2500
Seq 1
Seq 2
Seq 3
Seq 4
Seq 5
Seq 6
Seq 7
Seq 8
Te s t 3
Orgs
Mean
0
500
1000
1500
2000
2500
Seq 1
Seq 2
Seq 3
Seq 4
Seq 5
Seq 6
Seq 7
Seq 8
Te s t 4
Orgs
Mean

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Page - 20 -
3.2 PHYTOPLANKTON (BELOW 50 µM AND ABOVE 10 µM IN
MINIMUM DIMENSION)
For the phytoplankton samples below 50 and above 10 micron in minimum dimension in test
2 the first, fourth and end sequences delivered higher living organism counts then the mean
value determined. In test 3 the first four sequences are above the mean organism
concentration and in test 4 the first, second and fifth sequence delivered higher organisms
compared to the mean value (see Figure 15).
No data for Test 1
Figure 15 – Blue columns show numbers of living organisms below 50 microns and above 10
micron in minimum dimension of the sequential samples, the red line shows the mean number
of living organisms in all sequential samples per test.
The highest organism count per test was made in sequence 1 (test 2), sequence 3 (test 3) and
sequence 5 (test 4). The sequences with the most distant cell counts from the mean were
scattered throughout the tests without a clear trend.
The sequences which show an organism count nearest to the mean value were in test 2 the
sequence 3, in test 3 the sequence 1 and in test 4 the sequences 4 and 6.
0
10
20
30
40
50
Seq
1
Seq
2
Seq
3
Seq
4
Seq
5
Seq
6
Seq
7
Seq
8
Te s t 2
Orgs
Mean
0
10
20
30
40
50
Seq
1
Seq
2
Seq
3
Seq
4
Seq
5
Seq
6
Seq
7
Seq
8
Te s t 3
Orgs
Mean
0
10
20
30
40
50
Seq 1
Seq 2
Seq 3
Seq 4
Seq 5
Seq 6
Seq 7
Seq 8
Te s t 4
Orgs
Mean

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The highest living cell numbers of all sequences from all tests were observed in test 2 after
approximately 1 day holding time. Considering the mean organism counts of all sequences
per test, the organism number decreases with increasing holding time, which was also
observed in the zooplankton samples.
For phytoplankton cell counts the sequences in comparison to the samples taken over the
entire time showed lower organism concentrations in sequential samples (in average 20-37%
lower).

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4 HOW TO TAKE A REPRESENTATIVE SAMPLE?
It was shown by this study that different approaches (i.e., short/long sampling times) in the
sampling process result in different living organism concentrations. Therefore the selection of
an inappropriate sampling approach will influence the compliance control result. As a
consequence the living organism concentrations in the ballast water discharge may therefore
be underestimated, so that an inefficient ballast water treatment system (BWTS) could be
recognised as compliant. In contrast, living organism concentrations may be also
overestimated, and a BWTS complying with the D-2 Standard may fail in compliance tests.
The sequential trials showed different organism numbers in each sequence of one test
indicating the patchy organism distribution inside the ballast tank. This was observed during
all sampling events and for both organism groups studied. Hence, sampling during ballast
water discharge is biased by tank patchiness of organisms.
4.1 ZOOPLANKTON (ABOVE 50 µM IN MINIMUM DIMENSION)
The samples taken over the entire time contained much lower living organism concentrations
compared to the living organism count in the sequences so that sequential sampling may
deliver more representative results.
In three out of the four tests the highest zooplankton count occurred in the last sequence so
that sampling at this time may “over sample” the organism concentration.
4.2 PHYTOPLANKTON (BELOW 50 µM AND ABOVE 10 µM IN
MINIMUM DIMENSION)
Cell counts of sequences in comparison to the samples taken over the entire time showed
lower living organism concentrations in sequential samples, which is in contrast to the
zooplankton results.
For phytoplankton no consistent trend could be identified in which sequence the highest living
cell count occurred.
4.3 SAMPLING RECOMMENDATIONS
As no common trend of larger and smaller organisms could be found we split our sampling
recommendations per organism group.

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4.3.1 ORGANISMS 50 µM AND ABOVE
The samples taken over the entire time contained fewer living organisms compared to the
sequential samples. We assume that the sequential samples deliver more representative results.
Our study has shown that sequential samples taken in the very beginning and end during a
ballast tank is emptied are unlikely to give representative results of the living organism
concentration because in these samples the organism count showed high variations, which
may result in under- or oversampling the organism concentration. These findings are in line
with an earlier study (Gollasch & David 2010a).
Assuming the mean organism count from all sequences of one test shows the true organism
concentration in the tank, we recommend to avoid taking a sample during the very beginning
and end of the discharge time of a tank or tanks. By avoiding these time windows the samples
taken are nearest to the mean organism count of all sequences.
Although the best sampling time may be in the middle of a ballast tank discharge, the living
organism concentration still seems to be patchy so that we recommend taking at least two
samples in this time window. The mean value of the living organism concentration in these
two samples may be taken to assume the real organism concentration.
4.3.2 ORGANISMS BELOW 50 AND ABOVE 10 µM
In contrast to the larger organisms, the continuous drip over the entire time samples contained
higher organism counts for the smaller organisms compared to the mean of sequential samples.
The comparison of the concentration of the smaller living organisms between the different
sequences of all tests showed that no clear trend can be identified during which time window
a more representative sample will be taken. Therefore we recommend taking at least two
samples during the discharge of a ballast water tank with avoiding taking a sample during the
very beginning and end of the discharge time of a tank or tanks. The mean organism count in
these two or more samples may be seen as the real living organism concentration in the ballast
water.

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5 ACKNOWLEDGEMENTS
The study was funded by the German Bundesamt für Seeschifffahrt und Hydrographie,
Hamburg, Germany and we in particular like to highlight the support and valuable comments
provided by Kai Trümpler and Stefan Kacan. This voyage would not have been possible
without permission and the essential support of Konstaninos Stampedakis (Environmental
Protection Engineering S.A., Perama, Greece, the developer of the ERMA FIRST ballast
water treatment system). We further like to express our grateful thanks to the vessel crew for
their outstanding support, especially to Captain Vakchos Charalampos, Chief Officer Dejan
Pantic, and Chief Engineer Asylanis Kypiakos. For analysis of the phytoplankton samples we
thank the expert team of the Royal Netherlands Institute for Sea Research (NIOZ), Texel, the
Netherlands, and especially Alex Blin and Louis Peperzak.

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LIST OF FIGURES
Figure 1 – Design of the in-line ballast water sampling point. .................................................. 5
Figure 2 - Flow splitter to generate two equal splits of ballast water enable a parallel
comparison of samples. .............................................................................................................. 6
Figure 3 – Sampling point set-up with three sampling bins with submersible pumps. .............. 6
Figure 4 – Zooplankton sampling nets, left the sample taken over the entire time, right the two
nets used for the sequential samples. .......................................................................................... 7
Figure 5 - Unscrewing the removable cod-end form the zooplankton sampling net. ................ 8
Figure 6 - Position of zooplankton net with a high water level in the water collecting bin. ...... 8
Figure 7 - Taking a “continuous drip sample” with the jar to collect the sample in the bucket
in front of the yellow bin. ........................................................................................................... 9
Figure 8 - Water buckets of one test run. In front of each bucket are the phytoplankton
samples which were taken in duplicate, one sample with living cells and another Lugol-
preserved. ................................................................................................................................. 10
Figure 9 - Counting of zooplankton organisms on board using a stereomicroscope. .............. 13
Figure 10 - The Pulse-Amplitude Modulation (PAM) instrument to measure phytoplankton
viability. .................................................................................................................................... 14
Figure 11 – Numbers of living organisms 50 µm and above in minimum dimension/m3 of
ballast water. The blue columns show numbers of living organisms in different sequential
samples, the red columns in OET = sample taken over the entire discharge time of the tank,
and the orange columns show mean numbers of living organisms in sequential samples per
test. ........................................................................................................................................... 16
Figure 12 - Numbers of living organisms below 50 and above 10 micron in minimum
dimension/m3 of ballast water. The blue columns show numbers of living organisms in
different sequential samples, the red columns in OET = sample taken over the entire discharge
time of the tank, and the orange columns show mean numbers of living organisms in
sequential samples per test. ...................................................................................................... 17
Figure 13 – Concentrated samples of organisms 50 micron and above of test 3. Top centered
is the OET sample, below the OET sample the 8 sequential samples are shown. ................... 18
Figure 14 – Blue columns show numbers of living organisms with 50 micron in minimum
dimension and above of the sequential samples, the red line shows the mean number of living
organisms in all sequential samples per test. ............................................................................ 19
Figure 15 – Blue columns show numbers of living organisms below 50 microns and above 10
micron in minimum dimension of the sequential samples, the red line shows the mean number
of living organisms in all sequential samples per test. ............................................................. 20

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LIST OF TABLES
Table 1 - Main dimensions of the vessel and tank details. ......................................................... 4
Table 2 – Ballast water sampling events and tank details. ......................................................... 5
Table 3 – List of the samples taken also showing the start and end time of each sampling
event with duration and the water quantity sampled. Quantity sampled in litres, flowrate in
litres per minute. ....................................................................................................................... 11
Table 4 - Sampling event duration (sequential samples in blue shading, the samples taken over
the entire time (OET) in grey shading). ................................................................................... 12
Table 5 - Number of living organisms in the samples (OET = sample taken over the entire
discharge time of the tank; S= sequence; ml= millilitre; m³ = cubicmeter; µm = micrometre).
.................................................................................................................................................. 15

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