ConclusionsFrom the discussion of the results from 12 birds over three seasons it appears that much further work remains to be done. The results of this experiment are descriptive of what occurs with normal birds, but further experiments are clearly necessary to find out why. One of the major points is the variability between individual birds and this and not the statistical averages requires closer attention.For example, it has been known for a long time that shell thickness decreases in summer and it is almost certainly a temperature effect, but from the present results for individual birds, it is evident that whereas one bird exhibits this effect, another does not. What is the factor present in the second bird and absent or present in a smaller degree in the first ? If eggs with thinner shells tend to crack more easily, then birds laying thick-shelled eggs are a better commercial proposition from this aspect, but the ability of a bird to resist the tendency to produce thinner shelled eggs in summer may be equally important.Similarly, it has been shown that not all eggs of a given thickness will crack and more work is required on this problem.The next stage in relation to all characteristics considered in this paper seems to be experiments in which environmental factors such as temperature and humidity are controlled so that changes due to environment can be separated from genetic effects, and relationships between any two characteristics be more easily seen.Procedures for the production of a semi-synthetic, copper-deficient rat diet are described. The final product contains between 0.3 and 0.4 p g . of Cu per g. When supplemented with copper, the diet promotes rapid growth and good reproductive performance. With slight modification the diet would provide a suitable basal ration for studies of deficiencies of iron, zinc and manganese.
Alizarin fluorine blue, [3-NN-di(carboxymethyl)aminomethyl]-1,2dihydroxyanthraquinone, has been modified by the introduction of a sulphonate group into the 5-position. The complex formed by lanthanum(II1) with this new compound demonstrates a reaction towards fluoride similar to that shown by the complexes formed by lanthanum and cerium(II1) with the original alizarin fluorine blue but with the added advantage of increased solubility. This allows a metal to reagent complex ratio of 2 : 1 to be used, which gives considerable increase in sensitivity towards fluoride.ALIZARIN fluorine blue { [3-NhT-di (carboxymethyl) aminomet hyl] -1, Z-dihydroxyanthraquinone; AFB} has been used for the determination of fluoride ions in a great variety of matrices since 1959.1-3 The complex formed by lanthanum with this reagent (A,,,, 500 nm) reacts with fluoride to give a ternary complex (A,,,.567 nm) rather than undergo decomposition to give alizarin fluorine blue and lanthanum fluoride. While the reagent is highly effective at the final concentration level of 5 x M, the insolubility of the lanthanum complex in water at the operating pH of 4.5 limits the range of fluoride concentrations that can be examined. This problem can be largely overcome by operating with mixed aqueous organic solvents or at a higher pH, or both, but undesirable spectral shifts then occur, which reduce the difference between the absorption spectra given by the alizarin fluorine bluelanthanum and alizarin fluorine bluelanthanumfluoride complexes. Additionally, even at 5 x 10-5 M concentration, the presence of a buffer system that possesses mildly complexing anions such as acetate or succinate appears to be necessary in order to hold the complexes in solution and produce results of satisfactory reproducibility. Such buffers compete with alizarin fluorine blue for the metal ion and reduce sensitivity, although this effect is not serious. Numerous thorough studies of the influence of solvent, pH and reagent to metal ratio on the efficacy of the reaction have been r e p ~r t e d . ~-l ~ For many years improved versions of alizarin fluorine blue have been sought but without success. 14-16 Recently, however, we have succeeded in synthesising the alizarin fluorine blue analogue of alizarin-5-sulphonic acid, the structural formula for the potassium salt of which {potassium sulphonate; AFBS) 3-[NN-di(carboxymethyl) aminomethyl] -1,Z-dihydroxqant hraquinone-5is shown below.
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