Bile samples from dab (Limanda limanda) were collected at five offshore and coastal sites and from flounder (Platichthys flesus) at five coastal and estuarine sites (Figure 1). The two fish species are complementary in their distribution: flounder is a common species in coastal waters and estuaries (Vethaak and Jol, 1996), whereas dab is a major species widely distributed in the North Sea (Rijnsdorp et al., 1992). Fish may migrate at the time scale of PAH uptake and metabolism (days), so the 1-OHPyr levels will reflect an average for fish in the area where they were caught.

Annual surveys were carried out from 1998 to 2006. In the period 2006 to 2012, the sampling of dab ceased and the number of sampling sites and sampling rate for flounder was reduced, essentially because of financial constraints. The sampling procedure for dab and flounder was largely based on guidelines by the International Council for Exploration of the Sea (ICES) and OSPAR (Ariese et al., 2005; Davies and Vethaak, 2012). Some variation in PAH metabolites in bile appears to be related to sex and size (Kammann, 2007; Hylland et al., 2012). Therefore, these factors were accounted for in the sampling design.

Dab were caught in March, during their spawning migration period (January–April; Rijnsdorp et al., 1992) and flounder in September, at the end of their non-migratory pre-spawning phase (Vethaak and Jol, 1996). 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 >25 cm). Flounder were caught by use of smaller research vessels and occasionally commercial trawlers. An exception is the Western Scheldt (F2) where standing nets were used in 2005, 2006, 2007, and 2008. 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 >30 cm). Bile was collected from the same 15 female fish used for chemical analysis, and from an additional 15 males from the middle length class sampled in 1998–2012.

Fish were killed with an overdose of MS222 (ethyl 3-amino benzoate methanesulfonate, Sigma-Aldrich, Steinheim, Germany). Bile samples were collected by piercing the exposed gall bladder with a 1-ml syringe and emptying it in microcentrifuge tubes. Bile samples of typically 0.1–1 ml were immediately frozen and stored at −80°C until analysis in the laboratory. Samples from different individuals were analyzed separately.

Additional data on bile metabolites in flounder in 1996 were obtained from Ariese et al. (1997) and Vethaak et al. (2002). These flounders were caught in 1996 along the Dutch coast using a commercial fish trawler, or in polluted harbor areas of the port of Amsterdam [IJmuiden near an industrial steel works site (HO) and Amerikahaven in the North Sea Canal (NZK)], and in the harbor of Rotterdam (RHS, RHB) using fyke nets. Samples collected in 1996 from the Wadden Sea (F4), Eastern Scheldt (F1), and Ems-Dollard (F5) were collected by means of a beam trawl and each sample relates to pooled bile samples of 3 individual fish (see also Table S1).

The data used in this study derived from a data bank for which fish samples were collected in framework of a national monitoring program as part of a larger integrated program. The fish sampling procedures and protocols have been approved since 2003 by the ethical committee for animal welfare in experiments of the National Institute for Coastal and Marine Management (RIKZ) and comply with Dutch laws.

To assess the PAH exposure of fish, 1-OHPyr was determined in bile samples after enzymatic hydrolysis by means of high performance liquid chromatography (HPLC) with fluorescence (FL) detection (as described in detail in RWS protocol Code: A5.217; see also Ariese et al., 1997, 2005 and for review, Beyer et al., 2010). Bile samples were thawed and two subsamples were taken. For the determination of 1-OHPyr under standard conditions a minimum of 20 μL bile volume is required. One sub-sample was incubated with β-glucuronidase/arylsulfatase, then the reaction was stopped with ethanol and the sample centrifuged. The levels of 1-OHPyr in the supernatant was directly analyzed by means of reversed-phase HPLC-FL using a standard solution for external calibration. The second sub-sample of bile, diluted 1:100 with demineralized water/ethanol 50:50, was used for the measurement of the absorbance at 380 nm (mostly due to biliverdin). The HPLC-FL method showed excellent sensitivity: 1-OHPyr could be detected at the 0.4 ng/ml level, which will be amply sufficient for monitoring studies in the Dutch marine environment. In 9% of the cases, there was insufficient volume of bile to perform the analysis or the analysis of the bile sample did not perform well with HPLC. These measurements were indicated as “missing values.”

Originally a different technique, synchronous fluorescence spectrometry (SFS) was used to determine the concentrations of PAH metabolites in the bile samples of flounder from 1996 to 1999 (historical data used for comparison, see Table S1). The SFS method is a more rapid and simple method for the determination of 1-OHPyr in non-hydrolyzed fish bile samples but produces similar results as the HPLC/FL method; the numbers may be compared directly provided the differences in detection sensitivity between 1-OHPyr and conjugated 1-OHPyr (predominantly pyrene-1-glucuronide) are properly corrected for (Ariese et al., 1993, 1997, 2005; Beyer et al., 2010).

A summary of numbers of fish (dab and flounder) caught and bile samples analyzed per sampling location per year is given in Table S1. In total 1855 bile samples were analyzed for 1-OHPyr levels: 417 bile samples of dab caught from 1998 to 2005 and 1438 bile samples of flounder caught from 1996 to 2012. The influence of the size of the fish within the selected length class is negligible: larger specimens would ingest and metabolize more PAH molecules from water, ingested sediment particles and food, but they would also have larger gall bladders, so the metabolite concentrations are not expected to be different. In the Dutch monitoring programme 15 males: 15 females were sampled at each site in order to minimize the effect of sex, if any, on the PAH metabolite concentrations. Unfortunately, the sex data of the fish was not included in the data set; and thus did not allow us to test the influence of this factor on our results. The sex of flounders (35 males and 37 females) from a subset of monitoring sampling sites in 2006 (the data were published in Schipper et al., 2009) did not significantly influence the concentration of 1-OHPyr in bile (t-test, n = 72, p = 0.25). Other studies also found no significant influence of sex on PAH metabolite concentrations in both dab (Dévier et al., 2013) and flounder (Kammann, 2007). However, the variability within the dataset may increase by combining male and female data, because some authors reported higher PAH metabolite concentrations in males than in females from several fish species including flounder (Vuorinen et al., 2006; Kammann, 2007). The possible influence of fish sex is probably minor if any compared to that of feeding status.

Mean 1-OHPyr concentrations per location per year (raw data, that is, without standardization to Abs380) are listed in Table S2. In order to shed light on the spatial distribution and to allow comparison with historic data, all data have been presented including those from several locations were fish were sampled at only few time points.

Lowest mean concentrations per location were determined in dab caught off-shore the Dutch coast (D1 in 2001; 17 ng/ml). The concentration of 1-OHPyr in dab decreases with increasing distance from the shore with lowest concentrations at locations D1, D5. Among the regular monitoring sites, the highest mean concentrations were determined in flounder from the Western Scheldt (F2 in 2007; 2416 ng/ml), with clearly lower levels at the four other flounder locations. However, by far the highest 1-OHPyr concentrations were found at the four industrialized locations that were sampled in 1996 and 1999.

The main statistics of the ¹⁰log transformed 1-OHPyr data per location are shown in Figure 2 (for data per year see Figure S1, upper graph). As is common for positively skewed data, after log-transformation the mean in most cases approaches the median, except for location HO (most likely due to the low number of samples on this location).

The general effect of standardization on the data is a lowering of the 1-OHPyr concentration (when Abs380 ≥ 1) and in only a few cases (2%) the effect is an increase in the numerical values (Figure S2). These increases are limited to a 10-fold increase (when Abs380 = 0.1) in the majority of cases. More importantly, however, standardization would be expected to reduce the variability within each location and sampling year, and therefore the spread in the y-dimension should be narrower than that in the x-dimension (see Figure S2). However, visual inspection shows that that is not the case for the date grouped per location, and also not per location per sampling year (not shown). In fact, standardization increases the number of high extremes by means of over-correction.

Moreover, the absorption spectra of the bile samples suggest that the Abs380 levels may not always act as a reliable measure for total bile pigment. The mean Abs380 values are much higher in the more PAH polluted sites than in the less polluted ones, which would not be expected if the Abs380 reading would only measure endogenous breakdown products. In principle, this could point at a starvation effect in fish caught by standing net in the Western Scheldt from 2005 to 2008 or fyke nets in the industrialized sites in 1996 and 1998, which would justify standardization of the raw data. However, it could also indicate that in such highly turbid waters the fish might have absorbed much more suspended organic materials that will contribute to the bile density and color. This would lead to an overcorrection of the data, resulting in dampening of differences, as indicated by the statistical analysis (Figure 2). In addition, the relative ratio of Abs380/455/655 should be approximately constant if they represent the main degradation products of heme. However, in the more polluted areas such as the Western Scheldt the ratio is different, with the Abs380 being extra high as compared to the other wavelengths (data not shown).

Previous studies advocated the use of the bile pigment biliverdin (indicated by Abs380) or biliary protein as a means of normalizing biliary OH-PAH metabolite concentrations to feeding status (Aas et al., 2000; Ruddock et al., 2003). For example, biliverdin was identified as a suitable parameter for the standardization of 1-OH pyrene and other PAH metabolites (measured by HPLC-FL) in bile samples from flounder and dab from the North Sea and Baltic Sea (Kammann, 2007). However, several authors have reported that when the concentrations of proteins were used to normalize residues of OH-PAH metabolites, the observed variability in the concentrations did not differ strongly from data expressed without normalization (Escartin and Porte, 1999b; Richardson et al., 2004; Vuorinen et al., 2006). Experimental work has confirmed that changes in feeding status over a 24-h period did not appear to be a significant confounding factor in PAH bile concentrations (Richardson et al., 2004).

In the Dutch monitoring programme, fish samples were in almost all cases obtained from beam trawling. Exceptions were the Western Scheldt (F2) in the period 2005–2008 where standing nets were used due to lack of commercial trawling activities at this site, and the industrialized sites (HO, NZK, RHS, RHB) where catches (in 1996 and 1999) could only be obtained from fyke fisheries (see Table S1). Fish derived from standing nets may have been starved for maximal 12 h and for fish from fyke nets this may be extended to several days. Considering the analysis of the data, a possible confounding effect on bile concentrations cannot be fully explained by the use of a different fishery gear. Flounder from the Western Scheldt exhibited also high 1-OHPyr concentrations in 2009 and 2010 when a beam trawl (instead of a standing net) was used (Tables S1, S2). However, a confounding effect of starvation on the results cannot be completely ruled out. Caution is advised when comparing the results from location F2 from 2005 to 2008 and from the historical sites with other years at this site and with the other flounder sampling locations.

The present study provides a number of arguments why Abs380 is not a suitable parameter for the standardization of 1-OHPyr levels in the bile of fish, and in this work we will therefore focus on the raw, non-standardized data. However, it still can be helpful in the interpretation of the results, for example to determine the lightest and darkest (non-representative) outliers and should therefore be reported as supporting data.

The results of the trend analysis are summarized in Table 1. The trend analysis of the annual medians resulted in only one statistically significant trends: a downward trend at location F4.

The trend analysis of all data mainly shows downward trends, as illustrated for dab in Figure 3 and for flounder in Figure 4. As explained before, some care should be taken using and interpreting the results of the trend analyses that use all the data.

Maintenance and deepening dredging works regularly occur in the Western Scheldt (F2) and this could have influenced the high levels of PAH contamination levels at the sampling site in this estuary. There is evidence that dredging processes might lead to PAH mobilization into the water column and hence significantly increase the bioavailability of PAH to organisms such as fish (Eggleton and Thomas, 2004). Therefore, release of historical PAH contamination from buried sediment through (upstream) dredging operations may also have contributed to the observed high levels without a clear downward trend at the sampling site in the Western Scheldt in 2006–2010. Much higher mud disposal volumes near this site (F2) and upstream in the lower Scheldt River were reported for this period (Depreiter et al., 2015).

The data on 1-OHPyr concentrations in flounder and dab can be interpreted against assessment thresholds for biological effects measurements, recommended by ICES and OSPAR: thresholds between background and elevated but acceptable levels (background assessment concentration, BAC), and between acceptable and unacceptable levels (environmental assessment concentration, EAC; Davies and Vethaak, 2012). Threshold values indicative of significant harm (EAC) are not (yet) set for flounder and dab. However, with some caution the EAC for 1-OHPyr of 745 ng/ml set for halibut (Hylland et al., 2012) could be used. Furthermore, in a 3-year mesocosm study with flounder exposed to contaminated harbor dredging, levels of 1-OHPyr of 510–1030 ng/ml coincided with DNA damage and the development of liver tumors and associated lesions (Vethaak et al., 1996) and alternatively this threshold level could be used. In this case also possible additive effects due to mixture toxicity of PAHs and other toxic contaminants have been accounted for. Comparison of our monitoring data with these threshold values (Table 2) show that in general only 9.6% of the individual dab values from 1998 to 2005 and 0.6% of the individual flounder values from 1996 to 2012 are below BAC for 1-OHPyr (for both species 16 ng/ml). 9.3% of individual flounder and 0.3% of individual dab exceeded the EAC threshold for halibut. The corresponding percentages for flounder and dab for the lower provisional threshold of PAH effects of 510 ng/ml are 15.6 and 0.6%, respectively. By far most of the individual bile values fall between BAC and EAC and exhibit elevated response levels. Comparison of our recent flounder monitoring data (last 5 years) with data from the whole period (1996–2012) shows a decreased risk of PAH contamination at most locations over time with the exception of location F2 that seems to show an increased risk. The assessment of the monitoring data indicates that the PAH contamination in the estuarine sites Western Scheldt (F2) and the Ems-Dollard (F5) are still a high cause for concern. In contrast, PAH exposure levels in the Dutch coastal waters (F3), Eastern Scheldt (F1) and Wadden Sea (F4), and also at the offshore North Sea monitoring sites probably will not cause adverse health effects in flatfish. There are no recent data on the severely polluted industrialized sites that were sampled in 1996 and 1999.

The concentrations of 1-OHPyr in flounder and dab from Dutch waters compare well with those reported for these species in Dutch waters and elsewhere in the North Sea. Kammann (2007) reported 1-OHPyr concentrations (measured by HPLC-FL) in individual fish from < 0.7 to 838 ng/ml in bile of dab and 18 to 200 ng/ml in bile of flounder caught between 1997 and 2004 in the North Sea and Baltic Sea. In the North Sea, highest mean values (n = 20) per station of 1-OHPyr were determined in dab from the German Bight (159 ng/ml) with lower values found in offshore regions. Mean levels of 1-OHPyr in flounder bile in the Baltic Sea ranged between 54 and 92 ng/ml (Kammann, 2007).

Bile metabolite data (measured by HPLC-FL) provided by the international workshop on Integrated Assessment of Contaminants Impacts on the North Sea (ICON), carried out in the autumn of 2008, showed lowest mean levels of 1-OHPyr in flounder from Icelandic waters (18.4 ng/ml). Highest mean levels of 1-OHPyr were found in flounder from the Seine estuary (France) and the Alde river (UK), respectively 316.5 and 373.2 ng/ml. Dab from the North Sea showed mean levels ranging from 7.6 (Iceland) to 345.4 ng/ml in the Seine estuary (Kammann et al., in press). The ICON workshop also included the Dutch flounder sampling location (F4), which showed mean values of 77.0 (min 44.5–max 148.4 ng/ml) (Kammann et al., in press). These values match with the mean values (75.4 ng/ml) independently measured in the present study.

Concentrations between 50 and 500 ng/ml 1-OHPyr (measured by SFS) in flounder from the Dutch Wadden Sea from 1989 to 1993 reported by Eggens et al. (1996) are in agreement with the results found in the present study (32–295 ng/ml in 1996–1999, measured by HPLC-FL).

It is clear that the highest values of 1-OHPyr were found in the more industrialized harbors in the Netherlands in 1996 and 1999. However, there are no recent data available for these sites. Van der Oost et al. (1994) reported highest 1-OHPyr bile concentrations of approximately 8000 ng/ml (measured by SFS) in eel (Anguilla anguilla) caught in 1991 from freshwater sites near Amsterdam. The mean level of 1-OHPyr in eel collected in 2011 from the river Rhine in Germany near the Dutch border was 3160 ng/ml (measured by HPLC-FL) (Kammann et al., 2014).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.