APPLIED ISSUES

Efficacy of ‘saltwater flushing’ in protecting the Great

Lakes from biological invasions by invertebrate eggs in

ships’ ballast sediment

ELIZABETA BRISKI*, SARAH A. BAILEY*, †, MELANIA E. CRISTESCU*

AND HUGH J. MACISAAC*

*Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON, Canada

†

Great Lakes Laboratory for Fisheries and Aquatic Sciences, Fisheries and Oceans Canada, Burlington, ON, Canada

SUMMARY

1. Mid-ocean exchange and saltwater flushing were implemented as management practices

to reduce the likelihood of new biological invasions in the Laurentian Great Lakes

associated with ships’ ballast water and sediments. Despite this, there has been no formal

assessment of the efficacy of these procedures. Here, we conduct a comparative analysis of

community composition of dormant taxa transported by ballast sediment before and after

regulations came into effect in 2006.

2. Ballast sediment samples were collected from 17 ships during the post-regulation

interval of 2007 and 2008. Invertebrate eggs were counted, hatched and species identified

in the laboratory. Results were compared to similar samples collected from 39 ships

between 2000 and 2002, prior to implementation of saltwater flushing regulations.

3. The estimated amount of residual ballast sediment transported by vessels was

significantly lower during the post-regulation period, ranging from <1 to 45 tonnes per

ship, with an average of 5 tonnes. Mean density and number of dormant viable eggs per

ship declined 91 and 81%, respectively.

4. Community composition also changed through time, with Rotifera accounting for 78%

of taxa transported prior to regulation, whereas Cladocera and Copepoda each accounted

for 38% of abundance post-regulation. Although the number of non-indigenous species

(NIS) declined 73% per ship after 2006, the reduction was not statistically significant;

however, the number of freshwater NIS – which pose the greatest risk of invasion for the

Great Lakes – was significantly lowered.

5. Our comparative analysis suggests that ballast management regulations enacted in 2006

markedly reduced the probability of introduction of NIS via dormant eggs carried in

ballast sediments.

Keywords: ballast sediment, biological invasions, dormant eggs, non-indigenous species, saltwater

flushing

Introduction

The introduction of non-indigenous species (NIS) into

habitats outside their native range is increasing in

frequency worldwide (Mack et al., 2000; Wonham &

Carlton, 2005; Ricciardi, 2006). The shipping industry

has played a major role in the spread of NIS globally.

Correspondence: Elizabeta Briski, Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON N9B

3P4, Canada. E-mail: briski@uwindsor.ca; elzabriski@yahoo.com

Freshwater Biology (2010) 55, 2414–2424 doi:10.1111/j.1365-2427.2010.02449.x

2414 2010 Blackwell Publishing Ltd
Ships’ ballast water and associated sediments are a

leading mechanism for NIS introductions into marine

ecosystems (Carlton, 1985; Ruiz & Carlton, 2003;

Molnar et al., 2008) and are particularly important

for the Laurentian Great Lakes (Holeck et al., 2004;

Ricciardi, 2006). Sediment has been implicated as a

vector for natural and human-assisted zooplankton

dispersal (Koste & Shiel, 1989; Hairston et al., 1999;

Bailey et al., 2003, 2005). Ballast sediments may contain very large numbers of active invertebrates as well

as their viable dormant stages (Bailey et al., 2005;

Duggan et al., 2005).

To decrease ballast-mediated invasions to the Great

Lakes, two different ballast water regulations have

been enacted. First, mid-ocean exchange was recommended in 1989 and made mandatory in 1993 to

reduce the number of propagules in ballast water

(Canadian Coast Guard, 1989; United States Coast

Guard, 1993). This regulation was augmented to

incorporate management of residual ballast water

and accumulated sediments through mandatory saltwater flushing, beginning in 2006 (Government of

Canada, 2006; SLSDC, 2008). Mid-ocean exchange

involves replacement of water in filled ballast tanks

with ocean water, while saltwater flushing involves

rinsing ballast tanks containing only residual ballast

water and sediments through the uptake and subsequent discharge of several tonnes of ocean water. To

assure compliance with the regulations, nearly every

ship entering the Great Lakes is inspected by Canadian and ⁄or American agencies (GLBWWG, 2009). In

theory, mid-ocean exchange and saltwater flushing

should reduce abundance (i.e. propagule pressure)

and species richness (i.e. colonisation pressure) by

purging coastal water, sediments and taxa from tanks

and, for low salinity taxa, by killing remaining

individuals with osmotic stress (MacIsaac, Robbins

& Lewis, 2002; Lockwood, Cassey & Blackburn, 2009).

Multiple studies have assessed the efficacy of midocean exchange and⁄or saltwater flushing on active

stages of biota (Locke et al., 1993; Rigby & Hallegraeff,

1994; Wonham et al., 2001; Choi et al., 2005; Gray et al.,

2007; Humphrey, 2008), while only one has examined

efficacy of the procedures on dormant eggs of invertebrate species resident in ballast sediment (Gray &

MacIsaac, 2010). Gray & MacIsaac (2010) reported

only partial effectiveness of mid-ocean exchange at

rendering dormant stages non-viable after performing

in situ tests with ballast sediment. Further, laboratory

tests have demonstrated that diapausing eggs are

resistant to short-term saltwater exposure (Bailey

et al., 2004; Gray et al., 2005; Bailey, Nandakumar &

MacIsaac, 2006). The above studies, however, had

small sample sizes and did not evaluate potential

cumulative effects of ongoing ballast management,

warranting a more substantial analysis of the effect of

the current ballast water regulations on dormant

stages in sediments.

Here, we test the effect of the 2006 regulations,

which mandated saltwater flushing, on the density

and diversity of invertebrate dormant stages in ballast

sediment of transoceanic and coastal vessels arriving

to the Great Lakes. We conducted a random survey of

ship sediments in 2007 and 2008, and compared our

results with those of a survey conducted between 2000

and 2002 (Bailey et al., 2005). As both studies collected

samples after mid-ocean exchange became mandatory, the results reflect only the influence of saltwater

flushing regulations; however, for simplicity, in this

paper, we refer to pre-regulation (2000–2002) and

post-regulation time periods (2007–2008). We tested

the hypotheses that post-regulation ships carry less

residual sediment, contain a lower abundance of

dormant eggs and have lower egg viability than did

pre-regulation ships.

Methods

Sediment collection, dormant stage counts and hatching

Ballast sediment was collected opportunistically from

19 ballast tanks on 17 ships, which originated from

European, South American and Atlantic ports in the

USA, arriving to the Great Lakes during 2007 and

2008. Approximately 6 kg of sediment was collected

from each ballast tank for laboratory analysis of the

density, diversity and viability of invertebrate dormant eggs. Methodology was consistent with that of

Bailey et al. (2003, 2005), allowing for comparison of

results pre- and post-regulation. We note that Bailey

et al. (2005) sampled ships that carried only residual

ballast water at the time of entry to the Great Lakes,

while this study sampled ships with full ballast tanks

that were discharged after entry to the Great Lakes

(Table 1). Following Bailey et al. (2005), results from

multiple tanks sampled from a single ship at a single

sampling event were averaged, while independent

trips into the Great Lakes by a single vessel were

Dormant eggs in ballast sediment 2415

2010 Blackwell Publishing Ltd, Freshwater Biology, 55, 2414–2424
considered independent samples since new ballast

had been held in tanks between sampling intervals.

Personal observations of sediment depth and per cent

cover inside ballast tanks, combined with architectural diagrams of ships’ tanks, were used to estimate

the amount of residual sediment carried by each ship.

We obtained data about each ship’s ballast history,

including total ballast capacity and previous dates

and locations of ballast uptake and discharge, from

ships’ crews and mandatory reporting forms submitted to Transport Canada.

Upon return to the laboratory, sediment was homogenised by thorough mixing. Four 40-g subsamples were

taken from each tank sediment sample for egg density

counts. Subsamples were preserved in 95% ethanol,

followed by washing through a 45-lm sieve to remove

fine sediment. Eggs were separated from the remaining

sediment using the colloidal silica Ludox HS 40

(Burgess, 2001). Dormant stages were enumerated

under a dissecting microscope, and the average density

of eggs from the four subsamples was extrapolated to

the number of dormant propagules per ship.

All remaining sediment was stored in the dark at

4 C for at least 4 weeks to break the diapause cycle of

dormant stages before hatching experiments commenced (Schwartz & Hebert, 1987; Dahms, 1995). Two

types of hatching experiments were conducted on all

19 tank sediments following the methodology of

Bailey et al. (2005). First, ‘maximum diversity experiments’ isolated eggs from sediments prior to hatching

to determine the number of viable species. Second, to

represent more realistic hatching conditions inside

ballast tanks, ‘whole sediment experiments’ were

conducted, which did not separate eggs from sediments (Table 1). All experiments were conducted

using a light : dark cycle of 16 : 8 h.

For maximum diversity experiments, diapausing

eggs were isolated from 40-g sediment subsamples

using a sugar flotation method (Hairston, 1996; Bailey

et al., 2003, 2005). Extracted eggs were placed into vials

containing sterile synthetic pond water [0 parts per

thousand (&) salinity; Hebert & Crease, 1980] or a

sterile seawater medium with salinity of 15 or 30&. The

seawater medium was prepared using mid-ocean

ballast water collected from a vessel transiting the

Great Lakes, filtered through 2.5-lm Whatman paper

filter and diluted to 15 or 30& with the sterile, synthetic

pond water. Four replicates were placed into each of

Table 1 Differences in methodology between the pre-regulation and the post-regulation periods

This study

(the post-regulation period)

Bailey et al. (2005)

(the pre-regulation period)

Sampling

Tank status Ballast water discharged after

entering the Great Lakes

Ballast water discharged

outside the Great Lakes

Total number of tanks sampled 19 69

Total number of ships sampled 17 39

Number of ships where two

tanks were sampled

2 26

Number of ships where three

tanks were sampled

0 2

Egg density counts

Number of tanks counted 19 69

Hatching experiments

Maximum diversity experiments 19 tanks; four replicates;

0&, 15 and 30&; at 20 C

Five tanks; four replicates; 0 and

8&; at 10 C and 20 C

and

50 tank; one replicate; 0&; at 20 C

Whole sediment experiments 19 tanks; four replicates;

0, 15 and 30&; at 20 C

19 tanks; four replicates; 0&; at 20 C

and

10 tanks; four replicates; 8, 16 and

32&; at 20 C

Identification of taxa

Molecular markers COI and 16S

applied to eggs

Yes No

Morphological identification applied

to hatched animals

Yes Yes

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2010 Blackwell Publishing Ltd, Freshwater Biology, 55, 2414–2424
the 0, 15 and 30& treatments at 20 C (Table 1).

Whole sediment experiments were conducted by

placing 40-g sediment subsamples directly into

500-mL glass vessels. Four replicates were placed into

each of the 0, 15 or 30& treatments, with 150 mL of

media added to each vessel before incubation at 20 C.

Three different salinities were used in both types

of hatching experiments in an attempt to match

unknown species to a fresh-, brackish- or salt water

habitat to promote maximum hatching (Table 1). The

list of species generated from hatching experiments

was used to estimate egg viability as well as the effect

of saltwater flushing on freshwater, brackish and

saltwater taxa. Hatching percentage (H%) was calculated by dividing the total number of animals hatched

by the total number of eggs isolated for hatching, and

multiplying by 100.

We use the number of hatched eggs as a proxy

measure of egg viability, although we acknowledge

that some eggs that did not hatch may have been

viable but did not receive appropriate hatching cues.

All NIS hatched in the 0& treatment were considered

high-risk taxa with potential to establish populations

under environmental conditions of the Great Lakes,

unless an established population of the species

already exists. The freshwater species Daphnia magna

was not considered a high-risk NIS, however, as the

species almost certainly has been introduced into the

Great Lakes multiple times by both shipping (Bailey

et al., 2003, 2005; Duggan et al., 2005) and natural

(Louette & De Meester, 2005) vectors but has not

established a self-sustaining population; biotic or

abiotic factors may preclude invasion by this species

(Lauridsen & Lodge, 1996).

Identification of dormant eggs was conducted

directly using molecular methods, as well as through

traditional morphological taxonomy of hatched individuals (Table 1). DNA was extracted directly from

diapausing eggs using a HotSHOT method (MonteroPau, Go ́mez & Mun ̃oz, 2008). Fragments of the

mitochondrial gene cytochrome c oxidase subunit I

(COI) and 16S rDNA gene were amplified from each

egg using the universal primers LCO1490 and

HCO2190 (Folmer et al., 1994) and S1 and S2 (Palumbi,

1996), respectively. PCR were performed in a total

volume of 25 lL using 5 lL of DNA extract, 1· PCR

buffer, 12.5 lL of 10% trehalose, 0.1 lM of each

primer, 2.5 mM MgCl2, 0.14 mM dNTPs and 0.4 U

Taq DNA polymerase. The thermal profile consisted of

a 1-min initial cycle at 94 C, followed by five cycles of

94 C (40 s), 45 C (40 s) and 72 C (1 min), 35 cycles of

94 C (40 s), 50 C (40 s) and 72 C (1 min) and a final

extension of 72 C for 5 min. PCR products were

sequenced by an ABI 3130XL automated sequencer

(Applied Biosystems, Foster City, CA, USA).

Although our post-regulation methodology followed that used during the pre-regulation period

(Bailey et al., 2005), there are some differences

(Table 1). We conducted both types of hatching

experiments on all 19 tank samples in a fully replicated

fashion using three growth media at a single temperature (0, 15 and 30&, at 20 C). In contrast, Bailey et al.

(2005) conducted experiments using four growth

media at two temperatures (0, 8, 16 and 32& at 10

and 20 C), but were unable to fully replicate all

experiments because of large sample size (69 tanks).

Furthermore, Bailey et al. (2005) used only traditional

morphological taxonomy to identify hatched individuals to species level. Morphological identification of

dormant eggs can be difficult, even to the class level,

and almost 10% of unhatched eggs were reported by

Bailey et al. (2005) as ‘indeterminate’ taxon (Table 2).

While the molecular methods used in this study were

not able to identify all eggs to species level, the number

of species identifications was double that of morphological methods (E. Briski, unpubl. data).

Statistical analysis

We tested for differences in the cumulative mean

density of diapausing eggs, the mean density of

viable (hatched) eggs and the mean density of eggs

of NIS in 40 g subsamples between the pre- and

post-regulation sampling periods using t-tests and

Mann–Whitney U-tests (SPSS 11.5.0; SPSS Inc., 1989–

2002, Chicago, IL, USA). A logarithmic transformation was applied to both datasets to meet assumptions of parametric tests. When a normal distribution

was not achieved, or if results of a Levene’s test for

homogeneity of variances was significant, the nonparametric Mann–Whitney U-test was used (Table 3).

After analysis of 40-g sediment subsamples was

completed, further tests were conducted on extrapolated total egg abundance, total number of viable

eggs and abundance of eggs of NIS by multiplying

the average of four 40-g subsamples from each ship

by the amount of sediment carried by that ship. The

extrapolated data were again compared to Bailey

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2010 Blackwell Publishing Ltd, Freshwater Biology, 55, 2414–2424
et al.’s (2005) samples using t-tests and Mann–Whitney U-tests (SPSS 11.5.0, SPSS Inc.) (Table 3). A

significance level of 95% was used for all statistical

analyses. Power analyses estimating the number of

ship samples required to differentiate significant

differences in abundance of NIS between the two

time periods were calculated using JMP 7.0.2 (2007

SAS Institute Inc., Cary, NC, USA).

To estimate species richness of the larger vessel

population based on findings from our sampled

vessels, we calculated Chao-1, an estimator of species

richness based on the number of rare species in a

sample (Chao, 1984; Chao & Shen, 2003). We compared Chao-1 species richness estimates for the preand post-regulation periods to examine the efficacy of

ballast water flushing. Sample-based species rarefaction curves were generated for both sampling periods

to determine whether a significant difference existed

given our small sample size. Confidence intervals

(95%) were generated to test for significant differences between the two sampling periods (Chao &

Shen, 2006; Gotelli & Entsminger, 2006). Chao-1

estimates were calculated using SPADE software

(Chao & Shen, 2006), while rarefaction curves were

generated with 5000 random iterations using ECOSIM

(Gotelli & Entsminger, 2006). Species richness comparisons were conducted for both total richness and

richness of NIS.

Table 2 Per cent occurrence and abundance of dormant stages collected before

(39 vessels) and after (17 vessels) implementation of Canadian ballast water

management regulations in 2006. Dormant stages are arranged phylogenetically

by taxon. Pre-regulation data modified

from Bailey et al. (2005)

Taxon

Pre-regulation period Post-regulation period

% Occurrence % Abundance % Occurrence % Abundance

Rotifera 100 77.9 95 19.9

Asplanchna spp. 66.7 1.0 0 0

Brachionus spp. 97.4 76.2 95 19.9

Conochilus spp. 5.1 <1 0 0

Filinia spp. 48.2 <1 0 0

Synchaeta spp. 5.1 <1 0 0

Bryozoa 61.5 <1 63.2 3.9

Cladocera 76.9 9.3 89.5 37.8

Bosmina spp. 51.3 <1 10.5 1.3

Chydoridae 5.1 <1 0 0

Daphnia spp. 46.2 7.9 89.5 19.5

Diaphanosoma spp. 2.6 <1 1 <1

Moina spp. 25.6 <1 15 12.8

Onychopoda 1 <1 21 3.3

Copepoda 76.9 2.6 100 37.8

Indeterminate* 100 9.8 26.3 <1

*Indeterminate represent eggs that were not identified to any taxonomic level.

Table 3 Significance levels for statistical comparisons of experimental data between the pre-regulation and the post-regulation

periods. Significant P-values are presented in bold. A significance level of 95% was used for all statistical analyses

Experiment type Treatment compared

Leven’s test for equality

of variances (P) t-test (P)

Mann–Whitney

U test (P)

Egg counts Number of eggs in 40-g subsample 0.925 0.019

Amount of sediment per ship 0.330 0.001

Number of eggs per ship 0.916 <0.001

Maximum

diversity

Number of hatched eggs from 40-g subsample 0.001 <0.001

Number of eggs of NIS in 40-g subsample 0.400 0.173

Number of high risk eggs of NIS in 40-g subsample <0.001 <0.001

Number of hatched eggs per ship 0.037 0.009

Number of eggs of NIS per ship 0.007 0.814

Number of high risk eggs of NIS per ship <0.001 <0.001

Whole sediment Number of eggs hatched from 40 g subsample <0.001 0.007

Number of hatched eggs per ship <0.001 <0.001

NIS, non-indigenous species.

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2010 Blackwell Publishing Ltd, Freshwater Biology, 55, 2414–2424
Results

The estimated amount of residual sediment per vessel

ranged from <1 to 45 tonnes, with the mean tonnage

(5 tonnes) being significantly lower than in the preregulation period (14 tonnes per vessel; P < 0.05,

Table 3). Similarly, total density of dormant eggs

during the post-regulation period, which ranged from

1 to 80 eggs per 40-g sediment with mean density 20.3

eggs per 40 g, was significantly lower than in preregulation samples (143.5 eggs per 40 g; Fig. 1;

Table 3). The density of viable eggs, which ranged

from 0 to 17 eggs per 40 g (mean density of 1.9 eggs

per 40 g; 6.2% hatching success), was also significantly lower than in pre-regulation period (24.4 eggs

per 40 g; 24.8%; P < 0.05; Table 3, Fig. 1). While the

mean density of viable eggs of NIS post-regulation

(1.3 eggs per 40-g sediment; 1.6% hatch rate) did not

decrease significantly (4.7 eggs per 40 g; <1% hatch

rate for pre-regulation period), there was a significant

difference if only eggs of high-risk NIS are considered

(Fig. 1; Table 3).

The identification of diapausing eggs using molecular markers COI and 16S resulted in 17 distinct taxa

(Appendix S1). Only eight of the 13 distinct morphological groups of dormant stages recorded by Bailey

et al. (2005) were also recorded in this study. While

Bailey et al. (2005) found that community composition

of dormant eggs was dominated by Rotifera (77.9%), we

found that dormant eggs of Cladocera and Copepoda

were most abundant, each representing 37.8% of total

abundance. Seven NIS were identified, including five

Cladocera, one Copepoda and one Ascidia (Table 4).

Fourof the NIS were freshwatertaxa (D. magna,Daphnia

galeata, Cercopagis pengoi and Acartia tonsa), although

only one is considered high risk (A. tonsa; Table 4).

Maximum diversity experiments confirmed the viability of 10 taxa, with species richness ranging from 0 to

5 viable taxa per 40-g sediment (median 2; no eggs were

hatched from eight tanks). Cladocera were the most

species-rich group, representing 80% of all taxa

hatched. The remaining species belonged to Rotifera

and Copepoda (see Appendix S1). Forty per cent of

ships sampled in the post-regulation period carried

viable dormant stages of NIS (0–2 eggs per 40 g

sediment), although only one taxon (D. magna) hatched

from only one of the 19 tanks during whole sediment

experiments. The estimated species richness of the

post-regulation vessel population was 27.2 species

including 8.3 NIS, which was significantly lower than

estimates for the pre-regulation period (126 and 33

species estimated for total richness and NIS richness,

respectively, for the pre-regulation period) (Fig. 2).

Extrapolation of post-regulation subsample results

to whole ships resulted in mean and median abundances of 3.5 · 106 and 9.0 · 104 eggs ship)1

, respectively. The mean and median numbers of viable eggs

per ship were estimated at 1.9 · 106 and 1.3 · 105

eggs ship)1, respectively. Finally, the mean and median abundances of dormant eggs of NIS were estimated

as 1.8 · 105 and 0 eggs ship)1

, respectively. The total

abundance of dormant eggs, number of eggs hatched

and number of eggs of high risk NIS per ship were each

significantly lower in the post- versus pre-regulation

period (P < 0.05; Table 3). The estimated average total

ballast capacity of ships in our study was 14532 m)3

,

and assuming that all eggs from sediment would hatch,

it could result in average of 130 individuals m)3 of

which 12.3 individuals m)3 are NIS. However, considering hatching results from whole sediment experiments, the average number of invertebrates released

from eggs may be as low as 0.014 individuals m)3

.

Discussion

Results from this study indicate that the ballast

management regulations implemented in 2006 have

markedly reduced the probability of introduction of

Fig. 1 Mean (±standard error of mean) and median (horizontal

line in bar) density of invertebrate dormant eggs (total), of viable

invertebrate eggs (hatched), of viable eggs of non-indigenous

species (NIS) and of eggs of high-risk NIS in ballast sediment

samples from pre-regulation (grey bars) and post-regulation

sampling periods (black bar). The asterisk denotes a significant

difference between paired bars.

Dormant eggs in ballast sediment 2419

2010 Blackwell Publishing Ltd, Freshwater Biology, 55, 2414–2424
invertebrates to the Great Lakes via dormant eggs.

Sediment accumulation has been significantly

reduced, and with it the abundance of dormant stages

of invertebrate species. On average, ships in the postregulation period carried a potential inoculum one

order of magnitude lower than those sampled in the

pre-regulation period. Egg viability was lower, and

fewer eggs of perceived high-risk NIS were present

during the post-regulation period. Estimated species

richness for the vessel population was also much lower

in the post-regulation period (27 versus 126 species).

The addition of saltwater flushing to the ballast

water management regime reduced the tonnage of

accumulated sediments in ballast tanks threefold.

Physical removal of sediments probably contributed

to the observed reduction in egg number, as eggs are

probably discharged along with sediments. Less

sediment accumulation in tanks could also impact

viability of retained eggs if they are more exposed to

saltwater exposure during mid-ocean exchange

and ⁄or saltwater flushing. Bailey et al. (2004, 2006)

determined that saltwater exposure was significantly

more detrimental to viability of eggs extracted from

sediments than for those retained within sediment.

Furthermore, Reid et al. (2007) reported a reduction in

oxygen concentration in ballast tank water owing to

decaying organic matter. Reduced sediment accumulation in tanks may also expose a larger proportion of

retained eggs to unfavourable oxygen concentration at

the sediment–water interface. Reduction in sediment

Table 4 Non-indigenous species transported as dormant eggs in residual ballast sediment to the Great Lakes. Species are listed in

order of decreasing frequency and abundance of resting eggs, and ability to tolerate freshwater habitats. Occurrence identifies the

number of ships that the species was collected from. Abundance is the cumulative mean number of eggs identified from 40-g sediment

for all ships in which each species was found. Species hatched in 0& medium during laboratory experiments were considered an

environmental match for the Great Lakes. Further, species identified by molecular markers that did not hatch were assigned habitat

match based on literature research (Cercopagis pengoi and Botrillus schlosseri). Pre-regulation data are modified from Bailey et al. (2005)

Species name

Pre-regulation period Post-regulation period

Occurrence Abundance Occurrence Abundance Habitat match

Daphnia magna (Straus, 1820) 4 6 2 20.5 Y

Filinia passa (Muller, 1786) 4 3.5 Y

Brachionus leydigi (Cohn, 1862) 4 3 Y

Filinia cornuta (Weisse, 1847) 3 3 Y

Asplanchna girodi (De Geurne, 1888) 2 1 Y

Cephalodella sterea (Gosse, 1887) 1 4.75 Y

C. pengoi (Ostroumov, 1891)* 2 0.75 Y

Bosmina maritima (Muller, 1867) 1 2 Y

Diaphanosoma orghidani (Negrea, 1982) 1 1.25 Y

Daphnia galeata (Sars, 1864) 1 2 Y

Brachionus forficula (Wierzejski, 1891) 1 1 Y

Brachionus nilsoni (Ahlstrom, 1940) 1 1 Y

Conochilus coenobasis (Skorikov, 1914) 1 0.5 Y

Diaphanosoma mongolianum (Ueno, 1938) 1 0.5 Y

Cephalodella cf. stenroosi (Wulfert, 1937) 1 0.3 Y

Alona rustica (Scott, 1895) 1 0.25 Y

Brachionus bennini (Leissling, 1924) 1 0.25 Y

Brachionus diversicornis (Daday, 1883) 1 0.25 Y

Diaphanosoma sarsi (Richar, 1894) 1 0.25 Y

Hexarthra intermedia (Wiszniewski, 1929) 1 0.25 Y

Moina affinis (Birge, 1893) 1 N ⁄A Y

Acartia tonsa (Dana, 1849) 1 0.25 Y

Synchaeta baltica (Ehrenberg, 1834) 1 2.75 N

Synchaeta bacillifera (Smirnov, 1933) 1 2.25 N

Evadne nordmanni (Love ́n, 1836) 1 0.5 N

Pleopis polyphemoides (Leuckart, 1859) 1 N ⁄A 1 0.5 N

Podon intermedius (Lilljeborg, 1853) 1 0.5 N

B. schlosseri (Pallas, 1766)† 1 0.25 N

*C. pengoi did not hatch in our experiments, although it is established in the Great Lakes. †

B. schlosseri did not hatch in our experiments; habitat matching designation based on Lambert (2005).

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2010 Blackwell Publishing Ltd, Freshwater Biology, 55, 2414–2424
accumulation could also negatively impact egg viability via desiccation. Dry sediments have been documented for ships arriving to the west coast of

Canada and to the Great Lakes (Sutherland, Levings

& Wiley, 2009; S. A. Bailey, unpubl. data), and long

periods of desiccation or repeated hydration–dehydration cycles can negatively impact viability of

dormant eggs (Lavens & Sorgeloos, 1987; Hagiwara

et al., 1997). Furthermore, egg viability may be

reduced if small amounts of sediments facilitate

gradual rather than rapid changes in abiotic conditions. For example, exposure to brackish water (8&)

has a greater effect on viability of dormant eggs of

freshwater species like Bosmina leideri De Melo and

Hebert, 1994 and Daphnia longiremis (Sars, 1861) than

does exposure to ocean water (32&; Bailey et al., 2004).

Similarly, egg viability is reduced more by exposure to

low levels of oxygen than to complete anoxia (Lutz,

Marcus & Chanton, 1994). Under extreme abiotic

conditions, such as complete anoxia or high salinity,

dormant eggs remain inactive, while sub-optimal

conditions can initiate termination of diapause (Clegg

& Trotman, 2002; Garcı ́a-Roger, Carmona & Serra,

2005; Pauwels et al., 2007). Eggs that begin to develop

under less-than-optimal conditions may allocate extra

energy to adjust their metabolism to environmental

conditions such that energy reserves are depleted

before development is complete and emergence

occurs (Van Stappen, 1996; Bailey et al., 2004). Whatever the mechanism, the results of this study indicate

that egg viability – as determined by hatching success

– was significantly reduced in vessels entering the

Great Lakes following implementation of ballast

regulations for residual sediment and water.

Current ballast water management activities seemingly exert differential impacts on different taxa, as

evidenced by the change in dormant egg community

dominance from Rotifera to Cladocera and Copepoda.

Dormant eggs of Rotifera are best preserved in

constant salinity with low amounts of organic matter

(Hagiwara et al., 1997), while Cladocera have a hard

ephippial structure around eggs that enhances resistance to desiccation and rapid abiotic changes (Altermatt, Pajunen & Ebert, 2009). It is unclear why

abundance of Copepoda would increase compared to

other taxa, but it is possible that a portion of

Copepoda eggs were classified as ‘indeterminate’ in

the pre-regulation period (see Methods; Table 2),

since species identifications did not include molecular

analysis in the earlier study.

While we observed a significant decrease in the

total abundance, viability and species richness of

dormant eggs, egg abundance of NIS did not decrease

in the post-regulation period. This observation is

almost certainly as a result of insufficient sample

size. Using power analysis, we estimated that up to

171 ship samples would be required to confirm a

(a)

(b)

Fig. 2 Sample-based rarefaction curves from the pre-regulation

(grey line, ±95% C.I.) and the post-regulation (black line, ±95%

C.I.) periods for: (a) all ships sampled and (b) ships containing

non-indigenous species. Also shown are species richness estimates for the vessel population (Chao-1 ± 95% C.I.) for the preregulation (grey bar) and the post-regulation period (black bar).

Note the difference in scales for each x and y-axis.

Dormant eggs in ballast sediment 2421

2010 Blackwell Publishing Ltd, Freshwater Biology, 55, 2414–2424
significant difference in NIS egg abundance between

the two studies. However, egg abundance of high-risk

taxa dropped from an average of 78% during the preregulation period to an average of 1% post-regulation.

Only four NIS capable of tolerating fresh water were

recorded during this study. Cladocera D. magna and

C. pengoi were recorded in two ships, while D. galeata

and Copepoda A. tonsa were observed in one vessel

each. Following our earlier line of reasoning, D. magna

appears to be a low risk for successful establishment

in the Great Lakes. Cercopagis pengoi and D. galeata

represent the next highest risk for introduction based

on propagule pressure, but both have already established in the system (Taylor & Hebert, 1993; MacIsaac

et al., 1999). Indeed, presence of viable eggs of these

species in ballast sediments highlights the possibility

that these species were vectored to the lakes in ballast

sediment rather than ballast water. As a result,

A. tonsa is the only species recorded during this study

which presents a relatively high risk for invasion for

the Great Lakes via retained eggs in treated ballast

sediments.

Preventing species introductions via dormant invertebrate eggs is a particularly challenging task, because

ballast sediments are not easily flushed from tanks

and because dormant eggs are resistant to a wide

array of adverse environmental conditions and treatment strategies (Bailey et al., 2005, 2006; Gray, Duggan

& MacIsaac, 2006). Our results from whole sediment

experiments indicate that the current propagule

pressure posed by dormant eggs hatched into ballast

water, estimated at 0.014 hatched individuals m)3

,

would make an insignificant contribution to the

median number of propagules typically carried in

exchanged ballast water (2672.9 ind. m)3

; S. A. Bailey,

unpubl. data). As the proposed international ballast

water discharge standard applicable to invertebrate

zooplankton stipulates that treated ballast water must

contain less than 10 viable individuals m)3 (IMO,

2004), our results suggest that the risk of introductions

via in situ hatching is adequately managed through

saltwater flushing. In the worst case scenario, if all

eggs in the sediment were to hatch and become

available for discharge, or if all eggs in the sediment

would be discharged directly, the estimated abundance of viable individuals in filled ballast tanks

would increase by 130 individuals m)3

, which would

be non-compliant with the proposed international

ballast water discharge standard.

Acknowledgments

We thank the Shipping Federation of Canada and the

multiple shipping companies that facilitated access to

vessels, and our ballast sampling teams. We are

grateful for taxonomic assistance from Drs S.I. Dodson, J.R. Cordell and P. Hudson, and laboratory

support from R. Tedla, S. Ross and H. Coker.

Comments from two anonymous reviewers are gratefully acknowledged. This research was supported by

NSERC’s Canadian Aquatic Invasive Species Network, and by an NSERC Discovery Grant and DFO

Invasive Species Research Chair to HJM.

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Supporting Information

Additional Supporting Information may be found in

the online version of this article:

Appendix S1. List of invertebrate taxa identified

during pre-regulation and post-regulation studies.

As a service to our authors and readers, this journal

provides supporting information supplied by the

authors. Such materials are peer-reviewed and may

be re-organised for online delivery, but are not copyedited or typeset. Technical support issues arising

from supporting information (other than missing files)

should be addressed to the authors.

(Manuscript accepted 8 April 2010)

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