Environmental effects on arsenosugars and arsenolipids in ...Senescence (%) Observations Oban-Ctrl...

©CSIRO 2016 ©CSIRO 2016Environ. Chem. 2016, 13, 21-33 doi:10.1071/EN14229_AC

Page 1 of 30

Supplementary material

Environmental effects on arsenosugars and arsenolipids in Ectocarpus (Phaeophyta)

Ásta H. Pétursdóttir,A,B,C,D

Kyle Fletcher,B Helga Gunnlaugsdóttir,

C Eva Krupp,

A Frithjof C. Küpper

B

and Jörg FeldmannA,D

ATESLA – Trace Element Speciation Laboratory, Department of Chemistry, University of Aberdeen,

Aberdeen, AB24 3UE, Scotland, UK.

BOceanlab, University of Aberdeen, Newburgh, AB41 6AA, UK.

CMatis, Food Safety, Environment and Genetics Department, Vinlandsleid 12,

IS-113 Reykjavik, Iceland.

DCorresponding authors. Email: [email protected]; [email protected]

Instrumental parameters

Table S1. Instrumental parameters for ICP-MS and ESIMS

Column Agilent Eclipse, XBD-C18; 4.8 mm × 150 mm

Column temperature 30 °C

Injection volume 85 µL

Buffer A 0.1 % formic acid (methanoic acid) in water

Buffer B 0.1 % formic acid in methanol Splitter ratio 1 : 3

Flow rate 1 mL min–1

Gradient 0–25 min: 5–100 % B

25 min 100 % B

ICP-MS Agilent 8800

Mode Organic mode

RF power 1600 W

Nebuliser gas 0.84 L min–1

Optional gas 6 %

ESIMS LTQ Orbitrap Discovery; Thermo Scientific

Mode Positive

Spray voltage 4.5 kV

Environ. Chem. 2016 ©CSIRO 2016 ©CSIRO 2016

doi:10.1071/EN14229_AC

Page 2 of 30

Growth of cultures

a) b)

c)

Fig. S1. (a) Hincksia sp. from Reykjavík, Iceland; (b) Ectocarpus sp. from Cruden Bay, UK; (c) Pylaiella

littoralis, Reykjavik, Iceland.

a) b)

c) d)

Fig. S2. (a) Ectocarpus 022-10 (St3) in cultures grown under different conditions: (a) control sample; (b)

H2O2 addition; (c) low nitrate; (d) low phosphate.


doi:10.1071/EN14229_AC

Page 3 of 30

a) b)

Fig. S3. The visual difference for cultures grown as (a) control compared with (b) under oxidative stress.

Fig. S4. Phylogenetic tree of the three strains (algorithm used: Neighbour Joining phylogeny of the three

strains; assembled using MEGA6; Tamura et al.[1]).


doi:10.1071/EN14229_AC

Page 4 of 30

Table S2. Percentage of senescence cells in the Ectocarpus cultures Senescence

(%) Observations

Oban-Ctrl 20–30 St1 Oban-N 50–60 Sporangia visible, enlarged round cells with unusual structure Oban-P 50–60 Irregular sporangia, unhealthy looking cells, enlarged circular cell Oban-H2O2 60 Senescent cell much more tightly packed around a layer of somatic cells 022–10-Ctrl 70–80 Sporangia visible St3 022–10-N 70 022–10-P 60 022–10-H2O2 80–90 022–10-Ctrl+ As 70–80 St3 + As 022–10-N + As 70 022–10-P + As 60–70 Some enlarged cells 022–10-H2O2 + As 80–90 007–04-Ctrl 40–50 Some sporangia visible, much healthier than N and P treatments St1 007–04-N 70 Unusual cells detected 007–04-P 55–60 007–04-H2O2 60


doi:10.1071/EN14229_AC

Page 5 of 30

Total arsenic concentration in the Ectocarpus

Table S3. Mass balance for field-collected Ectocarpales

All concentrations are given in milligrams per kilogram. The MeOH/DCM fraction evaporated and

redissolved in MeOH. For total arsenic (totAs) concentration, the number of replicates depended on

available sample material. Labels A,B and C refer to different measurement days of the samples,

which are all different replicates

LS WS RS totAs

MeOH: sum of all

peaks

MeOH fraction

Colum recovery

WS of MeOH

Hexane fraction

H2O: sum of

all peaks

H2O fraction

Column recovery

Residue digest

Sum all

Total

Label totAs (%) (void) totAs totAs (%) totAs As conc.

EC–rvk 12–8

ECr1–B 1.29 0.95 136 0.19 0.02 1.18 1.89 62 2.7 5.5 7.6 (n = 1) ECr2–B 1.03 0.75 138 0.17 0.02 0.95 1.42 67 2.7 4.9

EC–rvk–17–8

ECr3–B 2.03 2.19 93 1.2 0.03 2.07 2.62 79 3.8 8.6 9.2 ± 1.3 (n = 2) ECr4–B 0.59 0.57 103 0.22 0.02 1.92 2.33 82 3.4 6.3

ECr5–A 0.64 0.57 113 0.22 0.03 1.72 2.27 76 4.1 6.9

ECr6–C 3.51 3.38 104 0.20 0.18 0.70 nm – 3.3 –

Hincksia sp.

H–B 1.27 2.62 49 0.56 0.22 1.21 1.25 96 3.4 7.3 11.4 (n = 1)

EC–Abdn

ECa1–A 1.38 1.39 100 0.12 0.02 2.52 2.69 94 9.3 13 13.9 ± 0.4 (n = 3) ECa2–B 1.28 1.22 105 0.33 0.04 1.84 2.03 91 9.1 12

ECa3–C 0.91 0.82 111 0.16 0.21 1.00 1.29 78 5.5 7.8

ECa4–C 1.27 1.10 115 0.15 0.31 1.05 1.23 85 5.0 7.6

EC–CB ECc–B 0.50 1.00 50 0 0.06 3.86 4.41 87 5.0 10 11.5 (n = 1)

Elachista sp.

E1–B 4.56 4.9 93 3.0 0.04 25.14 26.98 93 7.5 39 34 ± 2 (n = 3) E2–B 4.61 4.72 98 3.2 0.05 25.55 27.19 94 7.4 39

E3–A 4.83 4.16 116 3.7 1.36 21.23 21.55 99 8.7 34

Pylaiella sp.

P1–B 2.01 1.53 131 0.56 0.01 8.15 9.57 85 10 21 21.5 ± 0.3 (n = 3) P2–B 2.58 3.02 85 0.56 0.01 8.20 9.12 90 9.1 21

P3–A 1.47 1.1 134 0.93 0.01 8.22 9.12 90 12 22

P4–C 1.23 1.22 101 0.51 2.99 5.82 8.09 72 7.6 20

The MeOH/DCM extraction was performed before the water extraction and a portion of the water-

soluble arsenic species was extracted into this fraction. Table S3 and Table S4 show the concentration

of this void volume of the MeOH/DCM fraction, which contains water-soluble arsenic species, such

as AsSugars, as well as a minor contribution of AsLp break-down products.[2] To portray more

realistically the LS fraction, Fig. 2 in the manuscript does not include this void volume of the MeOH

fraction.


doi:10.1071/EN14229_AC

Page 6 of 30

Table S4. Total As in MeOH fraction, and sum of species, sum of species in water fraction and

sum of water-soluble arsenic eluting in void of the MeOH fraction, total arsenic (totAs)

concentration in the residue and the sum of all the extracted arsenic

All concentrations are given in milligrams per kilogram. nm, not measured; autosampler failed and

not enough sample material for another injection

LS WS RS Sample (each replica) MeOH:

sum of all peaks

MeOH: totAs

Column recovery

(%)

Water: sum of all

peaks

WS of MeOH (void)

Residue Total sum

1 EC–Oban Blank A nm 0.88 – 0.36 nm 2.12 3.36 2 EC–Oban Blank B 0.644 1.07 60.07 0.45 0.57 2.99 4.51 3 EC–Oban Blank C nm 1.41 – 0.49 nm 1.96 3.86 4 EC–Oban N30 % A 0.662 1.26 52.42 0.39 0.56 2.37 4.02 5 EC–Oban N30 % A 0.780 1.29 60.56 0.38 0.69 2.37 4.04 6 St1 EC–Oban N30 % A 0.903 1.03 87.47 0.35 0.74 1.65 3.03 7 EC–Oban P30 % A 0.664 1.29 51.50 0.50 0.55 2.96 4.75 8 EC–Oban P30 % A 0.584 0.87 67.47 0.46 0.44 2.64 3.97 9 EC–Oban P30 % A 0.530 0.97 54.44 0.28 0.44 3.02 4.27 10 EC–Oban H2O2 A 1.116 2.05 54.53 0.80 0.88 2.70 5.55 11 EC–Oban H2O2 A 1.328 2.21 60.12 0.93 0.95 1.99 5.13 12 EC–Oban H2O2 A 1.450 1.94 74.79 0.82 1.14 2.74 5.5 13 EC–007–04 Blank A nm 1.55 – 0.17 nm 3.10 4.82 14 EC–007–04 Blank B 1.51 2.35 64.32 0.19 0.79 3.08 5.62 15 EC–007–04 Blank C 0.97 1.83 53.18 0.14 0.32 4.08 6.05 16 EC–007–04 N30 % A 0.72 1.59 45.48 0.19 0.30 2.92 4.7 17 EC–007–04 N30 % B 0.79 1.59 49.42 0.24 0.30 2.82 4.65 18 St2 EC–007–04 N30 % C 0.69 1.67 41.03 0.08 0.40 2.92 4.67 19 EC–007–04 P30 % A 0.56 1.28 43.76 0.09 0.24 3.19 4.56 20 EC–007–04 P30 % B 0.78 1.41 54.98 0.24 0.47 3.69 5.34 21 EC–007–04 P30 % C 0.73 1.54 47.40 0.12 0.40 3.49 5.15 22 EC–007–04 H2O2 A 0.90 1.35 66.78 0.20 0.36 4.25 5.8 23 EC–007–04 H2O2 B 1.03 1.76 58.56 0.37 0.41 4.03 6.16 24 EC–007–04 H2O2 C 0.93 1.85 50.22 0.22 0.28 4.48 6.55 25 EC–022–10 Blank A 0.49 1.11 44.40 0.15 0.21 4.81 6.07 26 EC–022–10 Blank B 0.57 1.28 44.49 0.10 0.27 4.81 6.19 27 EC–022–10 Blank C 0.62 0.98 63.48 0.08 0.34 3.98 5.04 28 EC–022–10 N30 % A 0.54 1.10 49.59 0.11 0.24 4.52 5.73 29 EC–022–10 N30 % B 0.56 1.42 39.06 0.13 0.28 4.01 5.56 30 EC–022–10 N30 % C 0.48 1.11 42.88 0.16 0.22 3.89 5.16 31 EC–022–10 P30 % A 0.67 1.31 50.96 0.19 0.36 4.73 6.23 32 EC–022–10 P30 % B 5.47 6.99 78.22 0.32 5.12 3.80 11.11 33 EC–022–10 P30 % C 0.91 1.55 59.17 0.34 0.55 4.13 6.02 34 EC–022–10 H2O2 A 0.93 1.35 69.21 0.12 0.48 4.22 5.69 35 EC–022–10 H2O2 B 6.95 6.27 110.88 0.14 6.50 4.44 10.85 36 St3 EC–022–10 H2O2 C 5.54 5.37 103.10 0.11 5.17 6.07 11.55 37 EC–022–10 AsA 2.28 2.69 85.06 0.44 1.16 6.42 9.55 38 EC–022–10 AsB 1.43 2.02 70.78 0.32 0.29 6.71 9.05 39 EC–022–10 AsC 1.61 2.05 78.59 0.57 0.52 5.98 8.6 40 EC–022–10 AsN30 % A 1.21 1.74 69.73 0.38 0.42 4.78 6.9 41 EC–022–10 AsN30 % B 1.31 1.90 68.96 0.51 0.54 5.89 8.3 42 EC–022–10 AsN30 % C 2.11 2.36 89.05 0.56 1.24 5.48 8.4 43 EC–022–10 AsP30 % A 4.35 4.01 108.40 0.38 3.44 7.04 11.43


doi:10.1071/EN14229_AC

Page 7 of 30

LS WS RS Sample (each replica) MeOH:

sum of all peaks

MeOH: totAs

Column recovery

(%)

Water: sum of all

peaks

WS of MeOH (void)

Residue Total sum

44 EC–022–10 AsP30 % B 4.97 4.68 106.11 0.52 4.15 5.52 10.72 45 EC–022–10 AsP30 % C 1.70 3.21 52.98 0.48 1.05 5.00 8.69 46 EC–022–10 As H2O2 A 2.92 3.52 83.13 0.35 1.68 7.17 11.04 47 EC–022–10 As H2O2 B 8.08 6.95 116.35 0.70 6.88 6.26 13.91 48 EC–022–10 As H2O2 C 3.34 3.05 109.54 0.51 2.28 4.96 8.52

Fig. S5. Mass balance for the EC cultures. Sum of all peaks in the LS fraction, includes the void volume (i.e.

water soluble species) (some replicates had high concentrations in the void which was supported by totAs

concentration of the fractions, Table S4). Error bars for the fractions are s.d. Error bars for totAs are percentage

combined uncertainty (dependent on amount of dry sample available).

There was not enough sample material to measure the totAs in the same samples as the different

fractions of the EC cultures. Instead, a small amount was grown further for 8 weeks (one replica only

for each set of conditions). The results tended to be in a similar range to the sum of the different

fractions; 105 % ± 31 of the totAs for St2 and St3, Fig. S5. The sum of fractions for St1 was higher

than the measured totAs (165 % ± 16).

0

2

4

6

8

10

12

14

16

18

20

conc

. (m

g / k

g)

WS: Sum all peaks LS: Sum all peaks RS totAs


doi:10.1071/EN14229_AC

Page 8 of 30

This variation could be due to the algae not being the same sample as for the sequential extraction,

hence not as comparable as, for example, for the field-collected algae, and also the sample material

obtained ranged from 1–10 mg depending on environmental conditions. Samples of 3 mg or less

tended to differ more from the sum of fractions than samples where more material was available.

Additionally, for some replicas, there was an increase of arsenic found in the void volume of the

MeOH fraction (see Table S4). This was mostly evident for low-phosphate and oxidative stress

conditions – reflected here in higher totAs concentrations, Fig. S5.

When considering the small amount of sample material – both for the totAs measurement and the

sequential extraction – it is clear that the mass balance is in good agreement.

Environ. Chem. 2016 ©CSIRO 2016

doi:10.1071/EN14229_AC

Page 9 of 30

Arsenolipids: quantification and identification

Table S6. Quantification of AsLps in the Ectocarpus cultures (n = 3)

All concentrations are given in milligrams per kilogram. Sum, sum of AsLps (total concentration)

Rt (min) St3-ctrl St3-N St3-P St3-OS Rt (min) St3-As St3-AsN St3-AsP St3-OS 1.8 0.274 ± 0.054 0.244 ± 0.024 2.01 ± 2.20 4.05 ± 2.58 1.8 0.65 ± 0.37 0.73 ± 0.36 2.88 ± 1.32 2.71 ± 2.32 21.3 0.005 ± 0.002 0.009 ± 0.002 0.007 ± 0.001 0.003 ± 0.003 25.3 0.180 ± 0.020 0.151 ± 0.005 0.049 ± 0.006 0.168 ± 0.027 25.2 0.906 ± 0.036 0.621 ± 0.045 0.290 ± 0.063 0.586 ± 0.075 30.2 0.073 ± 0.004 0.093 ± 0.011 0.059 ± 0.014 0.103 ± 0.005 30.3 0.135 ± 0.006 0.133 ± 0.010 0.066 ± 0.011 0.121 ± 0.007 – – – – 31.9 – – 0.009 ± 0.001 0.019 ± 0.001 32.5 – – 0.015 ± 0.002 0.013 ± 0.006 32.7 – – 0.020 ± 0.001 0.017 ± 0.004 33.8 – – 0.011 ± 0.001 0.029 ± 0.005 33.6 – – 0.022 ± 0.001 0.066 ± 0.009 35.3 0.019 ± 0.011 0.015 ± 0.002 0.120 ± 0.028 0.081 ± 0.012 35.3 0.029 ± 0.002 0.019 ± 0.002 0.25 ± 0.024 0.027 ± 0.007 39.1 0.007 ± 0.001 0.010 ± 0.001 0.034 ± 0.006 0.013 ± 0.004 39.1 0.020 ± 0.005 0.014 ± 0.002 0.061 ± 0.011 0.027 ± 0.007 44.5 0.008 ± 0.001 0.012 ± 0.002 0.045 ± 0.010 0.016 ± 0.003 44.7 0.023 ± 0.008 0.015 ± 0.004 0.075 ± 0.012 0.037 ± 0.003 Sum 0.288 ± 0.011 0.281 ± 0.018 0.332 ± 0.031 0.423 ± 0.039 Sum 1.12 ± 0.02 0.811 ± 0.037 0.79 ± 11 0.877 ± 0.077 1.8 0.573 0.665 ± 0.077 0.479 ± 0.055 0.997 ± 0.110 1.8 0.56 ± 0.23 0.331 ± 0.046 0.373 ± 0.097 0.352 ± 0.054 24.5 – – 0.006 ± 0.002 – 25.3 0.037 0.023 ± 0.003 0.043 ± 0.003 0.069 ± 0.007 25.0 0.496 ± 0.032 0.241 ± 0.032 0.185 ± 0.014 0.295 ± 0.029 26.7 – 0.008 ± 0.001 0.007 ± 0.002 – 27.6 – – 0.005 ± 0.001 – 29.3 0.014 0.020 ± 0.008 0.025 ± 0.007 0.036 ± 0.010 28.9 0.107 ± 0.007 0.081 ± 0.007 0.054 ± 0.026 0.157 ± 0.010 32.8 0.004 0.009 ± 0.006 0.005 ± 0.001 0.023 ± 0.007 32.6 0.016 ± 0.001 0.020 ± 0.001 0.030 ± 0.025 0.047 ± 0.002 35.2 0.015 0.044 ± 0.023 0.023 ± 0.009 0.136 ± 0.036 35.0 0.029 ± 0.001 0.031 ± 0.001 0.019 ± 0.007 0.063 ± 0.005 36.0 0.014 ± 0.001 0.015 ± 0.001 0.014 ± 0.005 0.028 ± 0.004 38.4 0.004 0.009 ± 0.002 0.006 ± 0.001 0.015 ± 0.003 38.4 0.010 ± 0.004 0.009 ± 0.004 0.008 ± 0.002 0.005 ± 0.001 43.7 0.002 0.013 ± 0.004 0.007 ± 0.002 0.016 ± 0.002 43.2 0.008 ± 0.002 0.009 ± 0.002 0.006 ± 0.001 0.006 ± 0.001 Sum 0.076 0.105 ± 0.032 0.117 ± 0.022 0.282 ± 0.059 – 0.681 ± 0.033 0.401 ± 0.033 0.316 ± 0.011 0.601 ± 0.046


doi:10.1071/EN14229_AC

Page 10 of 30

Table S7. Identified AsHCs in Ectocarpus cultures

C19 H42 O As and C21 H46 O As identified by ESIMS accurate mass and retention time, compared

with As75 ICP-MS signal. Found in one to three of the replicas for each set of conditions. MSMS,

AsHC fragmentation pattern observed, example given in Fig. S5. Δm/z, (m/zfound – m/zcalc) × 106/m/zcalc

AsHC360 Δm/z (ppm)

Rt (min) MSMS

AsHC388 Δm/z (ppm)

Rt (min)

C19 H42 O As [M+H]+

measured

C21 H46 O As [M+H]+

measured

St1-ctrl 361.2436 –2.75 25.1 No 389.2762 0.76 25.7 St1-N 361.2438 –2.25 25.1 No

St1-P 361.2439 –1.89 25.1 Yes 389.2771 2.97 25.8 St1-OS 361.2439 –2.06 25.1 Yes

St2-ctrl 361.2442 –1.23 24.8 Yes 389.2759 –0.16 25.5 St2-N 361.2441 –1.48 24.9 Yes 389.2754 –1.27 25.6 St2-P 361.2441 –1.40 24.9 Yes 389.2755 –1.04 25.6 St2-OS 361.2439 –2.06 25.1 Yes 389.2752 –1.96 25.7 St3-ctrl 361.2445 –0.20 25.1 Yes 389.2761 0.38 25.7 St3-N 361.2443 –0.81 25.2 Yes 389.2757 –0.47 25.9 St3-P 361.2443 –0.98 25.3 Yes 389.2766 1.71 25.9 St3-OS 361.2440 –1.64 25.2 Yes 389.2752 –1.89 25.8 St3-As 361.2443 –0.81 25.1 Yes 389.2755 –1.19 25.8 St3-AsN 361.2442 –1.23 25.1 Yes 389.2753 –1.50 25.7 St3-AsP 361.2444 –0.65 25.2 Yes 389.2755 –1.12 25.8 St3-AsOS 361.2443 –0.90 25.2 Yes 389.2757 –0.63 25.8


doi:10.1071/EN14229_AC

Page 11 of 30

Fig. S6. MS2 chromatogram for St1-OS for AsHC360 (C19 H42 O As, 361.2444, Δm –0.647) showing the

main fragments: C2 H4 As (102.9522 Δm –1.73 ppm), C2 H6 As (104.9680, Δm –0.078), C2 H8 O As

(122.9785, Δm –0.918).

Table S8. Tentative identification of AsPLs in Ectocarpus cultures

C44 H87 O14 As P and C46 H91 O14 As P identified by ESIMS accurate mass and retention time,

compared with As75 ICP-MS signal. Found in one to three of the replicas under each set of conditions

AsPL944 Δm (ppm)

Rt (min)

AsPL972 Δm (ppm)

Rt (min) C44 H87 O14 As P C46 H91 O14 As P

St1-N 945.5058 1.49 35.5 St1-P 945.5048 0.45 35.3 St2-ctrl 945.5035 –0.90 33.4 St2-N 945.5023 –2.25 33.3 St2-P 945.5054 1.04 33.2 St2-OS 945.5070 2.78 33.3 St3-As 945.5070 2.78 35.1 St3-AsN 945.5054 1.10 34.5 St3-AsP 945.5010 –3.61 34.1 973.5349 –0.865 35 St3-AsOS 945.5046 0.20 34.1

ec10 #1578 RT: 25.13 AV: 1 NL: 5.06E5F: FTMS + c ESI d w Full ms2 [email protected] [85.00-375.00]

100 120 140 160 180 200 220 240 260 280 300 320 340 360

m/z

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Re

lative

Ab

un

da

nce

361.24

227.22

104.97122.98

268.30

102.95 249.08 317.77223.62 336.92125.76 193.64168.61


doi:10.1071/EN14229_AC

Page 12 of 30

(a) (b)

Fig. S7. Identified AsPL944 in (a) St1-P; (b) St2-OS; (c) St3As-P. Column recovery and further information

for the samples in Tables S9–S13 is given in Table S3.

Table S9. Quantities of AsLs in Ectocarpus found in Cruden Bay, UK and Hincksia sp. found

in Reykjavík, Iceland EC Cruden Bay (EC-C) Hincksia

(H1)

Rt (min)

Concentration (mg kg–1)

Rt (min)

Concentration (mg kg–1)

25.7 0.019 25.4 0.070 26.6 0.136 26.4 0.335 – – 27.2 0.060 27.8 0.026 28.0 0.052 – – 30.1 0.054 – – 30.8 0.055 – – 32.0 0.031 33.8 0.006 34.0 0.037 36.3 0.008 36.7 0.017 Sum AsLps 0.195 Sum 0.711

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

0.005

0.007

0.009

0.011

0.013

0.015

0.017

0.019

24 34 44

Inte

nsity

(ESI

MS)

Rel

ativ

e in

tens

ity (I

CPM

S)

Time (min)

As75 AsPL944

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

0.003

0.013

0.023

0.033

0.043

0.053

0.063

0.073

0.083

0.093

24 34 44

Inte

nsity

(ESI

MS)

Rel

ativ

e in

tens

ity (I

CPM

S)

Time (min)

As75

-2000

0

2000

4000

6000

8000

10000

12000

0

0.005

0.01

0.015

0.02

0.025

0.03

22 27 32 37 42

Inte

nsi

ty (

ESIM

S)

Rel

ativ

e in

ten

sity

(IC

PM

S)

Time (min)

As75

AsPL972

AsPL944


doi:10.1071/EN14229_AC

Page 13 of 30

Table S10. Ectocarpus from Aberdeen: identification and quantification of species ECa2 (n = 1) ECa2 acid (n = 1) ECa2 base (n = 1) ECa1 (n = 1) ECa3–4 (n = 2) Formula Rt

(min) Conc.

(mg kg–1) m/z

[M + H]+ Δm/z (ppm)

Rt (min)

Conc. (mg kg–1)

Δm/z (ppm)

Rt (min)

Conc. (mg kg–1)

Δm/z (ppm)

Rt (min)

Conc. (mg kg–1)

Rt (min)

Conc. (mg kg–1)

Δm/z (ppm)

1.8 0.118 1.8 0.883 1.8 0.827 1.8 0.33 1.8 0.155 – – 2.8 0.052 22.1 0.002 – – – – – – 3.2 0.014 23 0.005 – – – – – – 4.9 0.002 24.3 0.002 – – – – U (unknown) 25.1 0.002 23.2 0.001 24.6 0.008 – – – – U 26 0.006 25.2 0.01 25.2 0.079 25.6 0.008 22.5 0.003 C23 H38 O As 26.9 0.858 405.2150 4.19 25.9 0.014 – – 26.1 0.003 24.6 0.645 –0.45

C19 H42 O As 27.8 361.2439 –2.00 26.4 0.577 –1.45 26.4 0.633 –0.04 26.5 0.798 25.3 –1.3

C20 H44 O As 27.8 375.2595 –2.14 –1.50 –1.00 –2.4

C21 H46 O As 27.9 389.2750 –2.37 27.2 0.066 –0.81 27.2 0.072 –1.04 –0.6

C22 H48 O As 28 403.2908 –1.97 –1.30 U 28.7 0.025 28 0.012 28 0.031 U 29.3 0.034 29.9 0.011 29.9 0.012 28.8 0.007 28.4 0.06 U 29.6 0.062 30.9 0.012 – – 29.3 0.003 – – U 30.5 0.011 – – – – 29.7 0.02 – – U 31.6 0.013 – – – – 31.4 0.003 30.3 0.009 C43 H85 O14 As P 32.4 0.018 931.4864 –2.52 – – – – 32.3 0.005 31.2 0.01 C44 H87 O14 As P 32.9 0.027 945.5035 –0.95 – – – – 32.7 0.029 32.2 0.07 –2.6

U 33.4 0.072 – – – – – – – – C46 H91 O14 As P 35.8 0.128 973.5352 –0.48 – – – – C45 H89 O14 As P 959.5185 –1.62 – – – C47 H91 O14 As P 985.5364 0.76 – – – – 35.1 0.051 34.7 0.08 –4.1

C48 H95 O14 As P 39.2 0.01 1001.5635 –3.51 – – – – 38.5 0.01 37.8 0.02 43.5 0.004 42.9 0.0021

Sum AsLps 1.27 0.69 0.81 0.95 0.93


doi:10.1071/EN14229_AC

Page 14 of 30

Table S11. Quantified and identified AsLps in Ectocarpus from Reykjavik, Iceland ECr6 (n = 1) ECr3 (n = 1) ECr4 (n = 1) ECr5 (n = 1) ECr1-2 (n = 2) Formula Rt

(min) Conc.

(mg kg–1) m/z


Rt (min)

Conc. (mg kg–1)

Rt (min)

Conc. (mg kg–1)

Rt (min)

Conc.. (mg kg–1)

Rt (min)

Conc. (mg kg–1)

U (unknown) – – – – – – 22.2 0.003 23.0 0.009 22.1 0.0012 ± 0.0005 U – – – – 24.3 0.029 23.1 0.006 23.8 0.003 23.1 0.0019 ± 0.0001 U – – – – 24.9 0.017 25.5 0.027 24.5 0.010 25.4 0.061 ± 0.009 U 24.2 – 405.2132 –0.2 25.9 0.112 26.0 0.009 25.7 0.021 25.9 0.02 C19 H42 O As 25.3 2.179 361.2443 –0.8 26.6 0.558 26.4 0.291 26.5 0.504 26.4 0.756 ± 0.107 C20 H44 O As 375.2596 –1.7 C21 H46 O As 389.2754 –1.4 U 27.5 0.016 – – 27.8 0.054 – – 27.8 0.017 27.1 0.056 ± 0.009 U 28.1 0.014 – – – – 28.8 0.011 27.8 0.037 ± 0.001 U 28.6 0.121 – – 30.2 0.006 29.4 0.010 30.1 0.016 ± 0.001 U 30.4 0.009 – – 30.6 0.011 30.8 0.006 29.9 0.016 30.8 0.022 ± 0.002 C47 H89 O14 As P 32.3 0.156 983.5170 –3.1 – – 31.4 0.005 31.5 0.003 31.8 0.005 ± 0.001 C45 H89 O14 As P 34.3 0.502 959.5181 –2.0 – – 33.3 0.014 32.7 0.003 ± 0.001 C46 H91 O14 As P 973.5342 –1.6 – – 34.0 0.004 35.3 0.020 33.8 0.006 ± 0.001 C47 H93 O14 As P 37.8 0.163 987.5590 7.8 – – 36.5 0.005 38.7 0.003 36.3 0.008 ± 0.001 C48 H95 O14 As P 1001.5685 1.5 – – – – – – – – C49 H97 O14 As P 42.8 0.134 1015.5821 –0.5 – – – – – – – – U 50.2 0.020 – – – – – – – – – – Sum AsLps 3.31 0.78 0.36 0.64 0.98 ± 0.12


doi:10.1071/EN14229_AC

Page 15 of 30

Table S12. Quantification and identification of AsLps in Elachista E1-E2 (n = 2) E3 (n = 1) Chemical formula Rt

(min) Conc. (mg kg–

1) Δm/z (ppm)

m/z [M + H]+

Rt (min)

Conc. (mg kg–1)

Δm/z (ppm)

m/z [M + H]+

C23 H38 O As – – 25.5 0.008 2.113 405.2142 U (unknown) 26.6 0.002 ± 0.001 26.0 0.013 C19 H42 As O 27.4–

27.7 0.703 ± 0.081 –1.063 361.2442 26.4 0.748 –3.166 361.2435

C20 H44 As O –1.982 375.2595 –4.248 375.2587 C21 H46 As O –1.808 389.2752 –4.249 389.2743 C22 H48 As O –1.895 403.2908 U 28.8 0.02 28.1 0.038 U 29.4 0.025 ± 0.001 28.9 0.015 U 29.9 0.050 ± 0.001 29.4 0.019 U 31.2 0.041 ± 0.006 29.8 0.028 U – – 31.1 0.027 U 32.6 0.025 ± 0.001 32.3 0.011 C43 H85 As O14 P 33.1 0.064 ± 0.001 1.018 931.4897 32.8 0.027 –2.653 931.4863 C44 H87 As O14 P –0.382 945.5040 –0.509 945.5039 C47 H89 As O14 P 33.9 0.108 ± 0.008 –1.384 983.5187 33.6 0.045 C45 H89 As O14 P 35.5–

35.9 0.314 ± 0.008 –0.273 959.5198 35.3 0.112 –3.774 959.5164

C46 H91 As O14 P –1.429 973.5343 –4.316 973.5315 C47 H91 As O14 P –0.638 985.5363 –2.65 985.5331 C47 H93 As O14 P 39.6 0.099 ± 0.018 –0.63 987.5507 38.6 0.042 –3.222 987.5482 C48 H95 As O14 P –1.929 1001.565 –3.876 1001.563 C49 H97 As O14 P 45.2 0.034 1.761 1015.5844 – – Sum AsLps 1.49 1.13

For EC Rvk and Pylaiella, a single replica was much higher in concentration than the others, Table

S11 and Table S13. The higher concentration was supported by the totAs concentration measured

from the extract (Table S3); this could be due to homogeneity issues. These samples were used for

identification of the lipids because of their higher concentrations. They were excluded from average

calculations for the sum of AsLps, given in the main text in Fig. 2a, c.


doi:10.1071/EN14229_AC

Page 16 of 30

Table S13. Quantified and identified AsLps in Pylaiella P2 (n = 1) P4 (n = 2) P1 (n = 1) P3 (n = 1) Formula Rt

(min) Conc.

(mg kg–1) m/z


Rt (min)

Conc. (mg kg–1)

m/z [M + H]+

Δm/z (ppm)

Rt (min)

Conc. (mg kg–1)

Rt (min)

Conc. (mg kg–1)

24.9 0.002 24.9 0.004 26.8 0.038 24.0 0.003 ± 0.001 25.9 0.011 25.9 0.017 C23 H38 O As 27.7 0.176 405.2129 –0.9 24.7 0.012 ± 0.004 405.2127 –1.6 26.8 0.033 C19 H42 O As 28.3 361.2442 –1.1 25.9 0.24 ± 0.02 361.2435 –3.1 27.6 0.173 26.6 0.156 C20 H44 O As 375.2598 –1.3 375.2591 –3.2 C21 H46 O As 28.5 0.216 389.2753 –1.5 26.2 0.155 ± 0.008 389.2747 –3.1 27.9 0.121 27.2 0.061 C22 H48 O As 403.2910 –1.5 403.2903 –3.2 27.8 0.028 29.2 0.062 27.5 0.024 ± 0.007 28.8 0.023 28.9 0.025 29.6 0.181 28.0 0.042 ± 0.003 29.3 0.037 29.3 0.027 30.4 0.027 28.4 0.106 ± 0.005 29.7 0.085 29.7 0.073 31.6 0.043 29.3 0.005 ± 0.001 30.6 0.015 30.5 0.012 30.4 0.0168 ± 0.0001 31.7 0.024 31.4 0.013 C44 H87 O14 As P 32.5 0.048 945.5029 –1.6 31.3 0.014 ± 0.001 32.5 0.019 32.2 0.009 C45 H87 O14 As P 33.5 0.344 957.5036 –0.8 31.9 0.14 ± 0.01 957.5017 –2.9 33.6 0.107 33.2 0.058 C47 H89 O14 As P 983.5183 –1.8 32.3 983.5211 1.1 C45 H89 O14 As P 35.6 0.425 959.5177 –2.4 34.4 0.13 ± 0.03 959.5173 –2.8 35.7 0.076 35.2 0.053 C46 H91 O14 As P 973.5342 –1.5 973.5333 –2.5 C47 H91 O14 As P 985.5338 –1.9 C47 H93 O14 As P 39.4 0.102 987.5496 –1.8 38.1 0.02 ± 0.01 39.5 0.010 38.6 0.010 C48 H95 O14 As P 1001.5644 –2.6 C49 H97 O14 As P 44.8 0.038 1015.5802 –2.4 43.4 0.011 ± 0.007 43.4 0.004 Sum AsLps 1.70 0.90 0.72 0.53


doi:10.1071/EN14229_AC

Page 17 of 30

Table S14. Fragmentation patterns (MS-MS data) of selected samples AsHC360 AsHC374 AsHC388 AsHC402

Pylaiella (P2)

C19 H42 O As (361.24512, ppm 1.401). C2 H8 O As (122.9786, ppm 0.221). C2 H6 As (104.9680, ppm 0.303). C2 H4 As (102.9523, ppm –0.37)

C20 H44 O As (375.2608, ppm 1.35). C19 H40 As (343.2342, ppm 0.498) = M – MeOH + H. C3 H10 Oas (136.9943, ppm 0.344). C2 H6 As (104.968, ppm 0.303)

C21 H46 O As (389.2762, ppm 0.606). C3 H10 O As (136.994s, ppm 0.49). C2 H8 O As (122.9784, ppm –1.57). C2 H6 As (104. 9679, ppm –0.745)

C22 H48 O As (403.2918, ppm 0.684). C21 H 44 As (371.2647, ppm –1.72) = M – MeOH + H. C3 H10 O As (136.9942, ppm –0.094). C2 H6 As (104.9678, ppm –2.36)

EC rvk (ECr6)

C19 H42 O As (361.2448, ppm 0.377). C2 H8 O As (122.9785, ppm –0.836). C2 H6 As (104.9681, ppm 1.07). C2 H4 As (102.9523, ppm –0.47)

EC Abdn (ECa3)

C19 H42 O As (361.2443, ppm –0.897). C2 H8 O As (122.9782, ppm –2.71). C2 H6 As (104.9679, ppm –0.745). C2 H4 As (102.9522, ppm –1.24)

C20 H44 O As (375.2597, ppm 1.58). C19 H40 As (343.2239, ppm –29.54) = M – MeOH + H. C3 H10 O As (136.9939, ppm –1.99).

Elachista

(E1) C19 H42 O As (361.2445, ppm –0.343). C2 H8 O As (122.9783, ppm –2.06). C2 H6 As (104.9678, ppm –1.51). C2 H4 As (102.9521, ppm –2.6)

C20 H44 O As (375.2604, ppm –0.878). C3 H10 O As (136.9942, ppm –0.313). C2 H8 O As (122.9780, ppm –4.3) C2 H6 As (104.9679, ppm –0.745)


doi:10.1071/EN14229_AC

Page 18 of 30

The following graphs show the As75 signal from ICP-MS as well as the different signals from the

ESIMS (specific m/z) where individual signals have been elevated for clarity.

Fig. S8. Identified peaks in EC from Rvk (a) AsHCs; (b) AsPLs.

0

5000000

10000000

15000000

20000000

25000000

30000000

0

0.5

1

1.5

2

2.5

23 28 33 38 43

Inte

nsi

ty E

SIM

S

Re

lati

ve in

ten

sity

Time (min)

As75

AsHC360

AsHC374

AsHC388

0

10000

20000

30000

40000

50000

0

0.1

0.2

0.3

0.4

0.5

28 33 38 43

Inte

nsi

ty E

SIM

S

Re

lati

ve in

ten

stiy

IC

PM

S

Time (min)

As75 AsPL982 AsPL958 AsPL972 AsPL986 AsPL1000 AsPL1014


doi:10.1071/EN14229_AC

Page 19 of 30

a)

b)

c)

Fig. S9. (a) Chromatogram of Elachista: (b) identified AsHCs; (c) identified AsPLs.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 10 20 30 40 50

Re

lati

ve in

ten

sity

IC

PM

S

Time (min)

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

50000000

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

20 25 30 35 40 45 50

Inte

nsi

ty E

SIM

S

Re

lati

ve in

ten

sity

IC

PM

S

Time (min)

Elachista

AsHC360

AsHC374

AsHC388

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

0

0.1

0.2

0.3

0.4

0.5

30 35 40 45 50

Elachista

AsPL982

AsPL958

AsPL972

AsPL984

AsPL986

AsPL1000

AsPL1014

AsPL930


doi:10.1071/EN14229_AC

Page 20 of 30

a)

b)

c)

Fig. S10. Identified arsenolipids peaks in Pylaiella: (a) sample P2: AsPLs; (b) sample P2: AsHCs; (c) sample

P4: AsHC404.

0

100000

200000

300000

400000

500000

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

30 35 40 45

Inte

nsity

ESI

MS

Rel

ativ

e in

tens

ity IC

PMS

Time (min)

As75 AsPL956 AsPL982 AsPL958 AsPL972 AsPL984 AsPL986 AsPL1000 AsPL1014

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

0

0.2

0.4

0.6

0.8

1

1.2

1.4

25 26 27 28 29 30 31 32 33

Inte

nsity

ESI

MS

Rel

ativ

e in

tens

ity IC

PMS

Time (min)

As75 AsHC360 AsHC375 AsHC388 AsHC402

0

50000

100000

150000

200000

250000

300000

350000

400000

0

0.05

0.1

0.15

0.2

0.25

0.3

24 25 26 27 28 29 30

Inte

nsi

ty E

SIM

S

Rela

tive in

ten

sity

(IC

PM

S)

Time (min)

As75 AsHC404 AsHC360


doi:10.1071/EN14229_AC

Page 21 of 30

a)

b)

Fig. S11. (a) Overlay of Ectocarpus from Rvk and Abdn; (b) strain St3 under the different conditions with

Pylaiella. (A) Pylaiella; (B) St3 ctrl; (C) St3-N; (D) St3-P; (E) St3-OS. Identified peaks: 1, AsHC360 and

AsHC375; 2, AsHC388 and AsHC402; 3, AsPL956; 4, AsPL982; 5, AsPL972, AsPL984 and AsPL958; 6,

AsPL986 and AsPL1000; 7, AsPL1014.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

24 29 34 39 44

Rel

ativ

e in

ten

sity

(IC

PM

S)

Time (min)

EC Rvk

EC Abdn

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

-0.1

-0.05

0

0.05

0.1

0.15

0.2

24 29 34 39 44

Rel

ativ

e in

tens

tiy E

C n

atur

e

Rel

ativ

e in

tens

ity E

C c

ultu

res

Time (min)

A

B

C

D

E

3

4

5

6 7

1

2


doi:10.1071/EN14229_AC

Page 22 of 30

Fig. S12. Pylaiella measured on different analysis days (signal from ICP-MS).

Fig. S13. The Ectocarpus seaweed (black) from Abdn after acid (red) and base (blue) hydrolysis.

0

0.5

1

1.5

2

2.5

23 28 33 38 43

Rel

ati

ve

inte

nsi

ty

Time (min)

Day 1a

Day 1b

Day 2

Day 3

Day 4

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30 35 40

Rel

ati

ve

inte

nsi

ty

Time (min)


doi:10.1071/EN14229_AC

Page 23 of 30

Hydrolysis

Acid (HCl) and base (KOH) (both from Fisher Scientific) hydrolysis was performed on an

Ectocarpus sample from Aberdeen Beach. The samples were incubated for 24 h at room temperature

at 1 M concentration for both acid and base hydrolysis (900 µL of sample + 100 µL 10 M acid or

base). Samples were centrifuged if required. This was done for additional confirmation that the peaks

eluting after ~30–32 min were hydrolysable AsLps (e.g. AsPLs). The AsHCs were identified in the

sample as well as the acid and base hydrolysates (Table S10), although the AsHC peaks were shifted

to earlier retention time (pH difference). Small peaks eluting before and after the AsHCs could be

fragments (still containing the arsenic moiety) after hydrolysis.

Arsenosugars: identification and quantification

Fig. S14. Water fraction of Pylaiella, signal from the ICP-MS (solid: As75 as AsO+ m/z 91) and identified

Assugars with ESIMS (a–c), iAs = inorganic arsenic.

Table S15. Identified arsenosugars in Pylaiella with parallel HPLC-ICP-MS/ESIMS

Δm/z, (m/zfound – m/zcalc) × 106/m/zcalc

M + H Formula Δm/z (ppm)

329.0569 C10 H22 O7 As –0.741

483.0595 C13 H29 O12 As P –1.221

393.0187 C10 H22 O9 As S –0.781


doi:10.1071/EN14229_AC

Page 24 of 30

Table S16. Quantification of AsSugars in the Ectocarpales found in nature

All concentrations are given in milligrams per kilogram. Potentially some AsV contamination from

glassware

AsSugarOH (328) Rt 2.4

min

DMA Rt 3.3 min

AsSugarPO4 (482) Rt 3.9

min

AsSugarSO3 (392) Rt 7.0

min

AsV 14.1 min

Sum % column

recovery EC-Rvk12 (n = 2) 0.26 ± 0.02 0.041 ± 0.001 0.07 ± 0.02 0.19 ± 0.03 0.52 ± 0.05 1.06 64 ± 2 EC-Rvk17 (n = 3) 0.33 ± 0.04 0.047 ± 0.003 0.49 ± 0.07 0.48 ± 0.04 0.56 ± 0.06 1.90 73 ± 3 EC-Abdn (n = 2) 1.0 ± 0.2 0.052 ± 0.008 0.28 ± 0.04 0.35 ± 0.05 0.54 ± 0.08 2.18 81 ± 1 EC-Rvk (n = 1) 0.22 0.07 0.15 0.16 0.42 0.37 88 Hincksia (n = 1) 0.22 0.00 0.10 0.14 0.75 0.59 94 EC Cruden bay (n = 1) 0.24 0.04 0.31 0.69 2.58 3.9 87 Pylaiella (n = 3) 0.70 ± 0.01 0.134 ± 0.005 1.38 ± 0.01 5.65 ± 0.03 0.33 ± 0.01 8.2 82 ± 4 Elachista (n = 3) 7.9 ± 1.2 0.19 ± 0.03 6.2 ± 0.3 8.9 ± 0.7 0.68 ± 0.08 24.0 87 ± 2

Fig. S15. Comparison of iAs concentrations in Ectocarpales found in nature measured with HPLC-ICP-MS

and HG-ICPMS.

0

0.5

1

1.5

2

2.5

3

s1 s2 s3 s4 s6 s7 s8 s9 s10 s11 s12 s13 s14 s1a s2a s3a s4a

Rel

ati

ve

inte

nsi

ty

EC found in nature

iAs HPLC-ICPMS

iAs HG-ICPMS


doi:10.1071/EN14229_AC

Page 25 of 30

Table S17. Quantification of AsSugars in Ectocarpus in cultures (n = 3)

All concentrations are given in milligrams per kilogram. Estimated peak identifications by retention time: peak at 2.5 min, AsSugar328; peak at 3.4 min,

DMA; peak at 4.1 min, AsSugar482; and peak at 7.3 min, AsSugar 392. There was an AsV peak at 14.8 min; however, because of contamination from As in

glass, it was not possible to estimate concentration

Rt (min)

St1-ctrl St1-N St1-P St1-OS Rt (min)

St2-ctrl St2-N St2-P St2-OS

1.4 – – – 0.0019 ± 0.0007 1.5 0.006 ± 0.002 0.0092 ± 0.0002 0.013 ± 0.002 0.073 ± 0.037

2.5 0.081 ± 0.013 0.050 ± 0.014 0.138 ± 0.028 0.148 ± 0.014 2.6 0.089 ± 0.009 0.067 ± 0.002 0.081 ± 0.042 0.043 ± 0.051

3.4 0.0088 ± 0.0015 0.011 ± 0.002 0.012 ± 0.004 0.016 ± 0.002 3.4 0.010 ± 0.001 0.011 ± 0.002 0.012 ± 0.001 0.048 ± 0.037

4.1 0.094 ± 0.008 0.055 ± 0.011 0.005 ± 0.002 0.102 ± 0.01 4.1 0.012 ± 0.004 0.065 ± 0.018 – 0.079 ± 0.024

7.2 0.247 ± 0.034 0.254 ± 0.013 0.262 ± 0.064 0.581 ± 0.053 7.4 0.049 ± 0.01 0.060 ± 0.010 0.043 ± 0.02 0.0094 ± 0.0066

11.4 – 0.0033 ± 0.0005 – – 11.2 0.0022 ± 0.0007 0.007 ± 0.002 – –

Sum 0.43 ± 0.05 0.37 ± 0.02 0.42 ± 0.10 0.85 ± 0.06 Sum 0.17 ± 0.02 0.22 ± 0.03 0.15 ± 0.06 0.26 ± 0.08

1.4 0.027 ± 0.003 0.040 ± 0.004 0.026 ± 0.006 0.05 ± 0.01 1.4 0.097 ± 0.015 0.076 ± 0.013 0.08 ± 0.01 0.11 ± 0.02

2.5 0.047 ± 0.014 0.043 ± 0.010 0.010 ± 0.01 0.040 ± 0.003 2.5 0.16 ± 0.04 0.16 ± 0.02 0.16 ± 0.03 0.16 ± 0.05

3.2 0.0015 ± 0.0014 0.006 ± 0.003 0.008 ± 0.003 0.003 ± 0.002 3.4 0.009 ± 0.002 0.0070 ± 0.0007 0.013 ± 0.004 0.03 ± 0.01

4.0 0.00007 ± 0.00003 0.018 ± 0.015 – 0.010 ± 0.004 4.2 0.021 ± 0.005 0.095 ± 0.045 – 0.059 ± 0.056

7.4 0.034 ± 0.013 0.030 ± 0.008 0.15 ± 0.05 0.019 ± 0.001 7.3 0.16 ± 0.05 0.15 ± 0.02 0.20 ± 0.04 0.16 ± 0.07

Sum 0.11 ± 0.03 0.135 ± 0.022 0.28 ± 0.06 0.123 ± 0.016 Sum 0.44 ± 0.10 0.48 ± 0.08 0.46 ± 0.06 0.52 ± 0.14


doi:10.1071/EN14229_AC

Page 26 of 30

Morphology and microscopy

Table S18. The mean and standard deviation of the chloroplast size for the different

treatments of the EC cultures and the statistical significance of the chloroplast size increase or

decrease

OS, oxidative stress

Mean n s.d. Chloroplast Significance

EC-022-10-ctrl 24.8 21 12.1 St3 EC-022-10-N 34.7 20 15.9 Increase P < 0.001 EC-022-10-P 41.6 25 14.5 P = 0.284 EC-022-10-OS 29.7 20 15.7 Minor increase P = 0.033 EC-022-10+As-ctrl 15.5 22 9.6 St3+As EC-022-10+As-N 29.1 12 17.4 Increase P = 0.18 EC-022-10+As-P 40.1 16 17.9 Increase P < 0.001 EC-022-10+As+OS 13.6 11 19.5 P = 0.74 EC-007-04-ctrl 35.5 8 17.9 St2 EC-007-04-N 15.1 11 9.9 Decrease P = 0.004 EC-007-04-P 19.5 13 13.7 Decrease P = 0.18 EC-007-04+OS 19.5 8 17.1 Decrease P = 0.33 EC-Oban-ctrl 40.2 11 10.0 St1 EC-Oban-N 45.3 10 7.2 P = 0.11 EC-Oban-P 45.6 14 8.5 P = 0.19

EC-Oban+OS 34.1 10 8.5 P = 0.13


doi:10.1071/EN14229_AC

Page 27 of 30

TEM imaging (EC-022-10; St3)

Fig. S16. EC-022-10 TEM images of control sample.

For the control, Fig. S15, fibrils (F) associated with the cell membrane were seen; these have an

unknown function. Further research is required. Chloroplasts (Ch) appear to take up a large amount of

the cell. Mitochondria (Mi) are small and irregular. The membrane is difficult to discern from the rest

of the organelle. Mitochondria were defined by the dark spherical ultrastructure.

Ch

Ch Ch

Ch Ch Nu

Mi

F

F Mi


doi:10.1071/EN14229_AC

Page 28 of 30

Fig. S17. EC-022-10 TEM images of low-nitrate sample.

For the low-nitrate sample, there is an increase in chloroplast size compared with the control. The

mitochondria are irregular again but visible. The fibrils are not as dense as for the control.

Fig. S18. EC-022-10 TEM images of low-phosphate sample.

Fewer somatic cells are visible in the section. New sections are being produced to try to resolve

this. Chloroplasts are enlarged.

Dense body structures are visible as in the H2O2 treatment. No mitochondria are visible. Fibrils are

greatly reduced.

Ch

Nu Ch

DB DB

Ch

Ch

F

Ch

Ch

Nu


doi:10.1071/EN14229_AC

Page 29 of 30

Fig. S19. EC-022-10 TEM images of added-H2O2 sample. Fibrils still present associated with the cell

membrane. No large differences in chloroplasts and mitochondria are obvious. The mitochondria again seem to

be an irregular shape. Dense body structures can be seen occupying the cell.

F

Ch

DB

DB

Nu F

DB

DB

Mi?

Ch F


doi:10.1071/EN14229_AC

Page 30 of 30

References

[1] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: molecular evolutionary genetics

analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725. doi:10.1093/molbev/mst197

[2] R. C. Starr, J. A. Zeikus, UTEX – the culture collection of algae at the University of Texas at Austin. J.

Phycol. 1987, 23 (Suppl.), 1. doi:10.1111/j.0022-3646.1993.00001.x

http://dx.doi.org/10.1093/molbev/mst197

http://dx.doi.org/10.1111/j.0022-3646.1993.00001.x

Date post:	25-Apr-2020
Category:	Documents
Upload:	others
View:	1 times
Download:	0 times

Environmental effects on arsenosugars and arsenolipids in ...Senescence (%) Observations Oban-Ctrl...

Documents