Helgol Mar Res (2003) 57:262–271 DOI 10.1007/s10152-003-0159-x
ORIGINAL ARTICLE
V. Schmidt · S. Zander · W. K rting · K. Broeg · H. von Westernhagen · H. Dizer · P. D. Hansen · A. Skouras · D. Steinhagen Parasites of flounder (Platichthys flesus L.) from the German Bight, North Sea, and their potential use in biological effects monitoring C. Pollution effects on the parasite community and a comparison to biomarker responses
Received: 13 March 2003 / Revised: 11 June 2003 / Accepted: 3 July 2003 / Published online: 15 August 2003 Springer-Verlag and AWI 2003
Abstract In the frame of an integrated biological effect measures species richness and number of heteroxenous monitoring programme, the parasite community of floun- species decreased with increasing concentrations of der (Platichthys flesus) was investigated at different individual heavy metals or hydrocarbons in sediment locations in the German Bight from 1995 to 2000. In and blue mussel samples. Most of the parasitological order to assess the impact of environmental contamination parameters significantly reflected the established site- caused by anthropogenic activities on the parasite com- specific contamination gradient, when data were pooled munity, selected parasitological parameters that displayed over all sampling campaigns. Significant correlations significant differences between the sampling sites were were also found with the contamination level of individ- subjected to correlation analyses with site-specific con- ual flounder. The parasitological parameters included the tamination and individual pollution loads of their fish parasite species Lepeophtheirus pectoralis and Lernaeo- hosts. In addition, correlation analyses were conducted cera branchialis, which were not correlated to site- with the responses of selected genetic, biochemical, specific contamination. Several biomarkers were signif- histopathological, physiological and immunological pa- icantly correlated to the abundance of parasitic copepods rameters of fish, used as potential biomarkers. In total, A. cornuta, Lernaeocera branchialis and Lepeophtheirus 802 flounder were analysed for these parameters. Infor- pectoralis and to parasite community parameters. The mation on the chemical background at the sampling sites results showed that the abundance of several metazoan was derived from sediment samples and from 120 parasite species, species richness and parasite diversity samples of blue mussel (Mytilus edulis) tissue, collected were reduced in contaminated habitats, and that differ- at each of the sampling sites. Based on chemical data ences between sites were not only related to natural available from the sediment and blue mussel samples, a factors, such as salinity, but also to pollution-induced pollution gradient could be established between the stress. Thus, it can be concluded that the parasite sampling sites for individual contaminants. The relative community of fish responds to the level of pollution at abundance of Acanthochondria cornuta, Cucullanus het- a specific site as well as to residues of xenobiotics in erochrous and Zoogonoides viviparus, and the community individual fish. These findings give indications that the parasite community of fish is a valuable parameter for the Communicated by H. von Westernhagen, A. Diamant assessment of ecological consequences of chemical V. Schmidt · S. Zander · W. K rting · A. Skouras · contamination in aquatic habitats. D. Steinhagen ()) Fish Disease Research Unit, Keywords Pollution monitoring · Platichthys flesus · School of Veterinary Medicine, Parasite community · Biomarker · Bioindicator B nteweg 17, 30559 Hannover, Germany e-mail: [email protected] K. Broeg · H. von Westernhagen Introduction Alfred-Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany Field studies on bioassessment of environmental chal- lenge should include a combination of rapidly responding, H. Dizer · P. D. Hansen Institute of Ecological Research and Technology, sensitive biomarkers of pollution exposure and more Department of Ecotoxicology, ecologically relevant bioindicators, which integrate the Berlin Technical University, effects of various stressors (Adams 2002). Berlin, Germany 263 In general, biomarkers are used to indicate exposure of Methods an organism to a stressor and help to identify the mechanistic basis of causal relationships between stres- Sampling sors and their effects. They are mainly measured at the During spring and autumn of 1995–1997 and 1999–2000, 802 suborganism level, are sensitive to stressors and show a individuals of European flounder (Platichthys flesus L.) were direct, often highly variable response to these stressors on sampled by research vessel trawl catches at five different locations a short timescale (van der Oost et al. 2003). Bioindicators (Elbe estuary, Helgoland, Outer Eider estuary, Inner Eider estuary, respond at higher levels of biological organisation, like Spiekeroog) in the German Bight, North Sea. The stations were determined by their geographical positions. the population or community level. They are of high To avoid size or age effects, only individuals of a narrow size ecological significance but, due to the complexity of range (18–25 cm total length) were included in the analysis. Ecto- processes in population and community dynamics, pro- and endoparasites were collected from the fish. In addition, vide little information for helping to understand the biometric, biochemical and immunological measurements were underlying causal mechanisms between stressors and taken from the same individuals. Chemical analysis was performed on the filet of the same individuals. Details of sampling, effects. Their sensitivity to specific stressors is low and examination of flounder and numbers of evaluated fish specimen they tend to respond to effects of multiple stressors over a for each sampling period at each location have been described large spatial and temporal scale (Adams 2002). A elsewhere (Schmidt et al. 2003a). A list of the parasite taxa combined use of biomarkers and bioindicators, with a recorded during the course of the study, information about their infection levels and parasite community characteristics at the focus on the organism level, could provide a link between sampling locations have also been presented in detail previously mechanistic understanding and the ecological conse- (Schmidt et al. 2003a, 2003b). quences of pollution exposure, and increase the ability to interpret biological data (Adams 2002). The use of fish parasites as indicators for marine Parameters under study pollution has been widely and controversially discussed In the present study, those parasitological parameters which over the last few years. Distribution and infection levels indicated differences between the sampling locations, and were of parasites are not only influenced by environmental therefore used for correlation analyses, are the following: abun- contaminants but also by a variety of natural factors dance of Trichodina spp., Acanthochondria cornuta, Lepeoph- theirus pectoralis, Lernaeocera branchialis, metacercaria of an un- (M ller 1978; Khan and Thulin 1991; MacKenzie et al. determined trematode species, Zoogonoides viviparus, Cucullanus 1995; Kennedy 1997; Overstreet 1997). Therefore, it is heterochrous and the community measurements species richness, often recommended (Gelnar et al. 1997; Khan and Payne number of heteroxenous species, number of macroparasite individ- 1997; Overstreet 1997) that in pollution monitoring uals, Shannon-Wiener index of diversity and inverse Simpson’s index of diversity. All measurements were calculated for individual studies, parasitological data should be accompanied by fish, the indices were used according to Magurran (1988), and other types of data such as analytical chemistry or abundance according to Bush et al. (1997). biochemical biomarkers. The same fish individuals which were used for parasitological In the present study, environmental deterioration was investigations were also analysed for genetic, biochemical, phys- iological, histopathological and immunological parameters. The assessed in the frame of an integrated biological effect following parameters were incorporated into the correlation monitoring programme. The parasite community of analysis of this paper (detailed description of the parameters and flounder (Platichthys flesus L.) was investigated at the applied methods are given in the respective references): different sites in the German Bight over a period of 6 years and tested as a potential indicator of anthropogenic 1. Choline esterase activity in flounder brain (ChE-Br) (Dizer et al. 2003) impact at the population and community level. 2. Choline esterase activity in flounder muscle (ChE-f) (Dizer et The basic assumptions were that biotic diversity is al. 2003) highest in undisturbed habitats, whereas man-made stress, 3. DNA-unwinding (DNA-f), DNA strand breaks in hepatocytes such as chemical pollution, may lead to a loss of species (Dizer et al. 2003) 4. EROD activity (EROD), the activity of cytochrome P450- and a reduction of diversity (Kennedy 1997). dependent monooxygenase ethoxyresorufin-O-deethylase (Broeg In the present monitoring programme, standard chem- et al. 1999) ical parameters as well as a set of genetic, biochemical, 5. Lipids: accumulation of neutral lipids in flounder liver (Broeg physiological and immunological biomarkers, which were et al. 1999) 6. Lysosomal stability, the membrane stability of hepatocyte partly recommended as standard methods for general lysosomes. LY1: type of lysosomes which has an early monitoring of the biological effects of contaminants (van membrane breakdown; LY2: late membrane breakdown (Broeg der Oost et al. 2003), were sampled and evaluated from et al. 1999) the same individual fish, in addition to an assessment of 7. Lysozyme activity (Lys) in flounder plasma (Skouras et al. infection parameters of the parasite community. In this 2003) 8. Macrophage aggregate area (MAA), the mean size of macro- paper, the parasitological data were compared to site- and phage aggregates in flounder liver (Broeg et al. 1999) host-specific residue analyses and to responses of the 9. Macrophage aggregate activity (MAM), the activity of acid biomarkers under study. phosphatase in macrophage aggregates of flounder liver (Broeg et al. 1999) 10. Pinocytosis (Pin): uptake of neutral red by head kidney phagocytes (Skouras et al. 2003) 264 Table 1 Correlation analysis of parasite infection parameters of chodina spp.; Zoog Zoogonoides viviparus; N number of macropar- male and female flounder to residues in sediments and blue mussel asite individuals; S species richness; DDD 1,1-dichloro-2.2-bis(p- (Mytilus edulis) tissue from the same geographic location. Corre- chlorophenyl) ethane; DDE 1,1-dichloro-2.2-bis(p-chlorophenyl) lation coefficients and the level of significance are given. ethylene;P HCH hexachlorcyclohexane; PCB polychlorinated biphe- Significant correlations are additionally marked in bold. Acan nyls; PCBs sum of PCB 28, 52, 101, 118, 138, 153, 180; 1/D Acanthochondria cornuta; Cuc Cucullanus heterochrous; Lep inverse Simpson’s Index of diversity; H0 Shannon-Wiener Index of Lepeophtheirus pectoralis; Lern Lernaeocera branchialis; Met diversity, Hsp number of heteroxenous species Metacercaria of an undetermined trematode species; Trich Tri-
Sediment residues Abundance of: Community measurements 0 Residues n (samples) Trich Acan Lep Lern Meta Zoog Cuc SNHsp H 1/D Mercury a 6 0.54 0.60 0.37 0.03 0.09 0.94** 0.94** 0.89* 0.66 0.89* 0.77 0.77 Cadmium b 14 0.38 0.24 0.14 0.02 0.06 0.70** 0.44 0.40 0.35 0.39 0.14 0.06 Copper b 14 0.54* 0.63* 0.04 0.26 0.08 0.49 0.74** 0.74** 0.69 * 0.75** 0.56* 0.34 Lead b 14 0.39 0.58* 0.01 0.21 0.02 0.34 0.64* 0.68** 0.57 * 0.67** 0.51 0.36 Iron a 6 0.49 0.54 0.47 0.09 0.09 0.89* 0.89* 0.94** 0.60 0.94** 0.83* 0.83* Manganese a 6 0.54 0.60 0.37 0.03 0.09 0.94** 0.94** 0.89* 0.66 0.89* 0.77 0.77 Zinc a 6 0.49 0.54 0.49 0.09 0.09 0.89* 0.89* 0.94** 0.60 0.94** 0.83* 0.83* Nickel a 6 0.49 0.54 0.49 0.09 0.09 0.89* 0.89* 0.94** 0.60 0.94** 0.83* 0.83* Aluminium a 6 0.49 0.54 0.49 0.09 0.09 0.89* 0.89* 0.94** 0.60 0.94** 0.83* 0.83* Chromium b 14 0.36 0.48 0.02 0.19 0.01 0.11 0.62* 0.54* 0.46 0.57* 0.41 0.40 PCB 138 c 8 0.31 0.83* 0.05 0.24 0.43 0.76* 0.40 0.80* 0.36 0.74* 0.64 0.33 PPCB 153 c 8 0.34 0.88** 0.12 0.19 0.36 0.79* 0.50 0.85** 0.31 0.79* 0.74* 0.45 PCBs c 8 0.12 0.71* 0.00 0.21 0.43 0.63 0.38 0.78* 0.21 0.62 0.52 0.38
M. edulis residues Abundance of: Community measurements 0 Residues n (samples) Trich Acan Lep Lern Meta Zoog Cuc SNHsp H 1/D >Cadmium d 18 0.62** 0.73*** 0.38 0.25 0.04 0.48* 0.58* 0.51* 0.40 0.34 0.65** 0.54* b-HCH c 12 0.74** 0.60* 0.22 0.44 0.26 0.59* 0.56 0.58* 0.78** 0.73** 0.62* 0.47 p.p-DDE c 12 0.63* 0.56 0.00 0.55 0.52 0.51 0.33 0.51 0.77** 0.64* 0.41 0.19 Pp,p DDD c 12 0.68* 0.75** 0.14 0.34 0.25 0.68* 0.64* 0.72** 0.49 0.76** 0.59* 0.51 PCBs c 12 0.87*** 0.88*** 0.33 0.38 0.38 0.49 0.82*** 0.74** 0.68* 0.80*** 0.90*** 0.78** * P<0.05, ** P<0.01, *** P<0.001 a Samples were taken in 1996 b Samples were taken in 1996, 1999 and 2000 c Samples were taken during 1999 to 2000 d Samples were taken from 1997 to 2000
Table 2 Correlation analysis of parasite infection parameters to chodina spp.; Zoog Zoogonoides viviparus; N number of macropar- residues in the muscle of male and female flounder (n=185) over asite individuals; S species richness; DDD 1,1-dichloro-2.2-bis(p- the years 1999 to 2000. Parasite infection parameters and chemical chlorophenyl) ethane; DDE 1,1-dichloro-2.2-bis(p-chlorophenyl) analysis were performed on the same flounder individuals. ethylene; HCB hexachlorobenzene;P OCS octachlorostyrole; PCB Correlation coefficients and the level of significance are given. polychlorinated biphenyls; PCBs sum of PCB 28, 52, 101, 118, Significant correlations are additionally marked in bold. Acan 138, 153, 180; 1/D inverse Simpson’s Index of diversity; H0 Acanthochondria cornuta; Cuc Cucullanus heterochrous; Lep Shannon-Wiener Index of diversity, Hsp number of heteroxenous Lepeophtheirus pectoralis; Lern Lernaeocera branchialis; Met species Metacercaria of an undetermined trematode species; Trich Tri-
Abundance of: Community measurements 0 Trich Acan Lep Lern Meta Zoog Cuc SN Hsp H 1/D Mercury 0.01 0.18* 0.23** 0.30*** 0.16 0.06 0.18* 0.27** 0.28*** 0.15 0.11 0.05 HCB 0.26*** 0.49*** 0.37*** 0.43*** 0.21** 0.17* 0.27*** 0.29*** 0.50*** 0.22** 0.30*** 0.21** p.p-DDE 0.13 0.29*** 0.22** 0.21** 0.24** 0.11 0.18* 0.17* 0.34*** 0.17* 0.21** 0.15* p.p-DDD 0.17* 0.37*** 0.19** 0.37*** 0.26*** 0.19* 0.21** 0.22** 0.47*** 0.20** 0.16* 0.08 POCS 0.07 0.33*** 0.09 0.15* 0.07 0.13 0.21** 0.26*** 0.20** 0.18* 0.21** 0.17* PCBs 0.10 0.21** 0.18* 0.22** 0.17* 0.04 0.04 0.03 0.29*** 0.08 0.09 0.05 * P<0.05, ** P<0.01, *** P<0.001
11. Production of reactive oxygen species (ROS) by head kidney immature adult male flounder were used for all calculations (see leucocytes (baseline, and induced by phorbol myristate acetate, Broeg et al. 1999). ROS-PMA) (Skouras et al. 2003) 12. Vitellogenin, concentration of vitellogenin in the plasma of male flounder (Dizer et al. 2003) Residue analyses
As some of these biomarkers, such as EROD and MAM, were For a characterization of the sampling sites by their chemical influenced by the sex of the flounder (Broeg et al. 1999), only pollution burden, samples of sediment and blue mussel (Mytilus edulis) tissue from the Elbe estuary, Eider estuary and Helgoland 265 “Tiefe Rinne” were analysed for their heavy metal (Hg, Pb, Cd, Cu) metals and polychlorinated biphenyls (PCBs) in the and chlorinated hydrocarbon (see Tables 1, 2) content. Analyses sediment from the sampling locations (Table 1). This were carried out by a commercial laboratory that had undergone an intercalibration exercise (Handels- und Umweltschutzlaboratorium finding was even more pronounced when species rich- Dr. Wietz, Hamburg). A description of all parameters and the ness or the number of heteroxenous species was consid- results of these analyses are given in Dizer et al. (2003). ered (Table 1). The community measures Shannon- For residue analyses of flounder, muscles samples were Wiener’s index of diversity and inverse Simpson’s index collected from the same flounder individuals that were also analysed for their parasite community and for biochemical of diversity were only correlated to some heavy metal biomarker responses. At each sampling site and during every residues in the sediment (Table 1). sampling period, muscle tissue was collected from 10 individuals When residues in the tissue of M. edulis were and analysed for the heavy metal (Hg, Pb, Cd, Cu) and chlorinated considered, the abundance of A. cornuta, Z. viviparus hydrocarbon content by a commercial laboratory (Labor f r and C. heterochrous in flounder was highly correlated Fischgesundheit, Professor Harz, Bremerhaven; for details see Broeg et al. 1999; Schmolke et al. 1999). to decreasing concentrations of cadmium, b-HCH, p.p.-DDD and total PCBs (see Table 1). In addition, the community measures species richness, number of Statistical analyses heteroxenous species, number of macroparasite indi- In order to test for a relationship between flounder parasite viduals, Shannon-Wiener Index of Diversity and infection parameters and site-specific contamination levels, sedi- inverse Simpson Index also decreased with increasing ment and blue mussel residue burdens from the sampling locations concentrations of contaminant residues in mussel were used as sentinels. Correlation analyses were carried out tissue (Table 1). In contrast to these findings, the between parasitological data and the sediment and blue mussel tissue residue measurements. For this analysis, the mean abundance abundance of Trichodina spp. increased on flounder of the parasite species and mean values of the community indices from sampling sites with increasing xenobiotica loads under study were calculated on the basis of infection parameters of (Table 1). 9–30 flounder individuals from the sampling site under study. Parasite infection parameters of flounder were only These data were then compared to measurements of sediment correlated to those contaminants which showed a clear residues and mean values of residue concentration in 5–10 blue mussels from the same location and collected during the same regional pattern. When the contamination data from sampling period. mussel tissue were combined over the sampling period, A relationship between parasitological parameters and contam- the concentrations of b-HCH, p.p-DDE and p.p.-DDD ination levels of individual fish was determined by correlation were significantly higher in mussels from the Elbe estuary analyses between parasitological data and the residue burden measured in muscle tissue of the same flounder individuals. than from the other sites (Fig. 1). The concentrations of All residue measurements with values below the detection limit cadmium and total PCBs in the tissue of M. edulis were replaced by 2/3 of the value of the detection limit, in order to decreased gradually in the order Elbe>Eider>Helgoland include them in calculations (Schmolke et al. 1999). (Fig. 1). To test whether a relationship between responses of contami- nation biomarkers and effects on the parasite community could be observed, correlation analyses of both data sets were performed, also based on measurements collected from individual fish. Flounder muscle All correlation analyses were done from pooled data collected over all the sampling periods and were carried out using When xenobiotic loads in the muscle of individual Spearman’s Rank Correlation (STATISTICA 6.0, StatSoft). Com- parisons of groups were done using the Kruskal-Wallis test and flounder were compared to infection parameters of the Dunn’s post hoc test (SigmaStat 2.0). To test for an influence of parasite fauna from the same individuals, correlations salinity, parasitological parameters from locations with differences were observed between the concentration of several in salinity as well as with similar salinity were compared using chi- xenobiotics and the abundance of parasite species. square tests as described previously (Schmidt et al. 2003a). With increasing concentrations of hexachlorobenzene (HCB), DDE, DDD, octachlor styrol (OCS) or PCBs in flounder filet, the abundance of A. cornuta, C. hete- Results rochrous, Lepeophtheirus pectoralis and Lernaeocera branchialis decreased (Table 2). In addition, commu- Parasite infection parameters and sediment nity measures such as the number of parasite species, or mussel residues the number of macroparasite individuals, and Shannon Wiener’s index of diversity indicated a decreasing A correlation analysis of parasite infection parameters, parasite diversity with increasingcontaminationloadin such as abundance of parasite species and component flounder muscle (Table 2). In contrast to observations community indices, to sediment or mussel tissue con- on hydrocarbon loads, infection with parasitic cope- tamination levels indicated that the parasite fauna of pods A. cornuta, Lepeophtheirus pectoralis and Ler- flounder was influenced by the concentration of heavy naeocera branchialis increased with increasing metals and several chlorinated hydrocarbons at the concentrations of mercury in flounder filet (Table 2). sampling locations. The relative abundance of A. The parasite infection data were correlated to those cornuta, Z. viviparus and C. heterochrous in flounder xenobiotics which showed regional differences in at a sampling site showed high negative correlation concentration (Fig. 2). Flounder from the Elbe estuary coefficients to the concentration of individual heavy had higher residues of DDD, DDE, HCB and OCS than 266 Fig. 1 Concentration of select- ed contaminants in blue mussel tissue (Mytilus edulis) at three sampling sites in the German Bight in the years 1999–2000. Given are box plots with medi- an values (white square), 25th and 75th percentiles (box) and range without outliers (lines). E Elbe, I Inner Eider, H Hel- goland. Stars above box plots indicate significant differences of the site to all other sites (n=20/location, * P<0.05, ** P<0.01, *** P<0.001)
Fig. 2 Concentration of select- ed contaminants in the muscle of flounder (Platichthys flesus) from five locations in the Ger- man Bight in the years 1999– 2000. Given are box plots with median values (white square), 25th and 75th percentiles (box), range (lines) and outliers (black circle). E Elbe, O Outer Eider, H Helgoland, S Spiekeroog, I Inner Eider (n=185)
individuals from other sites. In contrast to this, the considerably lower than correlation coefficients obtained mercury concentration was lower in the flounder filet when the chemical contamination of the sediment or from the Elbe sampling site compared to individuals mussel tissue from the sampling location were considered originating from other sampling locations (Fig. 2). (compare Tables 1 and 2). The abundance of the parasitic Correlation coefficients of parasite infection parame- copepods A. cornuta, Lepeophtheirus pectoralis and ters to xenobiotic loads in the muscle of flounder were Lernaeocera branchialis on individual flounder showed 267 Table 3 Correlation analysis of parasite infection characteristics species. Biomarkers: ChE-Br choline esterase activity in flounder and biomarker responses of male flounder. Measurements taken brain; ChE-f choline esterase activity in flounder muscle; DNA-f from the same flounder individual were compared. Correlation DNA-unwinding, DNA strand breaks in hepatocytes; EROD coefficients and the level of significance are given. Significant activity of cytochrome P450 dependent monooxygenase ethoxyre- correlations are additionally marked in bold. The majority of sorufin-O-deethylase; LY1, LY2 membrane stability of hepatocyte parameters were analysed from 1995 to 2000, see notes for lysosomes; Lys lysozyme activity in flounder plasma; MAA mean exceptions. Abundance of the parasites: Acan Acanthochondria size of macrophage aggregates in flounder liver; MAM activity of cornuta; Cuc Cucullanus heterochrous; Lep Lepeophtheirus pec- acid phophatase in macrophage aggregates of flounder liver; Pin toralis; Lern Lernaeocera branchialis; Met Metacercaria of an uptake of neutral red by head kidney phagocytes; ROS production undetermined trematode species; Trich Trichodina spp.; Zoog of reactive oxygen species by head kidney leucocytes, base line; Zoogonoides viviparus; N number of macroparasite individuals; S ROS-PMA production of reactive oxygen species induced by species richness; 1/D inverse Simpson’s Index of diversity; H0 phorbol myristate acetate; Vit concentration of vitellogenin in the Shannon-Wiener Index of diversity, Hsp number of heteroxenous plasma of male flounder
0 Bio- n Trich Acan Lep Lern Meta Zoog Cuc SNHsp H 1/D marker (fish) EROD 441 0.16 *** -0.34 *** -0.27 *** -0.08 -0.19 *** -0.10 * -0.10 * -0.22 *** -0.20 *** -0.22 ***-0.33 *** -0.28 *** MAM 445 0.12* 0.23*** 0.06 0.24*** 0.03 0.18*** 0.15** 0.24*** 0.22*** 0.25*** 0.17*** 0.13** Ly1 444 0.19*** 0.20*** 0.08 0.04 0.08 0.21*** 0.07 0.11* 0.18*** 0.06 0.11* 0.09 Ly2 444 0.21*** 0.29*** 0.16*** 0.13** 0.16*** 0.12** 0.10* 0.15** 0.22*** 0.09* 0.18*** 0.15** ChE-Br a 138 0.04 0.17* 0.33*** 0.25** 0.21* 0.13 0.03 0.14 0.14 0.06 0.28*** 0.29*** ChE-f 262 0.01 0.12 0.03 0.09 0.03 0.15 0.04 0.05 0.02 0.02 0.02 0.01 DNA-f 262 0.06 0.12 0.06 0.04 0.06 0.23*** 0.13* 0.19** 0.05 0.10 0.06 0.03 MAA 445 0.06 0.04 0.04 0.03 0.05 0.06 0.04 0.03 0.01 0.07 0.08 0.07 Vit b 162 0.01 0.004 0.23** 0.07 0.14 0.03 0.003 0.01 0.002 0.04 0.03 0.2 Pin b 158 0.13 0.16* 0.01 0.02 0.19* 0.07 0.06 0.22** 0.01 0.10 0.15 0.14 ROS b 165 0.16* 0.10 0.07 0.01 0.19* 0.11 0.09 0.07 0.003 0.07 0.12 0.11 ROS-PMA b165 0.10 0.18* 0.05 0.15 0.02 0.20* 0.12 0.03 0.18* 0.05 0.04 0.02 Lys b 181 0.12 0.30*** 0.34*** 0.18* 0.13 0.32*** 0.08 0.17* 0.24*** 0.13 0.21** 0.22** * P<0.05, ** P<0.01, *** P<0.001 a Samples taken from 1995 to 1997 b Samples taken from 1999 to 2000
Fig. 3 Selected biomarker re- sponses of flounder (P. flesus) at five locations in the German Bight in the years 1995–2000. ChE-Br were analysed from 1995–1997 and Lys from 1999– 2000 only. Given are box plots with median values (white square), 25th and 75th per- centiles (box), range without outliers (lines). E Elbe, O Outer Eider, H Helgoland, S Spieker- oog, I Inner Eider. Asterisks above box plots in the graphs for EROD, MAM and LY2 indicate significant differences of the marked station to Outer Eider, Helgoland and Spieker- oog, and above box plots in the graph for Lys of the marked station to Elbe, Outer Eider and Inner Eider (P<0.001). The number of fish used for calcu- lations is indicated to the right of each graph
a clear correlation to contamination concentrations in coefficients were much higher when the mean abundance flounder muscle (Table 2). The abundance of the gut- of these parasites on flounder from each sampling site dwelling parasites C. heterochrous and Z. viviparus was were compared to the presence of xenobiotics in the also lower in flounder individuals with increased HCB, sediment at that particular site (Table 1). DDD, DDE or OCS loads (Table 2), but correlation 268 Biomarkers polluted locations in the North Sea, contamination levels in the German Bight are low (Herut et al. 1999). Of the biomarkers studied, only EROD, MAM, LY2, Although the concentrations of a large number of ChE-Br and Lys showed correlation to more than half of chemicals ranged close to or were below the detection the parasite infection parameters (Table 3). While the limit at all sampling sites (Dizer et al. 2003), a regional abundance of most of the parasite species under study and pattern could be observed in the concentration of some the parasite diversity parameters decreased with increas- contaminants. Schmolke et al. (1999) confirmed a ing measurements of EROD or ChE-Br, the values of decreasing contamination gradient for heavy metals in parasite infection parameters were positively correlated to sediments and mussel tissue in the order Elbe>Eider>Hel- measurements of the indicators MAM, LY2 and Lys goland for the years 1996 and 1997. The same gradient (Table 3). The abundance of Trichodina spp., however, was also described for the PCB content of birds’ eggs in increased with elevated EROD activity and decreased the Wadden Sea (de Jong et al. 1999). These gradual with increasing levels of MAM, LY1, LY2 or ROS. differences in the contamination load of the sediment was When the EROD, MAM and LY2 data from all not clearly evident when the sampling locations were samples during 1995 to 2000 were combined, a clear compared to each other for 1999–2000 (Dizer et al. 2003). regional pattern was evident (Fig. 3). The EROD activity It could only be confirmed that sediments from Helgoland was significantly higher in flounder from the Elbe estuary were less contaminated than those from Elbe or Outer compared to individuals from all other sites, while MAM Eider. In contrast, in mussel tissue, a contamination and LY2 were significantly reduced in individuals from gradient with Elbe>Eider>Helgoland could be confirmed the Elbe sampling location (P<0.001). In this analysis, no for the cadmium and PCB contents. For other contami- significant differences between sampling sites were found nants, such as DDD, DDE and HCH, such a gradient for ChE-Br, ChE-f or DNA-F (Fig. 3). Levels of plasma could not be established. The concentrations of theses lysozyme activity were significantly lower in flounder contaminants were, however, significantly higher in the from the Elbe and Outer and Inner Eider estuary than in tissue of mussels from the Elbe estuary than from all other individuals from Helgoland (P<0.001), but these mea- locations. surements were only available from samples taken during Contaminants which exhibited spatial differences in 1999 and 2000. concentration could be correlated to infection parameters of flounder parasites. In particular, the following infection parameters were Discussion found to be correlated to residue data of sediment and mussel tissue: abundances of selected parasite species, An indispensable prerequisite for biological effect mon- species richness and diversity decreased with increasing itoring which aims at testing pollution effects is the contamination levels (Schmidt et al. 2003a, 2003b). When proper selection of sampling sites. They should be these infection parameters were considered, gradual situated in the same ecological area but should differ in differences between the sampling locations in the order their pollution levels. Elbe