A new technique for biological monitoring

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1 A new technique for biological monitoring FUKIKO UEDA 1) *, MARIKO MOCHIZUKI 2), MAKOTO MORI 3) and RYO HONDO 1) 1) Laboratory of Veterinary Public Health, School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Kyounan, Musashino, Tokyo , JAPAN 2) Department of Applied Science, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Kyounan, Musashino, Tokyo , JAPAN 3) Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka , JAPAN *Corresponding author: Fukiko Ueda at above address. Abstract: - We have attempted to develop a new index with which it is possible to assess the cadmium (Cd) contamination by the small quantity of samples. Twenty-seven previously published papers reporting Cd uncontaminated cases were selected, and a new Cd index (Cd standard regression line; CSRL) was obtained, which was based on 101 data points from samples of normal kidney and liver. The CSRL has a significant correlation, although seven data points may be rejected by the test of equal probability ellipse. The equal probability ellipse obtained from the 101 data points was named the Cd equal probability ellipse (CEPE). The data from Cd-contaminated animals and human patients diverged significantly from the CSRL and CEPE, although the data from uncontaminated animals and humans were in agreement with them. These results show that the CSRL and CEPE can be used as a reference for uncontaminated animals when assessing the levels of Cd contamination in animals. Comparison with these indexes requires only a small quantity of samples. Pollution with Cd should be suspected when the Cd contents of liver and kidney from the target sample diverge significantly from the CSRL and CEPE. Key-Words: - Wildlife, biological monitoring, cadmium, contamination, new index, regression line, Itai-Itai disease. 1 Introduction Biological monitoring using wildlife is a useful method of investigating environmental contamination, and many species, including arthropods, molluscs, fish, amphibia, reptiles, birds, mammals and plants, have been used for the monitoring. We have been measuring the levels of contamination in wild birds caused by exposure to several toxic elements, including cadmium (Cd), since 1992, in order to evaluate the degree of biological contamination. However, such studies are often limited by a lack of epidemiological information on wildlife, such as information on age, sex, migratory patterns and feeding habits/diet, and it is often not possible to compare the same species in different countries. For example, the Cd content in the kidneys of ponies increase with age [1], the proportion of feed derived from animals in the greater scaup varies between 45% and 97% [2], and anemonefish change sex during their lifetime [3]. In addition, we usually do not have a method to estimate the age of birds, except in a few species. Most biological monitoring studies that use wildlife have compared the mean levels of metal contaminants in the organs, in spite of the individual data obtained often representing a wide range of values. Extreme values found in wild populations may be identified statistically based on the results of experimental studies, but large numbers of results from both experimental and wild animal populations are needed to assess the significance of the level of environmental contamination. Therefore, this method opposes the ideals of animal welfare and conservation. How can we reduce the level of sacrifice? We intended to conduct the most exact investigation for environmental pollution possible using the smallest number of samples. Contamination ISSN: ISBN:

2 with Cd is still a problem in Japan [4, 5] and there are large amounts of data on Cd contamination. Therefore, by reviewing references that described previous episodes of Cd contamination, we developed a new index with which it is possible to assess the Cd contamination using samples of minimal size. 2 Problem Formulation We initially compared the data from wild bird populations with those from many references containing data from animals uncontaminated with Cd, including birds, to assess the level that represented significant contamination. In the process, we developed a new index that represents uncontaminated animals [6, 7]. This new Cd index is based on 101 data points selected from a total of 62 species (land birds and waterfowl, 10 species; seabirds, 27 species; terrestrial mammals, 17 species; marine mammals, 8 species) in 27 previously published references. The data points represent the Cd contents (natural logarithm of each mean value) of kidneys and livers from uncontaminated animals. The formula of this Cd index was: log(y)=0.902 log(x)-1.334, R 2 = 0.944, P <0.01 (1) Four mean values obtained from land birds and waterfowl, seabirds, terrestrial mammals and marine mammals fell on this line (Fig. 1). Thus, we named this regression line obtained from the 101 points (1) the Cd standard regression line, CSRL. However, the data from the references cited may contain samples contaminated with Cd, although there was no description of any such contamination in the references. We therefore needed to examine the data in detail. 3 Problem Solution Although we used the natural logarithm in formula (1), common logarithm is easier to use than natural logarithm. In this study, the formula was recalculated as a formula of common logarithm. The formula after the recalculation was, log 10 (Y)=0.900 log 10 (X)-0.580, R 2 =0.944, P<0.01 (2) There was no difference between the two correlation coefficients obtained using formula (1) and (2). Thus, here, we propose that formula (2) of CSRL is sufficient to use for biological monitoring of Cd. 3.1 Examination of Cd standard regression line ISSN: ISBN:

3 The test by equal probability ellipse was employed to examine the data in the CSRL. Seven points of the 101 fell outside the ellipse, in spite of the fact that there was no description of any contamination in the relevant references (Fig. 2). The formulas containing 101 points (2) and 94 points (3) were: X 2 /(0.354) 2 +Y 2 /(2.952) 2 =1, tan 2θ=12.66, degree of axis=0.75, r=0.971 (2) X 2 /(0.272) 2 +Y 2 /(2.895) 2 =1, tan 2θ=22.94, degree of axis=0.76, r =0.983 (3) Here, X=log(X) and Y=log(Y) The regression line from 94 points containing formula (3), excluding the seven outliers, was, log(y)=0.941 log(x)-0.649, R 2 =0.965, P<0.01 (4) When the formulas given in (1) and (4) were compared, there was no significant difference between the two lines (Table 1). We had no reason to eliminate these seven points because the formulas of (1) and (4) were not different and there were no descriptions of Cd contamination in the references cited. From these results, we decided to use the regression line obtained from the 101 points (1) and the equal probability ellipse (2) as the Cd standard regression line, CSRL, and the Cd equal probability ellipse, CEPE, respectively. 3.2 Verification of CSRL and CEPE The CSRL was compared with data obtained from animals administered Cd experimentally, and from humans, including patients with Itai-itai disease [6]. Experiments involving the administration of Cd to rats ISSN: ISBN:

4 were performed in our laboratory. A solution of CdCl 2 (0.1, 1, 2 mm) was continuously administered into femoral vein of Wister rat ( ) during 2hrs by the method of Bradbury & Deane [8]. Organs were then removed and put into Pyrex tubes. The organs were weighed, and a solution of a mixture of HNO 3 and HClO 4 was added. The organs were digested at 180 C. The Cd contents of the kidney and liver were analyzed using atomic absorption spectrophotometry (AAS). The other data used were cited in previous reports (see [6]). The data from Cd-contaminated animals diverged significantly from the CSRL, whereas the data from uncontaminated animals were in agreement with the CSRL (Fig. 3). The formulas from rats and rhesus monkeys to which Cd was administered were:, log(y)=1.037 log (X)+0.531, R 2 = 0.95, P<0.01 (5) log(y)=1.743 log (X)-2.857, R 2 =1.000, P<0.01 (6) Although the slope of formula (5) was not different from that of (1), the y intercept (absolute term) was significantly different. As shown in the figure, the slope of the regression line obtained from rats given Cd and control rats was also changed. A similar result was obtained from the data from rhesus monkeys given Cd, and a very good regression line (6) was obtained from these data. These results suggest that animals to which Cd is administered can be clearly distinguished from the normal population. The CSRL was next compared with data obtained from humans in uncontaminated areas (UK, Sweden, West Germany, Hungary, Poland and Japan) and in contaminated areas (Japan; Patients with itai-itai disease and healthy people). The data from humans, including patients, in the Cd-contaminated area also diverged significantly from the CSRL, whereas the data from humans in uncontaminated areas were in agreement with the CSRL (Fig. 4 and Fig. 5). In addition, the equal probability ellipse (7) from the humans, including patients, in the Cd-contaminated area diverged clearly from that of the normal population, although the regression line was not significant. X 2 /(0.422) 2 +Y 2 /(0.659) 2 =1, tan 2θ=0.51, degree of axis=0.24, r = (7) Here, X=log(X) and Y=log(Y) Data from several people from uncontaminated areas was seemed to fall on the regression line obtained from rhesus monkeys to which Cd was administered. These people may have been contaminated by Cd, although there were no descriptions of Cd pollution in the references cited. ISSN: ISBN:

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6 When Cd is taken into an animal, it is chemically bound to metallothionein (MT), a Cd-binding protein, and carried to the liver, then to the kidney. A portion of the MT Cd complex accumulates in the kidney cortex. Therefore, Cd content is high in the liver in the early stage of Cd poisoning, and Cd accumulation in the kidney is increased in the middle stage. Kidney function decreases as a result of the accumulation of Cd, and then the Cd content in liver becomes high again in the chronic stage. The upward shift of the regression line obtained using data from animals to which Cd had been administered (as seen for rats), a change in the slope (as seen for rhesus monkeys), and the results from patients with itai-itai disease probably indicate this process. Comparisons with the CSRL and CEPE require only a small sample size. Pollution with Cd should be suspected when the Cd contents of livers and kidneys from the target sample diverge significantly from these indexes. These indexes may be a useful tool for assessment of the levels of Cd contamination in animals. We are currently applying these indexes to data from Japanese wildlife. 3.3 Application of CSRL and CEPE to Japanese wild birds We show several results from wild birds in this report, although we are in the process of comparing data from wild bird populations with the CSRL and CEPE Origin of wild birds and the method of Cd analysis Birds with heavy oil contamination: - In 1993 a tanker accident occurred offshore of Tomakomai (Hokaido), in the northern area of Japan, and many diving ducks were killed by the outflow of heavy oil. Although the reason was unknown in 2007, many pacific loons were also killed by heavy oil offshore of the Island of Tsushima, which is located between the Korean Peninsula and Japan in the Japan Sea. These birds were supplied from the Japanese Ministry of the Environment and the Tsushima Wildlife Conservation Center, respectively. The birds in both areas were covered with heavy oil. The Cd contents in the kidney and liver were analyzed using AAS. Other birds: - Some birds were captured legally under other national surveys by the Japanese Ministry of the Environment. Common cormorants from Shiga Prefecture and ducks from several prefectures were captured between 1993 and 1995, and common cormorants from Tokushima Prefecture were collected in Birds from Chiba Prefecture were supplied between 1994 and 1998 by the Gyotoku Birds Observatory in Chiba Prefecture. Cd analytical method: - Dry weights (wt.) of kidney and liver samples were determined, a mixed solution of HNO 3 and HClO 4 were added to the samples, and the samples were digested at 180 C. Completely crystallized samples were diluted with 0.1N HNO 3 just before the analyses. The metal content was determined using AAS or inductively coupled plasma emission spectrometry (ICP-AES, Spectro A.I., Germany). Details of the methods of sample preparation, analytical methods and recovery rate have been described in our previous reports [9-14] Wild birds contaminated by heavy oil Table 2 shows the mean Cd contents in the kidneys and livers of oil-contaminated birds. The mean Cd contents were and 7 12 µg/g dry wt. in the kidney and liver, respectively, except for a sample from long-tailed duck. The variance was large in the data from Tomakomai, as shown by the large standard errors (S.E.) of the mean values, while the variance was small in the data from Tsushima. However, all measured contents of Cd were within the CEPE (Fig. 6a and 6b), except for that of the long-tailed duck, and the regression line was not different from the CSRL. These results show that these birds were not contaminated with Cd Healthy wild birds The mean Cd contents in the kidneys and livers of mallard, spot-billed duck, northern pintail, and great cormorant were also examined after classification by the prefecture of collection (Table 3). The mean Cd contents in the kidneys of mallard from Akita and Ishikawawa and of spot-billed ducks from Akita were more than 10µg/g dry wt., and these values were more ISSN: ISBN:

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8 than double those in other wild birds. Although the mean Cd contents in the livers of mallard from Akita, and of spot-billed duck from Akita and Miyagi were more than 4µg/g dry wt., other mean values were less than 2µg/g dry wt. In the usual method of comparison among mean values, these results would lead to the conclusion that mallard from Akita and Ishikawa and spot-billed duck from Akita and Miyagi were contaminated by Cd and that the other birds were not contaminated. However, when these means were inserted into the CEPE, the mean value from spot-billed duck from Akita was within the CEPE formula (2), and the mean of the contents from the mallard from Akita and Ishikawa were also within the CEPE formula (3) (Fig. 7). This result shows that we should not judge the birds in Akita and Ishikawa to have been subject to abnormal levels of Cd pollution, although the mean contents obtained were high. On the ISSN: ISBN:

9 other hand, we may judge that the spot-billed ducks from Miyagi were contaminated by Cd because the mean value was high and outside the CEPE formula (3). Furthermore, the mean content of Cd obtained from great cormorants in Tokushima was outside the CEPE in spite of its low value. The data from great cormorants of Tokushima and Shiga were examined in detail (Fig.8). The Cd contents of organs from great cormorant in Tokushima were different from those in Shiga, and a different cluster was formed. The regression lines (a, b) obtained from these groups were also significantly different from the CSRL. Line (c) joins the two means from Shiga and Tokushima. As shown in Figure 8, the degree of contamination of great cormorants in Shiga seemed to be higher than that in cormorants from Tokushima, although the means from both Shiga and Tokushima were inside and outside the CEPE, respectively. We are now in the process of comparing data from more wild bird populations with the CSRL and CEPE in order to confirm its usefulness in monitoring Cd levels in wild populations. 4 Conclusion New Cd indexes (CSRL and CEPE) based on 101 data points (from normal kidney and liver) selected from previously published references showed significant correlations. The data from Cd-contaminated animals and human patients diverged significantly from the CSRL and CEPE, although the data from uncontaminated animals and humans were in agreement with the indexes. These results show that the CSRL and CEPE can be used as references for uncontaminated animals for assessment of the level of Cd contamination. Pollution with Cd should be suspected when the Cd contents of livers and kidneys from the target sample diverge significantly from the CSRL and CEPE. References: [1] Kowalczyk DF, Gunson DE, Shoop CR, Ramberg CF Jr, The effects of natural exposure to high levels of zinc and cadmium in the immature pony as a function of age, Environmental Research, 40, 2, 1986, pp [2] Kaneda H, Greater scaup, in; Hidaka T, Higuchi H, Morioka H, Yamagishi S (eds), The encyclopedia of animals in Japan, volume 3; birds I, Heibonsha Limited, Publishers, 1996 (in Japanese). [3] Godwin J, Behavioural aspects of protandrous sex change in the anemonefish, Amphiprion melanopus, and endocrine correlates, Animal Behaviour, Vol 48 (3), Sept 1994, pp [4] Asami T, Toxic metal pollution in Japanese soil, Agune Gijyutu center, 2001 (in Japanese). [5] WHO, Environmental health criteria 134 cadmium, [6] Mochizuki M, Mori M, Hondo R, Ueda F, A new index for evaluation of cadmium pollution in birds and mammals, Environmental Monitoring and Assessment, 137, 1-3, 2008, pp [7] Mochizuki M, Mori M, Hondo R, Ueda F, Biological Monitoring Using a New Technique, in; Harris JD and Brown PL (eds), Wildlife: Destruction, Conservation and Biodiversity, Nova Science Publishers Inc., [8] Bradbury MW, Deane R, Rate of uptake of lead-203 into brain and other soft tissues of the rat at constant radiotracer levels in plasma, Annals of the New York Academy of Sciences, 481, 1986, pp [9] Mochizuki M, Ueda F, Sasaki S, Hondo R, Vanadium contamination and the relation between vanadium and other elements in wild birds, Environmental Pollution, 106, 2, 1999, pp [10] Mochizuki M, Ueda F, Sano T, Hondo R, Relationship between vanadate induced relaxation and vanadium content in guinea pig taenia coli. Canadian Journal of Physiology and Pharmacology, 78, 4, 2000, pp [11] Mochizuki M, Hondo R, Kumon K, Sasaki R, Matsuba H, Ueda F, Cadmium contamination in wild birds as an indicator of environmental pollution, Environmental Monitoring and Assessment, 73, 3, 2002, pp [12] Mochizuki M, Sasaki R, Yamashita Y, Akinaga M, Anan N, Sasaki S, Hondo R, Ueda F, The distribution of molybdenum in the tissues of wild ducks, Environmental Monitoring and Assessment, 77, 2, 2002, pp [13] Mochizuki M, Hondo R, Ueda F, Simultaneous analysis for multiple heavy metals in contaminated biological samples, Biological Trace Element Research, 87, 2002, 1-3, pp [14] Mochizuki M, Mori M, Akinaga M, Yugami K, Oya C, Hondo R, Ueda F, Thallium contamination in wild ducks in Japan, Journal of wildlife diseases, 41, 3, 2005, pp ISSN: ISBN: