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Supplementary Material
Total arsenic and arsenic species analysis
4 Reagents and Standards
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Nitric acid (HNO
3
; Aristar, BDH, West Chester, USA) was used for the digestion of D. tertiolecta and T. pseudonana tissue for total arsenic determination. Ammonium dihydrogen phosphate (Suprapur, Merck, Darmstadt, Germany) and pyridine (Extra Pure, Merck, Darmstadt,
Germany) were used in the preparation of high performance liquid chromatography (HPLC) mobile phases. Formic acid (Extra Pure, Fluka, Munich, Germany) and ammonia solution
(>99.9%, Aldrich, Munich, Germany) were used for the adjustment of mobile-phase pH.
Methanol (HiPerSolv, BDH West Chester, USA), acetone, (HPLC, BDH, West Chester, USA), chloroform (AR, BDH, West Chester, USA) and deionised water (18.2 mΩ, Millipore, Billerica,
USA ) were used for the extraction of arsenic species.
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Stock standard solutions (1000 mg L -1 ) were made by dissolving the appropriate arsenic standard in 0.01 M HCl–deionised water. Arsenous acid (As(III)) and arsenic acid (As(V)) were from
AJAX Laboratory Chemicals, methylarsonic acid (MA) and dimethylarsinic acid (DMA) were from ChemService. Arsenobetaine bromide (AB) was synthesised by the methods of Cannon et al
. (1981), Arsenocholine iodide (AC) was synthesised by the methods of Minhas
et al
.(1998)
Trimethylarsine oxide (TMAO) was synthesised by the methods of Merijanian and Zingaro
(1966), Tetramethylarsonium ion (TETRA) by the methods of Momplaisir
et al.
(1991),
dimethylarsinoylethanol (DMAE), and dimethylarsinoylacetate (DMAA) by the methods of
Edmonds et al .
(1982) and Francesconi
et al
. (1989). Glycerol arsenoriboside, sulfonate
arsenoriboside, phosphate arsenoriboside and sulfate arsenoriboside (Gly-ribose, SO
3
-ribose,
PO
4
-ribose, OSO
3
-ribose, respectively) were obtained from a previously characterised Fucus
extract (Madsen et al. 2000). Thio-Gly-arsenoriboside, thio-PO
4
-ribose, thio-SO
3ribose, thio-
OSO
3
-ribose, and thio-DMAE were synthesised by bubbling H
2
S (Aldrich, 99.5%) through an extract of previously characterised Fucus
(Madsen et al. 2000) or synthetic standard for
approximately thirty seconds to one minute with a ~100% conversion rate. Thio-DMA was synthesised by the methods of Raml et al
. (2006).

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Determination of total arsenic concentrations
E. radiata tissue was digested for total arsenic concentrations using a microwave digestion technique, outlined by Baldwin et al.
(1994) with modifications. Approximately 0.07 g of
homogenised lyophilised samples were accurately weighed and recorded into 7 mL Teflon polytetrafluroacetate digestion vessels (A.I. Scientific, Hallam, Australia) and 1 mL of concentrated nitric acid added (Aristar, BDH, West Chester, USA). Digestion vessels with sample and acid were left in the fume cupboard for approximately 1 hour prior to digestion.
Microwave digestion (MDS 81D, CEM, Indian Trail, NC, USA) with a program cycle of 2 min
600 W, 2 min 0 W, 45 min 450 W was used for each set of samples with certified reference materials and blanks. Samples were allowed to cool after digestion for ~60 minutes then diluted to 10 ml with deionised water in 10 mL polyethylene vials. Total arsenic and phosphorus concentrations in samples were analysed using a Perkin Elmer (Waltham, USA) DRC-e
Inductive Coupled Plasma-Mass Spectrometer (ICP-MS) with an AS-90 autosampler (Waltham,
USA). Internal standards were added on-line to compensate for any acid effects and instrument
drift (Maher et al. 2001). The potential interference to arsenic (m/z 75) from
40
Ar
35
Cl
+
was determined by measuring chloride at m/z 35,
35
Cl
16
O
+
at m/z 51,
35
Cl
17
O
+
at m/z 52, and
40
ArCl
+ at m/z 77. Selenium was monitored at m/z 82 as a cross check for
40
Ar
37
Cl
+
. Other elements were corrected for interferences as outlined in Maher et al
(Maher et al. 2001). Certified reference
material, Ulva lactuca L. (CRM 279, Community Bureau of Reference, Brussels, Belgium),
Sargasso (NIES 9, National institute for environmental studies, Environment Agency of Japan) and Citrus Leaves (NBS 1572, National Bureau of Standards) was routinely used for quality assurance, and was analysed in the same manner as E. radiata tissue for determination of total arsenic and phosphorus concentrations. Measured values for total arsenic in U. Lactuca (n = 13)
(2.6 ± 0.7 μg g -1 ) were in agreement than certified values (3.09 ± 0.20 μg g -1 ), as were values for total arsenic in Sargasso (n=14) (measured - 110 ± 21 μg g -1 ; certified - 115 ± 9 μg g -1 ).
Extraction of arsenic species from E. radiata tissue
Water-soluble arsenic species were extracted from E. radiata tissues by a microwave extraction
procedure developed by Kirby and Maher (Kirby and Maher 2002). Briefly; homogenized
freeze-dried tissues were extracted twice with acetone to remove fats. Acetone extracts were

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86 evaporated in a fume cupboard, the residues dissolved by heating in 5mL of 1% m/v nitric acid and arsenic measured by ICP-MS as described above. The acetone extracted pellet was extracted with 1:1 v/v methanol-water heated to 75 o
C for 10 min, three times. The supernatant was removed after centrifuging at 4500 g for 10 min at 25
o
C. Supernatants were combined, evaporated to dryness using a vacuum concentrator and stored in a freezer until analysis. Extracts were reconstituted in 4mL of deionised water before analysis.
Arsenic Species Measurement
All extracts were filtered through a 0.20 µm RC syringe filter (Millipore, Billerica, USA ).
Aliquots of 20 or 40 µL were injected onto a high pressure liquid chromatography (HPLC) system consisting of a Perkin Elmer Series 200 mobile phase delivery and auto sampler system
(Perkin Elmer; Waltham, USA). The eluant from HPLC columns was directed by PEEK
(polyether-ether-ketone) (i.d. 0.02 mm) (Supelco, Fluka, Munich, Germany) capillary tubing into a Ryton cross flow nebuliser of a Perkin Elmer Elan-6000 ICP-MS, which was used to monitor the signal intensity of arsenic at m/z 75. Potential polyatomic interferences were checked by monitoring for other ions, as described for total arsenic analysis. The column conditions used for separation of arsenic species are outlined in Supplementary Table 1, with extensive details
presented in published literature (Foster et al. 2008; Foster et al. 2007; Kirby et al. 2004).
External calibration curves for quantification of arsenic species were prepared daily by diluting
As(III) for anionic species and AB for cationic species to 0, 0.5, 1, 10, 100 µg L
-1
. Peak area
responses (n = 10) relative to AB and As(III) have been reported previously (Foster et al. 2006)
Purities of arsenic species were determined periodically by HPLC-ICP-MS. The accuracy of arsenic speciation procedure was determined by analysis of the certified reference material
DORM-2 (National Research Council, Canada). The concentrations (mean ± SD) of AB (16.8 ±
1.6 µg g -1 ) and TETRA (0.2 ± 0.1 µg g -1 ) measured in DORM-2 tissues (n = 7) were similar to certified values (AB, 16.4 ± 1.1 µg g -1 ; TETRA, 0.236 ± 0.001 µg g -1 ). HPLC Column recoveries for all samples are presented in Supplementary Table 2.
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The chromatography software package Total Chrom (Perkin Elmer; Waltham, USA) was used to quantify arsenic species by peak areas. Arsenic species were identified by spiking with known standards and retention time matching.

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Supplementary Table 1 . HPLC Column conditions for the separation and quantification of arsenic species.
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Column
Size
Particle size
Buffer pH
Flow rate
Temperature
Arsenic species
Hamilton PRP-X100
Supelocosil LC-
SCX
Supelocosil LC-
SCX
150 or 250 mm x 4.6 mm 250 mm x 4.6 mm 250 mm x 4.6 mm
10 µm 10 µm 10 µm
20 mM NH
4
H
2
PO
4
, 1%
CH
3
OH
20 mM pyridine 20 mM pyridine
5.6
1.5 ml min
-1
40 ºC
As(V), DMA, MA, PO
SO
3
- and OSO
3
- arsenoribosides
4
-,
2.6
1.5 ml min
-1
40 ºC
DMAE, glycerol trimethyl arsenoriboside,
TETRA, AC and
TMAP
3
1.5 ml min
-1
40 ºC
AB and OH arsenoriboside

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Supplementary Table 2.
Extraction efficiencies and HPLC Column recoveries (n=1) for arsenic species present in E. radiata tissues of various decomposition stages.
Sample
Lake Tabourie Live
Lake Tabourie water-column
Lake Tabourie intertidal
Burill Lake Live
Burill Lake Water-column
Burill Lake Intertidal
Ulladulla Live
Ulladulla Water-column
Ulladulla Intertidal
E. radiata tissue extraction efficiency (%)
80
138
34
39
70
40
100
116
90
E. radiata tissue column recovery (%)
77
58
78
69
66
138
61
64
150

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References
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