Quantitative Nontarget Analysis of CECs in Environmental Samples Can Be Improved by Considering All Mass Adducts.

Quantitative nontarget analysis (qNTA) for liquid chromatography coupled to high-resolution mass spectrometry enables a more comprehensive assessment of environmental samples. Previous studies have shown that correlations between a compound's ionization efficiency and a range of molecular descriptors can predict the compound's concentration within a factor of 5. In this study, the qNTA approach was further improved by considering all mass adducts instead of only the protonated ion. The model was based on a quantitative structure-property relationship (QSPR), including 216 contaminants of emerging concern (CECs), of which 80 exhibited adduct formation that accounted for >10% of the total peak intensity. When all mass adducts were included, the test set coefficient of determination improved to Q2 = 0.855 compared to Q2 = 0.670 when only the protonated ions were considered (test set median RF error factor 1.6). The inclusion of all adducts was also important to transfer the RF QSPR model reliably. It was assumed that RF variations are sequence-dependent; therefore, a second QSPR model for the prediction of the transferability factor was built for each sequence. For validation, samples were analyzed up to two years apart. The median prediction fold change was 1.74 for analytical standards (63 compounds) and 2.4 for enriched wastewater effluent samples (41 compounds), with 80% of the compounds predicted within a fold change of 2.4 and 3.3, respectively. The model was also validated on a second instrument, where 80% of the 26 compounds in wastewater effluent were predicted within a factor of 3.8.

Supercritical Fluid Chromatography Coupled to High-Resolution Mass Spectrometry Reveals Persistent Mobile Organic Compounds with Unknown Toxicity in Wastewater Effluents.

Broad screening approaches for monitoring wastewater are normally based on reversed-phase liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS). This method is not sufficient for the very polar micropollutants, neglected in the past due to a lack of suitable analytical methods. In this study, we used supercritical fluid chromatography (SFC) to detect very polar and yet-undetected micropollutants in wastewater effluents. We tentatively identified 85 compounds, whereas 18 have only rarely been detected and 11 have not previously been detected in wastewater effluents such as 17α-hydroxypregnenolone, a likely transformation product (TP) of steroids, and 1H-indole-3-carboxamide, a likely TP from new synthetic cannabinoids. Suspect screening of 25 effluent wastewater samples from 8 wastewater treatment plants revealed several distinct potential pollution sources such as a pharmaceutical company and a golf court. The analysis of the same samples with LC-HRMS showed clearly how SFC increases the ionization efficiency for low-molecular-weight micropollutants (m/z < 300 Da) by a factor 2 to 87 times, which significantly improved the mass spectra for identifying very polar compounds. In order to assess which micropollutants might be of environmental concern, literature and toxicological databases were screened. There was a lack of available hazard and bio-activity data for regulatory-relevant in vitro and in vivo assays for >50% of the micropollutants. Especially, 70% of the data were lacking for the whole organism (in vivo) tests.

Source-Supported Suspect Screening (4S) of Phytotoxins in Terrestrial and Aquatic Environments: A Field Study of Lupinus angustifolius L. (Blue Lupin).

Phytotoxins (PTs) are bioactive secondary metabolites produced by plants. More recently, they have been recognized as important aquatic micropollutants. Despite that, only a few PTs have been detected and reported in terrestrial and aquatic environments, while their source and leaching pathways remain largely unclear. Herein, we established a novel approach named source-supported suspect screening (4S) to discover PTs in different environments, investigate their environmental occurrences, identify their sources, and initiate discussions on their leaching mechanisms. The 4S-approach was demonstrated on a five-month Lupinus angustifolius L. (L. angustifolius) crop field experiment, where plant, topsoil, drainage water, and surface water were sampled and analyzed. As a result, 72 PTs (flavonoids and alkaloids) were identified at high confidence, with 10 PTs fully confirmed. Fifty-three PTs detected in soil or water were linked to L. angustifolius, among which 26 PTs were coherently detected in all three environmental compartments. The occurrence and abundance of PTs in terrestrial soil and aquatic environments were influenced by the plant growth stage and precipitation. Soil served as an intermedium when PTs leached from L. angustifolius to the drainage water, while the degree of retardation and eventual occurrence in the aquatic environment depended on both PTs and soil physico-chemical properties.

Ultra-high-performance supercritical fluid chromatography-mass spectrometry for the analysis of organic contaminants in sediments.

A nontarget screening method was developed based on D-optimal designs for ultra-high performance supercritical fluid chromatography with positive and negative electrospray ionization mode mass spectrometry. A mixture of organic contaminants such as pesticides, steroids, surfactants, phenolic and fatty acids, and polycyclic aromatic hydrocarbon derivatives, was used for the optimization. An aprotic mixture of dichloromethane and acetone [3:1] performed overall best as the injection solvent. The highest peak capacities (n) were accomplished at the shallowest gradient (1%B/min), ammonium formate (n = 378 in negative ionization mode), or ammonium acetate (n = 327 in positive ionization mode) in methanol as the modifier. Capillary voltage, make-up solvent flow rate, water, and additive concentration were the most significant factors for improving peak intensity: higher peak intensities were obtained at lower additive concentrations (5mM ammonium formate), and with 5% water in positive ionization mode. Conversely, water had detrimental effects in negative ionization mode. The optimized method was used to quantify organic contaminants in 17 freshwater sediment samples from Copenhagen, Denmark. Out of 50 monitored contaminants, 35 were detected in at least one sample. Further, the method has a potential for target and nontarget screening analysis of organic contaminants in solid matrices.

Correction of Matrix Effects for Reliable Non-target Screening LC-ESI-MS Analysis of Wastewater.

Matrix effects are well-known challenges for accurate and comparable measurements with liquid chromatography (LC) electrospray ionization mass spectrometry (ESI-MS). This study describes a three-step method to evaluate and compensate for matrix effects in enriched wastewater extracts using LC ESI-high-resolution MS (HRMS). As a first step, the "dilute and shoot" approach was used to determine the optimal relative enrichment factor (REF) for a direct comparison between wastewater influent (REF 10) and effluent (REF 50) extracts. However, the rapid decrease in the number of non-target compounds detected with increasing dilution leads to the need for a correction of the matrix effect for analyzing samples with higher REFs. As a second step, the observed matrix effect at higher REFs was corrected by the retention time-dependent matrix effect. A new scaling (TiChri scale) of the matrix effect was introduced, which demonstrates that the total ion chromatogram (TIC) predicts the matrix effect as effectively as post-column infusion (PCI) approaches; thus, the average median matrix effect was improved from -65 to 1% for influent (REF 100) and from -46 to -2% for effluent extracts (REF 250). The TIC traces for concentrated (REF 250) influent and effluent samples were successfully used to correct the matrix effects and allowed the extent of micropollutant degradation in three WWTPs to be quantified. As a final step, the residual structure-specific matrix effect was predicted and corrected by quantitative structure-property relationships (QSPR), which led to a further correction of the matrix effect to 0 ± 7% for 65 compounds.

Ethephon-induced changes in antioxidants and phenolic compounds in anthocyanin-producing black carrot hairy root cultures.

Hairy root (HR) cultures are quickly evolving as a fundamental research tool and as a bio-based production system for secondary metabolites. In this study, an efficient protocol for establishment and elicitation of anthocyanin-producing HR cultures from black carrot was established. Taproot and hypocotyl explants of four carrot cultivars were transformed using wild-type Rhizobium rhizogenes. HR growth performance on plates was monitored to identify three fast-growing HR lines, two originating from root explants (lines NB-R and 43-R) and one from a hypocotyl explant (line 43-H). The HR biomass accumulated 25- to 30-fold in liquid media over a 4 week period. Nine anthocyanins and 24 hydroxycinnamic acid derivatives were identified and monitored using UPLC-PDA-TOF during HR growth. Adding ethephon, an ethylene-releasing compound, to the HR culture substantially increased the anthocyanin content by up to 82% in line 43-R and hydroxycinnamic acid concentrations by >20% in line NB-R. Moreover, the activities of superoxide dismutase and glutathione S-transferase increased in the HRs in response to ethephon, which could be related to the functionality and compartmentalization of anthocyanins. These findings present black carrot HR cultures as a platform for the in vitro production of anthocyanins and antioxidants, and provide new insight into the regulation of secondary metabolism in black carrot.

Forensic Investigations of Diesel Oil Spills in the Environment Using Comprehensive Two-Dimensional Gas Chromatography-High Resolution Mass Spectrometry and Chemometrics: New Perspectives in the Absence of Recalcitrant Biomarkers.

Forensic investigations of oil spills aim to find the responsible source(s) of the spill. Oil weathering processes change the chemical composition of the spilled oil and make the matching of oil spill samples to potential sources difficult. Diesel oil spill cases are more challenging, because biomarkers recalcitrant to long-term weathering are absent. We developed and tested a new method for the analysis and matching of diesel oil spills using two-dimensional gas chromatography-high resolution mass spectrometry (GC × GC - HRMS) and 2D-CHEMSIC (2-Dimensional CHEMometric analysis of Selected Ion Chromatograms), an extension of the CHEMSIC method to GC × GC data. The 2D-CHEMSIC performs pixel-based analysis using chemometrics on concatenated sections of 2D extracted ion chromatograms to assess the overall chemical variability of the samples, with potential applications for matching spill-source pairs in forensic investigations. The method was tested on samples from a number of diesel oil spill cases, (i) distinguishing chemically similar source diesels, (ii) investigating weathering effects on spill samples to determine type and degree of weathering, and (iii) improving the matching of diesel oil spills affected by weathering. Positive matches for spill-source pairs were identified after excluding the signals from the hydrocarbons most susceptible to evaporation, and photo-oxidized spills were also matched due to the presence of unaffected hydrocarbons. Forensic diagnostics obtained by the 2D-CHEMSIC were validated by the conventional CEN-Tr method.

Biodegradation, Photo-oxidation, and Dissolution of Petroleum Compounds in an Arctic Fjord during Summer.

Increased economic activity in the Arctic may increase the risk of oil spills. Yet, little is known about the degradation of oil spills by solar radiation and the impact of nutrient limitation on oil biodegradation under Arctic conditions. We deployed adsorbents coated with thin oil films for up to 4 months in a fjord in SW Greenland to simulate and investigate in situ biodegradation and photo-oxidation of dispersed oil droplets. Oil compound depletion by dissolution, biodegradation, and photo-oxidation was untangled by gas chromatography-mass spectrometry-based oil fingerprinting. Biodegradation was limited by low nutrient concentrations, reaching 97% removal of nC13-26-alkanes only after 112 days. Sequencing of bacterial DNA showed the slow development of a bacterial biofilm on the oil films predominated by the known oil degrading bacteria Oleispira, Alkanindiges and Cycloclasticus. These taxa could be related to biodegradation of shorter-chain (≤C26) alkanes, longer-chain (≥C16) and branched alkanes, and polycyclic aromatic compounds (PACs), respectively. The combination of biodegradation, dissolution, and photo-oxidation depleted most PACs at substantially faster rates than the biodegradation of alkanes. In Arctic fjords during summer, nutrient limitation may severely delay oil biodegradation, but in the photic zone, photolytic transformation of PACs may play an important role.

Evaluation of dimethyl sulfoxide (DMSO) as a co-solvent for toxicity testing of hydrophobic organic compounds.

Toxicity testing of hydrophobic compounds with low aqueous solubility remains challenging. Dimethyl sulfoxide (DMSO) is widely used as a co-solvent for toxicity testing of hydrophobic chemicals, but it may modulate chemical toxicity patterns. In this study, we critically evaluated the suitability of DMSO as a co-solvent for toxicity testing of hydrophobic organic compounds in aqueous solutions. As the toxicity measure, we used growth inhibition of a natural bacterial community, and the test toxicants included phenol, BTEX (benzene, toluene, ethylbenzene and xylene) and transformation products of polycyclic aromatic hydrocarbons (PAHs). We found that dose-response curves for phenol were unaffected by DMSO concentrations up to 10% (v/v) and that DMSO (5% v/v) did not affect the degree of bacterial growth inhibition for any of the other test compounds in short-term experiments (3.5 h). By contrast, marked co-solvent effects of DMSO were observed in the long-term assay (25 and 27 h). We therefore conclude that DMSO has excellent co-solvent properties for short-term (≤3.5 h) toxicity testing of sparingly water-soluble compounds and its application provides a simple, inexpensive approach for screening of various environmentally relevant hydrophobic chemicals. Importantly, the use of DMSO allows for generation of full dose-responses that may otherwise not be attained.

Increasing Flexibility in Two-Dimensional Liquid Chromatography by Pulsed Elution of the First Dimension: A Proof of Concept.

This work demonstrates the development of an online two-dimensional liquid chromatography (2D-LC) method where the first dimension column is eluted by a sequence of pulses of increasing eluotropic strength generated by the LC pumps (pulsed-elution 2D-LC). Between the pulses, the first dimension is kept in a no-elution state using low eluent strength. The eluate from the first dimension is actively modulated using trap columns and subsequently analyzed in the second dimension. We demonstrate that by tuning the length and eluotropic strength of the pulses, peaks with retention factors in water, kw, above 150 can be manipulated to elute in 3-4 pulses. The no-elution state can be kept for 1-10 min with only minor changes as to which and how many pulses the peaks elute in. Pulsed-elution 2D-LC combined with active modulation tackles three of the main challenges encountered in 2D-LC and specifically online comprehensive 2D-LC: undersampling, difficulties in refocusing, and lack of flexibility in the selection of column dimensions and flow rates because the two dimensions constrain each other. The pulsed-elution 2D-LC was applied for the analysis of a basic fraction of vacuum gas oil. Peak capacity was 4018 for a 540 min analysis and 4610 for a 1040 min analysis.

Optimizing gradient conditions in online comprehensive two-dimensional reversed-phase liquid chromatography by use of the linear solvent strength model.

The linear solvent strength model was used to predict coverage in online comprehensive two-dimensional reversed-phase liquid chromatography. The prediction model uses a parallelogram to describe the separation space covered with peaks in a system with limited orthogonality. The corners of the parallelogram are assumed to behave like chromatographic peaks and the position of these pseudo-compounds was predicted. A mix of 25 polycyclic aromatic compounds were used as a test. The precision of the prediction, span 0-25, was tested by varying input parameters, and was found to be acceptable with root mean square errors of 3. The accuracy of the prediction was assessed by comparing with the experimental coverages. Less than half of experimental coverages were outside prediction ± 1 × root mean square error and none outside prediction ± 2 × root mean square error. Accuracy was lower when retention factors were low, or when gradient conditions affected parameters not included in the model, e.g. second dimension gradient time affects the second dimension equilibration time. The concept shows promise as a tool for gradient optimization in online comprehensive two-dimensional liquid chromatography, as it mitigates the tedious registration and modeling of all sample constituents, a circumstance that is particularly appealing when dealing with complex samples.

Removal of Polysorbate 80 by complexation prior to LC-MS analysis.

The presence of Polysorbate 80 in samples can challenge liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS) analysis as it is easily ionised and detected. In this study, we demonstrate that interference from Polysorbate 80 can be reduced by complexation with a metal ion followed by precipitation by thiocyanate. The precipitation procedure was tested on a mixture of low molecular weight compounds (e.g. amino acids and non-amino organic acids) and it was shown that none of the tested compounds were precipitated.

Measuring internal azole and pyrethroid pesticide concentrations in Daphnia magna using QuEChERS and GC-ECD--method development with a focus on matrix effects.

Pyrethroids are highly toxic towards aquatic macroinvertebrates such as Daphnia magna and can be synergized when co-occurring with azole fungicides. A sensitive analytical method for the measurement of azole-pyrethroid mixtures in aquatic macroinvertebrates is not available at present. We developed and validated an extraction, cleanup, and quantification procedure for four pyrethroid insecticides and four azole fungicides at the picograms per milligram wet weight level in D. magna using a QuEChERS approach and GC-ECD analysis. Short- and long-term matrix effects were analyzed by injection of a series of extracts from D. magna, and the best surrogate standards were identified through correlation analysis of analyte responses. The presence of matrix clearly stabilized the analyte responses (≤6% relative standard deviation of peak area compared to up to 22% when injected without matrix). The sensitivity was high with detection limits and limits of quantification between 58-168 and 119-571 pg mg(wet weight)(-1) for the azoles and 5.8-27 and 12-84 pg mg(wet weight)(-1) for the pyrethroids, respectively. Accuracy (% recovery) was between 95 and 111% and the precision (repeatability) below 10% relative standard deviation for all analytes. In the case of prochloraz, α-cypermethrin, and deltamethrin, normalization to surrogate standards led to a clear improvement of accuracy and precision by up to 8 and 4%, respectively. The method was successfully applied to the measurement of internal α-cypermethrin concentrations in D. magna under environmentally relevant exposure conditions (exposure to a pulse in the micrograms per liter range) with and without co-exposure to propiconazole.

Evaluation of chromatographic conditions in reversed phase liquid chromatography-mass spectrometry systems for fingerprinting of polar and amphiphilic plant metabolites.

Metabolic fingerprinting is a relatively young scientific discipline requiring robust, yet flexible and fit-for-purpose analytical methods. Here, we introduce a simple approach to select reversed phase LC systems with electrospray MS detection for fingerprinting of polar and amphiphilic plant metabolites. The approach does not rely on isotopic labeling or biological origin of sample constituent and can also be used for non-biological matrices (e.g., oil or sewage sludge) or for other optimization purposes (e.g., mass spectrometric source parameterization). The LC systems varied in column chemistry and temperature, mobile phase pH/additive, gradient steepness/eluotropic strength, and electrospray mode of operation. The systems were evaluated based on the number of features detected using the matchedFilter algorithm from XCMS and the repeatability of this detection across analytical replicates. For negative ion mode detection, the best performances were obtained with an HSS T3 column operated at low pH, which produced a 3-fold increase in the number of reliable features extracted compared with the worst system. The best system for positive ion mode (i.e., the BEH C18 column operated at intermediate pH) only produced a 50 % increase in the number of reliable features. The data also indicate that baseline removal is unavoidable for reliable intensity estimations using peak areas, and that peak heights may be a more robust measure of intensity when baselines cannot be completely removed or in case of coelution, fronting or tailing.

Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

BACKGROUND: Quenching in cold buffered methanol at -40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline. RESULTS: Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in Lactobacillus paracasei subsp. paracasei (L. paracasei). Membrane integrity assayed by propidium iodide revealed that approximately 100 % [Corrected] of the L. paracasei cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the L. paracasei culture, fast filtration took several minutes, which is far from ideal for metabolites with high intracellular turnover rates. CONCLUSION: The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

Polycyclic Aromatic Acids Are Primary Metabolites of Alkyl-PAHs-A Case Study with Nereis diversicolor.

Although concentrations of alkylated polycyclic aromatic hydrocarbons (alkyl-PAHs) in oil-contaminated sediments are higher than those of unsubstituted PAHs, only little attention has been given to metabolism and ecotoxicity of alkyl-PAHs. In this study we demonstrated that metabolism of alkyl-PAHs primarily forms polycyclic aromatic acids (PAAs). We generalize this to other alkyl-PAHs, based on literature and the present study of the metabolism of 1-methylphenanthrene, 3,6-dimethylphenanthrene, and 1-, 2-, 3-, and 6-methylchrysene related to their unsubstituted parent PAHs. Also, we observed that body burdens and production of PAAs was related to the position of the methyl group, showing the same isomer specific preferences as for microbial degradation of alkyl-PAHs. We detected a high production of PAAs, and larger metabolism of alkyl-PAHs than their unsubstituted parent PAHs. We therefore propose that carboxylic acid metabolites of alkyl-PAHs have the potential of constituting a new class of contaminants in marine waters that needs attention in relation to ecological risk assessments.

Metabolic fingerprinting of Lactobacillus paracasei: a multi-criteria evaluation of methods for extraction of intracellular metabolites.

An untargeted multi-criteria approach was used to select the best extraction method among freeze-thawing in methanol (FTM), boiling ethanol (BE) and chloroform-methanol (CM) for gas chromatography mass spectrometry (GC-MS) metabolic fingerprinting of Lactobacillus paracasei subsp. paracasei (CRL-431®). The following results were obtained: (i) coverage and efficiency, measured by the number of features extracted and the sum of feature intensities, showed that FTM extraction resulted in the largest compound coverage with a total number of features 8.9 × 10(3) ± 0.5 × 10(3), while merely 6.6 × 10(3) ± 0.9 × 10(3) and 7.9 × 10(3) ± 0.8 × 10(3) were detected in BE or CM, respectively; (ii) the similarity of extraction methods, measured by common features, demonstrated that FTM yielded the most complementary information to BE and CM; i.e. 17 and 33 % of the features of FTM extracted were unique compared to CM and BE, respectively; and (iii) a clear-cut separation according to extraction method was demonstrated by assessment of the metabolic fingerprints by pixel-based data analysis. Indications of metabolite degradation were observed under the elevated temperature for BE extraction. A superior coverage of FTM together with a high repeatability over nearly the whole range of GC-amenable compounds makes this the extraction method of choice for metabolic fingerprinting of L. paracasei.

Pixel-based analysis of comprehensive two-dimensional gas chromatograms (color plots) of petroleum: a tutorial.

We demonstrate how to process comprehensive two-dimensional gas chromatograms (GC × GC chromatograms) to remove nonsample information (artifacts), including background and retention time shifts. We also demonstrate how this, combined with further reduction of the influence of irrelevant information, allows for data analysis without integration or peak deconvolution (pixel-based analysis).

Instrumental and theoretical advancements in pulsed elution-LC × LC: Investigation of pulse parameters and application to wastewater effluent.

Due to the decoupling of the first (1D) and second (2D) dimension in pulsed elution-LC × LC (PE-LC × LC), method development is more flexible and straightforward compared to fast comprehensive LC × LC where the dependencies of key parameters between the two dimensions limits its flexibility. In this study we present a method for pulse generation, which is based on a switching valve alternating between one pump that delivers the gradient and a second pump that delivers low eluotrophic strength for the pause state. Consequently, the dwell volume of the system was circumvented and 7.5, and 3.75 times shorter pulse widths could be generated at flow rates of 0.2, and 0.4 mL/min with satisfactory accuracies between programmed and observed mobile phase composition (relative deviation of 6.0 %). We investigated how key parameters including pulse width and step height, 2D gradient time and flow rate affected the peak capacity in PE-LC × LC. The conditions yielding the highest peak capacity for the PE-LC × LC- high-resolution mass spectrometry (HRMS) system were applied to a wastewater effluent sample. The results were compared to a one dimensional (1D)-LC-HRMS chromatogram. The peak capacity increased with a factor 34 from 112 for the 1D-LC run to 3770 for PE-LC × LC-HRMS after correction for undersampling. The analysis time for PE-LC × LC-HRMS was 12.1 h compared to 67.5 min for the 1D-LC-HRMS run. The purity of the mass spectra improved for PE-LC × LC-HRMS by a factor 2.6 (p-value 3.3 × 10-6) and 2.0 (p-value 2.5 × 10-3) for the low and high collision energy trace compared to the 1D-LC-HRMS analysis. Furthermore, the signal-to-noise ratio (S/N) was 4.2 times higher (range: 0.06-56.7, p-value 3.8 × 10-2) compared to the 1D-LC-HRMS separation based on 42 identified compounds. The improvements in S/N were explained by the lower peak volume obtained in the PE-LC × LC-HRMS.

Is Fucus a suitable biomonitoring organism for polycyclic aromatic hydrocarbon contamination? A study from the Faroe Islands.

To evaluate seaweed as a biomonitoring organism, Fucus was sampled in the Faroe Islands. Nineteen PAHs, including the EPA 16, and four groups of alkylated PAHs were quantified using GC-MS analysis of extracts obtained using a modified QuEchERS method with ultrasonication in acetonitrile, back-extraction into hexane, and Florisil® cleanup. Samples from the harbor of Tórshavn collected at high tide were the most polluted with PAH concentrations between 1.3 × 102 and 1.7 × 102 ng/g wet weight. All samples contained a factor 10 higher concentrations of alkylated PAHs compared to their parent compounds. These results suggest that Fucus might be suitable as a biomonitoring organism for PAH pollution. Differences between samples collected in close proximity and on different days were observed (same range of RSD 14-120% and 60-102%, respectively), suggesting that water exchange, tide levels, and direct exposure to surface diesel pollution have a strong influence on pollutant uptake in Fucus. The findings stress the need for further evaluation of the sampling strategy.