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Activities related to environmental specimen banking in Japan
The Science of the Total Environment, 139/140 (1993) 49-59 Elsevier Science Publishers B.V., Amsterdam
49
Activities related to environmental specimen banking in Japan Yoshinari Ambe Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan
ABSTRACT Some experiments on the preservability of chemicals in environmental samples such as atmospheric particulate matters and mussel meat have been conducted by National Institute for Environmental Studies (NIES), Japan, as a preliminary study for future establishment of environmental specimen bank. NIES also has been operating a small experimental sample bank, storing several kinds of environmental samples. Closely cooperating with NIES, Environment Agency of Japan has been performing several types of nationwide environmental monitoring of chemical substances and some parts of the collected samples have been stored in the cold storage room for future use. A retrospective analysis of TBT and TPT in the fish using these stored samples showed an availability and usefulness of specimen bank for the long term monitoring of pollutants.
Key words: environmental specimen banking; environmental monitoring; storage experiment; retrospective analysis; organic tin INTRODUCTION
In Japan, at present, there is no firm program to establish a full scale environmental specimen bank. Although the importance and usefulness of specimen banking for long term environmental monitoring of toxic chemicals has been recognized for several years by environmental scientists and administrative officials concerned, the realization of it is still not promising, mainly because of financial and man power difficulties. Some activities related to specimen bank have been performed by a few organizations in these years. The major ones are a preliminary study on the long term storage of environmental samples at the National Institute for Environmental Studies (NIES) and a program of Environmental Monitoring of Chemicals by the Environment Agency of Japan. Outlines of these two activities are given in this paper.
50
Y. AMBE
NIES RESEARCH ON THE LONG TERM STORAGE OF ENVIRONMENTAL SAMPLES
The activities concerning environmental specimen banking at NIES initiated in 1980 as part of a research program on the long term environmental monitoring and the major part of the program has already terminated in 1986 [1,2]. The research activities were divided into two groups. One was the study of preservability of pollutants in the environmental samples and the other was on the various problems such as collection and management of samples and countermeasures to the accidents which may occur in the operation of specimen bank. To accomplish these studies, a small scale specimen bank has been operated experimentally. Two types of sample storage experiments were designed as follows.
Stability of benzo[a]pyrene (B[a]p) in the stored atmospheric particulate matter samples An experiment to check the stability of B[a]p in atmospheric particulate matter [3] was conducted using samples collected on quartz glass fiber filters at an urban road side. Subgroups of each lot of samples were subjected to different conditions of storage and were analyzed at yearly intervals to check changes of the concentration of B[a]p during storage. Samples on the filters were stored in stainless steel boxes under the following conditions: (i) 20°C in air, (ii) 20°C in argon gas, (iii) -20°C in air and (iv) -20°C in argon gas. The concentration of B[a]p in the sample filters were measured after 0.5, 1, 2 and 4 years storage in each conditions. The trend of change expressed as percentage of the initial values for the two series of samples with different concentrations of B[a]p gave nearly similar results, both decreasing according to the storage conditions (Fig. 1). The rate of decrease of B[a]p during the test period was higher for the samples stored at 20°C decreasing by about 35% of the initial values. The samples stored in -20°C, on the other hand, reduced their B[a]p content by 12% showing significant effect of the difference of temperature. The difference of the atmosphere in the storage box did not show any remarkable effect in this experiment. A rapid decrease in the initial stage was remarkable for the storage both at 20 and -20°C. At -20°C, however, it showed no change after one year of storage, whereas under 20°C condition the concentration continued to decrease by several percents. From these results, it was concluded that the storage method applied in this storage program does not always completely preserve samples, but, as the rate of decrease of B[a]p after six months was quite low, at least 80% of the initial amount is expected to be preserved after more than 20 years of storage and the samples can be utilized for future retrospective analysis.
ENVIRONMENTAL SPECIMEN BANKING ACTIVITIES IN JAPAN
5l
Storage Time % 100
,,.
0.5
1
'
~
'
2
20Oc
Air Ar
,~,~o~,
Air
3
'
'
~
'
'
i
,
year
613
4O
20
0
Ar
,
i
i
21 1 •
1
•
i
-, ~ ....
Q. . . . . .
2
. . . . ~". . . . .
1~
.. . .. .. . .<> e . . . .. .. . .. .
i
,
Fig. 1. C h a n g e o f B[a]p in t h e a t m o s p h e r i c p a r t i c u l a t e m a t t e r s d u r i n g storage.
Stability test of spiked chemicals in biological samples An experimental study was designed to check changes in the concentration of chemicals spiked artificially into homogenized mussel meat under different conditions of storage. The tested samples were prepared by adding acetone solution of several kinds of organochlorine pesticides, PCB, hydrocarbons, and other organic chemicals (Table 1) into homogenized mussel meat. The mussel meat was homogenized again and distributed into small containers. The variables examined were materials of containers (glass bottle, glass ampoule and polyethylene bottle) and the temperature of storage (-20°C, -85°C, and liquid nitrogen temperature). After two years of storage, part of the stored samples were analyzed for the concentration of several kinds of spiked chemicals and it was observed that organochlorine pesticides did not change at all under the conditions examined. Phthalates, on the other hand, showed a small decrease of its concentration in the plastic bottle at -20°C and some organic chemicals such as No dimethyl-nitrosoamine reduced remarkably (by about 40%) at -20°C storage in every container.
52
Y. A M B E
TABLE 1 Chemicals spiked in the mussel meat sample p-Hydroxycinnamic acid Diphenylamine Hexachloethane n-Dimethylnitrosoamine Tributyltin acetate Ferrocene PCP 3-Methylchloranthrene Methylmercury chloride Benzo[alpyrene Polychlorobenzofuran Hydrocarbon mixture (Ci0-~8) Di-isobutylphthalate Diethylphthalate Dibutylphthalate Di-n-hepthylphthalate n-Butyl benzylphthalate
Di-2-ethylhexylphthalate Dimethylphthalate KC 300, 400, 500, 600 DDVP Sumithion Marathion Arochlor 5460 Aldrin c~-BHC /3-BHC 3,-BHC 6-BHC
o,p'-DDE
Dimethyltelephthalate Diethyltelephthalate Methylparathion Diptelex Endrin Dieldrin Kelthane Metoxychlor Hexachlorobenzene Heptachlor
p,p '-DDT p,p'-DDD p,p '-DDE o,p '-DDT o,p '-DDD
Unfortunately, storage test by using liquid nitrogen in this program was broken by a careless accident. The analysis of spiked chemicals in the stored samples in this experiment was conducted in cooperation with several university laboratories, and the remaining samples are still stored under the same conditions for future analysis.
Stability of certified reference materials NIES has prepared several types of certified environmental reference materials (Table 2) as references for chemical analysis. These reference materials have also been programmed to be tested for the long term stability in the room temperature. No significant change has been found so far in the certified concentrations of the elements in every reference materials and it was confirmed that these materials are usable at least for 5 years after the time of preparation.
The experimental specimen bank at NIES In connection with the studies outlined above on the preservability of sampies, a small scale experimental sample bank has been operated using storage facilities at NIES to examine practical problems related to specimen banking
ENVIRONMENTALSPECIMENBANKINGACTIVITIESIN JAPAN
53
TABLE 2 Environmental samples stored in the NIES experimental specimen bank (as of August 1991) Sample
Approximate number
Atmospheric samples Atmospheric particulate matter samples collected on filters Rain water and snow
200 50
Water samples Lake water
100
Sediment and soil samples Lake sediment Soil
100 50
Biological samples Plant leaves Moss and lichens Fish Shellfish Birds Human hair etc.
2000
Certified reference materials Pepperbush Pond sediment Mussel meat Rice flour Human hair Tea leaves Vehicle exhaust particles
Chlorella Sargasso Fish meat (for organic tin analysis)
such as collection and transportation of samples, registration and management system, and maintenance of storage facilities, etc. The storage facilities used in this program consist of 20 and -20°C storage rooms, -85 and -115°C deep freezers. The applied storage conditions are, although not always perfect, readily available and practically suitable, for the present, as selected for each sample type and the capacity of the storage rooms and freezers. Among the stored samples, atmospheric particulate matter samples from
54
¥. AMBE
a remote island, water samples of several polluted and unpolluted lakes, mussels and other marine organisms were collected under systematic long term monitoring programs. Samples, which were collected rather arbitrary during various environmental research works have also been stored - - as many as the capacity of the storage facilities allow. Table 2 shows the list of major groups of samples stored in the bank at present. Water, sediments, soil, atmospheric particle samples and part of biological samples are stored in the -20°C storing room and a part of precious biological samples are preserved in deep freezers. As regards human samples, due to the difficulty in the collection of human tissue because of ethical reasons and legal regulations in Japan, only human hair samples are stored in the bank. The number of stored samples is increasing yearly for the limited capacity of storing facilities and the stored samples are programmed to be replaced by new, more valuable ones, although aged samples grow more precious year by year. This tentative specimen bank has been working without any serious problem and is planned, for the present, to continue as long as possible in order to demonstrate the usefulness of specimen banking. ENVIRONMENTAL MONITORING OF CHEMICAL SUBSTANCES BY THE ENVIRONMENT AGENCY Several nationwide surveys and monitorings of the chemicals in the environment have been carried out since more than 10 years by the Environment Agency and parts of the samples collected during these surveys have been stored in the NIES sample bank and other places [4]. Some of them were utilized for retrospective analyses showing the availability and usefulness of specimen bank.
General inspection survey of chemical substances in the environment In order to check the appearance of persistent toxic chemicals which were discharged and diffused into the environment, the residues of more than 50 different kinds of chemicals in natural waters, sediments, fish and air at about 30 points have been surveyed every year since 1974. The chemicals surveyed were selected every year from the priority list of 2000 toxic chemicals compiled in 1978. By 1988, monitoring of 691 kinds of chemicals had finished and since 1989 a new survey program has started based on the new priority list including 1145 chemicals. As shown in Table 3, among the 691 chemicals, 105 were detected in the waters from more than one sampling site, 187 were detected
55
ENVIRONMENTAL SPECIMEN BANKING ACTIVITIES IN JAPAN
TABLE 3 Numbers of detected and non-detected chemicals in the 1974-1988 surveys Sample
Detected
Non-detected
Total
Water Sediment Fish Others (Air, etc.)
105 187 70 69
581 477 87 39
686 664 157 108
in the sediment reflecting the accumulation of chemicals in the sediment from the water. Number of the chemicals detected in the fish samples was 70, though the rate of occurrence and concentration were generally not high. In the waters, 581 types of chemicals were below the detection limit, 477 chemicals were not detected from sediments and no fish samples contained 8/7 kinds of chemicals examined. The details of the 1989 fiscal year environmental survey are as follows. Twenty-one chemicals as listed in the Table 4 were subjected to the survey in the water, bottom sediments and fish from 54 areas all over Japan. Among 21 substances, five substances including pyrene and phenyltin were detected from water. Twelve substances including pyrene and other PAHs were detected from bottom sediments and 10 chemicals were detected from fish. While the frequency of occurrence of PAHs in the waters and in fish samples was small, more than 60% of the sediment samples contained significant amounts of PAHs. It is also noticeable that the contamination by organic tin compounds spreads widely in waters, sediments and fish samples. Several compounds as shown in Table 4, on the other hand, were not detected in this survey.
Wildlife monitoring of chemicals The object of this program is to investigate the degree of pollution of wildlife by chemical substances on an annual basis. The species of wildlife investigated in this monitoring were chosen for their significance and usefulness as environmental indicators and considering possibilities for international comparison. Eight fish (chum salmon, angry rockfish, greenling, pacific saury, cod, sea bass, black porgy and dace), two shellfish (common mussel and Asiatic mussel) and two birds (gray starling and black-tailed gull) were collected from 18 areas (16 sea area, one fresh water area and one land area) in the 1989 fiscal year.
3/23 0/53 0/46 0/25 0/53
1/24 1/25 9/23 4/24 0/22
1/72 1/75 14/67 5/72 0/66
C/D
8/69 0/159 0/138 0/75 0/159
A/B
Water (t~g/ml)
0.00005 0.0001 0.00003-0.0473 0.00038-0.027
0.00001-0.000065
Detection range
0.00005 0.0001 0.00003 0.00006 0.00018
0.0001009 0.0001 0.0001 0.0001 0.0001
Detection limit
72/72 55/60 28/55 31/53 20/67
68/71 112/145 122/134 72/74 118/159
A/B
25/25 19/20 11/19 13/19 11/23
23/24 41/51 41/45 25/25 42/53
C/D
0.003-1.31 0.0081-0.34 0.019-1.1 0.007-0.5 0.015-0.16
0.02-3.9 0.0032-2.1 0.005-3.7 0.0009-1.8 0.01-5.5
Detection range
Bottom sediment (#g/g(dry))
0.003 0.006 0.015 0.005 0.015
0.006 0.003 0.005 0.0008
Detection limit
0.005 0.003 0,015 0.005
0.016 0.003 0.015-1.1 0.005-0.99
1/22 1/66 1/63 28/54 8/59
1/21 11/18 17/20
0.0013-0.0096 0.001 0.0012 0.001 0.008 0.003 0.003 0.004 0.003
6/21 1/37 1/41 0/22 1/40
Detection limit
10/63 1/111 1/123 0/66 1/120
Detection range
C/D
A/B
Fish (~.g/g(wet))
Non-detected chemicals: Tetrabromobiphenyl, Hexabromobiphenyl, Decabromobiphenyl, l,l-Bis(t-butylperoty)-3,3,5-trimethylcyclohexane (water, bottom sediments, fish), Dicyclopentadiene, 3Chloro-l,2-dibromopropane, o-Chlorotoluene, p-Chlorotoluene, Benzylchloride (water, bottom sediments).
Phenyltin compounds Diphenyltin compounds Anthraquinone
Dibenzola,h]anthracene
Pyrene Benzo[alanthracene BenzoIa]pyrene Benzo[elpyrene Benzo[blfluoranthene Benzo[[]fluoranthene total Benzo[k]fluoranthene Benzo[g,h,qperylene
Chemical substances
(A/B: detected samples/total samples, C/D: detected areas/investigated areas)
Result of environmental survey for chemical substances (1989)
TABLE 4
57
ENVIRONMENTALSPECIMEN BANKINGACTIVITIES IN JAPAN
Off-coast of Nemuro,
*,I
Suburb of Horloka City, Iwate Prefecture (Gray starling)
Jl
o/
Off-coast of Kushlro, Hokkaldo (Chum salmon)
Sea of Japsn (off-coast of Tohoku) ~
Noto Peninsula
Hokkaldo (Angry rockfish)
~
Yamada Bay
(Common mussel)
(Common mussel) (Greenllng)
\
Lake Biwa (Dace) Off-coast of Sanln ~ (Sea bass)
West Coast of sat . . . .
Peninsula (Sea bass)
\ ~ "x, 1
Q-
Off-coast of Joban
(Pacific aaury)
Y
Tokyo gay (Sea bass)
(Black-tailed
gull)
Ise Bay Miura Peninsula (Common mussel) (Common mussel) Osaka Bay (Sea bass)
Naruto (Asiatic mussel)
o9
Mouth of Riv. Shlmautc (Sea bass)
Sea of Japan (Sea bans)
0 ~Nagagusuku Bay Okinawn Pref. (Acanthopagrus Stvicolus)
Fig. 2. Areas and tested species in wildlife monitoring.
Taking into consideration the results of past survey, the following 36 substances are subject to this monitoring: PCBs and their analogues (3), Drin compounds (3), DDT compounds (6), Chlordane compounds (5), HCH compounds (4) chlorinated benzenes (10), phthalic esters (2), tributyl phosphate and organic tin compounds (2). This program started in 1978 and the results have been published every year. The levels of most of the chemical substances have been very low, except for a few cases, when for example the concentration of dieldrin in shellfish in some coastal areas showed unusually high values influenced by proximity to the land. The results can also be used to follow the long term behavior of chemical substances in the country as Fig. 3 shows the decreasing trend of DDT in the marine biosphere.
58
Y. AMBE
p,p'-DDT
in f i s h
I00
m
50
7,
37
cJ
/
/
/
/
z z /
11
•
/ / • ~ z 4
.
eJ
. '78
'79
'80
'81
'82
'83
'84
/
/
•
Z
/
'85
'86
'87
'88
'89
'90
Fig. 3. N u m b e r o f d e t e c t e d a n d n o n d e t e c t e d s a m p l e s a n d the ratio o f d e t e c t i o n for p , p ' - D D T in fish,
Storage of collected samples A part of the samples collected in the above survey and monitoring have been stored in the cold storage room at -20°C for the purpose of future analysis. About 500 g of the wet sample of fish, shellfish or sediment was crushed and homogenized and, 100 g of it was used to analyze the chemicals and the remaining 400 g was put into glass bottle to be stored in the -20°C storage room. One-hundred samples have been programmed to be stored every year since 1978. This storing program is rather arbitrary and a more systematic one should be considered in future.
A retrospective study of organic tin compounds in fish and shellfish In the course of the survey of chemicals in the environment, TBT (tributyltin) and TPT (triphenyltin), which are used as antifoulant paint for ship hulls and fishing nets, were detected in fish and shellfish samples in 1989. As the marine pollution by these chemicals is becoming one of the serious environmental problems, a retrospective analysis of them was carried out using the samples stored since 1980 to examine previous contamination by these substances. It was shown that contamination of fish by TBT and TPT in several bays had occurred in 1983. Figure 4 shows an example of the results for the contamination of sea bass by TPT in Tokyo Bay.
ENVIRONMENTAL SPECIMEN BANKING ACTIVITIES IN JAPAN
59
4 ppm Tokyo
Bay
( sea b a s s
)
3 ppm
2 ppm max. mean I ppm min.
'83
'84
'85
'86
'87
'88
'89
Fig. 4. Result of retrospective analysis of TPT in sea bass in Tokyo Bay.
Thus, usefulness o f stored sample in the e n v i r o n m e n t a l m o n i t o r i n g o f chemical substances is d e m o n s t r a t e d . CONCLUSION F r o m the experiences o f the activities related to sample banking, various i m p o r t a n t i n f o r m a t i o n is a c c u m u l a t i n g year by year. T h e necessity o f a large scale specimen b a n k is increasing m o r e and more. T h e m a j o r p r o b l e m s in the realization o f a big scale specimen b a n k are budget, m a n p o w e r and finally h o w to c o n t i n u e the system for a long time. REFERENCES 1 Y. Ambe, in R.A. Lewis, N. Stein and C.W. Lewis (Eds), Environmental Specimen Banking and Monitoring as Related to Banking. Martinus Nijhoff Publisher, The Hague, 1984, p. 33-44. 2 Y. Ambe, An overview of the research activities relating to environmental specimen banking. NBS Special Publication, 76 (1985) 22-25. 3 Y. Ambe, H. Mukai and K. Okamoto, Banking of atmospheric particulate matter samples for long term monitoring of atmospheric pollution and related reference material at the National Institute for Environmental Studies. NBS Special Publication, 740 (1988) 108-121. 4 Environment Agency of Japan, Chemicals in the Environment - - Report on Environmental Survey and Wildlife Monitoring of Chemicals in 1988 and 1989, Tokyo, 1991, p. 118.
49
Activities related to environmental specimen banking in Japan Yoshinari Ambe Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan
ABSTRACT Some experiments on the preservability of chemicals in environmental samples such as atmospheric particulate matters and mussel meat have been conducted by National Institute for Environmental Studies (NIES), Japan, as a preliminary study for future establishment of environmental specimen bank. NIES also has been operating a small experimental sample bank, storing several kinds of environmental samples. Closely cooperating with NIES, Environment Agency of Japan has been performing several types of nationwide environmental monitoring of chemical substances and some parts of the collected samples have been stored in the cold storage room for future use. A retrospective analysis of TBT and TPT in the fish using these stored samples showed an availability and usefulness of specimen bank for the long term monitoring of pollutants.
Key words: environmental specimen banking; environmental monitoring; storage experiment; retrospective analysis; organic tin INTRODUCTION
In Japan, at present, there is no firm program to establish a full scale environmental specimen bank. Although the importance and usefulness of specimen banking for long term environmental monitoring of toxic chemicals has been recognized for several years by environmental scientists and administrative officials concerned, the realization of it is still not promising, mainly because of financial and man power difficulties. Some activities related to specimen bank have been performed by a few organizations in these years. The major ones are a preliminary study on the long term storage of environmental samples at the National Institute for Environmental Studies (NIES) and a program of Environmental Monitoring of Chemicals by the Environment Agency of Japan. Outlines of these two activities are given in this paper.
50
Y. AMBE
NIES RESEARCH ON THE LONG TERM STORAGE OF ENVIRONMENTAL SAMPLES
The activities concerning environmental specimen banking at NIES initiated in 1980 as part of a research program on the long term environmental monitoring and the major part of the program has already terminated in 1986 [1,2]. The research activities were divided into two groups. One was the study of preservability of pollutants in the environmental samples and the other was on the various problems such as collection and management of samples and countermeasures to the accidents which may occur in the operation of specimen bank. To accomplish these studies, a small scale specimen bank has been operated experimentally. Two types of sample storage experiments were designed as follows.
Stability of benzo[a]pyrene (B[a]p) in the stored atmospheric particulate matter samples An experiment to check the stability of B[a]p in atmospheric particulate matter [3] was conducted using samples collected on quartz glass fiber filters at an urban road side. Subgroups of each lot of samples were subjected to different conditions of storage and were analyzed at yearly intervals to check changes of the concentration of B[a]p during storage. Samples on the filters were stored in stainless steel boxes under the following conditions: (i) 20°C in air, (ii) 20°C in argon gas, (iii) -20°C in air and (iv) -20°C in argon gas. The concentration of B[a]p in the sample filters were measured after 0.5, 1, 2 and 4 years storage in each conditions. The trend of change expressed as percentage of the initial values for the two series of samples with different concentrations of B[a]p gave nearly similar results, both decreasing according to the storage conditions (Fig. 1). The rate of decrease of B[a]p during the test period was higher for the samples stored at 20°C decreasing by about 35% of the initial values. The samples stored in -20°C, on the other hand, reduced their B[a]p content by 12% showing significant effect of the difference of temperature. The difference of the atmosphere in the storage box did not show any remarkable effect in this experiment. A rapid decrease in the initial stage was remarkable for the storage both at 20 and -20°C. At -20°C, however, it showed no change after one year of storage, whereas under 20°C condition the concentration continued to decrease by several percents. From these results, it was concluded that the storage method applied in this storage program does not always completely preserve samples, but, as the rate of decrease of B[a]p after six months was quite low, at least 80% of the initial amount is expected to be preserved after more than 20 years of storage and the samples can be utilized for future retrospective analysis.
ENVIRONMENTAL SPECIMEN BANKING ACTIVITIES IN JAPAN
5l
Storage Time % 100
,,.
0.5
1
'
~
'
2
20Oc
Air Ar
,~,~o~,
Air
3
'
'
~
'
'
i
,
year
613
4O
20
0
Ar
,
i
i
21 1 •
1
•
i
-, ~ ....
Q. . . . . .
2
. . . . ~". . . . .
1~
.. . .. .. . .<> e . . . .. .. . .. .
i
,
Fig. 1. C h a n g e o f B[a]p in t h e a t m o s p h e r i c p a r t i c u l a t e m a t t e r s d u r i n g storage.
Stability test of spiked chemicals in biological samples An experimental study was designed to check changes in the concentration of chemicals spiked artificially into homogenized mussel meat under different conditions of storage. The tested samples were prepared by adding acetone solution of several kinds of organochlorine pesticides, PCB, hydrocarbons, and other organic chemicals (Table 1) into homogenized mussel meat. The mussel meat was homogenized again and distributed into small containers. The variables examined were materials of containers (glass bottle, glass ampoule and polyethylene bottle) and the temperature of storage (-20°C, -85°C, and liquid nitrogen temperature). After two years of storage, part of the stored samples were analyzed for the concentration of several kinds of spiked chemicals and it was observed that organochlorine pesticides did not change at all under the conditions examined. Phthalates, on the other hand, showed a small decrease of its concentration in the plastic bottle at -20°C and some organic chemicals such as No dimethyl-nitrosoamine reduced remarkably (by about 40%) at -20°C storage in every container.
52
Y. A M B E
TABLE 1 Chemicals spiked in the mussel meat sample p-Hydroxycinnamic acid Diphenylamine Hexachloethane n-Dimethylnitrosoamine Tributyltin acetate Ferrocene PCP 3-Methylchloranthrene Methylmercury chloride Benzo[alpyrene Polychlorobenzofuran Hydrocarbon mixture (Ci0-~8) Di-isobutylphthalate Diethylphthalate Dibutylphthalate Di-n-hepthylphthalate n-Butyl benzylphthalate
Di-2-ethylhexylphthalate Dimethylphthalate KC 300, 400, 500, 600 DDVP Sumithion Marathion Arochlor 5460 Aldrin c~-BHC /3-BHC 3,-BHC 6-BHC
o,p'-DDE
Dimethyltelephthalate Diethyltelephthalate Methylparathion Diptelex Endrin Dieldrin Kelthane Metoxychlor Hexachlorobenzene Heptachlor
p,p '-DDT p,p'-DDD p,p '-DDE o,p '-DDT o,p '-DDD
Unfortunately, storage test by using liquid nitrogen in this program was broken by a careless accident. The analysis of spiked chemicals in the stored samples in this experiment was conducted in cooperation with several university laboratories, and the remaining samples are still stored under the same conditions for future analysis.
Stability of certified reference materials NIES has prepared several types of certified environmental reference materials (Table 2) as references for chemical analysis. These reference materials have also been programmed to be tested for the long term stability in the room temperature. No significant change has been found so far in the certified concentrations of the elements in every reference materials and it was confirmed that these materials are usable at least for 5 years after the time of preparation.
The experimental specimen bank at NIES In connection with the studies outlined above on the preservability of sampies, a small scale experimental sample bank has been operated using storage facilities at NIES to examine practical problems related to specimen banking
ENVIRONMENTALSPECIMENBANKINGACTIVITIESIN JAPAN
53
TABLE 2 Environmental samples stored in the NIES experimental specimen bank (as of August 1991) Sample
Approximate number
Atmospheric samples Atmospheric particulate matter samples collected on filters Rain water and snow
200 50
Water samples Lake water
100
Sediment and soil samples Lake sediment Soil
100 50
Biological samples Plant leaves Moss and lichens Fish Shellfish Birds Human hair etc.
2000
Certified reference materials Pepperbush Pond sediment Mussel meat Rice flour Human hair Tea leaves Vehicle exhaust particles
Chlorella Sargasso Fish meat (for organic tin analysis)
such as collection and transportation of samples, registration and management system, and maintenance of storage facilities, etc. The storage facilities used in this program consist of 20 and -20°C storage rooms, -85 and -115°C deep freezers. The applied storage conditions are, although not always perfect, readily available and practically suitable, for the present, as selected for each sample type and the capacity of the storage rooms and freezers. Among the stored samples, atmospheric particulate matter samples from
54
¥. AMBE
a remote island, water samples of several polluted and unpolluted lakes, mussels and other marine organisms were collected under systematic long term monitoring programs. Samples, which were collected rather arbitrary during various environmental research works have also been stored - - as many as the capacity of the storage facilities allow. Table 2 shows the list of major groups of samples stored in the bank at present. Water, sediments, soil, atmospheric particle samples and part of biological samples are stored in the -20°C storing room and a part of precious biological samples are preserved in deep freezers. As regards human samples, due to the difficulty in the collection of human tissue because of ethical reasons and legal regulations in Japan, only human hair samples are stored in the bank. The number of stored samples is increasing yearly for the limited capacity of storing facilities and the stored samples are programmed to be replaced by new, more valuable ones, although aged samples grow more precious year by year. This tentative specimen bank has been working without any serious problem and is planned, for the present, to continue as long as possible in order to demonstrate the usefulness of specimen banking. ENVIRONMENTAL MONITORING OF CHEMICAL SUBSTANCES BY THE ENVIRONMENT AGENCY Several nationwide surveys and monitorings of the chemicals in the environment have been carried out since more than 10 years by the Environment Agency and parts of the samples collected during these surveys have been stored in the NIES sample bank and other places [4]. Some of them were utilized for retrospective analyses showing the availability and usefulness of specimen bank.
General inspection survey of chemical substances in the environment In order to check the appearance of persistent toxic chemicals which were discharged and diffused into the environment, the residues of more than 50 different kinds of chemicals in natural waters, sediments, fish and air at about 30 points have been surveyed every year since 1974. The chemicals surveyed were selected every year from the priority list of 2000 toxic chemicals compiled in 1978. By 1988, monitoring of 691 kinds of chemicals had finished and since 1989 a new survey program has started based on the new priority list including 1145 chemicals. As shown in Table 3, among the 691 chemicals, 105 were detected in the waters from more than one sampling site, 187 were detected
55
ENVIRONMENTAL SPECIMEN BANKING ACTIVITIES IN JAPAN
TABLE 3 Numbers of detected and non-detected chemicals in the 1974-1988 surveys Sample
Detected
Non-detected
Total
Water Sediment Fish Others (Air, etc.)
105 187 70 69
581 477 87 39
686 664 157 108
in the sediment reflecting the accumulation of chemicals in the sediment from the water. Number of the chemicals detected in the fish samples was 70, though the rate of occurrence and concentration were generally not high. In the waters, 581 types of chemicals were below the detection limit, 477 chemicals were not detected from sediments and no fish samples contained 8/7 kinds of chemicals examined. The details of the 1989 fiscal year environmental survey are as follows. Twenty-one chemicals as listed in the Table 4 were subjected to the survey in the water, bottom sediments and fish from 54 areas all over Japan. Among 21 substances, five substances including pyrene and phenyltin were detected from water. Twelve substances including pyrene and other PAHs were detected from bottom sediments and 10 chemicals were detected from fish. While the frequency of occurrence of PAHs in the waters and in fish samples was small, more than 60% of the sediment samples contained significant amounts of PAHs. It is also noticeable that the contamination by organic tin compounds spreads widely in waters, sediments and fish samples. Several compounds as shown in Table 4, on the other hand, were not detected in this survey.
Wildlife monitoring of chemicals The object of this program is to investigate the degree of pollution of wildlife by chemical substances on an annual basis. The species of wildlife investigated in this monitoring were chosen for their significance and usefulness as environmental indicators and considering possibilities for international comparison. Eight fish (chum salmon, angry rockfish, greenling, pacific saury, cod, sea bass, black porgy and dace), two shellfish (common mussel and Asiatic mussel) and two birds (gray starling and black-tailed gull) were collected from 18 areas (16 sea area, one fresh water area and one land area) in the 1989 fiscal year.
3/23 0/53 0/46 0/25 0/53
1/24 1/25 9/23 4/24 0/22
1/72 1/75 14/67 5/72 0/66
C/D
8/69 0/159 0/138 0/75 0/159
A/B
Water (t~g/ml)
0.00005 0.0001 0.00003-0.0473 0.00038-0.027
0.00001-0.000065
Detection range
0.00005 0.0001 0.00003 0.00006 0.00018
0.0001009 0.0001 0.0001 0.0001 0.0001
Detection limit
72/72 55/60 28/55 31/53 20/67
68/71 112/145 122/134 72/74 118/159
A/B
25/25 19/20 11/19 13/19 11/23
23/24 41/51 41/45 25/25 42/53
C/D
0.003-1.31 0.0081-0.34 0.019-1.1 0.007-0.5 0.015-0.16
0.02-3.9 0.0032-2.1 0.005-3.7 0.0009-1.8 0.01-5.5
Detection range
Bottom sediment (#g/g(dry))
0.003 0.006 0.015 0.005 0.015
0.006 0.003 0.005 0.0008
Detection limit
0.005 0.003 0,015 0.005
0.016 0.003 0.015-1.1 0.005-0.99
1/22 1/66 1/63 28/54 8/59
1/21 11/18 17/20
0.0013-0.0096 0.001 0.0012 0.001 0.008 0.003 0.003 0.004 0.003
6/21 1/37 1/41 0/22 1/40
Detection limit
10/63 1/111 1/123 0/66 1/120
Detection range
C/D
A/B
Fish (~.g/g(wet))
Non-detected chemicals: Tetrabromobiphenyl, Hexabromobiphenyl, Decabromobiphenyl, l,l-Bis(t-butylperoty)-3,3,5-trimethylcyclohexane (water, bottom sediments, fish), Dicyclopentadiene, 3Chloro-l,2-dibromopropane, o-Chlorotoluene, p-Chlorotoluene, Benzylchloride (water, bottom sediments).
Phenyltin compounds Diphenyltin compounds Anthraquinone
Dibenzola,h]anthracene
Pyrene Benzo[alanthracene BenzoIa]pyrene Benzo[elpyrene Benzo[blfluoranthene Benzo[[]fluoranthene total Benzo[k]fluoranthene Benzo[g,h,qperylene
Chemical substances
(A/B: detected samples/total samples, C/D: detected areas/investigated areas)
Result of environmental survey for chemical substances (1989)
TABLE 4
57
ENVIRONMENTALSPECIMEN BANKINGACTIVITIES IN JAPAN
Off-coast of Nemuro,
*,I
Suburb of Horloka City, Iwate Prefecture (Gray starling)
Jl
o/
Off-coast of Kushlro, Hokkaldo (Chum salmon)
Sea of Japsn (off-coast of Tohoku) ~
Noto Peninsula
Hokkaldo (Angry rockfish)
~
Yamada Bay
(Common mussel)
(Common mussel) (Greenllng)
\
Lake Biwa (Dace) Off-coast of Sanln ~ (Sea bass)
West Coast of sat . . . .
Peninsula (Sea bass)
\ ~ "x, 1
Q-
Off-coast of Joban
(Pacific aaury)
Y
Tokyo gay (Sea bass)
(Black-tailed
gull)
Ise Bay Miura Peninsula (Common mussel) (Common mussel) Osaka Bay (Sea bass)
Naruto (Asiatic mussel)
o9
Mouth of Riv. Shlmautc (Sea bass)
Sea of Japan (Sea bans)
0 ~Nagagusuku Bay Okinawn Pref. (Acanthopagrus Stvicolus)
Fig. 2. Areas and tested species in wildlife monitoring.
Taking into consideration the results of past survey, the following 36 substances are subject to this monitoring: PCBs and their analogues (3), Drin compounds (3), DDT compounds (6), Chlordane compounds (5), HCH compounds (4) chlorinated benzenes (10), phthalic esters (2), tributyl phosphate and organic tin compounds (2). This program started in 1978 and the results have been published every year. The levels of most of the chemical substances have been very low, except for a few cases, when for example the concentration of dieldrin in shellfish in some coastal areas showed unusually high values influenced by proximity to the land. The results can also be used to follow the long term behavior of chemical substances in the country as Fig. 3 shows the decreasing trend of DDT in the marine biosphere.
58
Y. AMBE
p,p'-DDT
in f i s h
I00
m
50
7,
37
cJ
/
/
/
/
z z /
11
•
/ / • ~ z 4
.
eJ
. '78
'79
'80
'81
'82
'83
'84
/
/
•
Z
/
'85
'86
'87
'88
'89
'90
Fig. 3. N u m b e r o f d e t e c t e d a n d n o n d e t e c t e d s a m p l e s a n d the ratio o f d e t e c t i o n for p , p ' - D D T in fish,
Storage of collected samples A part of the samples collected in the above survey and monitoring have been stored in the cold storage room at -20°C for the purpose of future analysis. About 500 g of the wet sample of fish, shellfish or sediment was crushed and homogenized and, 100 g of it was used to analyze the chemicals and the remaining 400 g was put into glass bottle to be stored in the -20°C storage room. One-hundred samples have been programmed to be stored every year since 1978. This storing program is rather arbitrary and a more systematic one should be considered in future.
A retrospective study of organic tin compounds in fish and shellfish In the course of the survey of chemicals in the environment, TBT (tributyltin) and TPT (triphenyltin), which are used as antifoulant paint for ship hulls and fishing nets, were detected in fish and shellfish samples in 1989. As the marine pollution by these chemicals is becoming one of the serious environmental problems, a retrospective analysis of them was carried out using the samples stored since 1980 to examine previous contamination by these substances. It was shown that contamination of fish by TBT and TPT in several bays had occurred in 1983. Figure 4 shows an example of the results for the contamination of sea bass by TPT in Tokyo Bay.
ENVIRONMENTAL SPECIMEN BANKING ACTIVITIES IN JAPAN
59
4 ppm Tokyo
Bay
( sea b a s s
)
3 ppm
2 ppm max. mean I ppm min.
'83
'84
'85
'86
'87
'88
'89
Fig. 4. Result of retrospective analysis of TPT in sea bass in Tokyo Bay.
Thus, usefulness o f stored sample in the e n v i r o n m e n t a l m o n i t o r i n g o f chemical substances is d e m o n s t r a t e d . CONCLUSION F r o m the experiences o f the activities related to sample banking, various i m p o r t a n t i n f o r m a t i o n is a c c u m u l a t i n g year by year. T h e necessity o f a large scale specimen b a n k is increasing m o r e and more. T h e m a j o r p r o b l e m s in the realization o f a big scale specimen b a n k are budget, m a n p o w e r and finally h o w to c o n t i n u e the system for a long time. REFERENCES 1 Y. Ambe, in R.A. Lewis, N. Stein and C.W. Lewis (Eds), Environmental Specimen Banking and Monitoring as Related to Banking. Martinus Nijhoff Publisher, The Hague, 1984, p. 33-44. 2 Y. Ambe, An overview of the research activities relating to environmental specimen banking. NBS Special Publication, 76 (1985) 22-25. 3 Y. Ambe, H. Mukai and K. Okamoto, Banking of atmospheric particulate matter samples for long term monitoring of atmospheric pollution and related reference material at the National Institute for Environmental Studies. NBS Special Publication, 740 (1988) 108-121. 4 Environment Agency of Japan, Chemicals in the Environment - - Report on Environmental Survey and Wildlife Monitoring of Chemicals in 1988 and 1989, Tokyo, 1991, p. 118.