A sensitive method to determine 210Po and 210Pb in environmental samples by alpha spectrometry using CuS micro-precipitation

A new sensitive method to determine polonium-210 (210Po) and lead-210 (210Pb) in a diversity of environmental samples was developed. For fresh and marine waters, Po was pre-concentrated using a titanium (III) hydroxide (Ti(OH)3) co-precipitation. Solid environmental samples were digested with nitric acid (HNO3) and hydrogen peroxide (H2O2). The alpha thin layer source was prepared using CuS micro-precipitation and 210Po was measured by alpha spectrometry. Lead-210 was left to decay for up to a year and indirectly measured via its progeny, 210Po. The chemical recoveries for 210Po and 210Pb were high, 90% and 97%, respectively, for a large variety of samples and a very low minimum detectable activity (MDA) was obtained. The method was validated using standardized solutions and certified reference materials.

Certified standard solutions of 210 Po, 209 Po and 210 Pb were purchased from Eckert & Ziegler Isotope Products (Valencia, CA).In addition, reference materials (CLV1 and CLV2) containing 210 Po and 210 Pb, in equilibrium, were purchased from the International Atomic Energy Agency (IAEA; Vienna, Austria) 24 .The accuracy of the certified reference materials was tested by an inter-laboratory program established by Natural Resources Canada 25 .A certified stable lead standard was purchased from Millipore Sigma (Oakville, ON).

Analytical equipment
Radiological activity concentrations ( 209 Po, 210 Po and 210 Pb) were detected using an Alpha Analyst alpha spectrometer (Mirion Technologies (Canberra), Meriden, CT) with a counting efficiency superior to 17% and a background of < 1 count/hour in the energy regions of interest.Apex Alpha counting productivity software was used for detector setup and control, quality assurance, sample analysis and elaboration.The Pb chemical yield was verified by inductively coupled mass plasma spectrometry (ICP-MS) (8900 ICP-MS Triple Quad, Agilent Technologies, Santa Clara, CA).

Surface water
To test the applicability of this method for a range of water types, surface waters were collected from marine (n = 4) and fresh water (n = 13) systems representing a gradient of ionic content (Supplementary Fig. S1, S1).Marine surface waters (4 L) were collected within the St. Lawrence Estuary and Gulf of St. Lawrence in eastern Canada.Freshwater samples (4 L) were collected from Perch Lake in Ontario,Canada located on the Chalk River Laboratories (CRL) site.Immediately following collection, samples were filtered using a 0.45 µm filter (FHT-45, Waterra, Mississauga, ON) and acidified to 1% HNO 3 .The electrical conductivity of the samples was measured using an Oakton 450 Meter (Thomas Scientific, Swedesboro, NJ), and the elemental content was measured by ICP-MS (Supplementary Table S2).The range of conductivity, calcium mass concentration, and chloride mass concentration for the samples collected are shown in Table 1.

Biota
Biota from selected trophic levels were collected in the Lac Granet and Lac Camille-Roy systems (main lake and connected streams) of western Québec, Canada.Biotic samples collected included algae (n = 3), macrophytes (n = 7), plankton (n = 2), invertebrates (n = 9), molluscs (n = 6) and fish (n = 18).Plankton samples were collected with plankton nets (WildCo, Saginaw, MI) mesh sizes 153 µm and towed behind a boat.Algae, macrophytes, invertebrates and fish were collected using dip and kick nets (WildCo, Saginaw, MI).Immediately after collection, invertebrates and molluscs were placed in UPW and allowed to purge their gut contents for approximately 12 hours 26 .All samples were thoroughly rinsed in lake water and kept frozen until further processing.

Sample processing and digestion
The samples were processed within 30 days of collection to reduce 210 Po loss from radioactive decay and 210 Po ingrowth from 210 Pb.For the water samples, a one litre subsample was weighed into a glass beaker and acidified to pH 2 using concentrated HNO 3 to stabilize the sample and keep the Po in solution.Next, a known amount of 209  was filtered again through a 0.22 µm polystyrene filter (Fisher Scientific, Fair Lawn, NJ) to remove remaining particulate matter and colloids.Biota samples, were processed wet and were minced using a scalpel and scissors and homogenized before distributing 1 to 2 g (wet weight) into a 15 mL polypropylene digestion tube.A known amount of 209 Po (approximately 15 mBq) was added to each sample before digestion.To digest the sample, 3.3 mL of concentrated HNO 3 were added to the digestion tube and allowed to sit for approximately 1 h followed by the addition of 2.0 mL of 30% H 2 O 2 .The sample solution was then heated to 50 °C for 48 h.Once removed, the dissolved sample solution was allowed to cool and it was transferred to a 50 mL polypropylene conical tube (Cole-Parmer, Montreal, QC).The sample was centrifuged at 3500 rpm for 1.5 min.The supernate was transferred to a new 15 mL polypropylene conical tube (Cole-Parmer, Montreal, QC) and diluted to a volume of 10 mL with UPW.

Water samples pre-concentration
For the water samples only, Po was co-precipitated with Ti(OH) 3 using 0.6 mL of 10% m v −1 TiCl 3 , and the pH was adjusted between 10 and 12 using about 30 mL of 40% mv −1 sodium hydroxide (NaOH) solution.The fresh and marine water samples were left aside for 1 and 4 h, respectively, which allowed the Ti(OH) 3 to settle and to easily remove most of the supernate by decantation.The precipitate was then recovered by centrifugation, dissolved with 3.3 mL of concentrated HNO 3 and diluted to a volume of 10 mL with UPW.

CuS microprecipitation
. The 10 mL, 5 mol l −1 HNO 3, supernate collected following the water pre-concentration and biota sample digestion, was filtered through a 0.1 µm Resolve™ filter (Eichrom Technologies Inc., Lisle, IL) using a multi-hole vacuum box and recovered in a 50 mL polypropylene conical tube.Then, 0.2 mL of 500 µg mL −1 Cu(II) solution was added and mixed, followed immediately by the addition of 0.2 mL of 10% m v −1 Na 2 S, at which point a visible (brown) colloidal precipitate formed.The sample was then filtered immediately through a 0.1 µm Resolve™ filter.After rinsing the filter with 1 to 2 mL of UPW, the filtrate was set aside for future 210 Pb determination and replaced with a 50 mL polypropylene conical waste tube.The filter was rinsed with 1 to 2 mL of 80% ethanol.The precipitate retained on the filter was air-dried and mounted on a stainless steel disc (AF Murphy Die & Machine Co Inc., North Quincy, MA) for 210 Po determination by alpha spectrometry.Water samples were counted for 48-96 h, while biota samples were counted for 24 to 48 h.The in situ 210 Po activity concentrations were calculated by Eq. (1): where A' Po-210 is the 210 Po activity at the time of extraction, A' Po-210 ingrowth is a correction factor for 210 Po loss as well as 210 Po ingrowth from 210 Pb decay from the time of sampling and extraction, λ Po is the decay constant for 210 Po and T is time between sample collection and extraction 27,28 .

Pb determination
Lead-210 activity concentrations were determined by measuring the ingrowth activity of its daughter, 210 Po, using alpha spectrometry.Following the CuS micro-precipitation step, an additional aliquot of approximately 15 mBq of 209 Po yield tracer and 2.0 µg of stable Pb carrier was added to the final filtrate.The sample was then left aside for at least 4 months to allow 210 Po ingrowth.Finally, the CuS micro-precipitation was repeated a second time and the 210 Pb activity was calculated using Eq. ( 2): where A Pb-210 in situ is the activity concentration of the sample at the time of collection, A mPo is the activity of 210 Po at the time of the second CuS micro-precipitation, t 2 is a correction factor for the decay of 210 Pb from the time of sample collection and second extraction, t 1 is a ingrowth factor for 210 Po from the decay of 210 Pb for time between first and second extraction, λ Po and λ Pb are decay constants for 210 Po and 210 Pb, respectively 27,28 .The n c parameter is the Pb chemical yield calculated by applying the following Eq.( 3):

Figures of merit
The MDA was determined by measuring 210 Po and 210 Pb in ten reagent blank samples following all the steps of the described method and using the Currie equation 28 ( 4): (1) where k is a constant (1.645) to reach the 95% confidence interval, B is the number of background counts for a defined time in seconds (T), ε is the counting efficiency, R is the chemical recovery, V is the volume in litres and F is a unit conversion factor which equals 10 −3 .The method accuracy and precision were determined by measuring 210 Po (n = 7) and 210 Pb (n = 3) in water samples spiked with a known activity of 210 Po and 210 Pb, in equilibrium, using the developed method.The relative bias (B ri ) and the relative precision (S B ) were calculated using Eqs. 5 and 6. 29,30 : where A i is the measured activity, A ai is the added activity, B r is the mean relative bias and N is the number of replicates.
The method was validated for solid samples by determining the 210 Po and 210 Pb activity concentration in certified reference materials CLV-1 and CLV-2.The data followed a normal distribution and therefore a Student's t-test was used to evaluate differences in the 210 Po and 210 Pb activity concentrations measured in the certified reference materials by the inter-laboratory program and this method.

Animal care
The institutional review board that reviewed and approved the capture, handling and euthanasia of fish for this study was the Wildlife Management Branch of the province of Québec's Ministry of Forests, Wildlife and Parks (permit # 2021-07-14-070-08-GP).This approved and issued permit covered all aspects of fish collection, animal welfare and euthanasia methods.All methods were carried out in accordance with relevant guidelines and regulations.Euthanasia of fish was conducted following Guideline 113 of the Canadian Council on Animal Care guidelines on the care and use of fish in research, teaching and testing 31 .Specifically, fish euthanasia was conducted immediately after capture by swift blow to the head (destruction of brain tissue).Where relevant, all methods are reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org) for the reporting of animal experiments.

Recovery optimization
Initially, a chemical recovery of 37 ± 10% was obtained for environmental fresh water samples, which was much lower than test samples prepared with UPW (~ 85%).The average recovery increased to 89.8 ± 12.5% when the samples were re-filtered before the CuS micro-precipitation step using a 0.22 µm polyethersulfone filter (Fisher Scientific, Fair Lawn, NJ) to remove suspended particulate matter and colloids smaller than 0.45 µm in size.During the final filtration step, following the CuS micro-precipitation, the PoS precipitate as well as any particulate matter would be retained leading to a poor spectral resolution and recovery.This additional filtration step substantially improved the chemical yield and spectral resolution for field water samples.
Next, the method was tested on various types of samples and the results are shown in Fig. 1.The chemical recovery was high for all type of samples tested (90.7 ± 9.3%), which demonstrated that this method was versatile and robust.
Approximately 4 months after the initial 210 Po extractions, the same samples were analyzed again to indirectly measure the 210 Pb present in the sample.The chemical recovery following the ingrowth period was high (97.1 ± 8.2%) and consistent for all sample types as shown in Fig. 2.

Figures of merit
A very low MDA of 0.05, 0.02, and 0.01 mBq L −1 was obtained for 24, 48, and 96 h of counting, respectively (Table 2) The method was validated for water samples using water samples spiked with 210 Po and 210 Pb with a range of activities from 12 to 160 mBq and counted for 24, 48 and 96 h (Fig. 3).The mean 210 Po chemical recovery was 97 ± 7% over the 3 count times.Three samples were allowed to sit for approximately 4 months when the CuS micro-precipitation was repeated to validate the method for 210 Pb determination (Fig. 4) (mean 210 Pb chemical recovery was 102 ± 7% and mean stable Pb chemical recovery was 108 ± 6%).To further validate the method for solid samples, two certified reference materials (CLV-1 and CLV-2) with a range of 210 Po and 210 Pb activities from 74 to 660 mBq g −1 were chosen to represent a range of activity concentrations.Mean 210 Po chemical recoveries of 89 ± 3% and 80 ± 6% were calculated from the analyses of the two vegetation reference materials CLV-1 (n = 5) and CLV-2 (n = 4), respectively.A mean 210 Pb chemical recovery of 101 ± 9. % was calculated from the analyses of the vegetation reference material CLV-1 (n = 5) (Table 2 and Figs. 5, 6).Furthermore, the comparison of the expected and measured 210 Po and 210 Pb activities in reference materials using a two-tailed Student's t-test, assuming unequal variances (p-values > 0.05), indicates repeatability, confirming the method to be accurate for 210 Po and 210 Pb determination in environmental abiotic and biotic non-water samples.These results provided evidence that complex matrices, as well as reduced sample size and digestion temperatures, produced consistent Vol.:(0123456789) and high polonium and lead recoveries (78-101%) (Table 3).The developed method was also accurate and precise as evidenced by the mean relative bias and mean relative precision presented in Table 2.

Application and comparison of the method
The mean activity concentrations of 210 Po measured in different types of waters (freshwater and saltwater) and several types of freshwater organisms are shown in Fig. 7. Freshwater organism samples such as periphyton and invertebrates are often too small in mass to be able to accurately measure their 210 Po activity concentration.For example, sample size requirements and detection limits for other methods in the literature are summarized in Table 4.The sample quantity requirements and/or detection limits for these methods are not applicable to many environmental sample types.However, the developed method was successful in reducing sample quantities while maintaining sufficiently sensitive detection limits (Fig. 8).Furthermore, applying the CuS microprecipitation method to indirectly measure 210 Pb not only provided a more sensitive technique, it also allowed the same   www.nature.com/scientificreports/sample to be used for both the 210 Po and 210 Pb analyses, which reduced field sampling efforts and laboratory processing times.

Conclusion
Large data gaps exist in understanding the behavior of the 210 Po and 210 Pb in the environment, and this is largely due to challenges associated with measuring these radionuclides at the low-level activity concentrations typically encountered in the environment 14,18 .The developed method is simple and highly sensitive (MDA ~ 0.01 mBq L −1 ).Consistent and high chemical recoveries (> 80%) for abiotic and biotic environmental samples were obtained, thereby making it possible to adequately study the behavior of 210 Po and 210 Pb in the environment.
Supplementary Fig S2 and Supplementary Table

Figure 1 .
Figure 1. 209Po recovery for initial 210 Po extraction as a function of sample type.Error bars indicate the standard error of the mean.

Figure 2 .
Figure 2. 209 Po recovery as a function of sample type for indirect 210 Pb measurement via 210 Po extraction following a 4 month in growth period.Error bars indicate the standard error of the mean.

F()Figure 7 .
Figure 7. Mean 210 Po activity concentrations measured for water samples and solid samples.Error bars indicate the standard error of the mean.

Fish 20 gFigure 8 .
Figure 8. Mean MDAs measured for water samples and solid samples.Error bars indicate the standard error of the mean.

Table 1 .
Po yield tracer (approximately 15 mBq) and stable Pb standard (approximately 2.0 µg) was added.The sample Aqueous sample properties.

Table 2 .
Figure of merit for water method.

Table 3 .
Figures of merit for environmental samples.

Table 4 .
Overview of analytical methods for determining210Po in environmental samples.