Discussion
Biomagnification of BMAA along food chains was synthetically
demonstrated in the diatoms-dominated marine ecosystem. The
concentration of BMAA accumulated in zooplankton was generally higher
than that in phytoplankton (Fig. 2), and only in January the average
level of BMAA in zooplankton was slightly lower than that in
phytoplankton (Supplementary Fig. 3). We note that the average
concentration of BMAA in phytoplankton was much higher in January than
that in other seasons. The lowest density of phytoplankton
(> 20 µm) and the highest percent of diatoms (99.9%) also
occurred in January. Inversely, the lowest percent (87.2%) of diatom in
phytoplankton net samples occurred in June, meanwhile the average
concentration of BMAA was the lowest in four seasons, which hints that
the concentration of BMAA in phytoplankton was positively related to
diatoms. The diatom Bacillaria paxillifera was the dominant
species only in January, but it has not been reported as a producer of
BMAA in the previous studies17-19. Similarly, high
average concentrations of BMAA accumulated in zooplankton in January and
June, which may be related to the feeding behavior of zooplankton. Only
the
zooplanktonOikopleura dioica predominated in the zooplankton community as
the dominant species in both January and June (Supplementary Table 1).
The zooplankton O. dioicacreates
a mucus net ”house” for capturing phytoplankton during feeding. Water is
pumped through this house and phytoplankton are filtered out of the
water and then transferred into the mouth. We suggest that this feeding
method would result in an accumulation of BMAA by filtering large
amounts of diatoms, while Calanus sinicus and Acartia
pacificaare
omnivorous, primarily feeding on phytoplankton but also consuming
rotifers, ciliates, and their own eggs. Furthermore,Noctiluca
scientillans is a heterotroph, primarily engulfing fish eggs, bacteria,
in addition to plankton, diatoms and dinoflagellates. Therefore, the
feeding habit of zooplankton and the species composition of
phytoplankton are important factors for the accumulation of BMAA in
zooplankton populations.
The average concentration of BMAA in phytoplankton and zooplankton net
samples at all sampling stations in four seasons were 0.14 and 0.30 µg
g-1 DW, respectively. Compared across different
sampling stations, the highest
trophic
magnification factor (TMF) between phytoplankton and zooplankton trophic
levels are shown in Supplementary Fig. 4. The TMF values of BMAA ranged
from 0.45 to 11.82 in most of sampling stations except for C1 station in
all seasons, with an average value 4.58. The highest value occurred at
the sampling station C3 in September (11.82), and A3, A5, B2, D6 and D7
stations had relatively low TMF values (0~2). Compared
across different seasons, the highest TMF values for BMAA occurred in
different sampling stations was in September (8.50), followed by June
(3.98) and January (1.13). In a previous study, the concentration of
BMAA in cyanobacteria-enriched samples collected from Baltic Sea ranged
from 0.001 to 0.015 μg g-1 DW, and
zooplankton-enriched fraction contained 6-fold higher on average than
the cyanobacterial samples20, of which the TMF is in
the range of our study from phytoplankton to zooplankton
(1.13~8.50). For another case, average BMAA
concentrations were 4.12, 3.21, 3.76, and 6.05 µg g-1DW in cyanobacteria, mollusks, crustaceans and various fish species,
respectively, and the TMF values were below 1.5 along food chains in
Gonghu Bay of Lake Taihu, China46. A relatively low
biomagnification of BMAA, trophic magnification factor
~1.20, was also found in six freshwater aquaculture
products from Taihu Lake Basin in China21.
Comparatively, Jiaozhou Bay showed a relatively high biomagnification
along food chains from diatom-dominated phytoplankton.
In order to assess the risk of human exposure to BMAA, marine animals
including filter-feeding bivalve mollusks, carnivorous crustacea, and
saprophytic
gastropod mollusks, were collected together to monitor BMAA
accumulation. Results showed that BMAA was universally detected in
marine animals collected during the whole year. Average concentrations
of BMAA accumulated in bivalve, crustacea and gastropod mollusks were
0.84, 1.19, and 2.08 μg g-1 WW, respectively,
corresponding to 4.22, 5.95, and 10.42 μg g-1 DW based
on the conversion factor 5: 1 (wet weight: dry weight). The TMF of BMAA
from phytoplankton (average 0.14 μg g-1 DW) to these
animals were about 30.1, 42.5, and 74.4, respectively (Fig. 6). The
production ability of diatom-dominated phytoplankton in this study was
higher 1~2 orders of magnitude of that produced by
cyanobacteria in Baltic Sea20. The concentrations of
invertebrates (mussels and oysters, TMF 7~22) and
various vertebrates (fish, TMF up to 200 in some tissues of fish) also
demonstrated the magnification of BMAA along food
chains20, but a recent study questioned the
concentration of BMAA in the benthic fishes in the ecosystem of Baltic
Sea45. However, the detection of BMAA with higher
concentrations in gastropod mollusks living in the benthic ecosystem was
consistent with that of fishes living closer to the bottom of the Baltic
Sea which also contained higher BMAA concentrations than pelagic
fishes20. It is possible that BMAA settles to benthic
environments with the death of cyanobacteria and diatoms and
subsequently stored in marine sediments. More research is required to
discover the accumulation mechanism within marine animals living in the
benthic ecosystem. Compared to another study, although the total average
concentration of BMAA detected in freshwater cyanobacterial samples
(4.12
µg
g-1 DW) from Gonghu Bay, Taihu Lake,
China46 is about 30 times that detected in the
diatom-dominated phytoplankton from Jiaozhou Bay (0.14 µg
g-1 DW), the average concentration of BMAA detected in
the high-trophic N. didyma (21.4 μg g-1 DW) in
this study is about 5-times higher than that in the freshwater mollusks
and crustaceans (3.21 and 3.76 μg g-1 DW) reported in
the previous study46. This phenomenon demonstrated
that the TMF of BMAA from producer to benthic animals in the marine
ecosystem studied here is great higher than that in the freshwater
ecosystem of Gonghu Bay.
Results showed that BAMA was more widely present than BMAA and other
isomers in phytoplankton and zooplankton samples collected from Jiaozhou
Bay in this study. Unfortunately, the lack of commercialized BAMA
reference material restricted an accurate analysis of this compound.
Therefore, BAMA was verified by the precolumn AQC-derivatization method
using C18 column for most of samples because it does not have baseline
separation from BMAA on the HILIC column. In order to assess the
variation trend of BAMA in different trophic organisms, the
concentration of BAMA was estimated by the mass spectrometry response of
BMAA standard in this study. We note that the ratios of BAMA to BMAA in
different organisms roughly tended to decrease with increasing trophic
levels. The relationship between BAMA and BMAA requires further studies
to be explained in detail in the future.
The phytoplankton community (> 20 µm) was dominated by
diatoms in Jiaozhou Bay in all four seasons in 2019 in this study, which
was also consistent with some previous studies using 76
µm-net47-49.
The density ratio of dinoflagellate to diatom in most months was less
than 0.2, except in July, in 201047, and the annual
average ratio of dinoflagellate to diatom was less than 0.08 in
201148. Diatoms were the most dominant phytoplankton
group throughout the year in 2017, except in spring and winter, whenNoctiluca scintillans was co-dominant49. In
this study, only the dinoflagellate Ceratium kofoidii was
co-dominant species in June, and diatoms Skeletonema spp.,Chaetoceros spp., Thalassiosira spp. were co-dominant
species almost in the whole year. The positive detection of BMAA in
phytoplankton net samples suggested that diatoms may be the producer of
BMAA in Jiaozhou Bay. The production of BMAA in marine
diatoms17-19,50 and freshwater
diatoms51 have been reported in some previous studies.
Therefore, a total of 56 strains of diatoms pertaining to five genera
including Pseudo-nitzschia , Chaetoceros ,Thalassiosira , Planktoniella , and Minidiscus were
analyzed for BMAA, in which only Thalassiosira andChaetoceros were reported before to produce BMAA. In the previous
study by Jiang et al .17, five genera of diatoms
(Achnanthes sp., Navicula pelliculosa , Proboscia
inermis , Skeletonema marinoi , Thalassiosira sp.) were
cultured and tested to produce BMAA, in which the concentration of
protein-bound BMAA in T. sp. was 3.28
ng
g-1 DW. Seven species of marine diatoms
(Navicula pelliculosa , Chaetoceros socialis ,Coscinodiscus granii , Thalassiosira weissflogii ,Phaeodactylum tricornutum , Skeletonema marinoi ,Ditylum brightwellii ) were cultured and analyzed for BMAA, in
which the concentration of protein-bound BMAA in Ch. socialis andT. weissflogii were 0.62 and 2.19
µg
g-1 DW, respectively, and varied concentration of
free-form BMAA was also detected except for D.
brightwellii 50. The highest production of BMAA was
observed at the stationary growth phase of four diatom strains includingPh. tricornutum , Ch. sp., Ch. calcitrans , andT. pseudonana , at 1.4, 1.6, 1.8, and 0.28 µg
g-1 DW, respectively18. Large
discrepancy of BMAA concentration occurred in the laboratory cultures ofThalasososira and Chaetoceros in above different studies.
In this study, 37.5% (21/56) of the studied diatom strains contained
the protein-bound BMAA which belonged to four genera except forMinidiscus . Only three species of Minidiscus were cultured
and analyzed here (Supplementary Table 5), and we could not determine
that the diatoms of this genus are non-toxic for BMAA due to variation
of BMAA production in the same diatom genus. Interestingly, BMAA was
mainly present in the precipitated-bound fraction of the diatom
cultures, followed by the total soluble fraction. In the genusPseudo-nitzschia , a total of 9 strains detected BMAA, in whichPs. multiseries (0.41 μg g-1 DW) and Ps.
caciantha (0.30 μg g-1 DW) cultures contained total
soluble BMAA, and the other 7 strains were only found precipitated bound
BMAA ranging with 0.11~3.05 μg g-1 DW.
In the genus Thalassiosira , 8 strains detected BMAA in the
precipitated bound fraction ranging from 0.33~3.95 μg
g-1 DW, in which T. minima , T. alleniiand T. gravida also contained total soluble BMAA. In the genusChaetoceros , 3 strains contained precipitated bound BMAA below
0.3 μg g-1 DW. The precipitated bound BMAA was also
detected in a strain of Planktoniella at 0.16 μg
g-1 DW. The average production level of BMAA in
different diatom genera decreased in order of Thalasosira (2.07
μg g-1 DW) > Pseudo-nitzschia(1.00 μg g-1 DW) > Planktoniella(0.16 μg g-1 DW) > Chaetoceros(0.12 μg g-1 DW). Among the positively detected
strains of diatoms, 40-fold difference of BMAA concentration between the
highest (T. minima , 3.95 μg g-1 DW) and the
lowest (Ps. sp., 0.11 μg g-1 DW) level. An
additional batch culture of some BMAA-producing strains of diatoms was
conducted to confirm the BMAA-producing ability of diatoms was steady
and specific. The results showed
that diverse diatom strains isolated from Chinese coast could produce
BMAA dominated in the precipitated bound fraction. The distribution form
of BMAA in the cultures of isolated diatom strains was largely different
from that in the phytoplankton collected from Jiaozhou Bay.
Additionally, the isomer BAMA was not found in the laboratory cultures
although it was ubiquitous in the field diatom-dominated phytoplankton
samples. This discrepancy hints that the production of BMAA in diatoms
is affected by environmental factors such as nutrient, temperature,
predator pressure, etc. What is the real source of BAMA in the
phytoplankton samples collected from Jiaozhou Bay? How do the
environmental factors regulate the production of BMAA by diatoms? These
problems should be deeply explored in further studies.
Misincorporation of BMAA into proteins in the place of L -serine
has been considered as a bioaccumulation and pathogenic mechanism for
neurodegenerative diseases33,34,52, but some studies
also demonstrated that BMAA was not be miscoded into proteins in higher
organisms53,54 which was reviewed and proposed as a
result of differing methodologies55. In this study,
BMAA in diatoms was the highest in the protein-bound form. So far the
biosynthesis of BMAA is still unknown. However, the analytical results
of BMAA in the cultures of diatom strains demonstrated that the
production of BMAA in diatoms was species- or strain-specific, and
affected by nutrient conditions19. For example, the
production of BMAA varied in the strains MC4451, MC4185, and MC4423 ofPs. multiseries isolated from Taiwan Strait Y13, Taiwan Strait
E6, and Taiwan Strait Y23, respectively. The strain MC4451 produced
total soluble BMAA, while MC4185 produced precipitated bound BMAA and
MC4423 did not produce BMAA. The strains MC6394 and MC6056 ofPlanktoniella blanda were isolated from Taiwan Strait Y11 and
Hong Kong, respectively, in which the former produced precipitated bound
BMAA but the latter did not. The strains MC6475 (from Shantou Nan’ao
Treasure Island) and MC6447 (Tsing Yi, Hong Kong) of Thalassiosira
minima have the same pattern. As it
has been pointed above that the BMAA form in the field phytoplankton
collected from Jiaozhou Bay was different from that in the cultures of
isolated diatom strains under laboratory conditions. We hypothesize that
environmental stress or ecological predatory pressure from zooplankton
induce diatoms to release a free form BMAA from the protein-bound
fraction through the nitrogenous metabolism pathways.
In the present study, the benthic and carnivorous gastropod N.
didyma was identified again as a typical vector for BMAA in Jiaozhou
Bay ecosystem, of which the average concentration of BMAA reached 21.4
μg g-1 DW corresponding to the TMF of about 150 from
phytoplankton. In order to explore the production ability of BMAA by the
symbiotic bacteria in the gut of N. didyma , a total of 7 species
of symbiotic bacteria were cultured and identified for toxin analysis.
Results showed that none of these bacteria produced BMAA, but DAB was
detected in all samples, which supports the hypothesis that the
neurotoxin BMAA detected in N. didyma was accumulated from food
chains. In addition, DAB, as an isomer of BMAA, has been detected before
in diverse organisms such as cyanobacteria, phytoplankton, zooplankton,
mollusks, etc.19,24,25,30,38,40, but we hypothesize
that the marine bacteria isolated from the gastropod gut are possible
source of DAB detected in aquatic organisms. Interestingly, some unknown
compounds were also detected in these bacteria with the same transition
ions as DAB, which may be precursor compounds relative to DAB. If true,
DAB could also be produced by
some
heterotrophic prokaryotes in the environment, but no significant
biomagnification phenomenon has been reported for DAB in aquatic
ecosystems including this study. However, BMAA is known to be produced
only by autotrophic prokaryotes (cyanobacteria) or eukaryotes (diatom or
dinoflagellate) in aquatic environments. More research is needed to
determine the biosynthesis of BMAA, BAMA and DAB in marine environments.