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Bromoanisoles and methoxylated bromodiphenyl ethers in macroalgae from Nordic coastal regions.

Marine macroalgae are used worldwide for human consumption, animal feed, cosmetics and agriculture. In addition to beneficial nutrients, macroalgae contain halogenated natural products (HNPs), some of which have toxic properties similar to those of well-known anthropogenic contaminants. Sixteen species of red, green and brown macroalgae were collected in 2017-2018 from coastal waters of the northern Baltic Sea, Sweden Atlantic and Norway Atlantic, and analyzed for bromoanisoles (BAs) and methoxylated bromodiphenyl ethers (MeO-BDEs). Target compounds were quantified by gas chromatography-low resolution mass spectrometry (GC-LRMS), with qualitative confirmation in selected species by GC-high resolution mass spectrometry (GC-HRMS). Quantified compounds were 2,4-diBA, 2,4,6-triBA, 2'-MeO-BDE68, 6-MeO-BDE47, and two tribromo-MeO-BDEs and one tetrabromo-MeO-BDE with unknown bromine substituent positions. Semiquantitative results for pentabromo-MeO-BDEs were also obtained for a few species by GC-HRMS. Three extraction methods were compared; soaking in methanol, soaking in methanol-dichloromethane, and blending with mixed solvents. Extraction yields of BAs did not differ significantly (p > 0.05) with the three methods and the two soaking methods gave equivalent yields of MeO-BDEs. Extraction efficiencies of MeO-BDEs were significantly lower using the blend method (p < 0.05). For reasons of simplicity and efficiency, the soaking methods are preferred. Concentrations varied by orders of magnitude among species: ∑2BAs 57 to 57 700 and ∑5MeO-BDEs < 10 to 476 pg g-1 wet weight (ww). Macroalgae standing out with ∑2BAs >1000 pg g-1 ww were Ascophyllum nodosum, Ceramium tenuicorne, Ceramium virgatum, Fucus radicans, Fucus serratus, Fucus vesiculosus, Saccharina latissima, Laminaria digitata, and Acrosiphonia/Spongomorpha sp. Species A. nodosum, C. tenuicorne, Chara virgata, F. radicans and F. vesiculosus (Sweden Atlantic only) had ∑5MeO-BDEs >100 pg g-1 ww. Profiles of individual compounds showed distinct differences among species and locations.


Introduction
Macroalgae ("seaweeds", "sea vegetables") are used worldwide for human consumption (>80% in 2016), animal feed, cosmetics and agriculture. The global market is large and growing, $11.1 U.S. billion in 2016 and projected to reach $22.1 U.S. billion by 2024. 1 Macroalgae are a rich source of many nutrients, including vitamins, minerals, ber, phytochemical antioxidants (fucoxanthin, polyphenols) and omega-3s. [2][3][4][5] Several health benets are attributed to consumption of whole macroalgae and they exert a preservative effect when incorporated into foods, due to their antimicrobial activity. 3 On the other hand, caution for over-consumption has been advised due to the presence of accumulated heavy metals and other pollutants 2,3 and to excessive exposure to iodine, linked to hypothyroidism. 3 Many halogenated natural products (HNPs) are found in marine macroalgae. Bromophenolic compounds are a prominent subset of HNPs, comprising bromophenols (BPs) and their transformation products bromoanisoles (BAs), hydroxylated and methoxylated bromodiphenyl ethers (OH-BDEs, MeO-BDEs) and polybrominated dibenzo-p-dioxins (PBDDs). [6][7][8][9][10][11][12] BPs add characteristic and desirable avors to seafood, 13,14 and due to their high food quality macroalgae have been used to supplement the feed of aquacultured sh 15,16 and farm animals. 4 Several brominated polyphenols have antioxidant, antimicrobial, anticancer, antidiabetic and antithrombotic activities. 4,17 Bromophenolic compounds enter the human diet through macroalgae consumption and from compounds bioaccumulated in seafood, and proles of MeO-BDE congeners in human serum reect dietary exposure. 18 Toxic properties associated with bromophenolic compounds include disruption of hormone synthesis or activity (OH-BDEs and MeO-BDEs) 19 and oxidative phosphorylation (OH-BDEs), 20 and binding to the aryl hydrocarbon (Ah) receptor (PBDDs). 21,22 Bromophenolic compounds have been identied in several macroalgae species from the Baltic Sea: Dictyosiphon foeniculaceus, Ceramium tenuicorne, Polysiphonia fucoides and Pilayella littoralis, as well as in cyanobacteria Nodularia spumigena and Aphanizomenon os-aquae, [8][9][10][11] but quantitative data have only been reported for C. tenuicorne, 11,23-25 D. foeniculaceus 8 and N. spumigena. 8 Brominated polyphenols were isolated from the red alga Vertebrata lanosa collected on the coast of Norway. 26 Several simple BPs were identied in macroalgae species from the families Ceramiaceae, Delesseriaceae, Bonnemaisoniaceae, Rhodophyllaceae, Corallinaceae and Rhodomelaceae, collected on the Swedish west coast. 27 One of these compounds, lanosol (2,3-dibromo-4,5-dihydroxybenzyl alcohol) was also identied in seawater. To our knowledge, this is the only report of bromophenolic compounds in macroalgae from the west coast of Sweden.
Macroalgae and cyanobacteria are important sources of bromophenolic compounds to the Baltic ecosystem and they are transferred through the food web from macroalgae to invertebrate grazers to sh. 24 Several studies have reported bromophenolic compounds in Baltic mussels, which lter-feed on cyanobacteria 8,10,21,[28][29][30] and transferred from mussels to seaducks. 28 Macroalgae from the Nordic coastal region are being promoted as human food, with published recipes utilizing local species. 31 Consideration should be given to the bromophenolic compounds and other HNPs present in macroalgae used for human consumption, to place exposure in perspective with anthropogenic compounds.
In 2017-2018, we collected >30 species of macroalgae from the Bothnian Sea (northern Baltic), Sweden Atlantic coast (Skagerrak) and Norway Atlantic coast. Objectives are to establish analytical methods and determine the variability of

Macroalgae sampling
Macroalgae were collected from coastal sites in the Bothnian Sea (northern Baltic), Skagerrak (Sweden Atlantic) and Norway Atlantic. Amounts collected exceeded 20 g wet weight (ww) in most cases, except when limited by availability (Chara virgata 3 g, Ceramium tenuicorne 10 g ww). Aer species identication, the macrophytes were rinsed with deionized water, blot dried with laboratory tissues, and frozen at À20 C until analysis. A subset of 16 macroalgae species (18 specimens, some species from two locations), including one stonewort, was selected for the initial survey of BAs and MeO-BDEs. Collection details for these are given in Table 1 and locations are shown in Fig. 1. Species were chosen to represent different taxonomic groups (red, green, brown algae), locations, morphologies and anticipated content of BAs and MeO-BDEs.
Extracts from SOAK 1 or SOAK 2 were diluted with 60 mL deionized water (DIW), 3 mL saturated potassium chloride was added, and the mixture was partitioned three times with 20 mL DCM followed by 20 mL hexane. Combined organic layers were ltered through glass wool and concentrated by rotary evaporation and nitrogen blowdown into 5 mL hexane.
BLEND. 32,33 Macroalgae pieces were placed in a thick-walled glass jar and the above surrogates were added. A stainless steel  Table 1 for coordinates and sampling dates.
stick blender (Ultra Turrax T25, Janke & Kunkel GmbH, IKA Labortechnik, Staufen, Germany) was used to homogenize the macroalgae with 25 mL acetone + 10 mL hexane followed by two portions of 20 mL hexane + 3 mL diethyl ether, blending for one minute each time. The combined extracts were diluted with 60 mL DIW, 3 mL saturated potassium chloride was added and the mixture was partitioned. The organic layer was removed and the aqueous phase was extracted twice more with 20 mL hexane + 3 mL diethyl ether. Combined organic layers were ltered and concentrated as in the SOAK methods.
Of the 18 specimens, three were extracted in replicate (3-4) with each method, ve were extracted once with each method and ten were extracted only with SOAK 2 (Tables 3, 4 and S1 †).
Internal standard (59 ng 2,2 0 ,6,6 0 -tetrachlorobiphenyl, CB-54) was added to the macroalgae extracts, which were then vortexed for 1 min with 2 mL 99% sulfuric acid and placed in the refrigerator overnight to allow the phases to separate. Fucus serratus required a second sulfuric acid treatment. The upper hexane layer was transferred with hexane rinsings to a Pasteur pipet containing 0.5 g Florisil topped with 0.5 cm granular anhydrous sodium sulfate and the column was eluted with Table 3 Comparison of extraction methods, replicate analyses of three macroalgae, pg g À1 ww a
BDE68 and 6-MeO-BDE67 were identied using GC-LRMS and GC-HRMS by their retention times relative to authentic standards and ion ratios that fell within AE20% of those for standards, and quantied by GC-LRMS. Two tribromo-MeO-BDEs and one additional tetrabromo-MeO-BDE with unknown bromine substituent positions were also identied from their monitored ions by both LRMS and HRMS (Fig. 2). The unknown tribromo-MeO-BDEs were quantied by GC-LRMS versus the response factor (relative to CB-54) for a standard of 2 0 -MeO-BDE28 (which was not found in any of the macroalgae species) and the unknown tetrabromo-MeO-BDE was quantied by using the average of response factors for 2 0 -MeO-BDE68 and 6-MeO-BDE47. The two tribromo-MeO-BDEs appear to be the same ones previously identied in Baltic water and air samples. 34 Pentabromo-MeO-BDEs showed poor response by GC-LRMS, but three compounds were identied in some selected macroalgae species by HRMS using authentic standards: 6-MeO-BDE85, 6-MeO-BDE90 and 6-MeO-BDE99; 2-MeO-BDE123 was also sought but not found. Due to poor performance of the CB54 internal standard in GC-HRMS runs, semiquantitative results for pentabromo-MeO-BDEs were estimated without use of an internal standard and assuming a 200 mL sample volume. Four blanks were run by carrying solvents through the extraction and cleanup procedures. No target peaks were found, and blank quantities were estimated by integrating baseline noise in the vicinity of expected peaks. Blanks (mean AE SD) by compound class were: BAs 6.3 AE 2.6 pg, tribromo-MeO-BDEs 5.7 AE 3.4 pg, tetrabromo-MeO-BDEs 22 AE 9 pg. Some BAs and MeO-BDEs were absent from all macroalgae; 2,6-diBA and 2 0 -MeO-BDE28, and other MeO-BDEs were absent in some of the species. "Blanks" were also assessed using a larger data set from algae extractions and quantifying baseline signal in the absence of a noticeable target peak. The overall limits of detection, LOD ¼ (mean blank + 3 Â SD)/sample weight, from both types of blanks were: BAs 15 pg g À1 , tribromo-MeO-BDEs 10 pg g À1 and tetrabromo-MeO-BDEs 17 pg g À1 , based on a 2.5 g (ww) sample.
Percent recoveries of 2,4,6-triBA-d 5 , BDE-35 and BDE-119 surrogate spikes varied among the three extraction methods, with means ranging from 76% to 109% and relative standard deviations (% RSD) ranging from 8.7% to 21.7% ( Table 2). While mean recoveries of all surrogates using each method exceeded 75%, higher yields were generally obtained by SOAK 2 (MeOH-DCM) and BLEND methods than by SOAK 1 (MeOH). Differences in mean recoveries between SOAK 2 and SOAK 1 were signicant for all three surrogates (single factor ANOVA, post hoc Tukey HSD, p < 0.05), between SOAK 1 and BLEND for the two BDEs and not signicant between SOAK 2 and BLEND for any surrogate (p > 0.05). Concentrations of BAs and MeO-BDEs in macroalgae were corrected on a per-sample basis for surrogate yields (2,4,6-triBA-d 5 recovery for BAs, average of BDE-35 and BDE-119 recoveries for MeO-BDEs).

Comparison of extraction methods and reproducibility
Replicate extractions (3 to 4) of C. glomerata and F. radicans from the Bothnian Sea and F. vesiculosus from Skagerrak were done using SOAK 1 (MeOH), SOAK 2 (MeOH-DCM) and BLEND (mixed solvents) methods. Analytical results are summarized in Table 3 and elaborated in Table S1. † Percent relative standard deviations (% RSD) for replicate analyses of a particular species with a specied method ranged from 5% to 36% for BAs and 4% to 48% for MeO-BDEs, with overall average % RSDs of 18% and 23%, respectively ( Table 3). Sources of this variability are in the precision of the analytical method itself (the average % RSD of all surrogate recoveries was 13%, Table 2) and probably also in the reproducibility of removing water by blot drying with laboratory tissues. In most cases no signicant differences in extraction yields were found using SOAK 1 or SOAK 2 methods (one-way ANOVA, post hoc Tukey HSD test, p > 0.05). Differences between the SOAK and BLEND methods were sometimes signicant (p < 0.05) and lower with BLEND, especially for MeO-BDEs. The difference appears to be due to lower extraction efficiency of MeO-BDEs from the algal matrix, since surrogate recoveries with BLEND were high and not different from SOAK 2. Five other macroalgae species, U. intestinalis from the Bothnian Sea, and A. nodosum, F. serratus, R. confervoides and S. latissima from Skagerrak, were extracted once with each method. Here the extraction methods were compared by expressing yields relative to SOAK 2; i.e. SOAK 1/SOAK 2 and BLEND/SOAK 2. Results for these ve macroalgae plus the three species used in methods replication (Table 3) are summarized in Table 4. Averaged over all eight species and compounds, SOAK 1/SOAK 2 yielded ratios of BAs (0.99 AE 0.29) and MeO-BDEs (1.12 AE 0.26) that were not signicantly different from 1.00 (t-test, p > 0.05). BLEND/SOAK 2 ratios for BAs were slightly, but not signicantly, lower (0.90 AE 0.19, p > 0.05), while BLEND/ SOAK 2 ratios for MeO-BDEs were signicantly lower (0.61 AE 0.13, p < 0.001). Thus, the extraction efficiency of BAs from these test species did not differ signicantly by the three methods. Yields of MeO-BDEs were not signicantly different by SOAK 1 and SOAK 2. The extraction efficiency for MeO-BDEs was signicantly lower using BLEND, conrming results from replicate extractions of three species. For reasons of simplicity and efficiency, the soaking methods are preferred.
Six macroalgae species (A. nodosum, C. glomerata, F. radicans, F. serratus, F. vesiculosus and R. confervoides) that had been extracted by SOAK 1 or SOAK 2 received second week-long extractions with fresh solvents. Second extraction percentages were 6.6 AE 3.5% of rst-extraction yields for BAs and 21 AE 19% for MeO-BDEs. The MeO-BDE percentages are likely inated because second extraction quantities were oen near or below the LOD (LOD/2 was entered). Table 1 gives concentrations of the P 2 BAs (2,4-diBA + 2,4,6-triBA) and P 5 MeO-BDEs (2 0 -MeO-BDE68, 6-MeO-BDE47, one tetrabromo-MeO-BDE and two tribromo-MeO-BDEs) in pg g À1 ww. For those macroalgae receiving extractions by different methods, yields by all three methods were averaged for BAs, while only SOAK 1 and SOAK 2 results were averaged for MeO-BDEs. Composition details are shown in Fig. 3 and Table S2. † BAs were found in all sixteen macroalgae species examined, while eleven species contained one or more tribromo-or tetrabromo-MeO-BDEs.
Nine species were checked for pentabromo-MeO-BDEs by HRMS (Table S3 †). Due to poor performance of the CB54 internal standard in GC-HRMS runs, semiquantitative results were estimated without use of an internal standard and assuming a 200 mL sample volume. Only C. tenuicorne and F. radicans from the Bothnian Sea, and A. nodosum and F. vesiculosus from Skagerrak contained one or more of 6-MeO-BDE85, 6-MeO-BDE90 and 6-MeO-BDE99 at concentrations ranging from 1.3 to 18 pg g À1 ww.
Wide ranges of BAs and MeO-BDEs have been reported in 15 genera of red, green and brown macroalgae from Philippine waters, all collected in the same month: 2,4,6-triBA < 20 to 2200 pg g À1 ww, 2 0 -MeO-BDE68 < 20 to 229 000 pg g À1 ww and 6-MeO-BDE47 < 20 to 27600 pg g À1 ww. 7 Ranges of 2,4-diBA and 2,4,6-triBA in the red alga Polysiphonia sphaerocarpa from the littoral zone near Sydney, Australia were 100 to 300 and 200 to 700 pg g À1 ww. 36 Concentrations of BAs in our set of macroalgae are generally higher than those found in other investigations (Table 5). While BAs were highest in Fucus spp. and A. nodosum, which have not been analyzed by others, we also found higher levels in C. tenuicorne than previously reported. On the other hand, our levels of BAs in the brown alga D. foeniculaceus were comparable to those found by Löfstrand et al., 8 while their concentrations of tetrabromo-MeO-BDEs were higher than ours. The semiquantitative estimate of 17 pg g À1 ww 6-MeO-BDE85 in C. tenuicorne (Table S3 †) compares favorably with 35 pg g À1 ww reported in the same species. 11 In addition to variation among macroalgae species, 7 concentrations and proportions of bromophenolic compounds uctuate with the season. 6,11,23,24 The collection months of Nordic macroalgae specimens was July-August in the Bothnian Sea, October in Skagerrak and May in coastal Norway (Table 1), which may account for observed differences. Concentrations of 2,4-diBA and 2,4,6-triBA in C. tenuicorne, collected from the Baltic Proper between June and August, ranged from 4 to 70 and 47 to 390 pg g À1 ww, respectively, while ranges of 2 0 -MeO-BDE68 and 6-MeO-BDE47 were 3 to 29 and 5 to 71 pg g À1 ww (Table 5). 24 Each of these bromophenolic compounds peaked in August. Ratios of compounds in C. tenuicorne varied over the springsummer; 2,4,6-triBA/2,4-diBA from 5.5 to 17 and 6-MeO-BDE47/2 0 -MeO-BDE28 from 1.1 to 2.7. 24 Ratios of precursor 2,4,6-triBP/2,4-diBP concentrations also showed monthly uctuations. 23 In comparison, 2,4,6-triBA/2,4-diBA was 46, while 6-MeO-BDE47/2 0 -MeO-BDE28 was 0.70 in our specimen of C. tenuicorne, collected in August. Stresses from salinity variations, light intensity and grazing by predators also cause changes in concentrations of 2,4,6-triBP. 23 These factors hinder comparisons among species, locations and sampling times.
Both BAs and MeO-BDEs have been determined in macroalgae from Nordic ecosystems and the Philippines, with strikingly different results (Table 5). Even considering the spread of concentrations within each compound class and among species, the general trend is BAs > MeO-BDEs in Nordic ecosystems versus MeO-BDEs > BAs in the Philippines. Reasons are not apparent, and it would be interesting to conduct more comparisons between tropical and cold ecosystems.

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Methods of extraction varied in the other studies. SOAK 1 as used here was employed to extract macroalgae from the Philippines. 7 Variations of the BLEND method were used for Baltic specimens. 8,11,24 BPs in macroalgae from Hong Kong 6 and Australia 12,36 were isolated by combined steam distillation-solvent extraction. Free bromophenols (BPs) were also determined in the studies listed in Table 5, with levels similar or higher than those of BAs.
The more polar BPs and OH-BDEs were not considered in this initial survey. These are also abundant in macroalgae. [6][7][8][9][10][11][12]36 BPs and OH-BDEs are partially ionized at seawater pH, 37,38 but are nevertheless subject to bioaccumulation. 24,28,39 Diverse toxic effects of 2,4,6-triBP 40 and OH-BDEs [18][19][20] have been reported, and benecial antimicrobial activity has been attributed to 6-MeO-BDE47. 41 Many studies have demonstrated interconversion of BPs/BAs and OH-BDEs/MeO-BDEs through O-methylation/ demethylation reactions, 18,19,40 and it has been suggested that wildlife acquire OH-BDEs through O-demethylation of accumulated MeO-BDEs. 42 Thus, it is important to include both free phenolic and O-methylated forms in subsequent surveys, along with additional HNPs that have been reported in marine macroalgae; e.g. PBDDs, 8,9,11,21 BDEs substituted with multiple OH-or MeO-groups, hydroxylated and methoxylated bromobiphenyls 7 and brominated methylpyrroles. 43 The SOAK 1 (methanol) method has been used to extract compounds with free phenolic groups from macroalgae. 7 Non-target screening by tandem GCtime of ight mass spectrometry (GCxGC-ToF-MS) is very effective for identifying HNPs. 44 GCxGC-ToF-MS [45][46][47] and advanced data processing techniques based on GC-MS 48 and LC-MS 49,50 methods have identied hundreds to thousands of natural and anthropogenic halogenated compounds in marine mammals and sediments but so far these have not been applied to identi-cation of HNPs in macroalgae.

Conclusions
The data set for neutral bromophenolic compounds (BAs and MeO-BDEs) obtained in this survey is the largest for macroalgae in Nordic waters. A simple extraction procedure based on soaking the macroalgae in methanol (SOAK 1) or methanoldichloromethane (SOAK 2) gave equivalent yields of both compound classes and was signicantly more efficient for MeO-BDEs than blending with mixed solvents (BLEND) In retrospect, the sample sizes used in this survey are adequate for BAs, but somewhat low for MeO-BDEs, which were occasionally below or on the border of detection. Expanding the SOAK methods to accommodate larger samples is recommended.
Concentrations of BAs and MeO-BDEs in macroalgae were highly variable, spanning orders of magnitude, and compound proles differed according to the species. The concentrations and compound proles observed here represent only single sampling events, and it is likely that a more extensive survey would reveal seasonal and spatial variability. Other considerations for future research are expanding the list of target compounds to include the more polar BPs and OH-BDEs, search for other HNPs by nontarget screening, and examining the link between macroalgae and bioaccumulation of bromophenolic compounds and other HNPs in Nordic coastal waters.

Conflicts of interest
There are no conicts of interest to declare.