Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications

Summary

The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry.

Abstract

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model.

One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS).

ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.

Introduction

Metals and metal-based compounds continue to have pharmacological and toxicological relevance. The prevalence of heavy metal exposure and its impact on health has exponentially increased scientific investigation since the 1960s and reached an all-time high in 2021. The concentrations of heavy metals in drinking water, air pollution, and occupational exposure exceeds regulatory limits worldwide and remain an issue for arsenic, cadmium, mercury, chromium, lead, and other metals. Novel methods to quantify environmental exposure and analyze pathological development continue to be in high demand^1,2,3.

Conversely, the medical field has harnessed the physiochemical properties of various metals for clinical treatment. Metal-based drugs or metallodrugs have a rich history of medicinal purposes and have shown activity against a range of diseases, with the highest success as chemotherapeutics⁴. The most famous of metallodrugs, cisplatin, is a Pt-based anticancer drug deemed by the World Health Organization (WHO) as one of the world's essential drugs⁵. In 2010, cisplatin and its Pt derivatives had up to a 90% success rate in several cancers and were used in approximately 50% of chemotherapy regimens^6,7,8. Although Pt-based chemotherapeutics have had irrefutable success, the dose-limiting toxicity has set in motion investigations of alternative metal-based drugs with refined biological delivery and activity. Of these alternatives, Ru-based compounds have become the most popular^9,10,11,12.

Novel models and methodology are required to keep pace with the rate of need for metal pharmaco- and toxicokinetic studies. The zebrafish model lies at the intersection of complexity and throughput, being a high-fecundity vertebrate with 70% conserved gene homology¹³. This model has been an asset in pharmacology and toxicology, with extensive screenings for various compounds for lead discovery, target identification, and mechanistic activity^14,15,16,17. However, high-throughput screening of chemicals typically relies on waterborne exposures. Given that uptake can be variable based on the physicochemical properties of the compound in solution (i.e., photodegradation, solubility), this can be a major limitation of correlating dose delivery and response.

To overcome this limitation for comparison of dose to higher vertebrates, a methodology was designed to analyze trace metal concentrations in zebrafish larval tissue. Here, dose-response curves of lethal and sublethal endpoints were evaluated for cisplatin and novel Ru-based anticancer compounds. Lethality and delayed hatching were evaluated for nominal concentrations of 0, 3.75, 7.5, 15, 30, and 60 mg/L cisplatin. Pt accumulation in organism tissue was determined by ICPMS analysis, and organism uptake of respective doses were 0.05, 8.7, 23.5, 59.9, 193.2, and 461.9 ng (Pt) per organism. Additionally, zebrafish larvae were exposed to 0, 3.1, 6.2, 9.2, 12.4 mg/L of PMC79. These concentrations were analytically determined to contain 0, 0.17, 0.44, 0.66, and 0.76 mg/L of Ru. This protocol also allowed for the distinguishment of concentrations of Pt sequestered in the chorion of the larvae compared with the zebrafish tissue. This methodology was able to provide reliable, robust data for comparisons of pharmaco- and toxicokinetic activity between a well-established chemotherapeutic and a novel compound. This method can be applied to a wide range of metals and metal-based compounds.

Protocol

The AB strain zebrafish (Danio rerio) were used for all experiments (see the Table of Materials), and the husbandry protocol (#08-025) was approved by the Rutgers University Animal Care and Facilities Committee.

1. Zebrafish husbandry

1. Breed and maintain the zebrafish in a recirculating aquatic habitat system on a 14 h light:10 h dark cycle.
   1. Purify municipal tap water through sand and carbon filtration to obtain fish system water. Maintain the aquatic system water at 28 °C, <0.05 ppm nitrite, <0.2 ppm ammonia, and pH between 7.2 and 7.7.
   2. Feed the zebrafish a diet of hatched Artemia cysts, brine shrimp, and fish diet flake food.

2. Zebrafish dose-response protocol (Figure 1)

1. Prepare zebrafish egg water solution, either E3 medium or egg water made from stock sea salt at a concentration of 60 µg/mL dissolved in deionized water¹⁸. Avoid the use of methylene blue.
   NOTE: Through ICPMS, isobaric interference of zebrafish egg water was identified for strontium oxides, which overlapped with an isotope of ruthenium. Careful rinsing of the larvae before downstream analysis ameliorated this issue. E3 medium may be an easier choice for some due to the proprietary makeup of commercial sea salts.
2. Dissolve the metal or metal-based compounds in E3 or egg water. Vortex to break up any aggregate material and homogenize the solution.
   NOTE: In the experiment outlined below, PMC79 and cisplatin were dissolved at maximum concentrations of 12.4 mg/L and 60 mg/L with a maximum concentration of 0.5% dimethyl sulfoxide (DMSO) to resist precipitation.
   1. Dilute heavy metal or metal-based compounds, such as PMC79 and cisplatin, with E3 or egg water, preparing at least 5 concentration doses.
      1. Begin with low concentrations of stock solutions in pure vehicle (i.e., DMSO), then dilute with E3 or egg water. Consider the final vehicle concentrations carefully.
         NOTE: Certain metal-based compounds, such as cisplatin, degrade rapidly, and their solutions must be made fresh daily. CAUTION: Handle heavy metals and chemotherapeutics with care. Review the specific material safety data sheet (MSDS) for the metal of interest. Cisplatin may cause eye and skin irritation, be fatal if swallowed, and can cause toxicity in kidneys, blood, blood-forming organs, and fetal tissue. Avoid breathing fumes and contact with eyes, skin, and clothing. Wear impervious gloves and clothing, and safety glasses or goggles¹⁹.
3. Set up breeding tanks the afternoon prior to the experiment in the ideal ratio of 2 females to 1 male with a divider in place between the sexes²⁰.
   1. Pull the divider when the lights come on for the morning cycle.
   2. Allow the zebrafish to breed.
      NOTE: The length of time for breeding depends on the initial exposure stage required. For 3 h postfertilization, allow breeding for approximately 2 h. The eggs will reach 3 hpf after cleaning and segregating eggs.
   3. Move the breeding fish to a clean tank.
   4. Collect the eggs by pouring the tank water through a strainer.
   5. Invert the strainer over a Petri dish and use a squirt bottle filled with E3 or egg water to rinse the eggs into the dish.
   6. Clean the dish of food and waste prior to experimental use.
4. Randomize approximately twenty 3 hpf embryos per dose into individual glass vials using a transfer pipette and a small quantity of water.
5. After all embryos are in vials, remove all egg water and replace with enough dosing solution so that there is approximately 1 inch of solution above the height of the egg.
   NOTE: The necessity of dechorionation should be carefully considered. See the discussion section for more details.
6. Observe the embryos daily for lesions or lethality. Due to the rapid development of embryonic zebrafish, obtain daily images using any brightfield microscope/camera setup to identify minor lesions between days.
7. At the termination of the dose response (4-5 days post fertilization [dpf], per Organization Economic Cooperation and Development [OECD] guideline²¹), combine 3-5 larvae prior to euthanization for composite sampling. Euthanize by rapid cooling by snap-freezing in liquid nitrogen.
   NOTE: Euthanasia through an overdose of MS-222 or tricaine methanesulfonate may potentially interfere with ICPMS analysis downstream. Rapid-cooling euthanasia methods are encouraged for this protocol to reduce the possibility of interference.
8. Conduct 3 washes of tissue with high-purity water (such as reverse osmosis) to remove the excess compound from the exterior of the tissue.
9. Move the samples to acid- and microwave-safe 15 mL polypropylene centrifuge tubes. Be careful to remove all excess water as any remaining liquid may dilute the nitric acid, and therefore, the oxidizing potential of the acid during tissue degradation.
   NOTE: At this point, tissue may be stored at -20 °C until further analysis. For naturally abundant metals, cleaning the tubes in a 5% nitric acid bath prior to use will ameliorate ambient background levels.

3. Tissue digestion and ICPMS evaluation (Figure 2)

1. Add approximately 0.25 mL to the 15 mL polypropylene centrifuge tubes for up to 10 larvae (~100 µg) of high-purity nitric acid (69%). Ultrasonicate for 1 h to predigest the samples using the following settings: ultrasonic bath output: 85 W; 42 kHz ± 6%; temperature range: 19-27 °C.
   CAUTION: Wear ear protection during sonication. Nitric acid causes serious respiratory tract, eye, and skin burns. Wear full protective equipment and work in the fume hood or in places with adequate ventilation. It can be flammable with other materials. Nitric acid should be handled exclusively in a fume hood to prevent exposure to vapors produced during digestion. Do not inhale or ingest.
   1. Perform short cycles of tissue digestion (5 min intervals) in an acid-safe microwave digester until all tissue is visibly oxidized (i.e., uniform, clear-yellow solution).
      NOTE: The microwave protocol in Table 1 is recommended to be performed three times and includes a 5 min cooldown interval between each heating step.
   2. Monitor the integrity of the tubes carefully to avoid rupture and conduct short spins in the centrifuge (313 × g for 1 min) between cycles to move acid condensation to the bottom of the tube.
      NOTE: If the tissue is difficult to digest (especially chorions), 30% high-purity hydrogen peroxide can be used after acid digestion. Use the hydrogen peroxide (6.75 mL) to dilute the acid concentration to 3.5%, and allow the samples to sit overnight in a fume hood. Hydrogen peroxide will decompose to H₂O and is suitable for ICPMS analysis. Further, alternative metals may dissolve better in hydrochloric acid or a mixture of hydrochloric acid and nitric acid (i.e., aqua regia). Hydrogen peroxide is harmful if swallowed and causes serious eye damage. Wear skin and eye protection^22,23.
2. Once the tissue is visibly oxidized (i.e., uniform, clear-yellow solution), dilute the samples in a fume hood to 3.5% nitric acid using 6.75 mL of high-purity water and vortex to mix thoroughly.
   NOTE: At this point, the samples can be stored at room temperature. This step is unnecessary if hydrogen peroxide was added to aid digestion in step 3.1.
3. Conduct a matrix-matched, 7-point calibration curve (concentration range of 0.001-10 ppb) using a certified elemental standard (i.e., Pt or Ru, depending on the assay) with the metal of interest and optimal isotope(s) to account for any potential isobaric interferences.
   1. Using a stock concentration of the aqueous, certified elemental standard (Ru, Pt = 1000 ppb), take a 0.1 mL aliquot and pipet into a new 15 mL centrifuge tube. Dilute with 3.5% nitric acid to a final volume of 10 mL to produce a 10 ppb standard solution.
   2. Using the 10 ppb stock, make the following serial dilutions: 0.1, 1.0, and 5.0 ppb standard solutions in 3.5% nitric acid.
   3. Using the 0.1 stock, make the following serial dilutions: 0.001, 0.005, and 0.01 ppb standard solutions in 3.5% nitric acid.
4. Prepare the ICPMS instrument (see the Table of Materials) for sample analysis as follows:
   1. Prior to starting the instrument, ensure that the Argon gas valve is open, all tubing is securely connected, and clean 5% nitric acid is open for rinsing tubing and glassware between sample analyses.
   2. Check the condition of the torch and cones, and make sure the torch box is securely latched and the spray chamber drainage tube is properly connected to the peripump.
   3. Open the software (see the Table of Materials).
   4. Check the vacuum readings and ensure all turbo pumps are running at 100%.
   5. Click START in the Plasma Control System Status window to initiate the start sequence, turn on the plasma pump, plasma chiller, purge the nebulizer, and light the plasma. Wait for the plasma to be lit and stable when the status window will indicate that the startup sequence is complete. At this point, observe the green dots on the System State window that indicate that all Power supplies are on.
   6. In the menu bar, click on Control | autosampler in the dropdown menu. Enter the autosampler rack position for the tube containing 5% nitric acid. Allow the acid to enter the plasma.
   7. In the menu bar, click on Scans | Magnet in the dropdown menu. In the MagnetScan window, type 115 into the Marker Mass Position and click enter. Allow the magnet to scan across the mass range for ¹¹⁵In (114.6083 to 115.3749) for 30 min while the instrument warms up.
   8. After 30 min, use the autosampler control to move to the position of a 1 ppb multielement tuning solution. Aspirate the tuning solution and tune the instrument to optimize the signal reading. Adjust the torch position (X, Y, Z) such that the torch is aligned with the center of the cones and nebulizer flow rate (~ 30 psi) in the Plasma control window. Make the necessary adjustments in the Ion Optics Tuning window for the Source, Detector, and Analyzer.
   9. Once the signal reading is optimized (~1.2 × 10⁶ counts/s for 1 ppb on ¹¹⁵In), click Stop in the Magnet Scan window.
      1. Click Calibrate Magnet and select Low Resolution in the popup window.
      2. Click OK and open the "Mass Calibration (Low Resolution).smc" file to calibrate the Magnet.
         NOTE: The magnet calibration will measure the counts/s and fit a curve through the following mass range: ⁷Li to ²³⁸U.
      3. Click Save | Use to apply the current magnet calibration to the analyses. If measuring unknown samples with a huge range of concentrations, perform a Detector Calibration to compare ion pulse counts signals at low concentrations to attenuated ion signals produced at higher concentrations. Analyze the samples once tuning and calibration are complete.
   10. In the menu bar, click on Data Acquisition.
       1. Click on Method Setup in the dropdown menu. Use an existing method provided by the manufacturer, or create a method based on the elements of interest. If necessary, adjust the analysis mode, dwell time, switch delay, number of sweeps/cycles, resolution, detection mode, and the park mass for deflector settings.
       2. Click Save to record the method settings. Optimize the parameters for each metal and isotope. See Table 2 for the specific operation parameters used in this study.
   11. In the menu bar, click on Data Acquisition.
       1. Click on Batch Run in the dropdown menu. Alternatively, click on the BATCH Icon below the menu bar. Import the batch parameters from a spreadsheet, or create a sequence in the Batch Run window. Enter the sample type, the autosampler rack position, transfer time, wash time, replicates, sample ID, and method file.
   12. Arrange the batch run in the following order: standard solutions for the calibration curve (0.001-10 ppb Pt or Ru), followed by a quality control standard, then unknown samples.
       NOTE: Standard solutions are measured as counts/s on the ICPMS, and a linear regression is fit through the standards with a relative standard deviation (RSD) > 0.999. Unknowns are also measured as counts/s and solved for concentration in ppb using the linear regression of the calibration curve, y = mx + b. Data processing can also be completed using the referenced software.
   13. Monitor instrument drift and sample reproducibility by including a 0.5 ppb quality control standard every 5-10 samples.
       NOTE: Suitable quality control standards should be a certified standard reference material different from the standard used in the calibration curve.

Watts	Power	Minutes
300	50%	5
300	75%	5
300	0%	5
300	75%	5

Table 1: Microwave digestion protocol for larval tissue mass. Zebrafish larval samples were digested in 0.25 mL of nitric acid. This table has been modified from ²⁴.

Representative Results

These results have been previously published²⁴. Tissue uptake studies were conducted with waterborne exposures of cisplatin and a novel Ru-based anticancer compound, PMC79. Lethality and delayed hatching were evaluated for nominal concentrations of cisplatin 0, 3.75, 7.5, 15, 30, and 60 mg/L cisplatin. Pt accumulation in organism tissue was determined by ICPMS analysis, and organism tissue contained respective doses of 0.05, 8.7, 23.5, 59.9, 193.2, and 461.9 ng (Pt) per organism (Figure 3). Analytical determination of the nominal concentrations for cisplatin was not assessed, given the known stability of cisplatin.

Delayed hatching was observed at all cisplatin concentrations. Additional experiments were conducted for Pt concentrations with and without manual dechorionation. Post dechorionation, chorions were collected and analyzed for Pt separately. Nonlethal doses of cisplatin used for dechorionation studies determined that 93-96% of the total delivered dose of cisplatin had accumulated in the chorion with the remaining dose within the larval tissue (Figure 4).

Zebrafish larvae were exposed to 0, 3.1, 6.2, 9.2, 12.4 mg/L of PMC79. These doses were selected by determining the derivatives of an IC₅₀, as described previously¹⁶. These concentrations were analytically determined to contain 0, 0.17, 0.44, 0.66, and 0.76 mg/L of Ru. Unlike the cisplatin dose-response curve, delayed hatching was not observed in PMC79-exposed larvae. Chorions were not included in Ruthenium analysis as they naturally degraded prior to larval collection. Researchers may include chorion analysis without delayed hatching by dechorionating and collecting chorions at 24 dpf. The mass of metal within larval tissues analyzed at each concentration was 0.19, 0.41, and 0.68 ng (Ru) per larva (Figure 5). A summary of the toxicological endpoints, including lethal concentrations and/or doses for 50% of the population (LC₅₀/LD₅₀), effective concentrations, or doses for 50% of the populations (EC₅₀/ED₅₀), and the lowest observed adverse effect level (LOAEL) can be found in Table 3.

	Cisplatin			PMC79
	Nominal (mg/L)	µM	Pt (ng) / organism	Analytical Ru (mg/L)	µM	Ru (ng) / organism
LC50/LD50	31 (95% CI: 20.5-34.0)	158 (95% CI: 105-174)	193 (± 130)	0.79 (95% CI: 0.43-1.20)	7.8 (95% CI: 4.2-11.8)	NA
EC50	4.6	12.5	NA	NA	NA	NA
LOAEL	3.75	15.3	8.7 (± 4)	0.17	1.7	0.19 (± 0.05)

Table 3: Determination of solution and metallodrug uptake associated with toxicological endpoints. LD₅₀ was determined by metal equivalent analysis of Pt and Ru for cisplatin and PMC79, respectively. The LC₅₀ concentrations for PMC79 were analytically determined. However, analytical determination of nominal cisplatin concentrations was not conducted; given the known stability of cisplatin in solution, it was assumed that nominal and measured concentrations in solution would be equivalent. The delayed hatching endpoint for cisplatin exposure was evaluated in terms of ED₅₀ and LOAEL. The LOAEL concentrations of PMC79 were analytically determined. The LOAEL included lesions such as hemorrhaging along the caudal vein and tail artery, spinal curvature, and yolk sac edema. All 95% confidence intervals were calculated using the Litchfield Wilcoxon method. This table has been modified from ²⁴. Abbreviations: CI = confidence interval; LC₅₀ = Lethal Concentration for 50% of the population; LD₅₀ = Lethal Dose for 50% of the population; EC₅₀ = Effective Concentration for 50% of the population; LOAEL = lowest observed adverse effect level.

Figure 1: Zebrafish dose-response protocol. This protocol uses a modified approach adapted from the OECD FET. Made with Biorender. Abbreviation: OECD = Organization Economic Cooperation and Development; FET = fish embryo acute toxicity. Please click here to view a larger version of this figure.

Figure 2: Tissue digestion and ICPMS evaluation. The digestion protocol is effective for digesting a composite sample of zebrafish larvae. Abbreviation: ICPMS = inductively coupled plasma mass spectrometry. Created with Biorender. Please click here to view a larger version of this figure.

Figure 3: Cisplatin dose-response. (A) Percentage mean delayed hatching at 5 dpf correlated to the mean Pt equivalents determined per organism. (B) Percentage mean lethality at 5 dpf correlated to the mean Pt equivalents per organism. Percentage means: N = 40 per dose. Pt (ng) per organism: >4 composite samples per dose. Two experimental replicates were conducted, the ranges of which are displayed. This figure has been modified from ²⁴. Abbreviation: dpf = days post fertilization. Please click here to view a larger version of this figure.

Figure 4: Comparison of Pt (ng) present in the larvae and the chorion after exposure to 7.5 or 15 mg/L. Composite >3 larvae or chorions per sample; from left to right N = 13, 10, 10, and 11. Error bars represent standard deviation. Mann-Whitney rank-sum test P < 0.001 between larvae and chorion for both doses. This figure has been modified from ²⁴. Please click here to view a larger version of this figure.

Figure 5: PMC79 dose-response. (A) Percentage mean lethality was correlated to the analytically determined mean Ru equivalents in solution (mg/L). (B) Percentage mean lethality at 5 days post fertilization from the same experiment was correlated to the mean Ru equivalents per larva. Lethality: N = 40 per dose. Ru (mg/L): N = 6 composite samples per dose. Ru (ng) per larva >4 composite samples per dose. Two experimental replicates were conducted, the ranges of which are displayed. This figure has been modified from ²⁴. Please click here to view a larger version of this figure.

Dwell Time per Peak	4 ms
Switch Delay/ Peak (x10micros)	2
Number of Sweeps	350
Number of Cycles	1
Instrument Resolution	300
Detection Mode	Attenuated, Deflector Jump
Park Mass	98.90594
Element (isotopes)	Pt (192, 194, 195, 196), Ru (99, 100, 101, 102) Sr (84)

Table 2: ICPMS method parameters. Parameters for analysis of Pt and Ru isotopes to determine tissue concentrations of cisplatin and PMC79, respectively. Sr was included to monitor isobaric interferences associated with the tank water composition. This table has been modified from ²⁴. ICPMS = inductively coupled plasma mass spectrometry.

Discussion

The protocol described here has been implemented to determine the delivery and uptake of metal-based anticancer drugs containing either Pt or Ru. Although these methods have already been published, this protocol discusses important considerations and details to adapt this methodology for a range of compounds. The OECD protocol coupled with tissue digestion and ICPMS analysis allowed us to determine that PMC79 was more potent than cisplatin and resulted in disparate tissue accumulation, suggesting separate mechanisms. Furthermore, because the delivered dose of cisplatin was quantified, dose-response results were extrapolated to patient populations. Sublethal doses (e.g., LOAEL) were comparable to intravenous dosing concentrations in patients²⁴.

Although this method may be applied to a broad spectrum of metals and metal-based compounds, careful investigation of the physicochemical properties of the analyte must be taken into consideration. Metal-based compounds may be very difficult to dissolve, and various vehicles can be used to avoid this. Vehicle concentrations, such as DMSO, may need to be at higher concentrations than recommended in the OECD protocol. As such, it is important to maintain a nontoxic dose by closely monitoring the development of controls; continuously rocking the embryos during exposure mitigates precipitation. Additionally, organometallic compounds may not be stable in aqueous solution. If the degradation process is unknown, studies that involve 24 h solution renewal may be considered or compared to nonrenewal dose-response curves.

It is recommended to follow the OECD Fish Acute Embryo Toxicity Test (FET) Number 236²¹. However, modifications can be made to suit specific purposes. Glass containers avoid confounding toxicological variables, such as plastic and plasticizers, and do not adsorb metals as strongly, which would remove analytes from the egg water. For compounds that photodegrade, such as cisplatin, it may be beneficial to conduct the exposure without a light cycle.

There is much discussion in the literature regarding the need for dechorionation in zebrafish dose-response studies^25,26,27. Arguments for dechorionation at 24 hpf suggest that the chorion limits the permeability of compounds, thus generating false-negative results or augmented dose-response curves. Although these points have merit, conducting studies without dechorionation may provide mechanistic insight. These studies suggest cisplatin accumulates in the chorion of the embryos due to its alkylating activity (Figure 2). The resulting adducts reinforce the structure, resulting in delayed hatching. However, PMC79 and other Ru-based anticancer drugs did not cause this phenomenon²⁷. Although many chemotherapeutics enact their anticancer activity by alkylation, the lack of delayed hatching post PMC79 exposure indicated a disparate mechanism. Studies with or without dechorionation must be carefully considered or conducted in parallel.

Downstream tissue digestion and ICPMS analysis must be continuously considered. It is suggested to avoid using any reagents that may cause isobaric interferences and implement alternative methods. Reagents used during the dose-response studies may impact or react with the nitric acid and its oxidizing potential or contribute to isobaric interferences. It was discovered that the salt solution used to make egg water generated strontium (Sr) oxides, which overlapped with a specific isotope of Ru²⁴. Lowering salt concentrations or carefully cleaning the larvae can ameliorate this issue. For these reasons, it is suggested to avoid the antimicrobial methylene blue or the euthanizing agent, tricaine. Instead, autoclave and subsequently aerate the egg water to remove microbes or euthanize the larvae by rapid cooling. It is important at this step to achieve linear isotopic standard curves with minimal isobaric interferences for the analyte of interest.

An important limitation to this protocol is that organometallic compounds will be oxidized such that only the metal remains. As such, metabolism studies cannot be conducted. Although the protocol can be considered medium-throughput, the dose-response portion may be expedited with the aid of automatic chemical delivery systems and imaging. This protocol is a nascent methodology that may be modified and refined for a broad spectrum of metal and metal-based compounds for pharmaco- and toxicokinetic studies.

Materials

Name	Company	Catalog Number	Comments
AB Strain Zebrafish (Danio reri)	Zebrafish International Resource Center	Wild-Type AB	Wild-Type AB Zebrafish
ACS Grade Nitric Acid	VWR BDH Chemicals	BDH3130-2.5LP	Nitric Acid (68-70%); used to make 10% HNO3 acid-bath solution for soaking/pre-celaning centrifuge tubes
Aquatox Fish Diet (Flake)	Zeigler Bros, Inc.		Flake food to be mixed in a 1:4 ratio of Aquatox Fish Diet to TetraMin Tropical Flakes and used as feed
Artemia cysts, brine shrimp	PentairAES	BS90	Brine shrimp eggs sold in 15-ozz, vacuum-packed cans to be hatched and used as feed
ASX-510 Autosampler for ICPMS	Teledyne CETAC		Automatic sampler with conifgurable XYZ movement, flowing rinse station, and 0.3 mm inner dimension probe. Compatible with Nu AttoLab software for programmable batch analyses.
Centrifuge	Thermo Scientific	CL 2	Thermo Scientific CL 2 compact benchtop centrifuge with variable speed range up to 5200 rpm; used to bring sample and acid condensate to the bottom of the centrifuge tube bewteen microwave digestion intervals; aids in sample retention
Centrifuge tubes	VWR	21008-105	Ultra high performance polypropylene centrifuge tubes with flat cap; 15 mL volume; leak-proof with conical bottom
Class A Clear Glass Threaded Vials	Fisherbrand	03-339-25B	Individual glass vials for exposure containment
Dimethyl Sulfoxide	Millipore Sigma	D8418	Solvent or vehicle for hydrophobic compounds
Fixed Speed Vortex Mixer	VWR	10153-834	Vortex mixer; used to homogenize sample after acid digestion and dilution
High Purity Hydrogen Peroxide	Merk KGaA, EDM Millipore	1.07298.0250	Suprapur Hydrogen peroxide (30%); used for sample digestion
High Purity Nitric Acid	EDM Millipore	NX0408-2	Omni Trace Ultra Nitric Acid (69%); used for sample digestion
Instant Ocean Sea Salt	Spectrum Brands, Inc.	Instant Ocean® Sea Salt	Egg water solution contains instand ocean sea salt with a final concentration of 60 µg/ml
Mars X Microwave Digestion System	CEM, Matthews, NC		Microwave acid digestion system used to digest and homogenize samples under uniform conditions. For this methodology the open vessel digestion method was completed using single-use polypropylene centrifuge tubes at low power (300 W).
Multi-element Solution 3	SPEX CertiPREP	CLMS-3	Contains 10 mg/L Au, Hf, Ir, Pd, Pt, Fu, Sb, Sr, Te, Sn in 10% HCl/1% HNO3; used as a quality control standard for Pt and Ru analyses
Nu Instruments AttoM High Resolution Inductively Coupled Plasma Mass Spectrometer (HR-ICP-MS)	Nu Instruments/Amatek		Double focussing magnetic sector inductively coupled plasma mass spectrometer with flexible low to high resolution slit system, and dynamic range detector system. Data processing and quantification is done using NuQuant companion software.
Platinum (Pt) standard solution, NIST 3140	National Institute of Standards and Technology	3140	Prepared from ampoule containing 9.996 mg/g Pt in 10% HCl; ; used as a quality control standard for Pt analyses
Platinum (Pt) standard solution, single-element	High Purity Standards	100040-2	Contains 1000 mg/L Pt in 5% HCl
Ruthenium (Ru) standard solution, single-element	High Purity Standards	100046-2	Contains 1000 mg/L Ru in 2% HCl
TetraMin Tropical Flakes	Tetra	77101	Flake food to be mixed in a 1:4 ratio of Aquatox Fish Diet to TetraMin Tropical Flakes and used as feed
Trace Metal Grade Nitric Acid	VWR BDH Chemicals	87003-261	Aristar Plus Nitric Acid (67-70%); used for rinse solution in ASX-510 Autosampler
Ultrasonic water bath	VWR	B2500A-DTH	Ultrasonic water bath used to aid in acid digestion prior to microwave digestion