Biosynthesis of hydroxylated polybrominated diphenyl ethers and the correlation with photosynthetic pigments in the red alga Ceramium tenuicorne

Phytochemistry xxx (2016) 1e8

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Biosynthesis of hydroxylated polybrominated diphenyl ethers and the correlation with photosynthetic pigments in the red alga Ceramium tenuicorne Dennis Lindqvist a, *, Elin Dahlgren b, Lillemor Asplund a a b

Department of Environmental Science and Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden Legal Affairs, Swedish Environmental Protection Agency, SE-106 48 Stockholm, Sweden

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 May 2016 Received in revised form 18 August 2016 Accepted 13 October 2016 Available online xxx

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have been identified in a variety of marine organisms from different trophic levels indicating a large spread in the environment. There is much evidence pointing towards natural production as the major source of these compounds in nature. However, much is still not known about the natural production of these compounds. Seasonal trend studies have shown large fluctuations in the levels of OH-PBDEs in Ceramium tenuicorne from the Baltic Sea. Yet, even though indications of stimuli that can induce the production of these compounds have been observed, none, neither internal nor external, has been assigned to be responsible for the recorded fluctuations. In the present study the possible relationship between the concentration of pigments and that of OH-PBDEs in C. tenuicorne has been addressed. Significant correlations were revealed between the concentrations of all OH-PBDEs quantified and the concentrations of both chlorophyll a and Sxanthophylls þ carotenoids. All of which displayed a concentration peak in mid-July. The levels of OHPBDEs may be linked to photosynthetic activity, and hence indirectly to photosynthetic pigments, via bromoperoxidase working as a scavenger for hydrogen peroxide formed during photosynthesis. Yet the large apparent investment in producing specific OH-PBDE congeners point towards an targeted production, with a more specific function than being a waste product of photosynthesis. The OH-PBDE congener pattern observed in this study is not agreeable with some currently accepted models for the biosynthesis of these compounds, and indicates a more selective route than previously considered in C. tenuicorne. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Ceramium tenuicorne Ceramiaceae Biosynthesis OH-PBDE Bromophenols Pigments Chlorophyll

1. Introduction Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are ubiquitous in aquatic environments around the world. OH-PBDEs have for example been detected in biological samples from the great lakes basin of North America (Fernie and Letcher, 2010), the Pacific Ocean (Nomiyama et al., 2011), the Indian Ocean (Gribble, 2010), and the Atlantic Ocean (Strid et al., 2010). In the Baltic Sea the OH-PBDEs are equally ubiquitous throughout the food web, and € fstrand, 2011), fish have been identified in for example mussels (Lo €fstrand, 2011), seals (Routti et al., 2009), and predatory birds (Lo €f, 2012). Some congeners have even increased significantly in (Nordlo

* Corresponding author. E-mail address: [email protected] (D. Lindqvist).

concentration over the last 30 years, as observed in the livers from Baltic herring (Faxneld et al., 2014). This is of great concern due to the number of adverse effects that have been associated with OH-PBDEs, such as disruption of the oxidative phosphorylation (van Boxtel et al., 2008), genotoxicity (Ji et al., 2011), neurotoxicity (Dingemans et al., 2008), and hormonal disturbances (Meerts et al., 2001). Although OH-PBDEs can be formed by metabolic transformation of anthropogenic PBDEs, used as flame retardants (Hakk and Letcher, 2003), much evidence suggests natural production as the major source of these compounds in the Baltic Sea. Primary producers, such as algae, are known to synthesize brominated organic compounds (BOCs) like bromophenols (BPs) (Dahlgren et al., 2015; Flodin et al., 1999; Gribble, 2010). OH-PBDEs have been detected in € fstrand, 2011). However, a variety of alga species in the Baltic Sea (Lo contributions to the detected OH-PBDEs may also come from production in the epiphytical community on the surface of the algae

http://dx.doi.org/10.1016/j.phytochem.2016.10.009 0031-9422/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Lindqvist, D., et al., Biosynthesis of hydroxylated polybrominated diphenyl ethers and the correlation with photosynthetic pigments in the red alga Ceramium tenuicorne, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.009

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D. Lindqvist et al. / Phytochemistry xxx (2016) 1e8

(Unson et al., 1994). Recently, the production of 2,4,6tribromophenols (2,4,6-TBP) in isolated clonal material of Ceramium tenuicorne (Kützing) Waern (family Ceramiaceae) grown in a controlled laboratory setting, confirmed the hypothesis that this specie is a producer of brominated phenolic compounds (Dahlgren et al., 2015). Production of BOCs by algae is catalyzed by bromoperoxidases (BPOs) (Butler and Walker, 1993). BPOs are antioxidant enzymes that are abundant in marine organisms, and that are present in for example red, brown, and green algae (Butler and Walker, 1993). In plants and algae the processes of photosynthesis and respiration is linked to cellular production of reactive oxygen species (ROS) (Baroli and Niyogi, 2000; Veljovic-Jovanovic, 1998). Hydrogen peroxide (H2O2) formed as a result of photosynthetic and respiratory electron transport has been shown to be available as a substrate for BPO (Manley and Barbero, 2001). BPO uses H2O2 to oxidize bromide to e.g. hypobromous acid (HOBr), which is sequentially used to brominate organic substrates (Butler and Walker, 1993). The formation of BOCs by BPO may hence provide means of scavenging H2O2 before it reaches toxic concentrations (Pedersen et al., 1996). Besides haloperoxidases, several antioxidant systems have evolved to scavenge ROS, and to counteract ROS induced autotoxicity. These includes antioxidant molecules such as carotenoids, and antioxidant enzymes like superoxide dismutase (SOD) and ascorbate peroxidase (Baroli and Niyogi, 2000; Dummermuth et al., 2003; Veljovic-Jovanovic, 1998). BPO isolated from red algae have been shown to catalyze the bromination of phenol to 2,4,6-TBP in vitro (Shang et al., 1994). OHPBDEs may in turn be formed by dimerization of BPs. The dimerization can also be catalyzed by BPO as previously demonstrated in vitro (Lin et al., 2014). The dimerization proceeds via oxidation of BPs to bromophenoxy radicals (Lin et al., 2014; Neilson, 2003), which subsequently undergo biradical coupling (Lin et al., 2014; Neilson, 2003). OH-PBDEs may also be formed in a reaction between a bromophenoxy radical and a BP through loss of a bromine from the latter (Lin et al., 2014). Stressors such as exposure to pathogens and physical damage can result in a burst of ROS in algae (Collen and Pedersen, 1994; Kupper et al., 2001). Increased production of BOCs algae have in turn been associated with stress induced by ultra-violet (UV) exposure (Laturnus et al., 2004), temperature changes (Abrahamsson et al., 2003), high light intensity (Mtolera et al., 1996), as well as changed salinity and grazing (Dahlgren et al., 2015). As well as being a sink for hydrogen peroxide, the production of halometabolites is believed to contribute to enhanced survival and fitness of the alga through qualities such as antibiotic activities (Butler and Walker, 1993; McConnell and Fenical, 1980). The products of BPO activity in macroalgae have also been indicated as part of defense mechanisms against epiphyte settlement (Ohsawa et al., 2001) as well an anti-feeding deterrent (McConnell and Fenical, 1980). However, the exact role of OH-PBDEs in algal survival is still to be determined. This study was undertaken in response to an observation made during a previous investigation regarding the variation of OHPBDEs in C. tenuicorne over the summer season. During that investigation the alga samples were noted to possess a variation in their coloration, and seemingly darkened around the same time as the OH-PBDE concentrations peaked within the algae (Dahlgren et al., 2015). The aim of the present study was thusly to investigate the possible correlation between the concentrations of OHPBDEs and pigments in C. tenuicorne over the summer season. Ultimately to increase our knowledge on how and why these compounds are produced by the alga. The filamentous red macroalga, C. tenuicorne, is a common algae in the Baltic Sea. It grows as an epiphyte on other macroalgae, such

as Fucus vesiculosus, on rocks, or in loose lying algal mats €m and Bergstro € m, 1999), and are present from the surface (Bergstro down to below 10 m. C. tenuicorne is an opportunistic fast growing species that may benefit from the nutrient enrichment of the Baltic Sea (Schramm, 1996). This makes this species interesting in the context of OH-PBDEs in the Baltic Sea and their potential increase over time. The major endogenous xanthophylls, carotenoids, and chlorophylls of C. tenuicorne include b,b-carotene, lutein, zeaxanthin and chlorophyll a (Chl a). Chl b is not present in C. tenuicorne (Bianchi et al., 1997). C. tenuicorne from the northern Baltic Sea has also been shown to contain high levels of phycobiliprotein pig€ck and ments, with phycoerythtrin being the most abundant (Ba Likolammi, 2004). 2. Results 2.1. Concentrations and correlations of pigments and OH-PBDEs/ BPs Spectrophotometric analyses showed that the concentration of Sxanthophylls þ carotenoids (Sx þ c) in general was four times lower than that of Chl a in the algae over the summer season (see Table 1). The levels of Sx þ c also fluctuated in accordance with that of Chl a, with a clear statistical correlation between the concentrations of the two (see Fig. 1, data from the linear regression analysis is presented in the supplementary data (SD), Table S1). The concentrations of all seven OH-PBDE congeners quantified in this study, individually and as a sum (SOH-PBDE), showed statistical correlations with both the Sx þ c concentration and the Chl a concentration (with 95% confidence intervals). Overall the OHPBDE trends were better correlated with the Sx þ c trend than with the Chl a trend (SD, Table S1), and better correlated on wet weight basis than dry weight, while still significant on both (SD, Table S1). 2,4-dibromophenol (2,4-DBP) was also correlated with the pigment concentrations, while 2,4,6-TBP was not. Both the pigments and the OH-PBDEs showed two concentration peaks, one smaller on the 19th of June and one larger on the 19th of July (see Fig. 1). The two peaks were more pronounced for the general OHPBDE trend (depicted by the SOH-PBDE concentration trend in Fig. 1) than for the Sx þ c and Chl a trends. The congener pattern was relatively stable over the studied period and was dominated by 6-OH-BDE85 followed by 6-OHBDE137 (Table 1). The quantified seven OH-PBDEs included all (5) dominant peaks within the region of BP dimerization products containing 3e6 bromines in the gas chromatography mass spectrometry (GC-MS) chromatogram, and two minor peaks (see Fig. 2). The two quantified BPs were utterly dominant within the BP region throughout the sampling period. In the samples from mid-June another compound group of large brominated weak-acids was also detected, which appeared and then disappeared very rapidly, and seemingly independent of the pigments and OH-PBDEs. These compounds eluted later than the OH-PBDEs on the GC-MS system, and only one was of high enough concentration to produce a proper mass spectrum in full-scan mode. This mass spectrum showed an isotope pattern indicating six bromines in the molecule, but the molecular weight (m/z 734, for the methylated derivative) was not consistent with a BP dimer, in this case it weighed too much indicating a more complex backbone structure. 2.2. Correlations with external factors Acquired data for photosynthetic photon density (PPD) and UV could not be correlated with any measured concentrations of pigments or bromophenolic compounds in the algae (see Fig. 1). Recorded surface temperature at the sampling area increased from

Please cite this article in press as: Lindqvist, D., et al., Biosynthesis of hydroxylated polybrominated diphenyl ethers and the correlation with photosynthetic pigments in the red alga Ceramium tenuicorne, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.009

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Table 1 Measured wet weight concentrations of pigments (mg  g1), BPs and OH-PBDEs (pg  g1) as well as dry weight percentage used to correct for water content.

mg  g1

Chl a P xþc 2,4-DBP 2,4,6-TBP 20 -OH-BDE68 6-OH-BDE47 6-OH-BDE90 6-OH-BDE99 2-OH-BDE123 6-OH-BDE85 6-OH-BDE137 P 7OH-PBDEs Dry weight %a

pg  g1

110606

110613

110619

110627

110703

110719

110804

26 10 82 372 57 82 8.6 23 6.9 471 317 965 5.5

68 20 152 158 118 123 38 71 36 2790 2130 5300 5.8

99 31 203 298 148 234 145 239 108 4550 3490 8910 5.6

73 24 136 389 96 136 65 113 29 1790 1210 3440 5.4

116 29 57 859 74 211 63 164 76 1420 715 2720 5.8

350 81 584 1110 1120 1260 409 1080 511 9290 7040 20700 7.4

198 37 229 1550 97 169 65 146 80 1100 896 2560 8.6

100

100

Volumetric

100

2-OH-BDE123

A

6-OH-BDE85

MCounts 35

6-OH-BDE90 6-OH-BDE99

%

x+c 2,4-DBP UV Temp

2’-OH-BDE68 6-OH-BDE47

100

Chl a OH-PBDEs PPD 2,4,6-TBP

6-OH-BDE137

This value does not include the extractable organic content, corresponding to 0.22 ± 0.1%, based on previous analysis.

Surrogate

a

110530 30 11 2.1 87 6.5 23 1.8 5.2 2.6 114 53 206 6.0

0 19

22

24

Retention time (min) Fig. 2. GC-MS chromatogram (for m/z 79, 81) covering the elution area of BP dimers with 3e6 bromines. This sample was collected 110719.

0 100

B drawn, but the increase in potential grazers do coincide decently well to the increase in concentration of pigments and OH-PBDEs in these algae during the second week of July.

0 110619

110719

3. Discussion

Time (days) 3.1. Production of BOCs and photosynthesis/BPO activity Fig. 1. Dry weight concentration trends as well as light and temperature data. All trends are given on a percentage scale with the highest value of each trend set to 100%. A) Statistically correlated curves, stacked with an offset. B) Parameters not correlated to pigments and OH-PBDEs, stacked without offset. UV values are based on the measured levels, while PPD values are modelled.

<8  C in May, to 14e16  C between June 13 and July 3, and 20e23  C between July 12 and August 4 (Dahlgren et al., 2015). Although this could be correlated with the general increase in pigments and OHPBDEs up to the 19th of July, the drop in concentrations to the 4th of August spoils this correlation. Surface temperature could however be statistically correlated with 2,4,6-TBP concentration (p ¼ 0.037, R2 ¼ 0.93, dry weight basis). The grazer community in the sampling area was described by Dahlgren and co-workers as having little potential herbivores in mid-June to having an increasing number of potential herbivores in the beginning of July, including Idotea sp., Gammarus sp., and Theodoxus sp. (Dahlgren et al., 2015). No quantitative data on these observations exists and hence no mathematical correlations can be

Seasonal variation in 2,4,6-TBP content, with low levels in winter and high in summer, in the green alga Ulva lactuca has previously been associated with seasonal variation in BPO activity (Flodin et al., 1999). As an antioxidant enzyme BPO may potentially increase in algae during the summer as a result of higher rate of photosynthesis. It is imperative for the algae to strive for equilibrium between photosynthesis and ROS scavenging systems. If the levels of ROS exceed the capacity of the alga's antioxidant system, metabolic processes including photosynthesis will suffer oxidative damage (Baroli and Niyogi, 2000; Dummermuth et al., 2003). It has been observed that the brown seaweed Ascophyllum nodosum releases HOBr upon exposure to light, and it was speculated that the seasonal variation in bromine content of Arctic aerosols is related to photosynthetic activity of seaweeds present in the Arctic Ocean (Wever et al., 1991). Formation of volatile halocarbons, such as bromoform, in different species of marine algae has also been suggested to be dependent on photosynthesis and the production of H2O2 (Pedersen et al., 1996).

Please cite this article in press as: Lindqvist, D., et al., Biosynthesis of hydroxylated polybrominated diphenyl ethers and the correlation with photosynthetic pigments in the red alga Ceramium tenuicorne, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.009

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In U. lactuca linear relationships between light absorption and chlorophyll density, as well as between light level and photosynthesis (up to the point of saturation) have been established (SandJensen, 1988). Increased levels of photosynthetic pigments, whatever the cause, may thus lead to increased light absorption, assuming a fairly constant level of irradiance. Consequently this could supply a need for upregulation of ROS scavenging systems such as BPO. Two red algae species of the Rodomelaceae family has previously been suggested as examples of algae that mainly scavenge H2O2 by production of BPs (Pedersen et al., 1996). Formation of OH-PBDEs could be a sequential part of such scavenge-process. Oxidative coupling of BPs via phenoxy radical intermediates have been suggested as the primary route for OH-PBDE biosynthesis (Lin et al., 2014; Neilson, 2003). This coupling can be catalyzed by e.g. BPO in the presence of H2O2 (Lin et al., 2014), hence additionally relieving the organism of endogenous H2O2. Such process could explain the correlation between the levels of pigments and that of OH-PBDEs observed in this study (Fig. 1). As the PPD only fluctuate modestly over the studied period (see Fig. 1) an increase of photosynthetic pigments is likely to lead to enhanced light absorption within these algae. In this case however the OHPBDEs may be nothing more than a waste product of the algae's ROS scavenge system. 3.2. Mechanistic considerations Although OH-PBDEs can be formed by BPO mediated oxidative coupling of BPs, the congeners produced during in vitro experiments were non that have been found in nature (Lin et al., 2014). In the presence of H2O2, bromide, and isolated BPO, 2,4-DBP was converted to 2,4,6-TBP, which sequentially underwent homodimerization (Lin et al., 2014). In this study the concentration of 2,4,6-TBP did not correlate to that of the OH-PBDEs (Fig. 1), and based on the structure of the detected OH-PBDEs (Fig. 3) it is obvious that 2,4,6-TBP do not readily undergo neither homo- nor heterodimerization in C. tenuicorne. 2,4,6-TBP has also been observed not to correlated with OH-PBDEs in algae from Philippine waters (Haraguchi et al., 2010). Based on the lack of correlation it was suggested that the production of OH-PBDEs did not rely on 2,4,6-TBP as a substrate during the biosynthesis (Haraguchi et al., 2010). Oxidative dimerization of BPs can also be catalyzed by other peroxidases, and cytochrome P450 (Agarwal et al., 2014; Lin et al., 2015). However, formation of OH-PBDE via bacterial CYP450 only yielded one of the OH-PBDEs quantified in this study (20 -OHBDE68, Fig. 3) (Agarwal et al., 2014). In fact, the two dominant OHPBDEs within this study 6-OH-BDE85 (average 51 ± 4% of the SOHPBDE concentration on molar basis), and 6-OH-BDE137 (30 ± 5%) are thermodynamically unfavorable and would not be readily formed according to the prevailing model for OH-PBDE

biosynthesis. Furthermore, radical coupling of BPs can give a number of backbone structures due to the resonance structures of the phenoxy radicals. Including ortho-phenoxyphenols, para-phenoxyphenols, and dihydroxylated biphenyls. Yet only orthophenoxyphenols were identified among the major BP dimers in this study (see Figs. 2 and 3). In vitro dimerization with both BPO and CYP450 yielded significant amounts of para-OH-PBDEs (Agarwal et al., 2014; Lin et al., 2014). The selectivity in the dimerization witnessed in this study hints of possible involvement of a coordinating enzyme, i.e. an enzyme that coordinates the reactants in space during the dimerization. Secondly all major BP dimers observed in this study are ortho-(2,4-dibromophenoxy)phenols, which indicates a selective oxidation of 2,4-DBP. Neither of this can be achieved by BPO (Lin et al., 2014). Thus, not only does the congener pattern observed in this study not support the theory of formation of OH-PBDEs as a sink for H2O2, it does not support formation according to the prevailing model for OH-PBDE biosynthesis either. The high selectivity and apparent effort invested in forming thermodynamically unfavorable OH-PBDEs points towards a targeted (function selective) biosynthesis where potentially unidentified enzymes are involved. From a thermodynamic point of view it is unreasonable to expect the congener pattern observed here to be formed by simple oxidation reactions (i.e. formation of reactive bromine and subsequent formation of bromophenoxy radicals) and non-mediated chemical reactions. BPO can undeniably catalyze the formation of OH-PBDEs, and species lacking targeted routes for OHPBDE biosynthesis may still produce these via the action of BPO. In fact favorable structures such as for 20 -OH-BDE68 may even be formed as byproducts of BP synthesis under the right conditions, and hence also as byproducts of targeted OH-PBDE synthesis. 3.3. Production of BOCs and external factors In this study neither UV, PPD, nor temperature could be directly correlated with the concentrations of pigments and OH-PBDEs in the C. tenuicorne (see Fig. 1). Observations of an increase in grazers in the area preceding the larger concentration peak was however made during the sampling (see Dahlgren et al., 2015). Phenols and polyphenols suggestively produced as herbivory defense in the alga F. vesiculosus has previously been shown exhibit seasonal variations in concentration related to grazing pressure (Geiselman, 1980). 6OH-BDE137, one of the two dominant OH-PBDEs in the analyzed algae (Table 1), has been shown to be potent in the brine shrimp (Artemia salina) lethality test (Handayani et al., 1997). In comparison the LC50 value for 6-OH-BDE137 reported by Handayani et al. (1997) is orders of magnitude lower than those reported for e.g. aromatic carbamate insecticides in the corresponding bioassay (Barahona and Sanchez-Fortun, 1999). If the OH-PBDEs are

2’-OH-BDE68

6-OH-BDE47

6-OH-BDE90

6-OH-BDE99

2-OH-BDE123

6-OH-BDE85

6-OH-BDE137

Triclosan

Fig. 3. Structures of all seven quantified OH-PBDEs in this study, and the structure of triclosan for comparison.

Please cite this article in press as: Lindqvist, D., et al., Biosynthesis of hydroxylated polybrominated diphenyl ethers and the correlation with photosynthetic pigments in the red alga Ceramium tenuicorne, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.009

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produced as part of a defense against herbivores the correlation with photosynthetic pigments may stem from an increased need in energy to produce and maintain this defense, as well as a potential need for increased H2O2 to fuel the bromination process. Photosynthetic activity may vary in a supply-and-demand relationship with plant defense and may be increased to support formation of induced defense chemicals (Bolton, 2009; Tiffin, 2000). However, photosynthetic rate have also been shown to decrease in some plants during active defense (Bolton, 2009). The optimal defense tactic is to initiate all available defense mechanisms to increase the chance of one being effective against the attacking organism (Bolton, 2009). It is thus likely that an induction of OH-PBDE synthesis in this case only constitute a part of a larger activated defense apparatus. Production of other secondary metabolites have been observed to be positively or negatively correlated to photosynthetic rate depending on plant and product (Ibrahim et al., 2011; Mosaleeyanon et al., 2005). Increased grazing pressure during the first half of July could also explain the decreased coverage of C. tenuicorne observed by Dahlgren and co-workers at this time (Dahlgren et al., 2015). In such case the concentration peak in mid-July do not necessarily need to indicate an increased production of OH-PBDEs in the algae. Instead the increased concentration could be explained by selective grazing on algae containing lower levels of OH-PBDEs. When new algae start to colonize the area the abundancy of algae with high levels of OH-PBDEs gets diluted resulting in the observed subsequent drop in concentration. A regrowth of small C. tenuicorne was observed following the concentration peak in mid-July, and C. tenuicorne continued to grow in size and number throughout the rest of the summer (Dahlgren et al., 2015). The theory of a herbivory defense however struggles to explain the observed smaller concentration peak in mid-June, as the density of the inventoried herbivore species was considered low at this time (Dahlgren et al., 2015). However, 6-OH-BDE137 has also been shown to possess antibacterial and antifungal activity (Handayani et al., 1997). Overall the general structure of the quantified OHPBDEs possesses striking similarities to triclosan, a well-used antibacterial agent developed in 1960s (see Fig. 3). As the epiphyte coverage on the analyzed algae was not measured it cannot be excluded that the OH-PBDEs are produced as a defense against epiphyte settlement, and that the concentrations fluctuate in response to the density of this settlement. Pigmentation may in turn vary with the density of the biofilm to maintain adequate photosynthetic rate under conditions were light penetration through the film is reduced. While having proven antibacterial properties, OH-PBDEs have also been shown to be formed by bacteria (Agarwal et al., 2014; Gribble, 2010). As the bacterial coverage was not measured nor sampled separately, it cannot be entirely dismissed that the OHPBDE stem from bacteria. Bacteria found on other Baltic macro algae have been shown to produce antibiotic secondary metabolites (Goecke et al., 2013). Production of bioactive secondary metabolites by bacteria may be beneficial for algae in symbiotic relationships where the alga do not possess the ability to produce such compounds by itself. However, that the OH-PBDEs in this study were so well correlated to the alga pigments speaks against separate production inside and outside the alga. The production capacity of secondary metabolites is also be affected by the availability of nutrients, which vary over the summer season. The decreased coverage of C. tenuicorne preceding the OH-PBDE concentration could also have been beneficial for the remaining algae. OH-PBDE production could thus have increased at this time because of resource availability. The OH-PBDEs may then follow a trend of an overall production capacity in these algae. However, while the coverage of C. tenuicorne decreased at this time

5

the coverage of other alga species increased, particularly that of Pilayella littoralis (Dahlgren et al., 2015). 3.4. Production of BOCs and the algae's life cycles It has been shown that the levels of BPs in the red alga Lenormandia prolifera increased with increased age, suggestively due to partial continuously accumulation (Pedersen et al., 1979). According to the growth-differentiation balance hypothesis actively dividing and expanding cells in young plants may also be less able to produce secondary metabolites (Cronin and Hay, 1996). BPO activity has also been correlated with the life-cycle of the coralline alga Corallina pilulifera, and it was suggested that the enzyme level depended on the maturation of the alga (Itoh and Shinya, 1994). Considering the previously shown association between BP concentrations and BPO activity in algae (Flodin et al., 1999) it is possible that the levels of BP dimers may increase as a consequence of increased BPO activity during maturation. The observations on shifting C. tenuicorne coverage around the time of the concentration peak in mid-July, with decreasing coverage in the beginning of July and new recruitment in the second half of July may also indicate involvement of the natural lifecycle in the content of OH-PBDEs. On the other hand the concentration of 2,4,6-TBP did not decrease, but rather increased during the regrowth of C. tenuicorne (see Fig. 1). Although maturation may potentially account for changes in the concentration of OH-PBDEs it fails to explain the initial reason for producing these. The production of BOCs in C. tenuicorne may potentially also vary between the different isomorphic life stages, as previously discussed (Dahlgren et al., 2015). 4. Conclusion While the apparent correlation between OH-PBDEs and photosynthetic pigments supports the hypothesis of formation of OH-PBDEs as part of a H2O2 scavenge-system, the observed congener pattern among these OH-PBDEs do not. The lack of diversity in the backbone structure of the BP dimers and the high relative formation of thermodynamically unfavorable congeners questions the mechanistic model for OH-PBDE biosynthesis, and indicates a more selective route. The biosynthesis in C. tenuicorne seems targeted towards specific OH-PBDEs, which likely fills a particular function for the alga. The high bioactivity towards a range of organisms suggests production as defense chemicals. As a potential part of an inducible defense the correlation between OH-PBDEs and photosynthetic pigments may stem from the necessity for a high energy production to enable allocation of resources to generate and maintain the defense. The fluctuations in OH-PBDE concentration may also be related to the life-cycle of the alga, and increased production may occur under favorable conditions. The separate production of other complex BOCs peaking at a different time than the OH-PBDEs, as witnessed in these samples, further indicate that different brominated organic structures may fill different functions for the organism. The bromination mechanism may initially have evolved to fill one function, such as to scavenge H2O2, but once the system had been established it may over time have been intertwined with other systems such as chemical signaling (pheromones etc.) or chemical defenses (grazer deterrents and/or antibacterial agents), resulting in a variety of BOCs filling different functions. Fluctuations in OH-PBDEs concentrations observed in environmental samples of potential producers do not necessarily indicate changes in the production within the producer, but can also reflect changes in the population where algae with higher levels gets

Please cite this article in press as: Lindqvist, D., et al., Biosynthesis of hydroxylated polybrominated diphenyl ethers and the correlation with photosynthetic pigments in the red alga Ceramium tenuicorne, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.009

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D. Lindqvist et al. / Phytochemistry xxx (2016) 1e8

concentrated and diluted over time. Increased levels of brominated phenolic compounds within the algae could also potentially follow as a result of a release of previously biosynthesized compounds, stored in the algae in a form in which they go undetected by the chemical analysis used within this study. It is necessary to acknowledge the possible existence of several confounding factors that may cause the observed correlation between pigments and OH-PBDEs in this study. 5. Experimental 5.1. Sampling The analyzed samples of Ceramium tenuicorne (Kützing) Waern (family Ceramiaceae) were collected on eight occasions between the 30th of May and 4th of August 2011. Samples were collected from a depth of <1 m within a small defined area in a bay connected € Island, located in the Stockholm archipelago to N€ amdo (geographical coordinates latitude 59.18, longitude 18.7). Observations made on the general status and abundance of algae as well as on the community structure covering this sample period, has previously been reported for the bay area in which the point where these samples were harvested is located (Dahlgren et al., 2015). These samples were collected in parallel to the samples analyzed in the study by Dahlgren and co-workers. The algae were rinsed in seawater and associated visible organisms were removed. The samples were then frozen and stored at e20  C until analysis. 5.2. Analysis of pigments The pigments were extracted from roughly 0.7 g of alga material using ultrasound-assisted extraction with cold N,N-dimethylformamide (DMF) in a similar matter as previously described (Macias-Sanchez et al., 2009). To the greatest extent possible the algae matter and extracts were kept cold and in the absence of light, to avoid deterioration of pigments. The extracts were diluted to a proper level and filtered before spectrophotometric analyzes. Absorbance spectra of the alga extracts were obtained using a Hitachi U-3000 (Tokyo, Japan) UVeVIS spectrophotometer, with a slit width of 1 nm, working at a scanning rate of 60 nm  min1. The samples in DMF were scanned from 400 to 700 nm (see Fig. 4). Without Chl b influencing the absorbance of Chl a in the red spectrum region, the concentrations of Chl a (Ca) could be calculated directly using Lambert-Beer's law (Eq. (1)). The absorbance maxima of Chl a in the red region is at 664 nm (see Fig. 4), the specific absorption coefficient (a) for Chl a in DMF is

1.0

480

Absorbance

0.8

90.41 L  g1  cm1 (Wellburn, 1994), the path length of the cuvette was 1 cm.

Ca ¼

A664 a$l

(1)

The concentrations of total carotenoid pigments (Sx þ c), were calculated according to the equation proposed by Wellburn (1994) for CSxþc in DMF (Eq. (2)), with the absorbance measurement at 480 nm (Fig. 4). Eq. (2) adjusts for the absorbance of both Chl a and Chl b at 480 nm, however as mentioned in the introduction C. tenuicorne does not contain Chl b (concentration of Chl b expressed as Cb in Eq. (2)).

1000A480  0:89Ca  52:02Cb CP xþc ¼ 245

(2)

5.3. Analysis of bromophenolic compounds The OH-PBDEs and BPs were extracted from the alga samples (roughly 10 g each) using a solvent mixture of 2-propanol, diethyl ether and N-hexane. The extracts were washed with an acidic (0.2 M hydrochloric acid) sodium chloride (0.9%) solution prior to evaporation of the solvents (Jensen et al., 2003). Due to the low content of extractable organic material in these algae, the samples were not evaporated to complete dryness, as this may cause losses of low brominated BPs. After evaporation the volumes of the extracts were corrected to 3 mL in N-hexane, and the phenolic compounds were subsequently separated from neutral compounds using pH partitioning with potassium hydroxide in 50% ethanol (0.5 M, 2  1.5 mL). The alkaline fractions were acidified with hydrochloric acid and the phenolic compounds were extracted once more into N-hexane. Finally the OH-PBDEs and BPs were methylated with diazomethane and subsequently (after evaporation of excess diazomethane) treated with sulfuric acid (Hovander et al., 2000) before analyzes on gas chromatography mass spectrometry (GC-MS). Quantifications were made on a Varian GC triple quadrupole MS (Varian Inc., CA, USA) in negative ionization electron capture (ECNI) mode. The analyses were conducted using single ion monitoring (SIM) of the bromide ions m/z 79 and 81. Seven OH-PBDE congeners and two BPs were quantified and all were identified on the basis of their relative retention time in comparison with authentic reference standards. Procedural blanks were run in parallel to all samples, and all concentrations were recovery corrected according to the recovery of the surrogate standard 20 -OH-BDE28 (71 ± 14%, n ¼ 8). Quantifications were conducted against external calibration curves. Additional instrumental settings as well as sources for chemicals and standards used can be found in the SD.

664 5.4. Acquisition of external parameter data

0.6 0.4 0.2 0.0 400

500

600

700

Wavelength (nm) Fig. 4. Absorbance spectrum of a C. tenuicorne extract in DMF. This particular sample was collected 110719.

Modelled PPD data was extracted from the STRÅNG database (strang.smhi.se), governed by the Swedish Meteorological and Hydrological Institute (SMHI). The sum of the five daily PPD values (in mol  m2) leading up to each sample occasion was calculated and plotted against the concentrations of pigments and bromophenolic compounds in the collected algae. The same was done for UV (given in mWh  m2). However, measured data was also available for UV, from SMHI, albeit measured at some distance (roughly 140 km south-west) from the sampling location (the modelled data was acquired for the geographical coordinates of latitude 59.185, longitude 18.70).

Please cite this article in press as: Lindqvist, D., et al., Biosynthesis of hydroxylated polybrominated diphenyl ethers and the correlation with photosynthetic pigments in the red alga Ceramium tenuicorne, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.009

D. Lindqvist et al. / Phytochemistry xxx (2016) 1e8

Acknowledgment Thanks to Johan Eriksson and Ioannis Athanassiadis at Stockholm University for technical assistance during instrumental analysis. We also thank Henrik Dahlgren at the Museum of Natural € Island, History in Stockholm for collecting the algae from N€ amdo and Elena Gorokhova at Stockholm University for taking the time to discuss our finding with us. This research was partly funded by the Swedish Environmental Protection Agency. Financial support was also given by Stockholm University's strategic marine environment research funds, through the Baltic Ecosystem Adaptive Management (BEAM) project. The STRÅNG data used here are from the Swedish Meteorological and Hydrological Institute (SMHI), and were produced with support from the Swedish Radiation Protection Authority and the Swedish Environmental Protection Agency. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2016.10.009. References Abrahamsson, K., Choo, K.S., Pedersen, M., Johansson, G., Snoeijs, P., 2003. Effects of temperature on the production of hydrogen peroxide and volatile halocarbons by brackish-water algae. Phytochemistry 64, 725e734. Agarwal, V., El Gamal, A.A., Yamanaka, K., Poth, D., Kersten, R.D., Schorn, M., Allen, E.E., Moore, B.S., 2014. Biosynthesis of polybrominated aromatic organic compounds by marine bacteria. Nat. Chem. Biol. 10, 640e647. €ck, S., Likolammi, M., 2004. Phenology of Ceramium tenuicorne in the SW Gulf of Ba Finland, northern Baltic Sea. Ann. Bot. Fenn. 41, 95e101. Barahona, M.V., Sanchez-Fortun, S., 1999. Toxicity of carbamates to the brine shrimp Artemia salina and the effect of atropine, BW284c51, iso-OMPA and 2-PAM on carbaryl toxicity. Environ. Pollut. 104, 469e476. Baroli, I., Niyogi, K.K., 2000. Molecular genetics of xanthophyll-dependent photoprotection in green algae and plants. Phil. Trans. R. Soc. Lond. B 355, 1385e1394. €m, L., Bergstro €m, U., 1999. Species diversity and distribution of aquatic Bergstro macrophytes in the northern Quark, Baltic Sea. Nord. J. Bot. 19, 375e383. Bianchi, T., Kautsky, L., Argyrou, M., 1997. Dominant chlorophylls and carotenoids in macroalgae of the Baltic Sea (Baltic Proper): their use as potential biomarkers. Sarsia 82, 55e62. Bolton, M.D., 2009. Primary metabolism and plant defense-fuel for the fire. Mol. Plant-Microbe Interact. 22, 487e497. Butler, A., Walker, J.V., 1993. Marine haloperoxidases. Chem. Rev. 93, 1937e1944. Collen, J., Pedersen, M., 1994. A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta). Physiol. Plant 92, 417e422. Cronin, G., Hay, M.E., 1996. Within-plant variation in seaweed palatability and chemical defenses: optimal defense theory versus the growth-differentiation balance hypothesis. Oecologia 105, 361e368. Dahlgren, E., Enhus, C., Lindqvist, D., Eklund, B., Asplund, L., 2015. Induced production of brominated aromatic compounds in the alga Ceramium tenuicorne. Environ. Sci. Pollut. Res. 22, 18107e18114. Dingemans, M.M.L., de Groot, A., van Kleef, R.G.D.M., Bergman, A., van den Berg, M., Vijverberg, H.P.M., Westerink, R.H.S., 2008. Hydroxylation increases the neurotoxic potential of BDE-47 to affect exocytosis and calcium homeostasis in PC12 cells. Environ. Health Perspect. 116, 637e643. Dummermuth, A.L., Karstenb, U., Fisch, K.M., Koenig, G.M., Wiencke, C., 2003. Responses of marine macroalgae to hydrogen-peroxide stress. J. Exp. Mar. Biol. Ecol. 289, 103e121. €gifter i biota. Havet Faxneld, S., Nyberg, E., Danielsson, S., Bignert, A., 2014. Miljo 2013/2014 78e81. Fernie, K.J., Letcher, R.J., 2010. Historical contaminants, flame retardants, and halogenated phenolic compounds in peregrine falcon (Falco peregrinus) nestlings in the Canadian Great lakes basin. Environ. Sci. Technol. 44, 3520e3526. Flodin, C., Helidoniotis, F., Whitfield, F.B., 1999. Seasonal variation in bromophenol content and bromoperoxidase activity in Ulva lactuca. Phytochemistry 51, 135e138. Geiselman, J., 1980. Ecology of Chemical Defenses of Algae against the Herbivorous Snail, Littorina littorea, in the New England Rocky Intertidal Community. Dissertation. Massachusets institute of technology. Goecke, F., Labes, A., Wiese, J., Imhoff, J.F., 2013. Phylogenetic analysis and antibiotic activity of bacteria isolated from the surface of two co-occurring macroalgae from the Baltic Sea. Eur. J. Phycol. 48, 47e60. Gribble, G.W., 2010. Naturally occurring organohalogen compounds - a comprehensive update. Prog. Chem. Org. Nat. Prod. 91 http://dx.doi.org/10.1007/978-3211-99323-1. Hakk, H., Letcher, R.J., 2003. Metabolism in the toxicokinetics and fate of brominated flame retardants - a review. Environ. Int. 29, 801e828.

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