Biological effects methods for cod in the Inner Oslofjord

Biological effect parameters (BEM) are included in the monitoring program to assess the potential pollution effects on organisms. This cannot be done solely on the basis of tissue concentrations of chemicals. There are five BEM methods used (including analyses of degradation products of PAH in bile). Each method is in theory specific for individual or groups of chemicals. One of the

advantages of these methods used at the individual level is the ability to integrate biological and chemical endpoints, since both approaches are performed on the same individuals. The results can be seen in relation to newly established reference values (e.g. OSPAR 2013).

3.3.1 OH-pyrene metabolites in bile

Analysis of OH-pyrene in bile is not a measurement of biological effects, per se. It is included here, however, since it is a result of biological transformation (biotransformation) of PAHs, and is thus a marker of exposure. Quantification methods for OH-pyrene have been improved two times since the initiation of these analyses in the CEMP/MILKYS programme. In 1998, the

support/normalisation parameter was changed from biliverdine to absorbance at 380 nm. In 2000, the use of single-wavelength fluorescence for quantification of OH-pyrene was replaced with HPLC separation proceeding fluorescence quantification. The single wavelength fluorescence method is much less specific than the HPLC method. Although there is a good correlation between results from the two methods, they cannot be compared directly.

PAH compounds are effectively metabolized in vertebrates. As such, when fish are exposed to and take up PAHs, the compounds are biotransformed into polar metabolites which enhances the efficiency of excretion. It is therefore not suitable to analyse fish tissues for PAH parent

compounds as a measure of exposure. However, since the bile is a dominant excretion route of PAH metabolites, and since the metabolites are stored for some time in the gall bladder, the bile is regarded as a suitable matrix for analyses of PAH metabolites as a measure of PAH exposure.

In 2017 the median concentration of OH-pyrene metabolites in bile from cod in the Inner Oslofjord (st. 30B) were significantly higher than in 2016 (Tukey-Kramer HSD test), more than twice as high and the highest median the last 8 years. Median OH-pyrene bile concentration in 2017 was above the ICES/OSPAR assessment criterion (background assessment criteria, BAC) in this area as well as in fish from the Inner Sørfjord (st. 53B) and Skågskjera in Farsund (st. 15B). Furthermore, median OH-pyrene bile concentration in 2017 was slightly above the ICES/OSPAR assessment criterion also at Bømlo on the West coast (st. 23B, reference station), the station where concentrations were lowest. Note that the unit of the assessment criterion is ng/ml, without normalization to

absorbance at 380 nm. Also, in the Inner Sørfjord (st. 53B), the median concentration of OH-pyrene metabolites in bile from cod were significantly higher than in 2016 (Tukey-Kramer HSD test), by more than a factor of three, and the highest median since HPLC separation proceeding

fluorescence quantification was applied for this parameter. Among the four stations, OH-pyrene concentrations were significantly higher in the Inner Sørfjord (st. 53B) (Tukey-Kramer HSD test) however, no significant short-term trend could be observed in the Sørfjord (st. 53B) (Appendix F).

3.3.2 ALA-D in blood cells

Inhibited activity of ALA-D indicates exposure to lead. Although ALA-D inhibition is lead-specific, it is not possible to rule out interference by other metals or organic contaminants. Note that the protocol for ALA-D analysis was slightly altered (to avoid Hg-containing reagents) in 2017.

Trend analyses suggest a significant downward temporal trend in ALA-D activity over the last 10 years (n = 8) at the reference station (Bømlo area; 23B; Appendix F). The median ALA-D activity at this station appeared, however, slightly higher than the previous four years.

As previously noted, most years up to 2011 the activity of ALA-D in cod was somewhat inhibited in the Inner Oslofjord (st. 30B), compared to reference stations, i.e. Outer Oslofjord (st. 36B; only data to 2001), Bømlo in the Bømlo-Sotra area (st. 23B), and Varangerfjord (st. 10B; only data to 2001, not shown) (Green et al. 2016 – M-618|2016). The median ALA-D activity in the Inner Oslofjord (st. 30B) in 2017 was significantly lower (Tukey-Kramer HSD test) than in in the Bømlo-Sotra area (st. 23B, reference station). Also in the Inner Sørfjord (st. 53B), the median activity of ALA-D was significantly lower than at the reference station (st. 23B) (Tukey-Kramer HSD test). The often lower activities of ALA-D in cod from the Inner Oslofjord and Inner Sørfjord compared to the reference station (basis for comparison prior to 2007, 2009-2011 and 2013-2017) indicate the contamination of lead. Higher concentrations of lead in cod liver have generally been observed in the Inner Oslofjord and Inner Sørfjord compared to Bømlo, though with a relatively large individual variation. Median concentrations of lead in cod liver from the Inner Oslofjord (st. 30B) and the Sørfjord (st. 53B) were 0.145 mg/kg and 0.062 mg/kg, respectively, in 2017. In the Bømlo-Sotra area (st. 23B) the concentration was below the limit of detection (<0.03 mg/kg).

3.3.3 EROD-activity

High activity of hepatic cytochrome P4501A activity (EROD-activity) normally occurs as a response to the contaminants indicated in Table 5. It was expected that higher activity would be found at the stations that were presumed to be most impacted by planar PCBs, PCNs, PAHs or dioxins such as the Inner Oslofjord (st. 30B). In 2017, median EROD-activity in liver of cod from Bømlo (st. 23B), the Inner Oslofjord (st. 30B) and the Inner Sørfjord (st. 53B) were about30% higher than in 2016.

The median EROD-activity also were somewhat higher in the Oslofjord (st. 30B), than at stations 23B and 53B. Since 2000, the median EROD-activity has generally been higher in the Inner Oslofjord compared to the reference station on the west coast (Bømlo, st. 23B). in 2017. Statistically

significant downward trends in EROD activity were observed on a long-term basis (whole data series) at Bømlo (st. 23B) and the Inner Oslofjord (st. 30B) (Figure 64). Median EROD-activities were below the ICES/OSPAR assessment criterion (background assessment criteria, BAC).

No adjustment for water temperature has been made. Fish are sampled at the same time of year (September-November) when differences between the sexes should be at a minimum. Previous statistical analyses indicated no clear difference in activity between the sexes (Ruus et al. 2003 – TA-1948/2003). It has been shown that generally higher activity occurs at more contaminated stations (Ruus et al. 2003 – TA-1948/2003). However, the response is inconsistent (cf. Appendix F), perhaps due to sampling of populations with variable exposure history. Besides, there is evidence from other fish species that continuous exposure to e.g. PCBs may cause adaptation, i.e. decreased EROD-activity response.

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A

B

Figure 64. Median activitet (pmol/min/mg-protein) of EROD in cod liver from 1990 to 2017 in the Inner Oslofjord (st. 30B) (A) and from 1997 to 2017 in Bømlo (st. 23B) (B). The provisional high reference concentration (PROREF) and the factor exceeding PROREF are indicated with horizontal dashed lines (see Figure 5 and Appendix C).