**2. Introduction of CSRL and CEPE**

In this section, we explained about Cd standard regression line (CSRL) and Cd equal probability ellipse (CEPE). We selected previous publications that reported the content of Cd in samples of 46 mammals and 55 birds, and we used 101 data points from 27 reports in which the Cd contents were represented as arithmetic means. The 101 data points were plotted on a graph with the Cd content in the liver on the abscissa and the Cd content in the kidney on the ordinate. A significant correlation was obtained, as follows: Y=0.902X – 1.334, Y=log(y), X=log(x), R2=0.944, p<0.01. The regression line obtained after logarithmic transformation was log (Y)=0.900 log(X)-0.580 (R2=0.944, p<0.01)(1). When the outliers among the 101 data points were tested by equal probability ellipse, seven data points were identified as outliers as shown in Fig.2. After elimination of these seven points, the regression line obtained was: log(Y)= 0.941 log(X)-0.649, (R2=0.965, p<0.01)(2). There were no significant differences between the two lines (1&2) (Ueda et al., 2009a). In mention above, regression line obtained from 101 points and the equal probability ellipse were used as the Cd standard regression line, CSRL, and the Cd equal probability ellipse, CEPE, respectively.

The data from experimental animals to which Cd had been administered were distinct from the CSRL, as shown in Fig. 1.

Similarly, the data from humans who lived in a polluted area and from patients with Itai-itai disease were located outside the CEPE, as shown in Fig. 2. Although the values from humans who lived in non-polluted areas were high, the data were located within the CEPE, as shown in the figure (Fig.2). Detailed information on the references used (Mochizuki et al., 2008), the

Recently, we developed a solvent for use in biological monitoring using wildlife. This method was established using the significant regression lines obtained from the Cd content of kidney and that of liver (Mochizuki et al., 2008). Given that the data from animals were cited in various studies in which no particular contamination was described, we considered that these lines were indicative of normal metabolism in animals. This theory was supported by some evidence obtained from polluted animals, including humans (Mochizuki et al., 2008; Ueda et al., 2009a). Thus, the degree of contamination of humans (Mochizuki et al., 2008; Ueda et al., 2009a), experimental animals (Mochizuki et al., 2008; Ueda et al., 2009b), domestic animals (Ueda et al., 2011) and wild birds (Mochizuki et al., 2011a,c,d; Ueda et al., 2009a) has been analyzed using those indexes. Further, we developed a similar index for lead(Pb); the basis of this study was presented at an International Conference (Mochizuki et al., 2009), and the modified index has also

However, contamination with multiple elements is also an important problem in environmental science. Recently, we investigated the concentration of various elements in the urine of cats (Mochizuki et al., 2010c). In that study, a significant correlation was obtained among multiple elements in urine obtained from healthy cats, although a similar correlation was not observed in urine obtained from cats with urinary tract disease. A loss of balance and equilibrium among multiple elements had occurred in the urine of the diseased cats. This result suggested that similar indexes involving Cd and Pb can be obtained using

The new technique for biological monitoring is introduced in the first part of this chapter. Subsequently, we will attempt to establish an index to increase our understanding of the

In this section, we explained about Cd standard regression line (CSRL) and Cd equal probability ellipse (CEPE). We selected previous publications that reported the content of Cd in samples of 46 mammals and 55 birds, and we used 101 data points from 27 reports in which the Cd contents were represented as arithmetic means. The 101 data points were plotted on a graph with the Cd content in the liver on the abscissa and the Cd content in the kidney on the ordinate. A significant correlation was obtained, as follows: Y=0.902X – 1.334, Y=log(y), X=log(x), R2=0.944, p<0.01. The regression line obtained after logarithmic transformation was log (Y)=0.900 log(X)-0.580 (R2=0.944, p<0.01)(1). When the outliers among the 101 data points were tested by equal probability ellipse, seven data points were identified as outliers as shown in Fig.2. After elimination of these seven points, the regression line obtained was: log(Y)= 0.941 log(X)-0.649, (R2=0.965, p<0.01)(2). There were no significant differences between the two lines (1&2) (Ueda et al., 2009a). In mention above, regression line obtained from 101 points and the equal probability ellipse were used as the Cd standard

degree of contamination with multiple elements using multivariate analysis.

regression line, CSRL, and the Cd equal probability ellipse, CEPE, respectively.

The data from experimental animals to which Cd had been administered were distinct from

Similarly, the data from humans who lived in a polluted area and from patients with Itai-itai disease were located outside the CEPE, as shown in Fig. 2. Although the values from humans who lived in non-polluted areas were high, the data were located within the CEPE, as shown in the figure (Fig.2). Detailed information on the references used (Mochizuki et al., 2008), the

been submitted to a journal for publication.

measurement of multiple elements.

**2. Introduction of CSRL and CEPE** 

the CSRL, as shown in Fig. 1.

procedure for calculation of the indexes (Ueda et al., 2009a), and the data from humans and rushes monkeys (Mochizuki et al., 2008) have been described in our previous reports.

Fig. 1. Comparison of the data from laboratory animals. Original figure from Mochizuki et al. (2008) as modified by Ueda et al. (2009a).

A new development in the research area of biological monitoring has been introduced in this section. In the next section we describe the pilot study for establishment of a similar index using multiple elements.

Fig. 2. Comparison of human data. Original figure from Ueda et al. (2009a). Dot-line; equal probability ellipse by 101 data points, solid line; equal probability ellipse by 94 data points.

An Innovative Approach to Biological Monitoring Using Wildlife 161

1995. Other birds, which were protected in the Gyoutoku bird observatory in Chiba

Samples of kidney were removed from the birds, and about 200 mg of each sample was put into a Pyrex tube (Corning, USA), and dried in an oven at 70℃ to determine the dry weight of the sample. The appropriate volume of HNO3 : HClO4 (1:1, Wako Pure Chemical, Ltd., Japan) was added to the dried samples, and the samples were digested at 180℃. The contents of various elements in the kidneys of the birds were analyzed using inductively coupled plasma emission spectrometry (ICP-AES, FTP08, Spectro A.I., Germany). The eight target elements were: Cd, Cr, copper (Cu), lithium (Li), Mo, titanium (Ti), Tl and V. The standard additional method was employed for the analysis. The detailed methods of sample preparation and the analytical procedure have been described previously (Mochizuki et al.,

The statistical analyses used in the present study were carried out using computer software such as Lotus 2001 (Lotus Development), Excel 2003 (Microsoft Corporation), and JUMP (SAS Institute, Japan) to obtain the regression line, the confidence intervals, and the logarithmic transformation. Factor analysis was carried out using Excel add-in software

The contents of the eight elements measured in the kidney are shown in Table 1. The data were recalculated using factor analysis. The multiple variables used in the present study (contents of elements) were merged using factor analysis, a form of multivariate analysis. Thus, a higher factor score was thought to indicate more serious contamination by multiple elements. We obtained three significant factors, as shown in Table 2. No tendency for contamination was observed when the mean values of each category were compared. Thus, it is thought that the comparison using only mean values makes it difficult to understand

> **Anatidae Seabird Cormorant Ardeidae Others**  65 17 30 10 5

**Cd** 8.33±1.48 8.36±4.00 1.97±0.50 4.38±0.98 9.17±6.73 **Cr** 2.67±0.55 1.69±0.32 1.65±0.37 3.86±0.93 0.33±0.200 **Cu** 24.21±2.87 40.99±6.78 12.85±0.86 21.12±2.77 19.04±4.19 **Li** 1.85±0.52 1.24±0.36 1.62±0.43 3.04±0.92 0.32±0.23 **Mo** 6.88±1.04 3.80±0.94 4.62±0.43 5.35±1.39 12.57±8.98 **Ti** 2.07±0.56 0.80±0.34 1.32±0.34 2.94±0.88 0.83±0.83 **Tl** 9.17±2.14 2.87±0.49 3.33±0.49 5.07±1.45 1.97±1.85 **V** 2.50±0.60 1.10±0.32 2.35±0.48 3.04±0.92 1.75±1.16 Table 1. The contents of the elements in kidneys from birds of each category. The results are

represented as mean contents (μg/g dry wt.), and the standard error of the mean.

Prefecture, were transported to our laboratory after death.

**3.2.1 Establishment of an index for multiple elements** 

the degree of contamination by multiple elements.

**3.1.2 Analytical procedure** 

**3.1.3 Statistical methods** 

2002b).

(Esumi, Japan).

n

**3.2 Factor analysis** 
