Helgol Mar Res (2003) 57:181–189

DOI 10.1007/s10152-003-0143-5

ORIGINAL ARTICLE

Andreas Skouras · Thomas Lang · Michael Vobach ·

Dirk Danischewski · Werner Wosniok ·
J
rn Peter Scharsack · Dieter Steinhagen

Assessment of some innate immune responses

in dab (Limanda limanda L.) from the North Sea
as part of an integrated biological effects monitoring
Received: 14 September 2002 / Revised: 3 February 2003 / Accepted: 24 February 2003 / Published online: 8 April 2003

Springer-Verlag and AWI 2003

Abstract The marine flatfish dab (Limanda limanda), information about immunomodulatory effects associated
which lives in direct contact with contaminated sedi- with the exposure of fish to contaminants. In particular,
ments, is frequently used as a sentinel species in concentrations of plasma lysozyme, which can be anal-
international monitoring programmes on the biological ysed in an easy and inexpensive assay, are considered to
effects of contaminants. In this study, immune responses be an appropriate parameter for use in a battery of other
were recorded as indicators of sublethal chronic effects of bioindicators.
contaminants, in addition to measurement of the induc-
tion of mono-oxygenase ethoxyresorufin O-deethylase Keywords Biological effect monitoring · Fish diseases ·
(EROD) in liver cells, the inhibition of acetylcholin Innate immune response · North Sea · Pollution
esterase (AChE) in muscle and a quantification of grossly
visible diseases and parasites. In total, 336 dab were
analysed from five sampling areas in the North Sea, Introduction
including the German Bight, the Dogger Bank, the Firth
of Forth, and two locations close to oil and gas platforms The aquatic environment is being abused throughout the
(Ekofisk and Danfield). When considering plasma lyso- world by the introduction of a large number of xenobiotic
zyme levels, pinocytosis and respiratory burst activity of compounds derived from human activities in industry and
head kidney leucocytes, a clear gradient could be agriculture. Many of these substances have the potential
observed with decreased levels in individuals collected to impact on the ecosystem at relatively low concentra-
from the Firth of Forth and locations near the oil or gas tions (Connell et al. 1999). In order to assess the risk for
platforms compared with dab from the Dogger Bank or organisms posed by contaminant exposure and to classify
the German Bight. Individuals with induced EROD the environmental health of an ecosystem under chal-
activity displayed reduced lysozyme and respiratory burst lenge, various monitoring techniques have been used
activities. Lysozyme levels were also reduced in dab with (Van der Oost et al. 1997). These biomarkers are
lymphocystis or with nematodes. The data obtained biochemical, physiological or histopathological indicators
indicate that the assessment of innate immune parameters of either exposure to or early effects of anthropogenic
in a monitoring programme provides supplementary substances, and they become apparent at exposures to
concentrations less than those causing acute toxic effects.
Communicated by H. von Westernhagen and A. Diamant In various animals, the immune system appears to be
A. Skouras · J. P. Scharsack · D. Steinhagen ())
particularly sensitive to toxic effects of chemicals of
Fish Disease Research Unit, School of Veterinary Medicine, environmental concern. In the mammalian system, a
Bnteweg 17, 30559 Hannover, Germany battery of well-characterised immune assays to test for
e-mail: [email protected] functional or histopathological parameters is available
(Luster and Rosenthal 1993), and many of the same
T. Lang · M. Vobach · D. Danischewski
endpoints have been used in laboratory studies to
Federal Research Centre for Fishery,
Institute for Fishery Ecology, demonstrate chemically-induced immunotoxicity in fish
Deichstrasse 12, 27472 Cuxhaven, Germany (see recent reviews by Zelikoff et al. 2000; Bols et al.
2001).
W. Wosniok For field studies in “real-world” contaminated aquatic
Institute for Statistics, University of Bremen, environments, international panels such as the Interna-
PO Box 330440, 28334 Bremen, Germany
182

tional Council for the Exploration of the Sea (ICES)

recommended monitoring of the biological effects of
contaminants by means of, amongst other techniques,
biochemical parameters, such as the induction of mono-
oxygenase ethoxyresorufin O-deethylase (EROD) in liver
cells or the inhibition of acetylcholin esterase (AChE) in
muscle in addition to easily visible fish diseases and
parasites (see Stebbing and Dethlefsen 1992; Diamant and
Westernhagen 1999). As sentinel species, marine flatfish
are frequently used in international monitoring pro-
grammes. In the North Sea and the Baltic Sea, these are
mainly dab (Limanda limanda) and European flounder
(Platichthys flesus) (Secombes et al. 1997; Broeg et al.
1999; Lang and Mellergard 1999; Lang et al. 1999;
Grinwis et al. 2000; Lang 2002). For immune function
assessment, studies reveal that contaminants modulate
immune parameters in fish (Arkoosh et al. 1994; Fig. 1 Location of sampling sites for dab (Limanda limanda) in the
Secombes et al. 1995), but integrated studies, for instance North Sea. Danfield, oil and gas platforms (P01; 5520–56000 N,
correlating immune functions to measurements of bio- 04200 –05300 E); Ekofisk, oil platforms (P02; 56100 –56500 N,
chemical biomarkers, are scarce. 02400 –03500 E), Dogger Bank (N04, 54250 –54500 N, 02000 –
02310 E); Firth of Forth (N06; 56150 –56250 N, 01500 –02100
In the present study, innate immune functions were W); German Bight (JMP; 54150 –54300 N, 06580 –08270 E)
assessed in dab collected at various locations in the North
Sea along a contaminant gradient. From the same
individuals, biochemical biomarkers and grossly visible and frozen at 20C. The fish were then inspected for obvious signs
disease symptoms were recorded according to published of diseases and parasites using standardised methodologies as
described by Bucke et al. (1996). Briefly, the presence or absence
recommendations (see Stebbing and Dethlefsen 1992) and of the following diseases and parasites was registered: lymphocys-
the implications of the different measurements were tis, epidermal hyperplasia/papilloma, acute or healing skin ulcers,
compared. pigment anomalies, infections with nematodes and acanthocepha-
lans on the liver surface. The otoliths of individual fish were taken
for subsequent age determination.
The fish were then dissected and the head kidney was removed
Methods and transferred into a centrifugation tube filled with wash medium
(RPMI medium supplemented with 10,000 IU l1 sodium heparin,
Sampling medium; Biochrom, Berlin, Germany: heparin; Sigma, St. Louis,
Mo., USA) and stored at 4C for up to 24 h for further processing.
Sampling was carried out on board RV “Walther Herwig III” In addition, liver and muscle samples were collected from the
during cruises conducted in August/September 1999, 2000 and same individuals for biochemical analysis.
2001. Fishing was carried out by means of bottom trawling with
standard gear and methods (GOV or 140 ft bottom trawl, towing
time 1 h, towing speed 3–4 knots) at five different locations in the Biochemical parameters (EROD, CYP, ACHE, GST)
North Sea. The location of the sampling sites is shown in Fig. 1. In
the North Sea, regions at P01 (Danfield) and P02 (Ekofisk) are The following biochemical parameters were measured in dab in the
characterised by oil and gas platforms, station N06 is located in the framework of a routine biological effects monitoring conducted by
Firth of Forth, N04 at the Dogger Bank and JMP near Helgoland in the German Federal Research Centre for Fishery: 7-ethoxyre-
the German Bight. sorufin-O-deethylase (EROD) activity, total protein and levels of
Fish were sorted from the catches immediately and kept alive in cytochrome P450 1A (CYP1A) protein were measured in dab liver
tanks with permanent seawater flow-through and aeration. Further according to standard methods [for EROD see Burke and Mayer
processing took place within 1 h. In total, 336 female dab (Limanda (1974), protein content according to Lowry et al. (1951) and CYP
limanda L.) of the size class 20–24 cm were used for this 1A protein according to Goksoyr and Husoy (1992)]. From muscle
investigation. A maximum of 20 fish per site and campaign were tissue, cholinesterase activity (AChE) was measured colorimetri-
collected and processed. cally according to Ellmann et al. (1961). Glutathion-4-S-transferase
Sediment samples were taken only during the 1999 cruise at the (GST) activity in liver was determined with the method described
six different sites and analysed for organic contaminants. Methods by Bressler et al. (1999). The total protein content of liver samples
and results of this analysis are described in detail by Kammann et was measured according to Bradford et al. (1976).
al. (2001).
Leucocyte isolation
Examination procedure
Media and cells were kept on ice and washing procedures were
On board the research vessel, body length and weight were performed at 4C. Cell suspensions of head kidney leucocytes
measured for each fish. Blood was drawn from the caudal vein into (HKL) were prepared by forcing the tissues through a 100 m nylon
disposable syringes pre-filled with a lithium–heparin bead (Sar- screen (Swiss Silk Bolting Cloth Mfg, Zurich, Switzerland).
stedt, Germany). The haematocrit was determined from the blood Isolated HKL were washed three times with wash medium
according to standard procedures (Houston 1990). The remaining (10 min, 550 g) and resuspended in cell culture medium (RPMI-
blood was then transferred to centrifugation tubes, centrifuged at 1640 supplemented with 100,000 IU l1 penicillin, 100 mg l1
2,000 g for 15 min at 4C, and the supernatant plasma was collected streptomycin, 4 mM l-glutamine and 1% (v/v) carp serum
 183
(chemicals; Biochrom, Berlin, Germany: carp serum; serum from P<0.05. All calculations were done using the computer program
15 individual Cyprinus carpio L. was pooled, heat-inactivated for Sigma Stat (SPSS Science).
30 min at 56C, then 0.2 m was filtered and stored at 20C until Initially, the data were analysed for single campaigns separate-
use). Numbers of viable cells were determined by Trypan Blue ly. Because similar spatial differences were observed for all three
exclusion in a Neubauer haemocytometer. campaigns, the measurements were combined and analysed as a
pooled data set.

Production of reactive oxygen species by head kidney leucocytes

Generation of reactive oxygen species (ROS) by head kidney Results

leucocytes was measured by means of the Nitro Blue tetrazolium
salt (NBT) reduction assay. Cell suspensions were incubated in 96- Haematocrit
well flat-bottom microtitre plates (106 cells in a final volume of
175 l of cell culture medium) in triplicate and their ROS
production was induced by adding 0.15 mg l1 phorbol myristate The haematocrit value of fish is considered to represent a
acetate (PMA). The indicator NBT was added at 1 g l1. Wells simple, non-specific indicator of overall health (Blaxhall
without PMA served to determine the basal ROS generation of the 1972; Anderson 1990). Decreased haematocrit values
cells. After incubation for 2 h at 18C, the supernatants were were found in fish with nutritional deficiencies, infections
removed and the cells were fixed by adding 125 l of 100% with microorganisms or other health problems (Blaxhall
methanol. Each well was washed twice with 125 l of 70% (v/v)
methanol. Methanol was removed and the fixed cells were air-dried 1972). In the dab examined here, regional differences
overnight and stored in the dark for up to 2 weeks. The reduced (P<0.05) were found between individuals from the North
NBT (formazan) was dissolved in 125 l 2M KOH and 150 l Sea locations: individuals from the German Bight (JMP)
DMSO per well (all chemicals from Sigma-Aldrich, Germany). The had higher haematocrit values than dab from the areas
optical densities were recorded with a spectrophotometer at
650 nm. Ekofisk (P02), Dogger Bank (N04) and Firth of Forth
(N06) (P<0.05), and individuals from Danfield (P01) had
higher haematocrits than those from Dogger Bank (N04)
Endocytosis activity of head kidney phagocytes and Firth of Forth (N06) (P<0.05, see Fig. 2). In both
Endocytosis activity of HKL was measured by means of Neutral populations, length and age of the fish did not affect the
Red retention, as described by Matthews et al. (1990). This assay haematocrit value, while a small, but significant (R=0.29;
was adapted to microtitre plates. Briefly, 106 cells were incubated P<0.05) influence of weight was noted.
in a final volume of 175 l culture medium for 2.5 h at 18C with
10 mg l1 Neutral Red (NR; Sigma-Aldrich). All set-ups were made
at least in triplicate. After incubation, each well was washed twice
with 125 l of phosphate-buffered saline (PBS). After removing the Lysozyme
PBS, the cells were air-dried overnight and frozen at 20C for up
to 2 weeks. For spectrophotometric readings, the cells were lysed The mean lysozyme activity in the plasma of dab
with 100 l acid ethanol (3% HCl in 95% ethanol) and mixed with examined here was not affected by weight, length or
100 l PBS. The optical densities were recorded at 492 nm.

Lysozyme assay

Lysozyme activity of dab plasma was determined by means of a

turbidimetric assay according to Parry et al. (1965). A suspension of
0.2 g l1 Micrococcus lysodeikticus (Sigma-Aldrich) in 0.05 M
sodium phosphate buffer (pH 6.2) was mixed with 25 l of dab
plasma to give a final volume of 200 l per well. The optical density
was read in a spectrophotometer at 530 nm immediately after
mixing, after 0.5 min, and after 4.5 min at a temperature of 20€2C.
The decrease in absorbance was used to calculate the lysozyme
activity. One unit of lysozyme activity is defined as the amount of
sample causing a decrease in absorbance of 0.001 OD min1. Hen
egg-white lysozyme (Sigma-Aldrich) was used as external standard,
as described by Hutchinson and Manning (1996a).

Statistics

Normality of the data was tested with the Kolmogorov-Smirnov

test. To determine the significance of differences between groups,
data were compared using Student’s t test, the Mann-Whitney rank
sum test for not normally distributed data sets, or by a Kruskal- Fig. 2 Haematocrit in dab Limanda limanda collected at five
Wallis ANOVA and subsequent multiple comparison of means different sampling sites in the North Sea during three consecutive
using the Student-Newman-Keuls method at a probability of error sampling campaigns in August/September 1999–2001. Sampling
P<0.05. Correlations between data sets were tested with Pearson’s locations: P01 Danfield, N04 Dogger bank, P02 Ekofisk, N06 Firth
product moment correlation test, or with the Spearman rank of Forth, JMP German Bight. For geographical locations see Fig. 1.
correlation test when the data were not normally distributed. In the North Sea, haematocrit was significantly higher in dab at P01
Correlations were considered significant at a probability of error compared with individuals at P02, N06 and N04 (P<0.05). Dab
from JMP also had significantly higher haematocrit values than fish
from P02, N06 and N04 (P<0.05)
184

Fig. 3 Plasma lysozyme activity in dab Limanda limanda collected Fig. 4 Basal production of reactive oxygen species (ROS) by head
at five different sites in the North Sea during three consecutive kidney cells derived from dab (Limanda limanda) at five different
sampling campaigns in August/September 1999 –2001. Sampling locations in the North Sea. Sampling locations: Sampling locations:
locations: P01 Danfield, N04 Dogger bank, P02 Ekofisk, N06 Firth P01 Danfield, N04 Dogger bank, P02 Ekofisk, N06 Firth of Forth,
of Forth, JMP German Bight. For geographical locations see Fig. 1. JMP German Bight. For geographical locations see Fig. 1. Dab
Individuals at JMP had significantly higher lysozyme levels than from N04 had a significantly higher ROS production than
dab at P01, P02 and N06 (P<0.05). At N04, individuals had individuals from P01, P02 and N06 (P<0.05). Head kidney
significantly higher lysozyme values than dab collected from P01 leucocytes from JMP dab also showed a significantly higher ROS
and N06 (P<0.05) production than cells from individuals at P01 (P<0.05)

age of the individuals. Significant differences (P<0.05) in HKL was significantly (R=0.20, P<0.01) influenced by
activity, however, were found between dab from oil and fish weight.
gas platform areas (P01 and P02) and dab collected in the Clear differences in ROS production were observed
German Bight (JMP; see Fig. 3). Dab sampled at the between dab from different areas in the North Sea. HKL
Dogger Bank (N04) had significantly (P<0.05) higher from dab at the Dogger Bank (N04) had significantly
plasma lysozyme levels than dab from Danfield (P01) and higher (P<0.05) basal ROS production compared with
Firth of Forth (N06) (Fig. 3). individuals from the sites near gas or oil platforms (P01,
P02) and at the Firth of Forth (N06). Cells obtained from
dab collected near platforms at Danfield (P01) also had
Endocytosis by head kidney phagocytes significantly (P<0.05) lower basal levels of ROS produc-
tion than dab from the German Bight (JMP) (see Fig. 4).
The endocytosis activity of head kidney phagocytes was When HKL were stimulated with PMA, the cells
not affected by age or length of fish, but increased with responded with increased ROS production and showed
increasing weight of the individuals (P<0.05). Head slightly higher values in dab collected in the German
kidney phagocytes from dab collected in the German Bight (JMP) and at the Dogger Bank (N04) compared
Bight (JMP) showed a significantly higher endocytosis with fish from the oil and gas platforms. These differ-
activity than individuals from Ekofisk (P02) and Firth of ences, however, could not be confirmed as statistically
Forth (N06) (P<0.05). In addition, cells from dab significant (Fig. 5).
collected at N06 had a significantly higher endocytosis
activity than individuals from the Dogger Bank (N04) and
Danfield (P01) (P<0.05, data not shown). Correlation of innate immune responses
with physiological biomarkers
and grossly visible diseases
Production of reactive oxygen species
Measurements of innate immune responses and physio-
Both basal and stimulated ROS production of head- logical biomarkers were carried out on the same individ-
kidney-derived leucocytes were significantly influenced uals. When plasma lysozyme levels of individual North
by the age of fish (basal ROS production R=0.19, P<0.05; Sea dab were compared to endocytosis and ROS produc-
PMA-stimulated ROS production R=0.40, P<0.01). tion of HKL from the same individual, a positive
Weight or length had no effect on basal ROS production correlation was found for endocytosis and a negative
of HKL, while the PMA-stimulated ROS production of correlation for PMA-stimulated ROS production (see
Table 1). Dab with decreased haematocrits also had lower
plasma lysozyme levels (Table 1).
 185

The presence or absence of obviously visible diseases

or liver parasites had a marked impact on several of the
innate immune parameters measured, but the pattern
varied with the disease or parasitic infection (see Table 3).
Individuals with lymphocystis had reduced plasma lyso-
zyme levels with 892 (628–1,123) IU ml1 compared with
1,007 (797–1,337) IU ml1 in non-infected fish. In dab
with pigment anomalies, haematocrits were decreased, at
22 (19–24)% compared with 23 (20–27)% in non-affected
individuals, and basal as well as PMA-triggered ROS
production of HKL from affected dab was increased, with
0.156 (0.0985–0.387) OD values for basal and 1.085
(0.546–1.772) OD values for PMA-stimulated ROS
versus 0.105 (0.062–0.208) and 0.639 (0.375–1.169),
respectively, in unaffected individuals. In individuals with
skin ulcers or epidermal hyperplasia/papilloma, results of
measurements of innate immune responses were not
Fig. 5 Production of reactive oxygen species by head-kidney- different from those of unaffected dab.
derived dab (Limanda limanda) leucocytes upon stimulation with Infections with liver nematodes were accompanied by
the phorbol ester PMA. The dab were collected at five different
sampling sites in the North Sea during three consecutive sampling significantly reduced haematocrits (mean 22, range 19–
campaigns in August/September 1999–2001. Sampling locations: 24%, in infected and mean 23, range 21–27%, in
P01 Danfield, N04 Dogger bank, P02 Ekofisk, N06 Firth of Forth, uninfected dab), decreased plasma lysozyme levels (mean
JMP German Bight. For geographical locations see Fig. 1. 966, range 694–1,189 IU ml1 versus mean 991, range
Statistically significant differences were not found between the
sampling locations 797–1,369 IU ml1), reduced basal ROS production, but
increased endocytosis activity of HKL. Infections of dab
with liver acanthocephalans were accompanied by re-
When results of the measurements of the physiological duced endocytosis activity of HKL. Multiple regression
biomarkers of interest were compared with plasma analysis indicated that from the immune parameters
lysozyme levels, pinocytosis activity and ROS production considered here, haematocrits and endocytosis activity
of HKL of the same individual, the following correlations of HKL were mainly affected by nematode infections,
were found: dab with induced EROD activity had plasma lysozyme levels by lymphocystis, and ROS
decreased plasma lysozyme levels and decreased ROS production of HKL by pigment anomalies (Table 4).
production (P<0.01, Table 2) and individuals with
impaired ROS production of HKL also had lower AChE
and GST activities (P<0.01, Table 2). Individuals with Discussion
decreased plasma lysozyme levels had displayed induced
EROD and GST activities in liver cells (P<0.01, Table 2). Numerous studies have demonstrated that water contam-
Correlations between the haematocrit values of individual ination has an impact on innate as well as on adaptive
dab and responses of physiological biomarkers were not immune responses in fish (recently reviewed by Zelikoff
found. et al. 2000; Bols et al. 2001). This became very clear

Table 1 Cross-correlation be- Lysozyme Pinocytosis Basal ROS PMA activated ROS
tween immune parameters
measured in dab (Limanda li- Haematocrit 0.18** 0.12* 0.06 0.08
manda) collected in the North Lysozyme 1 0.24** 0.06 0.13*
Sea from five different loca- Pinocytosis 1 0.06 0.10
tions (see Fig. 1) Basal ROS 1 0.50**
Spearman’s correlation on ranks at *P<0.05, **P<0.01, N= 286–300

Table 2 Correlations between Biomarker Haematocrit Lysozyme Pinocytosis Basal ROS PMA activated ROS
biochemical parameters of dab
(Limanda limanda) and the im- EROD 0.00 0.26** 0.05 0.44** 0.29**
munological parameters ap- CYP 0.00 0.12 0.08 0.09 0.00
plied. The measurements were ACHE 0.10 0.03 0.06 0.35** 0.39**
done in the same individuals. GST 0.01 0.37** 0.16 0.23* 0.01
The dab were collected in
1999–2001 at five different lo- Spearman’s correlation on ranks, marked are correlations at *P<0.05, **P<0.01
cations in the North Sea (for Abbreviations: EROD 7-ethoxyresorufin-O-deethylase assay, CYP cytochrome P450 1A protein
locations see Fig. 1, N=84–200) concentration, ACHE cholinesterase activity, GST glutathion-4-S-transferase activity
186
Table 3 Comparison of the presence of grossly visible diseases and ments obtained from non-infected individuals by means of the
parasites in dab (Limanda limanda) and immunological parameters Mann-Whitney rank sum test. Listed are P values obtained from the
measured from the same individual. The dab were collected in test. Statistically significant differences in the immune response
1999 – 2001 at five different locations in the North Sea (see Fig. 1). between the groups at p<0.05 are marked in bold, n=286–300
Compared were immune parameters of affected dab to measure-
Disease Haematocrit Lysozyme Pinocytosis Basal ROS PMA activated ROS
Lymph 0.17 0.01() 0.18 0.47 0.32
Eppap 0.16 0.61 0.95 0.71 0.10
Ulc 0.17 0.73 0.47 0.19 0.36
Pigmel 0.01() 0.45 0.58 0.01(+) 0.01(+)
Nemato 0.01() 0.04() 0.01(+) 0.02() 0.69
Acanth 0.12 0.35 0.02() 0.17 0.64
(+) increased immune parameter in infected dab
() depressed immune parameter in infected individuals
Lymp lymphocystis, Eppap epidermal hyperplasia/papilloma, Ulc acute/healing skin ulceration, Pigmel anomaly in pigmentation, Nemato
nematodes (liver), Acanth acanthocephalans (liver)

Table 4 Multiple linear regres- Disease Haematocrit Lysozyme Pinocytosis ROS PMA activated ROS
sion between grossly visible
diseases and parasites in dab Lymph 0.42 0.04* 0.40 0.20 0.20
(Limanda limanda) and the im- Eppap 0.23 0.57 0.91 0.50 0.50
mune parameter measured in Ulc 0.14 0.72 0.72 0.74 0.74
the same individual. The pres- Pigmel 0.09 0.55 0.18 <0.01** <0.01*
ence of grossly visible diseases Nemato 0.04* 0.20 0.02* 0.14 0.84
or parasites is tested as ex- Acanth 0.86 0.60 0.12 0.72 0.88
plaining variable on the im-
mune responses applied. Given An influence was considered to be significant at *P<0.05, **P<0.001, n=286–300
are the P values obtained from Lymp lymphocystis, Eppap epidermal hyperplasia/papilloma, Ulc acute/healing skin ulceration, Pigmel
the calculation anomaly in pigmentation, Nemato nematodes (liver), Acanth acanthocephalans (liver)

when fish were exposed to various substances such as analysis was restricted to a defined size class of 20–24 cm
metals, pesticides or insecticides under laboratory condi- total length. These findings are consistent with results
tions, but could also be confirmed in studies on wild fish from field studies on flounder (Skouras et al. 2003),
collected from contaminated sites. Thus several authors Japanese medaka (Oryzias latipes, see Duffy et al. 2002)
(Dunier et al. 1991; Dunier and Siwicki 1993; Wester et and from mammals, which reveal a decreasing sensitivity
al. 1994; Zelikoff et al. 2000; Bols et al. 2001) to toxic insult with increasing age (Parkinson and Safe
recommended fish immune assays as useful techniques 1987). In the present study, only female dab were
for predicting toxicological risk associated with contam- collected during campaigns in August and September in
ination in aquatic environments. Innate immune responses order to reduce seasonal and sex-related variations, which
that protect an organism against infections without were described in detail by Hutchinson and Manning
depending on prior exposure to any particular pathogens (1996a). In addition, North Sea dab were collected at
are especially suitable biomarkers for assessing the locations with similar characteristics in respect of salinity,
adverse biological effects of contaminants (Wester et al. in order to reduce variations caused by this factor.
1994). When considering the North Sea sampling locations of
In wild fish, biological parameters such as enzyme the present study, a contamination gradient was discussed
activities or immune responses underlie natural fluctua- by Kammann et al. (2001) on the basis of PAH
tions and may be influenced by host-specific variation, contamination of the sediments. The highest contamina-
and when considering these responses as biomarkers of tion, expressed as S of 16 PAHs, was measured at Ekofisk
environmental degradation, contaminant-mediated effects (P02) with 35.87 ng g1 and the Firth of Forth (N06) with
have to be distinguished from these factors. Therefore, a 27.47 ng g1 dry matter, followed by Danfield (P01) with
sufficiently large number of individuals of comparable 13.78 ng g1. In the German Bight (JMP) (6.02 ng g1)
size should be collected, which is most desirable in a and at the Dogger Bank (N04) (5.79 ng g1) lower levels
long-term study (Anderson 1990). Thus in the present of PAHs were measured. Along with this pollution
study, 336 individuals were analysed during three sam- gradient, the plasma lysozyme level and the respiratory
pling campaigns in three consecutive years. When the burst activity of head kidney phagocytes was reduced in
data were analysed for the sampling campaigns separate- individuals from stations with higher contaminant levels
ly, general results were achieved (data not shown). (see Figs. 3, 4 and 5). In contrast, haematocrits and
Measurements of haematocrit, endocytosis, basal and endocytosis activity of HKL were not altered in dab from
PMA-stimulated ROS, however, were significantly influ- regions with increased PAH contamination. These find-
enced by body weight and age of dab, even though the ings substantiate observations from other studies in dab.
 187

In individuals caught after a major oil spill in the North the basis of programmes on the systematic monitoring of
Sea, serum lysozyme levels were negatively correlated the occurrence and prevalence of grossly visible diseases
with the PAH levels in the sediment (Secombes et al. in dab in the North Sea, which have been carried out by
1997). Dab exposed to oil-contaminated sediment or ICES member countries since the late 1970s (Dethlefsen
sewage sludge had lower serum lysozyme levels (Tahir et et al. 2000; Lang 2002). In the present study, the
al. 1993) and decreased ROS production by head kidney prevalence of grossly visible diseases was recorded along
phagocytes relative to control groups (Secombes et al. with an analysis of some innate immune responses.
1991; Tahir et al. 1993). In vivo exposure of dab to Individuals affected by lymphocystis, liver nematodes or
different concentrations of cadmium was also related to a acanthocephalans displayed altered plasma lysozyme or
reduction in the ROS production by head kidney phago- head kidney phagocyte activities compared with unaf-
cytes when compared with unexposed individuals (Hutch- fected dab. In parasite-infected individuals, lysozyme and
inson and Manning 1996b). respiratory burst activities were decreased, while in dab
The work reported here was part of an integrated field with lymphocystis, non-specific cellular responses ap-
study, which included a simultaneous assessment of other peared not to be affected. This is in contrast to observa-
biomarkers as recommended for monitoring programmes tions in American plaice, Hippoglossoides platessoides,
on biological effects (see Diamant and Westernhagen where head kidney cells displayed enhanced phagocytosis
1999), such as the induction of mono-oxygenase and respiratory burst activity in association with lympho-
ethoxyresorufin O-deethylase (EROD) or glutathion-4-S- cystis infection (Marcogliese et al. 2001). Other diseases,
transferase (GST) in liver cells, the inhibition of acetyl- such as epidermal hyperplasia/papilloma or skin ulcers,
cholin esterase (AChE) in muscle, and grossly visible fish were not observed to be associated with altered lysozyme
diseases and parasite infections. EROD is known to be a or head kidney phagocyte activity.
sensitive indicator of the exposure to lipophilic com- In conclusion, the data presented here indicate that
pounds such as PAHs, dioxins and coplanar PCB plasma lysozyme and head kidney phagocyte activities
congeners (Goksoyr and Frlin 1992; Boer et al. 1993; detected in North Sea dab display differences associated
Sleiderink et al. 1995). Cholinesterase (AChE) is widely with a sediment contamination gradient. Innate immune
used to estimate the neurotoxic impact of contaminants at responses were altered along with the physiological
the cellular level of marine organisms (Galgani et al. biomarkers GST or EROD and with the occurrence of
1992; Bressler et al. 1999) and the induction of fish diseases such as lymphocystis. The innate immune
glutathion-4-S-transferase (GST) activity indicates an parameters applied in the present study can easily be
adaptation of the organism to enhanced contaminant integrated into biological effects monitoring programmes
stress (Bressler et al. 1999). In the present study, and will provide supplementary information about im-
responses of the innate immune system and these munomodulatory effects associated with exposure to
biomarkers were recorded from the same individual dab, contaminants. In particular, plasma lysozyme, which can
which allowed us to compare responses of the different be analysed in an easy and inexpensive assay, is
parameters on the basis of individual fish. These considered as a suitable parameter in a battery of other
comparisons showed that dab with induced EROD or biomarkers.
GST activities also had lower lysozyme activity and
decreased phagocyte responses, which indicates that in Acknowledgements The authors thank Mrs. G. Piechotta and Mrs.
fish under contaminant stress, several functional systems Ursula Krschner for their skillful technical assistance. We also
thank the captain and the crew of RV “Walther Herwig III” for their
were affected. The observations made in dab confirm efforts during the sampling cruises and their excellent collabora-
findings in flounder from the German Bight (Skouras et tion. This study was funded in part by a BMBF grant in the MARS
al. 2003), where in individuals with decreased integrity of framework (BMBF code 03F0159A).
hepatocyte lysosomes, the EROD system was also
induced and innate immune responses were impaired. In
the present study on dab, correlation coefficients between References
biomarkers and immune parameters were much higher
than those found for flounder (Skouras et al. 2003), most Anderson DP (1990) Immunological indicators: effects of environ-
mental stress on immune protection and disease outbreak. Am
probably because a more pronounced contamination Fish Soc Symp 8:38–50
gradient existed between the sampling locations of the Arkoosh MR, Stein JE, Casillas E (1994) Immunotoxicology of an
present study compared with the locations in the German anadromous fish: field and laboratory studies of B-cell
Bight, where the xenobiotic load has decreased during the mediated immunity. In: Stolen JS, Fletcher TC (eds) Modula-
past decade (for details see De Jong 1999) and only a less tors of Fish Immune Responses, vol 1. SOS, Fair Haven, N.J.,
pp 33–48
pronounced contaminant gradient was found (Schmolke Blaxhall PC (1972) The haematological assessment of the health of
et al. 1999). In addition, the present study concentrated on freshwater fish: a review of selected literature. J Fish Biol
female dab collected during the same period in every 4:593–604
year, which probably reduced natural variation in the Boer J, Stronck CJN de, Traag WA, Meer J van der (1993) Non-
ortho and mono-ortho substituted chlorobiphenyls and chlori-
results. nated dibenzo-p-dioxins and dibenzofurans in marine and
A link between impaired immune functions of fish and freshwater fish and shellfish in the Netherlands. Chemosphere
disease susceptibility has been long suspected and forms 26:1823–1842
188
Bols NC, Brubacher JL, Ganassin RC, Lee LEJ (2001) Ecotoxi- Hutchinson TH, Manning MJ (1996a) Seasonal trends in serum
cology and innate immunity in fish. Dev Comp Immunol lysozyme activity and total protein concentration in dab
25:853–873 (Limanda limanda L.) sampled from Lyme Bay, UK. Fish
Bradford R (1976) A rapid and sensitive method for the quantifi- Shellfish Immunol 6:473–482
cation of microgram quantities of protein utilizing the principle Hutchinson TH, Manning MJ (1996b) Effect of in vivo cadmium
of protein-dye binding. Anal Biochem 72:248–254 exposure on the respiratory burst of marine flatfish (Limanda
Bressler V, Bissinger V, Abelson A, Dizer H, Sturm A, Kratke R, limanda L.) phagocytes. Mar Environ Res 41:327–342
Fishelson L, Hansen PD (1999) Marine molluscs and fish as Kammann U, Bunke M, Steinhart H, Theobald N (2001) A
biomarkers of pollution stress in littoral regions of the Red Sea, permanent fish cell line (EPC) for genotoxicity testing of
Mediterranean Sea and North Sea. Helgol Mar Res 53:219–243 marine sediments with the comet assay. Mutation Res 498:67–
Broeg K, Zander S, Diamant A, Krting W, Krner G, Paperna I, 77
Westernhagen H von (1999) The use of fish metabolic, Lang T (2002). Fish disease surveys in environmental monitoring:
pathological and parasitological indices in pollution monitor- the role of ICES. ICES Mar Sci Symp 215:202–221
ing. I. North Sea. Helgol Mar Res 53:171–194 Lang T, Mellergaard S (1999) The BMB/ICES sea-going workshop
Bucke D, Vethaak AD, Lang T, Mellergaard S (1996). Common “Fish diseases and parasites in the Baltic Sea”: introduction and
diseases and parasites of fish in the North Atlantic: training conclusions. ICES J Mar Sci 56:129–133
guide for identification. (ICES techniques in marine environ- Lang T, Mellergaard S, Wosniok W, Kadakas V, Neumann (1999)
mental sciences 19) International Council for the Exploration of Spatial distribution of grossly visible diseases and parasites in
the Sea, Copenhagen flounder (Platichthys flesus) from the Baltic Sea: a synoptic
Burke MD, Mayer RT (1974) Ethoxyresorufin: direct fluorimetric survey. ICES J Mar Sci 56:138–147
assay of microsomal O-dealkylation which is preferentially Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein
inducible by 3-methylcholanthrene. Drug Metab Dispos 2:483– measurement with the Folin phenol reagent. J Biol Chem
588 193:265–269
Connell D, Lam P, Richardson B, Wu R (1999) Introduction to Luster MI, Rosenthal GJ (1993) Chemical agents and the immune
ecotoxicology. Blackwell Science, Oxford response. Toxicology 92:229–243
De Jong F, Bakker JF, Berkel CJM van, Dankers NMJA, Dahl K, Marcogliese DJ, Fournier M, Lacroix, Cyr DG (2001) Non-specific
Gtje C, Marencic H, Potel P (1999) Wadden Sea quality status immune response associated with infections of lymphocystis
report. (Wadden Sea ecosystem no. 9) Common Wadden Sea disease virus in American plaice, Hippoglossoides platessoides
Secretariat, Trilateral Monitoring and Assessment Group, (Fabricius). J Fish Dis 24:121–124
Quality Status Report Group, Wilhelmshaven, Germany Matthews ES, Warinner JE, Weeks BA (1990) Assay of immune
Dethlefsen V, Lang T, Kves P (2000) Regional patterns in function in fish macrophages: techniques used as indicators of
prevalence of principal external diseases of dab Limanda environmental stress. In: Stolen JS, Fletcher DP, Anderson DP,
limanda in the North Sea and adjacent areas 1992–1997. Dis Robertson BS, Muiswinkel WB van (eds): Techniques in fish
Aquat Org 42:119–132 immunology. SOS, Fair Haven, N.J., USA, pp 155–163
Diamant A, Westernhagen H von (1999) Biological indicators for Parkinson A, Safe S (1987) Mammalian biologic and toxic effects
the detection of natural and man-made changes in coastal of PCBs. In: Safe S, Hutzinger O (eds) Environmental toxin
waters: an introduction to the MARS project. Helgol Mar Res series. 1. Polychlorinated biphenyls (PCBs): mammalian and
53:148–153 environmental toxicology. Springer, Berlin Heidelberg New
Duffy JE, Carlson E, Li Y, Prophete C, Zelikoff JT (2002) Impact York, pp 49–76
of polychlorinated biphenyls (PCBs) on the immune function of Parry RM, Chandau RC, Sahani RM (1965) A rapid and sensitive
fish: age as a variable in determining adverse outcome. Mar assay of muramidase. Proc Soc Exp Biol Med 119:384–386
Environ Res 54:559–563 Schmolke SR, Broeg K, Zander S, Bissinger V, Hansen PD, Kress
Dunier M, Siwicki AK (1993) Effects of pesticides and other N, Herut B, Jantzen E, Krner G, Sturm A, Krting W,
organic pollutants in the aquatic environment on immunity of Westernhagen H von (1999) Multivariate statistical approach to
fish: a review. Fish Shellfish Immunol 3:423–438 the temporal and spatial patterns of selected bioindicators
Dunier M, Siwicki AK, Damael A (1991) Effects of organophos- observed in the North Sea during the years 1995–1997. Helgol
phorus insecticides: effects of trichlorofon and dichlorvos on Mar Res 53:257–266
the immune response of carp (Cyprinus carpio). III. In vitro Secombes CJ, Fletcher TC, O’Flynn JA, Costello MJ, Stagg R,
effects on lymphocyte proliferation and phagocytosis and in Houlihan DF (1991) Immunocompetence as a measure of the
vivo effects on humoral response. Ecotoxicol Environ Safety biological effects of sewage sludge pollution in fish. Comp
22:79–87 Biochem Physiol 100C:133–136
Ellmann GL, Courtney KD, Andres V Jr, Featherstone RM (1961) Secombes CJ, White A, Fletcher TC, Stagg R, Houlihan DF (1995)
A new and rapid colorimetric determination of acetylcholines- Immune parameters of plaice, Pleuronectes platessa, L. along a
terase activity. Biochem Pharmacol 7:88–95 sewage sludge gradient in the Firth of Clyde, Scotland.
Galgani R, Bocquene G, Cadiou Y (1992) Evidence of variation in Ecotoxicology 4:329–340
cholinesterase activity in fish along a pollution gradient in the Secombes CJ, Tahir A, Stagg RM (1997) Immunocompetence in
North Sea. Mar Ecol Ser 19:17–82 flatfish as a measure of the biological effect of exposure to
Goksoyr A, Frlin L (1992) The cytochrome P450 system in fish, sewage sludge or hydrocarbon contaminated sediments. In:
aquatic toxicology and environmental monitoring. Aquat Tox- Zelikoff JT (ed) Ecotoxicology: response, biomarkers and risk
icol 22:287–312 assessment, an OECD workshop. SOS, Fair Haven, N.J., USA,
Goksoyr A, Husoy AM (1992) The cytochrome P 450 1A1 response pp 281–292
in fish: application and immunodetection in environmental Skouras A, Broeg K, Hansen PD, Westernhagen H von, Steinhagen
monitoring and toxicological testing. Mar Environ Res 35:147– D (2003) The use of innate immune responses as biomarkers in
150 a program of integrated biological effects monitoring on
Grinwis GCM, Vethaak AD, Wester PW, Vos JG (2000) Toxicol- flounder (Platichthys flesus) from the southern North Sea.
ogy of environmental chemicals in flounder (Platichthys flesus) Helgol Mar Res 57
with emphasis on the immune system: field, semi-field Sleiderink HM Everaarts JM, Goksoyr A, Boon JP (1995) Hepatic
(mesocosm) and laboratory studies. Toxicol Lett 112– cytochrome P450A induction in dab after oral dosing with the
113:289–301 polychlorinated biphenyl mixture Chlorphen A40. Environ
Houston AH (1990) Blood and circulation. In: Schreck CB, Moyle Toxicol Chem 14:679–687
PB (eds) Methods for fish biology. American Fisheries Society,
Bethesda, Md., USA, pp 273–334
 189
Stebbing ARD Dethlefsen V (1992) Introduction to the Bremer- pollution with feral eel (Anguilla anguilla). III. Statistical
haven workshop on biological effects of contaminants. Mar analyses of relationship between contaminant exposure and
Ecol Prog Ser 91:1–8 biomarkers. Aquat Toxicol 39:45–75
Tahir A, Fletcher TC, Houlihan DF, Secombes CJ (1993) Effect of Wester PW, Vethaak AD, Muiswinkel WB van (1994) Fish as
short-term exposure to oil-contaminated sediments on the biomarkers in immunotoxicology. Toxicology 86:213–232
immune response of dab, Limanda limanda (L.). Aquat Toxicol Zelikoff JT, Raymond A, Carlson E, Li Y, Beanman JR, Anderson
27:71–82 M (2000) Biomarkers of immunotoxicity in fish: from the lab to
Van der Oost R, Vindimian E, Brink PJ van den, Satumalay K, the ocean. Toxicol Lett 112–113:325–331
Heida H, Vermeulen NPE (1997) Biomonitoring aquatic