Environ Sci Pollut Res (2017) 24:28268–28276

https://doi.org/10.1007/s11356-017-0736-1

SHORT RESEARCH AND DISCUSSION ARTICLE

Comparison of bioaccumulation and elimination

of Escherichia coli and male-specific bacteriophages by ascidians
and bivalves
Ji Hoe Kim 1 & Kil Bo Shim 1 & Soon Beum Shin 2 & Kunbawui Park 1 & Eun Gyoung Oh 1 &
Kwang Tae Son 3 & Hongsik Yu 4 & Hee Jung Lee 2 & Jong Soo Mok 1

Received: 20 July 2017 / Accepted: 9 November 2017 / Published online: 20 November 2017
# Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract Levels of Escherichia coli and male-specific bacte- Keywords Ascidian . Bivalve . Filter feeder . Male-specific
riophages (MSBs) were determined in the filter feeders obtain- bacteriophage . Bioaccumulation . Elimination . Korea
ed from retail markets, commercial farms, and wild beds in
Korea. The accumulation and elimination of E. coli and MSBs
were compared between ascidians and bivalves (oysters and Introduction
mussels) during relaying and depuration. E. coli concentra-
tions in ascidians from retail markets ranged between < 20 The edible ascidian Halocynthia roretzi is a commercially
and 460 most probable number/100 g while MSBs were not important seafood species in Korea, Japan, and other Asian
detected. E. coli levels in bivalves from commercial farms and countries. Ascidians are cultured extensively in coastal re-
wild beds were not significantly different but bacterial levels gions of eastern and southern Korea and northeastern Japan
in ascidians were consistently lower. Ascidians exhibited (Mizuta et al. 2002). Similar to other tunicates, ascidians are
much lower ability than bivalves to accumulate E. coli and suspension feeders that filter phytoplankton and other
MSBs during relaying in a polluted coastal area. This study suspended particulate matter from the surrounding water
also shows that an equilibrium was developed between levels (Bone et al. 2003; Petersen 2007; Riisgård and Larsen
of microbes in water and ascidians and shellfish during relay- 2010). Besides ingesting phytoplankton and other food parti-
ing. E. coli and MSBs in ascidians decreased quickly during cles, filter-feeding animals (e.g., tunicates and bivalves) may
depuration in a clean seawater tank. However, after 1 day, also concentrate pathogenic bacteria and viruses in their guts,
E. coli in bivalves decreased by only 1.1–1.6 logs, and the so that consumption of filter feeders from polluted waters
elimination of MSBs was negligible. Therefore, depuration presents a risk to human health.
is an effective means to reduce the health risk of contaminated For many decades, the consumption of raw or undercooked
ascidians. bivalves, such as oysters and clams, has been implicated in
numerous food-poisoning outbreaks caused by pathogenic
microorganisms (Rippey 1994; Potasman et al. 2002; Lees
Responsible editor: Philippe Garrigues
et al. 2010; Sair et al. 2002; Iwamoto et al. 2010; Yang et al.
* Jong Soo Mok
2017). Fecal coliforms, including Escherichia coli, are used as
mjs0620@korea.kr fecal contamination indicators to assess the quality of bivalves
and to determine whether bivalves are safe for raw consump-
1
Food Safety and Processing Research Division, National Institute of tion (Mok et al. 2016a). Many countries have established reg-
Fisheries Science, Busan 46083, Republic of Korea ulatory limits using fecal indicators for bivalves and their
2
South Sea Fisheries Research Institute, National Institute of Fisheries growing areas (European Commission (EC) 2005; New
Science, Yeosu 59780, Republic of Korea Zealand Food Safety Authority (NZFSA) 2006; US Food
3
West Sea Fisheries Research Institute, National Institute of Fisheries and Drug Administration (US FDA) 2015; Korea Ministry
Science, Incheon 22383, Republic of Korea of Food and Drug Safety (KMFDS) 2016). Male-specific bac-
4
Southeast Sea Fisheries Research Institute, National Institute of teriophages (MSBs) have been also used for tracking pollution
Fisheries Science, Tongyeong 53085, Republic of Korea in marine estuaries and determining the risk of illness
Environ Sci Pollut Res (2017) 24:28268–28276 28269

associated with the consumption of raw bivalves (Burkhardt and mussels (Mytilus galloprovincialis) were also collected
et al. 1992b). Furthermore, in Korea, peeled whole ascidians from a commercial aquaculture farm and from wild beds.
are commonly consumed raw, including the hepatopancreas. The commercial farm was located in an area designated for
For this reason, as for bivalves, careful attention must be paid shellfish growing for export that met water quality standards
to the food safety of ascidians. of the National Shellfish Sanitation Program (NSSP) for ap-
Several experiments have been conducted to determine proved areas (US FDA 2015). The wild bed was located in a
the kinetics of uptake and elimination of fecal indicator shallow water coastal region near Busan City, which is sus-
bacteria and pathogens by shellfish. The accumulation ceptible to land-based pollutants. Samples of animals were
and elimination kinetics of enteric bacteria and viruses collected monthly from the commercial farm and from the
vary among bivalve species (Cabelli and Heffernan wild bed. All collected samples for microbiological analysis
1970) and depend on the type of microorganism were maintained below 10 °C during transport to the labora-
(Burkhardt et al. 1992a; Burkhardt and Calci 2000; Mok tory. Ascidians, oysters, and mussels for accumulation and
et al. 2016b) and environmental conditions (Burkhardt elimination experiments were taken from the designated
et al. 1992a). Although ascidians and bivalves are both shellfish-growing area.
filter feeders, they differ in size and in their particle cap-
ture mechanisms (Riisgård and Larsen 2010). It is well Analysis of fecal indicator bacteria
established that the filtration rates of ascidians vary with
the species and environmental conditions (Randløv and The levels of total coliforms, fecal coliforms, and E. coli in the
Riisgård 1979; Ribes et al. 1998; Petersen and Svane samples were enumerated by the most probable number
2002). Feed particle size and capture mechanisms have (MPN) method. The MPN method used was a five-tube test
been studied extensively in ascidians (Bone et al. 2003; using three tenfold serial dilutions; referring the numbers of
Petersen 2007; Riisgård and Larsen 2010). However, few tubes yielding positive results to published standard tables
studies have been conducted to evaluate the microbiolog- gave the concentrations of bacteria in the samples. The rec-
ical quality of live ascidians destined for raw consump- ommended procedures for the examination of seawater and
tion. Although bacterial contamination of ascidians can shellfish according to the American Public Health
occur at any step from pre-harvest through to final prep- Association (APHA) were applied for total coliform and fecal
aration, prevention of initial contamination at the harvest- coliform enumeration (APHA 1970). The presumptive test
ing area is important because, as noted, the animal is was performed using lauryl tryptose broth (Difco, Detroit,
commonly consumed raw in Asian countries. MI, USA) at 35 ± 0.5 °C. The confirmative test used brilliant
In the present study, microbiological quality was evaluated green-lactose broth (Difco) at 35 ± 0.5 °C for total coliforms,
based on the levels of indicators of fecal contamination, in- and EC medium (Difco) at 44.5 ± 0.2 °C for fecal coliforms.
cluding total coliforms, fecal coliforms, E. coli, and MSBs in In addition, the ISO/TS 16649-3 method (ISO 2015) was used
live bivalves (oysters and mussels) and ascidians collected for E. coli enumeration. Tubes of Minerals-modified
from retail markets or fields. The characteristics of bioaccu- Glutamate Broth (Oxoid, Basingstoke, UK) were incubated
mulation and elimination of E. coli and MSBs by H. roretzi at 37 ± 1 °C for 24 ± 2 h for the presumptive test. Each posi-
and by oysters and mussels were compared by relaying them tive culture tube showing yellow color was subsequently con-
at a polluted area and then depuration in seawater tanks to firmed by subculture onto a Tryptone Bile X-glucuronide
investigate the importance of ascidians as vectors in the trans- Agar (Oxoid) plate at 44 ± 1 °C for 22 ± 2 h. Results are
mission of pathogenic microorganisms. While levels of these expressed as MPN/100 mL for seawater and MPN/100 g for
microorganisms in bivalves (oysters and mussels) at retail ascidian and bivalve meat samples. The limits of detection
markets have been studied as well as bioaccumulation and (LODs) of these methods were the following: 18 MPN/
depuration, this is the first work that we are aware of that 100 g of meat and 1.8 MPN/100 mL of seawater for total
evaluates the levels of fecal indicators within ascidians as a and fecal coliforms, and 20 MPN/100 g of meat and 2.0
vector in the transmission of pathogenic microorganisms. MPN/100 mL of seawater for E. coli.

MSB analysis
Materials and methods
The MSB levels in ascidians and bivalves meat were assayed
Sample collection using the double-layer agar method described by Burkhardt
et al. (1992b). The E. coli culture HS[pFamp]R (ATCC
Live ascidians (H. roretzi) were obtained from local retail 700891) was obtained from the American Type Culture
markets in Tongyeong and Busan, Korea, during 2009– Collection through their Korean distributor and used as the
2010. Samples of H. roretzi, oysters (Crassostrea gigas), bacterial host strain for the MSBs. The bacterial host culture
28270 Environ Sci Pollut Res (2017) 24:28268–28276

was prepared as described in the US EPA method 1601 (US was stirred with an electronic stirrer for 1 h, and the mixture
EPA 2001). The assays used the antibiotics, streptomycin sul- was then subdivided into 30-L volumes. Prior to
fate, and ampicillin to prevent competing bacterial growth. subdividing, the levels of E. coli and MSBs in the mixture
The MSBs were quantified by plaque formation on the host were enumerated. Groups of 22 ascidians, 25 oysters, or 74
E. coli on the agar medium. The results are expressed as the mussels providing similar total net meat weights (ca. 400 g)
numbers of plaque-forming units (PFU) per 100 g or per were each placed in 30 L of the seawater mixture and aer-
100 mL of sample. The LODs were 17 PFU/100 g of meat ated (air filtered with a cotton plug). Seawater was sampled
and 10 PFU/100 mL of seawater. daily for analysis of E. coli and MSB concentration to in-
vestigate the water purification activities of the ascidians
Bioaccumulation experiments and bivalves. Water temperature and salinity were mea-
sured using an YSI 556 Multiprobe system (YSI Inc.,
Live ascidians, oysters, and mussels collected from the desig- Yellow Springs, OH).
nated shellfish-growing area were transferred to water near the
outflow of a sewage treatment plant to compare the accumu-
lation rates and final levels of E. coli and MSBs by ascidians
and bivalves. Ascidians, oysters, and mussels harvested from Results and discussion
the designated shellfish-growing area were used for relaying,
after confirming that they were free from E. coli and MSBs. Levels of fecal indicator bacteria and MSBs in ascidians
After scrubbing with a stiff brush to remove adhering detritus from retail markets
and fouling organisms, 37 animals (7 ascidians, 15 oysters,
and 15 mussels) were placed together into each of mesh bags Levels of total coliforms, fecal coliforms, E. coli, and MSBs in
(mesh size 10 mm). The bags were hung from buoys and live ascidians obtained from retail markets in Busan (Gijang
allowed to sink approximately 50 cm below the surface at market) and Tongyeong (Seoho market and Jungang market),
the relaying site. Duplicate samples of five ascidians and 12 Korea, are summarized in Table 1.
mussels and oysters were taken from each bag and assayed for Levels of fecal indicator bacteria in ascidians differed
the levels of E. coli and MSB. among the markets and sample batches. The maximum levels
recorded among the 30 samples taken from the three retail
Depuration experiments markets were 1100 MPN/100 g total coliforms, 230 MPN/
100 g fecal coliforms, and 460 MPN/100 g E. coli. The esti-
The depuration tank (100 × 100 × 70 cm high) had a working mated geometric means were 24 MPN/100 g for fecal coli-
volume of 400 L. Sand-filtered (Negatron Water Filter, Busan, forms and 22 MPN/100 g for E. coli. The MSBs were not
Korea), UV-irradiated (four 39-W lamps, Sungchang, detected in any of the retailed ascidians.
Anyang, Korea) seawater was used for the depuration exper- The levels of E. coli in most of the ascidians (n = 30) pur-
iments. Prior to the depuration experiments, no detection of chased from the three markets complied with the European
indicators in the seawater was confirmed. The water tempera- standard for live and raw seafood (bivalves, echinoderms,
ture was maintained at 12 ± 8 °C. Contaminated ascidians and tunicates, and gastropods) for human consumption (fewer
bivalves relayed for 5 days were washed thoroughly, and dam- than 230 E. coli per 100 g) (EC 2005), and no MSBs were
aged and moribund individuals were rejected. The contami- found. However, the level of E. coli (460 MPN/100 g) in one
nated animals were loaded into baskets without overlapping sample obtained from Seoho market, Tongyeong, exceeded
and the baskets were placed on the bottom of the depuration the European standard. The geometric mean value of E. coli
tank at a depth of approximately 8 cm. Seawater was supplied concentration in ascidians from the 30 retail samples (24
to the tank at a rate of 10 L/min. Duplicate samples of five MPN/100 g) was higher than that in retailed oysters from
ascidians, 10 oysters, or 10 mussels were retrieved for micro- the North Atlantic and Pacific coasts but similar to that of
biological analysis. Gulf and Mid-Atlantic coast oysters in the USA (DePaola
et al. 2010). Even in the ascidian sample that exceeded the
Water purification by filter feeders European E. coli standard of 230 MPN/100 g, the MSB con-
centration was below the detection limit of 17 PFU/100 mL.
Twenty similarly sized ascidians, oysters, or mussels were Although the ratios of MSBs and E. coli change during the
shucked (or peeled) and weighed for calculation of average sewage treatment process, E. coli are generally present in
net weight. Sewage containing bacteria and MSBs was fil- greater concentrations than MSBs in raw sewage (Doré et al.
tered with a paper pre-filter (Advantec No. 5A, Toyo Roshi, 2003). The sample that exceeded the European standard may
Japan) for the removal of particles. Filtered sewage (2 L) have been contaminated by untreated sewage at any stage of
was added to sand-filtered seawater (120 L), the mixture growing, harvesting, and handling.
Environ Sci Pollut Res (2017) 24:28268–28276 28271

Table 1 Levels of fecal pollution indicator bacteria and male-specific bacteriophages (MSB) in ascidians (Halocynthia roretzi) collected from three
retail markets in Korea during 2009–2010

Collected market Total coliform Fecal coliform E. coli (MPN/ No. of samples MSB (PFU/ No. of
bacteria (MPN/ bacteria (MPN/ 100 g) exceeded 230 100 g) sample
100 g) 100 g) E. coli MPN/100 g batches

Min Max GM Min Max GM Min Max GM Min Max Mean

Total < 18 1100 90 < 18 230 24 < 20 460 22 1 < 17 < 17 < 17 30
Gijang retail market, Busan < 18 790 101 < 18 78 25 < 20 80 22 0 < 17 < 17 < 17 9
Seoho retail market, Tongyeong < 18 1100 123 < 18 230 30 < 20 460 24 1 < 17 < 17 < 17 12
Jungang retail market, Tongyeong < 18 340 57 < 18 20 < 18 < 20 40 < 20 0 < 17 < 17 < 17 9

GM, geometric mean; MPN, most probable number; PFU, plaque-forming unit

Levels of fecal indicator bacteria and MSBs in ascidians 4900, and 2400 MPN/100 g, respectively, and the geometric
and bivalves collected from commercial farms and wild mean concentrations were 43, 221, and 102 MPN/100 g, re-
beds spectively. The MSBs were not detected in any of the samples.
No significant differences were observed in the levels of the
The levels of fecal indicator bacteria and MSBs in the seawa- indicative bacteria between oysters and mussels. The levels of
ter, ascidians, oysters, and mussels collected from commercial the indicator bacteria in ascidians were consistently lower than
farms and wild beds are summarized in Table 2. those observed in bivalves. The levels of MSBs could not be
Levels of fecal indicator bacteria in the seawater and in the compared among filter feeders because the MSBs were not
three filter feeders were significantly different among the sam- detected in any of the tested samples.
ple collection sites. Marked differences in bacterial levels were Korea is the world’s fourth largest producer of bivalves,
also observed among the ascidians, oysters, and mussels at the contributing almost 2.8% of the global harvest (Pawiro
same sampling site. Maximum and geometric mean concentra- 2010). Some of the shellfish-growing areas meet the approved
tions of total coliforms in ascidians from the commercial farm area criteria of the National Shellfish Sanitation Program
were lowest among the three filter feeders. However, there Guide for the Control of Molluscan Shellfish (US FDA
were no significant differences in fecal coliforms and E. coli 2015) for which the median, or geometric mean, of fecal co-
levels among ascidians, oysters, and mussels because the levels liforms in seawater should not exceed 14 MPN/100 mL, and
of fecal indicator bacteria in most of the samples were below the estimated 90th percentile should not exceed 43 MPN/
the detection limit. In the samples from wild beds, the maxi- 100 mL. Other shellfish-growing areas located close to pollu-
mum and geometric mean concentrations of three categories of tion sources are affected by the land-based fecal contaminants
indicative bacteria in ascidians were lower than those in oysters during wet weather (Ha et al. 2011).
and mussels. The maximum concentrations of total coliforms Most bivalve and ascidian farms are located in shallow
in ascidians, oysters, and mussels from wild beds were 230, coastal waters, which may be affected by the land pollution

Table 2 Levels of fecal pollution indicator bacteria and male-specific bacteriophages (MSB) in ascidians (Halocynthia roretzi), oysters (Crassostrea
gigas), and mussels (Mytilus galloprovincialis) collected from commercial shellfish-growing farms and wild beds in 2010

Sampling sites Samples Total coliform Fecal coliform E. coli (MPN/ No. of samples MSB (PFU/ No. of
(MPN/100 mL or g) (MPN/100 mL or g) 100 mL or g) exceeded 230 100 mL or g) sample
E. coli MPN/100 g batches
Min Max GM Min Max GM Min Max GM Min Max GM

Commercial Seawater < 1.8 22 2.5 < 1.8 4.0 1.8 - - - - < 10 < 10 < 10 12
farm Ascidians < 18 45 19 < 18 20 < 18 < 20 20 < 20 0 < 17 < 17 < 17 10
Oysters < 18 220 27 < 18 45 19 < 20 50 21 0 < 17 < 17 < 17 10
Mussels < 18 490 27 < 18 20 < 18 < 20 < 20 < 20 0 < 17 < 17 < 17 12
Wild bed Seawater < 1.8 220 17 < 1.8 79 4.1 < 2.0 13 2.3 - < 10 < 10 < 10 12
Ascidians < 18 230 43 < 18 45 19 < 20 20 < 20 0 < 17 < 17 < 17 12
Oysters < 18 4900 221 < 18 950 49 < 20 640 28 1 < 17 < 17 < 17 12
Mussels < 18 2400 102 < 18 330 29 < 20 130 22 0 < 17 < 17 < 17 12

GM, geometric mean; MPN, most probable number; PFU, plaque forming unit; -, not tested
28272 Environ Sci Pollut Res (2017) 24:28268–28276

sources. Our data showed that bivalves concentrated fecal a

indicator bacteria to a slightly higher degree than ascidians
did. Unfortunately, levels of MSBs in ascidians could not be 104

E. coli (MPN/100 mL or g)
compared with those in bivalves. Nevertheless, ascidians pro-
duced from coastal waters were apparently much safer for raw 103
consumption than oysters harvested from the same areas. The
bioaccumulation and elimination characteristics of these filter
feeders for E. coli and MSBs were compared in relaying and 102
depuration experiments to investigate possible reasons for the
differences in the levels of indicator bacteria in filter feeders 101
from the same growing water.

Bioaccumulation and elimination of E. coli and MSBs 100

0 1 2 3 4 5 6
by filter feeders Exposed Day

Bioaccumulation of E. coli and MSBs in filter feeders that b

were relayed from designated shellfish-growing areas to an
104

MSB (PFU/100 mL or g)
area contaminated by sewage is shown in Fig. 1. E. coli was
selected as the only test indicator because this bacterium was
recommended as the best biological indicator for contamina- 103
tion of bivalve flesh by fecal pollutants for public health pro-
tection (Edberg et al. 2000). Levels of E. coli and MSBs in
102
relaying waters fluctuated during the experiment in the ranges
33–170 MPN/100 mL and 10–110 PFU/100 mL, respectively.
The concentrations of E. coli in the filter feeders reached max- 101
imum levels after 1 or 2 days of relaying and fluctuated within
920–4900 MPN/100 g in mussels, 630–1400 MPN/100 g in
oysters, and 140–490 MPN/100 g in ascidians. Mussels were 100
0 1 2 3 4 5 6
consistently contaminated with higher levels of E. coli than Exposed Day
were oysters collected at the same time, and concentrations of
Fig. 1 Bioaccumulation of Escherichia coli (A) and male-specific
the bacteria in ascidians were much lower than those in oys- bacteriophages (MSBs) (B) in ascidians (●), oysters (□) and mussels
ters. In contrast to the relatively rapid accumulation of E. coli (■) during relaying in a polluted coastal area. Concentrations of E. coli
in filter feeders (1–2 days), MSB concentrations reached the and MSBs in the surrounding water are represented as open circles (○).
highest levels in each filter feeder after 4 days of relaying. Environmental conditions during relaying were: water temperature, 9.2–
10.7 °C; salinity, 31.8–32.6
Figure 1 also shows that an equilibrium was developed be-
tween levels of microbes in water and ascidians and shellfish
within 4 days for oysters and mussels.
Patterns of MSB accumulation differed among the three
species. MSBs were accumulated faster and to higher levels MSBs and are presented in Fig. 1. The accumulation factors
in oysters than in mussels or ascidians. A high level of MSBs for E. coli were 3.3 for ascidians, 11.4 for oysters, and 23.6 for
accumulated in both bivalves but low levels were observed in mussels. Accumulation factors for MSBs were 0.7, 60.9, and
ascidians. The MSBs in bivalve meat reached ca. 103 PFU/ 11.2, respectively. E. coli were most efficiently accumulated
100 g within 2 days after relaying in oysters, and within 4 days in mussels and MSBs were most efficiently accumulated in
in mussels, and remained at 103 PFU/100 g. In contrast, the oysters. The lowest accumulation factors for both E. coli and
highest level of MSBs recorded in ascidians was only 120 MSBs were observed in ascidians, especially for MSBs where
PFU/100 g. Thus, ascidians showed much poorer ability to the concentration was lower than in the surrounding water.
accumulate E. coli and MSBs compared with both bivalves. Many studies have been carried out on the accumulation of
In most cases, the MSB concentration in the flesh of ascidians enteric bacteria and viruses in various bivalve species
was lower than that in seawater. (Bedford et al. 1978; Timoney and Abston 1984; Doré and
The accumulation levels of bacteria and MSBs varied Lees 1995; Burkhardt and Calci 2000). Although bacteria and
among the three species. Accumulation factors for bacteria viruses are usually accumulated in bivalve tissue to levels
and MSBs in the ascidians, oysters, and mussels were calcu- much higher than in the surrounding water, interspecific dif-
lated from the geometric mean concentrations of E. coli and ferences among filter feeders in their ability to accumulate
Environ Sci Pollut Res (2017) 24:28268–28276 28273

microorganisms have been reported, with accumulation fac- a

106
tors varying from a fewfold to more than a hundredfold
(Kershaw et al. 2013; Burkhardt and Calci 2000). These dif-

E. coli (MPN/100 mL or g)
ferences are probably associated with the morphology of the 105
gills in bivalves and the branchial basket in ascidians (Kryger
and Riisgard 1988; Doré and Lees 1995; Galbraith et al. 2009; 104
Petersen and Svane 2002). Other biological factors may be
involved; for example, norovirus binds specifically to oyster
digestive tissues (Le Guyader et al. 2006), and Burkhardt and 103
Calci (2000) showed that F-specific coliphage was selectively
accumulated up to 99-fold in oysters (C. virginica). A number 102
of studies have shown that mussels accumulate enteric bacte-
ria, such as E. coli, faster and to higher levels than in oysters
101
(C. gigas). The average E. coli accumulation ratios in mussels 0 6 12 24 48 72 96 120
(Mytilus spp.) were from 1.3 to 3.4 times greater than those in Time (h)
oysters (C. gigas) (Berry and Younger 2009; Younger and
Reese 2013; Kershaw et al. 2013). In the present study, the b
106
geometric mean accumulation factor of E. coli in mussels was
2.1 times higher than that in oyster, which is consistent with

MSB (PFU/100 mL or g)
105
previous reports. However, accumulation factors of E. coli and
MSBs in tissues of the ascidian H. roretzi were lower than
those in mussels or oysters. 104
To investigate the cause of the lower accumulation factors
in ascidians, change patterns of E. coli and MSBs in a mixture 103
of filtered seawater and sewage for the three filter feeders were
assessed (Fig. 2). Species and animal size, water temperature,
and the size and concentration of particles are all known to 102
affect filtration activity by filter feeders (Vaughn and
Hakenkamp 2001; Petersen 2007). Thus, in the present study, 101
change patterns of E. coli and MSBs in ascidians and bivalves 0 6 12 24 48 72 96 120
were compared under the same environmental conditions. Time (h)
Species-specific differences were observed in decrease rates Fig. 2 Change patterns of Escherichia coli (a) and male-specific
of E. coli and MSBs. The level of E. coli in the water holding bacteriophages (MSBs) (b) in a mixture of filtered seawater and sewage
for ascidians (●), oysters (□), and mussels (■). Concentrations of E. coli
the ascidians declined more rapidly than that in the water and MSBs in control seawater are represented as open circles (○).
holding oysters and mussels. In contrast, MSBs were most Approximately 400 g net meat weight of filter feeders was placed in
slowly removed from the water holding ascidians. Although 30 L of seawater. Environmental conditions during examination were:
E. coli was rapidly removed from the water by the ascidians, water temperature, 8.8–10.5 °C; salinity, 32.1–32.7
the bacterium was accumulated to a lower concentration in
ascidian tissues than in bivalves (Fig. 1). However, MSBs in
ascidians in the surrounding water decreased slower than contaminated filter feeders are shown in Fig. 3. Different mi-
those in bivalves (Fig. 2) and accumulation of MSBs in ascid- crobial reduction patterns were observed between ascidians
ians was also lower than that in bivalves (Fig. 1). Bacteria are and bivalves. In the ascidians, the levels of microorganisms
an important food source for bivalves and tunicates. Stuart and declined quickly from high starting concentrations of E. coli
Klumpp (1984) observed that ascidians retained all particles (2200 MPN/100 g) and MSBs (310 PFU/100 g) to undetect-
larger than 0.6 μm with ca. 100% efficiency but the retention able levels (< 20 MPN/100 g for E. coli, < 17 PFU/100 g for
efficiency of the same-sized particles was only 20% in bi- MSB) within 1 day of depuration. However, E. coli levels in
valves. Thus, it was suggested that the bacteria were more bivalves, oysters, and mussels decreased by only 1.1 logs and
easily digested or inactivated by ascidians than by bivalves. 1.6 logs, respectively, and the elimination of MSBs was neg-
It was unclear whether MSBs were passed through the bran- ligible after 1 day of depuration. Bivalves were of class C
chial basket of ascidians or were not inactivated in hepatopan- (4600 to 46,000 E. coli MPN/100 g flesh) according to micro-
creas after uptake by animals. biological standards of EU legislation (EC 2005) before
Other experiments on the elimination of E. coli and MSBs depuration and improved to better than class Al (< 230
during depuration in a clean seawater tank from the E. coli MPN/100 g flesh) for raw consumption within 2 days
28274 Environ Sci Pollut Res (2017) 24:28268–28276

a economic reasons. Unlike bivalves, there have been few re-

Contamination in polluted Depuration
environment for 5 days for 3 days ports on the kinetics of accumulation and elimination of bac-
105
E. coli (MPN/100 mL or g)

teria and viruses by ascidians. In this study, we compared

microbiological quality and bioaccumulation and elimination
104
properties between ascidians and bivalves. We conclude that
103
the depuration process is an effective way to reduce the health
risk of contaminated ascidians but, similar to bivalves, ascid-
102
ians can be a vector of human disease when they are grown in
polluted coastal areas for raw consumption.
101

100
0 1 2 3 4 5 6 7 8 Conclusions
Day
In the present study, microbiological quality was evaluated on
b Contamination in polluted Depuration the basis of bacterial fecal contamination indicators and MSBs
environment for 5 days for 3 days in live ascidians collected from the growing areas and the
105
MSB (PFU/100 mL or g)

retail markets. In addition, the accumulation and elimination

104
characteristics of E. coli and MSBs by ascidian and bivalves
were compared during bioaccumulation by relaying them at a
103
polluted area and depuration in seawater tanks. The E. coli
concentrations of ascidians purchased from three retail mar-
102 kets ranged from < 20 MPN/100 g to 460 MPN/100 g, meet-
ing the European standard for live and raw seafood, with ex-
101 ception of one sample, and MSBs were not detected. No sig-
nificant differences were observed in the indicator levels for
100 bacteria in oysters and mussels; the indicator levels in ascid-
0 1 2 3 4 5 6 7 8 ians were consistently lower than those in bivalves collected
Day from commercial farms or wild beds.
Fig. 3 Bioaccumulation during relaying in a polluted area and E. coli was quickly accumulated in the filter feeders but
depuration in a clean seawater tank of Escherichia coli (a) and male-
MSB accumulation was delayed during relaying in a polluted
specific bacteriophages (b) by ascidians (●), oysters (□), and mussels
(■). Concentrations of E. coli and MSBs in the surrounding water coastal area. Mussels were consistently contaminated with
during contamination are represented as open circles (○). higher levels of E. coli than were oysters collected at the same
Environmental conditions of the relaying water: temperature, 10.5– time, and the bacterial levels in ascidians were even lower than
12.4 °C; salinity, 30.8–32.2. Environmental conditions of the
those in oysters. High levels of MSBs were accumulated in
depuration water: temperature, 11.2–12.8 °C; salinity, 32.3–33.4
bivalves but low levels of MSBs were found in ascidians.
These observations suggest that ability of ascidians to accumu-
late E. coli and MSBs is lower than that of mussels and oysters.
of treatment. In contrast, the MSB concentrations in bivalves Species-specific differences were also observed in the elim-
dropped by only 0.14–0.31 logs in the same period. ination rates of E. coli and MSBs by these filter feeders during
Several studies have revealed that viruses are retained by depuration in a clean seawater tank. The levels of microorgan-
bivalves for significantly longer periods than indicator bacte- isms in ascidians decreased quickly from starting concentra-
ria such as E. coli and fecal coliforms (Power and Collins tions of 2200 MPN/100 g of E. coli and 310 PFU/100 g of
1989; Doré and Lees 1995; Burkhardt and Calci 2000; MSBs to undetectable levels (< 20 MPN/100 g for E. coli, <
Oliveira et al. 2011). In particular, human norovirus has been 17 PFU/100 g for MSB) within 1 day of depuration. However,
shown to persist longer than feline calicivirus or poliovirus in E. coli levels in oysters and mussels decreased by only 1.1
oysters under depuration (Ueki et al. 2007; Mcleod et al. logs and 1.6 logs, respectively, and elimination of MSBs was
2009). Thus, although depuration is an efficient purification negligible after 1 day of depuration. Therefore, we conclude
procedure for bacterial contaminants in bivalves, the process that depuration is an effective means of reducing the health
has a limited effect on reducing viral levels. Our observations risk of contaminated ascidians.
are consistent with these earlier studies. Many studies on ac-
cumulation and elimination of enteric bacteria and bacterio- Funding information This work was supported by a grant from the
National Institute of Fisheries Science in Korea (R2017057).
phages for disease prevention have focused on bivalves for
Environ Sci Pollut Res (2017) 24:28268–28276 28275

References Iwamoto M, Ayers T, Mahon BE, Swerdlow DL (2010) Epidemiology of

seafood-associated infections in the United States. Clin Microbiol
Rev 23(2):399–411. https://doi.org/10.1128/CMR.00059-09
APHA (American Public Health Association) (1970) Recommended pro- Kershaw S, Campos CJA, Reese A, Mitchard N, Kay D, Wyer M
cedures for the examination of seawater and shellfish. American (2013) Impact of chronic microbial pollution on shellfish.
Public Health Association, Washington DC Cefas, Lowestoft, UK
Bedford AJ, Williams G, Bellamy AR (1978) Virus accumulation by the KMFDS (Korea Ministry of Food and Drug Safety) (2016) Korea food
rock oyster Crassostrea glomerata. Appl Environ Microbiol 35(6): code http://fse.foodnara.go.kr/residue/RS/jsp/menu_02_01_01.jsp.
1012–1018 Accessed 22 June 2016
Berry R, Younger A (2009) Interspecies comparison of E. coli accumu- Kryger J, Riisgard HU (1988) Filtration rate capacities in 6 species of
lation in bivalve shellfish using data obtained from official control European freshwater bivalves. Oecologia 77(1):34–38. https://doi.
monitoring under EU Regulation 854/2004. Presented at the 7th org/10.1007/BF00380921
International Conference on Molluscan Shellfish Safety (ICMSS09 Le Guyader FS, Loisy F, Atmar RL, Hutson AM, Estes MK, Ruvoën-
Nantes, France Clouet N, Pommepuy M, Le Pendu J (2006) Norwalk virus-specific
Bone Q, Carré C, Chang P (2003) Tunicate feeding filters. J Mar Biol Ass binding to oyster digestive tissues. Emerg Infect Dis 12(6):931–936.
U K 83:907–919 https://doi.org/10.3201/eid1206.051519
Burkhardt W, Calci KR (2000) Selective accumulation may account for Lees D, Younger A, Doré B (2010) Depuration and relaying. In: Rees G,
shellfish-associated viral illness. Appl Environ Microbiol 66(4): Pond K, Kay D, Bartram J, Santo Domingo J (eds) Safe manage-
1375–1378. https://doi.org/10.1128/AEM.66.4.1375-1378.2000 ment of shellfish and harvest waters. World Health Organization,
Burkhardt W, Rippey SR, Watkins WD (1992a) Depuration rates of IWA Publishing, London, pp 145–181
northern quahogs Mercenaria mercenaria and eastern oysters Mcleod C, Hay B, Grant C, Greening G, Day D (2009) Inactivation and
Crassostrea virginica in ozone- and ultraviolet light-disinfected sea- elimination of human enteric viruses by Pacific oysters. J Appl
water systems. J Shellfish Res 11:105–109 Microbiol 107(6):1809–1818. https://doi.org/10.1111/j.1365-2672.
Burkhardt W, Watkins WD, Rippey SR (1992b) Survival and replication 2009.04373.x
of male-specific bacteriophages in molluscan shellfish. Appl Mizuta S, Isobe S, Yoshinaka R (2002) Existence of two molecular spe-
Environ Microbiol 58(4):1371–1373 cies of collagen in the muscle layer of the ascidian (Halocynthia
Cabelli VJ, Heffernan WP (1970) Elimination of bacteria by the soft shell roretzi). Food Chem 79(1):9–13. https://doi.org/10.1016/S0308-
clam Mya arenaria. J Fish Res Board Canada 27(9):1579–1587. 8146(02)00167-X
https://doi.org/10.1139/f70-179 Mok JS, Lee KJ, Kim PH, Lee TS, Lee HJ, Jung YJ, Kim JH (2016a)
Bacteriological quality evaluation of seawater and oysters from the
DePaola A, Jones JL, Woods J, Burkhardt W, Calci KR, Krantz JA,
Jaranman-Saryangdo area, a designated shellfish growing area in
Bowers JC, Kasturi K, Byars RH, Jacobs E, Williams-Hill D,
Korea: impact of inland pollution sources. Mar Pollut Bull 108(1–
Nabe K (2010) Bacterial and viral pathogens in live oysters: 2007
2):147–154. https://doi.org/10.1016/j.marpolbul.2016.04.036
United States market survey. Appl Environ Microbiol 76(9):2754–
Mok JS, Lee TS, Kim PH, Lee HJ, Ha KS, Shim KB, Lee KJ, Jung YJ,
2768. https://doi.org/10.1128/AEM.02590-09
Kim JH (2016b) Bacteriological quality evaluation of seawater and
Doré WJ, Lees DN (1995) Behavior of Escherichia coli and male-specific
oysters from the Hansan-Geojeman area in Korea, 2011–2013: im-
bacteriophage in environmentally contaminated bivalve molluscs be-
pact of inland pollution sources. SpringerPlus 5(1):1412. https://doi.
fore and after depuration. Appl Environ Microbiol 61(8):2830–2834
org/10.1186/s40064-016-3049-9
Doré WJ, Mackie M, Lees DN (2003) Levels of male-specific RNA NZFSA (New Zealand Food Safety Authority) (2006) Animal products
bacteriophage and Escherichia coli in molluscan bivalve shellfish (Specifications for bivalve molluscan shellfish). http://www.
from commercial harvesting areas. Lett Appl Microbiol 36(2):92– foodsafety.govt.nz/elibrary/industry/Animal_Products-Applies_
96. https://doi.org/10.1046/j.1472-765X.2003.01268.x Anyone.pdf. Accessed 22 June 2016
EC (European Commission) (2005) Commission Regulation (EC) No. Oliveira J, Cunha A, Castilho F, Romalde JL, Pereira MJ (2011)
2073/2005 of 15 November 2005 on microbiological criteria for Microbial contamination and purification of bivalve shellfish: cru-
foodstuffs. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do? cial aspects in monitoring and future perspectives—a mini-review.
uri=CONSLEG:2005R2073:20071227:EN:PDF. Accessed 22 Food Control 22(6):805–816. https://doi.org/10.1016/j.foodcont.
June 2016 2010.11.032
Edberg SC, Rice EW, Karlin RJ, Allen MJ (2000) Escherichia coli: the Pawiro S (2010) Bivalves: global production and trade trends. In: Rees G,
best biological drinking water indicator for public health protection. Pond K, Kay D, Bartram J, Santo Domingo J (eds) Safe manage-
J Appl Microbiol 88(S1):106S–116S. https://doi.org/10.1111/j. ment of shellfish and harvest waters. World Health Organization,
1365-2672.2000.tb05338.x IWA Publishing, London, pp 11–19
Galbraith HS, Frazier SE, Allison B, Vaughn CC (2009) Comparison of Petersen JK (2007) Ascidian suspension feeding. J Exp Mar Biol Ecol
gill surface morphology across a guild of suspension-feeding 342(1):127–137. https://doi.org/10.1016/j.jembe.2006.10.023
unionid bivalves. J Molluscan Stud 75(2):103–107. https://doi.org/ Petersen JK, Svane I (2002) Filtration rate in seven Scandinavian ascid-
10.1093/mollus/eyn045 ians: implications of the morphology of the gill sac. Mar Biol 140:
Ha KS, Yoo HD, Shim KB, Kim JH, Lee TS, Kim PH, Ju JY, Lee HJ 397–402
(2011) Evaluation of the influence of inland pollution sources on Potasman I, Paz A, Odeh M (2002) Infectious outbreaks associated with
shellfish growing areas after rainfall events in Geoje Bay, Korea. bivalve shellfish consumption: a worldwide perspective. Clin Infect
Korean J Fish Aquat Sci 44:612–621 Dis 35(8):921–928. https://doi.org/10.1086/342330
ISO (International Organization for Standardization) (2015) ISO 16649- Power UF, Collins JK (1989) Differential depuration of poliovirus,
3:2015, Microbiology of the food chain—horizontal method for the Escherichia coli, and a coliphage by the common mussel, Mytilus
enumeration of beta-glucuronidase-positive Escherichia coli—part edulis. Appl Environ Microbiol 55(6):1386–1390
3: detection and most probable number technique using 5-bromo-4- Randløv A, Riisgård HU (1979) Efficiency of particle retention and fil-
chloro-3-indolyl-beta-D-glucuronide. International Organization for tration rate in four species of ascidians. Mar Ecol Prog Ser 1:55–59.
Standardization, Geneva https://doi.org/10.3354/meps001055
28276 Environ Sci Pollut Res (2017) 24:28268–28276

Ribes M, Coma R, Gili JM (1998) Seasonal variation of in situ U.S. FDA (Food and Drug Administration) (2015) National Shellfish
feeding rates by the temperate ascidian Halocynthia papillosa. Sanitation Program (NSSP), NSSP Guide for the Control of
Mar Ecol Prog Ser 175:201–213. https://doi.org/10.3354/ M o l l u s c a n S h e l l f i s h . h t t p : / / w w w. f d a . g o v / F o o d /
meps175201 GuidanceRegulation/FederalStateFoodPrograms/ucm2006754.htm.
Riisgård HU, Larsen PS (2010) Particle capture mechanisms in Accessed 20 April 2017
suspension-feeding invertebrates. Mar Ecol Prog Ser 418:255– Ueki Y, Shoji M, Suto A, Tanabe T, Okimura Y, Kikuchi Y, Saito N, Sano
293. https://doi.org/10.3354/meps08755 D, Omura T (2007) Persistence of caliciviruses in artificially con-
Rippey SR (1994) Infectious diseases associated with molluscan shellfish taminated oysters during depuration. Appl Environ Microbiol
consumption. Clin Microbiol Rev 7(4):419–425. https://doi.org/10. 73(17):5698–5701. https://doi.org/10.1128/AEM.00290-07
1128/CMR.7.4.419 Vaughn CC, Hakenkamp CC (2001) The functional role of burrowing
Sair AI, D'Souza DH, Jaykus LA (2002) Human enteric viruses as causes bivalves in freshwater ecosystems. Freshw Biol 46(11):1431–1446.
of foodborne disease. Compr Rev Food Sci Food Saf 1:72–89 https://doi.org/10.1046/j.1365-2427.2001.00771.x
Stuart V, Klumpp DW (1984) Evidence for food-resource partitioning by Yang JH, Mok JS, Jung YJ, Lee KJ, Kwon JY, Park K, Moon SY, Kwon
kelp-bed filter feeders. Mar Ecol Prog Ser 16:27–37. https://doi.org/ SJ, Ryu AR, Lee TS (2017) Distribution and antimicrobial suscep-
10.3354/meps016027 tibility of Vibrio species associated with zooplankton in coastal area
Timoney JF, Abston A (1984) Accumulation and elimination of of Korea. Mar Pollut Bull. https://doi.org/10.1016/j.marpolbul.
Escherichia coli and Salmonella typhimurium by hard clams in an 2017.07.054
in vitro system. Appl Environ Microbiol 47(5):986–988 Younger AD, Reese RA (2013) Comparison of Escherichia coli levels
U.S. EPA (Environmental Protection Agency) (2001) Method 1601: between bivalve mollusc species across harvesting sites in England
Male-specific (F+) and Somatic Coliphage in Water by Two- and Wales. J Shellfish Res 32(2):527–532. https://doi.org/10.2983/
step Enrichment Procedure. EPA 821-R-01-030, US EPA, 035.032.0232
Washington DC