Ocean Acidification-Mediated Food Chain Transfer of Polonium between Primary Producers and Consumers

Phytoplankton and zooplankton are key marine components that play an important role in metal distribution through a food web transfer. An increased phytoplankton concentration as a result of ocean acidification and warming are well-established, along with the fact that phytoplankton biomagnify 210Po by 3–4 orders of magnitude compared to the seawater concentration. This experimental study is carried out to better understand the transfer of polonium between primary producers and consumers. The experimental produced data highlight the complex interaction between the polonium concentration in zooplankton food, i.e. phytoplankton, its excretion via defecated fecal pellets, and its bioaccumulation at ambient seawater pH and a lower pH of 7.7, typical of ocean acidification scenarios in the open ocean. The mass of copepods recovered was 11% less: 7.7 pH compared to 8.2. The effects of copepod species (n = 3), microalgae species (n = 3), pH (n = 2), and time (n = 4) on the polonium activity in the fecal pellets (expressed as % of the total activity introduced through feeding) was tested using an ANOVA 4. With the exception of time (model: F20, 215 = 176.84, p < 0.001; time: F3 = 1.76, p = 0.16), all tested parameters had an impact on the polonium activity (copepod species: F2 = 169.15, p < 0.0001; algae species: F2 = 10.21, p < 0.0001; pH: F1 = 9.85, p = 0.002) with complex interactions (copepod x algae: F2 = 19.48, p < 0.0001; copepod x pH: F2 = 10.54, p < 0.0001; algae x pH: F2 = 4.87, p = 0.009). The experimental data underpin the hypothesis that metal bioavailability and bioaccumulation will be enhanced in secondary consumers such as crustacean zooplankton due to ocean acidification.


Introduction
There has been a growing interest in understanding the effect of ocean acidification (OA) in modulating the metal uptake among the phytoplankton and zooplankton in the coastal and marine ecosystems [1][2][3][4][5][6]. Marine phytoplankton are primary producers in the ocean, forming the base of the marine food web. The oceanic sequestration of carbon dioxide is leading to a lowering of pH. The average oceanic pH has dropped to~8.1, reaching the historical low for the past 2 million years [7]. With the increasing levels of atmospheric CO 2, oceanic pH is expected to drop by 0.3-0.4 units by the end of this century [8]. OA affects marine organisms in multiple ways, such as changes in acid-base balance, energy metabolism, redox balance, as well as the behavior of marine organisms [9][10][11].
One of the overarching effects of OA on phytoplankton communities is that phytoplankton will likely alter the chemical speciation of trace metals [4,[17][18][19][20]. Phytoplankton concentrate metals and transuranic elements from surrounding waters [21][22][23][24][25]. Some of these metals such as iron (Fe), copper (Cu), and zinc (Zn) are essential for the growth and development of phytoplankton. In contrast, others such as cadmium (Cd), mercury (Hg), lead (Pb), and polonium (Po) have no known biological functions but are highly toxic at elevated concentrations [26,27]. Considering increased oceanic productivity under climate change [28,29], and the reported polonium concentration factors (CF) of 10 3 -10 4 in microalgae from the Gulf [24,30], and even a higher CF of 10 5 among Gulf copepods [31], it is quite probable to observe a trophic transfer of polonium to higher trophic levels in marine food chains [30].
Zooplankton are an important link between primary producers and fish. Copepods are primary consumers in the ocean and the most numerous metazoans, supporting the marine food web and also acting as a biological pump of carbon [32]. Any effect on the abundance and diversity of copepods due to ocean acidification is likely to have serious implications on the proper function of marine ecosystems. Studies have shown the lethality of increasing pCO 2 levels on both surface and deeper copepod species [33]; however, the deeper copepods demonstrate greater tolerance to higher pCO 2 . It can be hypothesized that this higher tolerance is probably an adaptation to the high pCO 2 environment where they live. It has been reported that ocean acidification and warming causes oxidative stress and a reduction in the reproductive capacity of Acartia bifilosa [34]; however, the magnitude of these responses varies between species and also varies spatially in different regions [33,35,36]. The Gulf ecosystem is somewhat unique for its temperature, salinity, nutrient-loading, and pH and has shown resilience to these changes (i.e. corals are surviving at a higher temperature in turbid water; a breeding ground for various fin and shellfish, are in areas reported to be exposed to potentially stressing conditions) [15,24,37,38].
This study was designed to understand the polonium transfer among the primary producers and primary consumers under lower pH conditions that might prevail due to increased oceanic CO 2 sequestration considering the scenario of a 0.4 unit pH drop that is expected by the end of the century. The interest in understanding the behavior of polonium in a marine environment has been the focus of studies for the past five decades, since it results in an enhanced radiation dose to marine biota [39]. Studies have suggested that an anthropogenic contribution can enhance the 210 Po concentrations considerably in the marine environment [40][41][42][43][44]. Marine organisms are capable of concentrating radionuclides in their tissues [39,43,[45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63]; however, polonium elimination from zooplankton via their excretion of fecal pellets has been reported by Beasley et al. [64]. The concentration of polonium is of enormous interest because of its large contribution to the natural radiation dose and its high radiotoxicity [53,55].
In our previous experiment [4], we looked at polonium uptake in five microalgal species, i.e., Thalassiosira weissflogi, Tetrasemis suecica, Isochrysis galbana, Chaetoceros muelleri, and Dunaliella salina that are commonly used as a food source in the mariculture/aquaculture industry.
This experimental study is designed to understand, under future oceanic pH as per IPCC RCP8.5, likely changes in 210 Po concentration and possible uptake kinetics among the primary producers and consumers that are very important links in the marine food web. The microalgae with a known concentration of 209 Po were used for feeding copepods to understand the assimilation rates, transfer, and turnover of this radionuclide in microcrustacean zooplankton.

Sampling and Monoculture
The copepod samples were collected using a 50 µ plankton net with 0.6 m diameter and 2 m length. The samples were collected by vertical tows from 10-30 m depth to the surface in October 2019. All the samples obtained were placed in filtered seawater and immediately transported to the laboratory for sample identification and segregation to create a monoculture of Acartia pacifica, Euterpina acutifrons, and Parvocalanus crassirostis. The sample was emptied into a Bogorov counting chamber and examined under a stereomicroscope. Fine needles and pipettes were used to isolate the identified species for preparing a monoculture. Only adults were used for the experiment. Samples collected on a single day cruise from different areas were segregated at the lowest taxonomic level by specialists in copepod identification and following the characteristics of the species in an identification guide [65]. All the samples of single species from different areas were combined into a single beaker with filtered seawater. Once the monoculture was segregated, they were transferred into 1 L cylindrical enclosures in 50 L aquariums.

Carbonate Chemistry
The pH on the total scale, alkalinity (A T in µmol/kg), and salinity (in ‰) were measured at 12h, 24h, 36h, and 48h after the beginning of the experiment in each replicate. These data were used to calculate pCO 2 (in µatm) using CO2SYS with constants from Mehrbach et al. [66] refitted by Dickson and Millero [67] and a temperature of 25 • C. An ANOVA 2 statistical test was used to test the effect of target pH (8.2 and 7.7) and replicates (n = 3 per treatment) on the carbonate chemistry.

Experimental Exposure
The experiment was carried out in 50 L aquaria. Copepods were housed in a 1 L cylindrical enclosure with a mesh of 45 µm at the cod end and a sealed bottom, enabling easy segregation of excreted fecal pellets. The stocking density of the experiment was 1000 individuals per enclosure. The experiment was conducted in triplicate for each of the Parvocalanus crassirostis, Euterpina acutifrons, and Acartia pacifica monocultures. The copepods were maintained at 25 ± 0.5 • C on a 16:8 h light-dark cycle and the water was gently aerated. All beakers were maintained at the required pH for 48 h to assess the Po transfer in copepods.
Isochrysis galbana, Chaetoceros muelleri, and Dunaliella salina labeled with 209 Po were used as radiolabeled food to assess the transfer. The average 209 Po concentration in each phytoplankton species was determined (Table 1). Three replicates for each copepod and each algae ( Figure 1) were taken considering 8.2 pH as a control as it represents the present-day ambient pH of the Gulf and Mediterranean waters, and 7.7 pH mimicking the worst case scenario for 2100 in these waters. The pH was regulated using the IKS Aquastar system by bubbling CO 2 into the aquaria.  The experimental setup consists of six aquaria with nine enclosures each, and each aquarium has a single pH condition and an algae type for the three copepod types. The microalgal food was added slowly with 10 ml of food being added twice a day to each cylindrical enclosure. The water turns green but as the microalgae were consumed, it again becomes clear. It was observed that the maximum quantity of fecal pellets was obtained within 60 min of feeding. The experiment was terminated after 48 h and all the copepods were filtered on a 0.45 µ m filter paper and dried. The water was also analyzed.
Each sample of fecal pellets, phytoplankton, copepod, and water was digested using concentrated nitric acid, to allow determining the concentration of 208 Po and 209 Po. The samples were digested for at least 24 h, with occasional hydrogen peroxide additions to help in oxidizing the organic compounds. When a clear solution was obtained, it was allowed to evaporate to near dryness to remove HNO3. This concentrated residue was then dissolved in 100 ml of 0.5M HCl, and the solution placed on a magnetic stirrer at 30 o C for 24 h, following the procedure of Benoit and Hemond [68]. After the iron reduction with ascorbic acid, polonium ( 209 Po and 208 Po) in the solution were then spontaneously plated onto the surface of a 0.64-mm-thick silver disc (1.2 cm diameter) to be used as a thin alpha source for alpha spectrometry [69].
A Canberra forty-eight chamber alpha spectrometry system with passive ion-implanted silicon detectors (active area of 450 mm 2 , a background count of 2.3 per day, and a minimum depletion thickness of 90 µ m) was used for 208 Po and 209 Po determination using the 5.110 MeV energy of 208 Po and 4.877 MeV energy of 209 Po alpha particle emission. The counting time was 24 h. Upon plating, the solution was converted to 9M HCl and passed through an ion-exchange column (DOWEX 1 x 8 100-200 mesh) to ensure the complete removal of the polonium tracers. The samples were stored for six months to allow the in-growth of sufficient 210 Po from 210 Pb for performing a second 210 Po determination, however that is not reported here.
As part of the analytical quality assurance procedures, reagent blanks, and an IAEA certified reference material were also analyzed with each batch of samples. Several aliquots of the IAEA 446-Baltic Sea Seaweed certified reference material (CRM) were analyzed for 210 Po, and the concentration of 210 Pb (through the determination of 210 Po in secular radioactive equilibrium with 210 Pb) was found to vary between 10.1 and 10.7 Bq kg -1 with a median value of 10.5 Bq kg -1 . This result compared well with the 210 Pb ( 210 Po) information value of 10.9 Bq kg -1 and a 95% confidence interval of 10.2 to 12.0 Bq kg -1 provided in the The experimental setup consists of six aquaria with nine enclosures each, and each aquarium has a single pH condition and an algae type for the three copepod types. The microalgal food was added slowly with 10 mL of food being added twice a day to each cylindrical enclosure. The water turns green but as the microalgae were consumed, it again becomes clear. It was observed that the maximum quantity of fecal pellets was obtained within 60 min of feeding. The experiment was terminated after 48 h and all the copepods were filtered on a 0.45 µm filter paper and dried. The water was also analyzed.
Each sample of fecal pellets, phytoplankton, copepod, and water was digested using concentrated nitric acid, to allow determining the concentration of 208 Po and 209 Po. The samples were digested for at least 24 h, with occasional hydrogen peroxide additions to help in oxidizing the organic compounds. When a clear solution was obtained, it was allowed to evaporate to near dryness to remove HNO 3 . This concentrated residue was then dissolved in 100 mL of 0.5M HCl, and the solution placed on a magnetic stirrer at 30 • C for 24 h, following the procedure of Benoit and Hemond [68]. After the iron reduction with ascorbic acid, polonium ( 209 Po and 208 Po) in the solution were then spontaneously plated onto the surface of a 0.64-mm-thick silver disc (1.2 cm diameter) to be used as a thin alpha source for alpha spectrometry [69].
A Canberra forty-eight chamber alpha spectrometry system with passive ion-implanted silicon detectors (active area of 450 mm 2 , a background count of 2.3 per day, and a minimum depletion thickness of 90 µm) was used for 208 Po and 209 Po determination using the 5.110 MeV energy of 208 Po and 4.877 MeV energy of 209 Po alpha particle emission. The counting time was 24 h. Upon plating, the solution was converted to 9M HCl and passed through an ion-exchange column (DOWEX 1 × 8 100-200 mesh) to ensure the complete removal of the polonium tracers. The samples were stored for six months to allow the in-growth of sufficient 210 Po from 210 Pb for performing a second 210 Po determination, however that is not reported here.
As part of the analytical quality assurance procedures, reagent blanks, and an IAEA certified reference material were also analyzed with each batch of samples.  Seawater samples from the aquaria were analyzed after the exposure experiment using an established method [70]. The seawater sample was filtered using a 0.2 µm pore-size membrane filter and placed in a 1 L glass beaker. Ten mL of concentrated HCl was added to the sample to prevent radionuclides from adhering to the beaker wall. A spike of 100 µL of 208 Po tracer was added, and the sample stirred for 3 h to ensure radiotracer mixing. Five mL of each 0.2 M KMnO 4 and 0.3 M MnCl 2 were added, and the solution was adjusted to pH 9 with 25% NH 4 OH. This solution was stirred for 3 h, and then the precipitate was allowed to settle out for a day. Most of the supernatant was carefully decanted so as to not disturb the precipitate, and the remainder was centrifuged. The centrifuged precipitate was recovered with 10 mL of 1% H 2 O 2 in 5 M HCl from the centrifuge tube, taken to dryness on a hot plate, and the residue dissolved with 10 mL of 1% H 2 O 2 in 2 M HCl. Polonium in this solution was then plated on a silver disc. This silver disc was used as a thin alpha source for alpha spectrometry and determination of 209 Po in the aquaria sample.
Mean 209 Po concentrations in each microalgal species are given with the standard deviation of the sample (mean ± SD). Statistical analyses were performed using SAS. Differences between categories (copepod and algae species, pH, time, and replicate) were tested using ANOVA models followed by Scheffe's post-hoc tests when relevant. All data are presented as mean ± standard error of mean (SEM). We have used SEM as it indicates how different the population mean is from the sample mean, and how much the sample mean would vary if the study is repeated using a new sample within a single population. The pH was kept constant using IKS Aquastar computers as well as the addition of bicarbonates when required.

Carbonate Chemistry
A summary of the parameters of the carbonate chemistry over the course of the experiment is summarized in Table 2. Target pHs were reached (pH 8.20 ± 0.01 and 7.69 ± 0.01) and significant effects between pH treatments were observed for pH, alkalinity, and pCO 2 . No significant differences were observed between replicates ( Table 2). Table 2. Summary of the carbonate chemistry: measured pH on the total scale, alkalinity (A T in µmol/kg), salinity (in ‰), and calculated pCO 2 (in µatm). Difference between target the two target pHs and between replicates was tested using an ANOVA 2. Results are presented as mean ± SEM. The 209 Po recoveries were calculated by using 208 Po tracer. The measured activities at 8.2 and 7.7 pH in fecal pellets, organisms, seawater fractions, and the activity initially added are presented in Tables 3 and 4. The activity in fecal pellets, copepods, and water was subtracted from the total activity of algal food added to each experiment. The unaccounted activity is listed as loss, which might be due to adhesion on sides of the aquaria, losses in transfer, and possibly evaporation during the source preparation.
The statistical analysis revealed that the weight of the fecal pellet was only significantly different between copepod species (ANOVA 4, model: F 20,215 = 164.37, p < 0.0001; species: F 2 = 1611.63, p < 0.0001) but not the microalgae species (F 2 = 1.25, p = 0.29), pH (F 1 = 1.55, p = 0.22), or time (F 3 = 2.19, p = 0.09). A Scheffe's post-hoc test revealed that the fecal pellet weight was significantly different between the three species. The difference in dry weight was small between Acartia and Euterpina, however it was 25% in Parvocalanus as compared to the two other species (Figure 3).

Polonium
Effects of copepod species (n = 3), algae species (n = 3), pH (n = 2), and time (n = 4) on the polonium activity (expressed in % of the total activity introduced through feeding) in the fecal pellets were tested using an The average polonium activity in the fecal pellets collected from each of the three copepods show higher concentrations at lower pH of 7.7 with the exception of Parvocalanus on Isochrysis feed. The effect of pH for each combination of copepod and algal species is presented in Figure 4. A significant 3 to 4% increase in polonium activity is observed under pH 7.7 as compared to pH 8.2 across the experiment.
The average polonium activity in the fecal pellets collected from each of the three copepods show higher concentrations at lower pH of 7.7 with the exception of Parvocalanus on Isochrysis feed. The effect of pH for each combination of copepod and algal species is presented in Figure 4. A significant 3 to 4% increase in polonium activity is observed under pH 7.7 as compared to pH 8.2 across the experiment.
The relative activity of polonium in the copepods at the end of the experiment ranged between 0.97 and 1.64% of the total activity provided through food. This activity was significantly different between copepod species (ANOVA 3; model: F 17,53 = 27.48, p < 0.0001; copepod: F 2 = 56.14, p < 0.0001), algae species (F 2 = 99.62, p < 0.0001), pH (F 1 = 86.74, p < 0.0001), and the interaction between copepod and algae species (F 4 = 14.44, p < 0.0001). The general trend was a higher polonium concentration at lower pH of 7.7 with the exception of Acartia and Euterpina on Isochrysis. The effect of pH for each combination of copepod and algae species is presented in Figure 5.  The relative activity of polonium in the copepods at the end of the experiment ranged between 0.97 and 1.64% of the total activity provided through food. This activity was significantly different between copepod species (ANOVA 3; model: F17,53 = 27.48, p < 0.0001; copepod: F2 = 56.14, p < 0.0001), algae species (F2 = 99.62, p < 0.0001), pH (F1 = 86.74, p < 0.0001), and the interaction between copepod and algae species (F4 = 14.44, p < 0.0001). The general trend was a higher polonium concentration at lower pH of 7.7 with the exception of Acartia and Euterpina on Isochrysis. The effect of pH for each combination of copepod and algae species is presented in Figure 5. Significant difference between pH treatments are indicated with * (ANOVA; p < 0.05) and ** (p < 0.01).

Discussion
The average 209 Po concentration in fecal pellets of Parvocalanus crassirostis were 9.26 and 9.09 Bq g −1 , 7.50 and 7.58 Bq g −1 , and 5.01 and 5.06 for Isochrysis galbana, Chaetoceros muelleri, and Dunaliella salina feeding at 8.2 and 7.5 pH ( Figure 6). The average concentrations for Isochrysis galbana were skewed because of a single outlier, on the other three occasions the concentration were 8.80 Bq g −1 . The 209 Po excretion with fecal pellets was 1-2% higher at lower pH in Parvocalanus crassirostis.
The average 209 Po concentration in fecal pellets of Euterpina acutifrons, at 8.2 and 7.7 pH were 8.79 and 8.85 Bq g −1 , 7.50 and 7.56 Bq g −1 , and 5.01 and 5.03 Bq g −1 , for Isochrysis galbana, Chaetoceros muelleri, and Dunaliella salina ( Figure 6). The values were lower at 7.7 pH but the difference in 209 Po concentrations were between 0.5-0.8% among the algal food sources.
The average 209 Po concentration in fecal pellets was 0.5-2 % higher at 7.7 pH compared to 8.2 pH with the single exception of Parvocalanus crassirostis ingesting Isochrysis galbana ( Figure 6). The experimental data generated in this study supports the hypothesis that metal bioavailability is likely to change under an ocean acidification scenario. We Figure 5. Effect of pH on polonium activity (in %) in the copepod for each combination of copepod species (Acartia pacifica, Euterpina acutifrons, and Parvocalanus crassirostis) and microalgae species (Chaetoceros muelleri, Dunaliella salina, and Isochrysis galbana). Results are presented as mean ± SEM. Significant difference between pH treatments are indicated with * (ANOVA; p < 0.05) and ** (p < 0.01).

Discussion
The average 209 Po concentration in fecal pellets of Parvocalanus crassirostis were 9.26 and 9.09 Bq g −1 , 7.50 and 7.58 Bq g −1 , and 5.01 and 5.06 for Isochrysis galbana, Chaetoceros muelleri, and Dunaliella salina feeding at 8.2 and 7.5 pH ( Figure 6). The average concentrations for Isochrysis galbana were skewed because of a single outlier, on the other three occasions the concentration were 8.80 Bq g −1 . The 209 Po excretion with fecal pellets was 1-2% higher at lower pH in Parvocalanus crassirostis.
The average 209 Po concentration in fecal pellets of Euterpina acutifrons, at 8.2 and 7.7 pH were 8.79 and 8.85 Bq g −1 , 7.50 and 7.56 Bq g −1 , and 5.01 and 5.03 Bq g −1 , for Isochrysis galbana, Chaetoceros muelleri, and Dunaliella salina ( Figure 6). The values were lower at 7.7 pH but the difference in 209 Po concentrations were between 0.5-0.8% among the algal food sources.
Among Acartia pacifica fecal pellets the 209 Po concentration varied between 8.80 and 8.83 Bq g −1 , 7.50 and 7.54 Bq g −1 , and 5.01 and 5.04 Bq g −1 for Isochrysis galbana, Chaetoceros muelleri, and Dunaliella salina feeds at 8.2 and 7.5 pH, respectively. The 209 Po fecal pellet concentration at 7.7 exceeds 8.2 by~0.5% among different feed types. draw a parallel with Fowler [71] who reported surface-bound Pb is unassimilated in zoo-plankton and is therefore eliminated in fecal pellets. We are speculating that this observation might also be the case with polonium in our experiment, which led to higher 209 Po in fecal pellets in the experiment and could be due to the fact that much of the 209 Po was bound to the surface of the ingested phytoplankton and might not have gone in the cytoplasm of the phytoplankton. The 209 Po bioaccumulation was higher in Parvocalanus crassirostis, Euterpina acutifrons, and Acartia pacifica at 7.7 pH compared to 8.2, with only the two exceptions of Acartia pacifica and Euterpina acutifrons feeding on Isochrysis galbana (Figure 7). The polonium retention efficiency was 0.97-1.64%, suggesting a lower assimilation of this non-essential actinide. It is interesting to compare the 210 Po in these copepods, the 210 Po concentration in Euterpina acutifrons, Acartia pacifica, and Parvocalanus crassirostis were 0.17-0.18 Bq g −1 (dw), 0.26-0.27 Bq g −1 (dw), and 0.61-0.65 Bq g −1 (dw), respectively, which is substantially lower than those observed in this experiment; however, the concentration factors for these organisms were 10 5 . Drawing inference from the fact that higher bioaccumulation was observed at lower pHs, we assume that CFs will be higher under acidifying conditions.  The average 209 Po concentration in fecal pellets was 0.5-2% higher at 7.7 pH compared to 8.2 pH with the single exception of Parvocalanus crassirostis ingesting Isochrysis galbana ( Figure 6). The experimental data generated in this study supports the hypothesis that metal bioavailability is likely to change under an ocean acidification scenario. We draw a parallel with Fowler [71] who reported surface-bound Pb is unassimilated in zooplankton and is therefore eliminated in fecal pellets. We are speculating that this observation might also be the case with polonium in our experiment, which led to higher 209 Po in fecal pellets in the experiment and could be due to the fact that much of the 209 Po was bound to the surface of the ingested phytoplankton and might not have gone in the cytoplasm of the phytoplankton.
The 209 Po bioaccumulation was higher in Parvocalanus crassirostis, Euterpina acutifrons, and Acartia pacifica at 7.7 pH compared to 8.2, with only the two exceptions of Acartia pacifica and Euterpina acutifrons feeding on Isochrysis galbana (Figure 7). The polonium retention efficiency was 0.97-1.64%, suggesting a lower assimilation of this non-essential actinide. It is interesting to compare the 210 Po in these copepods, the 210 Po concentration in Euterpina acutifrons, Acartia pacifica, and Parvocalanus crassirostis were 0.17-0.18 Bq g −1 (dw), 0.26-0.27 Bq g −1 (dw), and 0.61-0.65 Bq g −1 (dw), respectively, which is substantially lower than those observed in this experiment; however, the concentration factors for these organisms were 10 5 . Drawing inference from the fact that higher bioaccumulation was observed at lower pHs, we assume that CFs will be higher under acidifying conditions. The experimental data indicate that the ingested polonium was rapidly defecated by all the three copepods, and the sinking fecal pellets would depurate surface waters of polonium. In addition, the question remains, will theses fecal pellets become part of the food chain of detritivores. Unfortunately, in this experiment, we have not looked at the residence time and retention of polonium in the fecal pellets, which will be interesting to examine under ocean acidification conditions. There is a likelihood of polonium being retained in fecal pellets, which would result in relatively short residence times in surface waters.

Conclusions
This experimental study has provided data to underpin possible 210 Po transfer processes at the base of the marine food chain under ocean acidification conditions. Considering increased oceanic productivity under climate change, and significant bioaccumulation of polonium in microalgae from the Gulf, and even a higher concentration among 9  The experimental data indicate that the ingested polonium was rapidly defecated by all the three copepods, and the sinking fecal pellets would depurate surface waters of polonium. In addition, the question remains, will theses fecal pellets become part of the food chain of detritivores. Unfortunately, in this experiment, we have not looked at the residence time and retention of polonium in the fecal pellets, which will be interesting to examine under ocean acidification conditions. There is a likelihood of polonium being retained in fecal pellets, which would result in relatively short residence times in surface waters.

Conclusions
This experimental study has provided data to underpin possible 210 Po transfer processes at the base of the marine food chain under ocean acidification conditions. Considering increased oceanic productivity under climate change, and significant bioaccumulation of polonium in microalgae from the Gulf, and even a higher concentration among Gulf copepods, it is quite probable to observe a polonium reduction in surface water due to the bioconcentration mechanism and abundance of the phytoplankton and zooplankton. On the contrary, the higher polonium concentration and significant quantities of fecal pellet production in mass is likely to result in polonium enrichment in the deeper levels and in bottom sediments due to the downward sinking of fecal pellets. The likelihood of deep water and bottom-sediment enrichment becoming part of the bottom-dwelling marine organism's and detritivore's food chains should be considered and could result in an enhanced dose to these benthic organisms.
A 0.5-2% enrichment of 209 Po at lower pHs might result in considerable vertical flux considering that with the amount of fecal pellets produced over the life time of a copepod will be at least 2-3 orders of magnitude greater than its mass. The assimilation fraction of ingested polonium was 0.97-1.64% and a slightly higher bioaccumulation was noted at a lower pH of 7.7 compared to 8.2; however, this study also supports the hypothesis of negligible retention of actinides, unlike the retention of other metals such as Zn (47%), Cd (30%), and Ag (17%) [80].
Assuming a similar biological behavior of 209 Po and 210 Po, it is quite interesting to note that significantly higher 209 Po concentrations in the Parvocalanus crassirostis, Euterpina acutifrons, and Acartia pacifica under the experimental conditions compared to 210 Po in same species in their natural environment, where the enrichment was 100,000 times.
Not only was the assimilation rate higher at a lower pH, the excretion of polonium was also higher at 7.7 pH compared to 8.2 pH. Polonium elimination in fecal pellets of zooplankton is a well-established phenomenon and has been known for decades. The higher concentration of polonium in copepod fecal pellets at a lower pH is likely to result in the efficient depletion of polonium in surface waters and its downward vertical flux through the water column. The retention of polonium in fecal pellets was roughly comparable to the concentration in the copepods, but the production rate of fecal pellets is quite high. This study supports the hypothesis that primary producers are likely to provide an enhanced dose of polonium to primary consumers, such as crustacean zooplankton. It would be quite interesting to assess the concentration of polonium bound to surfaces compared to that integrated in cytoplasm in phytoplankton, as the two fractions are likely to result in different assimilation rates in copepods. Data Availability Statement: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Conflicts of Interest:
The authors declare no conflict of interest.