4 Methods for chemical analyses
4.6 Quantification of silver in earthworms .1 Procedure .1 Procedure
To ensure a complete digestion of Eisenia fetida, it was necessary to perform cryogenic homog-enisation. To avoid contamination, homogenisation was performed under a laminar-flow hood.
All materials used as well as the worms were cooled in liquid nitrogen. The samples were ho-mogenised using a pestle and mortar. First a larger pestle was used for a coarser grinding fol-lowed by a smaller one. The resulting powder was transferred into cryo-proofed vials and stored directly above liquid nitrogen at approx. -150°C until lyophilisation.
Cryogenic homogenisation
The frozen samples were transferred into a Christ Alpha 1-2 freeze dryer (Martin Christ GmbH, Osterode am Harz, Germany) for lyophilisation. Freeze-drying was performed until samples reached constant weights.
Lyophilisation
Approximately 200 mg of homogenised and dried substance from each sample was weighed into a quartz digestion vessel, and 5 mL of concentrated nitric acid were added followed by the microwave digestion.
Microwave digestion
Program: heat for 25 min to maximum temperature of 220°C; hold at 220°C for 30 min; allow to cool for approx. 60 min; initial pressure 40 bar.
After digestion the vessels were filled up to an exact volume of 20 mL with ultrapure water.
A reference material (NIST 2977 Mussel Tissue) was digested along with the samples.
The digestion procedure is in accordance to the document ‘Guidelines for Chemical Analysis, Digestion of Environmental Samples’ from http://www.umweltprobenbank.de.
4.6.2 Analytical measurement
Nitric acid was of “Suprapur®” (supplied by Carl Roth, Karlsruhe). The water used was purified using an ELGA Pure Lab Ultra water purification system (purified water resistivity >18 MΩ∙cm).
Reagents for silver analysis
A commercially available silver ICP-standard containing 1000 mg/L Ag in nitric acid 2-3% was used to prepare appropriate stock solutions and respective calibration solutions. All prepared standard solutions had a final HNO3 concentration of 3%.
The reference material NIST 2977 Mussel Tissue was digested and analysed along with the samples to verify the procedures (purchased from LGC Standards, Wesel, Germany). Unfortu-nately, only a non-certified reference value of 4.58 mg Ag / kg is provided for this material.
Certified reference materials (chapter 21.1.6) and verifying the method
Furthermore, aqueous certified reference material TMDA-70 (purchased from Environment Canada, certified Ag conc. of 10.9 µg/L) was analysed along with the samples.
To additionally verify the analytical method a multi element Merck IV Standard (appropriately diluted to fit in the range of samples, lot HC957274, purchased from Merck, Darmstadt, Ger-many) was analysed along with the samples to verify the measured results.
The silver concentration in reagent blanks (n = 10) were below the limit of detection (< 2.6 µg/L), except one which was below the limit of quantification (< 8.8 µg/L)
All materials used for sample treatment were suitable for analyses of silver at trace levels. The glassware (beakers and volumetric flasks) was cleaned using a Miele washer “Automatic Disin-fector” combined with a water de-ioniser “Aquapurificator”, steamed out with HNO3, rinsed with ultrapure water and dried at approx. 60°C. The pipettes used were adjustable to variable vol-umes (50 - 250 µL, 200 - 1000 µL, 1000 - 5000 µL) and were purchased from Gilson (Abimed, Langenfeld, Germany) and Eppendorf (Wesseling, Germany).
Laboratory equipment
ICP-OES (raw data example: chapter 21.1.2)
Silver concentrations of aqueous samples were measured using an IRIS Intrepid II ICP-OES (Thermo Electron, Dreieich, Germany). Silver was detected at the wavelengths 328.068 nm, and 338.289 nm. Calibrations were performed before each measurement. Depending on concentra-tion range in samples the following calibraconcentra-tion soluconcentra-tions were used: blank, 1.0, 2.5, 5.0, 10, 25, 50, 100, and 250 µg/L.
The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument software. Due to spectral interferences at the wavelength of 338.289 nm, the ob-tained data from 328.068 were used for calculating concentrations. Correlation coefficient (r) was 0.99995. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.
The applied LOD/LOQ (Limit of detection / Limit of Quantification) calculations are:
LOD: 3 * method standard deviation from calibration line;
LOQ: 10 * method standard deviation from calibration line.
The information about the LOD/LOQ and correlation coefficient is compiled in Table 6.
The calibration line is shown in the raw data chapter 21.1.
Coefficient of determination (r) for respective calibration functions were taken from ICP-OES instrument outputs.
The resulting values are reported in Table 10.
Table 10: Silver in earthworms: LODs/LOQs, correlation.
Measurement date,
* Internal LOQ calculation was performed with more digits
Instrumental and analytical set-up of the ICP-OES:
-Thermo IRIS Intrepid II
-Thermo Electron Corporation, Germany -Analytical conditions
-Nebuliser: Concentric glass nebulizer, Thermo Electron Corporation, Dreieich, Germany -Spray chamber: Glass cyclonic spray chamber, Thermo Electron Corporation, Dreieich, Ger-many
-Nebuliser gas flow: 0.68 L/min -Make-up gas flow: 0.5 L/min -RF power: 1150 W
-Wavelengths: 328.068 nm, 338.289 nm (not evaluated due to spectral interferences)
The certified reference material TMDA-70 (certified as 10.9 µg Ag/L) was analysed as quality assurance sample with solution samples from the test. According to the quality assurance re-quirement, the silver recovery was in the range of ± 15% of the certified value. However, regard-ing Ag concentrations measured by ICP-OES, the mean recovery (accuracy) and precision of the non digested CRM TMDA-70 measurements were 109 ± 10% (n = 4).
Quality assurance measurements
The recovery for Merck IV solution samples containing 50 µg Ag / L was 104 ± 5% (n = 2).
For further quality assurance, recalibration samples were analysed along with the samples and the mean accuracy was determined to 101 ± 2% (n = 2) for an Ag concentration of 50 µg/L.
For collecting validation information of the digestion procedure of samples as well as the analyti-cal method, the mean recovery of silver in the reference material NIST 2977 Mussel Tissue was determined as 73.5 ± 6.4% (n =3), although only a non-certified reference value was given in the certificate.
