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German experience with the Japanese fish accumulation test
ECOTOXICOLOGY
German
AND
ENVIRONMENTAL
Experience
SAFETY
4, 17-20 (1980)
with the Japanese BRIGITTE Bayer
Ltd..
Test’
HAMBURGER
Leverkusen,
Received
Fish Accumulation
May
West Germany 13. 1979
Reports on accumulation tests and their results have appeared in literature since the end of the 1960s. In most cases the tests were carried out with inorganic salts of a wide variety of elements which were more or less readily accessible to analysis. As far as organic compounds are concerned the majority of the publications deal with PCB and associated materials, DDT and further pesticides as well as some other compounds. Good descriptions of test procedures are extremely rare; information on the highly important subject of analysis is given in very few cases only, although this subject, which financially is the most significant aspect of the tests, appears to be of major importance, in the same range like the evaluation. Accumulation, e.g., as mentioned in the Japanese law of 1974, is also termed bioaccumulation by a number of authors. It is distinct from biomagnification which results from tests concerning accumulation over the food chain. In this respect reference is made to the study by METCALF on the ecosystem model for the evaluation of pesticide biodegradability and ecological magnification which was described in 1971. More accurate details about the accumulation testing have been given for the first time in the Japanese Harmful Chemicals Law of 1974 which has been mentioned before. This law provides specifications for some parameters which must be accurately complied with. The technical and capital expenditure for the flow-through tests is quite remarkable since the maintenance of all individual parameters such as oxygen content, pH, hardness, and temperature calls for a separate control system for each of these. pH and hardness are not listed in the specifications, nor should they, since rigid regulation would necessitate enormous technical expenditure to maintain constant conditons. This expenditure is necessary for us in Leverkusen since, unfortunately, we have to regulate heavily fluctuating water hardness, and consequently as a result of aeration, pH fluctuations as well. Within our company we have set a German hardness of 15” (i.e., 18.8” E.H.) as a goal which complies with the hardness specified in other tests. I would recommend that hardness and pH are checked every day and entered in the test records. At a very low hardness, for example, the heavy metals, being more toxic in these ranges, are likely to give incorrect results because the separation mechanisms no longer function properly. On the other hand, extremely high hardnesses may lead to salt precipitation. This shows that test records are a valuable help when interpreting results. Nevertheless, ’ Presented at International Symposium, “Testing of Chemical Substances for Ecotoxicological Evaluation,” May 15-17, 1979, Auditorium GSF, Munich-Neuherberg. 17
0147-6513/80/010017-04$02.00/O Copyright All rights
‘B 1980 by Academic Press. Inc. of reproduction in any form reserved.
18
BRIGITTE
HAMBURGER
I wish to emphasize once more: No general specification of a given hardness should be demanded since the technical expenditure is very high. Hardness and pH should be determined after passing the active carbon filters, which have to be set up to free the water from chlorine. In our case, however, it was also necessary to install an ion exchanger which specifically eliminates copper, before these filters. The copper content should be below 0.1 mg/liter. Hardness is measured titrimetrically with photometric endpoint indication. From this stage the pulse is transmitted which opens the treated water valve for diluting our drinking water which is too hard. After adjustment of hardness and pH, the latter by adding small quantities of sulfuric acid, the water is aerated. This is done to adjust the oxygen content at a level of 7 mg oxygen/liter. The temperature specified for accumulation on carps (Cyprinus car,& LinnC) is 25 +- 2°C. This makes it necessary to raise the water temperature by heating. With a heavy flow volume it is no longer possible to maintain 25°C in the flow-through vessel merely by conditioning the temperature of the room, all the more as the drinking water has a temperature below 10°C during the winter months. Only when pretreated in this way is it possible to use the water for the actual test. The requisite quantities of water are pumped into the aquaria. The hose connections should be as short as possible and they should be replaced after each test. The quantity delivered by each individual pump is checked daily. The specified quantity of water is 3- 10 liter per fish, i.e., for carp which should have a weight of 20-40 g and a length of approx. 10 cm. The test should include 10 fish or more to provide adequate material for analysis. The Japanese specification stipulates: 2 fish must be analyzed every 2 weeks. We have selected loo-liter glass aquaria with outside silicone cementing. Water and product solution are metered by us separately. To maintain a constant product solution we use magnetic stirrers for the dissolving vessels. This procedure increases the safety of maintaining the specified concentrations. As an additional check, our Analytical Department determines the recovery rate twice a week. With a 25 liter/hr inflow it is necessary to treat 600 liters of water per tank. Apart from a blank tank two tanks with test concentrations are operated for 8 weeks. The law specifies that these two concentrations are to be calculated from the LCSO determined for red killifish (Orycius latipes), and they should preferably be l/lOOth and l/lOOOth of this, respectively. From Japanese colleagues we have heard, that now the concentrations 1 and 0.1 mg/liter were allowed to prove. This sentence was not established firmly in the law. Test records of the parameters mentioned before are drawn up daily. The fish are fed daily and in this respect it is of great importance to meter this feed accurately. Feed rest should preferably be avoided and, if they occur, be quickly removed. The excrement should be drawn off daily; in addition, the walls should be cleaned if there is any growth of algae. To be added to this high technical expenditure must be the special product-related analysis work. This is relatively simple where heavy metals are concerned. However, as a function of the constitution and properties of organic compounds it may become extremely difficult. The whole fish is subjected to analysis since it is consumed wholly in some areas. Test expenditure increases even more than described where compounds with radioactive markers are concerned. In this respect, it is also necessary to consider the trapping and subsequent treatment of the effluent.
JAPANESE
FISH
ACCUMULATION
TEST
19
Besides a high degree of technical expenditure, the described accumulation test also calls for considerable personnel. It is necessary to check up on controls. The daily test records also include the cleaning of the flow-through tanks. In this respect stock keeping must not be overlooked. The Japanese law specifies carp weighing 20-40 g and being approximately 10 cm long, and because of the difficulty of obtaining carp with this “ideal weight” the first tests were run by us with large golden orfe. The Japanese regulation recommends that the fish be bathed in water containing a drug where ecto- or endoparasites are present. With regard to “medical” treatment there is one point which should be mentioned: Which is the most suitable way of cleaning the aquaria? For this we have adopted the following procedure: Following strong rinsing the aquaria are first filled with a 0.1% formaldehyde solution which is left in them overnight and, after strong rinsing, they are treated overnight with an 0.01% potassium permanganate solution. We hope that this will eliminate any potential infection. All our tests have so far been carried out with substances which were readily soluble in water. While it appears essential to discuss the use of solubilizers, there are a few further points which should be dealt with before taking up this problem. We have now been carrying out accumulation tests for 4 years and, in addition, we have discussed the tests and their procedure within a German work group of the association of the German chemical industry. The resulting experience should be discussed here. We would suggest that the determination of the LC5,,, for which the Japanese law specifies Orycius latipes, be also permitted with test fish customary in the individual countries, e.g., zebra fish (Brachydunio rerio), a fish used in both static and flow-through tests by ISO. Rainbow trout [Salmo gairdneri) or golden orfe (Leucisus idus) could also be used. We have therefore already started tests to compare the individual species. First of all, however, it should be explained why we submit this proposal: We have tried to obtain in Germany red killifish from the association of killifish breeders. For the first 3 years it turned out to be impossible to obtain this species in Europe. In common with other colleagues in Germany and other countries we had so far red killifish flown in. However, we received only a few series which were in satisfactory condition, e.g., the first fish were infested with copepoda. The last two series suffered from fin rot, and the latest reply from Japan was that we still had to wait for some time until the fish would have reached the size specified by MITI. Such incidents quite considerably complicate the carrying out of accumulation tests in compliance with Japanese legal specifications in our country. On the other hand, on the strength of our 4 years of experience with zebra fish, we are now in a position to state that this species is always available in adequate quality, size, and health. Reference should also be made to the use of the local carp. So far we all have used Cyprinus carpio Linne. In 1978, just as our colleagues from BASF and Ciba-Geigy, we had carp flown in from Japan. The first results from BASF have shown that the data obtained are generally equal, and it was only at the beginning that the somewhat different endogenous rhythm appeared to play a part, which differs somewhat in spring. On completion of the trials, however, no more difference was found, and also during the summer in the course of a repeat, no difference was noticed. Our first comparison test which was run parallel with Japanese and German carp, shows no difference whatsoever. Both varieties differ outwardly by their scales and a somewhat differently shaped back. The fewer scales of the
20
BRIGITTE
HAMBURGER
European carp are probably to be attributed to the trend of meeting housewives’ wishes for fish with fewer scales. Obtaining carp with the ideal weight throughout the entire year is bound to introduce some difficulty-I wish to point out that this also holds true for Japan, because when we ordered fish in the spring we were asked to wait for some time since the fish still had to grow a bit bigger. Our own tests within our work group have shown that it is of advantage to retain the Japanese specifications regarding size and weight since a fish weighing 20-40 g is also within a favorable range for analysis work. A third subject of our considerations would be the length of the tests: are the 56 days necessary? We would offer for discussion whether or not a test of 42 days would be adequate. In such cases it would be possible for materials exhibiting an accumulation rate higher than 100 to give an additional week with pure water flowing in and corresponding analysis. Finally, to come to probably the most difficult problem, already referred to before, whether dispersants and solubilizers should be used. We definitely feel that the use of auxiliaries, whether as dispersants or solubilizers, should not be permitted. Knowledge on a distortion of the results when such auxiliaries are resorted to is so scarce that we can only warn against any specifications which are not adequately backed up by accurate scientific knowledge. Our Japanese colleagues, however, had expressed the wish of using only active materials. What could happen then would be that, for example, a water-insoluble dye could no longer be brought into solution because it is no longer present in the form of the technical dispersion. This would necessitate the use of a dispersant to carry out the accumulation test and such a dispersant had not been contained in the technical formulation of the dye. Yet, it is in all cases necessary to have stable dispersions for carrying out our tests and these must be obtainable with the dispersant used. This calls for stability checks and these should be carried out by experts with maximum specialization and should not be left to biologists and toxicologists only. Our Japanese colleagues have introduced further solubilizers in the discussion which have partly a true dissolving and partly a dispersing character. We propose that only such substances may be used which have an LCSO above 1000 mgiliter and which are not readily degradable. Moreover, there must be a guarantee that these auxiliaries do not lead to any increase in lipoid solubility or the toxicity. I believe that much effort still has to be invested into this field to arrive at truly satisfactory solutions. I may have mixed facts and wishes, yet under the auspices of a harmonizationfor which we all strive it ought to be permissible to say what in the light of our experience appears to be good and what appears to be less satisfactory. I hope that together we shall arrive at a specification which makes it possible for everybody to work economically. Finally, however, I wish to briefly touch upon the most expensive point of an accumulation test, that is the analysis work. An accumulation test, no matter with what organisms and if it was a static or dynamic experiment, should only be specified if the analysis work has been reproducibly proven under known or predictable concentrations. But is it really necessary to continue the testing of all materials with so much expenditure? Previous results have shown that all the materials tested had an accumulation factor below 100. We feel that another more simpler approach to selection should be found.
German
AND
ENVIRONMENTAL
Experience
SAFETY
4, 17-20 (1980)
with the Japanese BRIGITTE Bayer
Ltd..
Test’
HAMBURGER
Leverkusen,
Received
Fish Accumulation
May
West Germany 13. 1979
Reports on accumulation tests and their results have appeared in literature since the end of the 1960s. In most cases the tests were carried out with inorganic salts of a wide variety of elements which were more or less readily accessible to analysis. As far as organic compounds are concerned the majority of the publications deal with PCB and associated materials, DDT and further pesticides as well as some other compounds. Good descriptions of test procedures are extremely rare; information on the highly important subject of analysis is given in very few cases only, although this subject, which financially is the most significant aspect of the tests, appears to be of major importance, in the same range like the evaluation. Accumulation, e.g., as mentioned in the Japanese law of 1974, is also termed bioaccumulation by a number of authors. It is distinct from biomagnification which results from tests concerning accumulation over the food chain. In this respect reference is made to the study by METCALF on the ecosystem model for the evaluation of pesticide biodegradability and ecological magnification which was described in 1971. More accurate details about the accumulation testing have been given for the first time in the Japanese Harmful Chemicals Law of 1974 which has been mentioned before. This law provides specifications for some parameters which must be accurately complied with. The technical and capital expenditure for the flow-through tests is quite remarkable since the maintenance of all individual parameters such as oxygen content, pH, hardness, and temperature calls for a separate control system for each of these. pH and hardness are not listed in the specifications, nor should they, since rigid regulation would necessitate enormous technical expenditure to maintain constant conditons. This expenditure is necessary for us in Leverkusen since, unfortunately, we have to regulate heavily fluctuating water hardness, and consequently as a result of aeration, pH fluctuations as well. Within our company we have set a German hardness of 15” (i.e., 18.8” E.H.) as a goal which complies with the hardness specified in other tests. I would recommend that hardness and pH are checked every day and entered in the test records. At a very low hardness, for example, the heavy metals, being more toxic in these ranges, are likely to give incorrect results because the separation mechanisms no longer function properly. On the other hand, extremely high hardnesses may lead to salt precipitation. This shows that test records are a valuable help when interpreting results. Nevertheless, ’ Presented at International Symposium, “Testing of Chemical Substances for Ecotoxicological Evaluation,” May 15-17, 1979, Auditorium GSF, Munich-Neuherberg. 17
0147-6513/80/010017-04$02.00/O Copyright All rights
‘B 1980 by Academic Press. Inc. of reproduction in any form reserved.
18
BRIGITTE
HAMBURGER
I wish to emphasize once more: No general specification of a given hardness should be demanded since the technical expenditure is very high. Hardness and pH should be determined after passing the active carbon filters, which have to be set up to free the water from chlorine. In our case, however, it was also necessary to install an ion exchanger which specifically eliminates copper, before these filters. The copper content should be below 0.1 mg/liter. Hardness is measured titrimetrically with photometric endpoint indication. From this stage the pulse is transmitted which opens the treated water valve for diluting our drinking water which is too hard. After adjustment of hardness and pH, the latter by adding small quantities of sulfuric acid, the water is aerated. This is done to adjust the oxygen content at a level of 7 mg oxygen/liter. The temperature specified for accumulation on carps (Cyprinus car,& LinnC) is 25 +- 2°C. This makes it necessary to raise the water temperature by heating. With a heavy flow volume it is no longer possible to maintain 25°C in the flow-through vessel merely by conditioning the temperature of the room, all the more as the drinking water has a temperature below 10°C during the winter months. Only when pretreated in this way is it possible to use the water for the actual test. The requisite quantities of water are pumped into the aquaria. The hose connections should be as short as possible and they should be replaced after each test. The quantity delivered by each individual pump is checked daily. The specified quantity of water is 3- 10 liter per fish, i.e., for carp which should have a weight of 20-40 g and a length of approx. 10 cm. The test should include 10 fish or more to provide adequate material for analysis. The Japanese specification stipulates: 2 fish must be analyzed every 2 weeks. We have selected loo-liter glass aquaria with outside silicone cementing. Water and product solution are metered by us separately. To maintain a constant product solution we use magnetic stirrers for the dissolving vessels. This procedure increases the safety of maintaining the specified concentrations. As an additional check, our Analytical Department determines the recovery rate twice a week. With a 25 liter/hr inflow it is necessary to treat 600 liters of water per tank. Apart from a blank tank two tanks with test concentrations are operated for 8 weeks. The law specifies that these two concentrations are to be calculated from the LCSO determined for red killifish (Orycius latipes), and they should preferably be l/lOOth and l/lOOOth of this, respectively. From Japanese colleagues we have heard, that now the concentrations 1 and 0.1 mg/liter were allowed to prove. This sentence was not established firmly in the law. Test records of the parameters mentioned before are drawn up daily. The fish are fed daily and in this respect it is of great importance to meter this feed accurately. Feed rest should preferably be avoided and, if they occur, be quickly removed. The excrement should be drawn off daily; in addition, the walls should be cleaned if there is any growth of algae. To be added to this high technical expenditure must be the special product-related analysis work. This is relatively simple where heavy metals are concerned. However, as a function of the constitution and properties of organic compounds it may become extremely difficult. The whole fish is subjected to analysis since it is consumed wholly in some areas. Test expenditure increases even more than described where compounds with radioactive markers are concerned. In this respect, it is also necessary to consider the trapping and subsequent treatment of the effluent.
JAPANESE
FISH
ACCUMULATION
TEST
19
Besides a high degree of technical expenditure, the described accumulation test also calls for considerable personnel. It is necessary to check up on controls. The daily test records also include the cleaning of the flow-through tanks. In this respect stock keeping must not be overlooked. The Japanese law specifies carp weighing 20-40 g and being approximately 10 cm long, and because of the difficulty of obtaining carp with this “ideal weight” the first tests were run by us with large golden orfe. The Japanese regulation recommends that the fish be bathed in water containing a drug where ecto- or endoparasites are present. With regard to “medical” treatment there is one point which should be mentioned: Which is the most suitable way of cleaning the aquaria? For this we have adopted the following procedure: Following strong rinsing the aquaria are first filled with a 0.1% formaldehyde solution which is left in them overnight and, after strong rinsing, they are treated overnight with an 0.01% potassium permanganate solution. We hope that this will eliminate any potential infection. All our tests have so far been carried out with substances which were readily soluble in water. While it appears essential to discuss the use of solubilizers, there are a few further points which should be dealt with before taking up this problem. We have now been carrying out accumulation tests for 4 years and, in addition, we have discussed the tests and their procedure within a German work group of the association of the German chemical industry. The resulting experience should be discussed here. We would suggest that the determination of the LC5,,, for which the Japanese law specifies Orycius latipes, be also permitted with test fish customary in the individual countries, e.g., zebra fish (Brachydunio rerio), a fish used in both static and flow-through tests by ISO. Rainbow trout [Salmo gairdneri) or golden orfe (Leucisus idus) could also be used. We have therefore already started tests to compare the individual species. First of all, however, it should be explained why we submit this proposal: We have tried to obtain in Germany red killifish from the association of killifish breeders. For the first 3 years it turned out to be impossible to obtain this species in Europe. In common with other colleagues in Germany and other countries we had so far red killifish flown in. However, we received only a few series which were in satisfactory condition, e.g., the first fish were infested with copepoda. The last two series suffered from fin rot, and the latest reply from Japan was that we still had to wait for some time until the fish would have reached the size specified by MITI. Such incidents quite considerably complicate the carrying out of accumulation tests in compliance with Japanese legal specifications in our country. On the other hand, on the strength of our 4 years of experience with zebra fish, we are now in a position to state that this species is always available in adequate quality, size, and health. Reference should also be made to the use of the local carp. So far we all have used Cyprinus carpio Linne. In 1978, just as our colleagues from BASF and Ciba-Geigy, we had carp flown in from Japan. The first results from BASF have shown that the data obtained are generally equal, and it was only at the beginning that the somewhat different endogenous rhythm appeared to play a part, which differs somewhat in spring. On completion of the trials, however, no more difference was found, and also during the summer in the course of a repeat, no difference was noticed. Our first comparison test which was run parallel with Japanese and German carp, shows no difference whatsoever. Both varieties differ outwardly by their scales and a somewhat differently shaped back. The fewer scales of the
20
BRIGITTE
HAMBURGER
European carp are probably to be attributed to the trend of meeting housewives’ wishes for fish with fewer scales. Obtaining carp with the ideal weight throughout the entire year is bound to introduce some difficulty-I wish to point out that this also holds true for Japan, because when we ordered fish in the spring we were asked to wait for some time since the fish still had to grow a bit bigger. Our own tests within our work group have shown that it is of advantage to retain the Japanese specifications regarding size and weight since a fish weighing 20-40 g is also within a favorable range for analysis work. A third subject of our considerations would be the length of the tests: are the 56 days necessary? We would offer for discussion whether or not a test of 42 days would be adequate. In such cases it would be possible for materials exhibiting an accumulation rate higher than 100 to give an additional week with pure water flowing in and corresponding analysis. Finally, to come to probably the most difficult problem, already referred to before, whether dispersants and solubilizers should be used. We definitely feel that the use of auxiliaries, whether as dispersants or solubilizers, should not be permitted. Knowledge on a distortion of the results when such auxiliaries are resorted to is so scarce that we can only warn against any specifications which are not adequately backed up by accurate scientific knowledge. Our Japanese colleagues, however, had expressed the wish of using only active materials. What could happen then would be that, for example, a water-insoluble dye could no longer be brought into solution because it is no longer present in the form of the technical dispersion. This would necessitate the use of a dispersant to carry out the accumulation test and such a dispersant had not been contained in the technical formulation of the dye. Yet, it is in all cases necessary to have stable dispersions for carrying out our tests and these must be obtainable with the dispersant used. This calls for stability checks and these should be carried out by experts with maximum specialization and should not be left to biologists and toxicologists only. Our Japanese colleagues have introduced further solubilizers in the discussion which have partly a true dissolving and partly a dispersing character. We propose that only such substances may be used which have an LCSO above 1000 mgiliter and which are not readily degradable. Moreover, there must be a guarantee that these auxiliaries do not lead to any increase in lipoid solubility or the toxicity. I believe that much effort still has to be invested into this field to arrive at truly satisfactory solutions. I may have mixed facts and wishes, yet under the auspices of a harmonizationfor which we all strive it ought to be permissible to say what in the light of our experience appears to be good and what appears to be less satisfactory. I hope that together we shall arrive at a specification which makes it possible for everybody to work economically. Finally, however, I wish to briefly touch upon the most expensive point of an accumulation test, that is the analysis work. An accumulation test, no matter with what organisms and if it was a static or dynamic experiment, should only be specified if the analysis work has been reproducibly proven under known or predictable concentrations. But is it really necessary to continue the testing of all materials with so much expenditure? Previous results have shown that all the materials tested had an accumulation factor below 100. We feel that another more simpler approach to selection should be found.