- Email: [email protected]
Ship board testing of a deoxygenation ballast water treatment
Marine Pollution Bulletin 54 (2007) 1170–1178 www.elsevier.com/locate/marpolbul
Ship board testing of a deoxygenation ballast water treatment Tracy McCollin
b
a,*
, Gemma Quilez-Badia b,1, Kjell D. Josefsen c, Margaret E. Gill b, Ehsan Mesbahi d, Chris L.J. Frid b,2
a FRS Marine Laboratory, P.O. Box 101, 375 Victoria Road, Aberdeen, AB11 9DB, UK Dove Marine Laboratory, School of Marine Science and Technology, University of Newcastle upon Tyne, Cullercoats, North Shields NE30 4PZ, UK c SINTEF Materials and Chemistry, Sem Saelands v.2A, 7465 Trondheim, Norway d School of Marine Science and Technology, Armstrong Building, Newcastle University, Newcastle upon Tyne NE1 7RU, UK
Abstract A ship board trial of a deoxygenation method for treating ballast water was carried out during a voyage from Southampton (United Kingdom) to Manzanillo (Panama). A nutrient solution added to two ballast tanks encouraged bacterial growth, resulting in a gradual change to an anoxic environment. Samples were taken from two treated tanks and two untreated tanks to assess changes in the abundance and viability of zooplankton, phytoplankton and bacteria. The work was carried out before the International Maritime Organization (IMO) standard was agreed so only a broad indication of whether the results achieved the standard was given. For the zooplankton, the standard would have been achieved within 5 or 7 days but the phytoplankton results were inconclusive. The biological efficacy was the result of the combination of several factors, including the treatment, pump damage and an increase in the water temperature during the voyage. Crown Copyright 2007 Published by Elsevier Ltd. All rights reserved. Keywords: Phytoplankton; Zooplankton; Bacteria; Deoxygenation treatment; IMO standard; Ballast treatment
1. Introduction After many years of negotiation the International Maritime Organization (IMO) adopted an International Convention for the Control and Management of Ships’ Ballast Water and Sediments (the Convention) in February 2004. The aim of the Convention is to reduce the risk of introducing non-native species and, in order to achieve this, a ballast water treatment standard will be phased in over time based on the age and size of vessels (IMO, 2005). Until this standard was agreed the biological efficacy of ballast water treatment systems being developed could not be assessed and compared. *
Corresponding author. E-mail address: [email protected] (T. McCollin). 1 Present address: Smithsonian Environmental Research Center, 647 Contees Wharf Road, Edgewater, MD 21037, USA. 2 Present address: School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
Deoxygenation has been suggested as a treatment method and previous trials have included sparging with a gas stream of nitrogen (Mountfort et al., 1999; Tamburri et al., 2002), addition of glucose or sulphide (Mountfort et al., 1999), use of a vacuum chamber (Browning and Browning, 2003; Browning et al., 2004) and application of a Venturi Oxygen Stripping system to remove oxygen as ballast is loaded (Tamburri et al., 2004a,b). Initial results indicated that the addition of glucose or sulphide had little effect but that using nitrogen gas had some potential (Mountfort et al., 1999; Tamburri et al., 2002). On board trials with vacuum chambers had some success with zooplankton but the effect on phytoplankton was not clear (Browning and Browning, 2003; Browning et al., 2004). Experiments using a Venturi Oxygen Stripping system demonstrated it was effective at killing zooplankton but the results for phytoplankton were inconclusive. However, further shore based experiments using different methods of assessing viability of the phytoplankton have suggested
0025-326X/$ - see front matter Crown Copyright 2007 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2007.04.013
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
that this system may have the potential to achieve the new IMO standard (Tamburri et al., 2004b). The method used in this study was based on the addition of nutrients to stimulate the growth of bacteria, to consume the oxygen within the water and create a hostile environment for the aerobic organisms within the tank. This is similar to the method used by Mountfort et al. (1999) but uses a mix of nutrients designed to minimise the growth of sulphur reducing bacteria that may lead to increased corrosion and production of H2S. Literature reviews regarding the tolerance of different groups of organisms to anoxic conditions came to similar conclusions, i.e. that oxygen tolerance varied but that only a few organisms could tolerate hypoxia or anoxia for more than a few days and most could only survive a few hours (Assink et al., 2001; Tamburri et al., 2002). This treatment method was therefore designed to reduce and maintain the low oxygen levels over a number of days in order to ensure that even the most tolerant species were not able to survive. The treatment trial was carried out on the car carrier MV Don Quijote on a voyage between England and Panama. The purpose of the ship based trials was to test the efficiency of the method under operational conditions. 2. Materials and methods The trial was undertaken on board the pure car and truck carrier MV Don Quijote, a 22,625 metric tonne vessel with 17 ballast tanks and a ballast capacity of 8075 tonnes. The method was tested from the 21 to 28 June, 2003 during a voyage from Southampton (UK) to Manzanillo (Panama). Four ballast tanks were used during the trial and these were ballasted in line with normal operations (Table 1). Two upper wing tanks were used as treatment tanks (3U Port (3UP) and Starboard (3US)) and two double bottom tanks (DB3 Port (DB3P) and Starboard (DB3S)) were used as untreated control tanks. A nutrient solution (Table 2) was added via the sounding pipe to the two empty treatment tanks and the tanks were then filled with ballast water. 2.1. Sampling Samples of zooplankton, phytoplankton and bacteria were taken from each tank on day 0 (June 21st), i.e. when
1171
Table 2 Quantities of nutrients added to the treated tanks and calculated final concentration based on the tank volumes Compound
Tank 3US added
Tank 3UP added
Calculated conc. (g/m3)
Sucrose Glucose H2O NH4NO3 KNO3 Na2HPO4 Æ 2H2O Ship’s tap water (hot)
18.9 kg 7.5 kg 3.6 kg 3.6 kg 1.2 kg 26.4 l
16.6 kg 6.6 kg 3.2 kg 3.2 kg 1.0 kg 23.0 l
58 23 11 11 4
The volumes of tank 3US and 3UP were 326 and 285 m3, respectively.
the tanks were loaded and the nutrients added, and then after 3, 5 and 7 days. Sampling the tanks via the access manholes was not possible as they were covered with cargo so these samples were collected by pumping ballast water through the fire pump to the sampling equipment. The flow rate was 55–85 l min1 and the volume of water collected for each sample was calculated using a flow meter. The pipes were flushed for 15 min to remove water remaining in the pipe system before each new tank was sampled. Problems with the flushing meant that samples taken on the 24th (day 3) had to be disregarded. On the days that this sampling was not carried out, smaller samples for bacteria and chlorophyll a analysis were taken from the treated tanks via the sounding pipe by lowering a silicone tube connected to a peristaltic pump down the pipe and drawing the water up into a 1 l plastic bottle. The sample did not pass through the pump. The dissolved oxygen was measured with an oxygen electrode. Samples were taken by filling a plastic 1 l bottle with water from the tank, placing the probe in the bottle and sealing the top. The reading was taken once it had stabilised, which was usually after 0.5–1 h. A portable pH meter was used to determine pH. The concentration of H2S was assessed by a spectrophotometric method (Cline’s method) as described by Fonselius et al. (1999). The concentration of the mineral nutrients in the ballast water at the end of the study was determined according to NS (Norwegian Standard) 10304 (nitrate), NS 1189 (ortho-phosphate) and a modification of NS 4746 (ammonium) (available from www.standard. no).
Table 1 Details of the ballast tanks used during the trial and the position of the vessel when filling the tanks Tank
Capacity (m3)
Date/time
Start position
Water depth* (m)
Date/time
Stop position
Water depth* (m)
DB3 Star Port
513 513
21.06.03 01:00
50 26N 001 24W
50
21.06.03 02:00
50 21N 001 38W
55
3U Star Port
326 285
21.06.03 02:00
50 21N 001 38W
55
21.06.03 02:50
50 14N 002 01W
62
* Depth calculated from http://topex.ucsd.edu/WWW_html/mar_topo.html at Fisheries Research Services Marine Laboratory (Aberdeen, UK). DB = double bottom tanks and U = upper tanks.
1172
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
2.2. Zooplankton methods The zooplankton samples consisted of the organisms present in 1 m3 of water filtered using a 50 lm sieve. Once filtered, the plankton collected on the sieve was rinsed into a bottle with filtered sea water and stained with 0.1% neutral red solution in the ratio of 3 ml stain/100 ml sample. After staining for 30 min, 4 ml of 1 M sodium acetate solution was added per 100 ml of sample (Omori and Ikeda, 1984). The specimens were then fixed with 4% formalin in a volume equal to that of the sample (50/50) and stored overnight at 5 C. Thereafter, the samples were kept at room temperature until analysis. Prior to examination of the samples, glacial acetic acid was added drop by drop, until the colour of the solution changed to magenta. Live organisms turned a deep magenta after acidification, whereas dead specimens were light pink to white or transparent (when having an exoskeleton or shell). The sample was then filtered through a 48 lm sieve, washed with tap water and transferred to a Petri dish for sorting and counting using a stereomicroscope. When necessary, subsamples were taken by mixing the sample and taking a pre-determined volume that contained at least 100 individuals of one of the main groups (i.e. copepods and nauplii). The assessment of individual organisms included a morphological examination and whole organisms as well as identifiable body parts were taken into account and identified to the level of subclass or class. 2.3. Phytoplankton methods The phytoplankton sampling consisted of collecting samples for two types of analyses: chlorophyll a analysis and direct cell counts. For the chlorophyll a analysis, approximately 5 l of ballast water were pre-filtered through a 250 lm mesh into a bucket and mixed thoroughly, three replicates of 500 ml were collected and vacuum filtered using Whatman GF/A glass fibre filters (1.6 lm). Each filter was wrapped with aluminium foil and frozen immediately at 20 C until fluorometric chlorophyll a analysis based on an acetone extraction method was carried out (Arar and Collins, 1997). For the direct cell count samples the water from the tanks was pre-filtered through 250 and 100 lm sieves and the filtrate collected. Initially, 90 l were collected but from the second sampling day (day 3) 60 l were collected to reduce the time required for the next step. The filtrate was divided into three replicates of 30 or 20 l and each was filtered through a 10 lm plankton net. The collected sample was preserved with Lugol’s iodine (approximately 2 ml was added to each sample) and stored in a cool dark place until analysis. The analysis was carried out using standard counting procedures based on the Utermo¨hl sedimentation method (Utermo¨hl, 1958) and using an inverted microscope. The phytoplankton was identified to the level
of class, i.e. Bacillariophyceae (diatoms) and Dinophyceae (dinoflagellates) and enough of the sample was examined to ensure that a minimum of 100 cells from each class was counted. No assessment of viability could be made but only intact, undamaged cells were counted and these were assumed to be viable.
2.4. Bacterial methods Samples for bacterial analysis were collected in sterile polypropylene bottles (100 ml or 1 l) and kept cool until they could be processed further (usually within a few hours). The 100 ml samples were collected from the tube flushing water over the zooplankton sieve. On those days that zooplankton samples were not taken, a 1 l sample was pumped up from the treated tanks through the sounding pipe (see above). The concentration of viable bacteria in the samples was determined by a most probable number technique. The seawater was diluted in a tenfold dilution series in filtered (Whatman glass microfibre filter GF/A), heat sterilised (120 C, 20 min) seawater. Five 0.1 ml aliquots from selected dilutions were mixed with 0.1 ml medium in 5 wells in a pre-sterilised 96 microwell plate. The medium contained per litre: peptone, 10.0 g; yeast extract, 2.0 g; NaCl, 15.0 g; K2HPO4 Æ 3H2O, 20 mg; phenol red, 60 mg; filtered (glass microfibre filter GF/A) seawater, 500 ml, distilled water, 500 ml, pH 8.1 ± 0.1. The medium was heat sterilized (120 C, 20 min). The inoculated microwell plates were incubated at room temperature (22 ± 3 C) for 1–2 weeks. Positive cultures were scored by visible turbidity, and the most probable number determined from the tables given in Appendix II in the US Food and Drug Administration’s Bacteriological Analytical Manual online (Blodgett, 2001).
2.5. Statistical analysis for zooplankton The mortality of the zooplankton before and after treatment was calculated. For the control samples mortality was calculated as the dead organisms divided by the total number of organisms counted (dead plus alive). The mortality for treated samples was calculated based on the initial numbers of organisms found in each of the tanks on the first day and assuming that the missing organisms had been killed or destroyed by the treatment. Three–way nested ANOVA (tank was always nested to treatment) were conducted to examine mortality over time, between all tanks and between treatments; the rate at which copepods and nauplii disappeared from the treated and control tanks; the differences in concentrations of viable organisms in the treated and control tanks over time; and to compare the sensitivity between copepods and nauplii towards the treatment during the different days. Where necessary the data was transformed by squaring or using a Box-Cox or Log+1 transformation.
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
1173
2.6. Statistical analysis for Phytoplankton
3.3. Nutrients in treated ballast water
To examine any difference in chlorophyll a or cell concentrations between treated and control tanks and over time, two-way ANOVA tests were carried out. To test differences between particular days, tanks and samples taken via the fire pump and those taken via the sounding pipe, one-way ANOVA or a Kruskal–Wallis Test was used.
The nutrient solution added to the ballast water contained glucose, sucrose, ammonium, nitrate and phosphate to yield the concentrations given in Table 3. At the end of the trial the treated tanks contained slightly more ammonium than added at the start (105–128% of the original addition), less than 1% of the original addition of nitrate and 10–11% of the original addition of phosphate. The sugar content of the ballast water at the end of the trial was not analysed.
3. Results 3.1. pH and dissolved oxygen Bacterial growth in the treated tanks resulted in a decrease in pH, but remained constant in the control tanks (Fig. 1). The pH of the samples taken from the sounding pipe was slightly lower than for samples taken via the fire pump. Based on previous laboratory studies, the treated water was expected to become anoxic after 30 h (Josefsen, 2003) but the concentration of dissolved oxygen in the treated tanks was higher than expected for the first four sampling occasions (Fig. 2). This could be due to the electrode not stabilising before a reading was taken. The samples taken on the last day were anoxic but it is not possible to assess whether the water became anoxic before this. 3.2. Hydrogen sulphide No significant amounts of H2S were detected in the samples although the water from the treated tanks developed a pungent odour after a few days. As the analysis was performed under field conditions, an exact concentration could not be determined, but it was well below 1 lmol l1.
Fig. 1. pH in the treated and control tanks over the course of the voyage. Samples taken on the 22, 23, 25 and 27th June were taken via the sounding pipe, the remainder were sampled via the fire pump. The samples were taken at different times over the course of a day so the symbols from each day are not directly below one another. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
3.4. Zooplankton A large number of different taxa were found in the ballast tanks but as copepods and nauplii represented 38% and 60% of the organisms present respectively the results are based on these organisms. 3.5. Control samples As in previous trials carried out on the Don Quijote (Quilez-Badia et al., submitted for publication), high zooplankton mortality was observed in the untreated samples (Fig. 3). During the trial the zooplankton in the control tanks disappeared (assumed to be dead) at a rate of 20–30% a day (Fig. 4) so the 70–90% mortality rate at the beginning of the trial was assumed to be due to a combination of environmental conditions in the tanks and mechanical damage caused by the pump before they reached the sampling point. This complicated the assessment of the biological efficacy but as the water in all the tanks had been subjected to exactly the same conditions except that the untreated tanks had no nutrients added, it was assumed that any differences between the treated and control tanks over time was due to the treatment.
Fig. 2. Concentration of dissolved oxygen in water samples taken from the ballast tanks. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
1174
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
Table 3 Calculated concentration of sugar and nutrients immediately after addition of the solution to the treated tanks (3US and 3UP), and measured concentration of mineral nutrients in both treated and control tanks at the end of the trial Tank
Concentration (g/m3 ballast water) Glucose
DB3S DB3P 3US 3UP
NH4 þ ðas NÞ
Sucrose
NO3 ðas NÞ
PO4 3 ðas PÞ
Start
End
Start
End
Start
End
Start
End
Start
End
n.a n.a 20.9 20.9
n.a n.a n.a n.a
n.a n.a 58.0 58.1
n.a n.a n.a n.a
n.a n.a 1.93 1.94
<0.05 <0.05 2.47 2.03
n.a n.a 3.46 3.47
0.026 0.024 0.036 0.027
n.a n.a 0.65 0.65
<0.005 <0.005 0.071 0.068
n.a = Not analysed.
Fig. 3. The percentage mortality of the nauplii and copepods over the course of the voyage (mean ± SD). DB3P and DB3S = double bottom tank number 3 port and Starboard; 3US and 3UP = upper tank number 3 port and starboard.
Fig. 4. Concentrations of copepods and nauplii, i.e. both dead and alive over the course of the voyage, the vertical line represents the minimum and maximum concentration found in each sample and the symbol the average of three samples. The disappearance rates indicated by the dashed lines are based on the assumption that a given percentage of zooplankton disappear each day. The average concentration in the samples on the 21st is used as a starting level. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
3.6. Treated samples Zooplankton abundance decreased in all the tanks during the trial with differences between the treated and untreated tanks. The disappearance rate in the control tanks was 20–30% of the population per day for both copepods and nauplii, while in the treated tanks it was 45–55% per day for copepods and 50–65% per day for nauplii (Fig. 4). The decrease over time was significant for both treated and untreated tanks (three-way nested ANOVA,
for time, treatment and tank (nested to treatment), with the following interactions: time · treatment and time · tank (nested to treatment)) for both copepods (F = 62.08, p < 0.001) and nauplii (F = 52.99, p < 0.001). The overall abundance of copepods and nauplii was lower in the treated water compared to the untreated tanks (F = 42.46, p < 0.001, for copepods and F = 42.99, p < 0.001, for nauplii). Time increased the effect of the
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
treatment for nauplii (i.e. the interaction between time and treatment, F = 3.79, p < 0.05), the longer they had been in the treated tanks, the faster their numbers decreased. The mortality observed in the control tanks was significantly lower than in the treated tanks (three-way ANOVA (for time, treatment and live stage, with the following interactions: time · treatment, time · live stage, treatment · live stage and time · treatment · live stage), F = 34.43, p < 0.001). More live organisms were found in the samples from the control tanks than in the treated ones (three-way nested ANOVA, p < 0.001). No significant differences were found in terms of the mortality of copepods and nauplii suggesting they were equally sensitive to the treatment. The results are presented in relation to the IMO standard (i.e. <10 viable organisms P50 lm in minimum dimension per m3). However, as the standard was agreed after this work was carried out, this can only be considered a very broad comparison as these counts only include copepods and nauplii and will underestimate other groups that may have been present. Taking this into account the standard was achieved after 5 and 7 days in the treated tanks (3US and 3UP) (Fig. 5). The control tanks, which were subjected to all the same conditions except the addition of the nutrients, did not achieve the standard.
1175
Fig. 5. Concentration of viable zooplankton (mean ± SD). The line indicates the IMO standard of <10 viable organisms P50 lm in minimum dimension per m3. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
3.7. Phytoplankton 3.7.1. Chlorophyll a Samples taken from the treated tanks, particularly those sampled via the sounding pipe, tended to have a greater concentration of chlorophyll a than the controls (Fig. 6). On the last day there were significantly lower concentrations of chlorophyll a in the control tanks (below the limit of detection of 0.028 lg chl a l1) than in the treated tanks (2-way ANOVA p < 0.001). The samples taken via the sounding pipe suggested that the treated tanks had higher levels of chlorophyll a than those collected via the fire pump (Fig. 6) and for one tank (3US) there were significantly higher levels of chlorophyll a for samples taken on the 23rd in comparison to samples taken from the other tanks (Kruskal–Wallis, p < 0.05). 3.8. Cell counts On the first day of sampling there were no significant differences in the cell counts between the treated and control tanks (Fig. 7). On the second sampling date (26th) there was a higher concentration of dinoflagellates in one of the treated tanks (3US) (1-way ANOVA, p < 0.05) compared to the other tanks sampled on this day. On the final day of sampling higher concentrations of both diatoms (Kruskal–Wallis, p < 0.05) and dinoflagellates (1-way ANOVA, p < 0.001) were found in the control tanks. As with the zooplankton the IMO standard can only be compared very broadly as the phytoplankton cells counted during this trial were all >10 lm and <100 lm and the IMO standard states that <10 viable organisms P10 lm
Fig. 6. Chlorophyll a values for samples collected over the course of the voyage (mean ± SD). The star symbol represents samples that were taken via the sounding pipe, no control samples were taken on these occasions. All other samples were collected via the fire pump. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
and <50 lm in minimum dimension per ml should be discharged. Also, although only full, intact cells were counted no assessment of viability was made. Taking all this into account both the treated and untreated tanks achieved the standard on all occasions (Fig. 8), i.e. the water loaded into the tanks at the beginning of the trial was below the standard.
1176
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
Fig. 8. Combined concentration of dinoflagellates and diatoms from samples collected during the voyage (mean ± SD). The top of the scale represents the IMO standard of <10 viable organisms P10 lm and <50 lm in minimum dimension per ml. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
Fig. 7. Concentration of dinoflagellates and diatoms from samples collected with a 10 lm mesh (mean ± SD). DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
3.9. Bacteria The concentration of viable bacteria in the treated tanks increased rapidly, as expected, from around 1 · 104 GU (growth units) ml1 a few hours after filling the tanks to around 6 · 107 GU ml1 after 48 h (see Fig. 9). In contrast to previous experiments (Josefsen, 2003), instead of remaining at this level the concentration of viable bacteria in the treated tanks started to decline after about 5 days to a level of around 8 · 105 GU ml1 at the end of the experiment. The bacterial concentration also increased in the control tanks during the study. From a starting point of around 1 · 104 GU ml1 it increased about 10 times to around 1 · 105 GU ml1 at the end of the study. The high concentration recorded on the 26th was probably due to contamination with water from the previous sampling of the treated tanks. 4. Discussion Ship board testing of this deoxygenation technique followed on from promising laboratory trials (McCollin and Shanks, 2003; Quı´lez-Badia et al., 2003) and was an opportunity to assess whether this method could treat ballast
Fig. 9. Concentration of viable bacteria in the treated and control tanks over the course of the voyage. Samples taken on the 22, 23, 25 and 27th June were taken via the sounding pipe, the remainder were sampled via the fire pump. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard. The error bars indicate a theoretical 95% confidence interval as given by Blodgett (2001).
water effectively on a large scale under operational conditions. Previous testing of deoxygenation methods have been carried out at a variety of scales (Mountfort et al., 1999; Tamburri et al., 2004a,b; Browning and Browning, 2003; Browning et al., 2004) but there are limited data on the efficacy of these methods during ship board trials,
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
although testing of the Venturi Oxygen Stripping method on board vessels is currently underway (Smith et al., 2006). The method tested during this trial was based on the addition of nutrients to the ballast tanks to encourage the growth of bacteria, resulting in a gradual change to an anoxic environment. The method successfully reduced the oxygen level within the tanks and the water in the treated tanks was anoxic at the end of the trial. There was an unexpected decrease in the number of viable bacteria in the treated tanks after about 5 days. This could be owing to aerobic and facultative anaerobic bacteria starting to die and being replaced by obligate anaerobic bacteria (not detectable by the methods used), although in previous laboratory experiments obligate anaerobic bacteria have only constituted a small fraction of the flora (unpublished results). Increased predation by facultative or obligate anaerobic protozoa, which may have increased in number in response to the increased concentration of bacteria, or the bacteria starting to aggregate and being counted as one viable unit by the MPN method employed are other possible explanations for this decrease. Distinguishing between live and dead phytoplankton cells also caused problems. In this study only full, intact cells were counted and these were assumed to be viable. Methods such as assessing the condition of chloroplasts (Tamburri et al., 2004b) or using grow out techniques (Smith et al., 2006) are time consuming and it is likely that automated methods such as flow cytometry in combination with vital stains will be developed in the future (Veldhuis and Kraay, 2000; Veldhuis et al., 2004). When assessing the biological efficacy of this method it is important to consider that the organisms were affected by a number of factors such as reduced oxygen levels, lowered pH, mechanical damage by the pump and an increase in water temperature during the voyage from around 18 C to almost 30 C. There will also be a natural rapid decline of zooplankton and phytoplankton over the time of the voyage (Gollasch et al., 2000a; Lavoie et al., 1999; Olenin et al., 2000; Taylor and Bruce, 2000; Verling et al., 2005; Wonham et al., 2001) although on some occasions some species have been shown to increase during the voyage (Gollasch et al., 2000b). However, as the water in both treated and untreated tanks had been subjected to the same conditions except that the untreated tanks did not have the nutrient mix added it can be assumed that any difference between the treated and untreated tanks over time was due to the treatment. Also, the effect of the pumping will affect the biological efficacy of any ballast water treatment as, unless the tanks are loaded or unloaded by gravity, the ballast water will always have to pass through the pumps. The results of this study clearly demonstrated the abundance and viability of the zooplankton decreased more over time in the treated tanks compared to the untreated tanks. The phytoplankton results showed no clear treatment effect. There were fewer phytoplankton cells in the treated tanks on the last day of treatment compared to
1177
untreated tanks, suggesting that the treatment had successfully reduced the number of diatoms and dinoflagellates. However, the chlorophyll a concentrations were higher in the treated tanks than in the controls, suggesting that although the cells were present in lower numbers they contained a greater concentration of chlorophyll a. Even though these data cannot be conclusive, it would seem unlikely that this method of deoxygenation is an effective method of reducing diatoms and dinoflagellates. Only a very broad indication can be given as to whether the treatment method would have achieved the IMO ballast water treatment standard, which was agreed after this work was carried out. It is important to remember that only selected groups of organisms were counted, i.e copepods, nauplii, diatoms and dinoflagellates, so counts are likely to be underestimated and no assessment of viability could be made for the phytoplankton. When presented in comparison to the IMO standard the counts for the zooplankton showed that the treated tanks achieved the standard after 5 and 7 days and that the counts for the phytoplankton achieved the standard on all occasions, i.e the water that was loaded at the beginning of the voyage achieved the standard. For bacteria the IMO standard focuses on Escherichia coli and intestinal enterococci, both indicators of faecal contamination of the water, and toxicogenic Vibrio cholerae. None of these were analysed in this study. Overall, this trial demonstrated that in spite of the difficulties of determining a treatment effect when undertaking ship board trials, this treatment method and testing regime were able to show effects on zooplankton, with the deoxygenation method causing increased mortality in the treated tanks. However, the phytoplankton results were inconclusive and the methods of analysis would have to be adapted and improved if further ship board trials were to go ahead. This ballast water treatment method would only be suitable for vessels undertaking longer voyages as it relies on a gradual reduction in oxygen over time and if such a ballast water treatment were to be developed further the safe levels of nutrients that could be discharged would have to be ascertained (Ellis and van der Woerd, 2004). Acknowledgements This research was carried out as a European Union (EU) funded project (GRD1-2000-25383), the On Board Treatment of Ballast Water (Technologies, Development and Applications) and Application of Low-Sulphur Marine Fuel (MARTOB). The bacterial part of the project was also partly funded by Norsk Hydro ASA. We thank the officers and crew of the Don Quijote for all their help during the time of the trials and also Wallenius Wilhelmsen WW Lines and the shipping agents involved in ensuring all personnel and equipment arrived safely on board the vessel. Tony Vourdachas assisted with the sampling. The phytoplankton counts were carried out by Irina Olenina and her team at the Centre for Marine Research in Klaipeda.
1178
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
References Arar, E.J., Collins, G.B., 1997. Method 445.0. In vitro determination of chlorophyll a and phaeophytin a in marine and freshwater algae by fluorescence. Revision 1.2. US Environmental Protection Agency: Ohio. Assink, J., Ellis, J., Janssen, L., Josefsen, K., Mitropoulou, V., Sassi, J., Mesbahi, E., Vourdachas, A., 2001. Task 2.5 – Programme of requirements for ballast water treatement. MARTOB Report DTR2.5-12-01, pp. 35–53. Blodgett, R., 2001. Appendix 2. Most probable number from serial dilutions. In US Food and Drug Administration. Center for Food Safety and Applied Nutrition. Bacteriological Analytical Manual Online. Internet address: http://www.cfsan.fda.gov/ ~ ebam/bama2.html (as downloaded June 2003). Browning Jr., W.J., Browning, W.J., 2003. Ballast Water Treatment by De-oxygenation – The AquaHabiStarTM System. In: Raaymakers, S. (Ed.), 2003. 1st International Ballast Water Treatment R&D Symposium, IMO, London, 26–27 March 2001: Symposium Proceedings, GloBallast Monograph Series No.5. IMO: London, pp. 51–60. Browning Jr., W.J., Davis, J.P., Browning, W.J., 2004. Progress report on the AquaHabiStat deoxygenation system. In: Matheickal, J.T., Raaymakers, S. (Eds.), 2004. 2nd International Ballast Water Treatment R&D Symposium, IMO, London, 21–23 July 2003: Symposium Proceedings, GloBallast Monograph Series No.15. IMO: London, pp. 26–33. Ellis, J., van der Woerd, H., 2004. Environmental Impacts, Risk and Safety and Economic Aspects of Ballast Water Treatment Methods. MARTOB Report DTR-4.8-SSPA-03.04. Available from http://martob.ncl.ac.uk/Documents/. Fonselius, S., Dyrssen, D., Yhlen, B., 1999. Determination of hydrogen sulphide. In: Grasshoff, K., Kremling, K., Ehrhardt, M. (Eds.), Methods of Seawater Analysis. Wiley – VCH Verlag GmbH, Weinhiem. Gollasch, S., Rosenthal, H., Botnen, H., Hamer, J., Laing, I., Leppa¨koski, E., Macdonald, E., Minchin, D., Nauke, M., Olenin, S., Utting, S., Voigt, M., Wallentinus, I., 2000a. Fluctuations of zooplankton taxa in ballast water during short-term and long-term ocean-going voyages. International Review of Hydrobiology 85, 597–608. Gollasch, S., Lenz, J., Dammer, M., Andres, H.-G., 2000b. Survival of tropical ballast water organisms during a cruise from the Indian Ocean to the North Sea. Journal of Plankton Research 22, 923–937. International Maritime Organisation, 2005. Ballast Water Management Convention. International Maritime Organisation, London. Josefsen, K.D., 2003. Biological de-oxygenation. MARTOB Report DTR3.2.-SINTEF-09.03. Available from http://martob.ncl.ac.uk/Documents/. Lavoie, D.M., Smith, L.D., Ruiz, G.M., 1999. The potential for intracoastal transfer of non-indigenous species in the ballast water of ships. Estuarine, Coastal and Shelf Science 48, 551–564. McCollin, T.A., Shanks, A., 2003. Biological Assessment. Phytoplankton Results. MARTOB Report DTR-3.7.2-FRS-06.03. Available from http://martob.ncl.ac.uk/Documents/. Mountfort, D.O., Hay, C., Dodgshun, T., Buchanan, S., Gibbs, W., 1999. Oxygen deprivation as a treatment for ships’ ballast water-laboratory
studies and evaluation. Journal of Marine Environmental Engineering 5, 175–192. Olenin, S., Gollasch, S., Jonusˇas, S., Rimkute, I., 2000. En-route investigations of plankton in ballast water on a Ship’s Voyage from the Baltic Sea to the open Atlantic Coast of Europe. International Review of Hydrobiology 85, 577–596. Omori, M., Ikeda, T., 1984. Methods in Marine Zooplankton Ecology. John Wiley. Quı´lez-Badia, G. Gill, M., Frid, C.L.J., 2003. Biological Assessment. Zooplankton Results. MARTOB Report DTR-3.7.1-UNEW-08.03. Available from: http://martob.ncl.ac.uk/Documents/. Quilez-Badia, G., McCollin, T., Josefsen, K., Vourdachas, T., Gill, M., Mesbahi, E., Frid, C.L.J., submitted for publication. On board shorttime high temperature heat treatment of ballast water: A field trial under operational conditions. Smith, G., Mullady, T., Ruiz, G., Tamburri, M., 2006. Design and Implementation of Shipboard Tests of Ballast Water Treatment: The Case of Ventui Oxygen Stripping (VOS). In: Proceedings of 14th International Conference on Aquatic Invasive Species, May 14–19, 2006, Key Biscyane, Florida, p. 79. Taylor, M.D., Bruce, E.J., 2000. Mid Ocean Ballast Water Exchange: Shipboard Trials of Methods for Verifying Efficiency. Cawthron Institute in association with Battelle, Nelson. Cawthron Report 524. Tamburri, M.N., Wasson, K., Matsuda, M., 2002. Ballast water deoxygenation can prevent aquatic introductions while reducing ship corrosion. Biological Conservation 103, 331–341. Tamburri, M.N., Little, B.J., Ruiz, G.M., Lee, J.S., McNulty, P.D., 2004a. Evaluations of Venturi Oxygen StrippingTM as a ballast water treatment to prevent aquatic invasions and ship corrosion. In: Matheickal, J.T., Raaymakers, S. (Eds.), 2004. 2nd International Ballast Water Treatment R&D Symposium, IMO, London, 21–23 July 2003: Symposium Proceedings, GloBallast Monograph Series No. 15, IMO, London, pp. 34–47. Tamburri, M.N., Ruiz, G.M., McNulty, P.D., 2004b. Pilot-Scale Evaluations of Venturi Oxygen StrippingTM. In: Proceedings of 2nd International Conference and Exhibition on Ballast Water Management, Singapore, 19–21 May, 2004, pp. 87–88. Utermo¨hl, H., 1958. Sur Vervollkomming der quantitativen Phytoplankton-Methodik Mitteilungen. Internationale Vereiningung fuer Theoretische und Angewandte Limnologie 9, 1–38. Veldhuis, M.J.W., Bossink, M., Boon, J.P., 2004. Effect of secondary treatments on phytoplankton: a new technique to test the cell viability. In: Proceedings of 2nd International Conference and Exhibition on Ballast Water Management, Singapore, 19–21 May, 2004, pp. 87–88. Veldhuis, M.J.W., Kraay, G.W., 2000. Application of flow cytometry in marine phytoplankton research: current applications and future perspectives. Scientia Marina 64, 121–134. Verling, E., Ruiz, G.M., Smith, L.D., Galil, B., Miller, A.W., Murphy, K.R., 2005. Supply-side invasion ecology: characterizing propagule pressure in coastal ecosystems. Proceedings of the Royal Society B 272, 1249–1256. Wonham, M.J., Walton, W.C., Ruiz, G.M., Frese, A.M., Galil, B.S., 2001. Going to the source: role of the invasion pathway in determining potential invaders. Marine Ecology Progress Series 215, 1–12.
Ship board testing of a deoxygenation ballast water treatment Tracy McCollin
b
a,*
, Gemma Quilez-Badia b,1, Kjell D. Josefsen c, Margaret E. Gill b, Ehsan Mesbahi d, Chris L.J. Frid b,2
a FRS Marine Laboratory, P.O. Box 101, 375 Victoria Road, Aberdeen, AB11 9DB, UK Dove Marine Laboratory, School of Marine Science and Technology, University of Newcastle upon Tyne, Cullercoats, North Shields NE30 4PZ, UK c SINTEF Materials and Chemistry, Sem Saelands v.2A, 7465 Trondheim, Norway d School of Marine Science and Technology, Armstrong Building, Newcastle University, Newcastle upon Tyne NE1 7RU, UK
Abstract A ship board trial of a deoxygenation method for treating ballast water was carried out during a voyage from Southampton (United Kingdom) to Manzanillo (Panama). A nutrient solution added to two ballast tanks encouraged bacterial growth, resulting in a gradual change to an anoxic environment. Samples were taken from two treated tanks and two untreated tanks to assess changes in the abundance and viability of zooplankton, phytoplankton and bacteria. The work was carried out before the International Maritime Organization (IMO) standard was agreed so only a broad indication of whether the results achieved the standard was given. For the zooplankton, the standard would have been achieved within 5 or 7 days but the phytoplankton results were inconclusive. The biological efficacy was the result of the combination of several factors, including the treatment, pump damage and an increase in the water temperature during the voyage. Crown Copyright 2007 Published by Elsevier Ltd. All rights reserved. Keywords: Phytoplankton; Zooplankton; Bacteria; Deoxygenation treatment; IMO standard; Ballast treatment
1. Introduction After many years of negotiation the International Maritime Organization (IMO) adopted an International Convention for the Control and Management of Ships’ Ballast Water and Sediments (the Convention) in February 2004. The aim of the Convention is to reduce the risk of introducing non-native species and, in order to achieve this, a ballast water treatment standard will be phased in over time based on the age and size of vessels (IMO, 2005). Until this standard was agreed the biological efficacy of ballast water treatment systems being developed could not be assessed and compared. *
Corresponding author. E-mail address: [email protected] (T. McCollin). 1 Present address: Smithsonian Environmental Research Center, 647 Contees Wharf Road, Edgewater, MD 21037, USA. 2 Present address: School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
Deoxygenation has been suggested as a treatment method and previous trials have included sparging with a gas stream of nitrogen (Mountfort et al., 1999; Tamburri et al., 2002), addition of glucose or sulphide (Mountfort et al., 1999), use of a vacuum chamber (Browning and Browning, 2003; Browning et al., 2004) and application of a Venturi Oxygen Stripping system to remove oxygen as ballast is loaded (Tamburri et al., 2004a,b). Initial results indicated that the addition of glucose or sulphide had little effect but that using nitrogen gas had some potential (Mountfort et al., 1999; Tamburri et al., 2002). On board trials with vacuum chambers had some success with zooplankton but the effect on phytoplankton was not clear (Browning and Browning, 2003; Browning et al., 2004). Experiments using a Venturi Oxygen Stripping system demonstrated it was effective at killing zooplankton but the results for phytoplankton were inconclusive. However, further shore based experiments using different methods of assessing viability of the phytoplankton have suggested
0025-326X/$ - see front matter Crown Copyright 2007 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2007.04.013
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
that this system may have the potential to achieve the new IMO standard (Tamburri et al., 2004b). The method used in this study was based on the addition of nutrients to stimulate the growth of bacteria, to consume the oxygen within the water and create a hostile environment for the aerobic organisms within the tank. This is similar to the method used by Mountfort et al. (1999) but uses a mix of nutrients designed to minimise the growth of sulphur reducing bacteria that may lead to increased corrosion and production of H2S. Literature reviews regarding the tolerance of different groups of organisms to anoxic conditions came to similar conclusions, i.e. that oxygen tolerance varied but that only a few organisms could tolerate hypoxia or anoxia for more than a few days and most could only survive a few hours (Assink et al., 2001; Tamburri et al., 2002). This treatment method was therefore designed to reduce and maintain the low oxygen levels over a number of days in order to ensure that even the most tolerant species were not able to survive. The treatment trial was carried out on the car carrier MV Don Quijote on a voyage between England and Panama. The purpose of the ship based trials was to test the efficiency of the method under operational conditions. 2. Materials and methods The trial was undertaken on board the pure car and truck carrier MV Don Quijote, a 22,625 metric tonne vessel with 17 ballast tanks and a ballast capacity of 8075 tonnes. The method was tested from the 21 to 28 June, 2003 during a voyage from Southampton (UK) to Manzanillo (Panama). Four ballast tanks were used during the trial and these were ballasted in line with normal operations (Table 1). Two upper wing tanks were used as treatment tanks (3U Port (3UP) and Starboard (3US)) and two double bottom tanks (DB3 Port (DB3P) and Starboard (DB3S)) were used as untreated control tanks. A nutrient solution (Table 2) was added via the sounding pipe to the two empty treatment tanks and the tanks were then filled with ballast water. 2.1. Sampling Samples of zooplankton, phytoplankton and bacteria were taken from each tank on day 0 (June 21st), i.e. when
1171
Table 2 Quantities of nutrients added to the treated tanks and calculated final concentration based on the tank volumes Compound
Tank 3US added
Tank 3UP added
Calculated conc. (g/m3)
Sucrose Glucose H2O NH4NO3 KNO3 Na2HPO4 Æ 2H2O Ship’s tap water (hot)
18.9 kg 7.5 kg 3.6 kg 3.6 kg 1.2 kg 26.4 l
16.6 kg 6.6 kg 3.2 kg 3.2 kg 1.0 kg 23.0 l
58 23 11 11 4
The volumes of tank 3US and 3UP were 326 and 285 m3, respectively.
the tanks were loaded and the nutrients added, and then after 3, 5 and 7 days. Sampling the tanks via the access manholes was not possible as they were covered with cargo so these samples were collected by pumping ballast water through the fire pump to the sampling equipment. The flow rate was 55–85 l min1 and the volume of water collected for each sample was calculated using a flow meter. The pipes were flushed for 15 min to remove water remaining in the pipe system before each new tank was sampled. Problems with the flushing meant that samples taken on the 24th (day 3) had to be disregarded. On the days that this sampling was not carried out, smaller samples for bacteria and chlorophyll a analysis were taken from the treated tanks via the sounding pipe by lowering a silicone tube connected to a peristaltic pump down the pipe and drawing the water up into a 1 l plastic bottle. The sample did not pass through the pump. The dissolved oxygen was measured with an oxygen electrode. Samples were taken by filling a plastic 1 l bottle with water from the tank, placing the probe in the bottle and sealing the top. The reading was taken once it had stabilised, which was usually after 0.5–1 h. A portable pH meter was used to determine pH. The concentration of H2S was assessed by a spectrophotometric method (Cline’s method) as described by Fonselius et al. (1999). The concentration of the mineral nutrients in the ballast water at the end of the study was determined according to NS (Norwegian Standard) 10304 (nitrate), NS 1189 (ortho-phosphate) and a modification of NS 4746 (ammonium) (available from www.standard. no).
Table 1 Details of the ballast tanks used during the trial and the position of the vessel when filling the tanks Tank
Capacity (m3)
Date/time
Start position
Water depth* (m)
Date/time
Stop position
Water depth* (m)
DB3 Star Port
513 513
21.06.03 01:00
50 26N 001 24W
50
21.06.03 02:00
50 21N 001 38W
55
3U Star Port
326 285
21.06.03 02:00
50 21N 001 38W
55
21.06.03 02:50
50 14N 002 01W
62
* Depth calculated from http://topex.ucsd.edu/WWW_html/mar_topo.html at Fisheries Research Services Marine Laboratory (Aberdeen, UK). DB = double bottom tanks and U = upper tanks.
1172
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
2.2. Zooplankton methods The zooplankton samples consisted of the organisms present in 1 m3 of water filtered using a 50 lm sieve. Once filtered, the plankton collected on the sieve was rinsed into a bottle with filtered sea water and stained with 0.1% neutral red solution in the ratio of 3 ml stain/100 ml sample. After staining for 30 min, 4 ml of 1 M sodium acetate solution was added per 100 ml of sample (Omori and Ikeda, 1984). The specimens were then fixed with 4% formalin in a volume equal to that of the sample (50/50) and stored overnight at 5 C. Thereafter, the samples were kept at room temperature until analysis. Prior to examination of the samples, glacial acetic acid was added drop by drop, until the colour of the solution changed to magenta. Live organisms turned a deep magenta after acidification, whereas dead specimens were light pink to white or transparent (when having an exoskeleton or shell). The sample was then filtered through a 48 lm sieve, washed with tap water and transferred to a Petri dish for sorting and counting using a stereomicroscope. When necessary, subsamples were taken by mixing the sample and taking a pre-determined volume that contained at least 100 individuals of one of the main groups (i.e. copepods and nauplii). The assessment of individual organisms included a morphological examination and whole organisms as well as identifiable body parts were taken into account and identified to the level of subclass or class. 2.3. Phytoplankton methods The phytoplankton sampling consisted of collecting samples for two types of analyses: chlorophyll a analysis and direct cell counts. For the chlorophyll a analysis, approximately 5 l of ballast water were pre-filtered through a 250 lm mesh into a bucket and mixed thoroughly, three replicates of 500 ml were collected and vacuum filtered using Whatman GF/A glass fibre filters (1.6 lm). Each filter was wrapped with aluminium foil and frozen immediately at 20 C until fluorometric chlorophyll a analysis based on an acetone extraction method was carried out (Arar and Collins, 1997). For the direct cell count samples the water from the tanks was pre-filtered through 250 and 100 lm sieves and the filtrate collected. Initially, 90 l were collected but from the second sampling day (day 3) 60 l were collected to reduce the time required for the next step. The filtrate was divided into three replicates of 30 or 20 l and each was filtered through a 10 lm plankton net. The collected sample was preserved with Lugol’s iodine (approximately 2 ml was added to each sample) and stored in a cool dark place until analysis. The analysis was carried out using standard counting procedures based on the Utermo¨hl sedimentation method (Utermo¨hl, 1958) and using an inverted microscope. The phytoplankton was identified to the level
of class, i.e. Bacillariophyceae (diatoms) and Dinophyceae (dinoflagellates) and enough of the sample was examined to ensure that a minimum of 100 cells from each class was counted. No assessment of viability could be made but only intact, undamaged cells were counted and these were assumed to be viable.
2.4. Bacterial methods Samples for bacterial analysis were collected in sterile polypropylene bottles (100 ml or 1 l) and kept cool until they could be processed further (usually within a few hours). The 100 ml samples were collected from the tube flushing water over the zooplankton sieve. On those days that zooplankton samples were not taken, a 1 l sample was pumped up from the treated tanks through the sounding pipe (see above). The concentration of viable bacteria in the samples was determined by a most probable number technique. The seawater was diluted in a tenfold dilution series in filtered (Whatman glass microfibre filter GF/A), heat sterilised (120 C, 20 min) seawater. Five 0.1 ml aliquots from selected dilutions were mixed with 0.1 ml medium in 5 wells in a pre-sterilised 96 microwell plate. The medium contained per litre: peptone, 10.0 g; yeast extract, 2.0 g; NaCl, 15.0 g; K2HPO4 Æ 3H2O, 20 mg; phenol red, 60 mg; filtered (glass microfibre filter GF/A) seawater, 500 ml, distilled water, 500 ml, pH 8.1 ± 0.1. The medium was heat sterilized (120 C, 20 min). The inoculated microwell plates were incubated at room temperature (22 ± 3 C) for 1–2 weeks. Positive cultures were scored by visible turbidity, and the most probable number determined from the tables given in Appendix II in the US Food and Drug Administration’s Bacteriological Analytical Manual online (Blodgett, 2001).
2.5. Statistical analysis for zooplankton The mortality of the zooplankton before and after treatment was calculated. For the control samples mortality was calculated as the dead organisms divided by the total number of organisms counted (dead plus alive). The mortality for treated samples was calculated based on the initial numbers of organisms found in each of the tanks on the first day and assuming that the missing organisms had been killed or destroyed by the treatment. Three–way nested ANOVA (tank was always nested to treatment) were conducted to examine mortality over time, between all tanks and between treatments; the rate at which copepods and nauplii disappeared from the treated and control tanks; the differences in concentrations of viable organisms in the treated and control tanks over time; and to compare the sensitivity between copepods and nauplii towards the treatment during the different days. Where necessary the data was transformed by squaring or using a Box-Cox or Log+1 transformation.
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
1173
2.6. Statistical analysis for Phytoplankton
3.3. Nutrients in treated ballast water
To examine any difference in chlorophyll a or cell concentrations between treated and control tanks and over time, two-way ANOVA tests were carried out. To test differences between particular days, tanks and samples taken via the fire pump and those taken via the sounding pipe, one-way ANOVA or a Kruskal–Wallis Test was used.
The nutrient solution added to the ballast water contained glucose, sucrose, ammonium, nitrate and phosphate to yield the concentrations given in Table 3. At the end of the trial the treated tanks contained slightly more ammonium than added at the start (105–128% of the original addition), less than 1% of the original addition of nitrate and 10–11% of the original addition of phosphate. The sugar content of the ballast water at the end of the trial was not analysed.
3. Results 3.1. pH and dissolved oxygen Bacterial growth in the treated tanks resulted in a decrease in pH, but remained constant in the control tanks (Fig. 1). The pH of the samples taken from the sounding pipe was slightly lower than for samples taken via the fire pump. Based on previous laboratory studies, the treated water was expected to become anoxic after 30 h (Josefsen, 2003) but the concentration of dissolved oxygen in the treated tanks was higher than expected for the first four sampling occasions (Fig. 2). This could be due to the electrode not stabilising before a reading was taken. The samples taken on the last day were anoxic but it is not possible to assess whether the water became anoxic before this. 3.2. Hydrogen sulphide No significant amounts of H2S were detected in the samples although the water from the treated tanks developed a pungent odour after a few days. As the analysis was performed under field conditions, an exact concentration could not be determined, but it was well below 1 lmol l1.
Fig. 1. pH in the treated and control tanks over the course of the voyage. Samples taken on the 22, 23, 25 and 27th June were taken via the sounding pipe, the remainder were sampled via the fire pump. The samples were taken at different times over the course of a day so the symbols from each day are not directly below one another. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
3.4. Zooplankton A large number of different taxa were found in the ballast tanks but as copepods and nauplii represented 38% and 60% of the organisms present respectively the results are based on these organisms. 3.5. Control samples As in previous trials carried out on the Don Quijote (Quilez-Badia et al., submitted for publication), high zooplankton mortality was observed in the untreated samples (Fig. 3). During the trial the zooplankton in the control tanks disappeared (assumed to be dead) at a rate of 20–30% a day (Fig. 4) so the 70–90% mortality rate at the beginning of the trial was assumed to be due to a combination of environmental conditions in the tanks and mechanical damage caused by the pump before they reached the sampling point. This complicated the assessment of the biological efficacy but as the water in all the tanks had been subjected to exactly the same conditions except that the untreated tanks had no nutrients added, it was assumed that any differences between the treated and control tanks over time was due to the treatment.
Fig. 2. Concentration of dissolved oxygen in water samples taken from the ballast tanks. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
1174
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
Table 3 Calculated concentration of sugar and nutrients immediately after addition of the solution to the treated tanks (3US and 3UP), and measured concentration of mineral nutrients in both treated and control tanks at the end of the trial Tank
Concentration (g/m3 ballast water) Glucose
DB3S DB3P 3US 3UP
NH4 þ ðas NÞ
Sucrose
NO3 ðas NÞ
PO4 3 ðas PÞ
Start
End
Start
End
Start
End
Start
End
Start
End
n.a n.a 20.9 20.9
n.a n.a n.a n.a
n.a n.a 58.0 58.1
n.a n.a n.a n.a
n.a n.a 1.93 1.94
<0.05 <0.05 2.47 2.03
n.a n.a 3.46 3.47
0.026 0.024 0.036 0.027
n.a n.a 0.65 0.65
<0.005 <0.005 0.071 0.068
n.a = Not analysed.
Fig. 3. The percentage mortality of the nauplii and copepods over the course of the voyage (mean ± SD). DB3P and DB3S = double bottom tank number 3 port and Starboard; 3US and 3UP = upper tank number 3 port and starboard.
Fig. 4. Concentrations of copepods and nauplii, i.e. both dead and alive over the course of the voyage, the vertical line represents the minimum and maximum concentration found in each sample and the symbol the average of three samples. The disappearance rates indicated by the dashed lines are based on the assumption that a given percentage of zooplankton disappear each day. The average concentration in the samples on the 21st is used as a starting level. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
3.6. Treated samples Zooplankton abundance decreased in all the tanks during the trial with differences between the treated and untreated tanks. The disappearance rate in the control tanks was 20–30% of the population per day for both copepods and nauplii, while in the treated tanks it was 45–55% per day for copepods and 50–65% per day for nauplii (Fig. 4). The decrease over time was significant for both treated and untreated tanks (three-way nested ANOVA,
for time, treatment and tank (nested to treatment), with the following interactions: time · treatment and time · tank (nested to treatment)) for both copepods (F = 62.08, p < 0.001) and nauplii (F = 52.99, p < 0.001). The overall abundance of copepods and nauplii was lower in the treated water compared to the untreated tanks (F = 42.46, p < 0.001, for copepods and F = 42.99, p < 0.001, for nauplii). Time increased the effect of the
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
treatment for nauplii (i.e. the interaction between time and treatment, F = 3.79, p < 0.05), the longer they had been in the treated tanks, the faster their numbers decreased. The mortality observed in the control tanks was significantly lower than in the treated tanks (three-way ANOVA (for time, treatment and live stage, with the following interactions: time · treatment, time · live stage, treatment · live stage and time · treatment · live stage), F = 34.43, p < 0.001). More live organisms were found in the samples from the control tanks than in the treated ones (three-way nested ANOVA, p < 0.001). No significant differences were found in terms of the mortality of copepods and nauplii suggesting they were equally sensitive to the treatment. The results are presented in relation to the IMO standard (i.e. <10 viable organisms P50 lm in minimum dimension per m3). However, as the standard was agreed after this work was carried out, this can only be considered a very broad comparison as these counts only include copepods and nauplii and will underestimate other groups that may have been present. Taking this into account the standard was achieved after 5 and 7 days in the treated tanks (3US and 3UP) (Fig. 5). The control tanks, which were subjected to all the same conditions except the addition of the nutrients, did not achieve the standard.
1175
Fig. 5. Concentration of viable zooplankton (mean ± SD). The line indicates the IMO standard of <10 viable organisms P50 lm in minimum dimension per m3. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
3.7. Phytoplankton 3.7.1. Chlorophyll a Samples taken from the treated tanks, particularly those sampled via the sounding pipe, tended to have a greater concentration of chlorophyll a than the controls (Fig. 6). On the last day there were significantly lower concentrations of chlorophyll a in the control tanks (below the limit of detection of 0.028 lg chl a l1) than in the treated tanks (2-way ANOVA p < 0.001). The samples taken via the sounding pipe suggested that the treated tanks had higher levels of chlorophyll a than those collected via the fire pump (Fig. 6) and for one tank (3US) there were significantly higher levels of chlorophyll a for samples taken on the 23rd in comparison to samples taken from the other tanks (Kruskal–Wallis, p < 0.05). 3.8. Cell counts On the first day of sampling there were no significant differences in the cell counts between the treated and control tanks (Fig. 7). On the second sampling date (26th) there was a higher concentration of dinoflagellates in one of the treated tanks (3US) (1-way ANOVA, p < 0.05) compared to the other tanks sampled on this day. On the final day of sampling higher concentrations of both diatoms (Kruskal–Wallis, p < 0.05) and dinoflagellates (1-way ANOVA, p < 0.001) were found in the control tanks. As with the zooplankton the IMO standard can only be compared very broadly as the phytoplankton cells counted during this trial were all >10 lm and <100 lm and the IMO standard states that <10 viable organisms P10 lm
Fig. 6. Chlorophyll a values for samples collected over the course of the voyage (mean ± SD). The star symbol represents samples that were taken via the sounding pipe, no control samples were taken on these occasions. All other samples were collected via the fire pump. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
and <50 lm in minimum dimension per ml should be discharged. Also, although only full, intact cells were counted no assessment of viability was made. Taking all this into account both the treated and untreated tanks achieved the standard on all occasions (Fig. 8), i.e. the water loaded into the tanks at the beginning of the trial was below the standard.
1176
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
Fig. 8. Combined concentration of dinoflagellates and diatoms from samples collected during the voyage (mean ± SD). The top of the scale represents the IMO standard of <10 viable organisms P10 lm and <50 lm in minimum dimension per ml. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
Fig. 7. Concentration of dinoflagellates and diatoms from samples collected with a 10 lm mesh (mean ± SD). DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard.
3.9. Bacteria The concentration of viable bacteria in the treated tanks increased rapidly, as expected, from around 1 · 104 GU (growth units) ml1 a few hours after filling the tanks to around 6 · 107 GU ml1 after 48 h (see Fig. 9). In contrast to previous experiments (Josefsen, 2003), instead of remaining at this level the concentration of viable bacteria in the treated tanks started to decline after about 5 days to a level of around 8 · 105 GU ml1 at the end of the experiment. The bacterial concentration also increased in the control tanks during the study. From a starting point of around 1 · 104 GU ml1 it increased about 10 times to around 1 · 105 GU ml1 at the end of the study. The high concentration recorded on the 26th was probably due to contamination with water from the previous sampling of the treated tanks. 4. Discussion Ship board testing of this deoxygenation technique followed on from promising laboratory trials (McCollin and Shanks, 2003; Quı´lez-Badia et al., 2003) and was an opportunity to assess whether this method could treat ballast
Fig. 9. Concentration of viable bacteria in the treated and control tanks over the course of the voyage. Samples taken on the 22, 23, 25 and 27th June were taken via the sounding pipe, the remainder were sampled via the fire pump. DB3P and DB3S = double bottom tank number 3 port and starboard; 3US and 3UP = upper tank number 3 port and starboard. The error bars indicate a theoretical 95% confidence interval as given by Blodgett (2001).
water effectively on a large scale under operational conditions. Previous testing of deoxygenation methods have been carried out at a variety of scales (Mountfort et al., 1999; Tamburri et al., 2004a,b; Browning and Browning, 2003; Browning et al., 2004) but there are limited data on the efficacy of these methods during ship board trials,
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
although testing of the Venturi Oxygen Stripping method on board vessels is currently underway (Smith et al., 2006). The method tested during this trial was based on the addition of nutrients to the ballast tanks to encourage the growth of bacteria, resulting in a gradual change to an anoxic environment. The method successfully reduced the oxygen level within the tanks and the water in the treated tanks was anoxic at the end of the trial. There was an unexpected decrease in the number of viable bacteria in the treated tanks after about 5 days. This could be owing to aerobic and facultative anaerobic bacteria starting to die and being replaced by obligate anaerobic bacteria (not detectable by the methods used), although in previous laboratory experiments obligate anaerobic bacteria have only constituted a small fraction of the flora (unpublished results). Increased predation by facultative or obligate anaerobic protozoa, which may have increased in number in response to the increased concentration of bacteria, or the bacteria starting to aggregate and being counted as one viable unit by the MPN method employed are other possible explanations for this decrease. Distinguishing between live and dead phytoplankton cells also caused problems. In this study only full, intact cells were counted and these were assumed to be viable. Methods such as assessing the condition of chloroplasts (Tamburri et al., 2004b) or using grow out techniques (Smith et al., 2006) are time consuming and it is likely that automated methods such as flow cytometry in combination with vital stains will be developed in the future (Veldhuis and Kraay, 2000; Veldhuis et al., 2004). When assessing the biological efficacy of this method it is important to consider that the organisms were affected by a number of factors such as reduced oxygen levels, lowered pH, mechanical damage by the pump and an increase in water temperature during the voyage from around 18 C to almost 30 C. There will also be a natural rapid decline of zooplankton and phytoplankton over the time of the voyage (Gollasch et al., 2000a; Lavoie et al., 1999; Olenin et al., 2000; Taylor and Bruce, 2000; Verling et al., 2005; Wonham et al., 2001) although on some occasions some species have been shown to increase during the voyage (Gollasch et al., 2000b). However, as the water in both treated and untreated tanks had been subjected to the same conditions except that the untreated tanks did not have the nutrient mix added it can be assumed that any difference between the treated and untreated tanks over time was due to the treatment. Also, the effect of the pumping will affect the biological efficacy of any ballast water treatment as, unless the tanks are loaded or unloaded by gravity, the ballast water will always have to pass through the pumps. The results of this study clearly demonstrated the abundance and viability of the zooplankton decreased more over time in the treated tanks compared to the untreated tanks. The phytoplankton results showed no clear treatment effect. There were fewer phytoplankton cells in the treated tanks on the last day of treatment compared to
1177
untreated tanks, suggesting that the treatment had successfully reduced the number of diatoms and dinoflagellates. However, the chlorophyll a concentrations were higher in the treated tanks than in the controls, suggesting that although the cells were present in lower numbers they contained a greater concentration of chlorophyll a. Even though these data cannot be conclusive, it would seem unlikely that this method of deoxygenation is an effective method of reducing diatoms and dinoflagellates. Only a very broad indication can be given as to whether the treatment method would have achieved the IMO ballast water treatment standard, which was agreed after this work was carried out. It is important to remember that only selected groups of organisms were counted, i.e copepods, nauplii, diatoms and dinoflagellates, so counts are likely to be underestimated and no assessment of viability could be made for the phytoplankton. When presented in comparison to the IMO standard the counts for the zooplankton showed that the treated tanks achieved the standard after 5 and 7 days and that the counts for the phytoplankton achieved the standard on all occasions, i.e the water that was loaded at the beginning of the voyage achieved the standard. For bacteria the IMO standard focuses on Escherichia coli and intestinal enterococci, both indicators of faecal contamination of the water, and toxicogenic Vibrio cholerae. None of these were analysed in this study. Overall, this trial demonstrated that in spite of the difficulties of determining a treatment effect when undertaking ship board trials, this treatment method and testing regime were able to show effects on zooplankton, with the deoxygenation method causing increased mortality in the treated tanks. However, the phytoplankton results were inconclusive and the methods of analysis would have to be adapted and improved if further ship board trials were to go ahead. This ballast water treatment method would only be suitable for vessels undertaking longer voyages as it relies on a gradual reduction in oxygen over time and if such a ballast water treatment were to be developed further the safe levels of nutrients that could be discharged would have to be ascertained (Ellis and van der Woerd, 2004). Acknowledgements This research was carried out as a European Union (EU) funded project (GRD1-2000-25383), the On Board Treatment of Ballast Water (Technologies, Development and Applications) and Application of Low-Sulphur Marine Fuel (MARTOB). The bacterial part of the project was also partly funded by Norsk Hydro ASA. We thank the officers and crew of the Don Quijote for all their help during the time of the trials and also Wallenius Wilhelmsen WW Lines and the shipping agents involved in ensuring all personnel and equipment arrived safely on board the vessel. Tony Vourdachas assisted with the sampling. The phytoplankton counts were carried out by Irina Olenina and her team at the Centre for Marine Research in Klaipeda.
1178
T. McCollin et al. / Marine Pollution Bulletin 54 (2007) 1170–1178
References Arar, E.J., Collins, G.B., 1997. Method 445.0. In vitro determination of chlorophyll a and phaeophytin a in marine and freshwater algae by fluorescence. Revision 1.2. US Environmental Protection Agency: Ohio. Assink, J., Ellis, J., Janssen, L., Josefsen, K., Mitropoulou, V., Sassi, J., Mesbahi, E., Vourdachas, A., 2001. Task 2.5 – Programme of requirements for ballast water treatement. MARTOB Report DTR2.5-12-01, pp. 35–53. Blodgett, R., 2001. Appendix 2. Most probable number from serial dilutions. In US Food and Drug Administration. Center for Food Safety and Applied Nutrition. Bacteriological Analytical Manual Online. Internet address: http://www.cfsan.fda.gov/ ~ ebam/bama2.html (as downloaded June 2003). Browning Jr., W.J., Browning, W.J., 2003. Ballast Water Treatment by De-oxygenation – The AquaHabiStarTM System. In: Raaymakers, S. (Ed.), 2003. 1st International Ballast Water Treatment R&D Symposium, IMO, London, 26–27 March 2001: Symposium Proceedings, GloBallast Monograph Series No.5. IMO: London, pp. 51–60. Browning Jr., W.J., Davis, J.P., Browning, W.J., 2004. Progress report on the AquaHabiStat deoxygenation system. In: Matheickal, J.T., Raaymakers, S. (Eds.), 2004. 2nd International Ballast Water Treatment R&D Symposium, IMO, London, 21–23 July 2003: Symposium Proceedings, GloBallast Monograph Series No.15. IMO: London, pp. 26–33. Ellis, J., van der Woerd, H., 2004. Environmental Impacts, Risk and Safety and Economic Aspects of Ballast Water Treatment Methods. MARTOB Report DTR-4.8-SSPA-03.04. Available from http://martob.ncl.ac.uk/Documents/. Fonselius, S., Dyrssen, D., Yhlen, B., 1999. Determination of hydrogen sulphide. In: Grasshoff, K., Kremling, K., Ehrhardt, M. (Eds.), Methods of Seawater Analysis. Wiley – VCH Verlag GmbH, Weinhiem. Gollasch, S., Rosenthal, H., Botnen, H., Hamer, J., Laing, I., Leppa¨koski, E., Macdonald, E., Minchin, D., Nauke, M., Olenin, S., Utting, S., Voigt, M., Wallentinus, I., 2000a. Fluctuations of zooplankton taxa in ballast water during short-term and long-term ocean-going voyages. International Review of Hydrobiology 85, 597–608. Gollasch, S., Lenz, J., Dammer, M., Andres, H.-G., 2000b. Survival of tropical ballast water organisms during a cruise from the Indian Ocean to the North Sea. Journal of Plankton Research 22, 923–937. International Maritime Organisation, 2005. Ballast Water Management Convention. International Maritime Organisation, London. Josefsen, K.D., 2003. Biological de-oxygenation. MARTOB Report DTR3.2.-SINTEF-09.03. Available from http://martob.ncl.ac.uk/Documents/. Lavoie, D.M., Smith, L.D., Ruiz, G.M., 1999. The potential for intracoastal transfer of non-indigenous species in the ballast water of ships. Estuarine, Coastal and Shelf Science 48, 551–564. McCollin, T.A., Shanks, A., 2003. Biological Assessment. Phytoplankton Results. MARTOB Report DTR-3.7.2-FRS-06.03. Available from http://martob.ncl.ac.uk/Documents/. Mountfort, D.O., Hay, C., Dodgshun, T., Buchanan, S., Gibbs, W., 1999. Oxygen deprivation as a treatment for ships’ ballast water-laboratory
studies and evaluation. Journal of Marine Environmental Engineering 5, 175–192. Olenin, S., Gollasch, S., Jonusˇas, S., Rimkute, I., 2000. En-route investigations of plankton in ballast water on a Ship’s Voyage from the Baltic Sea to the open Atlantic Coast of Europe. International Review of Hydrobiology 85, 577–596. Omori, M., Ikeda, T., 1984. Methods in Marine Zooplankton Ecology. John Wiley. Quı´lez-Badia, G. Gill, M., Frid, C.L.J., 2003. Biological Assessment. Zooplankton Results. MARTOB Report DTR-3.7.1-UNEW-08.03. Available from: http://martob.ncl.ac.uk/Documents/. Quilez-Badia, G., McCollin, T., Josefsen, K., Vourdachas, T., Gill, M., Mesbahi, E., Frid, C.L.J., submitted for publication. On board shorttime high temperature heat treatment of ballast water: A field trial under operational conditions. Smith, G., Mullady, T., Ruiz, G., Tamburri, M., 2006. Design and Implementation of Shipboard Tests of Ballast Water Treatment: The Case of Ventui Oxygen Stripping (VOS). In: Proceedings of 14th International Conference on Aquatic Invasive Species, May 14–19, 2006, Key Biscyane, Florida, p. 79. Taylor, M.D., Bruce, E.J., 2000. Mid Ocean Ballast Water Exchange: Shipboard Trials of Methods for Verifying Efficiency. Cawthron Institute in association with Battelle, Nelson. Cawthron Report 524. Tamburri, M.N., Wasson, K., Matsuda, M., 2002. Ballast water deoxygenation can prevent aquatic introductions while reducing ship corrosion. Biological Conservation 103, 331–341. Tamburri, M.N., Little, B.J., Ruiz, G.M., Lee, J.S., McNulty, P.D., 2004a. Evaluations of Venturi Oxygen StrippingTM as a ballast water treatment to prevent aquatic invasions and ship corrosion. In: Matheickal, J.T., Raaymakers, S. (Eds.), 2004. 2nd International Ballast Water Treatment R&D Symposium, IMO, London, 21–23 July 2003: Symposium Proceedings, GloBallast Monograph Series No. 15, IMO, London, pp. 34–47. Tamburri, M.N., Ruiz, G.M., McNulty, P.D., 2004b. Pilot-Scale Evaluations of Venturi Oxygen StrippingTM. In: Proceedings of 2nd International Conference and Exhibition on Ballast Water Management, Singapore, 19–21 May, 2004, pp. 87–88. Utermo¨hl, H., 1958. Sur Vervollkomming der quantitativen Phytoplankton-Methodik Mitteilungen. Internationale Vereiningung fuer Theoretische und Angewandte Limnologie 9, 1–38. Veldhuis, M.J.W., Bossink, M., Boon, J.P., 2004. Effect of secondary treatments on phytoplankton: a new technique to test the cell viability. In: Proceedings of 2nd International Conference and Exhibition on Ballast Water Management, Singapore, 19–21 May, 2004, pp. 87–88. Veldhuis, M.J.W., Kraay, G.W., 2000. Application of flow cytometry in marine phytoplankton research: current applications and future perspectives. Scientia Marina 64, 121–134. Verling, E., Ruiz, G.M., Smith, L.D., Galil, B., Miller, A.W., Murphy, K.R., 2005. Supply-side invasion ecology: characterizing propagule pressure in coastal ecosystems. Proceedings of the Royal Society B 272, 1249–1256. Wonham, M.J., Walton, W.C., Ruiz, G.M., Frese, A.M., Galil, B.S., 2001. Going to the source: role of the invasion pathway in determining potential invaders. Marine Ecology Progress Series 215, 1–12.