Experimental Steps for the Determination of Total Organic Arsenic in Surface Water

Determining total organic arsenic in surface water is essential for accurate water quality evaluation, ecological risk assessment, and regulatory compliance under standards such as GB 3838, EPA 1632, and ISO 11969. This article presents a complete experimental protocol, covering sample collection, pretreatment, HPLC-ICP-MS speciation analysis, quantification, and quality control. It serves as a practical technical reference for environmental laboratories, researchers, and water quality engineers.

Technical Overview

Organic arsenic in surface water mainly originates from the microbial methylation of inorganic arsenic, agricultural runoff containing organoarsenical feed additives and pesticides, and industrial wastewater discharges. The principal organic species include monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), arsenobetaine (AsB), and arsenocholine (AsC). Because the toxicity, mobility, and bioavailability of arsenic depend strongly on its chemical form, measuring total arsenic alone is insufficient for a reliable risk assessment.

Total organic arsenic is defined either as the arithmetic sum of all identified organoarsenical species or, alternatively, as the difference between total arsenic and total inorganic arsenic, that is, the sum of arsenite As(III) and arsenate As(V). The preferred analytical technique is high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), which combines species-separating power with ultra-trace elemental detection. Liquid chromatography coupled with hydride generation atomic fluorescence spectrometry (LC-HG-AFS) is an accepted alternative for laboratories without ICP-MS access.

Typical method detection limits range from 0.02 to 0.5 micrograms per liter for individual species, which is far below the Class III arsenic limit of 50 micrograms per liter specified in GB 3838-2002 for surface water. The overall workflow comprises field sampling, filtration, preservation, chromatographic separation, MS detection, calibration, quantification, and rigorous quality control.

Testing Samples

Rivers and Streams

Flowing water bodies influenced by upstream mining, smelting, geothermal activity, or agricultural drainage. Representative sampling requires mid-channel, mid-depth collection away from riverbanks and discharge outfalls, ideally under both wet-season and dry-season conditions.

Lakes and Reservoirs

Stagnant or slow-exchange water bodies where arsenic speciation is governed by redox stratification. Samples should be collected at multiple depths, because anoxic bottom layers promote the reduction of As(V) to As(III) and subsequent biomethylation to MMA and DMA.

Other Surface Water Bodies

The protocol also applies to wetlands, estuaries, coastal waters, irrigation channels, ponds, and source waters for drinking water treatment plants. Estuarine samples with high salinity require special attention to chloride-induced spectral interference during ICP-MS measurement.

Testing Items

The standard testing scope for a comprehensive organic arsenic determination includes the following items:

• Arsenite, As(III), and arsenate, As(V), constituting total inorganic arsenic
• Monomethylarsonic acid, MMA
• Dimethylarsinic acid, DMA
• Trimethylarsine oxide, TMAO
• Arsenobetaine, AsB, and arsenocholine, AsC
• Total arsenic after microwave digestion
• Total organic arsenic, calculated by species summation or by subtraction of inorganic arsenic from total arsenic
• Method performance indicators, including detection limits, recovery rates, and repeatability

Testing Methods

Step 1: Sample Collection and Preservation

Collect samples in pre-cleaned 500 mL amber glass or high-density polyethylene bottles that have been soaked in 10 percent nitric acid and rinsed with ultrapure water. Observe the following precautions:

• Filter samples in the field through a 0.45 micrometer membrane if dissolved speciation is required; unfiltered aliquots represent the total recoverable fraction.
• Do not acidify samples intended for speciation analysis, because pH changes can interconvert arsenic species and invalidate the results.
• Fill bottles completely to minimize headspace, seal them tightly, and record sampling location, depth, time, temperature, pH, and conductivity.
• Store samples at 4 degrees Celsius in the dark and transport them in insulated containers with temperature monitoring.
• Analyze speciation samples within 7 days; a separate aliquot for total arsenic may be acidified with nitric acid to pH below 2 and held for up to 28 days.

Step 2: Sample Pretreatment

For speciation analysis, allow refrigerated samples to reach room temperature, mix gently, and filter through a 0.45 micrometer membrane if not already filtered in the field. Dilute samples with mobile phase where necessary to match the ionic strength of the chromatographic system, and transfer aliquots to autosampler vials. For total arsenic determination, digest a separate aliquot with nitric acid and hydrogen peroxide using microwave-assisted heating in sealed vessels according to HJ 700-2014 or EPA 200.8 procedures, then dilute the digest to a known volume.

Step 3: Chromatographic Separation by HPLC

Species separation is performed on a strong anion-exchange column, typically a polymer-based PRP-X100 type column of 250 millimeters length and 4.1 millimeters internal diameter with 10 micrometer particles. Recommended conditions are listed below:

• Mobile phase: 10 millimoles per liter ammonium phosphate buffer, adjusted to pH 6.0 with ammonia solution, filtered through a 0.22 micrometer membrane and degassed
• Flow rate: 1.0 milliliter per minute, isocratic elution
• Column temperature: 25 to 30 degrees Celsius
• Injection volume: 20 to 50 micrometers equivalent, typically 20 to 50 microliters
• Typical elution order under these conditions: AsB, As(III), DMA, MMA, As(V), with baseline resolution greater than 1.5 between adjacent peaks

Step 4: ICP-MS Detection Parameters

Couple the chromatographic outlet directly to the ICP-MS nebulizer through a low-dead-volume connection, and introduce an internal standard such as germanium-72, rhodium-103, or tellurium-125 via a post-column T-piece. Key instrument settings include:

• RF forward power: 1500 to 1550 watts
• Plasma argon flow: approximately 15 liters per minute; carrier gas: 0.9 to 1.0 liter per minute
• Monitored isotope: arsenic-75 at mass-to-charge ratio 75
• Collision cell: helium mode at 4 to 5 milliliters per minute to suppress the polyatomic argon-40 chloride-35 interference at mass 75
• Dwell time: 100 to 300 milliseconds per point to generate sufficient data density across chromatographic peaks
• Integration mode: chromatographic peak area quantification using time-resolved analysis software

Step 5: Calibration and Quantification

Prepare at least five calibration levels for each arsenic species over the range of 0.5 to 50 micrograms per liter, mixing individual stock standards of As(III), As(V), MMA, DMA, AsB, and AsC in the mobile phase. Verify linearity with a correlation coefficient of at least 0.995 for every species. Identify species by matching retention times within a tolerance of plus or minus 2 percent against calibration standards, confirm peak purity, and quantify each species from its calibration curve. Report total organic arsenic as the sum of all organic species peaks, or as total arsenic minus inorganic arsenic when the subtraction approach is used. Express results in micrograms per liter as arsenic, corrected for dilution factors.

Step 6: Alternative LC-HG-AFS Procedure

Laboratories equipped with atomic fluorescence spectrometers may perform LC separation followed by hydride generation, in which the column effluent is mixed online with hydrochloric acid and potassium borohydride solution, typically 0.5 to 2 percent potassium borohydride in 0.2 to 0.5 percent sodium hydroxide. Volatile arsines are swept by argon into a heated quartz atomizer and detected by AFS. Note that As(V), MMA, and DMA hydride generation efficiencies differ from As(III), so species-specific calibration and, where required, pre-reduction with potassium iodide and thiourea or L-cysteine must be applied.

Step 7: Quality Assurance and Quality Control

Apply the following QA/QC measures to every analytical batch:

• Method blank: one per batch of 20 samples, result must be below the detection limit
• Calibration verification: mid-level check standard every 10 injections, within plus or minus 10 percent of the true value
• Matrix spike and matrix spike duplicate: recovery between 80 and 120 percent, relative percent difference below 20 percent
• Laboratory duplicate: at least one per batch, relative deviation below 20 percent for concentrations above 5 times the detection limit
• Certified reference material, such as a trace-elements-in-water CRM, for total arsenic accuracy verification
• Continuing calibration blank to monitor carryover and memory effects
• Full documentation of instrument tuning, mass calibration, and collision cell performance before each sequence

Applications

The determination of total organic arsenic in surface water supports a wide range of environmental and regulatory activities:

• Routine surface water quality monitoring against GB 3838-2002 functional zone criteria and drinking water source protection requirements
• Environmental impact assessments and pollution source tracing near mining, smelting, geothermal, semiconductor, and agrochemical facilities
• Ecological and human health risk assessment based on species-specific toxicity data rather than total arsenic alone
• Research on arsenic biogeochemical cycling, biomethylation pathways, and redox transformation in lakes and reservoirs
• Verification of the effectiveness of wastewater treatment and arsenic removal processes at discharge outlets
• Baseline and trend studies for government environmental monitoring networks and accredited third-party laboratories

FAQ

Why must samples for speciation analysis not be acidified?

Acidification alters the redox equilibrium and can convert As(III) to As(V), promote demethylation of MMA and DMA, or cause adsorptive losses on container walls. Speciation samples must therefore be preserved only by refrigeration at 4 degrees Celsius in the dark, while a separate acidified aliquot is reserved for total arsenic determination.

How does total organic arsenic differ from total arsenic?

Total arsenic includes all chemical forms after digestion, whereas total organic arsenic covers only the organoarsenical species. It is obtained either by summing the chromatographic peaks of MMA, DMA, TMAO, AsB, AsC, and other organic species, or by subtracting inorganic arsenic, the sum of As(III) and As(V), from total arsenic.

What detection limits can be achieved?

With HPLC-ICP-MS, method detection limits of 0.02 to 0.5 microgram per liter per species are routinely achievable, depending on injection volume, matrix, and instrument sensitivity. LC-HG-AFS typically achieves detection limits near 0.1 to 1 microgram per liter.

How is chloride interference handled in saline surface water?

Argon-40 chloride-35 polyatomic ions interfere at mass 75, the sole arsenic isotope. Helium collision mode in the reaction cell, dilution of high-salinity samples, and chromatographic separation of chloride from the arsenic species peaks jointly eliminate this bias.

Which standards govern this analysis?

Relevant references include GB 3838-2002 for surface water quality classification, HJ 700-2014 and EPA 200.8 for total element determination by ICP-MS, EPA Method 1632 for arsenic speciation, and ISO 11969 for total arsenic determination. Accredited third-party laboratories should select validated methods appropriate to their scope of accreditation.

What holding time applies before analysis?

For speciation, analysis within 7 days of collection is strongly recommended because species transformation continues slowly even under refrigeration. For total arsenic in the acidified aliquot, a holding time of up to 28 days is generally acceptable.