Mercury is a toxic environmental pollutant that can be transformed into methyl mercury, a highly toxic organic compound, by both microorganisms and abiotic reactions in aquatic environments. As methyl mercury passes up the marine food chain it becomes increasingly concentrated by a process referred to as biomagnification.
The harmful nature of methyl mercury was notably documented in Japan after a chemical company released a significant quantity of it into Minamata Bay. The methyl mercury caused severe poisoning in local people, many of whom live on a diet of fish and shellfish.
More and more countries and global organisations are becoming increasingly concerned about the human health hazards caused by mercury in fish. Many already enforce strict regulations regarding it. Most stipulate maximum concentrations of mercury in fish of approximately 0.5mg/kg wet weight. However, there are differences in maximum mercury levels between countries and some variations depending on the type of fish. Most countries legislate specifically for methyl mercury, although there are some that provide guidelines for total mercury levels as well.
In the US, for example, the Food and Drug Administration (FDA) specifies a maximum level of 1ppm methyl mercury in edible portions of fresh, frozen or processed fish, shellfish, crustaceans and other aquatic animals. The European Food Safety Authority (EFSA) endorses the provisional tolerable weekly intake of 1.6mg/kg of methyl mercury. The European Commission has introduced the EC1881/2006 regulation, setting maximum levels for certain contaminants in foodstuffs.
Atomic absorption (AA) spectroscopy is an ideal tool for the measurement of low levels of mercury in fish. For laboratories interested in total mercury measurements this technique provides fast and accurate analysis of samples with detection limits below 0.07ppb (µg/L) in solution. This equates to 0.014mg/kg in the original fish sample, based on a 0.5g in 100mL preparative method. For laboratories analysing methyl mercury, AA spectrometers provide an excellent screening tool. Their cost-effectiveness and ease-of-use makes them a perfect partner to more complex and expensive techniques, such as HPLC-ICP-MS or GC-ICP-MS.
AA case study
For this particular application a Thermo Scientific iCE 3500 AA spectrometer and a Thermo Scientific VP100 vapour generation accessory were used. The continuous flow of reagents ensures that the system is self-cleaning, reducing memory effects and increasing sample throughput. The VP100 is entirely controlled by Thermo Scientific SOLAAR software, meaning that setting up a method and running an analysis is extremely simple.
A mercury cell (provided as standard with the VP100) was also used. This accessory provides an increased pathlength compared to a normal vapor cell and gives exceptionally low detection limits.
The sample preparation procedure is shown in Fig. 1. There are four main sections: sample drying, sample preparation, sample digestion and mercury reduction. The drying section may not be applicable for all situations, as it is only necessary if the final mercury concentration is needed as a dry weight value, for example mg/kg dry weight. Most countries and official regulatory bodies concentrations of mercury in a wet weight of sample.
1. Sample drying phase is not necessary if the final concentration of mercury is needed for a wet-weight sample.
2. Refer to the manufacturers guidelines when designing a digestion programme.
3. CARE: The reaction is exothermic and the flask may become hot. Also, make sure to add the hydroxylamine chloride slowly, otherwise the solution may foam and eject some sample from the flask.
Three different types of fish sample were used during the evaluation of this method: fresh fish (salmon), canned fish (sardine), and DORM-2 certified reference material (National Research Council of Canada, Institute for National Measurement Standards, Ottawa, Canada).
If dry weight measurements are needed then the fish samples should be homogenised and dried in an oven at 80°C until they reach a constant weight. Alternatively, the fish tissue can be freeze-dried and homogenised using a mortar and pestle. After drying, portions of approximately 0.5g should be accurately weighed out for digestion. For wet weight measurements the fresh fish should be homogenised in a food processor and a portion of approximately 0.5g should be accurately weighed and placed in a microwave digestion vessel. This provides a representative fish sample.
Following preparation in this manner, 1mL of 1000 ppb Hg standard solution was added to half of the salmon and sardine samples. This spike gave a concentration of 10 ppb Hg in the final 100mL sample. The other half of the samples did not have mercury added to them to allow the calculation of spike recoveries. The microwave digestion vessels containing the samples were placed in a fume extraction hood before adding 10mL concentrated HNO3. The vessels were left for at least 30 minutes without their lids on to allow gases to escape. After this time the vessels were placed into a microwave digestion system.
After digestion the samples were transferred to a 100mL graduated flask and 60mL of six per cent potassium permanganate solution was added. The sample vessels were left for at least two hours to ensure that all the mercury in the sample was reduced to Hg2+. It is very important to check that the vessels are not sealed during this stage, as gases are produced that could cause pressure to build up.
After the mercury was reduced, 15mL of 20 per cent hydroxylamine chloride solution was added to remove the excess potassium permanganate. Care was taken during the addition of the hydroxylamine chloride, as this produces an exothermic reaction and the vessel may become hot. It is essential to add the hydroxylamine chloride slowly during this stage and to gently mix the solution during the addition. Without these precautions a violent reaction may occur that could eject some sample from the flask, leading to inaccurate results. After allowing the solution to cool, deionised water was added to make the volume up to 100mL.
Preparation and results
Standards were prepared from a 1000 ppm (mg/L) mercury standard solution. This standard was first diluted to produce a 1000 ppb (µg/L) stock solution to allow simple preparation of a range of standards. To demonstrate the linear range of the iCE 3000 Series AA spectrometers a wide range of standards were used (1-100 ppb). The standards were matrix matched and prepared in the same order as the samples.
The VP100 requires both a reductant and an acid solution (50 per cent HCl) to perform the reactions that form the gaseous mercury. For this application the reductant was a solution of 7.5 per cent stannous chloride (SnCl2) stabilised in 10 per cent HCl.
The analysis was performed using the most sensitive absorption wavelength for mercury at 253.7 nm. Five re-samples were used, with each re-sample taking four seconds. This was done to thoroughly assess the short-term stability of the instrument during the development of the method. For normal use, three re-samples would be adequate. Deuterium background correction was used throughout the analysis.
The calibration curve showed excellent linearity up to 100 ppb (Fig. 2), which is equivalent to 20 mg/kg in a fish sample (assuming a sample weight of 0.5g) with an R2 value of 0.9989. This shows the superb performance of the iCE 3000 series over a wide concentration range. This calibration is equivalent to concentrations of 0-20mg/kg mercury in the original fish samples, assuming a sample mass of exactly 0.5g. The per cent relative standard deviations (per cent RSDs) for each of the standards were less than 2.5 per cent. This demonstrates the excellent stability of both the spectrometer and the VP100 accessory.
The method detection limit (MDL) and characteristic concentration were calculated using the automated 'Instrument Performance' Wizard in the SOLAAR software, automating all of the data processing, making the entire procedure quick and easy. The method was found to have a detection limit of 0.068 ppb (µg/L) in solution. This equates to an MDL of 0.014 mg/kg in the original fish sample (assuming a sample mass of 0.5 g). The MDL provides a measure of the noise and stability of the system. A lower detection limit allows you to confidently determine lower concentrations of mercury in your samples. The characteristic concentration is related to the sensitivity of the method. The characteristic concentration of this method was found to be 0.724 ppb in solution. This would be the equivalent of 0.145 mg/kg in the initial fish sample (assuming a sample weight of 0.5g).
Salmon and sardine samples were spiked with 10 ppb mercury prior to digestion and compared with unspiked samples to calculate recoveries. These 10 ppb spikes would correspond to a concentration of 2 mg/kg in normal fish samples (assuming a sample weight of 0.5 g) and demonstrate the accuracy of the analysis at levels appropriate to current legislation. The agreement with expected results is excellent, with the recovered values all falling within six per cent of the expected values. This demonstrates the repeatability and accuracy of both the sample digestion procedure and the vapour analysis using the Thermo Scientific iCE 3000 Series AA spectrometers (Fig. 3).
To ensure the accuracy of the sample preparation, digestion and analysis, three separate samples of the DORM-2 standard reference material were also analysed. The recoveries from these samples were also excellent, with an accuracy of +/-2 per cent or better. These results show that an AA spectrometer coupled with a vapour generation accessory offer excellent linear range, stability and accuracy during the analysis of trace levels of mercury in fish.
- Dr Andrew David Bowen is Applications Scientist and Hazel Dickson is Applications Chemist with Thermo Fisher Scientific, Cambridge, UK. www.thermo.com/ice.