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The quality of data generated for the analysis of environmental samples is critical to State and Federal regulatory agencies to ensure that decisions based upon the amounts of contaminants in environmental samples are truly protective of public health. The quality and validation of these measurements using the approved analytical methodology could be significantly enhanced by incorporating certified reference materials as part of the quality control protocols, yet this has not been widely implemented. Data from certified reference materials can provide critical information that can impact assessments regarding data use that is unavailable by other means. Using the analysis of Cr(vi) in soil extracts as the test case, some of the issues surrounding the use of certified reference materials for environmental applications are discussed in this paper, including the relative cost, and the availability and applicability regarding element coverage and content.

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A suite of three green tea-containing Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST): SRM 3254 Camellia sinensis (Green Tea) Leaves, SRM 3255 Camellia sinensis (Green Tea) Extract, and SRM 3256 Green Tea-Containing Solid Oral Dosage Form. The materials are characterized for catechins, xanthine alkaloids, theanine, and toxic elements. As many as five methods were used in assigning certified and reference values to the constituents, with measurements carried out at NIST and at collaborating laboratories. The materials are intended for use in the development and validation of new analytical methods, and for use as control materials as a component in the support of claims of metrological traceability.

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During the past seven years, several states within the US have enacted regulations that limit the amounts of selected non-nutritive elements in fertilizers. Internationally, several countries, including Japan, China, and Australia, and the European Union also limit the amount of selected elements in fertilizers. The elements of interest include As, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Se, and Zn. Fertilizer manufacturers and state regulatory authorities, faced with meeting and verifying these limits, need to develop analytical methods for determination of the elements of concern and to validate results obtained using these methods. Until now, there were no certified reference materials available with certified mass fraction values for all elements of interest in a blended, multi-nutrient fertilizer matrix. A new standard reference material (SRM) 695 trace elements in multi-nutrient fertilizer, has been developed to help meet these needs. SRM 695 has recently been issued with certified mass fraction values for seventeen elements, reference values for an additional five elements, and information values for two elements. The certificate of analysis includes an addendum listing percentage recovery for eight of these elements, determined using an acid-extraction inductively-coupled plasma optical-emission spectrometry (ICP-OES) method recently developed and tested by members of the Association of American Plant Food Control Officials.

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Analytical methods used for the isotope dilution inductively coupled plasma mass spectrometric (ID-ICP-MS) measurement of Cd at microg kg(-1) and sub-microg kg(-1) levels are described and applied to the certification of new dietary supplement, blood, and serum Standard Reference Materials (SRMs). The materials are: SRM 3240 Ephedra sinica Stapf Aerial Parts, SRM 3241 Ephedra sinica Stapf Native Extract, SRM 3243 Ephedra-Containing Solid Oral Dosage Form, SRM 3244 Ephedra-Containing Protein Powder, SRM 966 Toxic Metals in Bovine Blood, Level 1 (L1) and Level 2 (L2), and SRM 1598a Animal Serum. The concentration of Cd in the materials ranges from 120 microg kg(-1) down to 0.03 microg kg(-1). At these levels, the factors that most influence the accuracy of the ICP-MS data are the procedure blank and spectral and nonspectral interferences. Nonspectral interference, caused by the high concentration of dissolved solids in the matrices investigated, resulted in signal suppression. Matrix separation was used to enhance signal intensity and to reduce spectral interference for the accurate determination of Cd in SRM 1598a and SRM 3244. Chromatographic separation procedures using Chelex for SRM 1598a and anion exchange for SRM 3244 were optimized to achieve the desired separation characteristics without substantially increasing the procedure blank. Sensitivity for the determination of Cd in serum was additionally enhanced through the use of desolvation nebulization. We determined that separations were not required for the accurate ICP-MS determination of Cd in SRM 3240, SRM 3241, SRM 3243, and SRM 966 L2 under optimized analysis conditions. These samples were diluted to a minimum volume and introduced to the ICP-MS via low flow (40-100 microL/min) microconcentric nebulizers. SRM 966 L1 was also analyzed directly, but results were highly variable. The ID-ICP-MS sample preparation and ratio measurement protocols described here resulted in total expanded uncertainties of less than 1% for the determination of 90.85 microg kg(-1) Cd in SRM 3240, and less than 10% total expanded uncertainty for the determination of 0.0468 microg kg(-1) Cd in SRM 1598a.

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Freemartins are XX/XY chimaeras that develop as a result of the fusion of the placental circulation of at least one male and one female fetus. The pituitary glands of eight normal ewes at various stages of the oestrous cycle and three rams were compared with those of two male-type and three undifferentiated-type freemartins. The pituitaries were heaviest in the male-type freemartins, and their pattern of gonadotrophs, assessed by differential staining, was more intense than in the normal males. The pituitaries of the undifferentiated-type freemartins weighed less than those of the normal ewes but had more stained gonadotrophs than the normal ewes or rams. In both types of freemartins the pattern of cells resembled that of a castrated male.

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Freemartins are XX/XY chimaeras that develop as a result of the fusion of the placental circulation of at least one male and one female fetus. Of 19 freemartin ewes, 13 had testis-like structures, seven of them in an abdominal position and six in an inguinal position. Histologically, their gonads had structures resembling seminiferous tubules and interstitial cells, and grossly, most had structures derived from the mesonephric ducts (vasa deferentia, epididymides and vesicular glands). The other six freemartin ewes had small, undifferentiated gonads that lacked either follicles or seminiferous tubule-like structures. They also lacked any structures derived from the mesonephric ducts. No derivatives of the paramesonephric ducts were detectable in any of the freemartin ewes. The gonads of the male-type freemartins stained immunocytochemically for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and histochemically for alkaline phosphatase (AP) in a similar way to, but more extensively and intensely than, the gonads of normal rams, and the staining was confined to interstitial cell-like structures. The staining in the undifferentiated-type freemartins was weak, but both 3beta-HSD and AP were present in unidentified cell types.

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Freemartins are sterile XX/XY chimaeras that occur as a result of placental fusion between male and female fetuses during early pregnancy. Freemartins occur predominantly in cattle, although the prevalence of ovine freemartinism is increasing. In this study, the reproductive endocrinology of ovine freemartins was compared with that of normal sheep. Freemartins had significantly (P < 0.001) higher basal concentrations of LH and FSH than did normal ewes or rams, although the response of LH to GnRH (10 microg) was similar in freemartins, ewes and rams. Resting concentrations of oestradiol were similar in freemartins and ewes and were increased in both after eCG administration. Testosterone concentrations were higher in freemartins than in ewes, but were unresponsive to GnRH or eCG. Administration of 62.5 mg progesterone or 25 lg oestradiol twice a day for 3 days suppressed LH concentrations to baseline values in freemartins, ewes and rams. In ewes, 500 microg oestradiol administered twice a day caused preovulatory surges in LH concentrations, but suppressed LH in freemartins to baseline values. Thus, LH secretion can potentially be regulated in freemartins by gonadal steroids. FSH concentrations in freemartins were not suppressed by doses of inhibin that were effective in ewes and rams. Therefore, freemartins behave in part like castrated animals, as they have high basal concentrations of LH and FSH, which can be stimulated by GnRH and suppressed by gonadal steroids. Conversely, inhibin does not suppress FSH concentrations in freemartins, and freemartins have circulating concentrations of steroids intermediate between those of castrated and normal animals.

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The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.

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An isotope dilution cold vapor inductively coupled plasma mass spectrometry (ID-CV-ICPMS) method featuring gaseous introduction of mercury via tin chloride reduction has been developed and applied to the quantification and certification of mercury in various NIST standard reference materials: SRM 966 Toxic Metals in Bovine Blood (30 ng x mL(-1)); SRM 1641d Mercury in Water (1.6 microg x mL(-1)); and SRM 1946 Lake Superior Fish Tissue (436 ng x g(-1)). Complementary mercury data were generated for SRMs and NIST quality control standards using cold vapor atomic absorption spectroscopy (CVAAS). Certification results for the determination of mercury in SRM 1641d using two independent methods (ID-CV-ICPMS and CVAAS) showed a degree of agreement of 0.3% between the methods. Gaseous introduction of mercury into the ICPMS resulted in a single isotope sensitivity of 2 x 10(6) counts x s(-1)/ng x g(-1) for 201Hg and significantly reduced the memory and washout effects traditionally encountered in solution nebulization ICPMS. Figures of merit for isotope ratio accuracy and precision were evaluated at dwell times of 10, 20, 40, 80, and 160 ms using SRM 3133 Mercury Spectrometric Solution. The optimum dwell time of 80 ms yielded a measured 201Hg/202Hg isotope ratio within 0.13% of the theoretical natural value and a measurement precision of 0.34%, on the basis of three replicate injections of SRM 3133.

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The external phenotype and reproductive behaviour of 21 freemartin sheep and two male pseudohermaphrodite sheep were recorded with the aim of identifying any characteristics which might be used to predict a failure to breed. The size and shape of the vulva and clitoris, the length of the vagina, the size of the teats, the presence or absence of inguinal gonads, and the ultrasonographic characteristics of the inguinal gonads were recorded. A subjective assessment of the masculinity of each animal's body form was also made, and its behavioural responses to a virile ram and to an oestrus ewe were recorded. A number of physical and behavioural abnormalities were detected but the only consistent finding in all 23 animals was a short vagina which varied in length from 3.1 to 7.0 cm, compared with 10 to 14 cm in normal animals.

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Revised G-, Q- and R-banded karyotypes and ideograms for sheep chromosomes at the 420-band level of resolution are presented. The positions of landmark bands on the sheep chromosomes are defined by their distance relative to the centromere to facilitate comparison with equivalent cattle chromosomes. Chromosome-specific (reference) molecular markers that have been mapped to sheep chromosomes and their equivalent cattle chromosomes are proposed. Reference markers will facilitate genome comparisons between sheep and cattle and minimise confusion due to chromosome nomenclature. Numbering of the Robertsonian translocation chromosomes remains as previously reported.

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Abnormalities of the reproductive tract of female sheep were studied by examining 9970 reproductive tracts from cull ewes and 23,536 tracts from nulliparous sheep (prime lambs) over a period of 12 months in abattoirs in south-west England. Overall, 3.37 per cent of the tracts were pregnant (8.11 per cent of cull ewes, and 1.36 per cent of nulliparous sheep), with a peak incidence between September and December. A total of 655 ewes (6.57 per cent) and 459 nulliparous sheep (1.95 per cent) had acquired abnormalities of the reproductive tract. Within these totals, abnormalities of the ovaries accounted for 3.51 per cent (for the ewes) and 10.68 per cent (for the nulliparous sheep) of all the abnormalities, and abnormalities of the ovarian bursa and uterine tube accounted for 42.1 per cent (for the ewes) and 5.23 per cent (for the nulliparous sheep). In addition, uterine lesions (hydrometra and metritis) accounted for 9.92 per cent (for the ewes) and 13.51 per cent (for the nulliparous sheep); lesions of the cervix and vagina (total of 1.44 per cent) and Cysticercus tenuicollis cysts associated with the reproductive tract (total of 3.05 per cent) were less common. Among the ewes the most common ovarian lesions were ovulation tags, and follicular cysts were the most common in nulliparous animals. Lesions such as bursitis, parametritis and abscesses of the reproductive tract were much more common in cull ewes than in nulliparous sheep, probably having arisen from peripartum infections. Hydrosalpinx and hydrometra, in which the intraluminal fluid was clear, were present at relatively high incidence in nulliparous animals, but not in cull ewes. The proportion of tracts containing macerated fetal remnants (2.14 per cent of all abnormalities in cull ewes) was lower than expected. It was considered that the functional significance of many of the lesions, such as ovulation tags and C tenuicollis cysts, was likely to be low, although in some cases of the latter calcification of the cyst had occluded the uterine tubes. Other lesions, notably hydrosalpinx, bursitis and metritis were likely to have made the affected animals sterile. The acquired abnormalities were therefore more significant in terms of individual animal infertility than as a major cause of infertility in flocks.

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A questionnaire concerning the coat colour and sex of cats being vaccinated or neutered was sent to 2585 veterinary practices; 393 (15.2 per cent) were returned and information was obtained about 9816 cats. Of 4598 males, 20 were recorded as tortoiseshell (0.43 per cent). The frequency of the orange gene was 19.7 per cent assuming that male tortoiseshell cats had two X chromosomes. The chromosome complement and/or gonadal histology of 14 male tortoiseshell cats is described. Cytogenetic analysis of 11 animals revealed six with a 38,XX/38,XY complement, two with 39,XXY, two with 38,XX, and one with a 38,XY complement.

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Revised G-, Q- and R-banded karyotypes and ideograms for sheep chromosomes at the 420-band level of resolution are presented. The positions of landmark bands on the sheep chromosomes are defined by their distance relative to the centromere to facilitate comparison with equivalent cattle chromosomes. Chromosome-specific (reference) molecular markers that have been mapped to sheep chromosomes and their equivalent cattle chromosomes are proposed. Reference markers will facilitate genome comparisons between sheep and cattle and minimise confusion due to chromosome nomenclature. Numbering of the Robertsonian translocation chromosomes remains as previously reported.

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