Mobility Of Metals In Sediments Research Articles

Geochemical fractionation of trace metals and ecological risk assessment of surface sediments in Sepetiba Bay, Brazil.

The Sepetiba Bay (Southeast Brazil) is a known Cd- and Zn-contaminated site that received spills of a large slag pile leachate from a Zn smelter. With important harbors, Sepetiba Bay demands periodic dredging operations which affect the mobility of the metals. The main goal of this work was to assess metal mobility in sediments and its associated toxicity in a fictive dredging area, to evaluate the risks of the operation. To achieve this goal, 18 superficial sediment samples were collected and characterized for pH and Eh. Sediments were analyzed for grain size, organic carbon, and total nitrogen, and metal mobility was evaluated with a sequential extraction procedure, proposed by the European Community Bureau of Reference (BCR). The results demonstrate that Cd and Zn are mainly associated with the exchangeable fraction (mean concentrations 1.4mgkg-1 and 149.4mgkg-1, respectively) and reducible fractions (mean concentrations 0.3mgkg-1 and 65.5mgkg-1, respectively), while Fe, Cr, Cu, Ni, and Al were associated with the residual fraction. Metals in the residual fraction are probably associated with the mineral lattice of the sediment and should not represent an environmental risk for the biota. The application of the enrichment factor and three risk assessment indexes (Risk Assessment Code, Risky Pollution Index, and Bioavailability Risk Assessment Index) show that the sediments are considerably enriched in metals that constitute a relevant risk for the sediment biota. In the case of dredging operations, Cd and Zn should be released to the overlying waters and be available to organisms, threatening the whole ecosystem. The proposed approach was shown to be much more precise than what is frequently presented in the Environmental Impact Assessments that only consider the threshold limits of the legislation.

Distribution, speciation and mobility of metals in sediments of the Tianxiu hydrothermal field, Carlsberg Ridge, Northwest Indian Ocean

Tianxiu is the only ultramafic-hosted hydrothermal field that has been discovered on the slow-spreading Carlsberg Ridge (CR), northwest Indian Ocean. Sequential extraction has been performed on the surface sediments from the near-vent and far-field of Tianxiu to characterize the compositions of metalliferous sediments, the speciation of selected metals and the potential risks of metal mobility. The near-vent sediments of Tianxiu are mainly derived from hydrothermal inputs and are enriched in Fe, Zn, P, Pb, Mo and especially Cu, Mg, Co and Ni with significantly higher contents compared to metalliferous sediments formed in basalt-hosted hydrothermal systems. Most of the ore-forming elements (Fe, Cu, Zn, Mn, Co and Ni) and rare-earth elements (REEs) exist in the residual fraction and Fe Mn oxide fraction, followed by the carbonate fraction and organic fraction. The chondrite-normalized REE pattern in each phase is characterized by relative enrichment in light (L)REEs and positive Eu anomalies, consistent with the REE patterns of hydrothermal fluids. The far-field sediments of Tianxiu are mainly derived from background pelagic sedimentation while the sediments at some sites are influenced by a small amount of hydrothermal plume fallout. The investigated elements mainly exist in the Fe Mn oxide fraction, residual fraction and carbonate fraction. In particular, ore-forming elements Cu, Pb, Mn, Co and Ni are mostly enriched in the Fe Mn oxide fraction, which is attributed to their adsorption by hydrothermal particulates in the plumes. The potential mobility of each metal is evaluated in terms of its total content in the unstable phases, which reveals that most of the heavy metals (e.g., Cu, Zn, Mn and Pb) in the metalliferous sediments have high potential mobility (> 50%). • Heavy metals are particularly enriched in the near-vent sediments. • Heavy metals are mainly present in the residual fraction and Fe Mn oxide fraction. • Most of the heavy metals in the near-vent sediments have high potential mobility.

Mechanistic insights into trace metal mobilization at the micro-scale in the rhizosphere of Vallisneria spiralis

Mobilization of trace metals in the rhizosphere of macrophytes is controlled by root-driven chemical changes, especially the steep gradients of O2 and pH from the rhizosphere to bulk sediments. Here, the O2 and pH dynamics, and the distribution of trace metal, in the rhizosphere of Vallisneria spiralis were obtained using planar optodes and diffusive gradients in thin films, respectively. Radial O2 loss (ROL) and acidification occurred on all visible roots of V. spiralis and exhibited highly spatiotemporal dynamics depending on the root growth and various environmental conditions. Trace metals showed different mobilization mechanisms in the rhizosphere. ROL and produced Fe(III) (oxyhydr)oxides decreased the mobility of Fe, As, Co, V and W in the rhizosphere. However, Mn, Ni and Cu exhibited greater mobility in the rhizosphere than bulk sediments as a result of the oxidation of metal sulfide and proton-induced dissolution of minerals. In particular, Co and Ni presented increased activity at the interface between rhizosphere and bulk sediment, which was attributed to the redox dissolution processes of Fe and Mn as a result of ROL and rhizosphere acidification. These results provide new insights into the roles of macrophyte root-induced O2 and pH changes in controlling trace metal mobility in sediments.

Ecological risk assessment of trace metals in sediments under reducing conditions based on isotopically exchangeable pool

Determination of potential mobility of toxic trace metals in sediments under changing redox condition is important in ecological risk assessment. Current methods are limited in risk prediction in such dynamic environment. In this study, we have discussed the general disagreement from widely used methods (sediment quality guideline (SQGs), potential ecological risk index (PERI), risk assessment code (RAC) using BCR fraction information). In addition, the stable isotopic dilution method (IDM) was also modified to quantify metal lability in a microcosm experiment mimicking river bank sediment turning into anaerobic. The isotopically exchangeable Cd, Cu, Pb, and Zn quantified by IDM (%E incub) was used in the RAC to reveal the trend of risk during this process. Strong risks from Cd are suggested by the PERI and RAC as a result of high toxicity and mobility of the element, while SQGs suggests medium risk for Cu, Pb, and Zn in certain samples. The disagreement between the results of RAC assessed by metal lability (%E dry) and by BCR metal fractionation reflects the effect of sediment properties and source of metal contamination. The RAC based on the non-residual fractions is likely to overestimate the potential risk for most metals even there is a significant change in sediment Eh. The RAC assessed by %E incub reveals that the variability in risk in response to the reducing Eh is not consistent. Large fluctuation in %E incub for Cd (28.5%, 49.5%), Pb (27.6%, 18.2%), and Cu (14.4%, 24.7%) can shift the risks to a higher level in certain range of Eh in two sediments. In sediment with lower contents of metal binding phases (e.g. mineral oxides, organic matters), the release of metals can be more significant, thus higher ecological risk in changing redox condition.

High cadmium pollution from sediments in a eutrophic lake caused by dissolved organic matter complexation and reduction of manganese oxide

Eutrophication and metal pollution are global environmental problems. The risk of metal pollution is high in the eutrophic lakes because of high mobility of metal in sediments. However, the mechanism of cadmium (Cd) mobility in sediments is still unclear. Here we study the mobilization of Cd in sediments from the eutrophic Lake Taihu via monthly field monitoring of mobile Cd using diffusive gradient in thin films (DGT) and high resolution dialysis (HR-Peeper) techniques. We found a high mobility of Cd in sediments in February and March, resulting from reductive dissolution of Mn oxide mediation by high microbial activities, as shown by the similarities in distribution patterns of DGT-labile Cd and Mn. A two orders of magnitude increase in dissolved Cd concentrations (about 28 μg L−1) was observed in May and June, with dissolved Cd concentrations in overlying water about 110 times higher than the criteria continuous concentration set by Environmental Protection Agency. Hourly changes were found to coincide and correlate between dissolved Cd and dissolved organic matter (DOM) under simulated anaerobic conditions, strongly suggesting that the sudden outbreak of Cd pollution observed in the field resulted from the complexation of DOM with Cd in sediments. This was further supported by the NICA-Donnan model that more than 71% of dissolved Cd in the pore water in May and June was present as Cd-DOM complexes. Three components of DOM including humic-, tryptophan-, and tyrosine-like components in the sediments in June was identified using the fluorescence excitation emission matrix-parallel factor analysis. We found that Cd was stable complexed with tyrosine-like component. The Fourier transform infrared and two-dimensional correlation spectroscopy further revealed that Cd was bound to phenolic OH, alkene CC, alcoholic CO, aromatic CH, and alkene CH groups. Our study effectively promotes the understanding of Cd mobilization in sediments and highlights the risk of sudden Cd pollution events in the eutrophic lakes.

Sulfur Species, Bonding Environment, and Metal Mobilization in Mining-Impacted Lake Sediments: Column Experiments Replicating Seasonal Anoxia and Deposition of Algal Detritus

The oxidation state of sulfur [S] is a primary control on mobility of metals in sediments impacted by legacy mining practices. Coeur d’Alene Lake of northern Idaho, USA, has been impacted by upstream legacy mining practices that deposited an estimated 75 Mt of metal(loid)- and S-rich sediments into the lake. Future lake conditions are expected to include algal blooms, which may alter S and metal remobilization during the seasonal euxinic environment. Cores of the lake sediments were exposed to anoxic and anoxic + algal detritus conditions for eight weeks at 4.5 °C through introduction of a N2 atmosphere and addition of algal detritus. At a location 2.5 cm below the sediment-water interface, anoxic conditions promoted a shift in S species to continually larger concentrations of reduced species and an associated shift in the bonding environment reflective of increased S–metal bonds. Anoxic + algal detritus conditions suppressed the increasing trend of reduced S species and induced greater release of Mn compared to the anoxic-only conditions but did not appear to enhance the release of As, Cd, or Fe. The addition of algal detritus to the sediment-water interface of these Fe- and S-rich sediments enhanced mobilization of Mn likely because of dissimilatory metal reduction where the anaerobic oxidation of the algal detritus stimulated Mn reduction. Results of the study indicate that future metal release from the lake sediments will be altered with the likely deposition of algal detritus, but the effect may not enhance the release of acutely toxic metals, such as As or Cd, or substantially impact Fe cycling in the sediments.

Assessment of metal mobility in sediment, commercial fish accumulation and impact on human health risk in a large shallow plateau lake in southwest of China.

Sediment heavy metal pollution in the Dianchi Lake has been a long-term environmental problem of concern. This study investigated the lake sediment heavy metal contamination level, mobility, commercial fish metal accumulation and its impact on human health. The results show high As, Hg and Cd concentration in the sediment, while Pb and Cr contamination are insignificant. Sediment sequential extraction analysis shows that Hg in sediment has the highest portion of mobile fraction, followed by As, while the portion of mobile fractions of Cd, Pb and Cr in sediment is very low. The high concentrations of Hg and As in surface water and porewater were consistent with the chemical fraction composition of the two elements in sediment. Three major commercial fish species, Culterichthys erythropterus, Carassius auratus and Hypophthalmichthys molitrix, were collected for analysis of metal concentrations in their muscles. Among the same size of fish, C. auratus has the highest As concentration due to its bottom habitat and omnivorous feeding habits. On the other hand, C. erythropterus has the highest Hg concentration due to its relatively high trophic level position. The average THQ value of metals in fish tissue decrease in the order of As>Hg>Pb>Cd>Cr and the total THQ of average metal concentration in fish species decreased in the order of C. auratus>C. erythropterus>H. molitrix. Both THQ and total THQ is below 1, suggested no non-carcinogenic human health risk of fish consumption. However, TR of As in C. auratus was above 1.00E-04 threshold value, indicated potential carcinogenic human health risk. The results from this study indicate that although moderately to heavily contamination of Hg, As, and Cd occurred in Dianchi Lake sediment, only Hg and As tend to transport to surface water and accumulate in commercial fish due to their higher mobility in sediment.

Potential availability of trace metals in sediments in southeastern and southern Brazilian shipyard areas using the DGT technique and chemical extraction methods

Speciation and partitioning of trace metals, from solid to solution phases of sediments, control their bioavailability and thus their potential ecological risk to organisms. Therefore, in order to obtain a broad evaluation of their risk, it is necessary to couple methodologies that are able to assess metal mobility in sediment. In this study, the Diffusive Gradients in Thin Films (DGT) technique and the application of 0.1 M HCl acid extraction methods, together with solid-state voltammetric sensors, were used with the objective of assessing mobility and potential availability of Cr, Cu, Ni, Pb, V and Zn in sediment porewaters and solid sediments in southeastern and southern Brazilian shipyard areas. The highest labile metal concentrations were found in shipyards with the longest histories of operations. Trace metal distributions in porewater and in the solid phase of sediments (labile metals) and significant correlations among metals enabled to distinguish the contribution of anti-fouling paint components. The diffusive flux of every metal measured at the surface of the sediment indicated that CuDGT had the highest flux (3.66E−03 mmol·m−2 d−1) in the shipyard with the longest operating time. Therefore, enrichment was observed for Cu, Pb and Zn in sediments, indicating a possible ecological risk level of ‘Effects Range Median’ to ‘Apparent Effects Threshold’ for oyster larvae (Mollusca) (Cu), bivalves (Pb) and the infaunal community (Zn). Probable Effect Concentrations (PEC) to sediment-dwelling biota can be expected as well, related to high concentrations of Cu and Zn in sediment. This study allowed a comprehensive evaluation of potential bioavailability and ecological risk of trace metals in aquatic systems where there is continuous and specific input of these elements. The use of the DGT technique with solid-state voltammetry in the sediment of distinct Brazilian estuarine systems demonstrated its potential to be applied in future environmental network programs.

Mobility of Metals in Sediments Contaminated with Historical Mining Wastes: Example from the Tri-State Mining District, USA

The Tri-State Mining District (TSMD) of Kansas, Missouri, and Oklahoma was a world class zinc (Zn) and lead (Pb) producer. Mining ceased in the 1950s, leaving behind a large amount of mine wastes. Although much of the affected areas have been remediated, stream sediments may still contain toxic levels of these metals. The mobility of the metals was determined for sediment samples from Turkey Creek, Missouri. The median values of the metal content were 2700 mg/kg Zn, 161 mg/kg Pb, and 10.8 mg/kg Cd. These concentrations marginally surpass the sediment quality guidelines that differentiate between toxic and nontoxic conditions. Mobility was determined by sequential extraction of two phases—bioavailable and Fe-oxides. The fraction of the metal available to biota was 7.7% for Zn, 5.0% for Cd, and 0.4% for Pb, whereas the Fe-oxide fraction retained 25% of Zn, 21% of Pb, and 35% Cd. These values roughly agree with the values reported for other areas of the TSMD. Fractionation provides an estimate of the amount of metal available at the present conditions and gives the amount of metal available should the pH and/or Eh vary. The methodology puts an emphasis on ecosystem health and can be applied to other areas where Zn–Pb concentrations in soils and sediments are a concern.

Remediation of Toxic Metal Contaminated Sediment Using Three Types of nZVI Supported Materials.

Due to nZVI effectiveness in the removal of toxic metals as well as low-cost regarding its production, kaolinite, bentonite and carboxymethyl cellulose supported nZVI were chosen for in-situ remediation of river sediment. Small-scale laboratory studies have shown that the percentage of removed metal (Ni, Zn and Pb) ranged up to 80% depending on the nanomaterial used. The metal mobility in sediments was investigated using single extraction, which is proved to be better for quick estimation of metal mobility, and for highly contaminated sites both single and sequential extraction needs to be used. Risk assessment code indicated medium risk for Ni and high risk for Pb and Zn in untreated sediment. In-situ treatment in laboratory proved to be very effective, providing the choice of optimal doses of three different nanomaterials used towards the concentration of toxic metals in the sediment.

Environmental Risk Assessment of Sediments Deposited in Stormwater Treatment Facilities: Trace Metal Fractionation and Its Implication for Sediment Management

To gather further data about metal mobility in accumulated sediments in stormwater treatment facilities, metal mobility in sediments from sedimentation tanks, gully pot sediment traps, and sediment ...

Coupled effects of hydrodynamics and biogeochemistry on Zn mobility and speciation in highly contaminated sediments.

Porewater transport and diagenetic reactions strongly regulate the mobility of metals in sediments. We executed a series of laboratory experiments in Gust chamber mesocosms to study the effects of hydrodynamics and biogeochemical transformations on the mobility and speciation of Zn in contaminated sediments from Lake DePue, IL. X-ray absorption spectroscopy (XAS) indicated that the oxidation of surficial sediments promoted the formation of more mobile Zn species. Bulk chemical measurements of porewater, overlying water, and sediment also suggested that this process liberated aqueous metals to porewater and facilitated Zn efflux to the overlying water. In addition, sediment resuspension events increased the release of aqueous metals to both surficial porewater and the overlying water column. XAS analysis indicated that resuspension increased dissolution of Zn-sequestering mineral phases. These results show that both steady slow porewater transport and rapid episodic resuspension are important to the release of metal from fine-grained, low-permeability contaminated sediments. Thus, information on metals speciation and mobility under time-varying overlying flow conditions is essential to understanding the long-term behavior of metals in contaminated sediments.

Combined SEM/AVS and attenuation of concentration models for the assessment of bioavailability and mobility of metals in sediments of Sepetiba Bay (SE Brazil)

This study proposes a new methodology to study contamination, bioavailability and mobility of metals (Cd, Cu, Ni, Pb, and Zn) using chemical and geostatistics approaches in marine sediments of Sepetiba Bay (SE Brazil). The chemical model of SEM (simultaneously extracted metals)/AVS (acid volatile sulfides) ratio uses a technique of cold acid extraction of metals to evaluate their bioavailability, and the geostatistical model of attenuation of concentrations estimates the mobility of metals. By coupling the two it was observed that Sepetiba Port, the urban area of Sepetiba and the riverine discharges may constitute potential sources of metals to Sepetiba Bay. The metals are concentrated in the NE area of the bay, where they tend to have their lowest mobility, as shown by the attenuation model, and are not bioavailable, as they tend to associate with sulfide and organic matter originated in the mangrove forests of nearby Guaratiba area.

Influence of a salt marsh plant ( Halimione portulacoides) on the concentrations and potential mobility of metals in sediments

Influence of Halimione portulacoides, commonly found in temperate salt marshes, on sediment metal contents, speciation and potential mobility in case of sediment re-suspension was evaluated. Both colonized and non-colonized sediments were studied for total Cd, Cu, Pb and Zn contents and metal fraction exchangeable to water collected in situ. Sediment elutriates, prepared with water collected from each site, were used to simulate a sediment re-suspension phenomenon. As the characteristics and degree of contamination of sediments may influence system behaviour, salt marshes of two Portuguese estuaries, Cavado (NW coast) and Sado (SW coast), were studied. Cu, Pb and Zn contents higher than ERL (quality guideline, effect range-low) were observed, indicating potential risks for living organisms. Strong Cu-complexing organic ligands, also determined in both water and elutriates, were higher in rhizosediment elutriates, at concentrations similar, or even higher, to those of Cu. Such ligands condition metals speciation in the water column and probably also metal bioavailability. From rhizosediment significant amounts of Cu and Zn were transferred to the aqueous phase, concentrations 2–8 times higher than concentrations present in water. In contrast, elutriates of non-colonized sediment removed metals from water, Cu and Zn levels in elutriates being 2–6 times lower than initial ones. Cd and Pb levels in water and elutriates were not measurable in most cases. Results clearly indicate that metals potential solubility in the rhizosphere of plants was markedly higher than that in the surrounding sediment. The obtained results indicated that H. portulacoides presence (and probably other salt marsh plants) may cause a marked increase in metals concentrations in dissolved phase (pore water or even water column if rhizosediment is re-suspended). As salt marsh plants may be abundant in temperate and subtropical estuaries and costal lagoons, this phenomenon should not be disregard in future studies towards the sustainable management of such environments.

Comparison between BCR sequential extraction and geo-accumulation method to evaluate metal mobility in sediments of Dongting Lake, Central China

The form in which a metal exists strongly influences its mobility and thus, the effects on the environment. Operational methods of speciation analysis, such as the use of sequential extraction procedures, are commonly applied. The Dongting Lake, the second largest fresh-water lake in China, contains three China wetlands of international importance, the East Dongting Lake, South Dongting Lake, and West Dongting Lake. In this work, an optimized BCR sequential extraction procedure was used to assess the environmental risk of Cd, Cr, Cu, Ni, Pb and Zn in contaminated sediment of the Dongting Lake. The procedure was evaluated by using a certified reference material, BCR701. The results of the partitioning study indicated that in the lake sediments, more easily mobilized forms (acid exchangeable) were predominant for Cd, particularly in the samples from the East Dongting Lake. In contrast, the largest amount of Pb was associated with the iron and manganese oxide fractions and Cu, Zn, Cr, and Ni analyzed were mainly distributed in residual phase at an average percentage greater than 60% of the total metals. The potential risk to the lake’s water contamination was highest in the East Dongting Lake based on the calculated contamination factors. On the other hand, the total metal content was determined as well by inductively coupled plasma and mass spectrometry (ICP-MS) and assessed by using geo-accumulation index. The assessment results using geo-accumulation index were compared with the information on metal speciation. Both were correspondent with each other.

Metal concentrations and mobility in marine sediment and groundwater in coastal reclamation areas: A case study in Shenzhen, China

The concentrations of metals in the buried marine sediment and groundwater were differently affected by land reclamation. Nine metals (V, Cr, Mn, Co, Ni, Cu, Zn, Cd and Pb) in sediment and coastal groundwater from reclamation areas in Shenzhen were examined. The gradually decreased concentrations (V, Cr, Mn, Ni, Cu, Zn) in sediment and relatively higher concentrations (V, Cr, Mn, Co, Ni, Cu and Cd) in groundwater within reclamation areas were observed. The increase of V, Cr, Mn, Ni, Cu and Cd concentrations in groundwater within reclamation areas subsequently after land reclamation should be resulted from the mobilization of these metals accumulated in the sediment. These metals appear to be easily mobilized from solid phase to solution phase after reclamation. The physico-chemical changes such as reduction in pH and salinity in water environment induced by land reclamation appear to be responsible for metal mobility in the sediment–groundwater system.

Influence of Atmospheric Oxygen on Heavy Metal Mobility in Sediment and Soil (7 pp)

Background Aims. Pollution of sediment and soil by heavy metals is still an environmental problem. In order to assess the actual environmental risk, the mobile and biologically available content of heavy metals needs to be determined rather than the total content. This requires an analytical strategy preserving the actual binding forms of the heavy metals. Sampling and sample preparation are usually carried out in the presence of oxygen. As a consequence, oxidation of the metal and/or its binding partners is possible under these conditions, changing the original binding character. Therefore, sampling and sample preparation must be performed under inert conditions. The influence of atmospheric oxygen on the heavy metal mobility in sediment and soil for Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn is shown for samples of different origin. By means of a case study, an alternative for the extensive inert sampling and sample preparation is given; also, a mobility correction factor for the heavy metals mentioned is determined.

Spatial and temporal organic and heavy metal pollution at Mai Po Marshes Nature Reserve, Hong Kong

An intensive monthly sampling of water and sediments from 12 sites over 8 months covering wet and dry seasons at Mai Po Marshes Nature Reserve was conducted during June 1997–February 1998. Major organic (C, N and P) and heavy metal pollutants (Cd, Cr, Cu, Ni, Pb, Zn) water and sediment samples were examined. The results showed that Mai Po Marshes were severely polluted by organic matter and heavy metals, and the water from Deep Bay appeared to be the source of pollution. Up to 13–55% chance that the sediments of Mai Po Marshes were classified as moderately to seriously metal contaminated materials, according to the guideline set by Hong Kong Government. Empirical models describing organic matter and heavy metal spatial and seasonal dynamics in the water and sediments were formulated, based on data analysis. During wet season (June–October), more than 58% variations of total P can be explained by ortho-P in water, while ammonia-N explained up to 90% variations of total Kjeldahl nitrogen in water. Throughout the whole sampling period (June–February), there were significant correlations ( p<0.01) between total organic C in water. pH in the sediments and salinity in water appeared to be important factors determining heavy metal mobility in sediments, while potential metal release from the sediments is a concern when any oxidizing processes such as flooding or dredging are imposed on sediments.

Environmental mobility of trace metals in sediments collected in the Lake Balaton

In environmental pollution studies on solid materials sequential extraction techniques are used for the identification of the main binding sites of trace metals. A four-step sequential extraction procedure was applied to the determination of the distribution of elements like Cd, Cu, As, Pb, Cr, Ni, Zn, Al, Fe, and Mn in sediment samples collected in the Lake Balaton. The fractions were (1) exchangeable and bound to carbonate, (2) bound to Fe/Mn oxide, (3) bound to organic matter and sulfides, and (4) acid-soluble. Elements were mainly accumulated in the stable, acid-soluble fractions. Arsenic and Cd-ions were found in low concentration (1.2–13 mg/kg and < 1 mg/kg, respectively) in the samples. Based on the results determined at 15 sampling points it can be stated that the Lake Balaton has not yet been polluted by the elements investigated.