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Assessing the legacy of red mud pollution in a shallow freshwater lake: arsenic accumulation and speciation in macrophytes Justyna P. Olszewska, Andrew A. Meharg, Katherine Victoria Heal, Manus Patrick Carey, Iain D. M. Gunn, Kate R. Searle, Ian J. Winfield, and Bryan M. Spears Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00942 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 26, 2016
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Assessing the legacy of red mud pollution in a shallow freshwater lake: arsenic
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accumulation and speciation in macrophytes
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Justyna P. Olszewska,1,2 Andrew A. Meharg,3 Kate V. Heal,2 Manus Carey,3 Iain D. M.
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Gunn,1 Kate R. Searle,1 Ian J. Winfield,4 and Bryan M. Spears*,1
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1. Centre for Ecology & Hydrology (CEH Edinburgh), Bush Estate, Penicuik EH26 0QB,
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Scotland, UK
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2. School of GeoSciences, The University of Edinburgh, Crew Building, Alexander Crum
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Brown Road, Edinburgh EH9 3FF, Scotland, UK
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3. Institute for Global Food Security, Queen’s University Belfast, Belfast BT9 5HN, UK
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4. Lake Ecosystems Group, Centre for Ecology & Hydrology (CEH Lancaster), Lancaster
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Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK
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Abstract
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Little is known about long-term ecological responses in lakes following red mud pollution.
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Among red mud contaminants, arsenic (As) is of considerable concern. Determination of the
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species of As accumulated in aquatic organisms provides important information about the
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biogeochemical cycling of the element and transfer through the aquatic food-web to higher
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organisms. We used coupled ion chromatography and inductively coupled plasma mass
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spectrometry (ICP-MS) to assess As speciation in tissues of five macrophyte taxa in
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Kinghorn Loch, UK, 30 years following the diversion of red mud pollution from the lake.
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Toxic inorganic As was the dominant species in the studied macrophytes, with As species
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concentrations varying with macrophyte taxon and tissue type. The highest As content
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measured in roots of Persicaria amphibia (L.) Gray (87.2 mg kg-1) greatly exceeded the 3 –
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10 mg kg-1 range suggested as a potential phytotoxic level. Accumulation of toxic As species *
Corresponding author. Email:
[email protected], tel: +44 (0)131 4454343, fax: +44 (0)131 4453943 ACS Paragon Plus Environment
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by plants suggested toxicological risk to higher organisms known to utilise macrophytes as a
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food source.
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Introduction
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The estimated global production of red mud, a by-product of alumina production, is ~120
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million t a-1.1 In October 2010, failure of a containment reservoir in Ajka, Western Hungary,
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resulted in the release of ~1 million m3 of red mud waste, contaminating the Marcal River
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(catchment area 3078 km2) and entering the Danube River.2 The Hungarian red mud spill
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raised concerns about inappropriate storage of red mud and the impact of the waste on the
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receiving environment. However, little is known about the geochemical behaviour of red mud
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in freshwaters2 or of the short- and long-term ecological impacts and likelihood of recovery
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following its release into the environment. This is conspicuous given that many of its
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chemical constituents are redox sensitive and so likely to persist in aquatic depositional
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environments where they can represent an environmental and human health risk.
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Red mud is highly alkaline due to the addition of sodium hydroxide (NaOH) during the
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production process and contains metal oxides and elevated concentrations of a range of minor
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trace elements3,4,5 that can be toxic to aquatic organisms. Initial research after the Ajka
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accident focused on the short-term impact of the pollution on freshwaters. Mayes et al.6
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reported elevated concentrations of some contaminants, e.g. arsenic (As), vanadium (V),
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chromium (Cr) and nickel (Ni), in fluvial sediment downstream of the spill. The association
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of these elements mainly with residual phases suggests limited potential for their mobilisation
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in the environment. However, in depositional zones, release of these elements may be
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expected.6 Furthermore, river sediments contaminated with constituents of red mud can have
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pronounced toxic effects, for example, causing reduced bioluminescence of Vibrio fischeri
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and growth of Lemna minor and Sinapis alba.2 In aquatic ecosystems with short residence
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times, the long-term effects of pollution might be spatially limited to depositional zones,
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allowing system recovery.2 However, the residence time of pollutants in higher retention time
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aquatic ecosystems can exceed those in fluvial systems.7,8 Therefore, long-term monitoring of
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the red mud impact on aquatic life is particularly important in lakes that can have potentially
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longer time-scales of recovery.
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Among red mud contaminants, As is of considerable concern. It is a toxic element,9 with high
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concentrations in the aquatic environment posing an environmental and human health risk in
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many countries, e.g. in Pakistan, Bangladesh, India, Taiwan,10 Japan,11 China12 and the
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USA.13 Arsenic concentrations in contaminated groundwater were reported of up to 1 mg L-1
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in some areas of Bangladesh and 3 mg L-1 in Vietnam,14 whilst As concentration of 792 mg
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kg-1 was measured in surface sediment in Lake Biwa, Japan.11 In macrophytes, uptake of As
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can reduce phosphorus (P), nitrogen (N), potassium (K), chlorophyll a and protein content in
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tissues and, due to the chemical similarity of As to P, can interfere with some biochemical
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reactions.15
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Concentrations of contaminants accumulated by plants can be higher than in water, but lower
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than in bed sediments.16 The bioavailability of trace elements to plants can be regulated by a
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range of site specific factors including their concentration in the environment, trace element
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speciation, exposure time, and absorption mechanism.16 For example, uptake of the inorganic
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As species, arsenate, occurs through phosphate uptake pathways and has been shown to be
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negatively correlated with phosphate uptake by the macrophyte Spirodela polyrhiza L.9 In
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addition, the capacity of aquatic plants to sequester and accumulate metals is affected by
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many factors, such as plant growth rate, biomass accumulation and affinity for metal
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uptake.15 Previous studies have shown that As bioaccumulation varies among macrophyte
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species and among aquatic plant tissues.15-18 3 ACS Paragon Plus Environment
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Arsenic can occur in inorganic and organic chemical forms, or species, which are associated
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with different levels of toxicity.19 For example, the inorganic species arsenite (As(III)) is
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more toxic than organic arsenobetaine to plants and animals.19 The uptake mechanism20 and
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level of accumulation in plants19,21 also vary between As species. Significant differences
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between concentrations of different As species have been reported in rice (Oryza sativa L.),
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with the predominant form being inorganic As.21,22
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Determination of As speciation in plants is important for assessing the plant As toxicity to
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consumers at higher levels of the food chain.23-25 Most previous research on As species in
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plants has focused on crops and As speciation in plants in the aquatic environment has not
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been extensively studied, with most of the existing knowledge based on controlled laboratory
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experiments.9,18,20 Consequently, little is known about variation of individual As species
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between tissues of aquatic plants. Inorganic and organic As species have been determined
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previously in whole macrophytes9,19,26, but concentrations of As species have not been
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examined in different plant tissues. Determination of the species of As accumulated in
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macrophytes allows for more accurate assessment of the environmental risk that increases
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with the occurrence of inorganic As and is particularly important at sites where fish and
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waterfowl are used for human consumption.
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We assessed the impact of As on five macrophyte taxa - three submerged species
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(Potamogeton pectinatus L., Elodea nuttallii (Planch) H. St. John, Myriophyllum spicatum
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L.), one rooted, floating-leaved species (Persicaria amphibia (L.) Gray) and one multicellular
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algae (Chara spp.) in Kinghorn Loch 30 years after the diversion of red mud leachate.
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Arsenic speciation analysis was applied to quantify inorganic As (arsenite, As(III) and
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arsenate, As(V)) and four organic As species: arsenobetaine, dimethylarsinic acid (DMA),
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monomethylarsonic acid (MMA) and tetramethylarsonium ions (Tetra). Based on the results
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of previous studies on total As accumulation in macrophytes,15-17 As speciation in rice21 and 4 ACS Paragon Plus Environment
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terrestrial plants14 and As species uptake by macrophytes in controlled laboratory
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experiments,9,18,20 the following hypotheses were tested: (1) macrophytes in Kinghorn Loch
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accumulate higher concentrations of inorganic compared to organic As species, (2)
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accumulation of As species varies between above-sediment tissues (leaves and stems) of
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macrophytes and among macrophyte taxa, and (3) accumulation of As species in P. amphibia
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is greatest in the roots of the plant compared to the above-sediment tissues. This work
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provides the first comprehensive assessment of As accumulation and speciation in aquatic
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macrophytes following red mud pollution.
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Materials and Methods
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Study site
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Kinghorn Loch is a small lake of surface area 11.3 ha, mean depth 4.5 m and maximum
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depth 12.8 m, situated in Fife, Scotland (56o10ʹN; 3o11ʹW). The lake received highly alkaline
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leachate from a nearby red mud landfill site from 1947 to 1983 when the discharge was
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diverted. Caustic liquor reaching the lake consisted of a highly alkaline solution of NaOH and
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carbonate, with a mean pH of 12.1. It contained high concentrations of dissolved aluminium
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(Al; 137 mg L-1), As (3.56 mg L-1), vanadium (V; 5.3 mg L-1), phosphate (PO4-P; 2.95 mg L-
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1
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the loch sporadically, accounted for most of the elevated input of iron (Fe).4 The
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physicochemical pathways of pollutant transport within the bed sediments of the loch have
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been discussed in detail in Edwards (1985)4. Long-term monitoring of water chemistry of the
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loch was initiated in 1980 by the Forth River Purification Board and continued by the
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Scottish Environment Protection Agency (SEPA). The pollution led to intense algal blooms,
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fish kills and severe reduction of zooplankton, macroinvertebrate and macrophyte species
), sulfate (SO4-S; 154 mg L-1) and chloride (Cl; 67.8 mg L-1). Red mud solids, released to
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diversity and biomass.4 By 1985, two years after the diversion of the leachate from the lake, a
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collapse in the phytoplankton activity, development of zooplankton and an increase in
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abundance of benthic macroinvertebrates (though no increase in species richness) were
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observed.4 Bioaccumulation studies of the plankton and macroinvertebrate populations in
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1985 indicated no significant As bioaccumulation in Kinghorn Loch.4
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A pilot survey was conducted in 2013 to assess metal concentrations in surface waters and
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bed sediments.27 Total As concentrations in lake surface water and bottom water (1 cm above
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sediment; sampled July 2013; Site 1; Figure 1) were 14.9 µg L-1 and 14.8 µg L-1 respectively,
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and did not exceed Standards for Protection of Aquatic Life in the UK (50 µg L-1).28
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Concentrations of As in lake surface sediment at Site 1 (87 mg kg-1) and mean As
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concentration from 6 sites across the lake (160 mg kg-1)27 in July 2013 exceeded the
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Canadian Sediment Quality Guidelines for Protection of Aquatic Life, which predicts toxicity
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from sediment concentrations ≥ 17 mg kg-1 of As29. Study of As release from Kinghorn Loch
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sediment into water conducted in laboratory controlled experiments indicated seasonal
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mobilisation of As under reducing conditions.27
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Macrophyte sampling
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Macrophyte sampling was conducted on 18 July 2013 when whole plants were collected by
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hand and using a double-headed rake from a boat at three sites in Kinghorn Loch
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(56o40’26’’N; 3o11’34’’W (Site 1); 56o40’18’’N; 3o11’33’’W (Site 2); 56o40’22’’N; 3o11’16’’W
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(Site 3); Supporting Information Figure S1). The sites were chosen to represent areas at
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different distances (approximately 100, 250 and 430 m for Site 1, 2 and 3, respectively) and
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directions from the NW part of the lake, where the pollution entered the waterbody. Between
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three and ten plants per taxa (depending on the species specific volume of fresh plant
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material) were collected at each site. Samples represented the five most abundant macrophyte 6 ACS Paragon Plus Environment
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taxa in Kinghorn Loch – P. amphibia, P. pectinatus, E. nuttallii, M. spicatum and Chara spp.
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– as determined by a full lake survey conducted on 18 July 2013 following the procedure
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outlined in Gunn et al.30
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Sample preparation
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The macrophytes were stored in polyethylene bags at 4oC until processing in the laboratory
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within 24 hours. The plant tissues were placed into bags containing distilled water and
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manually shaken, repeatedly, to remove sediment, macroinvertebrates and epiphytic biofilms.
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We acknowledge that this approach may not have removed entirely epiphytic organisms
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although others have reported near complete removal of this community following similar
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treatments, as well as plant tissue damage under severe agitation31. To test for any variation
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in As accumulation in the different parts of macrophytes, P. pectinatus, E. nuttallii and M.
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spicatum samples were divided into leaves and stems, and samples of P. amphibia, the only
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collected species with well-developed roots, into leaves, stems and roots. Axes and branches
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of the multicellular algae Chara spp. were also separated and are referred to as stems and
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leaves, respectively, for the purpose of statistical analysis. Samples of each macrophyte tissue
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type for each species were combined by site from a number of individual plants to obtain
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enough dry material for As analyses. Samples were then oven dried at 80oC and ground using
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a Retsch mixer mill MM200.
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Chemical analysis
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Three replicate samples of each tissue type from each macrophyte species were analysed.
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Approximately 100 ± 5 mg of each powdered sample was digested with 1% nitric acid using
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a microwave digester (CEM Mars 6 1800W). Arsenic speciation was determined by ion
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chromatography (Thermo Dionex IC5000 Ion Chromatograph) coupled to inductively
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coupled plasma mass spectrometry (Thermo Scientific iCap Q ICP-MS). Arsenic species in
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the solutions were identified by comparing their retention times with those of standards
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including inorganic As (As(III) and As(V)), arsenite, DMA, MMA and Tetra and quantified
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by calibration curves with peak areas. Species with retention times not matching any of the
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standards were classified as unknown species. Total identified As species were calculated as
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the sum of identified inorganic and organic As species. The detection limit for all As species
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was 0.001 mg kg-1. The As species concentrations measured in duplicate analyses of the
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Certified Reference Material (CRM; NIST 1568b Rice flour) conducted in the same manner
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were within 25% of the certified values. The certified and measured values of As species in
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the CRM are listed in Table 1. The CRM did not contain arsenobetaine and Tetra. The
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method for determination of As speciation is described in detail in the Supporting
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Information.
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Statistical analyses
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The means of three replicate samples were used for all statistical analyses. Concentrations of
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different As species in P. amphibia tissues (leaves and stems) were analysed together with
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data for the other macrophytes species to examine across species variation in the above-
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sediment parts of macrophytes, and separately (concentrations in leaves, stems and roots) to
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test for differences between As concentrations in below- and above-sediment parts of P.
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amphibia.
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A one-sided two sample t-test was used to test whether the mean concentration of inorganic
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As was greater than the mean concentration of total organic As in the above-sediment tissues
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of macrophytes and also whether below-sediment surface tissues of P. amphibia contained
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higher concentrations of As species than above-sediment tissues.
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Mixed effect modelling was conducted to examine variations in concentrations of each As
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species across macrophyte species and tissue type (within shoots). Root samples were not
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included in these models, as they were only available for one species. Model validation was
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conducted on the final models. Homogeneity of variance was assessed by plotting residuals
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versus fitted values and the normality assumption was evaluated by plotting theoretical
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quantiles versus standardized residuals (Q-Q plots). In models with all macrophyte species,
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several response variables (i.e. inorganic As, arsenobetaine, total organic and the sum of all
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identified As species) were log10 transformed to meet homogeneity and normality
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assumptions. The random effect part of the model allowed for variations among sites and
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incorporated a split plot design of the data. The analysis started with the model containing
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both nominal fixed explanatory variables (i.e. macrophyte species and macrophyte tissue) and
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the two-way interaction between these two terms. The model selection process followed the
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procedure described in Zuur et al.32 Maximum likelihood ratio (ML) tests were performed,
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using the ANOVA function, to select the best-fit model by comparing AIC values and to
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determine the significance (P < 0.05) of dropped terms. The final model was re-run using
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restricted maximum likelihood (REML). Statistical analyses were conducted using R version
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3.0.1,33 using the ‘nlme’ package34 for the mixed modelling.
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Results
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Difference between inorganic and organic As concentrations across macrophyte taxa
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Figure 1 shows variations in As species concentrations across the five macrophyte taxa and
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different plant tissues of each of the taxa. The above-sediment parts of the five macrophyte
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species in samples combined across sampling sites contained significantly higher
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concentrations of inorganic As species than total organic As, with mean values of 8.80 mg
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kg-1 d.w. (s.d. 13.8) and 0.56 mg kg-1 d.w. (s.d. 0.44), respectively (Table 2). Higher
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concentrations of inorganic As were measured compared to total organic As concentrations in
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both tissues of all macrophytes species (Figure 1, Table S1). P. amphibia also contained
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significantly higher concentrations of inorganic As than total organic As, with mean values of
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40.0 mg kg-1 d.w. (s.d. 65.7) for inorganic and 0.25 mg kg-1 d.w. (s.d. 0.11) for organic forms
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(Table 2), respectively, calculated as the arithmetic mean of above and below-sediment
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surface tissues As concentrations.
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Differences in As species accumulation in above-sediment macrophyte tissues
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The lowest mean concentrations of inorganic As in the above sediment-surface tissues were
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measured in the leaves and stems of P. amphibia (0.76 mg kg-1 ± 0.14 and 1.07 mg kg-1 ±
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0.29, respectively) and the highest in leaves of E. nuttallii (45.1 mg kg-1 ± 20.7; Figure 1,
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Table S1). Two organic As species (MMA and Tetra) were below the limit of detection in all
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tissue types of Chara spp., with MMA also not detectable in leaves of M. spicatum. Mean
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concentrations of the four organic As species ranged from 0.01 mg kg-1 d.w. (DMA in Chara
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sp. branches, MMA in both tissues of P. amphibia, Tetra in P.amphibia leaves) to 1.16 mg
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kg-1 (arsenobetaine in Chara spp. branches). Unknown As species were present in the leaves
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of P. pectinatus and M. spicatum and in both tissues of Chara spp., with the highest mean
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concentration (1.04 mg kg-1 ± 0.47) measured in the latter species.
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The model selection process indicated a model with plant tissue, plant species and the
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interaction term included as an optimal one for inorganic As, arsenobetaine, MMA, Tetra,
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total organic As, and all identified As species (Table S2). Due to a lack of a significant effect,
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the interaction term was omitted from the model for unknown As species. None of the
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explanatory variables was found to have a significant association with plant DMA
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concentration.
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E. nuttallii had significantly higher (estimate: 1.79, S.E.: 0.27, t: 6.67, P:
