Environ. Sci. Technol. 2009, 43, 2151–2158
Long-Term Trends in the Prevalence of Cancer and Other Major Diseases Among Flatfish in the Southeastern North Sea as Indicators of Changing Ecosystem Health A . D I C K V E T H A A K , * ,† J O H A N G . J O L , ‡ AND JAN P. F. PIETERS§ Deltares, Marine and Coastal Systems, Post Office. Box 177, 2600 MH Delft, The Netherlands, Institute of Marine Resources and Ecosystem Studies, Post Office Box 77, 4400 AB Yerseke, The Netherlands, and Rijkswaterstaat Centre for Water Management, Post Office Box 17, 8200 AA Lelystad, The Netherlands
Received October 17, 2008. Revised manuscript received January 7, 2009. Accepted January 8, 2009.
This paper analyses and discusses spatial and temporal patterns in the prevalence of major skin diseases (lymphocystis, epidermal hyperplasia/papilloma, ulcers), intestinal parasite Glugea sp., and liver cancer in dab (Limanda limanda) and flounder (Platichthys flesus) in the Dutch section of the North Sea since the mid-1980s. We have attempted to relate disease prevalence trends in both species to chemical contaminant exposure and other relevant environmental factors including fish condition factor, population density, fishing activity, and water temperature. We observed a long-term decline in chemical-related liver cancer in the populations of both species since the early 1990s. Lymphocystis and skin ulcer (flounder only) have also displayed a significant decrease since then. We conclude that the widespread decline in the prevalence of several skin diseases and liver cancer in dab and flounder in Dutch waters in the past two decades is most likely due to the improved water quality and health conditions in this region.
Introduction A number of major diseases of different etiologies affecting flatfish populations have frequently been used as general indicators of marine environmental stress for several decades (1). Selection of these indicator diseases is primarily based on the known or suspected responsiveness of the disease to adverse environmental conditions (including contaminants), high susceptibility of sentinel species to macroscopically visible diseases, and the low impact of the conditions on fish mortality and wild populations, which makes their prevalence easy to measure. Regular and systematic monitoring of the occurrence of skin and liver diseases of flatfish in Dutch coastal waters and the southeastern part of the North Sea has been in progress since 1983. Until 1990, annual epidemiological surveys were * Corresponding author phone: +31 (0)152858659; fax: +31(0)152858710; e-mail:
[email protected]. † Deltares, Marine and Coastal Systems. ‡ Institute of Marine Resources and Ecosystem Studies. § Rijkswaterstaat Centre for Water Management. 10.1021/es8028523 CCC: $40.75
Published on Web 02/18/2009
2009 American Chemical Society
carried out as part of a specific study of the relationship between fish disease and pollution (1). In 1991, these surveys became part of the North Sea Task Force and of OSPAR’s Joint Assessment and Monitoring Program (JAMP), using standard protocols (2) and quality assurance procedures (3). The Dutch monitoring program on fish diseases took an integrated approach, including chemical contaminant exposure measurements in fish tissues and sediment, and supporting biological and hydrographical data. The work is largely coordinated through the International Council for the Exploration of the Sea (ICES) and its Environmental Data Centre, which holds the fish disease and chemical databases based on national reports. It is generally recognized that fish diseases have a multifactorial etiology (1, 4). Outbreaks of naturally occurring infectious diseases depend on the presence of infectious agents (viruses, bacteria, or parasites), but other host-related and environmental factors may be required before the disease can develop. These include the cohort history and the behavioral and migratory patterns of the host, the circumstances of exposure, temperature, nutrient enrichment, fisheries-induced stress, food supply, population density, etc. Host-related internal factors such as genetic makeup and physiological and immunological factors are equally important, and include sex, length/age, bioaccumulative capacity, exposure to chemical contaminants, and nutritional status. In principle, chemical contaminants and other environmental stressors may reduce disease resistance by causing physiological stress (4, 5), or they may facilitate the development and progression of infectious organisms (4). Alternatively, some noninfectious diseases, such as liver tumors, have a proven link to chemical contaminants (e.g., polynuclear aromatic hydrocarbons) (6, 7) and can be regarded as specific indicators of environmental genotoxins/ carcinogens (1, 4). Skin ulcerations in fish have been shown to be good indicators of marine environmental degradation, especially in estuarine waters and near freshwater outlets (8, 9). Finally, numerous studies confirm the value of parasites as potential indicators of pollution, including hydrocarbons, in the North Sea (10). Thus, fish diseases are ecologically relevant and integrative end points of chronic exposure to environmental stressors including contaminants. Elevated disease prevalence in fish (and other organisms) is considered one of the key indicators of ecosystem dysfunction, characteristic of ecosystem pathology (11). Monitoring disease status and changes in disease prevalence in marine fish can therefore provide valuable information on status and changes in environmental quality and can be useful in assessing the health of the North Sea ecosystem. In this paper, we analyze and discuss spatial and temporal patterns in the prevalence of major diseases in two flatfish species in the Dutch section of the North Sea since the mid 1980s. We have attempted to use the results of disease prevalence as an indicator of marine ecosystem status and health.
Materials and Methods Sentinel Species and Sampling Sites. Because of their high abundance, bottom-dwelling life style, and high disease frequency, the two flatfish species dab (Limanda limanda) and flounder (Platichthys flesus) have become major sentinels in the biological effect monitoring programs of OSPAR JAMP. The two species are complementary in their distribution: flounder is a common species in coastal waters and estuaries (12), whereas dab is a major species widely distributed in the North Sea (13). Dab were caught at five offshore and coastal VOL. 43, NO. 6, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Geographical position of sampling sites for dab (D1-D5) and flounder (F1-F5). Dogger Bank (D1); 150 km NW of Terschelling (D2); 70 km N of Borkum (D3); 60 km NW of Terschelling (D4); 90 km W of Callantsoog (D5); Eastern Scheldt (F1); Western Scheldt (F2); North Sea coastal zone (F3); Western Wadden Sea (F4); Ems/Dollard (F5). sites and flounder at five coastal and estuarine sites (Figure 1). Annual surveys were carried from 1991 to 1999. From 2000 to 2005 the number of sampling sites and sampling rate has been reduced, essentially because of financial constraints and the low observed prevalence of most indicator diseases. Fishing Operation and Sampling Procedure. The sampling procedure for dab and flounder was largely based on ICES guidelines (2). Dab were caught during their spawning migration period (January-April) (13) and flounder at the end of their nonmigratory prespawning phase (AugustSeptember) (12, 14). Dab were caught by the research vessel RV Tridens by means of a 6 m beam trawl. Two or more 15-min hauls were made along different trajectories through the center of the fixed coordinates of each station. After each haul, the fish were sorted into three length classes (15-19, 20-24, and g25 cm) and random samples from each class were examined for disease following the ICES guidelines as far as possible. The minimum sample sizes per site for recording visible diseases for each of the three length classes were 100, 100, and 50, respectively. A total sample of 250 fish per site would allow major spatial and temporal differences to be reliably detected (12). Internal examination for the presence of gross liver lesions was performed only on specimens from the longest length class, plus additional specimens from the middle length class, because neoplasms occur more frequently in large fish. The strategy of recording gross lesions probably underestimates the “true” prevalence of neoplastic lesions in fish because it does not incorporate microscopic lesions that are not visible by macroscopic examination. However, this strategy permitted screening of larger numbers of fish than using more laborious and 2152
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expensive histological investigation in all cases. In addition, the length-frequency distribution was recorded (total length to the nearest centimeter) and otoliths were taken for age determination for females and males separately. A gutted condition factor [(CF ) 100 × (somatic weight in milligrams) ÷ (total length in cubic millimeters)] was calculated for a subsample of at least 25 apparently healthy female dab from the middle length class (20-24 cm) at each site every year as indicators of the physiological status of the fish at the different stations. The catch per unit effort (expressed as number of fish per hectare) was used to estimate the relative population density of dab for each station and year. Flounder were caught by use of smaller research vessels and occasionally commercial shrimp trawlers. Sampling procedures were the same as those for dab, the only difference being that different target length groups were used (20-24, 25-29, and g30 cm). Inspecting Fish for Disease. Fish were examined for signs of the following conditions or diseases with specific diagnostic criteria for recording: lymphocystis (a viral skin disease), more than one surface nodule; epidermal hyperplasia/papilloma (dab only; a possibly viral etiology), one or more lesions g2 mm in diameter; acute/healing skin ulcers (mixed etiology, possibly bacterium-related), one or more open lesions; Glugea sp. in the intestine (dab only; a microsporidian parasite), one or more cysts; liver neoplasms (chemical etiology), one or more grossly visible liver nodules g2 mm in diameter. The analysis included only liver nodules that were histologically confirmed as neoplasms (hepatocellular adenomas/ carcinomas) according to criteria described elsewhere (14, 15). Chemical Analysis. The chemical analysis of biota in 1991-2005 was performed in the context of the OSPAR JAMP monitoring program according to standard protocols (16). Twenty-five individual female dab (20-24 cm) or male flounder (20-35 cm; stratified in five logarithmically equidistant length intervals, each containing five organisms) were taken from each sampling site in 1991-2005, their livers removed, and analyzed for concentrations of representative contaminants. For dab, the livers were pooled prior to analysis, and for flounder, all livers were analyzed individually [cadmium (Cd), polychlorinated biphenyl 153 (PCB153), and hexachlorobenzene (HCB) in liver]. Additional chemical data in flounder for 1986, 1989, and 1991 were obtained from Vethaak and Jol (12). We measured concentrations of biliary 1-OH pyrene, as a relative measure of total PAH uptake, using the synchronous fluorescence spectrometry (SFS) method (6). Bile was collected from the same 15 female fish used for CF and chemical analysis, and an additional 15 males from the middle length class sampled in 1996-2005. Data Presentation and Statistical Methods. We analyzed disease data by fitting a linear logistic model with disease occurrence as the dependent variable and site, year, length, and sex as independent variables (12, 14). The logistic model used predicts the log odds (logits) for the dependent variable as an additive function of the other variables, which enables odds ratios (ORs) to be adjusted for the influence of independent variables. For a disease, the odds are defined as the ratio between the number of fish showing signs of the disease and the number of fish without the disease. We estimated the parameters using the GLIM statistical package (17), in which a binomial error structure was assumed. We tested interaction effects by comparing the model with all main effects and the interaction effect under consideration to the model with the main effects only. The level of significance for each test separately was kept low (R ) 0.01) in order to ensure a low experimentwise error rate. Disease prevalence is expressed as the adjusted prevalence for a specific group of fish (females, middle length group). To assess the relationship between disease occurrence and age, disease
TABLE 1. Overall Observed Prevalences of Diseases in Dab (Limanda limanda) and Flounder (Platichthys flesus) at Each Sitea external diseases site
period
n
D1 D2 D3 D4 D5 all
1991-2005 1991-1999 1991-1999 1991-2005 1991-2005 1991-2005
8496 5441 6191 7208 8645 35 961
F1 F2 F3 F4 F5 all
1991-2004 1991-1999 1991-2004 1991-2005 1991-1999 1991-2005
3268 2548 3706 3834 1927 15 503
F1 F1 F3 F3 all
1983-2004 1985-2004 1983-2004 1985-2004 1983/5-2004
6800
% EP 1.2 1.4 2.3 2.0 1.6 1.7
(0.2-3.8) (0.2-4.4) (0.5-5.5) (0.4-5.0) (0.2-4.2)
internal diseases
% LY 3.6 1.8 1.2 1.0 0.9 1.8
% UL
Dab (data set 1) (1.9-6.4) 3.3 (0.9-5.9) (0.8-3.1) 0.6 (0.0-1.0) (0.7-2.0) 1.0 (0.0-1.8) (0.0-2.1) 0.6 (0.0-1.3) (0.0-2.2) 0.5 (0.0-0.9) 1.3
Flounder (data 1.1 (0.0-4.0) 0.9 (0.0-2.3) 2.3 (0.0-5.3) 1.9 (0.0-6.6) 1.3 (0.0-3.6) 1.6
set 0.4 0.7 1.7 6.7 0.7 2.5
2) (0.0-2.3) (0.0-1.4) (0.0-3.9) (0.3-10.6) (0.0-2.0)
n
% LN
4538 2343 3145 3555 4265 17846
0.8 0.8 0.5 0.7 1.1 0.8
(0.0-2.2) (0.0-1.7) (0.0-3.7) (0.0-5.6) (0.0-7.3)
1967 1435 2355 2232 915 9011
0.8 0.5 0.4 0.4 0.2 0.5
(0.0-2.6) (0.0-2.2) (0.0-1.7) (0.0-1.9) (0.0-0.8)
3574
0.5 (0.0-2.6)
6843 10 417
2.0 (0.0-5.6) 1.5
% GL 0.4 1.3 2.3 4.0 5.6 2.8
(0.0-1.4) (0.2-4.3) (0.7-8.3) (1.6-11.0) (1.8-23.3)
Flounder (data set 3) 2.7 (0.0-10.1) 0.9 (0.0-3.6)
11 457
15.5 (0.0-25.6)
3.1 (0.0-5.1)
18 257
10.7
2.3
a For overall prevalence (percent), all sizes, sexes and years were combined for each data set. LY, lymphocystis; EP, epidermal hyperplasia/papilloma; UL, skin ulcer; GL, Glugea infestation; LN, liver neoplasm; n, total number of fish examined; ne, not examined.
prevalences (in centimeter length classes) were translated into prevalences according to age class using age/length keys for the two sexes separately. We separately analyzed the following three data sets: (i) for spatial and temporal patterns in dab, data on all five diseases for 1991-2005; (ii) for spatial and temporal patterns in flounder, data on all three diseases for 1991-2005; (iii) for long-term temporal patterns in flounder, data on lymphocystis and skin ulcers for 1983-2004 and on liver neoplasms for 1985-2004 at the coastal waters site (site F3) and the Eastern Scheldt (site F1). We compiled data set 3 from data set 2 and additional data previously reported (12). Disease odds ratios were calculated relative to the following “baseline”: site D1 (dab); site F1 (flounder); female (both species), small length group (both species), 1991 (both species), 1983 (flounder, external diseases data set 3), 1985 (flounder, liver neoplasms, data set 3). Pearson correlation coefficients were calculated between adjusted disease prevalence (females, middle length group) and potential risk factors, using the mean value at each site. Because of multiple comparisons we set p < 0.01 to delineate variables worth further investigation. Trend analysis of disease odds ratios and liver and sediment contaminant concentrations was performed with a one-tailed nonparametric Mann-Kendall trend test (18) on a downward trend versus the null hypothesis of no trend with critical p < 0.05. All data were log-transformed to ensure normal distribution, and the analyses were performed with the SPSS statistical package (SPSS v15.0).
Results Overall Observed Disease Prevalence. In dab, Glugea, lymphocystis, and epidermal hyperplasia/papilloma were the most common diseases, followed by liver neoplasm. In flounder, lymphocystis was the predominant disease, followed by skin ulcer and liver neoplasm (Table 1). Spatial and Temporal Disease Patterns: 1991-2005 Data Sets for Dab or Flounder. Significant effects of some or all of the explanatory variables (site, year, length, and sex) were found, depending on the disease (Table 2, Figure 2). No
significant two-way interactions between these four variables were detected. We were unable to achieve a good statistical fit for liver neoplasms in flounder with any of the models using all four explanatory variables. This was largely due to low prevalence and the presence of zero values in many cells of the table. An examination of the age composition of fish at each site revealed a higher proportion of old dab (age 3 and older) at the sites located furthest offshore (D1, D2) compared to more in-shore sites and a slightly lower proportion of young flounder (age 1 and 2) at the coastal site (F3) compared to estuarine sites, particularly site F2 (Supporting Information, Figure S1). However, we observed no important changes in the age composition of dab and flounder over time. For both species, results from the loglinear analysis with age as an explanatory variable gave broadly similar results to the analysis based on length (Supporting Information, Tables S5-S7). Dab. There was significant spatial and temporal variation for all diseases investigated with the exception of liver neoplasms (Table 2). Whereas lymphocystis and skin ulcers showed highest prevalences at the site located furthest offshore on Dogger Bank (D1), the opposite was true of epidermal hyperplasia/papilloma, the prevalence of which was significantly lower at this site than at the other four sampling sites. For example, the estimated odds ratio (OR) of this disease was 2.1 times higher at the site north of Borkum (D3) than at the Dogger Bank site (D1). The prevalence of Glugea was highest at the sites closest to the coastline (sites D4 and D5 with estimated OR of 9.9 and 13.1 respectively). The prevalence of liver neoplasms and lymphocystis showed a significant downward trend over the entire survey area in 1991-2005, while the prevalence of skin ulcers showed a significant increasing trend in this period (Figure 2a). Pronounced fluctuations but no temporal trends were observed for epidermal hyperplasia/papilloma and Glugea sp. in 1991-2005 (Figure 2a). Large dab were more likely to have lymphocystis, skin ulcers, and liver neoplasms than small ones, and males were more likely to have lymphocystis than females (Table 2). VOL. 43, NO. 6, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Summary of Results of Logit Analysis Fitted to Three Data Setsa (a) Logistic Models Chosenb EP
LY
UL
GL
LN
flounder (data set 2)
year, site, length (dev ) 350.4; df ) 286) ne
year, length (dev ) 179.6; df ) 183) no good statistical fit
ne
year, site, length (dev ) 318.2; df ) 286) year, site, length (dev ) 335.6; df ) 321) year, site (dev ) 237.9; df ) 208)
year, site (dev ) 189.0; df ) 180) ne
flounder (data set 3)
year, site, length, sex (dev ) 305.1; df ) 285) year, site, length, sex (dev ) 312.0; df ) 320) year, site, length, sex (dev ) 242.5; df ) 205)
ne
year, site, length, sex (dev ) 101.8; df ) 116)
dab (data set 1)
(b) Estimated Odds Ratiosc effect
EP
site D2/site D1 site D3/site D1 site D4/site D1 site D5/site D1 large/small large/medium female/male
1.3 (0.9-1.7) 2.1 (1.6-2.7) 1.6 (1.2-2.1) 1.3 (1.0-1.7) 2.9 (2.2-3.8)
site F2/site F1 site F3/site F1 site F4/site F1 site F5/site F1 large/small large/medium female/male
ne
site F3/site F1 large/small large/medium female/male
ne
LY
UL
Dab (data set 1) 0.5 (0.4-0.6) 0.2 (0.1-0.2) 0.3 (0.3-0.4) 0.4 (0.3-0.5) 0.3 (0.2-0.4) 0.2 (0.1-0.2) 0.2 (0.2-0.3) 0.2 (0.1-0.2) 2.2 (1.6-3.0) 3.4 (2.5-4.4)
GL
LN
3.6 (2.0-6.3) 5.9 (3.6-9.8) 9.9 (6.2-15.8) 13.1 (8.3-20.7) 3.9 (2.8-5.5)
0.7 (0.5-0.8) Flounder (data set 2) 0.6 (0.4-1.1) 1.5 (0.8-3.1) 2.1 (1.4-3.1) 3.9 (2.2-7.0) 2.1 (1.4-3.2) 19.1 (11.2-32.7) 1.2 (0.7-2.0) 1.9 (0.9-4.1) 9.4 (6.1-14.4) 1.9 (1.5-2.5)
ne
0.4 (0.3-0.5) Flounder (data set 3) 3.9 (3.4-4.6) 3.2 (2.4-4.3) 7.8 (6.2-9.8) 0.4 (0.4-0.5)
ne
3.4 (2.9-4.0) 15.4 (7.1-33.2) 3.2 (1.9-5.3)
a Data set 1, 1991-2005 data for dab; data set 2, 1991-2005 data for flounder; data set 3, 1983/5-2004 data for flounder. LY, lymphocystis; EP, epidermal hyperplasia/papilloma; UL, skin ulcers; GL, Glugea infestation; LN, liver neoplasm; ne, not examined. b Chosen logistic models are shown with model terms included, deviances (dev), and associated residual degrees of freedom (df). c Estimated odds ratios (with 95% confidence intervals) are based on coefficient estimates of site, length, and sex effects where significant. Small, medium-sized, and large fish for data set 1 refer to length classes 15-19, 20-24, and g25 cm; for data sets 2 and 3 these values are 20-24, 25-29, and g30 cm.
Flounder. There was significant spatial and temporal variation for all diseases except liver neoplasms. The prevalence of lymphocystis in flounder was significantly higher in the North Sea coastal zone (F3), Western Wadden Sea (F2), and Ems Dollard (F5) than at other sampling sites in the southern part of the Dutch North Sea. Flounder showed higher prevalence of skin ulcers at all sites relative to the Eastern Scheldt (F1), especially in the Western Wadden Sea (F4), where the proportion of flounder affected with skin ulcers was 19.1 times higher than in the Eastern Scheldt (F1) (Table 2). The occurrence of lymphocystis and skin ulcers showed a significant downward trend in 1991-2005 (Figure 2b). Temporal Patterns: 1983/5-2004 Data Set for Flounder. Using the longer-term data sets did not substantially alter the adjusted odds ratios for the length and sex of lymphocystis. No significant effects of length and sex were found in the case of skin ulcers. Large flounder were more likely to have liver neoplasms than smaller ones, and females were more likely to have liver neoplasms than males (Table 2). All three diseases (lymphocystis, skin ulcer, and liver neoplasms) decreased significantly over the survey period (Figure 2c). In general terms, the prevalence of the three diseases was found to be a factor of 3.2-3.9 higher in the North Sea coastal zone compared to the Eastern Scheldt (the relatively clean site). Lymphocystis and liver neoplasms have reached near-zero 2154
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levels since 2000. Using age instead of length did not substantially alter the patterns obtained previously (Supporting Information, Table S8). Relationship to Condition Factor, Population Density, Water Temperature, and Contaminant Exposure. The temporal pattern in the condition factor (CF) of dab showed a significant increase at all sites up to 1995/1996, followed by a small decrease toward the end of the survey period. The mean CF values for all sites in 1991-2005 ranged between 0.696 and 0.920. Dab at Dogger Bank (D1) showed generally lower CF than those at the other sites. We found a significant, though moderate, negative correlation between CF and the adjusted prevalence of epidermal hyperplasia/papilloma (r ) -0.46, p ) 0.001) and liver neoplasms in dab (r ) -0.35, p ) 0.009). Differences in the mean CF of flounder were largely consistent over the years and sites. The mean CF values for all sites in 1991-2005 ranged between 1.023 and 1.236. The highest mean CF was found in the western Scheldt (site F2) in 1992 (1.236), when the figures differed significantly from almost all other sites and years. We did not identify any significant correlations between CF and the adjusted prevalence of any of the three diseases (p < 0.44). Relative population densities of dab, expressed as the catch per unit effort, were largely consistent between sites and years, with the exception of higher levels in 1991 and 2001 at the sites closest to the coast (D2 and D3). The mean
FIGURE 2. Year odds ratios with 95% confidence intervals for indicator diseases in (a) dab in 1991-2005 relative to 1991, (b) flounder in 1991-2005 relative to 1991, and (c) flounder long-term data set in the coastal zone (F3) in 1983/5-2005 relative to the first year. Data are adjusted for site, length, and sex (a, b) or for length and sex (c). number of dab caught for all sites in 1991-2005 ranged between 18 and 597 per hectare. The figures for flounder were between 2 and 114 fish per hectare. They showed a significant positive association with Glugea (r ) 0.56, p < 0.001), though not with any other disease in both species. The water temperature (mean winter values) showed a moderately positive correlation with the adjusted prevalence of Glugea in dab (r ) 0.34, p ) 0.008) but not with any other diseases in both species. Concentrations of Cd, PCB153, and HCB in dab and flounder liver showed considerable spatial and temporal variation. Overall, higher concentrations of contaminants were found in flounder from estuaries affected by industrial activities and harbors (F2 and F5) than in fish from more coastal waters (F1, F3, and F4). Concentrations of Cd, PCB153, and HCB were generally higher in dab liver at sites closer to the coast than in those from offshore sites. Analysis of the spatiotemporal data set on liver contaminants showed a complex pattern of weak to moderate but significant (positive and negative) correlations between mean contaminant level and adjusted disease prevalence. For dab, the following positive correlations (p < 0.01) were detected: PCB153 with epidermal hyperplasia/papilloma, Glugea, and liver neoplasm; HCB with liver neoplasm; and, Cd with lymphocystis and ulcers. For flounder, concentrations of HCB showed a positive correlation with lymphocystis (p < 0.01). Overall, statistically significant trends (p < 0.05) in mean contaminant concentrations were found in 13 out of 29 site-based time series, the large majority (85%) showing decreasing concentrations (Table 3). Exceptions were Cd in dab liver from the sites situated NW of Terschelling (D2 and D4), which showed a significant upward trend. The downward trends
included those for PCB153 and HCB in flounder liver from the coastal zone (site F3) which declined by a factor of 3-5 according to data collected in 1985-2003. Biliary polyaromatic hydrocarbon (PAH) metabolite levels in dab from 1998 to 2005 were higher at the more coastal sites (D2, D3, and D4), differing overall by about a factor of 2 (Table 2). Biliary PAH metabolite levels in flounder from the coastal sites (F1 and F3) in 1998-2003 showed lower levels of 1-OH pyrene equivalents (and thus lower PAH uptake) than those from the estuarine sites. We observed differences up to 2 orders of magnitude between the various sites. Intermediate levels were found in the western Wadden Sea (site F4). We found no consistent temporal patterns or trends in either species at the various sites, which may be due to low numbers of annual data points.
Discussion Systematically recorded long-term data on changes in disease prevalence in wild flatfish populations can provide valuable information on changes in North Sea ecosystem status. Our most striking finding was the dramatic decrease in the occurrence of chemical-related liver neoplasms in both species over the last two decades. A general downward trend toward background levels is also evident for lymphocystis in both species, with the exception of a significant peak value in dab in 2001. Skin ulcers in flounder also showed a downward trend. The only upward trend observed has been in skin ulcers in dab. The prevalence of liver neoplasms in dab and flounder in the Dutch North Sea region has been decreasing since the early 1990s. In the late 1980s, prevalences were locally as VOL. 43, NO. 6, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Concentration Ranges and Trends of Selected Contaminants in Dab and Flounder Liver and Biliary 1-OH Pyrene for Each Sitea contaminant ranges site
Cd, mg/kg of dry weight
PCB153, ng/g of lipid weight
D1 D2 D3 D4 D5 F1 F2 F3 F4 F5
0.42–0.98 (11) 0.39–0.53 (9) 0.31–0.56 (9) 0.19–0.70 (12) 0.21–0.49 (12) 0.08–0.12 (3) 0.15–0.78 (9) 0.07–0.21 (5) 0.03–0.12 (15) 0.09–0.46 (9)
65–176 (12) 132–297 (9) 164–380 (9) 123–467 (12) 160–429 (12) 83–350 (6) 1002–1788 (9) 188–830 (9) 142–442 (14) 229–517 (9)
direction of trend
HCB, ng/g of lipid weight
1-OH pyrene, ng/mL
Cd
PCB153
HCB
1-OH pyrene
15.2–29.4 (10) 18.2–27.5 (7) 16.5–33.1 (7) 14.5–29.0 (10) 13.4–30.9 (10) 6.0–34.0 (6) 11.6–43.1 (9) 19.3–131 (8) 3.7–38.4 (15) 30.7–228 (9)
1.4–5.1 (5) 7.4–9.6 (2) 9.7–13.6 (2) 5.7–16.7 (5) 4.4–8.3 (5) 3.1–14.5 (5) 22.2–65.2 (7) 2.1–35.0 (6) 5.0–20.0 (9) 13.7–63.9 (7)
0.05T 0.57*v 0.31T 0.52**v 0.18T na 0.37T –0.53T –0.26T 0.22T
–0.33T –0.54*V –0.39T –0.67**V –0.45*V –0.07T 0.17T –0.50*V –0.29T –0.11T
–0.56*V –0.24T 0.14T –0.51*V –0.82**V –0.47T –0.44*V –0.93**V –0.47**V –0.67**V
0.16T na na 0.20T 0.60T –0.60T 0.14T –0.07T –0.67**V –0.43T
a Concentrations are mean annual values (1-OH pyrene, normalized to 479 absorbance at 380 nm) with the number of sampling years given in parentheses. D1–D5 refer to sampling sites for dab, and F1–F5 refer to those for flounder. (V decreasing trend; v increasing trend; T no trend; *p < 0.05; **p < 0.01). na, no time series available.
high as 40% in flounder over 25 cm (older than 3-4 years) in Dutch coastal waters (12). Similarly, in 1986 liver neoplasms were found in 2.2-6.9% of dab over 20 cm in Dutch coastal waters [not included in our analyses because they were taken from different localities (1)] and have declined to low and near-zero values since the mid-1990s. These trends indicate a reduction in environmental exposure levels for fish to environmental genotoxins/carcinogens. A marked decline in the prevalence of liver neoplasms in various fish species has also been reported from U.S. waters. Liver neoplastic and neoplastic-related diseases in English sole from Puget Sound on the U.S. Pacific coast have shown a general decrease since 1999-2005, possibly due to sediment cleanup measures and better source controls (19). Because liver neoplasia develops slowly, an understanding of the movements of the populations under study is essential when attempting to link the occurrence of this condition with contaminant exposure indices. The spatial pattern of liver neoplasms in our study did not suggest a straightforward relationship with contaminants such as PAHs. It is possible that the occurrence of liver tumors in older dab from the less polluted open sea regions is due to the migration of individuals that spent their younger years in polluted coastal waters or relatively polluted offshore areas (13). Interestingly, liver neoplasms were only very rarely observed in flounder captured in the estuarine Western Scheldt and Ems-Dollard estuary (15), sites that are more heavily polluted with PAHs than the coastal zone (16) but where predominantly young fish occur. Vethaak and Jol (12) hypothesized that the high levels of liver neoplasms observed in older fish captured outside an estuary could be related to exposure to high levels of pollutants in the freshwater area in their earlier years. Thus, some aspects of the spatial patterns of liver neoplasms in flounder can be explained by the migratory behavior of fish between inshore feeding grounds and offshore spawning grounds. We would argue that, although tumor-initiating compounds such as genotoxic/carcinogenic PAHs are a necessary but not sufficient cause, the ultimate distribution patterns of liver neoplasms in older flatfish populations inhabiting coastal and offshore waters in the North Sea are primarily determined by a combination of tumor-promoting risk factors. It has been suggested that the significant increased risk of liver neoplasms in female flounder as compared to males could indicate a tumor-promoting role of 17-β-estradiol during sexual maturation (12, 20). It is clear from the above that reproduction-associated migration patterns play a critical role in explaining the distribution of liver neoplasms in flatfish, and at the same time limit the 2156
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straightforward use of this disease in biological effects monitoring. Another potential confounding factor in the use of liver neoplasms in surveys of disease arises from the possible elimination of diseased fish from the population by fishing pressure. Although a decrease in abundance of larger (and thus older) individuals over large parts of the North Sea during the last 30 years as reported by Daan et al. (21) results in a decrease in the absolute numbers of tumorbearing fish, we found no evidence in our 20- or 15-year time series of any shift in population age of both target species. The same authors concluded on the basis of longterm trawl surveys that fishing effort peaked in the mid1980s, declining slightly since (21). The effects of fishing can therefore be disregarded as a major contributing factor in the temporal decrease in liver tumor prevalence in both species. The observed spatial patterns of the microsporidian Glugea sp. in dab, with higher prevalences near the coast than in offshore waters, can be explained by the parasite’s ecophysiology and host-parasite relationships. It has been shown that infestations can be propagated by intermediate hosts such as shrimps and prawns and the infestation rate spreads when the temperature is fairly high, at 16 °C or over (22). Our results also show that CF (low CF indicates malnutrition and may enhance disease risk) and population density (high density may facilitate the transmission of disease) are unlikely to play an important role in the observed temporal variation in this or most of the other diseases examined. However, CF may have contributed to outbreaks of diseases in dab in offshore areas, particularly D1. Furthermore, of all diseases examined, only Glugea showed a significantly positive correlation with relative population density. Such an association is biologically plausible, as the transmission of diseases is likely to be facilitated by higher stock density. Fishery-induced skin injuries of fish from discards or escape from nets might have contributed to the higher prevalences of skin ulcers and lymphocystis in dab in heavily beam-trawled offshore areas (D1, D2, and D4) (12, 23). However, more research would be required to further establish such a link. The decreasing prevalence of ulcers in flounder from the western Wadden Sea since the early 1990s can largely be explained by the considerably improved habitat conditions for fish in this area, including adapted sluice management and measures to improve fish migration through the sluices (24).
In general, water quality in the Dutch aquatic environment has improved considerably over the last couple of decades (25). The pressures from direct inputs of nitrogen and phosphorus in 2003 had reduced in coastal waters by 25% and 60% respectively relative to 1985. Contaminants in Dutch waters have also shown a significant decrease over the last 20-25 years as a result of actions taken by the Dutch government to ban and control the amount of contaminants such as PCBs entering the aquatic environment. However, contaminants are still an issue in localized areas such as harbors and in most inshore waters (25). Our findings showing generally decreasing levels of PCB153 and HCB in fish in the last two decades, with higher levels in estuaries than in marine waters, fit this broad picture well. They are also in line with other recent and more extensive assessments covering larger regions of the North Sea and longer periods. A recent assessment report by OSPAR on data collected under the CEMP has shown generally downward trends in concentrations of hazardous substances, including Cd, Hg, Pb, HCB, the PAHs fluoranthene and benzo[a]pyrene, PCB153, HCB, and TBT in sediment or biota in the northeastern Atlantic, including the southeastern North Sea (26). A regional assessment showed that Cd and PCB levels in surface sediments in 1981-1996 fell by 71% and 70% in the Dutch coastal zone; PCB levels in the open sea area (>20 km from the coast) fell by 80%. The median PAH concentration in surface sediments decreased slightly in the coastal zone (by 20%) in 1986-1996, but a more recent analysis of 1986-2003 data revealed no trend (27). This agreed with our results for biliary 1-OH pyrene in flatfish. Overall, the majority of measurements show that concentrations of metals and most of the organic contaminants in the Dutch North Sea have decreased significantly in recent decades. Nevertheless, current concentrations of Cd, PCBs, and PAHs at our monitoring sitesseven the reference sites (D1 and F1)sstill exceed Dutch maximum permissible concentrations, suggesting that not only estuarine or coastal waters but also offshore waters may still suffer from a potential pollution problem, particularly from PAHs. Despite this, these concentrations appear not to exceed the risk levels for liver neoplasia and/or increase the risk of attracting diseases such as lymphocystis and epidermal hyperplasia/ papilloma in Dutch flatfish populations. We conclude that the widespread decline in major skin diseases and liver neoplasms in dab and flounder in Dutch waters in the past 15-20 years is most likely due to improved water quality in this region. Our assessment shows that readily visible fish diseases provide a suitable tool for marine environmental quality monitoring and can be used to evaluate one particular facet of North Sea ecosystem health. Although disease prevalence in flatfish from the Dutch North Sea appears to show a general decrease, this is not the case for skin ulcers in offshore waters. Therefore, continued monitoring in the North Sea is needed to identify consistently degraded sites and those with emerging problems. Fish disease monitoring and other biological effect monitoring techniques could make a useful contribution to the EU Marine Strategy Framework Directive that will apply an ecosystem approach. To give a sufficiently comprehensive assessment of ecosystem health for environmental management, however, other indicators will probably be required in addition to the chemical and pathological indicators.
Acknowledgments We thank to Victor Langenberg, Michiel Kotterman and Rob Bovenlander for coordinating the monitoring. Fish disease surveys and chemical analysis were carried out by the
National Institute for Fisheries Research (currently IMARES) in collaboration with Rijkswaterstaat/RIKZ.
Supporting Information Available Tables S1-S16, showing number of examined fish, logistic model estimates adjusted for length or age, and correlation matrices of disease occurrence with environmental and hostrelated risk factors, and Figure S1, showing the age composition of fish sampled. This information is available free of charge via the Internet at http://pubs.acs.org.
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