Is there a BMAA transfer in the pelagic and benthic food webs in the Baltic Sea?

The evidence regarding BMAA occurrence in the Baltic Sea is contradictory, with benthic sources appearing to be more important than pelagic ones. The latter is counterintuitive considering that pelagic primary producers, such as diatoms, dinoflagellates, and cyanobacteria, are the only plausible source of this compound in the food webs. To elucidate BMAA distribution in trophic pathways, we analyzed BMAA in the pelagic and benthic food webs sampled in summer 2010 in the Northern Baltic Proper. As potential BMAA sources, phytoplankton communities in early and late summer were used. As pelagic consumers, zooplankton, mysids and zooplanktivorous fish (herring) were used, whereas benthic invertebrates (amphipods, priapulids, polychaetes, and clams) and benthivorous fish (perch and flounder) represented the benthic food chain. To establish the trophic structure of the system, the stable isotope (δ13C and δ15N) composition of its components was determined. Contrary to the reported ubiquitous occurrence of BMAA in the Baltic food webs, only phytoplankton and lower consumers (zooplankton and mysids) of the pelagic food chain tested positive. Given that our analytical approaches were adequate, we conclude that no measurable levels of this compound occurred in the benthic invertebrates and any of the tested fish species in the study area. These findings indicate that widely assumed presence and transfer of BMAA to the top consumers in the food webs of the Baltic Sea and, possibly, other systems remain an open question. More controlled experiments and field observations are needed to understand the transfer and possible transformation of BMAA in the food web under various environmental settings.


Introduction
In the late 1960s, the increased incidence of amyotrophic lateral sclerosis-parkinsonism-37 dementia complex (ALS-PDC) among native Chamorro population (Guam, Micronesia) was 38 linked to β-N-methylamino-L-alanine (BMAA), a naturally produced non-proteinaceous 39 amino acid (Cox et al., 2003). A causative association between dietary exposure to BMAA 40 and this pathological condition has been broadly discussed, which stimulated research on 41 BMAA production and biomagnification in food webs and development of analytical 42 approaches for detection and quantification of BMAA and its natural isomers, 2,4-diamino 43 butyric acid (DAB), β-amino-N-methyl-alanine (BAMA) and N-(2-aminoethyl) glycine 44 (AEG) (Faassen, 2014;Lance et al., 2018). 45 The current view is that BMAA supposedly originates from microalgae and cyanobacteria. At 46 first, BMAA production was linked to aquatic and terrestrial cyanobacteria, both free-living 47 and symbiotic, which were considered the only producers of this compound (Cox et al., 2005; 48 Faassen, 2014). BMAA has been detected in a variety of aquatic environments where 49 cyanobacteria blooms can occur, such as oceans, lakes and desert springs, but also in 50 terrestrial environments, like desert mats (Cox et al., 2005(Cox et al., , 2009 webs has been greatly expanded as well as the production capacity. These findings have also 57 increased the possible variety of BMAA routes and bioaccumulation pathways in ecosystems 58 (Faassen, 2014). 59 In various freshwater and marine environments, invertebrate grazers and fish exposed to 60 blooms of potential BMAA producers have also been analyzed and, at least in some studies,    One would expect that pelagic fish that feed on zooplankton that is grazing on fresh diatoms, 94 dinoflagellates, and cyanobacteria (i.e., known BMAA producers) in the water column would 95 have higher BMAA concentrations in their body tissues compared to the bottom-dwelling 96 fish that feed primarily on deposit-feeding benthic animals that consume at least partially 97 degraded material. Indeed, even though spring diatom bloom is settled as a relatively fresh 98 material (Bianchi et al., 2002), the microbial activity in the sediment would decrease BMAA 99 5 content of the algae, and thus generally lower BMAA levels in the benthic food webs. The 100 settling of the summer bloom of cyanobacteria to the sediment has earlier been considered 101 negligible (Sellner et al., 1996), although more recent studies show that other cyanotoxins can  To elucidate BMAA distribution in the Baltic ecological pathways (Fig. 1) before and after a 108 cyanobacteria bloom, we analyzed BMAA levels in the pelagic and benthic food webs using 109 state-of-the-art analytical approach for BMAA detection and quantification. In parallel, to 110 confirm the trophic positions of the food web components used for the BMAA analysis, their 111 stable isotope (δ 13 C and δ 15 N) composition was determined; this is a standard method in 112 ecology and ecotoxicology surveys aiming to elucidate bioaccumulation pathways (Cabana 113 and Rasmussen, 1994). As potential BMAA sources, early-and late-summer phytoplankton communities were used.

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The pelagic samples (phytoplankton, zooplankton, and mysids) were collected in the  (Table 1). were sampled in Kvädöfjärden (58.0497° N, 16.7831° E). As a fish with pelagic feeding, we 162 used the Baltic herring Clupea harengus (Casini et al., 2004) sampled in the Landsort Deep. 163 All fish was length-measured, the epidermis and subcutaneous fatty tissue are carefully 164 removed, and muscle tissues were taken from the middle dorsal muscle layer for BMAA and 165 stable isotope measurements (Table 1).      invertebrates or fish species were found to contain BMAA. Moreover, no measurable BMAA 251 10 levels were detected in the sediment. Given that the analytical performance was adequate, 252 and both positive controls, i.e., the blue mussel and the cyanobacterium, tested positive, we 253 conclude that all samples that tested negative contained no measurable levels of this 254 compound.

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The blue mussel positive control yielded 3.1 µg g -1 wet weight, which is similar to the

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The BMAA concentrations measured in seston and zooplankton varied from 0.83 to 1.13 µg 265 g -1 wet weight, which corresponds to approximately 7.5-9.5 µg g -1 dry weight using common and open sea areas (Fig. 3). However, only one phytoplankton sample taken in the mid-June 294 contained BMAA (Fig. 4A, Fig. 5). The phytoplankton community composition at the time of  It is also possible that heterotrophic protists can accumulate BMAA and contribute to its bulk 309 concentration in seston. It has been suggested that picocyanobacteria, such as Synechococcus, 310 can contribute substantially to BMAA production and transfer to primary consumers whereas neither absolute nor relative amount of filamentous cyanobacteria contributed to the 317 BMAA levels (Fig. 4A). Analyzing more samples with different community structure and 318 linking BMAA levels to the community composition in the microbial loop would be one way 319 to identify the main BMAA producers and consumers in these communities. Another 320 approach would be to use culture-based studies with species that were putatively identified as 321 the BMAA producers in the field.

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Zooplankton and mysid samples were found to contain BMAA, both before and after the 323 cyanobacterial bloom, despite the lack of BMAA in the phytoplankton sampled in August 324 (Fig. 5). The composition of zooplankton communities differed substantially between the 325 sampling occasions, with cladocerans comprising >60% of the total zooplankton biomass in 326 June and copepods dominating (>70%) in August (data not shown). Moreover, BMAA levels 327 in mysids differed between the species, being 50-70% higher in M. mixta than in N. integer. 328 The difference could be related to the more herbivorous diet of N. integer and more 329 zooplanktivorous feeding of M. mixta (Rudstam et al., 1989). In line with this, the δ 15 N 330 values were 1.5‰ lower in N. integer compared to M. mixta (Fig. 6A), indicating more 331 herbivorous diet. Therefore, the stepwise increase in BMAA from phytoplankton to M. mixta 332 (Fig. 5) could be indicative of biomagnification, albeit more data are needed to substantiate 333 this suggestion.

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It has been repeatedly stressed that when collecting material for bioaccumulation studies, it is 335 essential to use an adequate temporal and spatial resolution and to ensure the pathway 336 identification and trophic relatedness of the ecosystem components (Walters et al., 2016). To 337 confirm the trophic relationships between the organisms involved in the bioaccumulation 338 assessment, a stable isotope approach has been commonly used (Cabana and Rasmussen,339 1994). We applied SIA to confirm the trophic linkages for the consumers, particularly the fish 340 species with a non-sedentary behavior. The SIA data (Fig. 6) confirmed that all animals 341 analyzed for BMAA were occupying the trophic positions as expected (based on their δ 15 N 342 values) and belonged to the same food web (δ 13 C values). In particular, the isotopic 343 signatures supported the assumption that herring collected in the area where zooplankton and 344 mysids were collected was indeed relying on these prey (Fig. 2); however, we found no 345 evidence of the BMAA presence in the fish. As no BMAA was found in the sediment and 346 benthic invertebrates (four species), it is not particularly surprising that all benthic fish that 347 were analyzed (two species) were negative. 348 The current view that in the food webs BMAA may be transferred from producers to 349 zooplankton and other filter-feeders, such as bivalves, and bioaccumulated in fish feeding on 350 these invertebrates is based only on a limited data. In particular, these patterns have been  Our findings suggest that BMAA levels in the Baltic food webs are much below the 376 suggested risk level considering effect concentrations reported for invertebrates  species, both zooplanktivorous and benthivorous, tested negative, which implies a low risk 379 for the top consumers, such as seal, birds, and humans. However, our sampling was limited to 380 a few occasions in the summer and a relatively small geographic area, whereas it is possible 381 that BMAA levels vary depending on environmental conditions and ecological responses of 382 its producers. Therefore, to understand the magnitude of this variability, it is important to 383 conduct a systematic sampling throughout a year and in different parts of the Baltic Sea.

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Sensitive and validated analytical methods should be used to ensure that the obtained results 385 are consistent. Quality control samples must be included in the surveys to evaluate the 386 performance of the methods, particularly the analyte recovery and accuracy of the results.   Table S1. Summary of samples used for BMAA analysis and SIA and collection methods. Organism size for the benthic invertebrates and mysids was determined as the average dry mass per individual in the SIA samples and the number of individuals used to prepare these samples, and for the fish, it is the total body length. For BMAA, on each sampling occasion, 2-3 replicate samples for invertebrates and 3 replicates for fish were analyzed; at least two technical replicates were used. Tows were taken with a small plankton net (10 µm) in the upper 20 m. Zooplankters (Acartia spp., Eurytemora affinis, and Bosmina coregoni) were separated by a light trap, picked with forceps under a compound microscope, and frozen at -20 °C. The rest of the material was filtered (5 µm) and used as phytoplankton samples. As detritus and heterotrophic protists were present, these samples were also referred to as seston.  Fig. 1. Conceptual diagram of the study design and the potential role of pelagic (invertebrates: zooplankton and mysids, and fish: herring) and benthic (invertebrates: amphipods, clams, priapulids, and polychaetes, and fish: flatfish and perch) food chains in the BMAA transfer from primary producers. mixta) collected before (June) and after (August) the cyanobacterial bloom. All samples were composite to integrate respective food web components over several sampling occasions; nd -not detectable.
24 Fig. 6. Food web structure determined by SIA (δ 15 N vs. δ 13 C; mean ± SD); pelagic (A) and benthic (B) compartments. See Table 1 for details on the food web components, sample origin, and the number of samples analyzed.