J. of Marine Env. Engg., Vol. 7, pp. 153-172
Reprints available directly from the publisher
Photocopying permitted by license only
© 2004 Old City Publishing, Inc.
Published by license under the OCP Science imprint,
a member of the Old City Publishing Group.
Effect Of UV Irradiation On Viability Of Micro Scale
And Resistant Forms Of Marine Organisms:
Implications For The Treatment Of Ships’ Ballast Water
D . O E M C K E 1 N . PA R K E R 2 A N D D . M O U N T F O RT 3 *
1Provisor
2CSIRO,
Pty Ltd, PO Box 219 Glen Osmond SA 5064, Australia
GPO Box 1538, Hobart, Australia
3Cawthron
Institute, Private bag 2, Nelson, New Zealand
The efficacy of UV in killing zoospores of
the seaweed, Undaria pinnatifida, a marine
bacterium (Vibrio alginolyticus), and cysts of
Gymnodinium catenatum have been determined in two laboratories. In experiments performed in New Zealand employing a flowthrough UV reactor in which the lamp was
positioned centrally along the length of the
reactor system between the inflow and outflow,
plots of log of viability versus dose (dose range,
50-350 mWscm-2) were negative log linear with
a maximum kill of 40% achieved in the case of
G. catenatum cysts. Over a lower dose range
(10-65 mWscm-2), negative log linear responses
were also obtained with cultures of Vibrio alginolyticus and with zoospores of U. pinnatifida,
in which kills of 99% (zoospores) and 99.9%
(V. alginolyticus) were achieved at doses of 60
and 37.5 mWs cm-2 respectively. Investigations
in Australia on the effects of UV irradiation on
cysts of G. catenatum using collimating beam
and continuous flow apparatus and semi-quantitative and quantitative methods to assess kill
confirmed the above results by showing survival (maximum kill, 70%) as determined by
hatching of planomeicytes even after irradiations up to 1,600 mWscm-2. This contrasts
with the findings with gametes of the same
organism which showed that doses of 110-220
mWscm-2 were sufficient to produce kills of >
99%. Lower doses (45 mWscm-2) could produce the same effect if cells were stored for a
period of time in the dark after treatment or
heated to 23oC. The results of these studies
demonstrate that while UV irradiation may be
an effective option for the treatment of bacteria, the asexual forms of micro-algae, and the
motile forms of macro-algae it is unlikely to be
____________________
*Corresponding author: Email: [email protected]
Phone: 64-03-54-82-319
Fax: 64-03-54-69464
154
OEMCKE, et al.
effective in the treatment of dinoflagellate
cysts. On this basis our recommendation
would be a dose of 60 to 120 mWscm-2
(excluding cysts) for UV treatment ballast
water. Because the dose range would be ineffective against the resistant forms of microscale organisms, and larger macro-ballast
organisms, these organisms would need to be
removed via an effective primary treatment.
To date no method has been demonstrated to
be effective in this respect.
Keywords: Ballast Water, UV Irradiation, disinfection, Gymnodinium catenatum, Undaria
pinnatifida, Vibrio alginolyticus
1. INTRODUCTION
This paper describes work undertaken in
Australia and New Zealand to examine the
efficacy of UV irradiation as a treatment
option and is focused on those organisms
that pose a challenge to treatment
technologies either through their size or their
ability to produce resistant forms.
It is estimated that the world’s shipping
fleet transports ten billion tonnes of ballast
water annually. Ships carry ballast water
when transporting little or no cargo in order
to maintain stability. The translocation of
exotic species in ships’ ballast was first
recorded by Medcof (1975) and is now a
recognised vector for the inter- and trans-continental translocation of marine species
including
toxic
dinoflagellate
algae
(Hallegraeff and Bolch 1991), macroalga
(Ribera 1995), Vibrio cholerae (McCarthy
and Khambaty 1994) and a range of zooplankton (eg. Carlton 1985). As a result of
transport in ballast water in a viable state
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
many species have now established themselves in foreign ports, where they have
achieved pest status. Worldwide, the economic and environmental cost of such pests is
many billions of dollars each year.
In 2004 the International Maritime
Organization adopted an international
convention for the control and managment
of ship’s ballast water. This means that the
30,000 to 40,000 ships trading
internationally will soon be required to
implement ballast water management.
Mandatory ballast water controls have been
introduced in New Zealand (1998) and
Australia (2001). Present “best practice”
management measures rely on ballast water
exchange, and there is currently no single
effective method for economical treatment of
all ships’ ballast water, nor a standard on
which to make comparisons of alternative
treatments.
Strategies being investigated to reduce
these introductions include ballast water
exchange at sea and treatment technologies.
Ballast exchange at sea can reduce the risk of
exotic species introductions, but not
effectively and can be dangerous. Rigby
(1994) suggested a series of ballast intake
management measures that can be used to
reduce the risk of taking in dinoflagellate
algae. Oemcke and van Leeuwen (2003a,b)
investigated the impact of ballast water
chemistry and conditions on methods that
could be used for ballast water disinfection.
Methodologies being developed for the
treatment of ship’s ballast water include
heating (Hallegraeff et al., 1997; Mountfort
et al., 1999, 2000, 2003; Rigby 1994; Rigby
et al., 1999; Taylor and Rigby 2001),
ozonation (Oemcke and van Leeuwen,
2003c,d; Dragsund et al., 2003), filtration
(Cangelosi et al., 2003; Matheickal et al.,
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
2003; Parsons et al., 1997), hydrocyclones
(Sutherland et al., 2003; Nilsen et al., 2003)
and UV irradiation. The effects of UV in
killing bacteria, protozoa and viruses are well
known (Campbell et al., 1995; Harris et al.,
1987; Liltved et al., 1995; Sobsey, 1989;
Sommer et al., 1995) and although UV
treatment systems have been installed on
several passenger liners as part of an
integrated system with other treatments (eg
the Optimar system aboard the M/S Sea
Princess) there has actually been very little
scientific appraisal on the efficacy of UV
irradiation on ballast organisms. These
include reports showing that UV irradiation
had variable effect on the germination of
cysts of phytoflagellate species in sediments
(Montani et al., 1995), and the effects of
ultraviolet irradiation on gametes of
Gymnodinium catenatum and the marine
algae, Amphidinium sp. (Oemcke and van
Leeuwen, in prep).
This paper describes the findings from the
first comprehensive studies examining the
effects of UV on ballast organisms. We detail
results on the effects of UV irradiation to
control dinoflagellate algal cysts and gametes
of Gymnodinium catenatum and on a marine
bacterial vibrio and zoospores of the seaweed, U. pinnatifida. The organisms were
selected on the basis of size (eg they would
pass through filter systems of 50 microns or
less) or ability to produce resistant forms (eg
G. catenatum). The results are discussed in
relation to other methods that are being
developed for the treatment of ships’ ballast
water. They also allow inferences to be made
on the practicality of developing UV irradiation systems to treat ships’ ballast.
155
2. METHODS
2.1 Continuous flow experiments on
Gymnodinium catenatum gametes
The Gymnodinium catenatum strain used
in these experiments (CS301/6) was selected
from three strains (CS301/6, CS301/8 and
CS302/10) on the basis of vigorous growth
and higher survival rates in stock cultures. All
cultures were obtained from the CSIRO
(Australia) Microalgae Culture Collection.
They were grown in sterile GSe media (CSIRO
Microalgae Culture Collection) and soil
extract purchased from the CSIRO Microalgae
Culture Collection. Oceanic seawater was
obtained from the Coral Sea, aged for six
months and autoclaved in teflon bottles for
media preparation (per Brand, 1986). All
glassware used in the preparation of media
and growth of G. catenatum was washed with
phosphate-free detergent, rinsed with hot tap
water, acetone, nitric acid and rinsed carefully
with high quality water (Milli-q).
Cultures were grown under white fluorescent light (Philips white), with overhead illumination, at a light intensity of 150 mEm-2s-1
with a 12:12 hr light:dark cycle and an average temperature of 18oC (per Blackburn et al.,
1989). Stock cultures were maintained in 24
well tissue culture plates to acclimatize them to
the conditions used in enumeration. Stock cultures were transferred to 20 to 30 mL of culture media in autoclaved 100 mL sterile
borosilicate glass erlenmeyer flasks, with cotton plugs, for two to four weeks before transfer to 100 mL of culture media in 500 mL sterile borosilicate glass erlenmeyer flasks with
cotton plugs. For UV testing 1 L of this vegetative culture was added to 4 L of aged, 0.2
mm filtered seawater obtained from the coast
near Townsville, Queensland, during winter.
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
156
OEMCKE, et al.
FIGURE 1
Sexual G. catenatum culture showing gametes, sexual cells and
a cyst (a) and inter-relationship between the sexual (diploid)
and vegetative (haploid) life cycle phases (b). Part b is modified from Blackburn et al., 1989.
Ultraviolet irradiation was performed
using a low pressure, single lamp, quartz
sleeve, ultraviolet reactor (UVS Ultra Violet
Pty Ltd.), with lamp output measured by the
manufacturer, using an International Light
IL700 radiometer, and dose calculated using
the area weighted average irradiance, UV
transmittance and flow rate through the reactor. Suspensions of the alga were pumped
through the UV reactor using a peristaltic
pump. The lamp and quartz sleeve were
cleaned with alcohol between tests.
Absorbance and transmittance at 254 nm
were measured in a Varian 635 UV-Vis spectrophotometer with 10 mm pathlength
matched pair quartz cells. The UV unit was
calibrated using Bacillus subtilis ATCC6633
as a test organism. The dose response was
comparable to published data for this organism (eg. Sommer et al., 1995).
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
Following irradiation, cells were either
immediately cultivated or stored for periods
of 1, 2, 3 or 5 days in acid washed 250 mL
bottles in the dark at room temperature
(average 23oC), in the dark in the culture area
(av. 18oC) or in 100 mL erlenmeyer flasks in
culture conditions.
All enumeration of G. catenatum gametes
was conducted by the MPN method (de Man,
1977). For G. catenatum all culturing was
conducted in 24 well tissue culture plates
with 1, 0.1, 0.01 and 0.001 mL of treated
solution added to 1 mL of culture media. An
initial test used 96 well tissue culture plates
for the smaller dilutions, but G. catenatum
would not grow in these plates. Viability of
gametes in the wells was assessed in two or
three ways: (i), presence of motile gametes
eight days after sub-sampling; (ii), gametes
that remained motile for seven or more con-
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
secutive days at any time following irradiation; and (iii), the presence of chains consisting of four gametes. To collect the data for
this assessment the wells were observed at
least every second day until all criteria being
checked on each test had either been met or
all cells were dead. Motility was not used as
UV affected cells did not lose motility immediately after treatment.
2.2 Batch experiments on Gymnodinium
catenatum cysts
2.2.1 Gymnodinium catenatum preparation
for collimating beam disinfection experiments
Gymnodinium catenatum hypnozygotes
(cysts) prepared by combining cultures of
strains CS301/8 and CS302/10 (CSIRO
micro-algae culture collection) using the
method of Blackburn et al. (1989). Cysts
were harvested for semi-quantitative and
quantitative collimating beam tests using a
micromanipulator, and ensuring that only
cysts were transferred. Figure 1a shows a sexual culture prepared this way, with a cyst seen
in the middle of the micrograph among
gametes and sexual cells. The inter-relationship of sexual (diploid) and asexual (vegetative, haploid) growth stages is shown in
Figure 1 b.
2.2.2 Collimating beam UV irradiation
apparatus
The collimating beam UV apparatus was
designed as per National Sanitation Foundation
standard ANSI/NSF 55-1991 (NSF, 1991).
Incident UV radiation was measured using a
UVP radiometer model UVX with a UVX-25
sensor, and was typically 50 µWcm-2. Tests
were conducted on very small numbers of
cysts (20 to 80) (eg. Hallegraeff et al., 1997) so
157
the petri dishes were not stirred, to avoid cysts
moving into areas affected by meniscus
(Sommer et al., 1995), or adhering to the walls
of the petri dish.
2.2.3 Enumeration and viability assessment
Semi-quantitative viability assessments
were conducted by placing sets of 10 cysts in
a 50 mm petri dish and irradiating them to
the required dose with the collimating beam
unit. After irradiation the cysts were either
enumerated immediately or stored in the dark
for a period of one to five days. To enumerate the cysts, the seawater medium they were
irradiated in was removed by pipette and
replaced with GSe growth medium. Cultures
were observed every second day until all cysts
had hatched, typically less than 25 days.
Positives were counted by the appearance of
pigmented, motile planomeiocytes followed
by the presence of motile gametes after 25
days.
Quantitative testing was conducted in the
same manner as the semi-quantitative tests,
except that cysts were individually plated into
1 mL of GSe growth media following treatment by transferring them with the micromanipulator. Individual cysts were observed
every second day until all cysts had hatched,
and the condition or state of the cyst recorded. Cysts which hatched formed motile, pigmented planomeiocytes or grey looking
planom-eiocytes. Any planomeiocytes which
remained surrounded by a pellicular layer
after hatching were not counted as positives
until they were free swimming. Cysts also
hatched to produce distorted planomeiocytes,
which were not counted as positives unless
they gained healthy appearance and motility.
Some cysts simply spilled their undifferentiated contents. Many cysts failed to hatch, lost
colour and the accumulation bodies disap-
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
158
OEMCKE, et al.
peared to leave a grey-black empty cyst.
Other cysts shrivelled without producing a
planomeiocyte. Survival (positives) was
determined by the presence of pigmented,
motile planomeiocytes and by the presence of
motile gametes.
2.2.4 Confirmation with continuous
flow reactor
A single confirmatory continuous flow test
was conducted on the cultures prepared for
batch testing using the UV apparatus
described in section 2.1. In this experiment
we used a low pressure, single lamp, quartz
sleeve, ultraviolet reactor (UVS Ultra Violet
Pty Ltd). Suspensions of the alga were
pumped through the UV reactor using a
peristaltic pump. Samples were irradiated
with 170 mWscm-2, then either immediately
enumerated or sub-jected to various storage
treatments. For the MPN tests, samples were
collected by stirring a storage bottle and
taking samples from the bulk liquid. Samples
were assessed either by MPN (de Man, 1977)
or individual culturing as for the quantitative
tests. Cultures were observed every second
day for eighteen days and then less frequently
until six weeks after irradiation. Positives
were determined by the presence of motile
cells for a period of seven or more
consecutive days during the period of
observation following reculture.
2.3 Continuous flow experiments on
G. catenatum cysts, U. pinnatifida and
V. alginolyticus
2.3.1. Organisms
The dinoflagellate, Gymnodinium catenatum, was obtained from the culture collection
at CSIRO, Hobart, Australia. Sporophylls of
the seaweed, U. pinnatifida, were obtained by
Mr T. O’Sullivan, from Oamaru Harbour on
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
the eastern coast of the South Island, New
Zealand. The bacterium Vibrio alginolyticus
was obtained from the culture collection at
the Cawthron Institute. It was isolated from
whole mussel preparations and its identity
conformed by J. Wright, Comm-unicable
Disease Centre, Wellington.
2.3.2. Incubation and germination of G.
catenatum cysts
Cysts of Gymnodinium catenatum after
purification by progressive dilutions, were
prepared to give a concentration of about 30
per ml in 2 L. The estimated purity of cysts in
relation to motile forms present was about
97%. Two hundred milliliters of untreated or
treated cyst solution was added to 200 ml of
germination GSe nutrient medium to take the
volume up to 400 ml in 1 L glass conical vessels. Flasks were incubated at 17oC at 100
µEm-2s-1 (12 h light-12 h dark) for 6 weeks
(Hallegraeff et al., 1997) and germination
was determined by fluorimetry.
2.3.3. Gametophyte production of
U. pinnatifida
Sporogenesis from mature sporophylls was
carried out as previously described (Mountfort
et al 1999). Released zoospores were filtered
(20 µm mesh) and 1 ml of filtrate added to 100
ml of standard seaweed medium (SSM; Hay
and Gibbs, 1996). The total contents were then
added to 10 L of SSM in a large vessel and the
contents were stirred. Aliquots (4 ml) of the
treated and untreated preparations were each
transferred in triplicate to wells of 12 well
chambers. These were incubated using a lighting regime as previously described (Mountfort
et al. 1999). Gametophyte production was
determined after 3 weeks incubation by direct
counting under a light microscope.
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
2.3.4. Bacterial culture methods
Vibio alginolyticus was maintained by the
Cawthron
Microbiological
Testing
Laboratory. A liquid inoculum was prepared
by adding a loop of a streak of bacteria on
marine broth agar to GP medium (Loeblich
and Smith, 1968). Bacterial counts were estimated by OD measurement against standard
curve of BaCl2 standards. The bacterial suspension was diluted to give approximately
1.5 × 109 organisms per ml. For experiments,
1 ml of suspension was added to 10 L of UV
and filter sterilised seawater which was thoroughly mixed. Aliquots of untreated and
experimental UV irradiated suspension (0.2
ml) were added to 1.8 ml of GP medium and
mixed. Five hundred microliters of suspension was spread onto a marine agar plates
(Marine Broth 2216 [Difco] on 2% agar)
which were incubated at 37oC. After 1 week
of incubation colony counts were determined
by using direct counting methods assisted by
using a colony counter (Scientifica and Cook
Ltd). Colonies were then recounted after 2
weeks incubation.
2.2.5. Irradiation by UV and determination
and effectiveness of treatment
Cysts of G. catenatum, zoospores of
U.pinnatifida or cells of V. alginolyticus were
passed through a UV reactor assembly using
a Masterflex variable speed peristaltic pump
(Cole Palmer Instrument Co.) set at flow
rates in the range, 0.24 to 6.6 Lmin-1. The
reactor was based on the Sterilflo 300 design
and built specifically for the study by
Contamination Control Ltd, Auckland, New
Zealand. It consisted of a stainless steel tube
(length, 434 mm; internal diameter, 29.35
mm) and an inside quartz sleeve (length, 450
mm; outside diameter, 24.5 mm), housing the
UV lamp (low pressure mercury lamp, output
159
at 253.7nm). The distance between the inlet
and outlet on the main body was 290 mm
giving an irradiated volume of 54 ml. For any
treatment, approximately 5 volumes of suspension was passed through the reactor
before collection of the fraction for viability
determination. Cyst survival was expressed
by fluorescence as a function of the control
(Ftr /Fcont × 100). Survival of zoospores was
expressed by the following relationship:
Gtr/Gk × 100 in which Gtr represent the number of germinated zoospores in the treated
system versus those in the control (Gk).
Survival of bacteria was expressed as Ctr/Ck ×
100 in which Ctr are the colony counts for the
treated system and Ck the counts for the control. The generic expression for survivorship
collectively used for all the test species was N
and N0 representing numbers in the test and
controls respectively.
3. RESULTS
3.1. Screening effectiveness of UV
irradiation in continuous flow experiments
3.1.1. Continuous flow experiments on
G. catenatum cysts, U. pinnatifida and
V. alginolyticus
The effects of UV irradiation were
screened using a flow-through assembly,
against G. catenatum cysts, the bacterium, V.
alginolyticus, and zoospores of U. pinnatifida. The results of tests on the marine vibrio
are shown in Figure 2a. The doses are higher
than suggested in the literature reviewed, but
are well within an acceptable range, with a 3
log (99.9%) removal achieved with a dose of
35 mWs cm-2. Inactivation results for
zoospores of U. pinnatifida are shown in
Figure 2b. A UV dose of 60 mWscm-2 effect-
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
160
OEMCKE, et al.
Log fluroescence -log (N/N0)
Log removal-log (N/N0)
Log removal-log (N/N0)
ed a >2 log (>99%) removal. The results on
the effects of UV irradiation on survivorship
of cysts are shown in Figure 2c. They show
that there was no significant effect on survival in the dose range 50 to 350 mWscm-2 (<
0.5 log removal). The kills for the former two
organisms could be attained within the limits
of the experimental design while those of G.
catenatum could not. Since the results for G.
catenatum were based on fluorescence
(indicative only) more unequivocal procedures were required to establish kill or survival. These are detailed in section 3.2.
FIGURE 2
Effect of UV irradiation on (a) V. alginolyticus cultures; (b),
zoospore suspensions of U. pinnatifida, and (c), cyst culture
of G. catenatum (measured by cell fluorescence)
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
3.1.2. Continuous flow experiments on
G. catenatum gametes
Figures 3 a, b and c show the effect of 3
UV doses on the disinfection of G. catenatum
gametes. The gametes were stored either in
photic culture conditions, in the dark within the
culture facility, or in the dark at room temperature. The results in these figures are for gamete
viability assessment by motility for seven days
or more post-irradiation. The results were similar to those for viability assessed by motility at
eight days after irradiation.
The figures demonstrate that increasing UV
dose not only increases the efficacy of UV irradiation, but also decreases the ability of the
organism to photoreactivate. At the UV dose of
220 mWscm-2 (Figure 3c), the G. catenatum
gametes are completely inactivated immediately
following irradiation, with no recovery from
photorepair. This is in contrast with the lower
dose of 110 mWscm-2 (Figure 3b), where the
dose is not high enough to immediately inactivate the gametes and they are still able to photorepair after one day of dark storage. At the
lowest dose of 46.5 mWscm-2 a period of dark
storage is required post-irradiation to ensure
that the organism is unable to photorepair. The
data also show the susceptibility of G. catena-
161
-log(n/No)
-log(n/No)
-log(n/No)
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
FIGURE 3
Effect of UV irradiation on gametes of G. catenatum, with the efficacy of UV irradiation measured by motility 8 days after UV
irradiation at doses of 46.5 mWscm-2 (a), and 110 mWscm-2 (b), and 220 mWscm-2 (c).
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
162
OEMCKE, et al.
FIGURE 4
Temporary resting cells formed in vegetative cultures after UV irradiation and dark storage: (a) photographed with no filters and
the microscope iris completely closed; (b) phase contrast and no filters; (c) no adjustment or filters
FIGURE 5
Temporary resting cells excysting: (a), (b) phase contrast, blue filter; (c) blue filter
tum gametes to dark storage at elevated temperatures, with an enhanced loss in the ability to
photorepair. The control cultures also showed a
negative response to elevated storage temperature, seen in all three figures. Elevated temperatures have been shown to speed the decline in
the ability of bacteria to photoreactivate
(Jagger, 1958).
Some of the irradiated G. catenatum gametes
were observed to enter a temporary resting stage.
The cells surrounded themselves with a pellicular
layer, which can be clearly seen in Figure 4 outlined around the gametes. Similar temporary resting cells have been observed in sexual culture
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
(Blackburn et al. 1989). The emergence of
gametes from the temporary resting stage is
shown in Figure 5. Some of the gametes which
emerged were viable, although many did not
emerge, or emerged in a non-viable state.
3.2. Irradiation of G.catenatum cysts in
collimating beam experiments
3.2.1. Effect of UV alone on G. catenatum cysts
Semi-quantitative testing obtained order of
magnitude estimates of the effective doses of
UV irradiation for the inactivation of G.
catanatum cysts. Table 1 shows results from
an experiment in which cysts were placed
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
163
TABLE I
Results from semi-quantitative tests for cells cultured immediately after inactivation, with appearance of a
planomeiocyte counted as a positive.
a
UV dose
(mWscm-2)
Control
inactivation
Experimental
inactivation
Number
of tests
100
200
400
800
< 1 log
< 1 log
< 1 log
< 1 log
< 1 log
< 1 log
< 1 log
< 1 log
1
2
2a
1
The second test contained three replicates, all with the same result.
TABLE II
Results from semi-quantitative tests for cells cultured immediately after inactivation, with motile gametes 25
days after irradiation used as a positive.
UV dose mWscm-2
100
200
400
800
a
Control cells motile at 25 days
+
+, -, +
-
Irradiated cells motile at 25 days
+
+, +
+, + a
+
The second test contained three replicates with the same result
into culture conditions immediately after
irradiation and survival assessed by the
appearance of a healthy, motile planomeiocyte. In each test, healthy, motile planomeiocytes were observed following irradiation.
Table 2 shows results from the same experiment as shown in Table 1, but with motility
at 25 days used to assess survival. All irradiated cultures contained motile cells at 25
days, but only 50% of the controls.
3.2.2. Effect of UV on G. catenatum cysts in
combination with heat and dark storage
The effect of temperature and dark storage
was investigated using both quantitative and
semi-quantitative experimental designs,
which are shown in Table 3. The quantitative
designs were used to investigate if there were
effects of UV which were too subtle for the
semi-quantitative tests, and to validate the
poor UV disinfection efficacy results from the
semi-quantitative tests. In the semi-quantitative tests, at UV doses of 200 and 400
mWscm-2, there was no effect of five days
storage at 23oC when the appearance of
planomeiocytes was used as a positive. There
was a response to 200 mWs cm-2 from 5 days
storage at 23o C, but this was not replicated
at 400 mWscm-2 and the control did not have
motility at 25 days for the test at 200
mWscm-2. The quantitative test showed no
efficacy of UV irradiation at 400 mWscm-2.
Quantitative tests were also conducted at
o
32 C with dark storage and UV doses of 100,
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
164
OEMCKE, et al.
TABLE III
UV inactivation in semi-quantitative and quantitative tests with dark storage following irradiation of G. catenatum cysts
UV dose
mWscm-2
200a
400a
400b
control
<1 log (-)
<1 log (+)
73 (45)c
immediate
cultivation
<1 log (+)
<1 log (+)
30 (20)
Days in the dark (23oC)
1
3
5
<1 log (+)
<1 log (+)
<1 log (-)
<1 log (+)
80 (60)
5 days in dark
(18oC)
<1 log (+)
64 (45)
a
Semi-quantitative tests (log reductions are for the appearance of a planomeiocyte; + and - are for the presence and absence, respectively,
of motile cells after 25 days).
b
Quantitative tests with 10 cysts (values are % showing planomeiocytes with the % achieving motility in parentheses).
c
11 cysts.
TABLE IV
Results of quantitative collimating beam tests on the UV inactivation of cysts (usually 10) followed by dark storagea
UV dose
mWscm-2
unirradiated
100
200
400
immediate
cultivation
46 (36)b
60 (20)
20 (0)
40 (20)
5 days in
dark (23oC)
40 (20)
40 (30)
70 (70)
80 (60)
Days stored in the dark (32oC)
1
3
5
c
17 (17)
0 (0)
0 (0)
—
0 (0)
0 (0)
d
9 (0)
0 (0)
0 (0)
30 (20)
10 (0)
0 (0)
a
Values shown are % with a motile, pigmented planomeiocyte, and values in parenthesis are for the % which hatched into free swimming gametes.
a-c
Refer to 28, 13 and 11 respectively, representing the numbers of cysts cultivated.
mWscm-2
200 and 400
(Table 4). The results
for immediate cultivation and five days dark
storage at 23 o C reflect the poor efficacy
recorded in Tables 1, 2 and 3. All treatments,
including unirradiated controls, showed no
viable gametes after storage at 32oC for three
days. Only the cultures exposed to 400
mWscm -2
had
viable,
pigmented
planomeiocytes after three days storage at
32 o C. A further quantitative test was
conducted with UV irradiation at doses of 800
and 1,600 mWscm-2 to determine if very high
UV exposures would have an effect on G.
catenatum cysts (Table 5).
Validation of the collimating beam tests
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
were conducted in the bench-scale reactor,
with an incident radiation flux of the order
10 mWcm-2. Validation at these higher fluxes
(but the same dose) were conducted as there
may be a response to radiation intensity in
germicidal (254nm) UV irradiation (Clancy
and Fricker, 1998) and as there is an intensity
response in the inhibition of photo-synthesis
of marine diatoms for UV-B (280-320 nm)
(Cullen and Lesser, 1991). Table 6 and Figure
6 show the results of exposures to 170
mWscm-2. Table 6 shows survival for cysts
cultured individually, as for the quantitative
collimating beam tests, and indicates no
effect of UV and/or dark storage with 54%
of cysts surviving in the control and 54 to
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
165
TABLE V
Results of quantitative collimating beam tests on the inactivation of G. catenatum cysts with cultivation
immediately after high doses of UV irradiation
UV dose
mWscm-2
Control
survival (%)a
Experimental
survival (%)a
800
1,600
70 (20)
70 (20)
50 (10)
30 (20)
a
Values are the percentage survival from 10 individually cultured cysts, with survival measured by the appearance of a planomeiocyte;
values in parenthesis are for survival measured by the presence of free swimming gametes.
TABLE VI
Results of bench scale tests on the UV inactivation of G. catenatum cysts with five days dark storage following
UV irradiation at a dose of 170 mWscm-2 (results from individually cultivated cysts)a
control
5 days in dark (18oC)
5 days in dark (23oC)
54 (19)b
54 (33)c
58 (20)c
a
Values shown are for % survival measured by the appearance of a planomeiocyte, and values in parenthesis are for % survival
measured by the appearance of gametes.
b, c
Refer to 72 and 24 respectively, representing the number of cysts individually cultivated.
58% surviving in the cysts subjected to UV
and dark storage.
Figure 6 shows results of MPN counts on
cyst survival in bench scale tests. The survival
increased above the level of cysts actually
present for cells cultured immediately after
inactivation, due to the survival of non-cyst
cells. This shows that sexual cells, such as
planozygotes and planomeiocytes and fusing
gametes may be more resistant to UV than
gametes but less resistant than cysts
(hypnozygotes). The result verifies the poor
disinfection efficacy results of the individual
cultures (Table 6), in which cyst survival after
UV treatment (dose, 170 mWs cm-2) and 5
days storage, was over 20% using this
assessment method.
4. DISCUSSION AND CONCLUSIONS
Until this study a serious evaluation of UV
irradiation and its potential for the treatment
of ship’s ballast had not been carried out.
Formerly this lack of information has led to
speculation sometimes tenuous on the potential of this method, some of which has been
based on either a paucity of fact or none at
all. In this study we have attempted to
appraise the method using organisms that
realistically represent those found in ballast
including those (eg micro-algal cysts) which
are resistant to many kinds of treatment, or
those that would pass through standard filters being developed to remove ballast organisms. Hence the selection of a bacterium and
zoospores of a seaweed.
Gametes of G. catenatum are relatively easy
to disinfect, with doses of less than 50 mWscm2 and five days dark storage, or 3 days if tem-
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
OEMCKE, et al.
-log(N/No)
166
FIGURE 6
Results from MPN tests on the disinfection efficacy of UV against G. catenatum cysts. The UV dose was 170mWscm-2. Symbols:
, maximum detectable removal by MPN count; , cells motile for a period of 7 days or more following reculture. Bars show
95% confidence interval of the MPN test.
perature is increased to 23oC. At UV doses of
between 110 and 220 mWscm-2 all G. catenatum gametes were inactivated, immediately
after irradiation. These results compare
favourably with Oemcke and van Leeuwen
(2003c) where cells of the marine dinoflagellate
alga Amphidinium sp. showed similar responses to UV dose, dark storage after UV exposure
and increasing storage temperature after UV
exposure. The gametes of Gym-nodinium catenatum were more sensitive to dark storage and
temperature rise following UV irradiation than
any of the cultures of Amphidinium reported
by Oemcke and van Leeuwen (2003c).
On equatorial crossings, in which the ballast water can reach 32oC, it would be very
surprising if gametes of this, or organisms with
similar temperature tolerances, could survive.
However, it is likely that gametes will survive
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
ballast transport, particularly if temperatures
do not change significantly, or on shorter voyages, such as the trip from Tasmania to New
Zealand or to Melbourne. Survival of gametes
of Alexandrium minitum have been found on
trips across the North Sea (MacDonald and
Davidson, 1998), several live vegetative species
on ships entering Canada (Subba Rao et al.,
1994) and live vegetative phytoplankton on
ships entering Washington (Kelly, 1992). The
combined/cargo ballast tanks of ships could be
a particular risk, as the large volumes of water
in these tanks, makes them more resistant to
fast heat transfer (eg. Ruiz et al., 1997).
Gametes of toxic dinoflagellates that are warm
water tolerant, such as Pfiesteria piscicida
(Burkholder et al. 1995) may be capable of
surviving in ballast water, without cysts necessarily being present. UV has potential to
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
remove these gametes from ballast water.
The decrease of log 0.5 or less after irradiation of cysts of G. catenatum with UV
demonstrates that these forms are resistant
to, and remain viable after UV treatment.
This particular result has been demonstrated
by testing in two separate laboratories using
commercial low pressure mercury vapour
lamps. The Australian studies also investigated the response of G. catenatum cysts to UV
in a collimating beam apparatus. The lack of
intensity effect between the collimating beam
and the continuous flow reactors implies that
technologies like pulsed UV and medium
pressure UV are unlikely to improve the efficacy of UV irradiation.
Experiments that included the effect of
heating showed that three to five days exposure to 32oC is sufficient for the inactivation
of G. catenatum cysts. This demonstrated
that temperature alone was the major factor
rather than a synergism between UV, dark
storage and temperature for cysts as evident
for vegetative (haploid) stages of both G.
catenatum and Amphidinium sp. This temperature response is comparable to those
observed by Hallegraeff et al. (1997), where
2 minutes at 38oC completely inactivated G.
catenatum cysts. They also survived 1 hour at
35oC, and no viable cultures could be
obtained after 1 hours treatment at 37.5oC.
Hallegraeff et al. (1997) also found that cysts
from
another
dinoflagellate
alga,
Alexandrium catenella, were inactivated by
4.5 hours at 38oC and maintained 20% viability after 8 hours at 36oC. The temperature
data recorded here contribute to understanding of the temperature sensitivity of dinoflagellate cysts at the lower boundary of lethal
temperatures, and are consistent with the predictions made for dinoflagellate cysts by
Mountfort et al (1999) on lethal tempera-
167
FIGURE 7
Compilation of data on approximate sizes of organisms and
the pore sizes of various filtration options
tures and times. They also suggest that at
least in some cases, cysts of dinoflagellates
would not survive ships passage through
equatorial waters.
Pelagic cultures of a marine vibrio (V. alginolyticus) were inactivated by a UV dose of
35 mWscm-2 (3 log) and pelagic zoospore
preparations of U. pinnatifida were inactivated by a dose of 60 mWs cm-2 (>2log). UV
doses of greater than 60 mWscm-2 are likely
to be effective for the removal of any bacteria
of concern in marine waters, based on these
results and a literature review. Most viruses
will also be removed. IPNV, which is a highly resistant virus, is more difficult to control
requiring >122 mWscm-2 for three log reduction. 120 mWscm-2 may be adequate for
reducing IPNV below infectious levels
(Munday et al., 1992). This level also controlled vegetative Amphidinium and is six
times the level required to control
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
168
OEMCKE, et al.
Cryptosporidium in potable water, which is
one of the most resistant human enteric parasites. We therefore suggest an initial test
level of 60 to 120 mWscm-2 for UV treatment
of ballast water, if dinoflagellate cysts are
excluded.
It must be stressed that these tests were
conducted under laboratory conditions. Real
ballast tanks contain a population of ambient
heterotrophic bacteria that will survive in
sediments and coatings including biofilms.
Bacteria in loose biofilms are likely to be
released with ballast as would be those in
sediment particularly after ship’s passage in
rough seas. Resident bacteria may also feed
on damaged algal cells, inhibiting their recovery. For example UV treated Amphidinium
sp. cells lose motility after treatment, and
sink (Oemcke and van Leeuwen, in prep). In
other cases algal cells may encyst (Ten Lohius
and Miller, 1998). Iron levels and potential
oxygen stress may also have an effect on
recovery and survivorship.
It is our view that UV irradiation may have
application in the treatment of ballast water
during ballasting, during deballasting and in
shore-based ballast water treatment plants. It
could not be used to treat ballast water whilst
vessels are in-transit due to the lack of a disinfection residual and the presence of iron (the
potential for reducing conditions are likely to
lead to fouling of UV lamps reducing their
effectiveness and requiring arduous cleaning
schedules). Treatment during deballasting is
unlikely as deballasting practices can mean that
very large quantities of ballast are discharged at
once, requiring very large treatment plants. If
water is pre-treated by filtration during de ballasting there will also be a problem with disposal of filter retentate. Treatment at shorebased plants appears unlikely to be practical
due to the difficulty of getting ballast ashore
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
and the need for large storage facilities or large
treatment plants to ensure that ships are not
delayed. The most feasible option for UV treatment, then, is a shipboard plant for treatment
during ballasting and this is worthy of serious
consideration. Combined with pre-treatment
by solids separation by filtration or hydrocyclone and the potential added benefit of dark
storage (in some organisms), the use of UV as a
ballast treatment option should be further
examined.
A compilation of data from various
sources on the approximate sizes of
organisms and removal sizes of filtration
alternatives is shown in Figure 7. Based on
the size range of the organisms it is clear that
50µm screens should remove zooplankton
while 20µm screens will remove
dinoflagellate cysts. Screens will also remove
some dinoflagellate algae and protozoans.
Twenty-five and fifty micron screens are
currently the subject of other pilot scale
testing (IMO 2000, Hillman et al., 2003).
Smaller screens and filters may be
appropriate, depending on the results of the
pilot scale tests. The 20 and 50 µm screens
(and even 5 µm filters) will not remove
bacteria and viruses, many diatoms,
protozoans or dinoflagellate algae. UV would
be an appropriate technology for
combination with screening to achieve ballast
disinfection. The screening will also remove
clumps, flocs and adsorbed particles which
reduce the effectiveness of UV irradiation.
Density-based treatment such as
hydrocyclones may be effective, due to the
specific gravity of cysts (Anderson et al.
1985). However the low density difference
between water and cysts probably rules out
hydrocyclones. Heat treatment at 37-38oC is
effective against a number of zooplankton
and dinoflagellate cysts (Hallegraeff et al.,
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
1997). Heat will not, however, be effective
against many viruses and bacteria and may
not be effective against warm water tolerant
species unless the treatment is amended to
very high temperatures. UV may be an
appropriate treatment to speed up the
disinfection of dinoflagellate cysts, depending
on the time taken for heat treatment. This
would involve UV treatment during
ballasting followed by in-transit heat
treatment.
Ultraviolet irradiation has potential for the
treatment of dinoflagellate algae (gametes
only), protozoa, bacteria and viruses in ballast
water following solids separation. UV
irradiation produces a synergism with heating
and dark storage, which may be exploitable
for treating organisms, which are not
susceptible to heat, and to speed up the
inactivation of organisms which are
susceptible in heat treatment. The most
significant risk with the use of UV is the need
to have adequate clarity of water for effective
treatment. In sewage treatment clarity is often
poor and UV remains an effective alternative if
correctly designed. It is considered that in
most port waters adequate design will ensure
suitable efficacy of treatment.
Further to the research described here the
development of a pilot plant to test the results
in the field is being actively pursued by an
Australian/New Zealand consortium of interested parties. The development of treatment
options was also endorsed at a workshop
held in Brisbane in May 1999 and reported in
The Ballast Water problem – Where to from
here? (Hillman [Ed], 1999).
The prime objective of the pilot plant is to
formulate the design criteria for the successful
development of a treatment plant capable of
economically treating ships’ ballast water in
tropical, subtropical and temperate waters.
169
This pilot plant will be portable and is specifically designed to be moved around Australia
and New Zealand to treat organisms in their
natural environment. For example, it will be
taken to Tasmania, Australia during Gymnodinium catenatum blooms and Asterias
amurensis reproduction to test efficacy on
those organisms in their natural environment.
Important objectives of the consortium
include: (i), provision of a test bed for potential ballast water treatment technologies meeting international standards; (ii), provision of a
greater understanding of the chemical, physical and biological properties of ballast water
contamination in order to improve the capability of ports and shipping management bodies to detect and more effectively respond to
introduced marine pests; (iii), pilot plant testing of real port conditions that target the key
introduced marine pest species that threaten
temperate, sub-tropical and tropical waters;
and (iv), identification of potential commercial
applications in the development of an important new service – ballast water treatment. The
proposed project will build on the extensive
results of earlier research including the findings described in this paper. It will move from
laboratory testing to the construction of a fully
functional pilot ballast water treatment plant
with approximately 50m3/day capacity. This
plant will, for the first time, combine the most
promising physical treatment procedures for
ballast water treatment in a single portable
system.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Lester
Sinton, ESR, Christchurch, for helpful advice
and discussions. The authors also wish to
acknowledge funding and support from the
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
170
OEMCKE, et al.
Ports Corporation of Queensland, the
Cooperative Research Centre for Ecologically
sustainable Development of the Great Barrier
Reef and the School of Engineering, James
Cook University. Prof J. (Hans) van Leeuwen
has been invaluable in his assistance with this
research. We also wish to acknowledge the
assistance provided by Mr Steve Warne,
Contamination Control Ltd., Auckland for
producing the flow-through UV irradiation
unit and express appreciation to the consortium members for their ongoing support.
Dragsund, E., Anderson, A. B. and Johannessen, B. A. (2003)
Ballast water treatment by ozonation. In: (Raaymakers,
S., ed) Proceedings of the Ist International Symposium of
ballast water treatment, IMO, London, pp. 21-30.
REFERENCES
Hillman, S. P. (Ed) (1999) The ballast water problem – where
to from here? Proceedings of a Workshop held 5-6 May
1999, Brisbane, Australia. EcoPorts Monograph Series
No 19. Ports Corporation of Queensland.
Anderson, D. M., Lively, J. L., Reardon, E. M. and Price, C. A.
(1985) Sinking characteristics of dinoflagellate cysts.
Limnol Oceanogr. 30, 1000-1009.
Hallegraeff, G. M. and Bolch., C. J. (1991) Transport of toxic
dinoflagellate cysts via ships’ ballast water. Mar. Poll.
Bull. 22, 27-30.
Hallegraeff, G.M., Valentine, J.P., Marshall, J. and Bolch, C. J.
(1997) Temperature tolerances of toxic dinoflagellate
cysts: application to the treatment of ships’ ballast water.
Aquatic Ecology 31, 47-52
Harris, G. D., Adams, V. D., Sorenson, D. L. and Curtis, M. S.
(1987) Ultraviolet irradiation of selected bacteria and
viruses with photoreactivation of bacteria. Water Res.
21, 687- 692.
Hay, C. and Gibbs, W. (1996) A manual for culturing Wakame
(Undaria pinnatifda) 1: Gametophytes. Cawthron Institute
Report No. 336, 1-51.
Hillman, S.P., Oemcke, D. and Schneider, P. (2003)
Development of a portable pilot plant to treat ships’ ballast water. Marine Scientist (submitted).
Blackburn, S. I., Hallegraeff, G. M., and Bolch, C. J. (1989)
Vegetative reproduction and sexual life cycle of the toxic
dinoflagellate, Gymnodinium catenatum from Tasmania,
Australia. J. Phycol. 25, 577-590
Jagger, J. (1958) Photoreactivation. Bacteriological Reviews
22, 99-142.
Brand, L.E. (1986) Nutrition and culture of autotrophic ultraplankton and picoplankton. In: (Platt, T. and Li W. K. W.
eds) Photosynthetic Picoplankton. Canadian Bulletin of
Fisheries and Aquatic Sciences 214, 205-233.
Kelly, J.M. (1992) Transport of non-native organisms in ballast sediments: an investigation of woodchip ships entering Washington state waters. The Northwest
Environmental Journal 8, 159-160.
Burkholder, J.M., Glasgow, H.B. & Hobbs, C.W. (1995) Fish
kills linked to a toxic ambush-predator dinoflagellate:
distribution and environmental conditions. Marine
Ecology Progress Series 124, 43-61.
Liltved, H., Hektoen, H. and Efraimsen, H. (1995)
Inactivation of bacterial and viral fish pathogens by
ozonation or UV irradiation in water of different salinity.
Aquacultural Engg. 14, 107-122.
Campbell, A. T., Robertson, L. J., Snowball, M. R. and Smith,
H.V. (1995) Inactivation of oocysts of Cryptosporidium
parvum by ultraviolet irradiation. Water Res. 29, 25832586.
Loeblich, A. R. and Smith, V. E. (1968) Chloroplast pigments
of the marine dinoflagellate, Gyrodinium resplendens.
Lipids 3, 5-15
Cangelosi, A. A., Knight, T. T., Balcer, M., Wright, D., Dawson, R.,
Blatchley, C., Reid, D., Mays, N. and Tacerna, J. (2003) Great
Lakes ballast technology demonstration project: Biological
effectiveness test program (including MV Regal Princess trials).
In: (Raaymakers, S., ed) Proceedings of the Ist International
Symposium of ballast water treatment, pp. 88-94.
Carlton, J. T. (1985) Transoceanic and interoceanic dispersal
of coastal marine organisms: the biology of ballast water.
Oceanogr. Mar. Biol. Ann. Rev. 23, 313-371.
Clancy, J. and Fricker, C. (1998) Control of Cryptosporidium
- how effective is drinking water treatment? Water
Quality International, July/August, 37-41.
Cullen, J. J. and Lesser, M. P. (1991) Inhibition of photosynthesis by ultraviolet radiation as a function of dose and
dosage rate. Mar. Biol. 111, 183-190.
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G
MacDonald, E. M. and Davidson, R. D. (1998) The occurrence of harmful algae in ballast discharges to Scottish
ports and the effects of mid-ocean exchange in regional
seas. In (Reguera, B., Blanco, J., Fernandez, M. L., Wyatt,
T., eds) Harmful Algae. Xunta de Galicia and
Intergovernmental Oceanographic Commission of
Unesco, pp220-223.
de Man, J.C. (1977) MPN tables for more than one test.
European Journal of Applied Microbiology 4, 307-316.
Matheickal, J. T., Waite, T. D. and Mylvaganam, S. T. (2002)
Ballast water treatment by filtration. In: (Raaymakers, S.,
ed) Proceedings of the Ist International Symposium of
ballast water treatment, pp. 7-15.
McCarthy, S. A. and Khambarty, F.M. (1994) International
dissemination of epidemic Vibrio cholerae by cargo ship
ballast water and other nonpotable waters. Appl. Enviro.
Microbiol. 60, 2597-2601.
U V I R R A D I AT I O N O N V I A B I L I T Y O F M I C R O S C A L E
Medcof, J. C. (1975) Living marine animals in a ship’s ballast
water. Proc. Natnl. Shellfisheries Assoc. 65, 11-12.
Montani, S., Meksumpun, S. and Ichimi, K. (1995) Chemical
and physical treatments for the destruction of phytoflagellate cysts. J. Mar. Biotechnol. 2, 179-181.
Mountfort, D. O., Hay, C., Dodgshun, T., Buchanan, S. and
Gibbs, W. (1999) Heat treatment of ship’s ballast water:
application of a model based on laboratory studies. J.
Mar. Environ. Engineer 5, 193-216.
Mountfort, D. O., Dodgshun, T., Gibbs, W and McCallin, B.
(1999) Towards a feasible heat treatment system for
ships’ ballast water. Proceedings of AAPMA meeting,
Brisbane, May 5-6, pp 125-126.
Mountfort, D. O., Dodgshun, T. and Taylor, M. (2003) Ballast
water treatment by heat: New Zealand laboratory and
shipboard trials. In: (Raaymakers, S., ed) Proceedings of
the Ist International Symposium of ballast water treatment, IMO, London, pp. 45-50.
Munday, B., Clark, A., Hine, M., Lester, R. and Whittington,
R. (1992) An epidemiological review of possible disease
introductions through ships’ ballast water. In: An
Epidemiological Review of Possible Introductions of Fish
Diseases, Northern Pacific Seastar and Japanese Kelp
Through Ships’ Ballast Water. Australian Quarantine and
Inspection Service, Ballast Water Research Series, Report
No. 3, pp 1-238.
Nilsen, B., Nilsen, H. and Mackey, T. (2003) The Optimar ballast system. In: (Raaymakers, S., ed) Proceedings of the
Ist International Symposium of ballast water treatment,
IMO, London, pp. 126-136
Oemcke, D.J and van Leeuwen, J. (2003a) Chemical and physical characterisation of ballast water part 1: Effects on
ballast water treatment processes. Journal of Marine
Environmental Engineering 7, 47-64.
Oemcke, D.J and van Leeuwen, J. (2003a) Chemical and physical characterisation of ballast water part I: determining
the efficiency of ballast exchange. Journal of Marine
Environmental Engineering 7, 65-76.
171
Rigby, G. (1994) Possible solutions to the ballast water problem. Ballast Water Symposium Proceedings, Canberra 1113 May, pp 87-106.
Rigby, G., Hallegraeff, G., Sutton, C. (1999) Novel ballast
water heating technique offers cost-effective treatment to
reduce risk of global transport of harmful marine organisms. Mar. Ecol. Progr Ser. 191, 298-293
Ruiz, G. M., Carlton, J. T. and Hines, A. H. (1997) Global
invasions of marine and esturarine habitats by nonindigenous species: mechanisms, extent and consequences. Amer. Zool. 37, 621-632
Sobsey, M. D. (1989) Inactivation of health related microorganisms in water by disinfection processes. Water Sci.
Tech. 21, 179-195.
Sommer, R., Cabaj, A., Schoenen, D., Gebel, J., Kolch, A.,
Havelaar, A.H. and Schets, F.M. (1995) Comparison of
three laboratory devices for UV inactivation of microorganisms. Water Science and Technology 31, 147-156.
Subba Rao, D.V., Sprules, W.G., Locke, A. and Carlton, J.T.
(1994) Exotic phytoplankton from ships’ ballast waters:
risk of potential spread to mariculture sites on Canada’s
east coast. Canadian Data Report of Fisheries and
Aquatic Sciences 937.
Sutherland, T., Levings, C., Peterson, S. and Hesse, W. (2003)
The influence of cyclonic separation and UV treatment
on the mortality of marine plankton. In: (Raaymakers, S.,
ed) Proceedings of the Ist International Symposium of
ballast water treatment, IMO, London, pp. 120-125
Taylor, A. H. and Rigby, G. (2001) Suggested designs to facilitate improved management and treatment of ballast
water in new and existing ships. Report prepared for the
Department of Agriculture, Fisheries and Forestry,
Australia. Report No 12, pp 1-58.
Ten Lohuis, M.R. and Miller, D.J. (1998) Genetic
transformation of dinoflagellates (Amphidinium and
Symbiodinium): expression of GUS in microalgae using
heterologous promoter constructs. The Plant Journal 13,
427-435.
Oemcke, D. and van Leeuwen, J. (2003c) Seawater ozonation
of Bacillus subtilis spores: implications for the use of
ozone in ballast water treatment. Ozone Science and
Engineering (accepted).
Oemcke, D. and van Leeuwen, J. (2003d) Ozonation of the
marine dinoflagellate alga Amphidinium sp. Implications for ballast water disinfection Water
Research (submitted).
Parsons, M. G., Harkins, R. W., Mackey, T. P., Munro, D. J.
and Cangelosi, A. (1997) Design of the Great Lakes
Technology Demonstration Project. Transactions
SNAME 105, 323-328
Ribera, M.A. (1995) Introduced marine plants, with special
reference to macroalgae: mechanisms and impact. Prog.
Phycological Res. 11, 217-268.
JOURNAL
OF
M A R I N E E N V I R O N M E N TA L E N G I N E E R I N G