Condition monitoring in the water column 2005: Oil hydrocarbons in fish from Norwegian waters

This report has been prepared by Institute of Marine Research (IMR) & University of Stockholm (UoS) on behalf on the offshore petroleum industry operators on the Norwegian Continental Shelf as part of the authority requirements in the Health, Safety and Environmental regulation (Activity regulation). The objectives for this study have been: 1. Determine to what extent fish from the oil installation areas at Tampen and the Halten Bank contain elevated levels of petroleum hydrocarbons compared with fish from reference areas at the Egersund Bank/Ling Bank and in the Barents Sea by measurements of NPD/PAH in fish muscle. 2. Comparison of NPD/PAH bile metabolites in fish from Tampen compared with fish from Egersund Bank/Ling Bank 3. Study possible genototoxic effects in fish from Tampen compared with fish from Egersund Bank/Ling Bank by measurements of hepatic DNA adducts. 4. Document to what extent discharges of alkylphenols in produced water cause estrogenic effects in fish from Tampen by measurements of alkylphenols in muscle and liver, and vitellogenin in plasma. Di- and polyaromatic hydrocarbons (NPD/PAH) have been analysed in muscle of cod and haddock caught in the North Sea at Ling Bank/Egersund Bank (reference), Tampen, Halten Bank and Barents Sea (reference), autumn 2005 and concentrations found to be below levels of quantification (LOQ) for fish sampled from all regions. Cod sampled at the Ling Bank/Egersund Bank in the Southern part of the North Sea had the same levels of PAH metabolites in bile as cod sampled from the Tampen region. Haddock demonstrated significantly higher levels of fluorescence for all three wavelength pairs measured, indicating a higher levels of 2-, 3-, 4- and 5-ring PAHs for haddock sampled in the Tampen region compared with haddock from the Ling Bank/Egersund Bank region. Saithe, on the other hand demonstrated higher levels of 2- and 3-ring compounds at the Ling Bank/Egersund Bank compared with fish sampled at Tampen. Overall, the highest levels of PAH metabolites in bile were measured in haddock. DNA adducts were analyzed in liver of cod, haddock and saithe at Tampen and from Ling Bank/Egersund Bank (reference site). In both areas the highest levels of DNA adducts were measured in haddock. The percentage of individuals with detectable adducts was also higher in haddock than for the other species. Haddock from Tampen had significant higher DNA adduct levels compared with haddock from Egersund Bank/Ling Bank (Mann-Whitney test), indicative of more PAH exposure in this region. Higher levels of DNA adducts in haddock caught at Tampen compared with the Egersund Bank have earlier been reported by Klungsøyr et al. (2003). Significant differences were not found for cod and saithe collected from the same areas. Analyses of alkylphenols in cod liver, haddock liver and herring muscle from Ling Bank/Egersund Bank and Tampen regions demonstrated levels below limits of detection (LOD) for all stations. The absence of alkylphenols in fish is in line with the results from the 2002 monitoring (Klungsøyr et al., 2003), and it supports the risk assessment carried out by Myhre et al. (2004) that stated that the risk for estrogenic and reproductive effects in fish after alkylphenols exposure from produced water discharges is very low. There were no differences in VTG concentration in plasma of cod caught at Tampen compared with Ling Bank/Egersund Bank that could not be explained by differences in size and sexual maturation

Condition monitoring in the water column 2008: Oil hydrocarbons in fish from Norwegian waters

The objectives for this study have been: 1. Measure NPD/PAH in haddock liver from the Egersund Bank, the Halten Bank andthe Barents Sea. 2. Determine to what extent fish from the oil installation areas at Tampen and the Halten Bank contain elevated levels of petroleum hydrocarbons compared with fish from reference areas at the Egersund Bank and in the Barents Sea by measurements of metabolites of PAH and alkylphenols in bile of haddock, saithe and long rough dab. 3. Measure vitellogenin levels in blood from male cod from the Egersund Bank, Tampen, the Halten Bank and the Barents Sea. 4. Study possible genotoxic effects in fish from Tampen compared with fish from the Egersund Bank/Ling Bank by measurements of hepatic DNA adducts. 5. Perform gonad histology on haddock and long rough dab to study whether differences can be observed between fish caught at Tampen compared with fish caught at the Egersund Bank and the Barents Sea

PAH and biomarker measurements in fish from condition monitoring in Norwegian waters in 2005 and 2008

Condition monitoring in fish from open seas are performed in Norway every third year. The objectives are to investigate whether fish from Norwegian seas contain elevated levels of components that originate from discharges from the petroleum activity. We present results from the samplings in 2005 and 2008 from four areas: The Egersund Bank (reference area North Sea), Tampen, the Halten Bank and the Barents Sea (reference area). NPD and PAH measured in fish muscle in 2005 were found to be below LOQ for all regions. In 2008 sum NPD were measured in haddock liver and found to be low for all regions (8-34 ng/g wet weight). Bile metabolites from haddock were measured by GCMS in 2008. The main contributor to sum PAH metabolites at Tampen and at the Egersund Bank was 1-hydroxy phenanthrene with levels of 510±814 and 133±207 ng/g bile, respectively. Levels of this metabolite in haddock from the Halten Bank and the Barents Sea were 43±71 and 19±14 ng/g bile, respectively. Levels of alkylphenols were found below LOQ. Levels of Vtg in blood of male cod were generally low from all regions. Measurements of DNA adducts in fish liver did not show changes for cod and saithe, while a significant increase were observed in haddock from Tampen compared to haddock from the Egersund Bank. Lipid content in the liver was significantly reduced in haddock from Tampen. Fatty acid profiles showed that haddock from Tampen had relatively high levels of arachidonic acid, and the ratio between omega-3 and omega-6 ((n-3)/(n-6)) poly unsaturated fatty acids were significantly lower in neutral lipids, free fatty acids and phosphotidylcholine/phosphotidylethanolamine, compared with haddock from the other regions

Feeding Activity and Xenobiotics Modulate Oxidative Status in Daphnia magna: Implications for Ecotoxicological Testing

To apply biomarkers of oxidative stress in laboratory and field settings, an understanding of their responses to changes in physiological rates is important. The evidence is accumulating that caloric intake can increase production of reactive oxygen species and thus affect background variability of oxidative stress biomarkers in ecotoxicological testing. This study aimed to delineate effects of food intake and xenobiotics on oxidative biomarkers in Daphnia magna. Antioxidant capacity measured as oxygen radical absorbance capacity (ORAC) and lipid peroxidation assayed as thiobarbituric acid reactive substances (TBARS) were measured. Food intake was manipulated by varying food densities or by exposing the animals to chemicals inhibiting feeding rate (pharmaceutical haloperidol and pesticide lindane). Feeding rate proved to affect both protein, ORAC, and TBARS in unexposed daphnids. However, there was no significant effect of feeding rate on the protein-specific ORAC values. Both substances affected individual protein and ORAC levels and changed their relationship to feeding rate. Our results show that inhibition of feeding rate influenced the interpretation of biomarker response and further emphasize the importance of understanding (1) baseline variability in potential biomarkers due to variations in metabolic state and (2) the contribution of feeding rate on toxic response of biomarkers

Disparate effects of antibiotic-induced microbiome change and enhanced fitness in Daphnia magna

It is a common view that an organism’s microbiota has a profound influence on host fitness; however, supporting evidence is lacking in many organisms. We manipulated the gut microbiome of Daphnia magna by chronic exposure to different concentrations of the antibiotic Ciprofloxacin (0.01–1 mg L-1), and evaluated whether this affected the animals fitness and antioxidant capacity. In line with our expectations, antibiotic exposure altered the microbiome in a concentration-dependent manner. However, contrary to these expectations, the reduced diversity of gut bacteria was not associated with any fitness detriment. Moreover, the growth-related parameters correlated negatively with microbial diversity; and, in the daphnids exposed to the lowest Ciprofloxacin concentrations, the antioxidant capacity, growth, and fecundity were even higher than in control animals. These findings suggest that Ciprofloxacin exerts direct stimulatory effects on growth and reproduction in the host, while microbiome- mediated effects are of lesser importance. Thus, although microbiome profiling of Daphnia may be a sensitive tool to identify early effects of antibiotic exposure, disentangling direct and microbiome-mediated effects on the host fitness is not straightforward

Sucralose Induces Biochemical Responses in <i>Daphnia magna</i>

<div><p>The intense artificial sweetener sucralose has no bioconcentration properties, and no adverse acute toxic effects have been observed in standard ecotoxicity tests, suggesting negligible environmental risk. However, significant feeding and behavioural alterations have been reported in non-standard tests using aquatic crustaceans, indicating possible sublethal effects. We hypothesized that these effects are related to alterations in acetylcholinesterase (AChE) and oxidative status in the exposed animals and investigated changes in AChE and oxidative biomarkers (oxygen radical absorbing capacity, ORAC, and lipid peroxidation, TBARS) in the crustacean <i>Daphnia magna</i> exposed to sucralose (0.0001–5 mg L⁻¹). The sucralose concentration was a significant positive predictor for ORAC, TBARS and AChE in the daphnids. Moreover, the AChE response was linked to both oxidative biomarkers, with positive and negative relationships for TBARS and ORAC, respectively. These joint responses support our hypothesis and suggest that exposure to sucralose may induce neurological and oxidative mechanisms with potentially important consequences for animal behaviour and physiology.</p></div

Generalized Linear Models for biomarkers of oxidative damage (lipid peroxidation, TBARS, pmol ind⁻¹), antioxidative defense (total oxygen radical absorbance capacity, ORAC, mg Trolox ind⁻¹) and neurotoxicity (acetylcholinesterase activity, AChE, nmol AcSCh min⁻¹ mg protein⁻¹) in <i>Daphnia magna</i> exposed to a range of sucralose concentrations (sucralose, 0.1–5000 µg L⁻¹); individual protein weight (protein, µg ind⁻¹) was used as a proxy for body size.

<p>(A) TBARS as a function of sucralose concentration, protein and ORAC; (B) ORAC as a function of sucralose concentration and protein; and (C) AChE as a function of sucralose concentration and ORAC. All data for the regression analysis were Box-Cox transformed; significant effects are in bold face.</p

Experimental concentrations of sucralose and biomarker values measured in <i>Daphnia magna</i>.

<p>A control using M7 medium was included in each of the four experiments (Exp I to IV). Data for the biomarkers within the experiment are shown as ranges (min-max) and for controls as mean values. Note that number of treatments differs among the experiments.</p

Relationships between AChE, ORAC and TBARS in <i>Daphnia magna</i> (all data from the Experiments 1–4 are combined).

<p>The data are Box-Cox transformed and normalized to the respective controls.</p

Disparate effects of antibiotic-induced microbiome change and enhanced fitness in Daphnia magna.

It is a common view that an organism's microbiota has a profound influence on host fitness; however, supporting evidence is lacking in many organisms. We manipulated the gut microbiome of Daphnia magna by chronic exposure to different concentrations of the antibiotic Ciprofloxacin (0.01-1 mg L-1), and evaluated whether this affected the animals fitness and antioxidant capacity. In line with our expectations, antibiotic exposure altered the microbiome in a concentration-dependent manner. However, contrary to these expectations, the reduced diversity of gut bacteria was not associated with any fitness detriment. Moreover, the growth-related parameters correlated negatively with microbial diversity; and, in the daphnids exposed to the lowest Ciprofloxacin concentrations, the antioxidant capacity, growth, and fecundity were even higher than in control animals. These findings suggest that Ciprofloxacin exerts direct stimulatory effects on growth and reproduction in the host, while microbiome- mediated effects are of lesser importance. Thus, although microbiome profiling of Daphnia may be a sensitive tool to identify early effects of antibiotic exposure, disentangling direct and microbiome-mediated effects on the host fitness is not straightforward