FEBS-1: Otolith certified reference material for trace metals

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FEBS-1 is a saggital otolith reference material procured from red snapper (Lutjanus campechanus). Certified values are based on unweighted mean results from data submitted by collaborating laboratories. The expanded uncertainty (U_CRM) in the certified value is equal to U = ku_c, where u_c is the combined standard uncertainty calculated according to the JCGM Guide ^Footnote1 and k is the coverage factor. The value of u_c is determined from the combined uncertainties of the vairous analytical methods (u_char) as well as uncertainties associated with homogeneity (u_hom).

It is intended that U_CRM encompasses every aspect that reasonably contributes to the uncertainty of the measurement ^Footnote2. A coverage factor k = 2 was applied for all elements. The table below lists those elements for which certified values have been established.

Table 1: Certified quantity values for FEBS-1

Element	Mass fraction - (mg/kg)
Barium (d,i,p)	5.09 ± 0.23
Lithium (d,i,n,p)	0.305 ± 0.044
Magnesium (d,i,n,p)	23.6 ± 1.3
Manganese(d,n)	0.686 ± 0.016
Sodium (d,i,n)	2594 ± 161
Strontium (d,i,p)	2055 ± 79
	Mass Fraction - (g/100g)
Calcium (d,i,n,x)	38.3 ± 1.4

Coding

The coding refers only to the instrumental method of analyte determination.

• d - Inductively coupled plasma mass spectrometry (ICP-MS)
• i - Inductively coupled plasma mass spectrometry
• n - Neutron activation (INAA)
• p - Isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS)
• x - X-ray fluorescence spectrometry (XRF).

Intended use

This reference material is primarily intended for use in the calibration of procedures and the development of methods used for the analysis of bulk elemental composition of otoliths and other marine aragonites.

Storage and sampling

This material should be kept tightly closed in the original bottle and should be stored in a cool location. The contents should be well mixed by rotation and shaking of the bottle prior to use. The bottle should be tightly closed immediately after use.

Instructions for drying

FEBS-1 can be dried to constant mass in an oven for 3 hours at 105 °C.

Preparation of FEBS-1

The otoliths were extracted from the fish, rinsed in ultrapure water, air-dried, and stored in polyethylene bottles. Surface contamination was later removed in a class 100 cleanroom by restrinsing all otoliths in 2% ultrapure HNO₃ for 5 minutes, followed by triple-rinsing in ultrapure water. Otoliths were subsequently sonicated in ultrapure water for 10 minutes, triple-rinsed again in ultrapure water and allowed to dry under a laminar flow hood for 72 hours. The otoliths were crushed using a ball mill and the resulting powder was sieved through 100 and 200 mesh stainless steel sieves (150 μm and 75 μm particle sizes, respectively). The 100+ and 200+ fractions were returned to the mill and ground until all material passed the 200 mesh sieve. The material was then radiation sterilized at a minimum dose of 25 kGy (Neutron Products Inc., Dickerson, MD, USA).

Stability

As a result of interest in only the total amount content of the major, minor and trace elements, coupled with the chemical and physical properties of the matrix, the need for any rigorous stability testing of this material under its recommended conditions of storage was obviated and, as such, contributions to a specific long-term stability component of the uncertainty were not included in the evaluation of the combined uncertainty presented in Table 2.

Expiry

The certified values for FEBS-1 are considered valid until April 2015, provided the CRM is handled and stored in accordance with instructions herein.

Certified value

The results from a sub-group of participants were used for the certification of FEBS-1. These laboratories were selected based on their previous experience analysing otolith samples. The certified values were calculated from the unweighted means of the results of the participating laboratories ^{Footnote3 Footnote4}. New guidelines for CRM producers suggest all sources relevant to the user of the material should contribute to the uncertainty of the certified value ^{Footnote 2 Footnote 3 Footnote 4}. Included in the overall uncertainty estimate are uncertainties in the batch characterization (u_char), uncertainties related to possible between-bottle variation (u_hom) as well as instability derived from effects relating to long-term storage and transport (u_stab). Expressed as standard uncertainties, these components can be combined as:

Equation 1:  $u_{\mathrm{c(CRM)}}^2=u_{\mathrm{char}}^2+u_{\mathrm{hom}}^2+u_{\mathrm{stab}}^2$

The various components used to calculate the certified values are shown in Table 2.

Characterization

The characterization uncertainties (u_char) were calculated from the standard deviation of the means ^{Footnote 4} .

Homogeneity

The material was tested for homogeneity at NRC using ICP-MS or ICP-OES. Results from triplicate sub-samples (0.250 g) from at least 8 bottles were evaluated using ANOVA ^{Footnote 5}.

Table 2 summarizes the resulting uncertainty components for homogeneity for each element at the 250 mg sub-sample size used for the testing.

Table 2: Uncertainty components for FEBS-1

Incertainty	u (wBa), mg/kg	u (wCa), g/100g	u (wLi), mg/kg	u (wMg), mg/kg	u (wMn), mg/kg	u (wNa), mg/kg	u (wSr), mg/kg
uchar	0.039	0.62	0.0211	0.395	0.006	72.9	21
uhom	0.108	0.35	0.0055	0.535	0.0055	23.5	34
uc	0.115	0.717	0.022	0.665	0.0081	80.3	40
U (k=2)	0.23	1.4	0.044	1.33	0.016	161	79

Information values

Significant heterogeneity was observed for many elements, precluding their certification. A mass fraction interval is presented in the following table, which simply summarizes both the highest and lowest determined of the element in any given subsample by the primary ID-ICP-MS technique along with corroboration by another methodology. These data may be of interest to users/readers and hence are included in this report.

Table 3:  Information values for FEBS-1

Element	Interval*	Technique
Cd, μg/kg	1.4 - 3.2	ID-ICP-MS, ICP-MS
Cu, mg/kg	4.2 - 6.8	ID-ICP-MS, ICP-MS
Ni, μg/kg	16 - 29	ID-ICP-MS
Pb, mg/kg	0.40 - 0.77	ID-ICP-MS, ICP-MS
Zn, mg/kg	3.2 - 6.3	ID-ICP-MS, INAA

* Extreme values of mass fractions obtained during sample characterization.

Metrological traceability

Results presented in this certificate are traceable to the SI through gravimetrically prepared standards of established purity and international measurement intercomparisons. As such, they serve as suitable reference materials for laboratory quality assurance programs, as outlined in ISO/IEC 17025. This CRM is registered at the Bureau International des Poids et Mesures (BIPM) in Appendix C of the Comité International des Poids et Mesures database listing Calibration and Measurement Capabilities accepted by signatories to the Mutual Recognition Arrangement of the Metre Convention.

Accreditation

The Chemical Metrology laboratory is compliant to ISO 17025 and ISO Guide 34, with approval by The Inter-American Metrology System (SIM). The certificate of approval is available upon request.

Updates

Users should ensure that the certificate they have is current. The NRC web site will contain any new information.

Acknowledgements

The following staff members of the Measurement Science and Standards portfolio at the NRC contributed to the production and certification of FEBS-1: V. Clancy, J. Lam, C. Brophy, R. Sturgeon, L. Yang and S. Willie.

The cooperation of the following is gratefully acknowledged:

R. Greenberg, R.O. Spatz, R. Vocke, G. Turk and K. Murphy National Institute for Standards and Technology, Chemical Science and Technology Laboratory, Gaithersburg, USA

Z. Chen, Laboratory for Isotope and Trace Element Research Department of Chemistry and Biochemistry Old Dominion University, Norfolk, VA 23529 USA

S. Thorrold, Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.

Date of issue: August 2005
Date of expiry: April 2015

Approved by:

Zoltan Mester, Ph. D.
Group Leader, Chemical Metrology
Measurement Science and Standards

Comments, information and inquiries should be addressed to:

National Research Council of Canada
Measurement Science and Standards
1200 Montreal Road
Building M-12
Ottawa, Ontario K1A 0R6

Telephone: 613-993-2359
Fax: 613-993-2451
Email: CRM-MRCInorganic-Inorganiques@nrc-cnrc.gc.ca