Comparative Biochemistry and Physiology, Part C 166 (2014) 59–64

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Effects of exposure to high concentrations of waterborne Tl on K and Tl concentrations in Chironomus riparius larvae Ryan Belowitz, Erin M. Leonard, Michael J. O'Donnell ⁎ Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

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Article history: Received 29 May 2014 Received in revised form 8 July 2014 Accepted 14 July 2014 Available online 19 July 2014 Keywords: Atomic absorption spectroscopy Chironomus riparius Gut Hemolymph LC50 Potassium Thallium Whole Animal

a b s t r a c t Thallium (Tl) is a non-essential metal which is released into the environment primarily as the result of anthropogenic activities such as fossil fuel burning and smelting of ores. The ionic radius of monovalent Tl+ is similar to that of K+ and Tl+ may thus interfere with K+-dependent processes. We determined that the acute (48 h) lethal concentration where 50% of the organisms do not survive (LC50) of Tl for 4th instar Chironomus riparius larvae was 723 μmol L−1. Accumulation of Tl by the whole animal was saturable, with a maximum accumulation (Jmax) of 4637 μmol kg−1 wet mass, and KD of 670 μmol Tl l−1. Tl accumulation by the gut appeared saturable at the lowest four Tl concentrations, with a Jmax of 2560 μmol kg−1 wet mass and a KD of 54.5 μmol Tl l−1. The saturable accumulation at the gut may be indicative of a limited capacity for intracellular detoxification, such as storage in lysosomes or complexation with metal-binding proteins. Tl accumulation by the hemolymph was found to be linear and Tl concentrations in the hemolymph were ~75% of the exposure concentration at Tl exposures N26.9 μmol L−1. There was not a significant decrease in whole animal, gut or hemolymph K during exposure to waterborne Tl at any of the concentrations tested (up to 1500 μmol L−1). The avoidance of hypokalemia by C. riparius larvae may contribute to survival during acute waterborne exposures to Tl. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Thallium (Tl) is an inorganic, non-essential metal that can be toxic at low concentrations (Zitko et al., 1975; Borgmann et al., 1998). In the natural environment, Tl is present in unpolluted freshwater at concentrations between 2.4 × 10− 5 and 4.9 × 10− 5 μmol L− 1 (Peter and Viraraghavan, 2005). In Canada, Tl concentrations in the Great Lakes range from 4.4 × 10− 6 to 2.4 × 10− 4 μmol L−1 (Cheam, 2001). In polluted sites that receive waste water, such as mining sites and power plants in Eastern Canada, Tl concentrations can be as high as 0.12 μmol L−1 (Cheam, 2001). 15,000 kg of Tl is produced worldwide annually (Kazantzis, 2000). Its uses include the production of fiber optic glass, semi conductors and specialized research equipment (Peter and Viraraghavan, 2005). However, the majority of Tl (2 × 106 to 5 × 106 kg) is released into the environment by anthropogenic activities (Kazantzis, 2000), of which the major contributors are fossil fuel burning and the refining and smelting of ores (Peter and Viraraghavan, 2005). Once Tl enters the environment it can accumulate in aquatic organisms. Metal toxicity occurs when metabolically available metal in an organism reaches a threshold concentration, where it can then exert its toxic effects. This concentration is dependent on the rate of uptake ⁎ Corresponding author. Tel.: +1 9055259140; fax: +1 9055226066. E-mail addresses: [email protected] (R. Belowitz), [email protected] (E.M. Leonard), [email protected] (M.J. O'Donnell).

http://dx.doi.org/10.1016/j.cbpc.2014.07.003 1532-0456/© 2014 Elsevier Inc. All rights reserved.

from the diet and environment, and the rate of detoxification and excretion by the organism (Rainbow, 2007). Less literature is available on Tl toxicity to aquatic species compared to more well studied essential (Cu, Zn) and non-essential (Pb, Cd) metals, and the exact mechanism of Tl toxicity is not well understood (Galván-Arzate and Santamarı́a, 1998). A possible mechanism of Tl toxicity is competition with the physiologically important ion K (Mulkey and Oehme, 1993), due to the same charge and similar ionic radii of the two ions: 1.33 and 1.4 Å for K and Tl, respectively (Zhou and MacKinnon, 2003). Competition between K and Tl has been shown to occur in plants (Siegel and Siegel, 1976), and Borgmann et al. (1998) reports that only waterborne K (and not Ca, Mg, Na or other ions) affect Tl toxicity and accumulation in Hyalella azteca. The LC50 is the lethal concentration where 50% of the organisms do not survive. LC50 values are important parameters for comparing the lethality of different metals, for identifying which species are more sensitive to exposure, and for setting water quality guidelines. Acute toxicity tests are also useful for explaining the mechanism of the toxic effect (Watts and Pascoe, 2000) Chironomus riparius are dipterans found in ecologically diverse habitats, and spend the majority of their life cycle as benthic larvae (Pinder, 1995). They are commonly found in extremely polluted aquatic environments, indicating that they are extremely tolerant towards a wide range of harmful xenobiotics (Pinder, 1995). First instar C. riparius larvae are the most sensitive life stage during Cd exposure, (Williams et al., 1986), yet their acute (24 h) LC50 values for waterborne exposures

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to Cd, Cu, Pb, Ni and Zn are well above the water quality guidelines set by the Canadian Council of Ministers for the Environment (CCME) and the US Environmental Protection Agency (USEPA) (Béchard et al., 2008). Fourth instar larvae are even more tolerant; their acute LC50 value for Cd is several orders of magnitude above CCME and USEPA guidelines (Gillis and Wood, 2008b). This indicates that C. riparius are both well protected and extremely tolerant towards acute waterborne metal exposure. However, toxicity values for invertebrates that are derived exclusively from acute waterborne exposures should be viewed with some caution, as (Mebane et al., 2008) have found that Chironomus dilutus are more sensitive to chronic exposures relative to vertebrates based on acute-to-chronic toxicity ratios. Poteat and Buchwalter (2014a) have also raised 4 key concerns in regard to traditional metal toxicity testing which uses acute dissolved-only exposures with aquatic insects. These concerns include the time to reach steady state tissue concentrations (which are much longer than acute exposure duration), the inapplicability of established acute toxicity mechanisms (i.e. the surface action of metals), and the importance of dietary metal sources which are both greater contributors to tissue burdens and provide more physiologically active metals. Several studies have examined the mechanism of acute metal tolerance in C. riparius. Metal-binding proteins, such as metallothionein, may bind non-essential metals (such as Cd) and render them inert, and thus contribute to tolerance (Gillis et al., 2002). There is also some evidence for an interaction between metallothionein and Tl, as exogenous metallothionein has been found to decrease Tl-induced oxidative damage in rat livers (Kılıç and Kutlu, 2010). A potential mechanism of acute Tl toxicity in C. riparius based on in vitro ion flux measurements using ion-selective microelectrodes has been suggested (Belowitz and O'Donnell, 2013). These authors found that acute exposures to waterborne Tl interfered with K transport at the anterior- and posteriormidgut, which would result in a hypothetical loss of total hemolymph K after ~40 min and total body K after ~ 2 h. In this study we investigated the tolerance of C. riparius towards waterborne exposures of Tl by determining the acute (48 h) LC50. Our objective was to determine accumulation of waterborne Tl by the whole animal, gut and hemolymph, and we have also measured corresponding K concentrations in these tissues with a view to determining if Tl exposure is associated with hypokalemia. 2. Materials and methods 2.1. Chironomid larvae C. riparius larvae were cultured at McMaster University, initiated from ropes obtained from Environment Canada. C. riparius egg ropes were hatched in Petri dishes, and 1st instar larvae were transferred to 10 L aquaria containing fine-grained silica sand and aerated dechlorinated Hamilton tap water (‘culture water’). The ionic composition of the culture water (in mmol L−1) was Na+ (0.6), Cl− (0.8), Ca2+ (1.0), K+ (0.4), Mg2 + (0.4) and Tl+ (b1.5 × 10− 5). The water was moderately hard (120–140 mg l− 1 CaCO3) with an alkalinity of 95 mg/L and pH was 7.8–8.0. The water was replaced at intervals of ~ 10 days. The larvae were fed ad libitum ground Nutrafin™ fish flakes (45% protein, 5% crude fat, 2% crude fiber, 8% moisture; Rolf C. Hagen Corp., Mansfield, MA) and maintained at 21 ± 2 °C under a 16:8 h light:dark photoperiod. Dissolved organic content (DOC) has been reported as 2.3 mg l−1 for our culture water (Leonard and Wood, 2013).

Tl was added from a stock solution of reagent grade TlNO3 (SigmaAldrich) dissolved in the culture water. After the Tl exposure solutions were prepared and placed in the beakers, they were covered and lightly aerated for 24 h. The seven concentrations used for this study were based on preliminary toxicity tests and selected in order to bracket the apparent LC50. To allow for gut clearance, 3rd and 4th instars C. riparius larvae were removed from the tank and placed in acid washed 250 ml beakers containing aerated culture water for 24 h prior to adding them to the exposure solution. After gut clearance, 15 larvae were added to each exposure or control solution of each of the three replicates (i.e. 7 Tl exposures + control × 3 replicates × 15 chironomid/beaker = 360 chironomids total). Chironomids in replicate 1 were used for whole-animal ion measurements, in replicate 2 for gut ion measurements, and in replicate 3 for hemolymph ion measurements. Water samples (5 ml) were collected for total (unfiltered) and dissolved (filtered) Tl measurements immediately after the addition of the larvae to the beakers, and at the 48 h time point. For measurements of dissolved Tl, the water samples were filtered through an Acrodisk™ 0.45 μm in-line-syringe-tip filter (Pall Corporation, Ann Arbor, MI, USA). The Tl concentration for each exposure was calculated as the mean of the dissolved Tl at 0 and 48 h. The average concentrations for the three replicates (in μmol L− 1) were 0.01, 3.4, 26.9, 277, 544, 730, 966 and 1558. K and Tl concentrations in the whole animal, gut and hemolymph were plotted against the Tl concentration from each replicate rather than the average Tl concentration obtained from the mean of the three replicates. Mortality was assessed after 24 and 48 h by gentle prodding of the larvae. Larvae that did not respond were considered dead and removed from the beaker. After 48 h, the surviving larvae were removed from the beaker and placed in culture water for 5 min to remove adsorbed Tl. The chironomids were then rinsed with deionized water (18.2 M Ω cm, Millipore Corporation, Billerica, MA, USA), and blotted dry on tissue paper. Larvae used for whole animal ion measurements were weighed to the nearest microgram on a balance (Mettler UMT2) and placed in 1.5 ml microcentrifuge tubes for tissue digestion. At the highest concentrations of Tl exposure (i.e. above the LC50), fewer larvae survived; thus, at the two highest concentrations the K and Tl values were based on analysis of 20% or fewer of the original exposure population of chironomids. Because of concerns that ion measurements in animals exposed to Tl at concentrations above the LC50 might be influenced by inclusion of moribund animals (i.e. survivorship bias), statistical analysis of Tl and K levels in the whole animal, gut and hemolymph were restricted to animals exposed to concentrations below 966 μmol L− 1. The K values from the two highest concentrations which were excluded from the statistical analysis are reported in text. The gut was removed from larvae under saline, and the gut and carcass were weighed to the nearest microgram after excision and transferred to individual 1.5 ml microcentrifuge tubes. The composition of the saline was based on C. riparius hemolymph ion measurements performed by (Leonard et al., 2009b) and contained (in mmol L−1) K+ (5), Na+ (74), Ca2+ (1), Mg2+ (8.5), HCO− 3 (10.2), HEPES (10), and glucose (20); saline pH was titrated to 7 before use. Hemolymph samples were collected by piercing the larvae under paraffin oil in a Sylgard-lined Petri dish. The hemolymph formed a sphere in the paraffin oil and the diameter of the sphere was measured using an eye-piece micrometer. The volume of the hemolymph sample was then was then calculated as

2.2. Acute thallium exposures for 48 h LC50 studies The protocol for acute metal exposures was based on that of Gillis and Wood (2008a). The Tl exposures were performed in the same room and under the same lighting conditions as the colony to prevent major environmental changes (e.g. temperature, humidity). Tests were performed in culture water (composition described above), and

  3 V ¼ πd =6  1000

ð1Þ

where V is the volume (in nl), and d is the diameter (in mm). Hemolymph samples were then transferred to 1.5 ml microcentrifuge tubes.

R. Belowitz et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 59–64

2.3. K and Tl analysis

Data are reported as mean ± SEM. Acute LC50 values were calculated by Probit analysis using ToxCalc — Toxicity Data Analysis Software v5.0.32 (Tidepool Scientific Software, McKinleyville, CA, USA). Comparisons between K and Tl concentrations in the tissues were performed using oneway analysis of variance (ANOVA) followed by Dunnett's and Tukey's post-hoc test if p b 0.05. All statistical analyses were performed using GraphPad Instat software (GraphPad InStat, GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was designated if p b 0.05. 3. Results 3.1. Tl accumulation and toxicity: concentration-dependent kinetics for the whole animal, gut and hemolymph of C. riparius larvae The 48 h LC 50 for dissolved Tl in moderately hard water was 723 μmol L − 1 (Fig. 1). There were no differences in total or dissolved Tl concentrations, and no differences in Tl concentrations at 0 or 48 h. Accumulation of Tl by whole animals was concentration-dependent and saturable, with a maximum accumulation (Bmax) of ~4600 μmol kg−1 wet mass and a KD of 670 μmol L−1 (Fig. 2A). Concentration factors

% Survival

75

50

25

0 0

200

400

600

800 1000 1200 1400 1600 1800

[Tl] dissolved (µmol l-1) Fig. 1. Acute (48 h) Tl toxicity in 3rd–4th instars C. riparius larvae. The LC50 calculated by Probit analysis was 723 μmol L−1. Error bars are standard error about the mean (SEM) of the three replicates with 15 starting larvae per replicate.

(CFs) of Tl in the whole animal were 18- and 5-fold at the two lowest Tl exposures (3.4 and 26.9 μmol L−1), and ~4-fold over the range of the higher exposure concentrations (26.9–730 μmol L−1).

[Tl]whole animal (µmol kg-1 wet weight)

A

3000

2000 Bmax = 4637 (µmol kg-1) ± 1025

1000

KD = 670 (µmol l-1) ± 264 r 2 = 0.88

0 0

250

500

750

1000

[Tl] dissolved (µmol l-1)

B

[Tl]gut (µmol kg-1 wet weight)

2.4. Statistical analysis

100

12000 9000 6000

Bmax: 2560 ± 374 KD: 54.5 ± 23.8

3000

r 2 =0.61

slope: 13.3 ± 2.1 Y-int: -258 ± 814 r 2 =0.91

0 0

250

500

750

1000

[Tl] dissolved (µmol l-1)

C [Tl]hemolymph (µmol l-1)

Concentrations of dissolved Tl in the exposure solutions were measured in filtered water samples by atomic absorption spectroscopy (AAS) using a Graphite Furnace (GFAAS; Varian SpectrAA-220 with graphite tube atomizer [GTA — 110], Mulgrave, Australia). The concentrations of Tl in the exposure solutions reported throughout this study are the dissolved Tl measurements. Tl concentrations in the whole-animal, gut and hemolymph were determined for control and Tl-exposed larvae by GFAAS. Method blanks (2) and Tl calibration standards (Sigma-Aldrich) were included in every run (25 samples). Metal recovery was ±15% from analytical reference material TM24 and TM25 (Environment Canada) and a maximum difference of 5% between duplicates was accepted. K concentrations were determined by flame atomic absorption spectroscopy (FAAS; Varian 220FS SpectaAA, Varian Techtron, Mulgrave, Australia). Method blanks (2) and K calibration standards (Fisher Scientific) were included in every run. Animal tissues were digested in 70 μl of concentrated nitric acid (trace metal grade, Fisher Scientific, Ottawa, ON, Canada) at room temperature for 6 days, after which 30 μl of 30% H2O2 was added (Fisher Scientific, Ottawa, ON, Canada). 24 h after the addition of the H2O2, 750 μl of 1% nitric acid was added to each sample. Samples that required further dilution for Tl measurements were diluted between 5- and 225fold with 1% nitric acid. For K measurements, all the samples were diluted an additional 3-fold with 0.1% CsCl (Sigma-Aldrich) in 1% nitric acid. The gut and hemolymph samples were digested in 30 μl of concentrated trace metal grade nitric acid at room temperature for 6 days, after which 10 μl of 30% H2O2 was added. 24 h after the addition of the H2O2, 100 μl of 1% nitric acid was added to each sample. Samples that required further dilution for Tl measurements were diluted between 3- and 100-fold with 1% nitric acid. For K measurements, the samples were diluted an additional 9- to 15-fold with 0.1% CsCl in 1% nitric acid. A concentration factor (CF) indicates how much more Tl is in the tissue relative to the aqueous exposure concentration. CFs were determined by dividing the concentration of Tl in the tissue (e.g. whole animal, gut or hemolymph) by the aqueous Tl exposure concentration. CFs N 1 indicate a higher concentration of Tl in the tissue relative to the aqueous exposure solution, whereas CFs b 1 indicate a lower concentration of Tl in the tissue relative to the aqueous exposure concentration.

61

750

500 slope: 0.75 ± 0.06 Y-int: 21.5 ± 31.4 r 2: 0.97

250

0 0

250

500

750

1000

[Tl] dissolved (µmol l-1) Fig. 2. Concentration-dependent uptake kinetics of Tl by the whole animal (A), gut (B), and hemolymph (C) in 3rd–4th instars C. riparius larvae. Values are means ± SEM.; N = 7–13 surviving larvae per exposure concentration.

R. Belowitz et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 59–64

3.2. The effect Tl exposure on K concentrations in the whole animal, gut and hemolymph of C. riparius larvae There was not a statistically significant decrease in whole animal, gut or hemolymph K concentration at any of the Tl exposure concentrations (Fig. 4A–C). Although there appears to be a decrease in K in the hemolymph (Fig. 4C) at the highest analyzed Tl exposure (~700 μmol L−1), this decrease was not significant (p = 0.06). Even at the highest Tl concentrations (≥966 μmol L−1) that were not analyzed due to survivorship bias and fewer surviving larvae (n ≤ 3), there was no obvious trend showing a decrease in K concentration. Whole animal K levels for these two highest Tl exposures of ~ 966 and 1558 μmol L− 1 were 33.1 ± 3.0 μmol g−1 wet mass (n = 3) and 44.5 μmol g−1 wet mass (n = 1), respectively. For the gut and hemolymph replicates, no larvae survived at the highest Tl exposure (~ 1558 μmol L−1). At the highest Tl exposure with surviving larvae (966 μmol L−1), K levels were

[K+] whole animal (µmol g-1 wet weight)

50 45 40 35

Whole animal 30 0

10 20 30 40 50

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500

700

[Tl] dissolved (µmol l-1)

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300

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100

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700

[Tl] dissolved (µmol l-1)

C

15

10

5

Hemolymph 0 0

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20

30

40

50

300

[Tl] dissolved (µmol

500

700

l-1)

Fig. 4. K concentrations in the whole body (A), gut (B) and hemolymph (C) in 3rd–4th instars C. riparius larvae exposed to waterborne Tl in acute (48 h) toxicity tests. Values are means ± SEM.; N = 7–13 surviving larvae per exposure concentration.

Whole Animal

198.2 μmol g− 1 wet mass (n = 2) and 10.1 mmol−1 ± 1.5 (n = 3) for the gut and hemolymph, respectively.

75

Carcass Gut

4. Discussion

50

Hemolymph

100

% of Total Tl

A

[K+]gut (µmol g-1 wet weight)

The accumulation of Tl appears to be non-saturable (i.e. linear) at the gut when all the Tl exposure concentrations, shown as the solid line in Fig. 2B, are included. However, there was evidence for saturation at the four lowest Tl concentrations, displayed in Fig. 2B as the dotted curve (Bmax = 2560 μmol L−1, KD = 54.5 μmol L−1; r2 = 0.61). CFs of Tl in the gut were 67- and 31-fold above the exposure concentrations of 3.4 and 26.9 μmol L−1, respectively. At higher Tl exposures (N26.9 μmol L− 1), CFs of Tl in the gut were ~ 8- to 15-fold above the exposure concentrations. Hemolymph Tl accumulation was linear (Fig. 2C), and CFs were lower (4.2- and 1.6-fold) compared to the gut and whole animal at the two lowest Tl exposures. Interestingly, CFs were b1 (i.e. less Tl in the hemolymph relative to exposure solution) at Tl exposures above 26.9 μmol L−1. In general, the CFs were inversely proportional to the exposure concentration in all tissues (whole animal, gut and hemolymph). Fig. 3 shows the percent of total Tl in the gut or hemolymph (relative to the whole animal). The total amount of Tl in the whole animal and gut (μmoles) was determined for each exposure concentration by multiplying the mass of the chironomid or gut (kg) by the concentration of Tl in the respective tissue (μmol kg−1 wet mass). Similarly, the total amount of Tl in the hemolymph was calculated as the product of hemolymph volume (l) and Tl concentration in the hemolymph (μmol L−1). The percent of Tl in the carcass was calculated as the Tl not accounted for by the gut and hemolymph (i.e. Tl in carcass = [whole animal Tl — (gut Tl + hemolymph Tl)]). Tl in the carcass was ~70% of total Tl, consistent with the proportion of chironomid body mass represented by the carcass (~60%). The role of the gut in accumulating 20% of total Tl was of interest, given that it represents such a small amount of total body mass (b10%). In contrast with the gut, the hemolymph accounted for a significantly smaller amount of total Tl given that it represents ~30% of the mass of the larvae.

[K+] hemolymph (mmol l-1)

62

25 0 3

30

280

545

730

[Tl] dissolved (µmol l-1) Fig. 3. The total amount of Tl in each body compartment, as a percent of the total Tl in the whole animal. The total thallium in the whole animal, gut and hemolymph were based on measured Tl concentrations and the recorded weight or volume. The % of Tl in the carcass was calculated as: [Tl]carcass = ([Tl]whole animal- ([Tl]Gut + [Tl]Hemolymph)). Values are means ± SEM.; N = 7–13 surviving larvae per exposure concentration.

4.1. Tl toxicity values in C. riparius larvae To our knowledge, this study is the first to measure the acute toxicity of waterborne Tl to C. riparius larvae and to examine the relationships between Tl exposure and concentration factors (CFs) in the whole animal, gut and hemolymph. We have also examined the effects of Tl exposure on K homeostasis. The 48 h LC50 of 4th instar C. riparius larvae (723 μmol L−1) indicates a high tolerance towards Tl compared to other aquatic organisms. For example, the 48 h LC50 of Tl in Daphnia magna is ~ 70-fold lower (LeBlanc, 1980), and in the rotifer Brachionus calyciflorus it is ~40-fold lower (Calleja et al., 1994), while (Zitko et al., 1975) reports that the

R. Belowitz et al. / Comparative Biochemistry and Physiology, Part C 166 (2014) 59–64

48 h LC50 for rainbow trout is 53.2 μmol L− 1. However, it should be noted that the Tl concentrations tested in this study are above what would normally be found in the environment. The lowest exposure that was tested (3 μmol L− 1) is still ~ 25-fold higher than polluted sites in Canada with the highest Tl levels (Cheam, 2001), but is within the range of contaminated mining sites found in some parts of China (Xiao et al., 2004). The testing at such high concentrations was necessary in order to bracket the LC50 of C. riparius, which are extremely tolerant to Tl. The high tolerance towards acute waterborne Tl exposures is consistent with the high tolerance of C. riparius to extremely high concentrations of many essential and non-essential metals (Béchard et al., 2008; Brix et al., 2011; Leonard and Wood, 2013). First instar C. riparius larvae are the most sensitive life stage (Williams et al., 1986) but still display remarkable tolerance. The 24 h LC50 concentrations of Pb (2.9 μmol L−1), Cu (32.9 μmol L−1), Cd (83 μmol L−1), Ni (N 425 μmol L−1) and Zn (N 382 μmol L−1) for 1st instar larvae are at least 25 times higher than CCME water quality guidelines (Béchard et al., 2008). The 48 h LC50 of Cd, a divalent non-essential metal, is ~ 10 mmol L−1 in 4th instar C. riparius larvae (Gillis and Wood, 2008a) which indicates that short term exposures to Tl are more toxic by ~ 13-fold. It should be noted that acute LC50 values derived exclusively from waterborne exposures may not accurately predict toxicity. A review of field and laboratory toxicity data (Brix et al., 2011) has identified several studies where dietary exposure is the dominant pathway for Cd uptake in most aquatic insects. Further investigation is required to determine if the same trend occurs for Tl in C. riparius larvae.

4.2. Tl accumulation There was some evidence for a saturable accumulation of Tl in the gut over the range of the lowest 4 concentrations (Fig. 2B, dotted curve). However, when all the Tl concentrations were included in regression analysis it appears that accumulation is linear and nonsaturable (Fig. 2B, solid curve). A third possibility is that there is both a saturable and a non-saturable component to Tl accumulation in the gut. For example, in Oncorhynchus mykiss, there is saturable accumulation of Ni at low exposure concentrations (0 to 30 μmol L−1 Ni) and a linear accumulation at higher concentrations (N100 μmol L−1 Ni2+) in the stomach, mid- and posterior- intestine (Leonard et al., 2009a). The possibility of the saturable accumulation of Tl at low concentrations may be indicative of detoxification by the intracellular compartmentalization of Tl within lysosomes, as observed in Mercenaria cytosomes for Hg accumulation (Fowler et al., 1975). The saturable accumulation may also be caused by the limited availability of metal-binding proteins, such as metallothionein, a cysteine-rich protein that binds excess metals to decrease the metabolically available fraction (Fowler, 1987). Metallothionein has been detected in C. riparius, and its levels increase in response to increased Cd exposure (Gillis et al., 2002). At higher concentrations, the accumulation of Tl appears to be by simple diffusion into the gut (i.e. the linear portion of the curve). In contrast, hemolymph Tl concentrations were lower, and more regulated at higher Tl exposures. The source of Tl in the gut lumen during waterborne exposure was not determined during this study. It is possible that Tl entered the gut lumen due to drinking, or as a result of C. riparius eating its fecal matter or dead larvae (which could contain adsorbed Tl). However, these potential dietary sources of Tl were minimized by gut clearing the larvae prior to exposure, and by removing fecal matter and dead larvae daily. Another possible source of Tl in the gut lumen could be the localization of Tl in the hindgut tissue due to excretory processes (e.g. transport of ingested Tl into the hemolymph, filtration by the Malpighian tubules and excretion via the hindgut). Further studies looking at the localization of Tl in the different parts of the gut and Malpighian tubules would be needed to explore this possibility.

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A previous study based on in vitro ion measurements with microelectrodes has reported that the gut of C. riparius transports Tl in the direction of the hemolymph from the gut lumen (Belowitz and O'Donnell, 2013). The results from the current study support this finding, as it was found the gut was not a perfect barrier to Tl entry into the hemolymph. We found that the hemolymph contained ~3-fold more Tl than the exposure concentrations at low Tl exposures, whereas at higher concentrations (above 26.9 μmol L−1) the hemolymph Tl concentration was only ~75% of the exposure concentration (Fig. 2C). Similar findings for hemolymph Cd concentrations in C. riparius have been reported (Leonard et al., 2009b). Our findings indicate that Tl gains access to the hemolymph, where it may be able to exert toxic effects on sensitive tissues such as the nervous system. CFs of Tl in the hemolymph were N 1 only at Tl exposures well below the LC50. It may be that at low exposures there are sufficient mechanisms for detoxification (e.g. metallothionein proteins), while higher exposure concentrations require increased elimination. The mechanism of elimination may involve secretion by the Malpighian (renal) tubules. The Malpighian tubules can eliminate both Cd (Leonard et al., 2009b), and Tl (Belowitz and O'Donnell, 2013) by secreting the ions into the tubule lumen. 4.3. The effect of Tl on K concentrations in the whole animal, gut and hemolymph There was no effect of Tl exposure on K concentration in the whole animal, gut or hemolymph, in contrast to findings with mammals (Mulkey and Oehme, 1993). Our previous study (Belowitz and O'Donnell, 2013) measured in vitro K+ flux along the gut after Tl exposure, and estimated that a net loss of hemolymph K+ would occur after ~40 min. We did not find evidence for loss of K from the hemolymph in our current study, which measured K concentrations after a 48 h exposure to Tl. The differences in these findings may be attributed to the methodology, as ion-fluxes determined by microelectrode measurements were performed for only ~60 min, after which it is possible homeostatic mechanisms may have been altered so as to compensate for the loss of K+. It may also indicate that hemolymph factors not present in the in vitro studies play an important role in ionoregulation. This current study suggests that the avoidance of hypokalemia may contribute to survival of C. riparius during acute exposures to Tl. It is worth noting that C. riparius may tolerate exposure to high concentrations of Cd by preventing hypocalcemia from occurring, possibly due to the lower affinity of Ca transporters for Cd relative to other aquatic organisms (Gillis and Wood, 2008b). Poteat and Buchwalter (2014b) also have shown that neither Zn nor Cd exhibits inhibitory effects towards Ca uptake in mayfly and caddisfly larvae. It thus appears that the uptake of toxic metals by aquatic insects during acute waterborne exposures does not occur at the expense of the essential metals that they are supposed to replace. Other potential mechanisms of Tl toxicity may involve inhibition of cellular respiration, interference with riboflavin and riboflavin-based cofactors and/or disruption of Ca homeostasis (Mulkey and Oehme, 1993). Since Tl is classified as a soft Lewis acid, it forms more stable bonds with soft ligand donors, such as those rich in sulfur containing compounds (House, 2008). This can cause Tl to bind to a wide range of sulfur rich proteins and interfere with their normal function. While the mechanism(s) of Tl toxicity could not be elucidated from this study, we were able to report some data that suggest mechanisms of tolerance. We found that Tl accumulation was proportionate to the exposure concentration, and CFs were inversely proportionate to the aqueous exposure concentration in the whole animal, gut and hemolymph. This observation has been well documented by (McGeer et al., 2003) and observed in Chironomus javanus by (Shuhaimi-Othman et al., 2011) for Cu, Cd, Zn, Pb, Ni, Fe and Al. It is revealing that the CFs were much larger in the gut relative to the whole animal, and that the CFs were less than one in the hemolymph at higher exposures. This suggests two things; first, that the gut is acting as a partial barrier to Tl entry

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into the hemolymph, and second, that access to the hemolymph needs to be more tightly regulated, perhaps to prevent exposure of sensitive tissues such as the nervous system to Tl. The high tolerance to metal exposure that has been observed in C. riparius may indicate unusual transport kinetics in the gut relative to other aquatic organisms. Future studies on C. riparius would benefit from measuring the presence of metals in the hemolymph, which presents a more sensitive site for determining accumulation of Tl, and perhaps other metals. Acknowledgements Supported by NSERC (38350-2010) grants to MJO. The authors are grateful to Prof. Chris Wood and Dr. Patty Gillis for critical comments on the original thesis, and to Prof. Edward Berkelar who provided extensive technical advice for measuring Tl using AAS. The authors are also grateful to an anonymous reviewer who provided suggestions that have been incorporated into the final manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpc.2014.07.003. References Béchard, K.,Gillis, P.,Wood, C., 2008. Acute toxicity of waterborne Cd, Cu, Pb, Ni, and Zn to first-instar Chironomus riparius larvae. Arch. Environ. Contam. Toxicol. 54, 454–459. Belowitz, R.,O'Donnell, M.J., 2013. Ion-selective microelectrode measurements of Tl+ and K+ transport by the gut and associated epithelia in Chironomus riparius. Aquat. Toxicol. 138, 70–80. Borgmann, U., Cheam, V., Norwood, W., Lechner, J., 1998. Toxicity and bioaccumulation of thallium in Hyalella azteca, with comparison to other metals and prediction of environmental impact. Environ. Pollut. 99, 105–114. Brix, K.V., DeForest, D.K., Adams, W.J., 2011. The sensitivity of aquatic insects to divalent metals: a comparative analysis of laboratory and field data. Sci. Total Environ. 409, 4187–4197. Calleja, M., Persoone, G., Geladi, P., 1994. Comparative acute toxicity of the first 50 multicentre evaluation of in vitro cytotoxicity chemicals to aquatic non-vertebrates. Arch. Environ. Contam. Toxicol. 26, 69–78. Cheam, V., 2001. Thallium contamination of water in Canada. Water Qual. Res. J. Can. 36, 851–877. Fowler, B.A., 1987. Intracellular compartmentation of metals in aquatic organisms: roles in mechanisms of cell injury. Environ. Health Perspect. 71, 121. Fowler, B.A., Wolfe, D.A., Hettler, W.F., 1975. Mercury and iron uptake by cytosomes in mantle epithelial cells of quahog clams (Mercenaria mercenaria) exposed to mercury. J. Fish. Res. Board Can. 32, 1767–1775. Galván-Arzate, S., Santamarı́a, A., 1998. Thallium toxicity. Toxicol. Lett. 99, 1–13. Gillis, P.L., Wood, C.M., 2008a. The effect of extreme waterborne cadmium exposure on the internal concentrations of cadmium, calcium, and sodium in Chironomus riparius larvae. Ecotoxicol. Environ. Saf. 71, 56–64. Gillis, P.L., Wood, C.M., 2008b. Investigating a potential mechanism of Cd resistance in Chironomus riparius larvae using kinetic analysis of calcium and cadmium uptake. Aquat. Toxicol. 89, 180–187.

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Effects of exposure to high concentrations of waterborne Tl on K and Tl concentrations in Chironomus riparius larvae.

Thallium (Tl) is a non-essential metal which is released into the environment primarily as the result of anthropogenic activities such as fossil fuel ...
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