Eight sampling sites were chosen throughout Manadas Creek to determine the levels of antimony, arsenic, and thallium in the water column, sediments, tissues, and shells (Figure 2). Sites were chosen based on location, accessibility, and presence of water (Table 1). Site 1 is located on the west side of a busy road near businesses and downstream from a residential and recreational area. Site 2 is along another heavily traveled road. The water in this tributary of the creek comes from storm drains as well as effluent from the North Laredo Wastewater Treatment Plant. This part of the creek contains building metals and broken pieces of cement slab. Site 3 is located on the west side of I-35, which is downstream from a cement ready mix plant. Site 4 is in a part of the creek that has been channeled below a large body of water and is surrounded by warehouses. Oil was observed on the water surface in this part of the creek. Site 5 islocated downstream from the inactive antimony plant and is known to be polluted by chemicals released from that plant. Site 6 is located downstream of site 5 and is located on the west side of Mines road. The bottom of the creek here contains pieces of asphalt, cement slab and large rocks. Site 7 is in a part of the creek that is located along a major road between a boat sales and service business and truck repair shop. Site 8 is upstream of site 7. Site 8 looks cleaner compared to site 7 as water from the North Creek Plaza pushes all the garbage to the back of the creek where it eventually gets caught in hanging trees or under the railroad bridge.

Note: Depths were recorded on sampling days.

In March 2013, 1000 clams were collected from a gravel bar above the confluence of the Rio Grande and Santa Isabel Creek (Figure 3). These clams were analyzed as controls and concentration of metals (As, Sb, and Tl) were below detection limits. An additional 400 mussels were collected on March 30, 2013, for the addition of Site 8 as well as to replace mussels at Site 7. When transporting the clams to the lab, they were placed in buckets filled with river water. In the lab, mussels were rinsed with deionized water to remove any particles from their shell. After they were air dried, the mussels were measured for their length (24 - 36 mm), width (14 - 19 mm), height (20 - 32 mm) and weight (6.3 - 10.4 g). The mussels were housed in aquariums with filtered river water until they were put in cages out in the field. About 150 clams were placed in stainless-steel cages (15 cm × 15 cm × 17 cm) at the different sites on March 16, 2013. In April, 200 mussels were added to the cages at Site 7 and Site 8 due to the high mortality at that part of the creek. Each cage was about 1/2 filled with gravel substrate. Cages were suspended midstream from T-post at the sites. At Sites 1 and 4, cages were placed on the sediment bed of the creek and secured with rebar due to the shallow water. Cages were checked once a week to ensure they were still in their location, submerged and that the mussels were alive.

For analysis, 3 specimens from each site were collected bi-weekly. Specimen samples were taken back to the lab in ice. Clam shells were cleaned with deionized water to remove any algae or sediments and then depurated in distilled water for 24 hours. After 24 hours, they were frozen at −80˚C until they were dissected.

Water samples were collected midstream at all sites using whirl-paks and then taken back to the lab in ice. Sediment samples were collected on one occasion at all sites. Grab samples were collected at three different spots at each site upstream of the cages. About 6 cm of the upper sediment layer was removed. Samples were stored in whirl-paks, mixed and placed in ice to be taken back to the lab.

Ultrapure water (Millipore, United States) was used to prepare the standards, blanks, and dilution of acid digested samples. Triton^TM X-100, Nitric acid (HNO₃, 70%), Hydrogen peroxide (H₂O₂, 30%) and Hydrofluoric acid (HF, 51%) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Standard reference material (SRM) 1566b oyster tissue, 2710a Montana I Soil, and 1643f -trace elements in water were purchased from National Institute of Science and Technology, Gaithersburg, MD, United States. A calibration curve method was used to quantify the heavy metal concentrations. 100 mg/L analytical grade stock solution of metals was purchased from VWR (Radnor, PA, USA) and used to prepare standards for calibration. Trace metal analysis was done with an Agilent ICP-720 Inductively coupled plasma-optical emission spectrometer (Agilent Technologies, Santa Clara, California, USA).

Tissue, shell, water, and sediment samples were collected for metal analyses. Clams were dissected frozen. The gills, mantle, foot, digestive tract, and gonads were each isolated using stainless-steel instruments. Once all the soft tissues were removed, the tissue was cleaned and rinsed. After dissection, the clam tissues and shell were put into crucibles and dried to a constant mass for 2 hours at 100˚C. Each of the tissue and shell samples were ground into a fine powder to evenly mix the samples before digestion.

Water samples were immediately filtered after being taken back to the lab using a syringe filter with a 0.45 µm pore diameter membrane filter and acidified with nitric acid before being stored. Sediment samples were evenly mixed, dried at 100˚C for 24 hours, and ground into a fine powder.

For water samples, 10 mL of acid mixture (7 mL HNO₃ and 3mLHF) was added to 100 mL of water sample in a beaker and digested using U.S. EPA method 3052 [34] . The beaker was covered with an evaporating disc, and allowed to reflux for 15 minutes on a hotplate at 80˚C. After 15 minutes, the beaker was allowed to cool and 5 mL of HNO₃ was added. The beaker was refluxed for 30 minutes and cooled again. 2 mL of water and 5 mL of hydrogen peroxide was added. The beaker was refluxed for 30 minutes, followed by removing the evaporating disc to let the solution evaporate to about 5 mL of volume. Once cooled the solution was diluted to 50 mL in a volumetric flask with MilliQ water. The solution was filtered with a 0.45 µm filter disc into a centrifuge tube and then capped for later analysis. Samples were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES). In the case of sediments, 0.5 g of prepared sediment was weighed into a beaker and 10 mL of acid mixture was added and digested using U.S. EPA method 3052 described above. Pooled tissue and shell samples of 3 - 5 individuals from each site were analyzed for As, Sb, and Tl using U.S. EPA method 3052. The samples were digested by adding 10 mL of acid mixture to an analytical amount of 10 - 500 mg of pooled sample and digested using U.S. EPA method 3052 as was done for water samples.

All glassware and plastic containers used were soaked in an acid-wash bath (0.1% Triton X-100 and 5% HNO₃) for 24 hours and then triply rinsed with deionized water. Analyses of the samples were carried out in triplicates. A calibration blank, and a reference standard were used to ensure the accuracy of the ICP-OES. Blanks and standards were run after every ten samples. To avoid memory effects between samples, MilliQ water was run through the analysis tubing to remove any traces of metals that remained. Certified SRM1566b oyster tissue, 2710 a Montana I Soil, and 1643 f-trace elements in water were digested using U.S. EPA method 3052 to check accuracy of the digestion protocol adopted. Method blanks were employed to check for background contamination. The analytical values were within the range of certified values and the recoveries of the metals across the sampling sites varied from a low of 90% to a high of 105%.

ANOVA was used to determine differences in the metal distribution in the water column of Manadas Creek at the different sites as well as to see differences in the metal distribution in the soft tissues of the clam (p < 0.05). A Kruskal-Wallis test was used to determine variability in the metal concentrations at the different sites. Also, Pearson correlation was used to determine any correlations between the metal concentrations in the mussel and the water column.

The metal pollution index (MPI) was used to assess the load of metals (As, Sb, and Tl) in the gills, DI tract, gonads, foot, and mantles of C. fluminea. MPI was calculated according to the equation (1) [35] .

M P I = ( M 1 × M 2 × M 3 × ⋯ × M n ) 1 / n (1)

M_n = The concentration of metal n (mg/kg) in a certain tissue.

Bioaccumulation factor (BAF) and biota-sediment accumulation factor (BSAF) were calculated for each site to evaluate the efficiency of As, Sb, and Tl accumulation in the tissues and shell. Water, sediment, and sample concentrations for a site were averaged together. BAFs and BSAFs were calculated using the following equations (2) and (3): [36] [37] [38] .

BAF = metalconcentrationintheorganism metalconcentrationinthewater (2)

BSAF = metalconcentrationintheorganism metalconcentrationinthesediment (3)

Figure 4 shows the mean concentrations of metals (As, Sb, and Tl) in the water column from each sampling site collected from March 2013 to August 2013. Statistical analysis reveals variability in the concentrations of As (ANOVA, df₁ = 5, df₂ = 66, F = 31.412, p < 0.000), Sb (ANOVA, df₁ = 5, df₂ = 66, F = 62.337, p < 0.000), and Tl (ANOVA, df₁ = 5, df₂ = 66, F = 5.373, p < 0.000) in the water at the sampling sites. The range of Sb (4.36 - 13.45 µg/L) and Tl (3.63 - 7.47 µg/L) across all eight sampling sites were above the US EPA maximum contaminant level (MCL) in water (Sb, 6 µg/L; Tl, 2 µg/L) [39] .

On the other hand, the range of As (5.73 - 10.33 µg/L) were all below the MCL (10 µg/L) except at Site 5 (10.33 µg/L) which is adjacent to the decommissioned antimony plant. Site 5 had the highest arsenic concentration (10.33 µg/L), while the lowest arsenic concentration (5.29 µg/L) was recorded at Site 2. The highest antimony concentration, 13.45 µg/L, was observed at Site 5, while the lowest antimony concentration, 4.36 µg/L, was recorded at Site 1. The highest thallium

concentration, 7.47 μg/L, was observed at Site 5, while the lowest concentration, 3.63 µg/L, was recorded at Site 2. Tl concentrations measured in this study exceed the concentration of Tl in uncontaminated waters (1 μg/L) [40] . Overall, the highest arsenic, antimony and thallium mean concentrations in the water column were observed at site 5, which is the site closest downstream to the decommissioned antimony smelter. This observation implies that activities at the smelting plant led to the release of heavy metals into the creek. The higher concentration of metals in the water around the decommissioned plant area points to the coexistence of other metals in antimony ores.

Table 2 shows results from this study compared with previous studies in the creek. A study conducted by the United States International Boundary and Water Commission (USIBWC) in March 1993 recorded mean concentrations of arsenic, antimony, and thallium as 10 μg/L, 78 μg/L, and 2.2 μg/L respectively [19] . Mean As and Tl concentrations were within the MCL set by the EPA. However, mean Sb concentration in that study exceeded the MCL by 12-fold. A follow up study by the same organization in May 1995 recorded concentrations of arsenic, antimony, and thallium in the water as 6.3 μg/L, 36.4 μg/L, and 1.9 μg/L respectively [19] .

The second study shows a reduction of metal concentration especially Sb which was recorded at about 50% reduction. A study in Feb-May 2008 recorded As and Sb concentrations in the creek as 20.6 and 219.3 μg/L respectively, and a study in in February 2010 recorded mean As and Sb concentrations 47.99 μg/L and 22.80 μg/L respectively [18] [41] . These results highlight fluctuations over the years of study which can be attributed to seasonal variations in geochemical conditions [42] . A trend of an overall reduction in the metal load in the creek is also observed. The reduction in mean As and Sb concentrations can be attributed to the effectiveness of the containment steps taken by the city to stop/reduce the release of Sb into the surrounding ecosystems.

- was not measured in the study.

Higher Sb concentrations observed points to the sediment acting as a sink and source of metal in the water column [43] [44] . Since metals are non-biodegradable, they are sorbed in particles and settle down on the sediment. Seasonal fluctuations in water flow and occasional disturbance can remobilize the metals trapped in the sediment and serve as source of metal. This was evidenced by the higher bioaccumulation of Sb in C. fluminea in the 2010 study by Addo-Mensah et al. [41] . The presence of antimony, arsenic and thallium in the creek are mostly due to the antimony smelter. Thallium and arsenic are by-products of these operations. These toxic chemicals are naturally found in very small amounts in the earth’s crust, but in higher concentrations, they are carcinogens and a threat to human health [45] . Antimony enters the water from weathering of rocks, effluents from agricultural, industrial, and mining/smelting processes [19] . Arsenic easily dissolves in water and enters the waters by erosion, use as a pesticide, and from industrial, municipal, and smelting effluents [19] . Thallium usually enters the water from effluents of smelting [19] .

Figure 5 shows the arsenic, antimony, and thallium concentrations in the sediments of Manadas Creek collected at the end of the sampling period, August 31, 2013. The highest mean concentration of arsenic (6.41 mg/kg) and antimony (5.77 mg/kg) in the sediments of Manadas Creek were observed at Site 5, while the highest mean concentration for thallium (3.00 mg/kg) was recorded at site 3. Site 2 had the lowest mean concentrations for arsenic (3.29 mg/kg) and antimony (1.77 mg/kg) in the sediment. Site 8 had the lowest mean concentration for thallium (1.95 mg/kg). Kruskal-Walllis test reveals differences in metal concentrations in the sediments for As (p < 0.025) and Sb (0.007) among the sites, while there is no variability of Tl (p > 0.315) among the sites. Site 5 had the highest concentration values of antimony (75.77 mg/kg) and arsenic (6.41 mg/kg). Site 3 had the highest concentration value for thallium (3.00 mg/kg). Site 2 had the lowest concentration values of arsenic and antimony (3.29 and 1.77 mg/kg), while Site 8 had the lowest concentration value for thallium (1.95 mg/kg). Concentration of Tl across the sampling sites were not significantly different from each other (P < 0.05). Concentration of As and Tl across the sampling sites were comparable to mean arsenic and thallium concentrations in rivers across the United States of America and other parts of the world [40] [46] [47] [48] . The concentration of As in the sediments in this study was comparable to As concentration

in previous study by Baeza et al., 2010 [18] . The concentration of all metals studied at site 1 - 4 were comparable to that observed in uncontaminated soils and sediments in the USA [48] [49] [50] [51] .

Site 5 which is adjacent to the antimony smelter had antimony concentrations higher than that of uncontaminated sediments but comparable to that observed in antimony mining sites or smelter plants [52] [53] . The Sb concentration gradient observed from Site 5 to 8 points to the smelting activities as a likely point source of contamination [54] .

The average concentration of As in the sediments at all the sampling sites (4.73 mg/kg) was not statistically different at the 0.05 level from the previous studies by USIBWC in 1993 and 1996 (5.2 mg/kg 7.6 mg/kg respectively [19] . The average Tl in the sediments of the sites studied is 2.42 mg/kg and it is not significantly higher at the 0.05 level than previous studies by USIBWC in 1993 and 1996 (0.25 mg/kg and 0.17 mg/kg respectively). Mean Sb concentration is 19.69 mg/kg, but the highest Sb concentration is 75.77 mg/kg. The concentration recorded is significantly lower than that observed in the study by Baeza et al., 2010 (470 mg/kg [18] . This general trend of decrease and stability of metal concentration in the sediment has been observed in European rivers since the 1970s. This trend is attributed to sound environmental policies. In the case of this smelter plant, remediation measures have been effective [55] .

MPIs of the tissues are represented in Figure 6. Gills and DI tract had the highest MPIs 13.92 and 13.66 respectively. The gills of bivalve clams like C. flumineaare used for respiration and filtering food [56] . These activities allow them to be in close contact with metal enriched sediments thereby having high MPI as seen in the results. The DI tract of the clam consists of the mouth, esophagus, and stomach (which is within the liver) is used for food digestion and waste excretion.

These two major metabolic organs had the highest MPI and the accumulated metals the most. The mantle which covers the visceral mass, and the foot are not involved in metabolic activities of the clam and hence have low MPI. Site 5 which is adjacent to the decommissioned plant had the highest MPI when compared with the other sampling sites. This supports the observation that abandoned smelting and processing sites are point sources of heavy metal pollution [57] [58] .

Distribution of the bioaccumulation of the metals in different parts of C. fluminea throughout the sampling period is shown in Table 3.

Analysis of the shell showed As, Sb and Tl values were all below the detection limit. The shells therefore did not bioaccumulate the metals studied. Also, mean thallium concentration in the organs of C. fluminea were below detection limit. The distribution of arsenic (ANOVA, df₁ = 4, df₂ = 355, F = 349.502, p < 0.000) and Sb (ANOVA, df₁ = 4, df₂ = 355, F = 100.313, p < 0.000), in the tissues of Corbicula fluminea varied. The average antimony concentrations recorded in the tissues of the clam were the highest followed by arsenic. The DI tract tissue had the highest arsenic concentration, followed by the gills, then gonads, then foot, and finally the mantle. For antimony, the gills had the highest concentration, followed by the DI tract, then gonads, then mantle, and finally the foot. This order mirrors that which was observed in the MPI. Based on the results, the organotropism for arsenic is DI tract > gills > gonads > foot > mantle > shell and the organotropism for antimony is gills > DI tract > gonads > mantle > foot > shell. The organotropism order agrees with similar studies where they also observed that metabolically active organs accumulated most metals whereas muscular organs like the foot had low metal affinity [59] .