Effectiveness and potential toxicological impact of the PERACLEAN® Ocean ballast water treatment technology

ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 71 (2008) 355–369 www.elsevier.com/locate/ecoenv

Effectiveness and potential toxicological impact of the PERACLEANs Ocean ballast water treatment technology Yves de Lafontainea,, Simon-Pierre Despatiea, Chris Wileyb a

Aquatic Ecosystem Protection Research Division, Environment Canada, St. Lawrence Center, 105 McGill Street, Montreal, QC, Canada H2Y 2E7 b Department of Fisheries and Oceans, Central and Arctic Region, 201 N. Front Street, Sarnia, Ont., Canada N7T 8B1 Received 13 October 2006; received in revised form 27 September 2007; accepted 21 October 2007 Available online 20 February 2008

Abstract The efficacy and the potential toxicological impact of a proposed ballast water treatment (PERACLEANs Ocean) using peracetic acid (PAA) as active substances to control species introduction was assessed in both fresh- and salt water experiments at very cold water temperatures (1–2 1C). Levels of PAA gradually declined over the 5-day experiments, while levels of hydrogen peroxide remained relatively stable. The rate of decay of both the PAA and hydrogen peroxide in water was accelerated in the presence of sediments. Water quality properties varied significantly with treatment level with a maximum reduction of pH by 2.0 units and a concomitant 20-fold increase in dissolved organic carbon levels. Living biomass of organisms in treated water was reduced by 99% after 2 days. Results from six toxicological tests revealed very steep dose–response curves of the treatment. The toxic response of treated waters was higher in fresh water than in salt water. The PERACLEANs Ocean treatment may represent an effective technology to treat ballast waters under a wide range of temperature and salinity conditions. The discharge of treated fresh water may however pose some toxicological risk to fresh water receiving environments and to cold waters in particular. Crown Copyright r 2007 Published by Elsevier Inc. All rights reserved. Keywords: Ballast water treatment; PERACLEAN Ocean; Biocide; Peracetic acid; Bioassays; Aquatic invasive species

1. Introduction The discharge of ship’s ballast water is the principal vector for the introduction and transfer of nonindigenous aquatic species throughout the world (Carlton, 1985; Olenin et al., 2000; Ruiz et al., 2000). In order to reduce the risk of species introduction, ships are asked to conduct ballast water exchanges (BWE) at sea before entering the coastal and inland waters of many countries (International Maritime Organization, 2004). BWE is, however, not always fully effective in eliminating species introductions and transfer (Locke et al., 1993; Hay and Tanis, 1998; Hu¨lsmann and Galil, 2001), and various treatment technologies have been proposed as an alternative to BWE (Hay et al., 1997; SWRCB-CEPA, 2002; Matheickal and Raaymakers, 2004). The use of these technologies is Corresponding author. Fax: +1 514 496 7398.

E-mail address: [email protected] (Y. de Lafontaine).

highly dependent on their demonstrated effectiveness in treating ballast waters under the variety of environmental conditions typically encountered during a ship’s voyage. These technologies must be effective in treating both freshand salt water at temperatures ranging from near 0 to 30 1C. They must be environmentally safe inasmuch as the disposal of treated ballast waters meets environmental regulations at the point of discharge and that there is no impact on receiving waters. This is particularly important when chemical biocides (referred as ‘‘active substances’’ by the IMO convention) are considered as a treatment option. Treatment processes should therefore be evaluated in laboratory tests and on-board ship trials to assess their effectiveness and their potential environmental impact before making recommendations on their use to replace BWE. Fuchs and de Wilde (2004) indicated that the PERACLEANs Ocean chemical treatment can effectively kill organisms and bacteria in ballast water. PERACLEANs

0147-6513/$ - see front matter Crown Copyright r 2007 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.10.033

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Y. de Lafontaine et al. / Ecotoxicology and Environmental Safety 71 (2008) 355–369

Ocean is a commercially available liquid biocide whose active ingredients are peracetic acid (PAA) and hydrogen peroxide (H2O2), the latter used as a bacteriostatic agent to prevent regrowth of microorganisms. Inert ingredients of the mixture are water and acetic acid. At pHo8.2, PAA predominantly occurs in its undissociated form, which makes it a potentially active biocide in fresh water. Being very effective against bacteria, yeasts, moulds, protozoa, and algae (Baldry, 1983) and leaving non-toxic residues (Swern, 1970), PAA is widely used in food and health industries as a powerful sterilizer and sanitizer at concentrations up to 100 mg/L. PAA is also a promising disinfectant for municipal wastewater and sewage effluent (Baldry and French, 1989; Wagner et al., 2002). Being not persistent and not bioaccumulable, PAA in aqueous solutions rapidly decomposes into acetic acid, oxygen, and water, due to abiotic decomposition, hydrolysis, or reaction with organic compounds, and these processes are affected by pH, salinity and temperature (ECETOC, 2001). Its rate of decay is inversely related to temperature (Kunigk et al., 2001). Waters disinfected with PAA are not mutagenic (Monarca et al., 2002). In laboratory experiments and on-board ship trials, PERACLEANs Ocean resulted in 100% mortality of a large spectrum of aquatic organisms, including vegetative microalgae, dinoflagellate cysts, protozoans, ostracods, nematodes, rotifers, bivalves, and copepod nauplii and adults, when added at concentrations between 50 and 400 ppm for 24 or 48 h (Fuchs and de Wilde, 2004; Gregg and Hallegraeff, 2006). Preliminary toxicological assays in seawater treated with PERACLEANs Ocean indicated potent toxicity for up to 28 days after initiation of the treatment (Nautilus Environmental, 2005). Gregg and Hallegraeff (2006) reported that a 200-ppm concentration of the product degraded to non-toxic levels for marine algae in 3–6 weeks when prepared in filtered seawater. The effectiveness and the potent toxicity of the PERACLEANs Ocean treatment remained to be verified for fresh water environments and at cold temperatures. The objective of the present study was to assess the effectiveness of the PERACLEANs Ocean treatment technology and the potential toxicological impact on both fresh- and salt water at the very cold water conditions (o4 1C) typically encountered in winter in temperate waters and high latitudes. Specifically, the following hypotheses were tested: (1) the degradation rate of the PERACLEANs Ocean treatment is similar in both freshand salt water, (2) the PERACLEANs Ocean treatment effectively eliminates living aquatic organisms in very cold environments, (3) treated waters bear no residual toxicity within 5 days of treatment, and (4) the degradation of hydrogen peroxide residues by the addition of fungal catalase (Kikuchi-Torii et al., 1982) eliminates any residual toxicity in cold waters. The results of these experiments will help to determine the potential toxicological impact of discharging PERACLEANs Ocean treated waters into natural aquatic systems.

2. Materials and methods 2.1. Test product The sample of PERACLEANs Ocean used for experimentation contained 15% PAA (CH3COOOH), 14.3% hydrogen peroxide (H2O2), 26.5% acetic acid (CH3COOH) and water. A 40-L plastic container of the solution was received from Degussa AG Laboratories in New Jersey, USA, and immediately stored at cold temperatures (2 1C). The fungal catalase (Aspergillus niger) was made by Genencor International, Inc. According to the technical specifications sheet, the product contains o1% catalase in addition to sodium chloride, sodium citrate and sodium biphosphate, with a pH between 5.1 and 5.4 and a specific gravity between 1.15 and 1.19. The solution was not sterile and such yeast extracts may contain viable organisms at variable densities (usually o50 000 cfu/mL) (Barrette et al., 1999). The product was stored at cold temperatures upon reception.

2.2. The degradation-rate experiment The effect of water salinity and sediments on the rate of decay of the PERACLEANs Ocean treatment was determined in a laboratory experiment using 20-L plastic containers. The experimental design consisted of four series of six containers filled with 15 L of water at six different levels of salinity spanning a gradient from fresh- to salt water (0, 3.1, 7.1, 14.0, 21.1, 27 PSU). This was achieved by mixing natural, unfiltered salt water at 27 PSU collected at Mont-Joli in the St. Lawrence lower estuary with natural, unfiltered fresh water (0 PSU) from the St. Lawrence River near Montreal. Two series of containers were treated with PERACLEANs Ocean at a level of 150 ppm and two series were left untreated. For one series of both treated and untreated containers, approximately 1 kg of sediments taken from a ballast tank aboard the Canadian Provider (a ship plying the Great Lakes—St. Lawrence system) was added to the bottom of each container prior to filling with water. The sediments contained large amounts of rust particles and debris from ballast tank corrosion. All containers were placed in a controlledtemperature chamber kept at 6 1C. Water samples were taken from each container twice daily for 7 days and immediately analysed for PAA and hydrogen peroxide levels (see later in the text). Water temperature, salinity and pH were measured at the beginning and at the end of the experiment.

2.3. The cold water experimental design The cold water experiments, one with fresh water and one with salt water, were conducted in four large-volume (4.5 m3) cylindrical polyethylene tanks (1.83 m diameter and 1.80 m high) in the fish culture room of the Parc Aquarium du Que´bec (Quebec City), on the shores of the St. Lawrence River. This was done to verify the homogeneity of coverage of the treatment. To minimize heat transfer and eliminate light penetration, the tanks were covered with insulation and a 5-cm-thick styrofoam sheet was also placed at the water surface. Three feedthrough holes were made 10 cm from the top, in the middle and 10 cm from the bottom of each tank. Feedpipes ran from the holes to a midway point inside each tank. On the outside, each tank was fitted with a ball-valve with a short piece of Tygon tubing. The system was designed to collect water samples from the centre of the tank at three different depths. Ambient room temperature was maintained at between 1 and 8 1C during the experiments. For the fresh water experiment, tanks were filled with natural (unfiltered) water from the St. Lawrence River using the pumping station facility of the Parc Aquarium du Que´bec. The salt water experiment was conducted using filtered synthetic seawater (Instant Oceans), with a salinity of 32 PSU, supplied by the Parc Aquarium du Que´bec. Synthetic seawater was used because of the logistic problems encountered with the collection and transport of large volumes of natural seawater from possible sampling sites located 300 km downstream. In each experiment, one tank remained untreated and served as a control while

ARTICLE IN PRESS Y. de Lafontaine et al. / Ecotoxicology and Environmental Safety 71 (2008) 355–369 three tanks were treated with PERACLEANs Ocean at concentrations of 100, 200, and 300 ppm for the fresh water experiment and 100, 200, and 400 ppm for the salt water one. The treatment was fed into the tanks by injecting four equal quantities of the stock solution at equal time intervals when filling the tanks to ensure complete mixing. The experiments ran 5 days each and were conducted between March 7 and 19, 2005. Seventy-two hours after treatment application, the fungal catalase was added at a concentration of 5, 10, and 15 ppm (or 20 ppm in the salt water experiment), depending on the initial treatment concentration. To ensure its complete and homogeneous distribution, the catalase was stirred into the tank by means of a small submersible pump with tubing extending to the bottom of the tank. No catalase was added to the control tank.

2.4. Sample collection Water temperature was monitored at 30-min intervals by sensors (70.01 1C) suspended halfway inside each tank (total number of records per tank ¼ 249). The ambient air temperature was also continually recorded. Water temperature and specific conductivity (or salinity) were measured twice daily near the surface and near the bottom of each tank with an in situ sensor probe. Near surface and near bottom water samples were drawn twice daily to measure pH, dissolved oxygen, PAA and hydrogen peroxide concentrations. Samples were also taken daily at middepth in each tank for analysis of chemical and biological water properties. For ecotoxicological assays, samples were taken immediately (within 2 h) after treatment application, on day 3 (71 h after application, immediately before the addition of catalase) and on day 4 (96 h after application, 24 h after catalase addition).

2.5. Laboratory analyses After collection, pH of water was measured by a laboratory pH-meter and dissolved oxygen was determined by the Winkler titration method (70.1 mg/L). Total suspended solids (TSS) in 1-L samples were estimated by weighing with a detection limit of 1 mg/L (70.1 mg/L). Samples from the fresh water experiment were prepared for analysis of nitrates (NO2 NO3), ammonia (NH+ 4 ), orthophosphates (PO4 ), total phosphorus (TP), dissolved organic carbon (DOC), particulate organic carbon (POC) and particulate organic nitrogen (PON). Only DOC was monitored for the salt water experiment. All samples were analysed following the standard protocols of Environment Canada—Quebec Region. The analytical detection limits for the various parameters were as follows: NO2-NO3 ¼ PO TP ¼ 0.01 mg/L, 0.04 mg/L, NH+ 4 ¼ 0.01 mg/L, 4 ¼ 0.01 mg/L, DOC ¼ 0.25 mg/L, POC ¼ 0.05 mg/L, PON ¼ 0.08 mg/L. Levels of PAA and hydrogen peroxide were determined within 2 h after sampling using spectrophotometric methods (Pinkernell et al., 1997; Wagner and Ruck, 1984). The detection limit for both compounds was 0.5 mg/L. The analysis of synthetic seawater samples for PAA was problematic due to the production of a precipitate and values below 10 mg/L were thus considered below the detection limit. The biomass of living microorganisms, including bacteria, phytoplankton and microzooplankton, was estimated by ATP levels in particulate matter (Parsons et al., 1984). Water samples drawn for ATP determination were prefiltered on a series of three on-line filters using 53- and 10-mm mesh Nitex netting and 0.7 mm GF/F filters, respectively, in order to obtain living biomass estimates for three size classes (0.7–10, 10–53, and 453 mm). ATP levels were determined with a Turner Designs model TD20/20 luminometer using the luceferin–luciferase assay with a detection limit of 1017 mol/mL. The biomass of living phytoplankton in water was estimated by fluorometric determination of chlorophyll a concentrations in 400 mL water samples (Parsons et al., 1984). The concentration of phaeopigments was also determined and the ratio of chlorophyll to phaeopigments was calculated as an index of phytoplankton vitality. Ratio values 42 are usually interpreted as evidence of healthy and active phytoplankton cells, while values approaching 1 indicate increased amounts of phaeopigments relative to chlorophyll, which correspond to decaying phytoplankton (Hendry et al., 1987; Pena et al., 1991).

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The potent toxicity of treated waters on various components of the food web was assessed by a battery of six bioassays from bacteria to fish, including the Microtox (Vibrio fischeri) test, the algal (Selenastrum capricornatum) test, the cladoceran (Daphnia magna) test, the microcrustacean (Thamnocephalus platyurus) test, the Coelenterate (Hydra attenuata) test and the trout (Oncorhynchus mykiss) test. The Microtox and algal tests are two sublethal tests measuring the inhibitory effect of toxic compounds on cell physiology and growth. The four others are lethal tests measuring the proportion of dead organisms resulting from toxic exposure over different time periods. Although their experimental protocols vary slightly, all assays are based on a common procedure that consists of exposing, under controlled laboratory conditions, living organisms to a dilution series of water samples and to note and quantify the observed effects. The Microtox test was conducted using a bacterial concentration of 1E6 per ml in four replicates for each of the 10 diluted concentrations with an exposure time of 15 min. The algal test consisted of 10 diluted concentrations with five replicates, each one with 10 000 cells/ml for a 72 h exposure time at 24 1C. The Daphnia test used five newly hatched (o24 h old) individuals in three replicates for the six dilutions tested and the contact time was 48 h at 20 1C. For the microcrustacean test, three replicates of 10 larvae were used for each of the six diluted samples at dark at 25 1C for 24 h. The Hydra test was conducted for 96 h using seven diluted concentrations with three replicates and three organisms per replicate. The fish test consisted in exposing 10 specimens to each of the 10 diluted samples during 96 h. Data were analysed to compute various endpoint values (LC50—50% lethal concentration; IC50—50% inhibitory concentration; or NOEC—no observed effect concentration) depending on the assay. The results are expressed in toxic units (TU), which correspond to the inverse of the dilution factor required to reduce potent toxicity at the selected endpoint concentration. All the toxicity tests were conducted according to standard procedures and protocols currently used by Environment Canada (Environment Canada, 1995; St-Laurent et al., 1992; Trottier, 1996). All bioassays using experimental animals during this study were conducted following approved protocols in accordance to guidelines provided by the GLLFAS/NWRI animal care committee of Environment Canada.

2.6. Statistical processing Differences between surface and bottom samples for the chemical and biological composition in the large-volume tanks were tested by paired t-tests to verify water column homogeneity. The effect of the treatment on the various water properties was assessed by calculating the differences between treated and control samples and significant differences from 0 were tested by paired t-tests. Temporal trends in parameters were tested by a linear regression model and variations between treatment levels were examined by comparing slope coefficients and by an ANCOVA. The effect of the addition of catalase was tested by an ANOVA of the differences in values before and after catalase addition. The toxic responses of the ecotoxicological assays (expressed in TU) were analysed by a two-way ANOVA without replication to test differences between treatment levels and between sampling times. All statistical tests were performed with the significance threshold set at the 0.05 probability level.

3. Results 3.1. The degradation-rate experiment PAA and H2O2 degraded much faster in salt water than in fresh water, and even more so in the presence of sediments (Fig. 1). Without sediments, PAA and H2O2 were reduced by 30% and 12.5%, respectively, in fresh water (0 PSU) after 7 days, but by 92% and 52% in waters at salinity of 7 PSU and above. In the presence of sediments, PAA was degraded by 91% in fresh water 7

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Without sediments

With sediments 24

24 22

20

7 PSU 14PSU 21 PSU 27 PSU

18 16 14

Peracetic acid (ppm)

Peracetic acid (ppm)

22

0 PSU 3 PSU

20

12 10 8 6

18 16 14 12 10 8 6

4

4

2

2

0

0 0

20

40

60

80

100

120

140

160

180

0

20

40

80

100

120

140

160

180

140

160

180

Without sediments

24

24

22

22

20

20

Hydrogen peroxide (ppm)

Hydrogen peroxide (ppm)

With sediments

60

18 16 14 12 10 8 6 4 2

18 16 14 12 10 8 6 4 2

0

0 0

20

40

60

80

100

120

140

160

180

Elapsed time (hours)

0

20

40

60 80 100 120 Elapsed time (hours)

Fig. 1. Rate of degradation of peracetic acid and hydrogen peroxide in water at different salinity levels and with and without sediments.

days post treatment and became undetectable (o0.5 mg/L) in brackish water after 3 days. Correspondingly, the degradation rate of H2O2 was 62% in fresh water and 93% in brackish water (47 PSU). Irrespective of the presence of sediments, the rate of decay of both PAA and H2O2 changed rapidly with the increasing salinity of the water from fresh (0 PSU) to brackish (7 PSU), but remained very similar for water 47 PSU. The water temperature during this experiment varied from 6.3 to 7.0 1C. The mean pH of the treated waters without sediments (5.7070.11) was lower than the pH level in the presence of sediments (6.1570.16). 3.2. The cold water experiments The level of PAA in each tank was proportional to the initial treatment concentration. Concentrations in fresh water gradually declined by 51–66% over the course of the experiment, but were still being detected 5 days after application (i.e. 120 h) (Fig. 2a). The calculated degradation rate (linear regression slope) of PAA averaged 2.186,

3.505, and 4.626 mg/L/day at the 100, 200, and 300 ppm treatment levels, respectively. The percent decline in PAA thus varied from 13.7% to 11.2% per day and was inversely related to the initial treatment concentration. PAA measurements in salt water were above the detection limit (10 mg/L for synthetic salt water samples) at the highest treatment concentration (400 ppm) only and were not detected by day 3. Levels of hydrogen peroxide (H2O2) were directly proportional to the initial treatment concentration in each tank and remained stable over time until the addition of catalase, which rapidly reduced levels to near zero in both the fresh- and salt water experiments (Figs. 2b and c). Whatever the treatment level, hydrogen peroxide was always more concentrated in fresh water samples than in salt water. There were no significant differences in the concentrations of PAA and H2O2 in the surface and bottom samples taken from each tank (paired t-tests, p40.05), indicating the water column was well-mixed in all cases. The average water temperature varied between 0.78 and 1.22 1C in the experimental fresh water tanks (Table 1) and

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Fig. 2. Temporal variations in concentrations of peracetic acid and hydrogen peroxide at different treatment levels in fresh water and salt water experiments. Closed and open symbols indicate surface and bottom samples, respectively. Vertical dashed lines show when catalase was added.

between 2.4 and 2.7 1C in the salt water experiment (Table 2). Temperature remained stable over time, with no pronounced temporal trends and did not differ significantly with water depth (paired t-tests, p40.05), revealing no thermal stratification of the water column during the experiments. The specific conductivity of treated fresh water decreased slightly (by 2–5 units only) depending on the treatment level, but later increased after adding catalase (Table 1). Values did not vary significantly with water depth (paired t-tests, p40.05). Water salinity in the salt water experiment ranged from 32.0 to 32.5 PSU, but was independent of treatment level (Table 1). In fresh water, the average pH dropped from 7.5 (natural St. Lawrence River water in control tank) to 6.7, 6.16, and 5.65 in the 100, 200, and 300 ppm in the treated tanks, respectively (Table 1). The pH of salt water dropped from 8.2 (untreated synthetic seawater) to 7.3, 6.75, and 6.2 with the addition of 100, 200, and 400 ppm of the treatment solution, respectively. The difference in pH (relative to the

control) as a function of PERACLEAN treatment concentration (in ppm) was similar in both the fresh- and salt water experiments (Fig. 3) and was empirically modelled by fitting an exponential decay function: pH difference ¼  2:4794 þ 2:4860 eð0:004626 concentrationÞ ðr2 ¼ 0:995; n ¼ 16Þ. The pH of the treated waters remained stable over time and did not vary significantly with either water depth or the addition of catalase. Very high levels of dissolved oxygen, indicative of supersaturation conditions (420 mg/L), were noted in all treated tanks across both experiments (Table 1). The treatment resulted in a significant propor tional increase in NO2-NO3, NH+ 4 , PO4 and TP in fresh water, as well as in DOC in both fresh- and salt water. The increase in DOC averaged 14.8 mg/L per 100 ppm of treatment added. In the presence of catalase, levels of NH+ 4 were significantly reduced by approximately 33%,

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Table 1 Mean and standard deviation of physico-chemical water properties at different treatment levels in both freshwater and saltwater experiments p-Value

Treatment level (ppm)

Freshwater experiment Temperature (1C) Specific conductivity (mS/cm) PH DO (mg/L) NO2-NO3 (mg N/L) NH4 (mg N/L) PO4 (mg P/L) TP (mg P/L) DOC (mg C/L)

0

100

200

300

0.77 (70.24) 286.4 (70.5) 7.62 (70.06) 12.9 (70.1) 0.44 (70.02) 0.018 (70.001) 0.015 (70.002) 0.024 (70.002) 4.14 (70.77)

1.05 (70.13) 285.1 (70.3)-287.4 (70.2) 6.69 (70.05) 20.3 (71.6) 0.44 (70.07) 0.059 (70.005)-0.043 0.090 (70.002)-0.142 0.194 (70.005) 18.94 (71.18)

1.22 (70.13) 283.5 (70.6)-287.8 (70.4) 6.15 (70.06) 28.3 (71.8) 0.48 (70.02) 0.076 (70.003)-0.052 0.170 (70.008)-0.238 0.353 (70.011) 33.43 (70.83)

0.91 (70.10) 282.0 (70.3)-288.0 (70.1) 5.66 (70.05) 35.6 (73.5) 0.51 (70.01) 0.092 (70.002)-0.061 0.249 (70.004)-0.299 0.549 (70.018) 46.65 (70.89)

p-Value

Treatment level (ppm)

Saltwater experiment Temperature (1C) Salinity (PSU) PH DO (mg/L) DOC (mg C/L)

o0.01-n.s. o0.001 o0.001 n.s. o0.001 o0.001 o0.001 o0.001

0

100

2.45 (70.20) 32.34 (70.06) 8.19 (70.02) 10.3 (70.5) 1.00 (70.04)

2.68 32.34 7.32 16.65 5.25

(70.11) (70.06) (70.02) (71.0) (70.10)

200

400

2.69 (70.06) 32.24 (70.12) 6.72 (70.05) 25.0 (71.85) 9.16 (70.07)

2.62 (70.06) 32.31 (70.04) 6.16 (70.03) 31.7 (76.1) 53.27 (70.26)

n.s. o0.001 o0.001 o0.001

Arrows indicate change in values before and after the addition of catalase.

0.0

Table 2 Percent reduction (%) in total ATP concentrations (mol/mL) at different treatment levels and sampling times in the freshwater experiment

Y = - 2.4794 + 2.4860 e

(-0.004626 X)

, r = 0.995, n = 16

-0.2

Fresh water Salt water

-0.4

2.6 25.7 54.5 72.0 78.2 (with catalase) 97.4 (with catalase)

Total ATP in untreated tank 6.74  1014 1.15  1013 7.60  1014 1.60  1013 1.30  1013 1.09  1013

Treatment level (ppm) 100

200

300

89.80 98.84 99.99 99.99 99.63 98.78

90.45 99.60 99.99 99.52 99.94 99.83

92.88 96.94 92.33 99.99 99.48 99.53

-0.6

pH variation

Sampling time (h)

-0.8 -1.0 -1.2 -1.4 -1.6 -1.8 -2.0

while PO 4 levels increased by 0.05 mg P/L. The other parameters did not vary over time. TSS were low ranging from 1.5 to 1.7 mg/L at the start of the fresh water experiment, dropped to 0.9–1.2 mg/L on day 3 and rose again to 1.8–1.9 mg/L with the addition of catalase. POC varied from 0.19 to 0.81 mg C/L irrespective of the treatment level, but mean values declined gradually from 0.5 (at time 0) to 0.25 mg C/L (on day 4) over the course of the experiment. PON was never detected (o0.08 mg N/L). Due to winter conditions that do not usually support a high phytoplankton biomass in the St. Lawrence River, initial levels of chlorophyll a in the fresh water tanks were quite low (0.10.3 mg/L) (Fig. 4a). The concentrations in the control tank remained relatively constant over time and were higher (ANCOVA, po0.05) than levels measured in all treated tanks, where levels decreased by 50–60% over time. Although the differences in chlorophyll levels were

0

100

200

300

400

Treatment concentration (ppm) Fig. 3. Variation in water pH level by treatment concentration.

slight, levels were consistently lower in the 300-ppm treatment tank than in the other treated tanks. The average ratio of chlorophyll to phaeopigment concentrations increased from 5 to 9 in the control tank, while values for all treated tanks were consistently below 2.0 after 24 h and approached 1 on day 4 (Fig. 4b). The addition of catalase had no effect on chlorophyll a levels (ANOVA, p40.05). Levels of ATP in the treated tanks declined very rapidly for the three size classes analysed (Fig. 5). Concentrations had already dropped by one order of magnitude (490%) 2 h after application and became undetectable

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Fig. 4. Mean (and standard error) concentration of chlorophyll a (top panel) and chlorophyll/phaeopigment ratio (bottom panel) in treated and untreated tanks during the fresh water experiment.

(1017 mol/mL) in the two smallest size fractions (o50 mm) after 25 and 54 h, respectively (Fig. 5). Levels in the largest size fraction tended to be more variable over time, particularly in the 300-ppm treatment tank. Following the addition of catalase, ATP levels of the smallest size fraction increased in all three treatment levels. The increase in ATP in the presence of catalase was also noted in the other size fractions, but not in every tank. ATP concentrations in the untreated tank remained fairly stable during the experiment and decreased with increasing size class, as expected. Summing up the ATP content of the three size fractions, we estimated that the total biomass of living microorganisms in the treated tanks was reduced by 90% within 2 h of treatment application and by 99.99% after 54 h (Table 2). This corresponds to an overall reduction of four orders of magnitude in the quantity of living matter within 2 days of treatment. 3.3. Ecotoxicological tests A positive toxic response for each of the six ecotoxicological assays was noted in all treated samples collected immediately (2 h) and up to 96 h after treatment application in fresh water (Fig. 6). The toxic effect was strongest

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against bacteria (Microtox test ¼ 135–714 TU) and weakest against the coelenterate Hydra attenuata (Hydra test ¼ o1–11 TU) and generally decreased with the size and trophic level of the test organism. The toxic response increased proportionally with the initial treatment level (two-way ANOVA, treatment effect, po0.05 for all assays except the trout test with p ¼ 0.10). It varied relatively little over time, but the trend for slightly lower toxic responses after catalase addition on day 4 (97.4 h) (Fig. 6) was statistically significant (two-way ANOVA, time effect, po0.05) for four assays, but not for the Microtox or trout tests. This residual toxicity was successfully eliminated (as indicated by Microtox testo2 TU) by adding sodium thiosulfate to neutralize the solution by quenching residual PAA (Liberti et al., 1999) before draining the tanks at the end of the experiment. In an attempt to develop a dose–response curve of the treatment in fresh water, toxicity measurements of each assay were pooled and plotted as a function of the residual amount of PAA at the time of sampling (Fig. 7). Given that a positive toxic response was measured for every assay in the absence of hydrogen peroxide (after catalase addition), the calculation of a dose–response curve as a function of hydrogen peroxide content was meaningless. The dose– response curve of every assay was very steep, with toxicity ranging from near 0–100% over a corresponding change of less than one order of magnitude in PAA concentration in treated waters. The negative values of the algal test suggest that low PAA concentrations in the treatment may result in some algal growth. The calculated IC50 and LC50 values (Table 3) varied from 0.08 to 4.2 mg/L PAA and increased moving from bacteria to fish, indicating that larger organisms tend to be less sensitive to PAA. The results of the Microtox tests revealed that, 2 h after treatment application, the toxicity of treated salt water (5–91 TU; Table 4) was 10–20 times lower than that of fresh water (135–714 TU; Fig. 7). Moreover, the potent toxicity of the treatment in salt water declined very rapidly over time, yielding no toxic response (Microtox testo2.0 TU) after 71 and 96 h. 4. Discussion 4.1. Rate of degradation and chemical water properties Fuchs and de Wilde (2004) previously indicated that the half-life of PERACLEANs Ocean treatment would be calculated in minutes to hours in seawater, but much longer (between 2 and 24 h) in fresh water. This is supported by our experimental results, which show that both PAA and H2O2 degrade faster in salt water than in fresh water, each compound having its own dynamic of decay depending on environmental conditions (Figs. 1 and 2). The effect of water salinity was most pronounced within the 0–7 PSU salinity range, suggesting that the rate of decay of the treatment may change very rapidly in brackish and estuarine waters. A compilation of our experimental

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10 -12 0.7-10 micron size class

ATP (mole/mL)

10 -13 10 -14

0 ppm 100 ppm 200 ppm 300 ppm

10 -15 10 -16

D.L.

10 -17 0 10

24

48

96

72

-12

Catalase addition

10-50 micron size class

ATP (mole/mL)

10 -13 10 -14 10 -15 10 -16 10 -17 0

24

48

72

96

48

72

96

10 -12 > 50 micron size class ATP (mole/mL)

10 -13 10 -14 10 -15 10 -16 10 -17 0

24

Time after treatment (hours) Fig. 5. ATP concentrations for three size classes of particles in treated and untreated tanks in the fresh water experiment. Vertical line indicates time of catalase addition and horizontal dashed line is the analytical detection limit (1  1017 mol/mL).

results and those obtained from literature was made to calculate and compare the half-life time of PAA, the time reach a background level of 0.5 mg/L and the percent of loss in PAA at day 5 of treatment under different experimental conditions (Table 5). This clearly showed the major effects of water types and the presence of sediments on the degradation of PAA. The half-life time of PAA was much shorter in salt water (o24 h) than in fresh water (4100–170 h) environment, so that the percent loss of PAA after 5 days usually varied between 90% and 99% in salt water as opposed to between 46% and 90% in fresh water solution. Characterized by a very steep curvilinear degradation curve, the concentrations of PAA in salt water

(47 PSU) declined rapidly and became merely undetected (o0.5 mg/L) after 48 h in the presence of sediments (Fig. 1). A similar steep curvilinear decay of PAA in salt water was also observed during a field trial on the island of Texel (Netherlands), where 150 ppm of PERACLEANs Ocean completely degraded (o0.5 mg/L PAA) within 12 h after being mixed with natural seawater (ca. 8 1C) during a massive bloom of Phaecystis globosa, corresponding to a total suspended matter of 4.8–6.0 mg/L (Veldhuis et al., 2006). In absence of sediments, PAA residues in salt water levelled off at 1.5–2 mg/L indicating that organic matter will facilitate the complete degradation of PAA. The minimal amount of organic matter needed to achieve a

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1000

After 2 h After 71 h After 96 h + catalase

363

100 ppm 200 ppm 300 ppm

Toxic units

100

10

1 Microtox

Algal

Microinvert.

Daphnia

Hydra

Trout

Toxicological tests Fig. 6. Results of six toxicological assays for water samples at three treatment levels (100, 200, and 300 ppm) in the fresh water experiment. Measurements were taken at the beginning of treatment (2 h), immediately before catalase addition (71 h) and 1 day after catalase addition (96 h). Toxic units were calculated as the inverse of the percent dilution of the sample corresponding to the selected endpoint of each test.

complete degradation of PAA is unknown and should be quantified. The very short half life time (o0.5 h) of PAA in synthetic seawater (Table 5) would preclude the use of synthetic seawater for future laboratory testing of the efficacy of PERACLEANs Ocean as a ballast water treatment method. In fresh water condition, the degradation curve of PAA was almost linear and characterized by a much slower rate of decay (Figs. 1 and 2). Similarly, a previous experiment showed that the degradation of 100 mg PAA/L in natural fresh water from a pond and a stream was only 66% after 96 h and took 3 weeks to reach 99% (Table 5; ECETOC, 2001). The half-life time and the percent loss of PAA in demineralized or in distilled water indicated values of degradation rate much slower than those in natural fresh water (Table 5). The presence of organic matter will therefore speed up the decay of PAA in fresh water, as was found for salt water. Yuan et al. (1997) showed that PAA degrades primarily by spontaneous decomposition at pH between 5.5 and 8.2. Given the kinetics constants reported by Kunigk et al. (2001), the decomposition rate of PAA in pure water at 1 1C would be very slow, with less than a 2% loss in 5 days. Presumably, the low pH and low temperature of our fresh water experiment (Fig. 3) may have contributed to slowing down the degradation of PAA relative to that previously observed in pond water (Table 5). Further experiments in fresh water over a range of water temperatures would be required to determine which of these two factors contributed most to this process. The H2O2 content in the PAA formulation provides a synergistic action to the disinfection activity over the

longer term because it is more stable (Alasri et al., 1992; Liberti et al., 1999). This stability was evident in our experiments, where levels tended to level off rapidly during the first 3 days of application (Figs. 1 and 2). The overall loss of H2O2 in sediment-free fresh water was relatively slight (10–15%) after 5 days and reached nearly 50% in seawater. This was supported by other studies showing that the H2O2 concentration in fresh water from a pond and a stream dropped by 8% after 96 h (ECETOC, 2001) and by 30% after 50 h in seawater (Veldhuis et al., 2006). Although the presence of sediments accelerated quite significantly the decay of H2O2 in both fresh- and salt water, peroxide residues were still easily detected one week after treatment (Fig. 1). However, these residues were easily and completely eliminated (o0.5 mg/L) within a few hours (o4 h) in both fresh- and salt water with the addition of catalase, thus confirming that catalase activity was not halted or impaired by cold water temperatures. The addition of catalase had no effect on PAA levels. The effective use of catalase to quench hydrogen peroxide residues in municipal wastewater treatment was demonstrated by Wagner et al. (2002) and could eventually be required to similar purpose during ballast water treatment with PERACLEANs Ocean. The introduction and mixing of catalase into full ballast tanks might be technically difficult, however, and perhaps even cost prohibitive. Our results clearly showed, however, that the presence of sediments enhanced the decay of PERACLEANs Ocean in fresh water (Fig. 1). The sediments used in our experiment came from ship ballast tanks and contained large, undetermined amounts of rust and other metal

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Fig. 7. Dose–response curves for six bioassays as a function of levels of peracetic acid in PERACLEANs Ocean treatment.

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Table 3 Estimated effect concentrations (at two different endpoints) for various test organisms exposed to peracetic acid contained in PERACLEANs Ocean treatment Assay

Trophic position

Type of toxicity test

Effect measured

IC50 or LC50

IC10 or LC10

Microtox TM (Vibrio fischeri) Algal test (Selenastrum capricornatum) Cladoceran test (Daphnia magna) Microcrustacean test (Thamnocephalus platyurus) Hydra test (Hydra attenuata) Trout test (Oncorhynchus mykiss)

Decomposing bacteria Primary producers Herbivores Herbivores

Acute sublethality (15-min) Chronic sublethality (24 h) Acute lethality (48 h) Acute lethality (24 h)

Light inhibition (IC) Growth inhibition (IC) Mortality (LC) Mortality (LC)

0.076 0.21 0.29 0.85

0.016 0.084 0.113 0.55

Herbivores Carnivores

Acute lethality (96 h) Acute lethality (96 h)

Mortality (LC) Mortality (LC)

4.2 1.8

2.7 0.60

Table 4 Time variation of the toxic response (in toxic units) of the Microtox assay for saltwater initially treated at three different concentrations of PERACLEANs Ocean Time after treatment application (h)

2 71 96 (with catalase added)

Treatment level (ppm) 100

200

400

5 o2 o2

40 o2 o2

91 o2 o2

The toxic unit is calculated as the inverse of the percent dilution of the sample corresponding to the selected endpoint (IC50).

particles resulting from tank corrosion. Our results suggest that treatment effectiveness and degradation can be quite variable depending on turbidity and on sediment quantity, as well as the extent of corrosion in ballast tanks filled with fresh water. Given that PAA decomposition can be initiated by metal catalysts such as iron, copper or chromium (ECETOC, 2001), the rusty metallic surface of the ballast tanks (including the walls, dividers and supports) may therefore favour the rapid degradation of the treatment in fresh water conditions. Variables such as the amount of sediment or rust in salt water would appear to be less problematic to treatment effectiveness. Overall, salt water containing large quantities of sediment may thus provide the best conditions for rapid and effective degradation of the treatment’s two active ingredients. On the other hand, relatively clear fresh water (without sediments) appears to represent the worst condition for rapid degradation of the PERACLEANs Ocean treatment. Consequently, the biological efficacy of the treatment at a given concentration may be extended in fresh water ballast tanks relative to tanks containing salt water. The treatment had a significant effect on all water quality parameters measured in our experiments and the observed changes were directly proportional to the initial concentration of the treatment (Table 1). Changes in some properties (conductivity, nitrates) were minor and environmentally benign, but others (PO 4 , DOC and low pH) could be cause for concern. The levels of PO 4 and DOC in treated waters increased by a factor of 16 and 20,

respectively. The increase in PO 4 and TP is linked to the presence of a stabilizer used to make the PERACLEANs Ocean formulation (Bernd Hopf, Degussa AG, pers. comm.). Increased DOC is mainly due to the presence of PAA and acetic acid (Collivignarelli et al., 2000). The measured increase of 14.8 mg C/L in DOC per 100 ppm of treatment solution in our experiments is indeed very close to the expected theoretical value of 15.6 mg C/L given that PAA is equivalent to a 39% solution of acetic acid having a carbon fraction of 40%. High levels of orthophosphates and DOC in treated waters may eventually represent a nutritive source favouring eutrophication of fresh water ecosystems. With regard to pH, Canadian guidelines for protection of aquatic life recommend that water pH should not be less than 6.5, while wastewaters with pHo5.0 cannot be discharged into natural systems. All other parameters measured in the treated waters were within the range of Canadian water quality guidelines. It should be noted, however, that values for the various water quality parameters may vary according to the initial quality of the water to be treated and that the values reported here should not be generalized to all applications of this chemical treatment. 4.2. Toxicology of treated waters The PERACLEANs Ocean treatment was found to be extremely effective in eliminating living aquatic forms in less than 3 days in both fresh- and salt water experiments. Two lines of evidence support this conclusion. First, the efficacy of the treatment was demonstrated by the rapid and highly significant decline in ATP levels in the treated waters (Fig. 5). Within 2 h, the biomass of living microorganisms in treated tanks was lowered by one order of magnitude, confirming the very rapid action of the treatment. The fact that ATP in the 0.7–10 mm size fraction was not detected 24 h post treatment suggests that most microorganisms (including bacteria, yeasts, moulds and small algae) died because of treatment activity. ATP in the intermediate size fraction (10–53 mm) were also undetected 48 h after initiating treatment, indicating the less rapid action of the treatment on larger organisms. Traces of ATP in the 300-ppm treatment level (Fig. 5) are not easily

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Table 5 Summary of results on the degradation rates of PAA in different experimental conditions Water type

Temp. (1C)

Total suspended solids (mg/L)

Sediments

Initial PAA concentration (in mg/L)

Half-life time (h)

Time (h) to reach 0.5 mg/ L

% loss of PAA at day 5 (%)

Source

Freshwater Freshwater Freshwater Freshwater Freshwater Freshwater

1 1 1 6–7 6–7

1.5–1.7 1.5–1.7 1.5–1.7 n.d. (2) n.d. n.d.

No No No No Yes No

15 (100)a 30 (200)a 45 (300)a 22.5 (150)a 22.5 (150)a 100

98 110 110 4170 1

194 224 226 454a 4400a 504

55–62 52–55 46–50 25 92.5 67

0

No

100

0

No

95

This study This study This study This study This study Chalkley (1991) cited in ECETOC (2001) Chalkley (1991) cited in ECETOC (2001) Pierre et al. (2000) cited in ECETOC (2001) This study This study Veldhuis et al. 2006 This study

Demineralised water Distilled water

25

Saltwater 47 PSU Saltwater 47 PSU Saltwater 415 PSU Synthetic seawater 32 PSU

6–7 6–7 8 1

n.d. (2) n.d. 4.8–6 0

No Yes No No

Synthetic seawater 33 PSU

n.d.

0

Synthetic seawater 20 PSU

n.d.

0

10 432 a

13.5

10 1 2 o2

No

22.5 (150) 22.5 (150)a 22.5 (150)a 15–60 (100–400)a 105

0.11

499

No

105

0.33

499

4400 55–80 20 48

91 499 499 499

Kuhn (2000) cited in ECETOC (2001) Kuhn (2000) cited in ECETOC (2001)

The half-life time of PAA and the calculated time to reach to undetected level of PAA (o0.5 mg/L) are expressed in hours. a Values in parentheses indicate the corresponding initial concentration of the PERACLEANs Ocean treatment.

explained, although they appear to suggest that some organisms occurring at low densities remained present in the water and would require longer exposure time to be killed. The 100% mortality response measured in the ecotoxicological tests on larger organisms exposed for 48–96 h tend to support this hypothesis (Fig. 7). The possibility of contamination during sample processing and filtration cannot be entirely ruled out. The significant reduction in chlorophyll a concentrations and in the chlorophyll/phaeopigment ratios of treated waters (Fig. 4) indicates that the treatment can effectively eliminate living fresh water phytoplankton in ballast tanks. Veldhuis et al. (2006) also noted a 490% reduction in chlorophyll a concentrations and the complete elimination of Phaeocystis cells in seawater treated with 150 ppm PERACLEANs Ocean. They claimed that residual traces of chlorophyll a in treated waters would not be associated with intact phytoplankton cells. Indeed, the decline in the phytoplankton vitality of treated waters as opposed to the increase observed in the control tanks (Fig. 4) points to the very poor physiological state of living cells, if any remain. Although further tests on higher biomasses of larger plankton would be desirable, our results agree with those of Fuchs and de Wilde (2004), who demonstrated that the PERACLEANs Ocean treatment effectively killed a variety of marine organisms, including protozoans, ostracods, bivalves, polychaetes, and copepods. The second line of evidence on the efficacy of the treatment comes from the results of the ecotoxicological bioassays. High toxic responses were shown in all six toxicity tests and within a very short time (2 h) after

treatment application (Fig. 6). Efficacy appeared to be greatest against bacteria (Microtox test) and tended to drop with increasing organism size and trophic position. Positive toxic responses were measured for all three treatment levels being tested (Fig. 6), suggesting that a low treatment concentration should preferably be used to minimize the possible toxic impact of treated waters at discharge. The pronounced steepness of the dose–response curves for all tests revealed rapid changes in toxicity within a small range of test concentrations (Fig. 7), which is generally considered advantageous for a biocide. Our estimates of the IC50 or LC50 concentrations for the various tests were indeed very similar to results yielded by toxicity tests conducted in the laboratory (summarized in ECETOC, 2001). The IC50 value for the algal test in our experiment was 0.21 mg PAA/L, which is almost identical to the value of 0.18 mg PAA/L previously observed using a different PAA formulation (Hick et al., 1996 cited in ECOTOC, 2001). Values of the EC50 for the Daphnia test in other studies ranged from 0.50 to 1.1 mg PAA/L (four studies cited in ECETOC, 2001), which encompass our estimate of 0.85 mg PAA/L for the PERACLEAN formulation. Finally, our calculated LC50 value (1.8 mg PAA/L) for the trout test is also within the range of concentrations (0.912.0 mg PAA/L) measured in four previous laboratory tests (ECETOC, 2001). The toxic response of treated fresh water samples changed little over time (Fig. 6) due to the slow degradation rate of both PAA and H2O2. This implies that the discharge of treated fresh water from ballast tanks would represent a toxic risk to natural fresh water

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environments. The addition of catalase did not eliminate the toxic response of the treated waters, indicating that this toxicity cannot be attributed to hydrogen peroxide residues. The toxicity tests can be reproduced if concentrations are expressed as PAA levels regardless of H2O2 concentrations. This suggests, indeed, that PAA concentrations might explain the toxicity of the PAA formulations and that the levels of hydrogen peroxide are less relevant (ECETOC, 2001). Our results on toxic response before and after catalase addition confirmed this assumption. The great stability of pH in treated fresh waters that remained acidic over time (even after adding the catalase) might be due to acetic acid in solution (as also indicated by elevated DOC levels). The complete elimination of the toxic response, as indicated by Microtoxo2.0 TU (Table 4), was achieved by adding sodium thiosulfate to neutralize the solution (Wagner et al., 2002). The minor but consistent increase in ATP levels after adding catalase might be explained by the presence of living organisms in the original catalase extract (Barrette et al., 1999). The survival and growth of these organisms would be possible in the absence of hydrogen peroxide. In summary, our experimental results revealed significant residual toxicity in treated waters, most probably associated with residual PAA causing low pH, and not related to the presence of hydrogen peroxide residues. By contrast, the salt water samples exhibited no toxic response 3 days post treatment (Table 4), consistent with the apparent rapid degradation of PAA measured in synthetic salt water, where PAA was not even detected. This demonstrated the major difference in efficacy and potential toxic impact of the PERACLEANs Ocean treatment in fresh- versus salt water environments. The toxic response of the Microtox test on samples taken 2 h after treatment application varied greatly between the two water types. The lower toxicity values of the salt water samples relative to the fresh water samples reflects the higher degradation rate of PAA in salt water. The nontoxic response of the Microtox test in seawater samples collected immediately before catalase addition (71 h post treatment) provided further evidence that levels of H2O2 present in solution (Fig. 2) were of no concern. 4.3. Practical applications of the treatment The results of the present study show that the PERACLEANs Ocean technology can effectively treat both fresh water and salt water in ships’ ballast tanks. Moreover, this chemical treatment was found to be effective in very cold water, thus making it applicable across a wide range of water temperatures and salinity conditions. Because of the relatively high toxicity responses measured in our experiments, consideration must be given to the type of receiving water to which PERACLEANs Ocean-treated ballast waters are discharged. Discharging fresh water ballast treated with a high concentration of PERACLEANs Ocean into a fresh water harbour may

367

present a potential toxicological risk to the receiving environment. Four days after treatment application, waters treated at the lowest level (100 ppm) indicated a 5.6 TU toxic response for the trout test (Fig. 6), suggesting that a 1:6 dilution would be the lowest necessary to reduce toxicity to the equivalent of the LC50 endpoint (50% mortality) at discharge. A 1:16 dilution would be required to reduce toxicity to equal the LC10 endpoint. Given the half-life time of PAA measured in fresh water (Table 5) and assuming a similar degradation rate in ballast tanks, ships would have to retain their ballast waters for 15–20 days after treatment to reduce PAA levels below the LC50 threshold. Although warmer temperatures and higher organic matter content of water during treatment application or longer periods between applications and discharge may help to reduce the residual toxicity of treated waters, such an assumption remains to be investigated. Both PAA and hydrogen peroxide can be degraded by temperature, metal ions (from corrosion) and organic matter (planktonrich waters or sediments), suggesting that lower residual concentrations of these chemicals could be expected under real ship conditions. The cold-temperature experiments in this study were designed to reproduce the conditions for ballast waters in ships transiting the cold environments of the world, including some Canadian fresh water systems such as the St. Lawrence Seaway and the Great Lakes in winter. Our experimental set-up was shown to adequately generate and maintain extremely low water temperatures in largevolume tanks over extended time periods, thus making possible more realistic testing of ballast water treatment technologies. Despite the relatively large size of the tanks and the lack of any water mixing, no vertical stratification was observed for the parameters measured, either in the control or the treated tanks. Given the small amount of suspended matter in the water (TSS 1 mg/L), sedimentation was not a problem during our experiments. The near-freezing temperature (1–21C) and the short duration (o6 days) of our experiments may correspond to extreme conditions for evaluating ballast water treatment (Hunt et al., 2005), but they are not unrealistic. Such are the conditions under which many ships transit the North Atlantic Ocean between Europe and North America, and in less than a week. Furthermore, they may also correspond to the situation faced by ships heading toward the Laurentian Great Lakes in late fall and early spring. This large fresh water ecosystem has been invaded by more than 180 nonindigenous species, many of which were introduced via the discharge of ballast waters (de Lafontaine and Costan, 2002; de Lafontaine and Simard, 2004; Ricciardi, 2006). The great majority of ships entering the Great Lakes do not carry ballast onboard (NoBOB ships), so that only residual waters and sediments are typically found on the bottom of their ballast tanks (Colautti et al., 2003; Grigorovich et al., 2003). On the voyage, these NoBOB ships may call at various ports and take up fresh water that will be discharged into a different harbour a few days later.

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This procedure is believed to contribute to the mixing and transfer of organisms surviving on the bottom of ballast tanks. Because of its rapid efficacy, the PERACLEANs Ocean treatment technology represents a possible solution to the NoBOB problem. However, the toxic response of treated fresh water, as demonstrated in the present study, would limit the application of this treatment throughout the Great Lakes. 5. Conclusions In conclusion, our evaluation of the effectiveness and the potential toxicological impact of the PERACLEANs Ocean treatment showed that the technology may be suitable to treat ballast waters under a wide range of temperature and salinity conditions. Living biomass of organisms in treated water was reduced by 99% after 2 days. Chemical properties of treated water were significantly altered during treatment and levels of PAA in fresh water 5 days after treatment yielded residual toxicity so that the discharge of treated ballast water may however pose some toxicological risk to fresh water receiving environments. This residual toxicity could be reduced either by lowering the initial treatment concentration or by extending the treatment duration to bring PAA concentration of treated water down to background level of 0.5 mg/ L. Given the very steep toxicity curves of PAA and its slow decay rate in fresh water, a reduced of the initial treatment concentration during applications would still provide sufficient exposure time to eliminate organisms present in ballast fresh water tanks. Acknowledgments The authors thank Manon Harwood, Sylvie Roberge, Yan Chambers, and Jasmin Perrier for their unflagging effort and support during this study and Michel Lagace´ for providing the facilities at the Parc Aquarium du Que´bec. Sincere appreciation goes to our colleague Joe Lally, whose expertize and rigour, not to mention kindness, greatly contributed to the success of our experiments. Funding for this project was provided by Transport Canada and Environment Canada. Funding sources and animal care: This study was funded by special research support from Transport Canada and by the Water Science and Technology Direction of Environment Canada. All bioassays using experimental animals during this study were conducted following approved protocols in accordance to guidelines provided by the GLLFAS/NWRI animal care committee of Environment Canada. The experimental study design was reviewed by local scientific committee for approval. References Alasri, A., Rocques, C., Michel, G., Cabassud, C., Aptel, P., 1992. Bacterial properties of peracetic acid and hydrogen peroxide, alone

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