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Use of lanthanum for water treatment A matter of concern?
Chemosphere 239 (2020) 124780
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Use of lanthanum for water treatment A matter of concern? Geert J. Behets a, Kayawe Valentine Mubiana b, Ludwig Lamberts a, Karin Finsterle c, Nigel Traill d, Ronny Blust b, Patrick C. D'Haese a, * a
Laboratory of Pahophysiology, Department Biomedical Sciences, University of Antwerp, Belgium Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Belgium c Limno Solutions International, Australia d Phoslock® Environmental Technologies Ltd, Australia b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Lanthanum uptake after exposure to lanthanum modified bentonite (LMB) treated water is marginal. Gastrointestinal absorption of lanthanum (La) does not depend on its chemical composition. The slightly increased liver La levels in a worst case scenario do not hold a risk for hepatotoxicity. Exposure to LMB treated water should not be considered a risk to animal and/or human health. As for its therapeutic use, La is considered a safe tool in surface water eutrophication management.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2019 Received in revised form 8 August 2019 Accepted 4 September 2019 Available online 6 September 2019
Among several other eutrophication management tools, Phoslock®, a lanthanum modified bentonite (LMB) clay, is now frequently used. Concerns have been raised as to whether exposure to Phoslock®treated water may lead to lanthanum accumulation/toxicity in both animals and humans. In the present experimental study, rats were administered lanthanum orally as either lanthanum carbonate, lanthanum chloride or Phoslock® at doses of either 0.5 or 17 mg/L during 10 weeks. Controls received vehicle. The gastrointestinal absorption and tissue distribution of lanthanum was investigated. Extremely strict measures were implemented to avoid cross-contamination between different tissues or animals. Results showed no differences in gastrointestinal absorption between the different compounds under study as reflected by the serum lanthanum levels and concentrations found in the brain, bone, heart, spleen, lung, kidney and testes. At sacrifice, significant but equally increased lanthanum concentrations versus vehicle were observed in the liver for the highest dose of each compound which however, remained several orders of magnitude below the liver lanthanum concentration previously measured after long-term therapeutic administration of lanthanum carbonate and for which no hepatotoxicity was noticed in humans. In conclusion, (i) the use of LMB does not pose a toxicity risk (ii) gastrointestinal absorption of lanthanum is minimal and independent on the type of the compound, (iii) with exception of the liver, no significant increase in lanthanum levels is observed in the various organs under study, (iv) based on
Handling Editor: Martine Leermakers
* Corresponding author. University of Antwerp Universiteitsplein 1, B-2610, Wilrijk, Belgium. E-mail address: [email protected] (P.C. D'Haese). https://doi.org/10.1016/j.chemosphere.2019.124780 0045-6535/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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previous studies, the slightly increased liver lanthanum levels observed in a worst case scenario do not hold any risk of hepatotoxicity. © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Eutrophication is considered the main water quality issue worldwide. The enrichment of surface waters with nutrients, mainly nitrogen (N) and phosphorus (P) negatively affect the multiple functions and services of freshwater ecosystems by accelerating the primary production and increasing the risk of nuisance cyanobacteria blooms [Lürling et al., 2016; Schindler et al., 2016]. The consequences of such blooms are surface scums and accumulation of cyanobacteria-toxins in the water which presents a severe threat to the ecosystem itself, pets and human health [Chorus et al., 2000; Carmichael 2001; World Health Organization, 2003; Lürling and Faassen, 2013; Salmaso et al., 2015; BoumaGregsona et al., 2017]. Subsequently, the decomposition of increased amounts of organic matter leads to oxygen depletion and a marked decline of the redox potential especially in the hypolimnion and close to the sediment. A complex interaction between biogeochemical processes coupled with low oxygen concentrations or anoxia, induces the release of legacy phosphorus and toxic €chter substances from the sediment and can provoke fish kills. [Ga and Müller 2003; Matzinger et al., 2010; La Van and Cooke, 2011; Small et al., 2014]. Notwithstanding extensive external load management, the recovery of lentic freshwater ecosystems is often delayed due to the internal phosphorus load. In lake management, actions using specific materials to counteract persistent eutrophication by targeting particularly the internal phosphorus load are described as geoengineering [Spears et al., 2014; Lürling et al., 2016]. Whilst in the past aluminum sulphate (Alum, Al2(SO4)3) was most frequently applied to inactivate the internal phosphorus pool [Welch and Cooke, 2005], the use of the lanthanum (La) modified bentonite (LMB, Phoslock®), currently is rapidly emerging as an effective eutrophication management tool to prevent blue-green algae blooms by controlling the internal phosphorus load (Copetti et al., 2016). Compared to Alum, Phoslock® is far less susceptible to resuspension than alum flocs, the formed lanthanum-phosphate bound is stable over a wide pH-range and its use does not affect the pH of the water [Ross et al., 2008; Egemose et al., 2010; Spears et al., 2013a, 2013b]. Hence, the risk due to the presence of La3þ ions in LMB treated lake water is much lower compared to the risk related to the formation of soluble aluminum species after an alum application [Reitzel et al., 2013; Zamparas and Zacharias, 2014; Lürling et al., 2016, D'Haese et al., 2019]. Phoslock® is a lanthanum (La) modified bentonite clay designed by the Commonwealth Scientific and Industrial Research Organization (CSIRO) in the 1990s for the control of oxyanions, especially phosphate, in water (Douglas, 2002). Phoslock® is commonly applied from a barge, as a slurry, where it acts to strip dissolved phosphorus en route through the water column. Once settled onto the sediment surface, the product can enhance the capacity of lake bed sediments to retain phosphorus by forming LaPO4.(H2O), a particulate, natural occurring mineral known as rhabdophane. Due to its very low solubility (Ksp 1024.7 to 1025.7 mol2l2) LaPO4.(H2O) is not available to phytoplankton, and is stable under a wide range of environmental conditions, commonly reported in eutrophic lakes [Liu and Byrne 1997; Robb et al., 2003; Haghseresht et al., 2009; Ross et al., 2008; Meis
et al., 2012]. Interestingly, before putting possible increased exposure to lanthanum in perspective it is worth mentioning that a pharmaceutical compound based on lanthanum carbonate (La2(CO3)3; Fosrenol®), was developed by Shire Pharmaceuticals for use in patients with chronic renal failure. Due to the loss of renal function, these patients accumulate phosphate. Dietary restrictions can alleviate this only modestly, thus additional treatment is necessary, usually in the form of phosphate binding agents. These phosphate binders, which are taken orally (several grams/day) with the meals, bind phosphate in the gut, thus rendering it unavailable for absorption in the gastrointestinal (GI) tract [Persy et al., 2006; Persy et al., 2009; Tonelli et al., 2010]. Related to the utilization of Phoslock® the common operational assumptions are (1) lanthanum is not released from the bentonite carrier under natural environmental conditions occurring in lakes and (2) the formation of LaPO4 is the dominant mechanism of phosphate removal and phosphorus retention in Phoslock® treated lakes [Dithmer et al., 2015, 2016; Copetti et al., 2016]. However, there is still some concern that small amounts of lanthanum may be liberated from the carrier as filterable lanthanum (FLa), in particular in low alkalinity water, and be present in the water in either its ionic state (La3þ) or as small particulates [Lürling & Tolman, 2010; Van Oosterhout & Lürling, 2011; Reitzel et al., 2017]. This would, in turn, expose consumers of Phoslock®-treated drinking water to a potential risk related to lanthanum accumulation/toxicity. The production process of Phoslock® fulfils strict international standards (NSF/ANSI 60). However, due to the occurrence of small amounts of FLa species in LMB treated water it is possible that the gastrointestinal absorption, tissue distribution/accumulation and potential toxicity properties of Phoslock® derived lanthanum might resemble those of LaCl3 more closely than those of the wellstudied and therapeutically used phosphate binding agent lanthanum carbonate (Fosrenol®). Although gastrointestinal absorption of lanthanum after oral administration of lanthanum carbonate was demonstrated to be extremely low (0.00127±0.00080% in humans) and therapeutic use of this compound has proven to be safe when administered at doses up to 1e3 g/day during several years [Pennick et al., 2006; Damment and Pennick 2008; Hutchison et al., 2018], health authorities have questioned to which extent fractional intestinal absorption of lanthanum after exposure to lanthanum chloride or Phoslock® derived lanthanum may be higher than that of lanthanum carbonate. It has indeed been argued that because of its higher solubility constant intestinal absorption of lanthanum chloride might be higher as compared to less soluble lanthanum compounds such e.g. lanthanum carbonate. This might be true when lanthanum should be present at extremely high concentrations; i.e. those exceeding equimolar concentrations of phosphate and/or other oxyanions (e.g. oxalate) in the intestine which are well known strong binding ligands for lanthanum. However, given the highest lanthanum concentrations ever observed in Phoslock® treated water one may reasonably hypothesize that in view of the very high phosphate-oxyanion/lanthanum ratio intestinal binding of lanthanum to these ligands will occur at the same extent for the different lanthanum compounds under study leaving no ionic lanthanum available for intestinal absorption which in turn will dramatically limit gastrointestinal absorption of lanthanum after
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exposure to either of the above mentioned lanthanum-containing compounds to the same extent. As data on this topic in the literature are highly limited, further evidence for this has to be provided from experimental studies. The aim of the current study was to obtain insight into the gastrointestinal absorption, tissue distribution/accumulation of lanthanum after oral gavage of either lanthanum chloride, lanthanum carbonate or LMB at doses which for each compound corresponded to 17 and 0.5 mg elemental lanthanum/L respectively. 2. Materials and methods Experimental procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals 85-23 (1996) and were approved by the University of Antwerp Ethics Committee (Approval number 2016e22). 2.1. Animals and study set-up Animals were housed two per cage for the duration of the study in a temperature-controlled (25 C) room with a strict 12 h light/ dark cycle at the central animal facility of the University of Antwerp. Food and water consumption were recorded once a week, and animals were weighed weekly. The animals were inspected daily for morbidity. Animals had free access to a standard rodent diet (SSNIFF Spezialdi€ aten, Soest, Germany) and tap water (pH 7.8). Study set up (Fig. 1). During a 2-week equilibration period, rats were adapted to the diet. Treatment by oral gavage was then started for a 10-week period. A constant dose volume of 10 mL/kg was used and the volume was individually adjusted weekly, according to the most recent body weight. For dose calculations, it was assumed that healthy animals drink approximately 75 mL/kg/day, and it was assumed that Phoslock® contained 5% lanthanum (van Oosterhout and Lürling, 2013). Fifty six male (56) Wistar rats (225e250 g body weight at start of the study, Charles River, France) were randomly divided into 7 study groups consisting of 8 animals each, and were exposed to (i) vehicle, (ii) 2 doses of lanthanum chloride, (iii) 2 doses of lanthanum carbonate and (iv) 2 doses of LMB, respectively. 2.2. Dosing With regard to the choice of the dosing, it was aimed that rats should be exposed to either lanthanum chloride, lanthanum carbonate and LMB at doses corresponding to the worst case scenario
Fig. 1. Study scheme. WCS: Worst case Scenario, corresponding to 17 mg/L of lanthanum; RLL: Reference Lower Limit, corresponding to 0.5 mg/L lanthanum.
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(WCS; 17 mg/L) and the reference lower limit (RLL; 0.5 mg/L). The WCS is a hypothetical worst case situation and is based on the highest Phoslock® dose ever applied to a lake [Spears et al., 2013a, 2013b] and assuming that all lanthanum contained within Phoslock® would be released from the bentonite matrix and would be present as ionic lanthanum. Initially it was intended to set the RLL at 0.01 mg lanthanum/L which refers to the lower range of filterable lanthanum concentrations measured after a Phoslock® application. Prior testing however, showed that the food of the animals contained up to 0.4 mg of lanthanum/g dry weight. Since healthy animals eat approximately 25e30 g/day, this would correspond to a lanthanum intake of 10e12 mg/day from the food. The planned RLL dose thus would have added only 0.25 mg lanthanum to the daily dietary intake. It was therefore decided to increase the low dose to 0.5 mg lanthanum/L, corresponding to an additional daily intake of approximately 11 mg. All dosing stock solutions were freshly prepared every week. Appropriate dilutions to administer to the animals were made daily. Lanthanum carbonate and Phoslock® were suspended in 1% carboxymethylcellulose. Lanthanum chloride was dissolved in tap water, since it forms cloudy precipitates in carboxymethylcellulose which might affect gastrointestinal absorption. The vehicle group received 1% carboxymethylcellulose only, while an additional 4 animals, which were gavaged with tap water were also included. The lanthanum content in the dosing solutions of the different lanthanum compounds was measured before the start of the experiment to check whether it effectively corresponded to the nominal dose which theoretically was 127.5 mg/L. We found concentrations of 122.9, 99.8 and 80.6 mg/L for lanthanum chloride, lanthanum carbonate and Phoslock® respectively. Differences most probably were due to the degree of hydration of lanthanum carbonate in particular, or the amount at which lanthanum was present in Phoslock® which was assumed to be 5%. Based on these concentrations appropriate correction was made to obtain the nominal doses. We also measured the lanthanum concentration in the water and in the 1% carboxymethylcellulose used for gavage which was as low as 0.26 mg/L and 0.18 mg/L and thus negligible. To avoid cross-contamination during dosing, the following sequence was used: vehicle groups, low dose groups and lastly the high dose groups.
2.3. Sampling At weeks 0, 4, 7 and 10, animals were housed individually in a metabolic cage for 24 h to collect urine samples followed by blood sampling. The urinary volume was recorded and the samples divided in 2 fractions and frozen at 20 C pending analysis. Blood was drawn from the tail vein in restrained, conscious animals. A first few drops of blood were collected for immediate analysis using the i-Stat Point of Care technology (Abbott) for measurement of blood pH and bicarbonate content. Afterwards, a 1.5 mL blood sample was collected and allowed to clot on ice (approximately 2e3 h). After centrifugation, serum was divided in aliquots and stored at 80 C until further analysis. After 10 weeks of dosing, animals were sacrificed by exsanguination through the retro-orbital plexus after anesthesia with Animale, 60 mg/kg sodium pentobarbital (Nembutal, Ceva Sante France) via intraperitoneal injection. In addition to blood and urine, the following tissues were taken: brain, liver, spleen, lungs, heart, kidney, testes and bone. Tissue samples were immediately wet weighed and stored at 80 C in plastic containers pending further analysis. During sacrifice, care was taken to avoid crosscontamination between tissues and animals. The same sequence as for dosing was used (i.e. firstly vehicle groups, then low dose
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groups and lastly the high dose groups). Similarly, tissues were removed in the same order in all animals (brain, testes, kidney, spleen, bone, heart, liver, lungs). All non-disposable materials were thoroughly cleaned between animals. All materials used for tissue collection, blood and urine sampling were checked in advance for possible lanthanum contamination by soaking them for 24 h in demineralized water and measuring lanthanum content. No lanthanum was detectable in any of the disposables used.
0.6 mg/L in the lanthanum carbonate group at week 7), but group averages remained below 0.2 mg/L in all treatment groups, versus 0.1 mg/L in the vehicle group (Fig. 2). An increased variability in the urinary lanthanum excretion was noted. Urinary excretion of lanthanum was minimal, with a maximal excretion of approximately 25 ng/24 h. At week 10, a statistically significant increase in urinary lanthanum excretion was seen in the Phoslock® WCS group, and at week 4 in the lanthanum chloride WCS group, versus vehicle groups (Fig. 3).
2.4. Lanthanum measurement and biochemical analyses 3.2. Tissue lanthanum levels Lanthanum was determined in serum and the various tissues by High Resolution Inductively Coupled Mass Spectrometer (HR-ICPMS; Element XR, Thermo Scientific, Finnigan Element 2, Bremen, Germany). Briefly, serum and urine samples were freeze-dried, dissolved in 200 mL ultrapure HNO3 (Optima Grade, Fluka, Brussels, Belgium) and diluted with ultrapure demineralized/reverse osmosis treated water to a final volume of 5 mL. Tissue samples were digested in 1 mL ultrapure HNO3 at 110 C for 45 min and adjusted to 10 mL with ultrapure demineralized water. Prior to measurement with HR-ICP-MS, the samples were further diluted to bring the acid concentration in the solutions to approximately 2%. In order to overcome matrix interferences, lanthanum concentrations were accurately quantified by applying the standard addition calibration technique, whereby a series of aliquot sample solutions were spiked with increasing lanthanum concentrations. Method validation was achieved by analysis of a biological tissue reference material BCR 668 (European Commission e Joint Research Centre, Geel, Belgium) which was processed in a similar manner as the tissue samples. During analysis, a NIST reference standard (SRM1640a, USA National Institute of Standards and Technology, Gaithersburg, USA) was used as analytical QC check. In the brain samples, iron, copper, zinc and calcium were determined by means of electrothermal atomic absorption spectrometry (AAnalyst 800, PerkinElmer, USA). Here the same digestion and analytical procedure as previously described for bone analysis were applied [D'Haese et al., 1999]. 2.5. Statistical analysis Statistical analysis was performed using SPSS (Version 22, IBM). The Kruskall-Wallis test was used to test for differences between groups, followed by a Mann-Whitney U test with Bonferroni correction when appropriate. A p-value < 0.05 was considered statistically significant. Values which were larger than the 75th percentile þ 3x the Interquartile range or lower than the 25th percentile e 3x the Interquartile range were considered outliers and eliminated from further statistical analysis. 3. Results Throughout the study, no statistically significant differences were noted in body weight gain (Supplementary Fig. 1), food or water consumption between the different treatment groups (results not shown). Serum pH (Supplementary Fig. 2) and serum bicarbonate (HCO 3 ) (Supplementary Fig. 3) levels did not show any statistically significant differences between treatment groups. There was no mortality over the study period. 3.1. Serum and urine lanthanum levels Serum lanthanum levels showed no statistically significant differences between treatment groups, or between treatment and vehicle. Occasionally, some animals showed higher levels (up to
In the animals treated with the low dose of all three compounds, lanthanum levels did not show any increase versus vehicle treated animals. In all three high dose groups, total lanthanum content of the liver was increased, showing levels up to 10 ng/g wet weight. No differences between the three compounds were noted (Fig. 4). Accumulation of lanthanum in heart, spleen, lung, kidney and testes was highly limited. Heart lanthanum levels were below 1 ng/ g wet weight in all treatment groups, and no differences were found between compounds or doses. Interestingly, some vehicle treated animals had slightly higher lanthanum levels up to 2 ng/g wet weight. Spleen lanthanum levels were below 0.1 ng/g wet weight in all treatment groups. Lanthanum levels in the lung were slightly higher (up to 7 ng/g wet weight), and also showed some higher variability. Testes and kidney lanthanum levels were below 4 ng/g in all treatment groups, and again no statistically significant difference between treatment groups or vehicle was found (Fig. 5). Some accumulation of lanthanum in bone can be expected, given the chemical similarity between lanthanum and calcium. In the current study, we found bone lanthanum levels to remain below 35 ng/g. Values were similar in all lanthanum-treatment groups which did not differ from the vehicle group (Fig. 6). Brain lanthanum levels showed no statistically significant differences between treatment groups and vehicle, and were generally below 0.5 ng/g. In the lanthanum carbonate group treated with the highest dose, two animals showed higher levels, but were still below 2 ng/g. In addition to lanthanum we also measured some essential elements; i.e. iron, copper, zinc and calcium in the brain. For none of these elements significant differences were found between the different treatment groups (Fig. 7). 4. Discussion This study was performed in order to evaluate the possible accumulation of lanthanum following oral dosing with Phoslock®, lanthanum carbonate or lanthanum chloride, and investigate whether the gastrointestinal absorption properties of these compounds might differ from one another. The highest dose used corresponds to the WCS (‘Worst Case Scenario’) levels of lanthanum related with the use of Phoslock® (17 mg/L), while the lowest dose was intended to correspond to the RLL (‘Reference Lower Limit’, 0.01 mg/L). Our initial testing of the animal diet, however, revealed that this dose was below the daily dietary intake of lanthanum by the animals. Indeed, the regular diet used for the animals contained 0.4 mg lanthanum/g. This corresponds to an intake of lanthanum of 10e12 mg/day per animal from only the food. The WCS dose in this study corresponded to approximately 383 mg lanthanum/day per animal, while the RLL dose would correspond to only 0.25 mg lanthanum/day per animal, well below the daily intake with the food. It was therefore decided to increase the lowest dose to correspond with lake water containing 0.5 mg lanthanum/L. During the course of the study, all animals showed normal weight gain, and no morbidity or mortality was observed over the course of the 10-week treatment. Serum lanthanum levels showed
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Fig. 2. Serum lanthanum levels in the different treatment groups. Dots show data from individual animals while the bars show the mean value for the treatment groups. No statistically significant differences were found between treatment groups, or within one group over time.
Fig. 3. Urinary lanthanum levels in the different treatment groups. Urinary lanthanum excretion (ng/24 h). Dots show data from individual animals, while bars show the mean value for the treatment groups. *: p < 0.05 vs vehicle (same time-point).
no differences, neither between groups nor over time, and generally remained below 0.1 mg/L, even after 10 weeks of exposure. In previous studies [Behets et al., 2004; Bervoets et al., 2006 & 2009] using lanthanum carbonate at doses as high as 1000 mg/kg/day (i.e. 780-fold higher compared to the doses used in the current study), they found plasma lanthanum levels to increase from 0.4 mg/L to 2.5 mg/L, during the first four weeks of treatment after which they stabilized for the remainder of the 12-week treatment period. Compared to the cited papers the doses used in the current study thus are too low to cause a significant response in the serum lanthanum levels.
Although in our study urinary lanthanum excretion showed statistically significant increases in two groups, this is unlikely to be physiologically or scientifically relevant, since the time-points before and after the observed increase show no differences. The most likely explanation for these findings is accidental contamination during collection of the urine samples. When housing rats in metabolic cages for 24 h urine collection, possible minor contamination from faeces cannot be ruled out completely [Damment et al., 2009], especially in the highest dosing groups where lanthanum levels in the faeces can be up to 106 higher than those found in urine. As expected, because of the highly limited gastrointestinal
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Fig. 4. Lanthanum levels in the liver of the different treatment groups. Liver lanthanum content (ng/g wet weight). Dots show data from individual animals, Lines show group median values. *: p < 0.05 vs vehicle group; **: p < 0.01 vs vehicle group.
absorption of lanthanum (<0.0007% in rats) and the fact that less than 2% of the absorbed lanthanum is eliminated by the kidneys [Damment and Pennick 2008] the overall urinary excretion of lanthanum is very low and did not exceed 1 ng/24 h. The minute gastro-intestinally absorbed amounts of lanthanum readily bind to proteins, mainly transferrin, and are cleared from the body through the liver and bile [Damment and Pennick, 2007]. It is therefore not surprising that liver lanthanum levels are increased after lanthanum exposure. Consistent with this, liver lanthanum levels were increased in all three WCS groups as compared to RLL and vehicle groups. To which extent the somewhat, albeit not satistically significantly higher liver lanthanum levels in the WCS lanthanum chloride dose group versus the other groups might have been due to the use of water as vehicle, is difficult to assess and cannot be deduced from the present study. It should be noted however, that carboxymethylcellulose at doses up to 5% is a commonly accepted vehicle for oral dosing in experimental studies evaluating absorption, toxicity and efficacy of therapeutics and other compounds is also used in food industry. Interestingly in the low-dose (RLL) groups, lanthanum levels were indistinguishable from those found in vehicle-treated animals. In all groups, liver lanthanum levels were <10 ng/g wet weight. Although no liver function analysis was performed in the current study, a disturbance of liver function due to lanthanum is unlikely, since in previous studies of our group (using a 780-fold higher dose compared to the WCS dose of the present study), we have shown that liver lanthanum levels plateauing at 1 mg/g wet weight (i.e. 100 to 300-fold higher than those observed in the present study) did not have any effect on liver function parameters [Bervoets et al., 2009]. Another tissue in which some accumulation of lanthanum might be expected is bone. In the current experiment, we found bone lanthanum levels up to 35 ng/g wet weight. Using the 780-fold higher therapeutic doses used in our previous studies, bone lanthanum content reached levels of 0.5e1 mg/g wet weight, without any negative effects on bone metabolism/-histology [Behets et al., 2004 & 2005; Bervoets et al., 2006]. Results thus allow us to conclude that with the concentrations used in the
present study no negative effects on the bone are to be expected. The other tissues analyzed were the heart, spleen, lung, testes and kidney. For all these tissues, lanthanum levels were in the ng/g range and no statistically significant differences between treatment groups, or versus vehicle were found. Given the very low concentrations we encountered in the tissues under study with the doses used, increased risk for harmful effects reasonably should not be a matter of concern. Concerns have been raised as to whether lanthanum might accumulate in the brain. In the present study brain lanthanum levels in general remained below 0.2 ng/g wet weight, and did not differ between treatment groups nor versus vehicle treated animals. In our previous studies, we found brain lanthanum levels to be < 30 ng/g in animals treated with therapeutic doses of 1000 mg/ kg/day of lanthanum carbonate. In these studies, we did not observe gross neurological effects in the animals. In contrast, Feng et al., [2006] have reported neurological effects in rats treated with lanthanum chloride at a dose of 40 mg/kg/day during 6 months. They found brain levels up to 40 ng/g dry weight. A likely explanation for this discrepancy might be contamination issues. Indeed, given the extremely low levels of lanthanum that are absorbed in the gut, tissue lanthanum levels as demonstrated above are extremely low. However, during animal sacrifice, particular care has to be taken to avoid cross contamination of the sampled tissue with the lanthanum content in the gut (which is several orders of magnitude higher) and/or blood. In the present study, very strict procedures were implemented to avoid cross-contamination between tissues or animals. In their study, Feng et al., [2006], using synchrotron radiation X-ray fluorescence analysis (SRXRF), also reported a significant decrease in calcium, iron and zinc levels in the brain of animals treated with lanthanum chloride. Therefore, we also measured the concentration of these elements in the brain tissue using quantitative electrothermal atomic absorption spectrometry, however, found no differences between treatment groups and/or vehicle. The reason for the results to be conflicting between both studies is not clear and difficult to explain. Given the fact that calcium, iron and zinc levels in the brain, respectively are up to 83.300, 6.000 and 7.100 times higher than the brain lanthanum levels measured in rats of the present study receiving the WCS dose, it is hard to believe that such low lanthanum concentrations would be able to deplete the brain from the above mentioned essential elements. As discussed below administration of lanthanum chloride at doses used in the Feng et al., [2006] study might theoretically induce hyperchloremic acidosis. To which extent this condition may be responsible for the disturbed concentration of the above mentioned elements in the brain cannot be answered at this moment. The primary objective of this study was to investigate whether there was any difference in gastrointestinal absorption and/or tissue distribution of lanthanum after dosing as the chloride salt, the carbonate salt or the LMB compound. When comparing the same dosage groups of the different compounds, no differences in tissue distribution of lanthanum were seen. This is not unexpected, since the only factor that differs when ingesting different lanthanum salts is the counterion (in this case, the carbonate or chloride ions). In the acid environment of the upper gastrointestinal tract, the soluble lanthanum salts (like chloride and nitrate) but also the less soluble carbonates, dissociate into their respective ionic forms. Once dissociated, lanthanum ions readily bind to any available phosphate to form insoluble lanthanum phosphate within the lumen of the gut after which it is removed through fecal excretion. It is worth noting that in the gut, phosphate concentrations are several orders of magnitude higher than those of lanthanum. In a regular rat diet, the phosphorus content is approximately 0.70%, corresponding to an average daily intake of 200 mg, or 7 mmol of
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Fig. 5. Lanthanum levels in the heart, spleen, lung, kidney, testes and bone (ng/g wet weight) of the different treatment groups. No statistically significant differences were found between treatment groups and vehicle.
phosphate. In humans, average phosphate intake is approximately 14 mmol/day. In contrast, the lanthanum intake in the highest dose groups of the present study was 380 mg or 2.7 mmol/day. Theoretically, this implies complete binding of lanthanum to phosphate within the gastrointestinal tract, thus leaving an almost negligible amount of free lanthanum available for absorption.
Discussion is ongoing as to whether, independent of suggested differences in gastrointestinal absorption, ingestion of lanthanum chloride might be more toxic than lanthanum carbonate - in particular at the level of the brain. Indeed, in experimental studies in which lanthanum chloride was administered daily via the drinking water to rats at doses up to 0.5% of lanthanum (i.e.
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Fig. 6. Brain lanthanum levels (ng/g wet weight) of the different treatment groups. No statistically significant differences were found between treatment groups or vehicle.
20.4 mmol/L lanthanum chloride [Hu et al., 2018], corresponding to 2.8 g/L of lanthanum (i.e. 1200 times the highest levels observed in Phoslock® treated water and 165 times the highest dose in the present study) neurological effects as assessed by measurement of some biochemical parameters and behavior studies have been reported [Feng et al., 2006; He et al., 2008; Zheng et al., 2013; Jin et al., 2017]. It must be noted however, that with these doses the chloride concentration in the drinking water is as high as 2.2 g/L. Chloride ions are absorbed from the gastrointestinal tract through anion exchangers, whereby bicarbonate is released. Since the CO2/bicarbonate equilibrium is the main pH buffering system in the body, excess chloride intake can result in low plasma bicarbonate levels, representing metabolic acidosis (Seifter and Chang, 2016). This was observed in patients treated with the phosphate binding agent, Sevelamer hydrochloride. During therapeutic use of this compound, a dose-dependent decrease in bicarbonate levels due to the chlorine load resulting in metabolic acidosis was repeatedly reported (De Santo et al., 2006; Oka et al., 2008 & 2014). Interestingly, this was no longer seen when the formulation was changed to Sevelamer carbonate. This thus indicates that the counterion (i.e. chloride), rather than the active compound itself, was responsible for the observed acidosis. Although no differences in serum pH or bicarbonate levels were observed in the present study at the doses
Fig. 7. Iron (Fe), copper (Cu), zinc (Zn) and calcium (Ca) content in the different treatment groups. No statistically significant differences were found between treatment groups or vehicle for the respective elements under study.
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used, development of acidosis might reasonably have occurred in the above mentioned experimental studies in which rats were exposed to 165 times higher lanthanum chloride concentrations. To the best of our knowledge, pH and/or metabolic acidosis were not measured in the above mentioned studies (Hu et al., 2018; Feng et al., 2006; He et al., 2008; Zheng et al., 2013; Jin et al., 2017) and the causal role of increased chloride levels and acidosis is therefore difficult to prove. Nevertheless, it should be noted that studies have shown that a low pH is deleterious for the cerebral microenvironment whilst temporary pH changes could be responsible for cerebrovascular damage and cholinergic cell death [Pirchl et al., 2006]. In the present study dosing was based in relation to the use of LMB as a solid-phase phosphorus sorbent material to manage eutrophication and prevent cyanobacteria blooms by controlling the internal phosphorus load in aquatic systems (Mucci et al., 2018). In order to evaluate the safety margin and the potential risks for humans related to the treatment of waterbodies with LMB in the present study a worst case scenario (WCS) was adopted based on the highest LMB application rate reported in the literature (Spears et al., 2013a, 2013b). Given (i) the lanthanum concentration measured in surface waters as either filterable, total or predicted ‘free’ (uncomplexed) ionic lanthanum following the highest LMB application rate (Spears et al., 2013a, 2013b), (ii) the duration and magnitude of exposure through the use of lanthanum-treated drinking water, consumption of biota (fish, crustaceans, plants), dermal exposure via water or sediment or through the inadvertent consumption of sediments particularly in young children, (iii) the ability of ‘free’ lanthanum to directly bind phosphate and other oxyanions in the intestine resulting in a low bioavailability and, (iv) the absence of significant toxic effects when used therapeutically during years as a phosphate binding agent in patients with chronic kidney disease at daily doses which are up to three orders of magnitude higher than the WCS dose applied in the present study (Hutchison et al., 2016), one may reasonably accept that exposure to lanthanum via the drinking water or leisure activities will pose no increased risk for toxicity in humans. Although out of the scope of the present study should dermal contact to LMB treated water be acknowledged as another important exposure pathway. The European Food Safety Authority (EFSA) reports for bentonite the absence of irritation and sensitization properties (EFSA, 2012). A weak sensitization potential for lanthanum chloride hexahydrate (LaCl3.(H20)6) has been reported by the European Chemicals Agency (ECHA, 2013). Sensitization potential is, however, reported for a higher percentage compared to the assumed 5% lanthanum content in LMB (van Oosterhout & Lürling, 2013) and therefore we consider that lanthanum would not be sensitizing at the level presented in the LMB, the more since no evidence is provided so far for lanthanum to be present herein as lanthanum chloride hexahydrate. Overall, weighing off the potential severe health effects to humans and animals of cyanobacteria blooms and accumulation of cyano-toxins in water used for recreational activities, irrigation and drinking water supply (Chorus et al., 2000; Carmichael, 2001;; World Health Organization, 2003; Lürling and Faassen, 2013; Zamyadi et al., 2012, 2013) versus the negligible risk for any toxic effect of LMB derived lanthanum the use of LMB should be considered a powerful safe tool to prevent the serious health risks linked to cyanobacteria. 5. Conclusion In conclusion, accumulation of lanthanum in rats after dosing the WCS dose of Phoslock®, lanthanum carbonate or lanthanum chloride was limited to a slight increase in liver lanthanum to levels
9
at which it has previously been demonstrated that no harmful effects are to be expected. Other tissues (including the brain) did not show any accumulation compared to vehicle treatment. There was no difference in accumulation between Phoslock®, lanthanum carbonate and lanthanum chloride, suggesting that gastrointestinal uptake of lanthanum is similar for all these compounds at the doses administered in the present study. In view of the long-term experience with lanthanum carbonate for phosphate binding treatment and the absence of any reported negative effects due to accumulation of lanthanum at levels being several orders of magnitude higher than those observed in the present study, the potential, minimal uptake of lanthanum through exposure to Phoslock®treated water is unlikely to pose a health risk to animals and/or humans. CRediT authorship contribution statement Geert J. Behets: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing original draft. Kayawe Valentine Mubiana: Formal analysis, Methodology. Ludwig Lamberts: Formal analysis, Methodology. Karin Finsterle: Conceptualization, Resources, Writing - review & editing. Nigel Traill: Conceptualization, Resources, Writing - review & editing. Patrick C. D'Haese: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing - original draft, Writing - review & editing. Acknowledgements This study was supported by a grant from Phoslock® Environmental Technologies Ltd, United Kingdom/Australia. NT and KF are employees of Phoslock® Environmental Technologies Ltd, United Kingdom/Australia. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.124780. References Behets, G.J., Dams, G., Vercauteren, S.R., Damment, S.J., Bouillon, R., De Broe, M.E., D'Haese, P.C., 2004. Does the phosphate binder lanthanum carbonate affect bone in rats with chronic renal failure? J. Am. Soc. Nephrol. 5 (8), 2219e2228. Behets, G.J., Verberckmoes, S.C., Oste, L., Bervoets, A.R., Salome, M., Cox, A.G., Denton, J., De Broe, M.E., D'Haese, P.C., 2005. Localization of lanthanum in bone of chronic renal failure rats after oral dosing with lanthanum carbonate. Kidney Int. 67 (5), 1830e1836. Bervoets, A.R., Oste, L., Behets, G.J., Dams, G., Blust, R., Marynissen, R., Geryl, H., De Broe, M.E., D'Haese, P.C., 2006. Development and reversibility of impaired mineralization associated with lanthanum carbonate treatment in chronic renal failure rats. Bone 38 (6), 803e810. Bervoets, A.R., Behets, G.J., Schryvers, D., Roels, F., Yang, Z., Verberckmoes, S.C., Damment, S.J., Dauwe, S., Mubiana, V.K., Blust, R., De Broe, M.E., D'Haese, P.C., 2009. Hepatocellular transport and gastrointestinal absorption of lanthanum in chronic renal failure. Kidney Int. 75 (4), 389e398. Bouma-Gregsona, K., Powera, M.E., Bormans, M., 2017. Rise and fall of toxic benthic freshwater cyanobacteria (Anabaena spp.) in the Eel river: buoyancy and dispersal. Harmful Algae 66, 79e87, 2017. https://doi.org/10.1016/j.hal.2017.05. 007. Carmichael, W.W., 2001. Health effects of toxin-producing cyanobacteria: “the CyanoHABs”. Hum. Ecol. Risk Assess. 7 (5), 1393e1407. Chorus, I., Falconer, I.R., Salas, H.J., Bartram, J., 2000. Health risks caused by freshwater cyanobacteria in recreational waters. J. Toxicol. Environ. Health Part B. 3 (4), 323e347. Copetti, D., Finsterle, K., Marziali, L., Stefani, F., Tartari, G., Douglas, G., Reitzel, K., Spears, B.M., Winfield, I.J., Crosa, G., D'Haese, P., Yasseri, S., Lürling, M., 2016. Eutrophication management in surface waters using lanthanum modified bentonite: a review. Water Res. 15 (97), 162e174. Review. D'Haese, P.C., Couttenye, M.M., Lamberts, L.V., Elseviers, M.M., Goodman, W.G., Schrooten, I., Cabrera, W.E., De Broe, M.E., 1999. Aluminum, iron, lead, cadmium,
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copper, zinc, chromium, magnesium, strontium, and calcium content in bone of end-stage renal failure patients. Clin. Chem. 45 (9), 1548e1556. D'Haese, P.C., Douglas, G., Verhulst, A., Neven, E., Behets, G.J., Vervaet, B.A., Finsterle, K., Lürling, M., Spears, B., 2019. Human health risk associated with the management of phosphorus in freshwaters using lanthanum and aluminium. Chemosphere 220, 286e299. Damment, S.J., Pennick, M., 2007. Systemic lanthanum is excreted in the bile of rats. Toxicol. Lett. 171 (1e2), 69-7. Damment, S.J., Pennick, M., 2008. Clinical pharmacokinetics of the phosphate binder lanthanum carbonate. Clin. Pharmacokinet. 47 (9), 553e563. Damment, S.J., Cox, A.G., Secker, R., 2009. Dietary administration in rodent studies distorts the tissue deposition profile of lanthanum carbonate; brain deposition is a contamination artefact? Toxicol. Lett. 188 (3), 223e229. De Santo, N.G., Frangiosa, A., Anastasio, P., Marino, A., Correale, G., Perna, A., Di Stazio, E., Stellato, D., Santoro, D., Di Meglio, E., Iacono, G., Ciacci, C., Savica, V., Cirillo, M., 2006. Sevelamer worsens metabolic acidosis in hemodialysis patients. J Nephrol Suppl 9, S108eS114. Dithmer, L., Lipton, A.S., Reitzel, K., Warner, T.E., Lundberg, D., Nielsen, U.G., 2015. Characterization of phosphate sequestration by a lanthanum modified bentonite clay: a solidstate NMR, EXAFS, and PXRD study. Environ. Sci. Technol. 49, 4559e4566. Dithmer, L., Nielsen, U.G., Lürling, M., Spears, B.M., Yasseri, S., Lundberg, D., Moore, A., Jensen, N.D., Reitzel, K., 2016. Responses in sediment phosphorus and lanthanum concentrations and composition across 10 lakes following applications of lanthanum modified bentonite. Water Res. 97, 101e110. Douglas, G.B., 2002. Remediation Material and Remediation Process for Sediments. US Patent, p. 6350383. ECHA, 2013. Lanthanum chloride, anhydrous [Skin sensitization]. In: Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACh) [Database]. European Chemicals Agency (ECHA), Helsinki, Finland. http://echa.europa.eu/ registration-dossier/-/registered-dossier/14555/7/5/2. EFSA, 2012. Scientific opinion on the safety and efficacy of bentonite as a technological feed additive for all species. EFSA J. 10 (7), 2787. Egemose, S., Reitzel, K., Andersen, F.O., Flindt, M.R., 2010. Chemical lake restoration products: sediment stability and phosphorus dynamics. Environ. Sci. Technol. 44, 985e991. https://doi.org/10.1021/es903260y. Feng, L., Xiao, H., He, X., Li, Z., Li, F., Liu, N., Zhao, Y., Huang, Y., Zhang, Z., Chai, Z., 2006. Neurotoxicological consequence of long-term exposure to lanthanum. Toxicol. Lett. 165 (2), 112e120. €chter, R., Müller, B., 2003. Why the phosphorus retention of lakes does not Ga necessarily depend on the oxygen supply to their sediment surface. Limnol. Oceanogr. 48, 929e933. Haghseresht, F., Wang, S.B., Do, D.D., 2009. A novel lanthanum modified bentonite, Phoslock®, for phosphate removal from wastewaters. Appl. Clay Sci. 46 (4), 369e375. He, X., Zhang, Z., Zhang, H., Zhao, Y., Chai, Z., 2008. Neurotoxicological evaluation of long-term lanthanum chloride exposure in rats. Toxicol. Sci. 103 (2), 354e361. Hu, X., Yang, J., Sun, Y., Gao, X., Zhang, L., Li, Y., Yu, M., Liu, S., Lu, X., Jin, C., Wu, S., Cai, Y., 2018. Lanthanum chloride impairs memory in rats by disturbing the glutamate-glutamine cycle and over-activating NMDA receptors. Food Chem. Toxicol. 113, 1e13. Hutchison, A.J., Wilson, R.J., Garafola, S., Copley, J.B., 2016. Lanthanum carbonate: safety data after 10 years. Nephrology 21 (12), 987e994. Hutchison, A., Whelton, A., Thadhani, R., Achenbach, H., Vergani, A., Wu, J., Hall, G., 2018. Long-term mortality and bone safety in patients with end-stage renal disease receiving lanthanum carbonate. Nephron 23, 1e10. Jin, C., Gao, L., Li, Y., Wu, S., Lu, X., Yang, J., Cai, Y., 2017. Lanthanum damages learning and memory and suppresses astrocyte-neuron lactate shuttle in rat hippocampus. Exp. Brain Res. 235 (12), 3817e3832. La Van, T., Cooke, S.J., 2011. Advancing the science and practice of fish kill investigations. Rev. Fish. Sci. 19 (1), 21e33. https://doi.org/10.1080/10641262. 2010.531793. Liu, X., Byrne, R.H., 1997. Rare earth and yttrium phosphate solubilities in aqueous solution. Geochem. Cosmochim. Acta 61 (8), 1625e1633. Lürling, M., Faassen, E.J., 2013. Dog poisonings associated with a microcystis aeruginosa bloom in The Netherlands. Toxins 5, 556e567. Lürling, M., Mackay, E., Reitzel, K., Spears, B.M., 2016. Editorial - a critical perspective on geo-engineering for eutrophication management in lakes. Water Res. 15 (97), 1e10. Editorial. Lürling, M., Tolman, Y., 2010. Effects of lanthanum and lanthanum-modified clay on growth, survival and reproduction of Daphnia magna. Water Res. 44 (1), 309e319. Matzinger, A., Müller, B., Niederhauser, P., Schmid, M., Wüst, A., 2010. Hypolimnetic oxygen consumption by sediment-based reduced substances in former eutrophic lakes. Limnol. Oceanogr. 55 (5), 2073e2084. https://doi.org/10.4319/ lo.2010.55.5.2073. Meis, S., Spears, B.M., Maberly, S.C., O'Malley, M.B., Perkins, R.G., 2012. Sediment amendment with Phoslock® in Clatto Reservoir (Dundee, UK): investigating changes in sediment elemental composition and phosphorus fractionation. J. Environ. Manag. 93 (1), 185e193. Mucci, M., Maliaka, V., Noyma, N.P., Marinho, M., Lürling, M., 2018. Assessment of possible solid-phase phosphate sorbents to mitigate eutrophication: influence
of pH and anoxia. Sci. Total Environ. 619e620, 1431e1440. Oka, Y., Miyazaki, M., Takatsu, S., Oohara, T., Toda, K., Uno, F., Matsuda, H., 2008. A review article: sevelamer hydrochloride and metabolic acidosis in dialysis patients. Cardiovasc. Haematol. Disord. - Drug Targets 8 (4), 283e286. Oka, Y., Miyazaki, M., Matsuda, H., Takatsu, S., Katsube, R., Mori, T., Takehara, K., Umeda, Y., Uno, F., 2014. Sevelamer hydrochloride dose-dependent increase in prevalence of severe acidosis in hemodialysis patients: analysis of nationwide statistical survey in Japan. Ther. Apher. Dial. 18 (1), 37e43. Pennick, M., Dennis, K., Damment, S.J., 2006. Absolute bioavailability and disposition of lanthanum in healthy human subjects administered lanthanum carbonate. J. Clin. Pharmacol. 46 (7), 738e746. Persy, V.P., Behets, G.J., Bervoets, A.R., De Broe, M.E., D'Haese, P.C., 2006. Lanthanum: a safe phosphate binder. Semin. Dial. 19 (3), 195e199. Review. Persy, V.P., Behets, G.J., De Broe, M.E., D'Haese, P.C., 2009. Management of hyperphosphatemia in patients with end-stage renal disease: focus on lanthanum carbonate. Int. J. Nephrol. Renovascular Dis. 2, 1e8. Pirchl, M., Marksteiner, J., Humpel, C., 2006. Effects of acidosis on brain capillary endothelial cells and cholinergic neurons: relevance to vascular dementia and Alzheimer's disease. Neurol. Res. 28 (6), 657e664. Reitzel, K., Jenses, H.S., Egemose, S., 2013. pH dependent dissolution of sediment aluminium in six Danish lakes treated with aluminium. Water Res. 47, 1409e1420. Reitzel, K., Balslev, K.A., Jensen, H.S., 2017. The influence of lake water alkalinity and humic substances on particle dispersion and lanthanum desorption from a lanthanum modified bentonite. Water Res. 125, 191e200. Robb, M., Greenop, B., Goss, Z., Douglas, G., Adeney, J., 2003. Application of PhoslockTM, an innovative phosphorus binding clay, to two Western Australian waterways: preliminary findings. Hydrobiologia 494 (1), 237e243. Ross, G., Haghseresht, F., Cloete, T.E., 2008. The effect of pH and anoxia on the performance of Phoslock®, a phosphorus binding clay. Harmful Algae 7 (4), 545e550. Salmaso, N., Capelli, C., Shams, S., Cerasino, L., 2015. Expansion of bloom-forming Dolichospermum lemmermannii (Nostocales, Cyanobacteria) to the deep lakes south of the Alps: colonization patterns, driving forces and implications for water use. Harmful Algae 50, 76e87. https://doi.org/10.1016/j.hal.2015.09. 008. Schindler, D.W., Carpenter, S.R., Chapra, S.C., Hecky, R.E., Orihel, D.M., 2016. Reducing phosphorus to curb lake eutrophication is a success. Environ. Sci. Technol. 50, 8923e8929. Seifter, J.L., Chang, H.Y., 2016. Disorders of acid-base balance: new perspectives. Kidney Dis. 2 (4), 170e186 (Review). Small, K., Kopf, R.K., Watts, R.J., Howitt, J., 2014. Hypoxia, blackwater and fish kills: experimental lethal oxygen thresholds in juvenile predatory lowland river fishes. PLoS One 9 (4), e94524. https://doi.org/10.1371/journal.pone.0094524. Spears, B.M., Meis, S., Anderson, A., Kellou, M., 2013a. Comparison of phosphorus (P) removal properties of materials proposed for the control of sediment P release in UK lakes. Sci. Total Environ. 442, 103e110. https://doi.org/10.1016/j.scitotenv. 2012.09.066. Spears, B.M., Lürling, M., Yasseri, S., Castro-Castellon, A.T., Gibbs, M., Meis, S., McDonald, C., McIntosh, J., Sleep, D., Van Oosterhout, F., 2013b. Lake responses following lanthanum-modified bentonite clay (Phoslock®) application: an analysis of water column lanthanum data from 16 case study lakes. Water Res. 47 (15), 5930e5942. Spears, B.M., Maberly, S.C., Pan, G., Mackay, E., Bruere, A., Corker, N., Douglas, G., Egemose, S., Hamilton, D., Hatton-Ellis, T., Huser, B., Li, W., Meis, S., Moss, B., Lürling, M., Phillips, G., Yasseri, S., Reitzel, K., 2014. Geo-engineering in lakes: a crisis of confidence? Environ. Sci. Technol. 48, 9977e9979. Tonelli, M., Pannu, N., Manns, B., 2010. Oral phosphate binders in patients with kidney failure. N. Engl. J. Med. 362 (14), 1312e1324. Van Oosterhout, F., Lürling, M., 2011. Effects of the novel ‘flock & lock’ lake restoration technique on Daphnia in Lake Rauwbraken (The Netherlands). J. Plankton Res. 33 (2), 255e263. van Oosterhout, F., Lürling, M., 2013. The effect of phosphorus binding clay (Phoslock®) in mitigating cyanobacterial nuisance: a laboratory study on the effects on water quality variables and plankton. Hydrobiologia 710, 265e277. Welch, E.B., Cooke, G.D., 2005. Internal phosphorus loading in shallow lakes: importance and control. Lake Reservoir Manag. 21, 209e17. World Health Organization, 2003. Guidelines for safe recreational water environments. In: Coastal and Fresh Waters, ume 1, p. 253. https://www.who.int/ water_sanitation_health/publications/srwe1/en/. Zamparas, M., Zacharias, I., 2014. Restoration of eutrophic freshwater by managing internal nutrient loads. 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Use of lanthanum for water treatment A matter of concern? Geert J. Behets a, Kayawe Valentine Mubiana b, Ludwig Lamberts a, Karin Finsterle c, Nigel Traill d, Ronny Blust b, Patrick C. D'Haese a, * a
Laboratory of Pahophysiology, Department Biomedical Sciences, University of Antwerp, Belgium Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Belgium c Limno Solutions International, Australia d Phoslock® Environmental Technologies Ltd, Australia b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Lanthanum uptake after exposure to lanthanum modified bentonite (LMB) treated water is marginal. Gastrointestinal absorption of lanthanum (La) does not depend on its chemical composition. The slightly increased liver La levels in a worst case scenario do not hold a risk for hepatotoxicity. Exposure to LMB treated water should not be considered a risk to animal and/or human health. As for its therapeutic use, La is considered a safe tool in surface water eutrophication management.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2019 Received in revised form 8 August 2019 Accepted 4 September 2019 Available online 6 September 2019
Among several other eutrophication management tools, Phoslock®, a lanthanum modified bentonite (LMB) clay, is now frequently used. Concerns have been raised as to whether exposure to Phoslock®treated water may lead to lanthanum accumulation/toxicity in both animals and humans. In the present experimental study, rats were administered lanthanum orally as either lanthanum carbonate, lanthanum chloride or Phoslock® at doses of either 0.5 or 17 mg/L during 10 weeks. Controls received vehicle. The gastrointestinal absorption and tissue distribution of lanthanum was investigated. Extremely strict measures were implemented to avoid cross-contamination between different tissues or animals. Results showed no differences in gastrointestinal absorption between the different compounds under study as reflected by the serum lanthanum levels and concentrations found in the brain, bone, heart, spleen, lung, kidney and testes. At sacrifice, significant but equally increased lanthanum concentrations versus vehicle were observed in the liver for the highest dose of each compound which however, remained several orders of magnitude below the liver lanthanum concentration previously measured after long-term therapeutic administration of lanthanum carbonate and for which no hepatotoxicity was noticed in humans. In conclusion, (i) the use of LMB does not pose a toxicity risk (ii) gastrointestinal absorption of lanthanum is minimal and independent on the type of the compound, (iii) with exception of the liver, no significant increase in lanthanum levels is observed in the various organs under study, (iv) based on
Handling Editor: Martine Leermakers
* Corresponding author. University of Antwerp Universiteitsplein 1, B-2610, Wilrijk, Belgium. E-mail address: [email protected] (P.C. D'Haese). https://doi.org/10.1016/j.chemosphere.2019.124780 0045-6535/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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previous studies, the slightly increased liver lanthanum levels observed in a worst case scenario do not hold any risk of hepatotoxicity. © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Eutrophication is considered the main water quality issue worldwide. The enrichment of surface waters with nutrients, mainly nitrogen (N) and phosphorus (P) negatively affect the multiple functions and services of freshwater ecosystems by accelerating the primary production and increasing the risk of nuisance cyanobacteria blooms [Lürling et al., 2016; Schindler et al., 2016]. The consequences of such blooms are surface scums and accumulation of cyanobacteria-toxins in the water which presents a severe threat to the ecosystem itself, pets and human health [Chorus et al., 2000; Carmichael 2001; World Health Organization, 2003; Lürling and Faassen, 2013; Salmaso et al., 2015; BoumaGregsona et al., 2017]. Subsequently, the decomposition of increased amounts of organic matter leads to oxygen depletion and a marked decline of the redox potential especially in the hypolimnion and close to the sediment. A complex interaction between biogeochemical processes coupled with low oxygen concentrations or anoxia, induces the release of legacy phosphorus and toxic €chter substances from the sediment and can provoke fish kills. [Ga and Müller 2003; Matzinger et al., 2010; La Van and Cooke, 2011; Small et al., 2014]. Notwithstanding extensive external load management, the recovery of lentic freshwater ecosystems is often delayed due to the internal phosphorus load. In lake management, actions using specific materials to counteract persistent eutrophication by targeting particularly the internal phosphorus load are described as geoengineering [Spears et al., 2014; Lürling et al., 2016]. Whilst in the past aluminum sulphate (Alum, Al2(SO4)3) was most frequently applied to inactivate the internal phosphorus pool [Welch and Cooke, 2005], the use of the lanthanum (La) modified bentonite (LMB, Phoslock®), currently is rapidly emerging as an effective eutrophication management tool to prevent blue-green algae blooms by controlling the internal phosphorus load (Copetti et al., 2016). Compared to Alum, Phoslock® is far less susceptible to resuspension than alum flocs, the formed lanthanum-phosphate bound is stable over a wide pH-range and its use does not affect the pH of the water [Ross et al., 2008; Egemose et al., 2010; Spears et al., 2013a, 2013b]. Hence, the risk due to the presence of La3þ ions in LMB treated lake water is much lower compared to the risk related to the formation of soluble aluminum species after an alum application [Reitzel et al., 2013; Zamparas and Zacharias, 2014; Lürling et al., 2016, D'Haese et al., 2019]. Phoslock® is a lanthanum (La) modified bentonite clay designed by the Commonwealth Scientific and Industrial Research Organization (CSIRO) in the 1990s for the control of oxyanions, especially phosphate, in water (Douglas, 2002). Phoslock® is commonly applied from a barge, as a slurry, where it acts to strip dissolved phosphorus en route through the water column. Once settled onto the sediment surface, the product can enhance the capacity of lake bed sediments to retain phosphorus by forming LaPO4.(H2O), a particulate, natural occurring mineral known as rhabdophane. Due to its very low solubility (Ksp 1024.7 to 1025.7 mol2l2) LaPO4.(H2O) is not available to phytoplankton, and is stable under a wide range of environmental conditions, commonly reported in eutrophic lakes [Liu and Byrne 1997; Robb et al., 2003; Haghseresht et al., 2009; Ross et al., 2008; Meis
et al., 2012]. Interestingly, before putting possible increased exposure to lanthanum in perspective it is worth mentioning that a pharmaceutical compound based on lanthanum carbonate (La2(CO3)3; Fosrenol®), was developed by Shire Pharmaceuticals for use in patients with chronic renal failure. Due to the loss of renal function, these patients accumulate phosphate. Dietary restrictions can alleviate this only modestly, thus additional treatment is necessary, usually in the form of phosphate binding agents. These phosphate binders, which are taken orally (several grams/day) with the meals, bind phosphate in the gut, thus rendering it unavailable for absorption in the gastrointestinal (GI) tract [Persy et al., 2006; Persy et al., 2009; Tonelli et al., 2010]. Related to the utilization of Phoslock® the common operational assumptions are (1) lanthanum is not released from the bentonite carrier under natural environmental conditions occurring in lakes and (2) the formation of LaPO4 is the dominant mechanism of phosphate removal and phosphorus retention in Phoslock® treated lakes [Dithmer et al., 2015, 2016; Copetti et al., 2016]. However, there is still some concern that small amounts of lanthanum may be liberated from the carrier as filterable lanthanum (FLa), in particular in low alkalinity water, and be present in the water in either its ionic state (La3þ) or as small particulates [Lürling & Tolman, 2010; Van Oosterhout & Lürling, 2011; Reitzel et al., 2017]. This would, in turn, expose consumers of Phoslock®-treated drinking water to a potential risk related to lanthanum accumulation/toxicity. The production process of Phoslock® fulfils strict international standards (NSF/ANSI 60). However, due to the occurrence of small amounts of FLa species in LMB treated water it is possible that the gastrointestinal absorption, tissue distribution/accumulation and potential toxicity properties of Phoslock® derived lanthanum might resemble those of LaCl3 more closely than those of the wellstudied and therapeutically used phosphate binding agent lanthanum carbonate (Fosrenol®). Although gastrointestinal absorption of lanthanum after oral administration of lanthanum carbonate was demonstrated to be extremely low (0.00127±0.00080% in humans) and therapeutic use of this compound has proven to be safe when administered at doses up to 1e3 g/day during several years [Pennick et al., 2006; Damment and Pennick 2008; Hutchison et al., 2018], health authorities have questioned to which extent fractional intestinal absorption of lanthanum after exposure to lanthanum chloride or Phoslock® derived lanthanum may be higher than that of lanthanum carbonate. It has indeed been argued that because of its higher solubility constant intestinal absorption of lanthanum chloride might be higher as compared to less soluble lanthanum compounds such e.g. lanthanum carbonate. This might be true when lanthanum should be present at extremely high concentrations; i.e. those exceeding equimolar concentrations of phosphate and/or other oxyanions (e.g. oxalate) in the intestine which are well known strong binding ligands for lanthanum. However, given the highest lanthanum concentrations ever observed in Phoslock® treated water one may reasonably hypothesize that in view of the very high phosphate-oxyanion/lanthanum ratio intestinal binding of lanthanum to these ligands will occur at the same extent for the different lanthanum compounds under study leaving no ionic lanthanum available for intestinal absorption which in turn will dramatically limit gastrointestinal absorption of lanthanum after
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exposure to either of the above mentioned lanthanum-containing compounds to the same extent. As data on this topic in the literature are highly limited, further evidence for this has to be provided from experimental studies. The aim of the current study was to obtain insight into the gastrointestinal absorption, tissue distribution/accumulation of lanthanum after oral gavage of either lanthanum chloride, lanthanum carbonate or LMB at doses which for each compound corresponded to 17 and 0.5 mg elemental lanthanum/L respectively. 2. Materials and methods Experimental procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals 85-23 (1996) and were approved by the University of Antwerp Ethics Committee (Approval number 2016e22). 2.1. Animals and study set-up Animals were housed two per cage for the duration of the study in a temperature-controlled (25 C) room with a strict 12 h light/ dark cycle at the central animal facility of the University of Antwerp. Food and water consumption were recorded once a week, and animals were weighed weekly. The animals were inspected daily for morbidity. Animals had free access to a standard rodent diet (SSNIFF Spezialdi€ aten, Soest, Germany) and tap water (pH 7.8). Study set up (Fig. 1). During a 2-week equilibration period, rats were adapted to the diet. Treatment by oral gavage was then started for a 10-week period. A constant dose volume of 10 mL/kg was used and the volume was individually adjusted weekly, according to the most recent body weight. For dose calculations, it was assumed that healthy animals drink approximately 75 mL/kg/day, and it was assumed that Phoslock® contained 5% lanthanum (van Oosterhout and Lürling, 2013). Fifty six male (56) Wistar rats (225e250 g body weight at start of the study, Charles River, France) were randomly divided into 7 study groups consisting of 8 animals each, and were exposed to (i) vehicle, (ii) 2 doses of lanthanum chloride, (iii) 2 doses of lanthanum carbonate and (iv) 2 doses of LMB, respectively. 2.2. Dosing With regard to the choice of the dosing, it was aimed that rats should be exposed to either lanthanum chloride, lanthanum carbonate and LMB at doses corresponding to the worst case scenario
Fig. 1. Study scheme. WCS: Worst case Scenario, corresponding to 17 mg/L of lanthanum; RLL: Reference Lower Limit, corresponding to 0.5 mg/L lanthanum.
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(WCS; 17 mg/L) and the reference lower limit (RLL; 0.5 mg/L). The WCS is a hypothetical worst case situation and is based on the highest Phoslock® dose ever applied to a lake [Spears et al., 2013a, 2013b] and assuming that all lanthanum contained within Phoslock® would be released from the bentonite matrix and would be present as ionic lanthanum. Initially it was intended to set the RLL at 0.01 mg lanthanum/L which refers to the lower range of filterable lanthanum concentrations measured after a Phoslock® application. Prior testing however, showed that the food of the animals contained up to 0.4 mg of lanthanum/g dry weight. Since healthy animals eat approximately 25e30 g/day, this would correspond to a lanthanum intake of 10e12 mg/day from the food. The planned RLL dose thus would have added only 0.25 mg lanthanum to the daily dietary intake. It was therefore decided to increase the low dose to 0.5 mg lanthanum/L, corresponding to an additional daily intake of approximately 11 mg. All dosing stock solutions were freshly prepared every week. Appropriate dilutions to administer to the animals were made daily. Lanthanum carbonate and Phoslock® were suspended in 1% carboxymethylcellulose. Lanthanum chloride was dissolved in tap water, since it forms cloudy precipitates in carboxymethylcellulose which might affect gastrointestinal absorption. The vehicle group received 1% carboxymethylcellulose only, while an additional 4 animals, which were gavaged with tap water were also included. The lanthanum content in the dosing solutions of the different lanthanum compounds was measured before the start of the experiment to check whether it effectively corresponded to the nominal dose which theoretically was 127.5 mg/L. We found concentrations of 122.9, 99.8 and 80.6 mg/L for lanthanum chloride, lanthanum carbonate and Phoslock® respectively. Differences most probably were due to the degree of hydration of lanthanum carbonate in particular, or the amount at which lanthanum was present in Phoslock® which was assumed to be 5%. Based on these concentrations appropriate correction was made to obtain the nominal doses. We also measured the lanthanum concentration in the water and in the 1% carboxymethylcellulose used for gavage which was as low as 0.26 mg/L and 0.18 mg/L and thus negligible. To avoid cross-contamination during dosing, the following sequence was used: vehicle groups, low dose groups and lastly the high dose groups.
2.3. Sampling At weeks 0, 4, 7 and 10, animals were housed individually in a metabolic cage for 24 h to collect urine samples followed by blood sampling. The urinary volume was recorded and the samples divided in 2 fractions and frozen at 20 C pending analysis. Blood was drawn from the tail vein in restrained, conscious animals. A first few drops of blood were collected for immediate analysis using the i-Stat Point of Care technology (Abbott) for measurement of blood pH and bicarbonate content. Afterwards, a 1.5 mL blood sample was collected and allowed to clot on ice (approximately 2e3 h). After centrifugation, serum was divided in aliquots and stored at 80 C until further analysis. After 10 weeks of dosing, animals were sacrificed by exsanguination through the retro-orbital plexus after anesthesia with Animale, 60 mg/kg sodium pentobarbital (Nembutal, Ceva Sante France) via intraperitoneal injection. In addition to blood and urine, the following tissues were taken: brain, liver, spleen, lungs, heart, kidney, testes and bone. Tissue samples were immediately wet weighed and stored at 80 C in plastic containers pending further analysis. During sacrifice, care was taken to avoid crosscontamination between tissues and animals. The same sequence as for dosing was used (i.e. firstly vehicle groups, then low dose
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groups and lastly the high dose groups). Similarly, tissues were removed in the same order in all animals (brain, testes, kidney, spleen, bone, heart, liver, lungs). All non-disposable materials were thoroughly cleaned between animals. All materials used for tissue collection, blood and urine sampling were checked in advance for possible lanthanum contamination by soaking them for 24 h in demineralized water and measuring lanthanum content. No lanthanum was detectable in any of the disposables used.
0.6 mg/L in the lanthanum carbonate group at week 7), but group averages remained below 0.2 mg/L in all treatment groups, versus 0.1 mg/L in the vehicle group (Fig. 2). An increased variability in the urinary lanthanum excretion was noted. Urinary excretion of lanthanum was minimal, with a maximal excretion of approximately 25 ng/24 h. At week 10, a statistically significant increase in urinary lanthanum excretion was seen in the Phoslock® WCS group, and at week 4 in the lanthanum chloride WCS group, versus vehicle groups (Fig. 3).
2.4. Lanthanum measurement and biochemical analyses 3.2. Tissue lanthanum levels Lanthanum was determined in serum and the various tissues by High Resolution Inductively Coupled Mass Spectrometer (HR-ICPMS; Element XR, Thermo Scientific, Finnigan Element 2, Bremen, Germany). Briefly, serum and urine samples were freeze-dried, dissolved in 200 mL ultrapure HNO3 (Optima Grade, Fluka, Brussels, Belgium) and diluted with ultrapure demineralized/reverse osmosis treated water to a final volume of 5 mL. Tissue samples were digested in 1 mL ultrapure HNO3 at 110 C for 45 min and adjusted to 10 mL with ultrapure demineralized water. Prior to measurement with HR-ICP-MS, the samples were further diluted to bring the acid concentration in the solutions to approximately 2%. In order to overcome matrix interferences, lanthanum concentrations were accurately quantified by applying the standard addition calibration technique, whereby a series of aliquot sample solutions were spiked with increasing lanthanum concentrations. Method validation was achieved by analysis of a biological tissue reference material BCR 668 (European Commission e Joint Research Centre, Geel, Belgium) which was processed in a similar manner as the tissue samples. During analysis, a NIST reference standard (SRM1640a, USA National Institute of Standards and Technology, Gaithersburg, USA) was used as analytical QC check. In the brain samples, iron, copper, zinc and calcium were determined by means of electrothermal atomic absorption spectrometry (AAnalyst 800, PerkinElmer, USA). Here the same digestion and analytical procedure as previously described for bone analysis were applied [D'Haese et al., 1999]. 2.5. Statistical analysis Statistical analysis was performed using SPSS (Version 22, IBM). The Kruskall-Wallis test was used to test for differences between groups, followed by a Mann-Whitney U test with Bonferroni correction when appropriate. A p-value < 0.05 was considered statistically significant. Values which were larger than the 75th percentile þ 3x the Interquartile range or lower than the 25th percentile e 3x the Interquartile range were considered outliers and eliminated from further statistical analysis. 3. Results Throughout the study, no statistically significant differences were noted in body weight gain (Supplementary Fig. 1), food or water consumption between the different treatment groups (results not shown). Serum pH (Supplementary Fig. 2) and serum bicarbonate (HCO 3 ) (Supplementary Fig. 3) levels did not show any statistically significant differences between treatment groups. There was no mortality over the study period. 3.1. Serum and urine lanthanum levels Serum lanthanum levels showed no statistically significant differences between treatment groups, or between treatment and vehicle. Occasionally, some animals showed higher levels (up to
In the animals treated with the low dose of all three compounds, lanthanum levels did not show any increase versus vehicle treated animals. In all three high dose groups, total lanthanum content of the liver was increased, showing levels up to 10 ng/g wet weight. No differences between the three compounds were noted (Fig. 4). Accumulation of lanthanum in heart, spleen, lung, kidney and testes was highly limited. Heart lanthanum levels were below 1 ng/ g wet weight in all treatment groups, and no differences were found between compounds or doses. Interestingly, some vehicle treated animals had slightly higher lanthanum levels up to 2 ng/g wet weight. Spleen lanthanum levels were below 0.1 ng/g wet weight in all treatment groups. Lanthanum levels in the lung were slightly higher (up to 7 ng/g wet weight), and also showed some higher variability. Testes and kidney lanthanum levels were below 4 ng/g in all treatment groups, and again no statistically significant difference between treatment groups or vehicle was found (Fig. 5). Some accumulation of lanthanum in bone can be expected, given the chemical similarity between lanthanum and calcium. In the current study, we found bone lanthanum levels to remain below 35 ng/g. Values were similar in all lanthanum-treatment groups which did not differ from the vehicle group (Fig. 6). Brain lanthanum levels showed no statistically significant differences between treatment groups and vehicle, and were generally below 0.5 ng/g. In the lanthanum carbonate group treated with the highest dose, two animals showed higher levels, but were still below 2 ng/g. In addition to lanthanum we also measured some essential elements; i.e. iron, copper, zinc and calcium in the brain. For none of these elements significant differences were found between the different treatment groups (Fig. 7). 4. Discussion This study was performed in order to evaluate the possible accumulation of lanthanum following oral dosing with Phoslock®, lanthanum carbonate or lanthanum chloride, and investigate whether the gastrointestinal absorption properties of these compounds might differ from one another. The highest dose used corresponds to the WCS (‘Worst Case Scenario’) levels of lanthanum related with the use of Phoslock® (17 mg/L), while the lowest dose was intended to correspond to the RLL (‘Reference Lower Limit’, 0.01 mg/L). Our initial testing of the animal diet, however, revealed that this dose was below the daily dietary intake of lanthanum by the animals. Indeed, the regular diet used for the animals contained 0.4 mg lanthanum/g. This corresponds to an intake of lanthanum of 10e12 mg/day per animal from only the food. The WCS dose in this study corresponded to approximately 383 mg lanthanum/day per animal, while the RLL dose would correspond to only 0.25 mg lanthanum/day per animal, well below the daily intake with the food. It was therefore decided to increase the lowest dose to correspond with lake water containing 0.5 mg lanthanum/L. During the course of the study, all animals showed normal weight gain, and no morbidity or mortality was observed over the course of the 10-week treatment. Serum lanthanum levels showed
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Fig. 2. Serum lanthanum levels in the different treatment groups. Dots show data from individual animals while the bars show the mean value for the treatment groups. No statistically significant differences were found between treatment groups, or within one group over time.
Fig. 3. Urinary lanthanum levels in the different treatment groups. Urinary lanthanum excretion (ng/24 h). Dots show data from individual animals, while bars show the mean value for the treatment groups. *: p < 0.05 vs vehicle (same time-point).
no differences, neither between groups nor over time, and generally remained below 0.1 mg/L, even after 10 weeks of exposure. In previous studies [Behets et al., 2004; Bervoets et al., 2006 & 2009] using lanthanum carbonate at doses as high as 1000 mg/kg/day (i.e. 780-fold higher compared to the doses used in the current study), they found plasma lanthanum levels to increase from 0.4 mg/L to 2.5 mg/L, during the first four weeks of treatment after which they stabilized for the remainder of the 12-week treatment period. Compared to the cited papers the doses used in the current study thus are too low to cause a significant response in the serum lanthanum levels.
Although in our study urinary lanthanum excretion showed statistically significant increases in two groups, this is unlikely to be physiologically or scientifically relevant, since the time-points before and after the observed increase show no differences. The most likely explanation for these findings is accidental contamination during collection of the urine samples. When housing rats in metabolic cages for 24 h urine collection, possible minor contamination from faeces cannot be ruled out completely [Damment et al., 2009], especially in the highest dosing groups where lanthanum levels in the faeces can be up to 106 higher than those found in urine. As expected, because of the highly limited gastrointestinal
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Fig. 4. Lanthanum levels in the liver of the different treatment groups. Liver lanthanum content (ng/g wet weight). Dots show data from individual animals, Lines show group median values. *: p < 0.05 vs vehicle group; **: p < 0.01 vs vehicle group.
absorption of lanthanum (<0.0007% in rats) and the fact that less than 2% of the absorbed lanthanum is eliminated by the kidneys [Damment and Pennick 2008] the overall urinary excretion of lanthanum is very low and did not exceed 1 ng/24 h. The minute gastro-intestinally absorbed amounts of lanthanum readily bind to proteins, mainly transferrin, and are cleared from the body through the liver and bile [Damment and Pennick, 2007]. It is therefore not surprising that liver lanthanum levels are increased after lanthanum exposure. Consistent with this, liver lanthanum levels were increased in all three WCS groups as compared to RLL and vehicle groups. To which extent the somewhat, albeit not satistically significantly higher liver lanthanum levels in the WCS lanthanum chloride dose group versus the other groups might have been due to the use of water as vehicle, is difficult to assess and cannot be deduced from the present study. It should be noted however, that carboxymethylcellulose at doses up to 5% is a commonly accepted vehicle for oral dosing in experimental studies evaluating absorption, toxicity and efficacy of therapeutics and other compounds is also used in food industry. Interestingly in the low-dose (RLL) groups, lanthanum levels were indistinguishable from those found in vehicle-treated animals. In all groups, liver lanthanum levels were <10 ng/g wet weight. Although no liver function analysis was performed in the current study, a disturbance of liver function due to lanthanum is unlikely, since in previous studies of our group (using a 780-fold higher dose compared to the WCS dose of the present study), we have shown that liver lanthanum levels plateauing at 1 mg/g wet weight (i.e. 100 to 300-fold higher than those observed in the present study) did not have any effect on liver function parameters [Bervoets et al., 2009]. Another tissue in which some accumulation of lanthanum might be expected is bone. In the current experiment, we found bone lanthanum levels up to 35 ng/g wet weight. Using the 780-fold higher therapeutic doses used in our previous studies, bone lanthanum content reached levels of 0.5e1 mg/g wet weight, without any negative effects on bone metabolism/-histology [Behets et al., 2004 & 2005; Bervoets et al., 2006]. Results thus allow us to conclude that with the concentrations used in the
present study no negative effects on the bone are to be expected. The other tissues analyzed were the heart, spleen, lung, testes and kidney. For all these tissues, lanthanum levels were in the ng/g range and no statistically significant differences between treatment groups, or versus vehicle were found. Given the very low concentrations we encountered in the tissues under study with the doses used, increased risk for harmful effects reasonably should not be a matter of concern. Concerns have been raised as to whether lanthanum might accumulate in the brain. In the present study brain lanthanum levels in general remained below 0.2 ng/g wet weight, and did not differ between treatment groups nor versus vehicle treated animals. In our previous studies, we found brain lanthanum levels to be < 30 ng/g in animals treated with therapeutic doses of 1000 mg/ kg/day of lanthanum carbonate. In these studies, we did not observe gross neurological effects in the animals. In contrast, Feng et al., [2006] have reported neurological effects in rats treated with lanthanum chloride at a dose of 40 mg/kg/day during 6 months. They found brain levels up to 40 ng/g dry weight. A likely explanation for this discrepancy might be contamination issues. Indeed, given the extremely low levels of lanthanum that are absorbed in the gut, tissue lanthanum levels as demonstrated above are extremely low. However, during animal sacrifice, particular care has to be taken to avoid cross contamination of the sampled tissue with the lanthanum content in the gut (which is several orders of magnitude higher) and/or blood. In the present study, very strict procedures were implemented to avoid cross-contamination between tissues or animals. In their study, Feng et al., [2006], using synchrotron radiation X-ray fluorescence analysis (SRXRF), also reported a significant decrease in calcium, iron and zinc levels in the brain of animals treated with lanthanum chloride. Therefore, we also measured the concentration of these elements in the brain tissue using quantitative electrothermal atomic absorption spectrometry, however, found no differences between treatment groups and/or vehicle. The reason for the results to be conflicting between both studies is not clear and difficult to explain. Given the fact that calcium, iron and zinc levels in the brain, respectively are up to 83.300, 6.000 and 7.100 times higher than the brain lanthanum levels measured in rats of the present study receiving the WCS dose, it is hard to believe that such low lanthanum concentrations would be able to deplete the brain from the above mentioned essential elements. As discussed below administration of lanthanum chloride at doses used in the Feng et al., [2006] study might theoretically induce hyperchloremic acidosis. To which extent this condition may be responsible for the disturbed concentration of the above mentioned elements in the brain cannot be answered at this moment. The primary objective of this study was to investigate whether there was any difference in gastrointestinal absorption and/or tissue distribution of lanthanum after dosing as the chloride salt, the carbonate salt or the LMB compound. When comparing the same dosage groups of the different compounds, no differences in tissue distribution of lanthanum were seen. This is not unexpected, since the only factor that differs when ingesting different lanthanum salts is the counterion (in this case, the carbonate or chloride ions). In the acid environment of the upper gastrointestinal tract, the soluble lanthanum salts (like chloride and nitrate) but also the less soluble carbonates, dissociate into their respective ionic forms. Once dissociated, lanthanum ions readily bind to any available phosphate to form insoluble lanthanum phosphate within the lumen of the gut after which it is removed through fecal excretion. It is worth noting that in the gut, phosphate concentrations are several orders of magnitude higher than those of lanthanum. In a regular rat diet, the phosphorus content is approximately 0.70%, corresponding to an average daily intake of 200 mg, or 7 mmol of
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Fig. 5. Lanthanum levels in the heart, spleen, lung, kidney, testes and bone (ng/g wet weight) of the different treatment groups. No statistically significant differences were found between treatment groups and vehicle.
phosphate. In humans, average phosphate intake is approximately 14 mmol/day. In contrast, the lanthanum intake in the highest dose groups of the present study was 380 mg or 2.7 mmol/day. Theoretically, this implies complete binding of lanthanum to phosphate within the gastrointestinal tract, thus leaving an almost negligible amount of free lanthanum available for absorption.
Discussion is ongoing as to whether, independent of suggested differences in gastrointestinal absorption, ingestion of lanthanum chloride might be more toxic than lanthanum carbonate - in particular at the level of the brain. Indeed, in experimental studies in which lanthanum chloride was administered daily via the drinking water to rats at doses up to 0.5% of lanthanum (i.e.
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Fig. 6. Brain lanthanum levels (ng/g wet weight) of the different treatment groups. No statistically significant differences were found between treatment groups or vehicle.
20.4 mmol/L lanthanum chloride [Hu et al., 2018], corresponding to 2.8 g/L of lanthanum (i.e. 1200 times the highest levels observed in Phoslock® treated water and 165 times the highest dose in the present study) neurological effects as assessed by measurement of some biochemical parameters and behavior studies have been reported [Feng et al., 2006; He et al., 2008; Zheng et al., 2013; Jin et al., 2017]. It must be noted however, that with these doses the chloride concentration in the drinking water is as high as 2.2 g/L. Chloride ions are absorbed from the gastrointestinal tract through anion exchangers, whereby bicarbonate is released. Since the CO2/bicarbonate equilibrium is the main pH buffering system in the body, excess chloride intake can result in low plasma bicarbonate levels, representing metabolic acidosis (Seifter and Chang, 2016). This was observed in patients treated with the phosphate binding agent, Sevelamer hydrochloride. During therapeutic use of this compound, a dose-dependent decrease in bicarbonate levels due to the chlorine load resulting in metabolic acidosis was repeatedly reported (De Santo et al., 2006; Oka et al., 2008 & 2014). Interestingly, this was no longer seen when the formulation was changed to Sevelamer carbonate. This thus indicates that the counterion (i.e. chloride), rather than the active compound itself, was responsible for the observed acidosis. Although no differences in serum pH or bicarbonate levels were observed in the present study at the doses
Fig. 7. Iron (Fe), copper (Cu), zinc (Zn) and calcium (Ca) content in the different treatment groups. No statistically significant differences were found between treatment groups or vehicle for the respective elements under study.
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used, development of acidosis might reasonably have occurred in the above mentioned experimental studies in which rats were exposed to 165 times higher lanthanum chloride concentrations. To the best of our knowledge, pH and/or metabolic acidosis were not measured in the above mentioned studies (Hu et al., 2018; Feng et al., 2006; He et al., 2008; Zheng et al., 2013; Jin et al., 2017) and the causal role of increased chloride levels and acidosis is therefore difficult to prove. Nevertheless, it should be noted that studies have shown that a low pH is deleterious for the cerebral microenvironment whilst temporary pH changes could be responsible for cerebrovascular damage and cholinergic cell death [Pirchl et al., 2006]. In the present study dosing was based in relation to the use of LMB as a solid-phase phosphorus sorbent material to manage eutrophication and prevent cyanobacteria blooms by controlling the internal phosphorus load in aquatic systems (Mucci et al., 2018). In order to evaluate the safety margin and the potential risks for humans related to the treatment of waterbodies with LMB in the present study a worst case scenario (WCS) was adopted based on the highest LMB application rate reported in the literature (Spears et al., 2013a, 2013b). Given (i) the lanthanum concentration measured in surface waters as either filterable, total or predicted ‘free’ (uncomplexed) ionic lanthanum following the highest LMB application rate (Spears et al., 2013a, 2013b), (ii) the duration and magnitude of exposure through the use of lanthanum-treated drinking water, consumption of biota (fish, crustaceans, plants), dermal exposure via water or sediment or through the inadvertent consumption of sediments particularly in young children, (iii) the ability of ‘free’ lanthanum to directly bind phosphate and other oxyanions in the intestine resulting in a low bioavailability and, (iv) the absence of significant toxic effects when used therapeutically during years as a phosphate binding agent in patients with chronic kidney disease at daily doses which are up to three orders of magnitude higher than the WCS dose applied in the present study (Hutchison et al., 2016), one may reasonably accept that exposure to lanthanum via the drinking water or leisure activities will pose no increased risk for toxicity in humans. Although out of the scope of the present study should dermal contact to LMB treated water be acknowledged as another important exposure pathway. The European Food Safety Authority (EFSA) reports for bentonite the absence of irritation and sensitization properties (EFSA, 2012). A weak sensitization potential for lanthanum chloride hexahydrate (LaCl3.(H20)6) has been reported by the European Chemicals Agency (ECHA, 2013). Sensitization potential is, however, reported for a higher percentage compared to the assumed 5% lanthanum content in LMB (van Oosterhout & Lürling, 2013) and therefore we consider that lanthanum would not be sensitizing at the level presented in the LMB, the more since no evidence is provided so far for lanthanum to be present herein as lanthanum chloride hexahydrate. Overall, weighing off the potential severe health effects to humans and animals of cyanobacteria blooms and accumulation of cyano-toxins in water used for recreational activities, irrigation and drinking water supply (Chorus et al., 2000; Carmichael, 2001;; World Health Organization, 2003; Lürling and Faassen, 2013; Zamyadi et al., 2012, 2013) versus the negligible risk for any toxic effect of LMB derived lanthanum the use of LMB should be considered a powerful safe tool to prevent the serious health risks linked to cyanobacteria. 5. Conclusion In conclusion, accumulation of lanthanum in rats after dosing the WCS dose of Phoslock®, lanthanum carbonate or lanthanum chloride was limited to a slight increase in liver lanthanum to levels
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at which it has previously been demonstrated that no harmful effects are to be expected. Other tissues (including the brain) did not show any accumulation compared to vehicle treatment. There was no difference in accumulation between Phoslock®, lanthanum carbonate and lanthanum chloride, suggesting that gastrointestinal uptake of lanthanum is similar for all these compounds at the doses administered in the present study. In view of the long-term experience with lanthanum carbonate for phosphate binding treatment and the absence of any reported negative effects due to accumulation of lanthanum at levels being several orders of magnitude higher than those observed in the present study, the potential, minimal uptake of lanthanum through exposure to Phoslock®treated water is unlikely to pose a health risk to animals and/or humans. CRediT authorship contribution statement Geert J. Behets: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing original draft. Kayawe Valentine Mubiana: Formal analysis, Methodology. Ludwig Lamberts: Formal analysis, Methodology. Karin Finsterle: Conceptualization, Resources, Writing - review & editing. Nigel Traill: Conceptualization, Resources, Writing - review & editing. Patrick C. D'Haese: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing - original draft, Writing - review & editing. Acknowledgements This study was supported by a grant from Phoslock® Environmental Technologies Ltd, United Kingdom/Australia. NT and KF are employees of Phoslock® Environmental Technologies Ltd, United Kingdom/Australia. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.124780. References Behets, G.J., Dams, G., Vercauteren, S.R., Damment, S.J., Bouillon, R., De Broe, M.E., D'Haese, P.C., 2004. Does the phosphate binder lanthanum carbonate affect bone in rats with chronic renal failure? J. Am. Soc. Nephrol. 5 (8), 2219e2228. Behets, G.J., Verberckmoes, S.C., Oste, L., Bervoets, A.R., Salome, M., Cox, A.G., Denton, J., De Broe, M.E., D'Haese, P.C., 2005. Localization of lanthanum in bone of chronic renal failure rats after oral dosing with lanthanum carbonate. Kidney Int. 67 (5), 1830e1836. Bervoets, A.R., Oste, L., Behets, G.J., Dams, G., Blust, R., Marynissen, R., Geryl, H., De Broe, M.E., D'Haese, P.C., 2006. Development and reversibility of impaired mineralization associated with lanthanum carbonate treatment in chronic renal failure rats. Bone 38 (6), 803e810. Bervoets, A.R., Behets, G.J., Schryvers, D., Roels, F., Yang, Z., Verberckmoes, S.C., Damment, S.J., Dauwe, S., Mubiana, V.K., Blust, R., De Broe, M.E., D'Haese, P.C., 2009. Hepatocellular transport and gastrointestinal absorption of lanthanum in chronic renal failure. Kidney Int. 75 (4), 389e398. Bouma-Gregsona, K., Powera, M.E., Bormans, M., 2017. Rise and fall of toxic benthic freshwater cyanobacteria (Anabaena spp.) in the Eel river: buoyancy and dispersal. Harmful Algae 66, 79e87, 2017. https://doi.org/10.1016/j.hal.2017.05. 007. Carmichael, W.W., 2001. Health effects of toxin-producing cyanobacteria: “the CyanoHABs”. Hum. Ecol. Risk Assess. 7 (5), 1393e1407. Chorus, I., Falconer, I.R., Salas, H.J., Bartram, J., 2000. Health risks caused by freshwater cyanobacteria in recreational waters. J. Toxicol. Environ. Health Part B. 3 (4), 323e347. Copetti, D., Finsterle, K., Marziali, L., Stefani, F., Tartari, G., Douglas, G., Reitzel, K., Spears, B.M., Winfield, I.J., Crosa, G., D'Haese, P., Yasseri, S., Lürling, M., 2016. Eutrophication management in surface waters using lanthanum modified bentonite: a review. Water Res. 15 (97), 162e174. Review. D'Haese, P.C., Couttenye, M.M., Lamberts, L.V., Elseviers, M.M., Goodman, W.G., Schrooten, I., Cabrera, W.E., De Broe, M.E., 1999. Aluminum, iron, lead, cadmium,
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