Testing algal-based pCO2 proxies at a modern CO2 seep (Vulcano, Italy)

Understanding long-term trends in atmospheric concentrations of carbon dioxide (pCO2) has become increasingly relevant as modern concentrations surpass recent historic trends. One method for estimating past pCO2, the stable carbon isotopic fractionation associated with photosynthesis (Ɛp) has shown promise over the past several decades, in particular using species-specific biomarker lipids such as alkenones. Recently, the Ɛp of more general biomarker lipids, organic compounds derived from a multitude of species, have been applied to generate longer-spanning, more ubiquitous records than those of alkenones but the sensitivity of this proxy to changes in pCO2 has not been constrained in modern settings. Here, we test Ɛp using a variety of general biomarkers along a transect taken from a naturally occurring marine CO2 seep in Levante Bay of the Aeolian island of Vulcano in Italy. The studied general biomarkers, loliolide, cholesterol, and phytol, all show increasing depletion in 13C over the transect from the control site towards the seep, suggesting that CO2 exerts a strong control on isotopic fractionation in natural phytoplankton communities. The strongest shift in fractionation was seen in phytol, and pCO2 estimates derived from phytol confirm the utility of this biomarker as a proxy for pCO2 reconstruction.

Scientific RepoRtS | (2020) 10:10508 | https://doi.org/10.1038/s41598-020-67483-8 www.nature.com/scientificreports/ where Ɛ f is the maximum isotopic fractionation due to CO 2 -fixation via the enzyme Rubisco, which has shown a sum range from 25 to 28‰ [17][18][19] . It should be noted that the very few in vivo Rubisco fractionation studies have much lower values 20,21 , which Wilkes and Pearson 22 suggest there may be due to multiple stages of fractionation instead of the singular Rubisco fractionation step. Several other studies have expanded on Eq. (1) for specific consideration, particularly in calculating b, e.g. instantaneous cell growth rate accounting for differences in photoperiod 23,24 and CO 2 fixation rate 25 .
Using the knowledge obtain from culture studies 26,27 , the measurement of Ɛ p in algal biomarkers preserved in the geologic record can be used to reconstruct past pCO 2 . These biomarkers are almost exclusively alkenones, long-chain unsaturated methyl and ethyl n-ketones produced by haptophytes 8,28,29 . Although this proxy has generated a large number of pCO 2 records [30][31][32] , there are several limitations, such as the exceptionally low Ɛ f recorded for the alkenone-producer Emiliania huxleyi of 11‰ 20 , a potential insensitivity of this proxy at low CO 2 levels 24,33 , and difficulties in constraining the b factor over time 34 . One other limitation is the fact that alkenones first commonly appeared in the geologic record ca. 45 million years (Ma) ago 35 , prohibiting pCO 2 reconstructions prior to this time.
As an alternative, the isotopic fractionation of general phytoplankton biomarkers, compounds that are produced by a multitude of species, may offer some solutions to the limitations of the alkenone pCO 2 proxy such as more spatial ubiquity and temporal longevity. This general biomarker approach has been poorly explored; however, though there are some examples of this being applied to phytane, a diagenetic product of omnipresent chlorophyll-a, for periods extending beyond the alkenone record, i.e. in the Cretaceous [36][37][38] and in a Phanerozoic compilation 39 . However, this general biomarker approach has not been extensively tested in laboratory cultures or present-day environments.
For modern studies of the general biomarker approach, naturally-occurring phytoplankton communities are necessary to mimic the widespread contributors to general phytoplankton biomarkers, as opposed to the typical single-species approach of laboratory cultures. Mesocosm experiments may offer more natural environmental conditions and communities, though none have been conducted on general phytoplankton biomarkers for pCO 2 reconstructions. Alkenones and particulate organic carbon (POC) have been explored in one mesocosm experiment using natural communities, i.e. under three pCO 2 conditions in a contained area for ca. 21 days 40 . These authors suggested the minor changes they observed in δ 13 C values for alkenones and POC indicate that fractionation is not primarily controlled by CO 2 concentrations but instead by algal growth rate and carbon-uptake mechanisms. However, these experiments are inherently difficult to set-up, reproduce, and control.
Here we expand this new approach to testing pCO 2 response in natural phytoplankton communities, by analyzing the response of isotopic fractionation in general phytoplankton biomarkers across a CO 2 gradient at a naturally occurring CO 2 seep. CO 2 seeps, which consistently bubble CO 2 into the surrounding environment and thus have very high CO 2 concentrations near the seep, have hardly been explored for biological studies due to the assumed high sulfide concentrations, toxic to many organisms, typically associated with volcanic degassing 41 . However, Hall-Spencer et al. 42 used these extremely high pCO 2 environments for ocean acidification experiments, which lead to studies at other seep sites, i.e. Italy 43 , Papua-New-Guinea 44 , New Zealand 45 , and Japan 46 . The new approach was initially tested with a 3-point transect (high, mid, and control pCO 2 ) of a marine CO 2 seep site on Shikine Island, Japan, covering a range of CO 2 concentrations that offer an analogue for past oceans 39 . However, this specific site proved to have confounding factors where the imprint of CO 2 on Ɛ p measured in general biomarkers of surface sediment was masked by extreme weather events (i.e. typhoons) that caused sediment transport.
Here, we more thoroughly explore this new approach at a different marine CO 2 seep system approximately 30 m into Levante Bay at Vulcano Island, Italy, a location with much more stable weather conditions than Japan. We collected surface sediments in a high-resolution 16-point transect from high CO 2 towards ambient CO 2 values. Here, we analyzed the Ɛ p of several general phytoplankton biomarkers, compounds that have been virtually unstudied in modern phytoplankton communities, deposited in surface sediments and tested their response to the CO 2 gradient at sixteen sites throughout the bay.

Results
For this study, we collected surface sediments in May and October from close to the seep site (ca. 3 m distance) to a control site unaffected by the seep 47 at a constant depth of ca. 1.5 m at the time of sampling (Fig. 1). The δ 13 C of DIC measured in seawater collected in May from the bay does not show notable change over the gradient of CO 2 (Table S1), which confirms that lack of change noted in the literature 48 . For this reason, we averaged the δ 13 C of DIC measured in our study with that of Cornwall et al. 49 across all sites (0.7‰ ± 0.4‰ s.d.) and assumed this to be representative for the bay region.
All three biomarkers show a steady increase in δ 13 C values over the transect from the CO 2 seep towards the control site ( Fig. 3; Table S2). The exceptions are the more depleted δ 13 C values at Site 2 and Site 9, where we observed some minor gas bubbling in the sediment, suggesting the release of small amounts of CO 2 at these sites. Over the transect from Site 1 (the seep) to Site 16 (the control), the δ 13 C of loliolide ranges from − 27.4 to − 21.6‰ (Fig. 3A). From the seep to around Site 10, the δ 13 3C). There is a relatively consistent increase in the δ 13 C of phytol over the entire transect, except for a small decrease at Site 9, where we observed minor additional gas bubbling in the sediment. The δ 13 C of phytol shows minor variation between seasons (ca. 0.5%), except for the control site which showed a difference of 1.4‰.

Discussion
The three most abundant biomarkers, loliolide, cholesterol, and phytol, are all derived from phytoplankton and represent broad phytoplankton groups [51][52][53] . Composition of the diatom assemblages and cyanobacteria in this bay are further described in Johnson et al. 43 All become increasingly enriched in 13 C over the transect from high CO 2 concentrations near the seep to the control Mediterranean values. The observed isotopic depletion that occurs with increasing CO 2 concentrations matches theory 5,6,54 . Furthermore, this pattern closely follows the results observed at Shikine Island, i.e. a consistent depletion δ 13 C of the same biomarkers with increasing proximity to the CO 2 seep 50 , but here offered in a 16-point transect instead of the 3-points at the Japan site. Given that CO 2 was the major variable over the transect in Italy, as well as Shikine Island, this strongly suggests that CO 2 concentrations indeed have a strong impact on isotopic fractionation of general phytoplankton biomarkers, suggesting their potential as a pCO 2 proxy. Although the general trends between the two CO 2 seep sites are similar, there is a difference in the magnitude and consistency in isotopic changes between the two sites. In the Shikine Island study, loliolide showed the largest isotopic shift over the transect (− 7.9‰) as compared with phytol (− 5.2‰) and cholesterol (− 5.2‰). However, in the Vulcano Island surface sediments, phytol had the most pronounced isotopic shift (− 8.0‰) as compared with loliolide (− 5.8‰) and cholesterol (− 5.1‰). Furthermore, the changes in loliolide over the Vulcano Island transect are more variable compared with the consistent trends in isotopic values observed in phytol and cholesterol. Here, we will explore these differences.
The δ 13 C profile of loliolide at Vulcano Island (Fig. 3A) has the least consistent trend among the three biomarkers, fluctuating between − 27.4 and − 25.0‰ from Site 1 (the seep) to Site 13. Loliolide is derived from the major xanthophyll fucoxanthin and is considered a biomarker for diatoms, especially in the absence of haptophyte algae 51,55 , based on its predominance at sites with substantial diatom communities, although some other non-diatom species also produce fucoxanthin 56 . Light microscopy analysis of selected sediments across the transect showed that Site 2 contains nearly no diatom frustules, Site 5 had abundant centric diatoms as well as some pennate diatoms, while Site 9 is characterized by a great diversity especially among pennate diatoms though with relatively low overall abundance, and Site 13 and Site 16 (control site) had both high abundance and high diversity of both centric and pennate diatoms (Stoll H. and Mejía Ramírez L. M., personal communications). Decreased diversity in increased proximity to the seep has previously been observed in periphytic diatom www.nature.com/scientificreports/ assemblages at this site 43 , though with a drastic increase abundance in chlorophyll-a by ca. fivefold from Site 6 to 16. Johnson et al. suggest that the increase abundance but decreased diversity is due to some diatoms benefitting from increasing CO 2 through a reduction in the energetic costs of their CCMs 43 . The different composition of diatoms at each site, particularly between centric and pennate diatoms, may explain why we observe a high δ 13 C variability in loliolide. Different species may have slightly different isotopic fractionation due to e.g. different cell geometry and morphologies 11 or different bicarbonate pumping strategies that has been observed in diatom species [57][58][59] . This concept may be further supported by the stronger increase in δ 13 C values observed between sites 13 and 16, where the higher diversity of species may yield a more robust overall δ 13 C signal through averaging biosynthetic differences among species. This complexity in the signal of loliolide may weaken the potential of this biomarker for past pCO 2 reconstructions. The δ 13 C profile of cholesterol (Fig. 3B) showed a more consistent decline over the transect than loliolide, though with a smaller difference in absolute values than phytol and loliolide from the seep towards the control. Because cholesterol is produced by all eukaryotes, such as phytoplankton or by heterotrophs, which modify ingested sterols 52,60 , terrestrial input, in addition to the algal input, can potentially dilute the autochthonous isotopic signal. However, the lack of terrestrial triterpenoids and long-chain (> C 22 ) even carbon number fatty alcohols (Fig. 2) suggest minimal input of terrestrial biomarkers in the bay. Another explanation for the smaller isotopic change is that the cholesterol has contributions from heterotrophs, which produce cholesterol by modifying ingested phytoplanktonic sterols. Although this does not yield large isotopic fractionation 61 , the zooplankton often have stronger mobility than their photoautotroph counterparts; they may consume phytoplankton from various locations (and consequently various CO 2[aq] concentrations) throughout the bay. This idea is supported by the notable δ 13 C differences in cholesterol between the two seasons, where the offsets are not consistently in one direction. Based on these observations the δ 13 C of cholesterol must be considered carefully when used in reconstructing past CO 2 concentrations.
The δ 13 C profile of phytol had the most robust trend across the transect (Fig. 3C) with an δ 13 C enrichment of ca. 8‰ from the seep to the control. Phytol, derived from chlorophyll-a, is found in all oxygenic photoautotrophs 53 . Terrestrial input may affect the signal of phytol but, as discussed above, there is no evidence for this here. Based on these results, phytol shows the greatest sensitivity to the CO 2 gradient, and thus the most promise for reconstructing past pCO 2 . The phytol results from Shikine, Japan 50 likewise show great promise for reconstructing past pCO 2 .
To test the validity of using the δ 13 C of the general biomarkers to estimate past pCO 2 , we used phytol, the most promising of the various general phytoplankton biomarkers explored here with the most consistent trend and the strongest δ 13 C shift over the gradient. We calculated the stable carbon isotopic photosynthetic fractionation (Ɛ p ) using the δ 13 C of phytoplankton biomass (δ p ) and the δ 13 C of CO 2 (δ d ): The δ p is calculated from the offset between phytol and biomass, which is 3.5‰ ± 1.3 standard deviation based on the average of 23 representative marine phytoplankton species grown in cultures 39 . The δ d is calculated from the δ 13 C of DIC (0.7‰ ± 0.4‰ s.d.) correcting for temperature and pH (Table S1). The mean annual sea surface temperature for Vulcano Island (20.2 ºC ± 0.5 °C s.d.; https ://www.seate mpera ture.info) was used to calculate the temperature-dependent carbon isotopic fractionation of CO 2[aq] with respect to HCO 3 -62 . The pH gradient, ranging from 5.5 pH near the vent to 8.2 pH in the control 63 , was used to define the relative contribution of different inorganic carbon species to the measured DIC 64 (Table S1). Uncertainty was calculated using Monte Carlo simulations which consider the culmination of each individual parameter with its associated uncertainty, as described by Witkowski et al. 39 , here including δ 13 C of phytol ± 0.5‰ s.d., offset between biomass and phytol ± 1.3‰ s.d., δ d ± 0.4‰ s.d., and T °C ± 0.5 °C (Table S2). This uncertainty has an equal effect on the final uncertainties in calculated ε p , i.e. 0.1‰ error in the δ d will lead to a 0.1‰ error in ε p 39 . Phytol-derived Ɛ p ranges from 22.2 to 8.2‰ ± 1.4‰ s.d. (Fig. 4A) and shows a consistent decline in fractionation from the seep towards the control site. This includes Site 2 where measured δ 13 C values are higher than at Site 1, but Ɛ p now shows the expected trend of more fractionation closer to the vent. This is attributed to the strong shift in pH between these two sites (5.5 pH at the vent and 6.25 pH at Site 2 63 ) which we have here corrected for. The highest Ɛ p value of 22.2‰ near the seep is approaching maximum isotopic fractionation due to CO 2 -fixation (Ɛ f ), which has been shown to range between 25 and 28‰ in laboratory cultures 18 , but still does not quite reach full expression of Ɛ f . This is somewhat unexpected given the constant bubbling of CO 2 at this site and thus very high CO 2 concentrations, i.e. up to ca. 3× modern CO 2[aq] 43 . Several possibilities may explain why the full expression of Ɛ f has not been reached. For one, given the relatively small area of the bay, it is possible that surface sediment has moved around the bay over time due to tidal actions and bottom water currents, which dampens the overall signal by allochthonous organic matter transported from area's outside of the bay, as also inferred for Shikine Island 50 . Furthermore, algae are unlikely to grow and deposit in precisely same location and given that the impact of the CO 2 seep noticeably changes over tens of meters 43 , this likely leads to some mixed signal among sites, resulting in a suppressed signal. Another alternative is that the calculated Ɛ f of the phytoplankton community in Levante Bay may be lower than that inferred from the many culture studies 11,17,65 . Indeed, several recent studies show that Ɛ f of the different Rubisco types may be lower than previously assumed 66 .
In order to see how well Ɛ p of phytol can reconstruct CO 2[aq] , we estimated CO 2[aq] and pCO 2 from the δ 13 C of phytol using the equation adapted from the high plant model 5 for algae 7 , and described in Eq. (1) 17 , where b reflects species carbon demand per supply 8 and Ɛ f reflects the maximum isotopic fractionation due to CO 2 -fixation. The value of b is a complicated catchall for factors influencing isotopic fractionation such growth rate and cell-size 67 , light intensity and membrane leakiness 24,68 , further complicated due to the multitude www.nature.com/scientificreports/ of sources for general phytoplankton biomarkers. Studies have suggested an empirical average 170‰ kg µM −1 ± 43 kg µM −1 s.d. for b based on a compilation of δ 13 C values of bulk organic matter in marine surface sediments, as well as some limited phytol studies 39,50 . Furthermore, we use an average Ɛ f for phytoplankton species of 26.5‰ ± 1.5‰ uniform distribution 39 based on the 25 to 28‰ range observed in laboratory cultures 69 . As described above, uncertainty was calculated using Monte Carlo simulations, considering each individual parameter with its associated uncertainty, as described by Witkowski et al. 39 . Here, we include the uncertainties associated with Ɛ p plus the new additional uncertainties associated with b ± 43 kg µM −1 s.d., Ɛ f ± 1.5‰ uniform distribution, T °C ± 0.5 °C s.d., and sea surface salinity ± 1‰ s.d.
The resulting phytol-based CO 2[aq] values range from 9.3 to 39.4 µM (Fig. 4B). The highest value of 39.2 µM (+ 20.6/− 11.0 µM) is near the vent at Site 1, dropping to 23.7 µM (+ 7.1/− 5.2 µM) at Site 2, then to 14.3 µM (+ 3.0/− 2.7 µM) at Site 3, before gently declining to 9.6 µM (± 1.8 µM) at the control Site 16. If we calculate the pCO 2 from CO 2[aq] using Henry's Law constant K 0 , which considers salinity and temperature 70 , the resulting pCO 2 reconstruction range from 280 to 1,182 µatm (Fig. 4C) Comparison of CO 2[aq] estimates with those reported for sites 63 equivalent of our Site 2, 9, and 16 (30 µM ± 7, 14 µM ± 1, and 12 µM ± 1, respectively; Fig. 4B), show that these estimates agree within uncertainty, suggesting that our approach yields reasonable estimates. Only at the control site there is a slight underestimation of CO 2 concentrations. One possible explanation is an incorrect assumption for the b value. However, this seems unlikely given that (i) b values would need to be increased beyond any known b value thus observed to account for this underestimation, and (ii) this would lead to even higher past pCO 2 estimations which are based on b values compiled from laboratory cultures and natural experiments 39 . A more likely explanation is the change in phytoplankton community over the bay, where the control community is dominated by high affinity CCM species as observed for macroalgae 49 . Given that these species actively pump bicarbonate under low CO 2 conditions, this may explain the lessened Ɛ p , yielding lower CO 2 estimations. This effect has also been observed in the mesocosm experiments with different CO 2 concentrations 40 , especially if there is limited carbon dioxide leakage from cells. Recent studies have shown lower sensitivity of Ɛ p to CO 2 in laboratory cultures and in glacialinterglacial reconstructions caused by the upregulation of phytoplankton CCMs 24,33 , which suggest using this Ɛ p based proxy with caution in reconstructing low-CO 2 worlds. In contrast, the proxy seems to do well in estimating pCO 2 concentrations similar to some of the higher concentrations that have been reconstructed over the past 455 Myr 39 , suggesting it may be applicable for past greenhouse worlds.

conclusion
We tested three general phytoplankton biomarkers in surface sediments in a transect from a naturally occurring CO 2 seep located in Levante bay, Vulcano Island, Italy, towards the open Tyrrhenian Sea. The δ 13 C of the biomarkers showed a distinct increase with increasing distance from the CO 2 seep, in agreement with the idea that CO 2 concentrations have a strong control on isotopic fractionation. In particular, the δ 13 C of phytol yielded a strong and consistent trend throughout the transect, and the agreement between estimated and measured CO 2 concentrations demonstrates the promise of this biomarker for paleo pCO 2 reconstructions. Our results show that CO 2 seep environments may prove a useful testing ground for new CO 2 proxies.

Materials and methods
Sample site. Levante Bay (Fig. 1) is located on the northeast of Vulcano Island, an Aeolian Island north of Sicily. Volcanic activity on the island started in the upper Pliocene 71 , where the cooling of magmatic and hydrothermal fluid mixing into the crater fumeroles is believed to have created the pocket of CO 2 , which outgasses into the bay 72 . Located at ca. 1 m depth at 38.41694° N 14.96° E, the main underwater venting gas field outputs ca. 3.6 tons of gas per day 73 . This gas is composed of 97-98% CO 2 and ca. 2% H 2 S 63 . The sea water temperature 63 of ca. 19.7 °C and salinity 43 of ca. 38‰ is homogenous throughout the small bay. Currents are mostly wind-driven, with minimal tidal range (< 40 cm) and depths throughout the entire bay, and thus all sample sites, ranged between 1 and 2 m. Precipitation varies throughout the year, with the dry months (May-August) averaging 16 mm/month and the wet months (October-January) averaging 87 mm/month. The input of CO 2 gas intensely influences the geochemical composition of the bay's waters, as seen by the strong pH gradient starting at the seep to across the bay from pH 5.5 to 8.2 in April and from pH 6 to 8 in September. For more details on the geochemistry, see Boatta et al. 63 .

Materials
Samples were collected in 23-24 May and 16-17 October of 2017. A preliminary study was conducted in May using one site with a high CO 2 concentration, two sites with a middle CO 2 concentration, and one control site (i.e. not affected by the CO 2 seep) as defined in Johnson et al. 43 , where seawater was collected for the δ 13 C of dissolved inorganic carbon (DIC) and surface sediments were collected for the δ 13 C of biomarker lipids. Seawater for DIC analysis was collected by overfilling glass vials and adding mercury chloride (0.5%) before sealing the vials closed with Apiezon M grease and securing the stopper with rubber bands. Surface sediments were collected by diving, scooped into geochemical bags, and immediately frozen; once back in the lab, these sediments were freeze-dried and kept refrigerated. All surface sediments were collected in triplicate at each site within a square of 2 by 2 m. The same sediment sampling method was used again in October, when a higher-resolution transect of sixteen sites was collected (Fig. 1). Given that the results of the δ 13  www.nature.com/scientificreports/ throughout the bay (see Table S1), as also revealed by another study in this region 48 , seawater samples were not collected in October.

Methods.
The δ 13 C of DIC of seawater collected in May was measured on a gas bench coupled to an isotope ratio mass spectrometer (IRMS) in duplicate. Samples were prepared using 100 µL of 85% H 3 PO 4 then flushed with He. Seawater (500 µL) was injected to each vial, left to react for 1 h, and then the headspace was measured. Standards prepared with 0.3 mg of Na 2 CO 3 and 0.4 mg of Ca 2 CO 3 were flushed with He, injected with 100 µL of 85% H 3 PO 4 , and reacted for 1 h. The standards were run at the start and end of each sequence, as well as every six runs. Sediments were freeze-dried and homogenized using a mortar and pestle. Sediments were then extracted using a Dionex 250 accelerated solvent extractor at 7.6 × 106 Pa at 100 °C using dichloromethane (DCM): MeOH (9:1 v/v). Extracts were transferred to centrifuge tubes to be refluxed with 1 N KOH in MeOH and the resulting base hydrolyzed extracts were neutralized to pH 5 using 2 N HCl in MeOH. The hydrolyzed extract was separated into apolar (hexane: DCM, 9:1 v/v), ketone (DCM), and polar (DCM: MeOH, 1:1 v/v) fractions, respectively, over an alumina column. Polar fractions were silylated with pyridine: N,O-Bis(trimethylsilyl) trifluoroacetamide (1:1 v/v) and heated for 1 h at 60 °C. The δ 13 C values of loliolide, cholesterol, and phytol were corrected for the addition of three C atoms in the trimethylsilyl group using the known δ 13 C value of BSTFA (− 32.2‰).
Silylated polar fractions were then injected on gas chromatography-flame ionization detector (GC-FID) to determine relative abundances and general quality of chromatography before analyzing it on a gas chromatography-mass spectrometer (GC-MS) to identify compounds and on gas chromatography-isotope ratio-mass spectrometer (GC-IRMS) to measure the isotopic composition of specific compounds. GC-FID, GC-MS, and IRMS instrumentation all had starting oven temperatures of 70 ºC ramped at 20 ºC/min to 130 ºC and then ramped at 4 ºC/min to 320 ºC for 10 min. Separation was accomplished using a CP-Sil 5 column (25 m × 0.32 mm; df 0.12 μm) with He carrier gas. System performance on all three instruments was conducted daily using the same in-house mixture of n-alkanes and fatty acids. Additional standards were run on the IRMS using perdeuterated n-alkanes (C 20 and C 24 ) with known δ 13 C values (− 32.7 and − 27.0‰, respectively) and were limited to uncertainty within the standard of ± 0.5‰; if outside this range, the machine was conditioned until it was within this limit. The IRMS was also oxidized regularly, with a daily oxidation of 10 min, backflushed with He for 10 min, and purged for 5 min; a shorter version of this sequence was conducted in post-sample seed oxidation, which includes 2 min oxidation, 2 min He backflush, and 2 min purge conditioning line and a longer version of this sequence was conducted at the end of each week with 1 h oxidation, 1 h He backflush, and 10 min purge conditioning line.

Data availability
All data are present in the paper and/or the Supplementary Materials.