- Email: [email protected]
Age as a factor influencing results in the acute daphnid test with Daphnia magna Straus
PII: S0043-1354(99)00296-1
Wat. Res. Vol. 34, No. 5, pp. 1419±1424, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
AGE AS A FACTOR INFLUENCING RESULTS IN THE ACUTE DAPHNID TEST WITH DAPHNIA MAGNA STRAUS BERTHOLD KLEIN* Lower Saxony State Agency for Ecology, PO Box 10 10 62, D-31110, Hildesheim, Germany (First received 1 March 1999; accepted in revised form 1 July 1999) AbstractÐContrary to assumptions which have been current up to the present, juvenile daphnids of the species D. magna Straus exhibit a very varied sensitivity towards toxic chemicals between the ages of 0± 24 h. During this period the greatest susceptibility towards potassium dichromate is reached between the ages of 22±24 h; younger daphnids are up to four times less sensitive. For this reason it is impossible to arrive at a reliable prognosis with potassium dichromate in de®ned concentration as reference substance using test groups of unknown age structure. However, positive controls for verifying correct testing procedure rely on comparable sensitivity amongst the test organisms and are essential for validating the test results. The range of 0.6±1.7 mg/l for the 24 h-EC50 of potassium dichromate, which the ISO norm 6341 propagates as validity criterium, does not take sucient account of the connection between dosage and eectiveness of this substance and thereby masks age related inequalities in the test animals. Therefore it cannot substitute the positive control which is missing in the test speci®cations. As the toxicity of potassium dichromate is pH-dependent a speci®c carbonate hardness for the test medium should be determined. # 2000 Elsevier Science Ltd. All rights reserved Key wordsÐDaphnia, toxicity test guidelines, potassium dichromate acute toxicity
INTRODUCTION
Biotests with Daphnia magna Straus have been used and accepted throughout the world for decades as instruments for the estimation of the acute toxicity of xenobiotics in aquatic environments. One advantage often mentioned in respect of this organism results from the fact that identical animals can be bred through acyclic parthenogenesis and used for testing. However, one aspect of this circumstance has not yet been given due consideration. In genetically identical cohorts a particularly high degree of consistency may be anticipated in the reactions of individuals to de®ned chemical stimuli. Nonetheless, to date no successful process has been developed to establish a reliable prognosis for the acute daphnid toxicity test using a reference substance of de®ned concentration which could serve as a validity criterion, such as is the case in other biotesting procedures implementing a positive control. The ISO standard 6341 (1996) tends to obscure this dilemma. Occasional EC50 determinations with a regression analysis of the dosage-eectiveness of potassium dichromate are used to attest that the test material is in satisfactory condition. This procedure is considered as sucient to ensure the validity of all tests carried out between such EC50 determinations. The *Tel.: +49-5121-509638; fax: +49-5121-509196; e-mail: [email protected]
present paper indicates a reason why it is not possible to guarantee the plausibility of a test result using a single nominal concentration of potassium dichromate within the EC50 range under the conditions prescribed by the ISO 6341.
MATERIALS AND METHODS
The parent animals (clone 5) were reared and the juvenile daphnids were tested (test duration 24 h) in climatised rooms (208C 2 18C) under continuous ¯uorescent light (Universal White). The breeding medium was the M4 medium according to Elendt (1990); the testing medium was dilution water according to the German ®sh test standard (constituents for 5 l: 1.6172 g CaCl22H2O, 0.6163 g MgSO47H2O, 42 mg NaHCO3, 9.6 mg KCl). The parent animals were kept (and the young daphnids were tested) in glass beakers (50 ml, tall form). They were fed daily with Scenedesmus subspicatus at the same time as the medium (40 ml) was changed. The feed algae were taken from batch cultures which were cropped once a week and kept refrigerated at 48C in suspension after being regulated to a TOC content of circa 70 mg mlÿ1. The feed was administered at 1 ml per daphnid. Those juvenile daphnids taken for testing were maintained until the test in M4 medium with added feed. To avoid changes in the nominal concentration of the chemicals involved which may have aected results where the dosage-eectiveness was particularly marked, test animals were always put into a intermediate bath of the appropriate concentration before being transferred into the actual test vessel. For further details and speci®c deviations from the procedure described here refer to the text and legends of the ®gures.
1419
1420
Berthold Klein
Fig. 1. Acute toxicity of potassium dichromate (0.5 mg lÿ1) to 0±24 h old group bred and irregularly fed daphnids. RESULTS
The initial situation Neither the ISO 6341 nor its national precursors contain detailed instructions for the maintenance of the daphnids. Therefore it may be assumed that unde®ned numbers of individuals, irregular feeding and water changing at only approximately regular
intervals are the rule and therefore from one laboratory to the next dierences in maintenance practices exist. Figure 1 shows an example of what happens when the available range of interpretation is used to the full. Clearly, the eect of dichromate at 0.5 mg lÿ1 cannot be ascertained when only the age of the test animals (as being less than 24 h) and the breeding and testing medium are de®ned. A test
Fig. 2. Age-dependent sensivitity of Daphnia magna: 0±24 h old daphnids and 24±48 h old daphnids (0.5 mg lÿ1 K2Cr2O7; group bred and irregularly fed daphnids).
Age-dependent sensitivity of Daphnia magna
1421
Fig. 3. Age-dependent sensivitity of 2±26 h old daphnids (0.4 mg lÿ1 K2Cr2O7; individually reared parent animals).
design whose results for a de®ned agent allow every possibility between no eect and 100% eect is patently inadequate. Figure 1 shows nine obviously false negative results. The age of the test animals as a factor in¯uencing results Having established according to the results shown in Fig. 1 that the age of the parent animals is unrelated to the daily variations in the sensivity of their 0±24 h old progeny, so that it appears to be of no importance whether the test animals are taken from a ®rst, second or other clutch, it remained to investigate the age of the test animals themselves with a view to its relevance to test results. The question is, whether the assumptions can be accepted that daphnids less than a day old are the most sensitive and that they are during this period equally sensitive, which the standard presents as being more or less self-evident. The ®rst assumption is discredited by the series of experiments shown in Fig. 2. If the 0±24 h old juveniles, obtained daily by means of sieving them out from among the parents, are divided into two groups, of which one is tested immediately and the other after 24 h, it can be seen, that the 24±48 h age group is highly signi®cantly more sensitive than the 0±24 h age group. This is of course reason enough to investigate the in¯uence of age within the time period up to 24 h as prescribed by the standard. For this purpose individual rearing appeared to be advantageous. On the one hand the eects of population density on number and ®tness of the progeny (Goser, 1997) could be excluded; on the
other hand reproductive events can be more easily registered and exploited in any time interval as desired. For instance, extracting juveniles from amongst a group of parent animals would always represent a disturbance for the majority of parents not involved with the brood. Through regular observation at 2 h intervals juveniles were obtained between 0±2 h old, which were then tested in 0.4 mg lÿ1 K2Cr2O7 after 2 h or a factor of 2 h up to the age of 24±26 h. The results of these tests, shown in Fig. 3, refute the assumption that the sensivity of the animals is uniform in the age range 0±24 hours. In fact, the dierences are so great that, in respect of test groups of inde®nite age structure, neither the scattered results of Fig. 1 nor the very loose range of 0.6±1.7 mg lÿ1 in ISO 6341 for the EC50 for potassium dichromate are a source of surprise. Table 1. Age-dependent sensitivity of single clutches from single parent animalsa Age (h)b
Test number 2±4 1 2 3 4 5 6 7 8 9 10 11 12 13 a
0/7 0/7 0/5 6/16 0/9 0/9 0/8 0/10 0/11
4±6
6±8
20±22
2/10 5/7 0/14 0/10
0/10
22/22
10/11 13/13 13/13 15/26
0.4 mg lÿ1 K2Cr2O7. (n/n=n immobilized of n tested daphnids).
b
22±24
21/21 10/10 11/11 7/7
24±26 10/10 10/10 6/6
1422
Berthold Klein
Fig. 4. Medium-dependent toxicity of K2Cr2O7 (group bred, parent animals transferred daily; 16 h/8 h light/dark photoperiod).
Although Fig. 3 is self explanatory and practically excludes any doubts as to the depency of sensitivity on age, the ®ndings can be further con®rmed by dividing a single brood of one parent animal and testing the individuals after varying lengths of time. The test material thus obtained is as uniform as it can possibly be, namely clones with identical genetic material{; this is the case even if the culture from which the individuals for single rearing were extracted contained males capable of sexual reproduction, so that the culture may not be genetically pure. Table 1 contains the results of such extreme tests and con®rms, that animals older than 22 h have practically no chance of survival in 0.4 mg lÿ1 potassium dichromate, whereas as far as 2±8 h old animals are concerned immobilized daphnids are very rare (test 6, 2±4 h old daphnids).
General relevancy of the age dependent sensitivity It remains to establish that these ®ndings can be generalised. Are they limited to the special case of the chemical dichromate, particularly related to the parents being kept individually or to the unusual test condition of continuous lighting? None of these limitations apply. We found that the factor age exercised the same in¯uence on test results with 2,4-Dichlorophenol: 3 mg/l of this organic chemical are absolutely lethal to 20±24 h old daphnids in much less than 24 h, whereas 2±6 h old daphnids survive with a probability of over 90%. These ®ndings were produced with group-reared animals in a 16-to-8 hour light/dark photoperiod, {``Most authors agree that there is no true pairing of homologous chromosomes in Daphnia and so there is no chance of gene recombination'' (Zaagnini, 1987)
and the same maintenance conditions did not lead to any other eect in the case of dichromate. In¯uence of the medium on the sensivity towards potassium dichromate Those who have gained experience over many years using the daphnid test and the reference substance potassium dichromate will view our laboratory with understandable scepticism, as the high degree of sensitivity with EC50 values around 0.2 mg lÿ1 documented in our experiments has never been observed elsewhere. Nonetheless, our values are correct and reproducible. They have come about on the basis of the premise, falsely stated in the German standard DIN 38412 Teil 31 (1989), that the dilution water for the ®sh test is identical to that of the daphnid test according to DIN 38412 Teil 30 (1989) and thereby to ISO 6341. In fact the ®sh water only contains 13% of the amount of NaHCO3 described in ISO 6341 (and in addition only 33% of the amount of KCl, but 110% CaCl2). This dierence alone causes the higher toxicity of potassium dichromate as documented in Fig. 4; it does not aect the general conclusions to be drawn from the age dependent sensitivity and the steep gradient of the dosage-eect correlation curve. DISCUSSION
The results presented here explain beyond doubt why under the premises of the various national and international norms concerning the acute daphnid test it has remained impossible to introduce a positive control with potassium dichromate into the test. The aim of a positive control would consist in the demonstration of uniform sensitivity, and aim of a positive control would consist in the demonstration of uniform sensitivity, and this is not the
Age-dependent sensitivity of Daphnia magna
case in the age range of 0±24 h with Daphnia magna. Curiosly enough Fig. 3 suggests that with test groups of unknown age structure it is rather possible to estimate the average age after the test than to predict the test result in advance. For this reason the EC50 determination for potassium dichromate as prescribed by the ISO 6341 cannot undertake the function of a validity criterion. The steep gradient of the dosage-eect correlation curve for potassium dichromate (Fig. 4) obliges us to recognise that EC50 values in the wide range of 0.6±1.7 mg lÿ1 allow no more exact conclusions than that the daphnids used in the determination were between 0±24 h old. As this is required by the standard anyway, the inference is trivial and provides no more information than that an EC50 value of 0.6 would show that mainly older daphnids were involved, whereas a value of 1.7 indicates a population with a large proportion of daphnids only a few hours old. But in a concrete test situation this is irrelevant; the point at issue is to show that the test animals are uniformly sensitive. This can only be achieved with reference substances in de®ned concentrations. Potassium dichromate exhibits a number of advantages as a reference substance: for instance very good solubility in water, stability and commercial availability as titration solution. Its only disadvantage in the acute daphnid test is normally seen in the steep dosage-eect correlation curve. Small errors in producing the reference solution can result in the tolerance range required by the validity criterion being failed. On the other hand, the steep correlation curve de®nes two very proximate concentration bounds above which nothing and below which everything occurs. Thus, in the same way that Enserink et al. (1990), following on from Stephan (1977), used these two bounds as 99% con®dence limits for LC50 values in case no partial mortality occured, two de®ned concentrations of potassium dichromate could function as positive control and enable collectives with comparable sensitivity to be established. The choice of a reference substance with a less steep dosage-eect correlation curve would not solve the problem of the missing positive control. The validity criterion could be too easily satis®ed and would lose the necessary property of demonstrating uniform sensitivity. And as in addition uniform sensitivity can be more eectively demonstrated in the range of highest sensitivity than in a range of less sensitivity, in using dichromate as reference substance the age group 20±24 h would be the most suitable. The consequence of demanding a uniformly sensitive test animal collective is of course that the test can no longer be established relatively easily in a laboratory, as appeared to be the case up to now. If the age of the test animals has to be taken in account because of its signi®cance for the results,
1423
only those born during normal working hours will be suitable, or in other words two thirds of the progeny from a daphnid culture will be unusable. Nonetheless, the new situation will have to be taken into account, as a biotest with individuals of demonstrably variable sensitivity can only be used for screening purposes. In any attempt to employ the method in comparative tests on chemicals (ranking), and certainly in formulating legal requirements for waste water evaluation, the lack of a positive control, of a proof of uniform sensitivity, will preclude a plausible and legally watertight test result. The very dierent degrees of sensitivity in the test animals, unobserved up to now even though the variation occurs within a few hours, probably constitutes the main reason for the unacceptably wide distribution of the test results using de®ned concentrations of reference substances. They are certainly sucient to explain the divergence documented in Fig. 1. Nevertheless, this is no guarantee that there may not be other in¯uences which have not yet been taken into sucient consideration and which must be investigated and incorporated into the standard, so that a bona ®de positive control can function as validity criterion. In this connection we mention the in¯uence of quantity and quality of the feed used in the culture, often mentioned in papers (Belanger et al. 1989; Enserink et al. 1990; Goser 1997; Vigano 1993) but not yet addressed in concrete standard recommendations, as well as the in¯uence of the quantity of feed available to the juvenile daphnids prior to the test, which recent trials suggest should not be left out of account (Klein, in preparation). However, it is not a matter for speculation, but a de®nite necessity as con®rmed by Fig. 4, that the test medium mentioned in ISO 6341 can no longer be considered as merely exemplary, but must be exactly de®ned if potassium dichromate is to continue to be used as a reference substance. For the test medium which we used also ful®lled the generalised requirements of the ISO 6341 concerning the Ca/Mg ratio and the resulting hardness. As the German standard DIN 38412 Teil 30 contains no mention of the pH-value of the dilution water, using the ®sh water of DIN 38412 Teil 31 was quite legitimate according to the German norm, even if it were erroneously equated to the daphnid water. The signi®cant dierence compared with the daphnid water according ISO 6341 consists in the lower degree of carbonate hardness, which lowers the pHvalue by circa 0.5 units, thereby shifting the dissociation balance chromate/dichromate in favor of dichromate, which appears to be the more toxic constituent. AcknowledgementsÐThis paper is dedicated to Prof. Dr. Horst Neumann, who instituted the biotest laboratory in the State Agency, on the occasion of his 70th birthday.
1424
Berthold Klein REFERENCES
Belanger S. E., Farris J. L. and Cherry D. S. (1989) Eects of diet, water hardness, and population source on acute and chronic copper toxicity to Ceriodaphnia dubia. Arch. Environ. Contam. Toxicol. 18, 601±611. DIN 38412 Teil 30 (1989) Determination of the non-acutepoisonous eect of waste water to Daphnia by dilution limits (L30). (Bestimmung der nicht akut giftigen Wirkung von Abwasser gegenuÈber Daphnien uÈber VerduÈnnungsstufen). DIN 38412 Teil 31 (1989) Determination of the non-acutepoisonous eect of waste water to ®sh by dilution limits (L31). (Bestimmung der nicht akut giftigen Wirkung von Abwasser gegenuÈber Fischen uÈber VerduÈnnungsstufen). Elendt B. P. (1990) Nutritional quality of a microencapsulated diet for Daphnia magna. Eects on fatty acid composition and midgut ultrastructure. Archiv. fuÈr Hydrobiologie 118, 461±475. Enserink L., Luttmer W. and Maas-Diepeveen H. (1990) Reproductive strategy of Daphnia magna aects the sensivity of its progeny in acute toxicity tests. Aquatic Toxicology 17, 15±26.
Goser B. (1997) Density-dependent changes of development and reproduction of Cladocera: causes and ecological signi®cance. In Diss. RWTH Aachen. Shaker Verlag Aachen, [DichteabhaÈngige AÈnderungen der Entwicklung und Reproduktion bei Cladoceren: Ursachen und oÈkologische Bedeutung. ISBN 3-82652938-3]. ISO 6341 (1996) Water qualityÐDetermination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea)ÐAcute toxicity test. Stephan C. E. (1977) Methods for calculating an LC50. In Aquatic Toxicology and Hazard Evaluation, ASTM STP 634, eds F. L. Mayer and J. L. Hamelink, pp. 65±84. American Society for Testing and Materials, Philadelphia, Pa. Vigano L. (1993) Reproductive strategy of Daphnia magna and toxicity of organic compounds. Wat. Res. 27(5), 903±909. Zaagnini F. (1987) Reproduction in Daphnia. Memorie dell' Istituto Italiano di Idrobiologia ``Dott. Marco De Marchi'' 45, 245±284.
Wat. Res. Vol. 34, No. 5, pp. 1419±1424, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
AGE AS A FACTOR INFLUENCING RESULTS IN THE ACUTE DAPHNID TEST WITH DAPHNIA MAGNA STRAUS BERTHOLD KLEIN* Lower Saxony State Agency for Ecology, PO Box 10 10 62, D-31110, Hildesheim, Germany (First received 1 March 1999; accepted in revised form 1 July 1999) AbstractÐContrary to assumptions which have been current up to the present, juvenile daphnids of the species D. magna Straus exhibit a very varied sensitivity towards toxic chemicals between the ages of 0± 24 h. During this period the greatest susceptibility towards potassium dichromate is reached between the ages of 22±24 h; younger daphnids are up to four times less sensitive. For this reason it is impossible to arrive at a reliable prognosis with potassium dichromate in de®ned concentration as reference substance using test groups of unknown age structure. However, positive controls for verifying correct testing procedure rely on comparable sensitivity amongst the test organisms and are essential for validating the test results. The range of 0.6±1.7 mg/l for the 24 h-EC50 of potassium dichromate, which the ISO norm 6341 propagates as validity criterium, does not take sucient account of the connection between dosage and eectiveness of this substance and thereby masks age related inequalities in the test animals. Therefore it cannot substitute the positive control which is missing in the test speci®cations. As the toxicity of potassium dichromate is pH-dependent a speci®c carbonate hardness for the test medium should be determined. # 2000 Elsevier Science Ltd. All rights reserved Key wordsÐDaphnia, toxicity test guidelines, potassium dichromate acute toxicity
INTRODUCTION
Biotests with Daphnia magna Straus have been used and accepted throughout the world for decades as instruments for the estimation of the acute toxicity of xenobiotics in aquatic environments. One advantage often mentioned in respect of this organism results from the fact that identical animals can be bred through acyclic parthenogenesis and used for testing. However, one aspect of this circumstance has not yet been given due consideration. In genetically identical cohorts a particularly high degree of consistency may be anticipated in the reactions of individuals to de®ned chemical stimuli. Nonetheless, to date no successful process has been developed to establish a reliable prognosis for the acute daphnid toxicity test using a reference substance of de®ned concentration which could serve as a validity criterion, such as is the case in other biotesting procedures implementing a positive control. The ISO standard 6341 (1996) tends to obscure this dilemma. Occasional EC50 determinations with a regression analysis of the dosage-eectiveness of potassium dichromate are used to attest that the test material is in satisfactory condition. This procedure is considered as sucient to ensure the validity of all tests carried out between such EC50 determinations. The *Tel.: +49-5121-509638; fax: +49-5121-509196; e-mail: [email protected]
present paper indicates a reason why it is not possible to guarantee the plausibility of a test result using a single nominal concentration of potassium dichromate within the EC50 range under the conditions prescribed by the ISO 6341.
MATERIALS AND METHODS
The parent animals (clone 5) were reared and the juvenile daphnids were tested (test duration 24 h) in climatised rooms (208C 2 18C) under continuous ¯uorescent light (Universal White). The breeding medium was the M4 medium according to Elendt (1990); the testing medium was dilution water according to the German ®sh test standard (constituents for 5 l: 1.6172 g CaCl22H2O, 0.6163 g MgSO47H2O, 42 mg NaHCO3, 9.6 mg KCl). The parent animals were kept (and the young daphnids were tested) in glass beakers (50 ml, tall form). They were fed daily with Scenedesmus subspicatus at the same time as the medium (40 ml) was changed. The feed algae were taken from batch cultures which were cropped once a week and kept refrigerated at 48C in suspension after being regulated to a TOC content of circa 70 mg mlÿ1. The feed was administered at 1 ml per daphnid. Those juvenile daphnids taken for testing were maintained until the test in M4 medium with added feed. To avoid changes in the nominal concentration of the chemicals involved which may have aected results where the dosage-eectiveness was particularly marked, test animals were always put into a intermediate bath of the appropriate concentration before being transferred into the actual test vessel. For further details and speci®c deviations from the procedure described here refer to the text and legends of the ®gures.
1419
1420
Berthold Klein
Fig. 1. Acute toxicity of potassium dichromate (0.5 mg lÿ1) to 0±24 h old group bred and irregularly fed daphnids. RESULTS
The initial situation Neither the ISO 6341 nor its national precursors contain detailed instructions for the maintenance of the daphnids. Therefore it may be assumed that unde®ned numbers of individuals, irregular feeding and water changing at only approximately regular
intervals are the rule and therefore from one laboratory to the next dierences in maintenance practices exist. Figure 1 shows an example of what happens when the available range of interpretation is used to the full. Clearly, the eect of dichromate at 0.5 mg lÿ1 cannot be ascertained when only the age of the test animals (as being less than 24 h) and the breeding and testing medium are de®ned. A test
Fig. 2. Age-dependent sensivitity of Daphnia magna: 0±24 h old daphnids and 24±48 h old daphnids (0.5 mg lÿ1 K2Cr2O7; group bred and irregularly fed daphnids).
Age-dependent sensitivity of Daphnia magna
1421
Fig. 3. Age-dependent sensivitity of 2±26 h old daphnids (0.4 mg lÿ1 K2Cr2O7; individually reared parent animals).
design whose results for a de®ned agent allow every possibility between no eect and 100% eect is patently inadequate. Figure 1 shows nine obviously false negative results. The age of the test animals as a factor in¯uencing results Having established according to the results shown in Fig. 1 that the age of the parent animals is unrelated to the daily variations in the sensivity of their 0±24 h old progeny, so that it appears to be of no importance whether the test animals are taken from a ®rst, second or other clutch, it remained to investigate the age of the test animals themselves with a view to its relevance to test results. The question is, whether the assumptions can be accepted that daphnids less than a day old are the most sensitive and that they are during this period equally sensitive, which the standard presents as being more or less self-evident. The ®rst assumption is discredited by the series of experiments shown in Fig. 2. If the 0±24 h old juveniles, obtained daily by means of sieving them out from among the parents, are divided into two groups, of which one is tested immediately and the other after 24 h, it can be seen, that the 24±48 h age group is highly signi®cantly more sensitive than the 0±24 h age group. This is of course reason enough to investigate the in¯uence of age within the time period up to 24 h as prescribed by the standard. For this purpose individual rearing appeared to be advantageous. On the one hand the eects of population density on number and ®tness of the progeny (Goser, 1997) could be excluded; on the
other hand reproductive events can be more easily registered and exploited in any time interval as desired. For instance, extracting juveniles from amongst a group of parent animals would always represent a disturbance for the majority of parents not involved with the brood. Through regular observation at 2 h intervals juveniles were obtained between 0±2 h old, which were then tested in 0.4 mg lÿ1 K2Cr2O7 after 2 h or a factor of 2 h up to the age of 24±26 h. The results of these tests, shown in Fig. 3, refute the assumption that the sensivity of the animals is uniform in the age range 0±24 hours. In fact, the dierences are so great that, in respect of test groups of inde®nite age structure, neither the scattered results of Fig. 1 nor the very loose range of 0.6±1.7 mg lÿ1 in ISO 6341 for the EC50 for potassium dichromate are a source of surprise. Table 1. Age-dependent sensitivity of single clutches from single parent animalsa Age (h)b
Test number 2±4 1 2 3 4 5 6 7 8 9 10 11 12 13 a
0/7 0/7 0/5 6/16 0/9 0/9 0/8 0/10 0/11
4±6
6±8
20±22
2/10 5/7 0/14 0/10
0/10
22/22
10/11 13/13 13/13 15/26
0.4 mg lÿ1 K2Cr2O7. (n/n=n immobilized of n tested daphnids).
b
22±24
21/21 10/10 11/11 7/7
24±26 10/10 10/10 6/6
1422
Berthold Klein
Fig. 4. Medium-dependent toxicity of K2Cr2O7 (group bred, parent animals transferred daily; 16 h/8 h light/dark photoperiod).
Although Fig. 3 is self explanatory and practically excludes any doubts as to the depency of sensitivity on age, the ®ndings can be further con®rmed by dividing a single brood of one parent animal and testing the individuals after varying lengths of time. The test material thus obtained is as uniform as it can possibly be, namely clones with identical genetic material{; this is the case even if the culture from which the individuals for single rearing were extracted contained males capable of sexual reproduction, so that the culture may not be genetically pure. Table 1 contains the results of such extreme tests and con®rms, that animals older than 22 h have practically no chance of survival in 0.4 mg lÿ1 potassium dichromate, whereas as far as 2±8 h old animals are concerned immobilized daphnids are very rare (test 6, 2±4 h old daphnids).
General relevancy of the age dependent sensitivity It remains to establish that these ®ndings can be generalised. Are they limited to the special case of the chemical dichromate, particularly related to the parents being kept individually or to the unusual test condition of continuous lighting? None of these limitations apply. We found that the factor age exercised the same in¯uence on test results with 2,4-Dichlorophenol: 3 mg/l of this organic chemical are absolutely lethal to 20±24 h old daphnids in much less than 24 h, whereas 2±6 h old daphnids survive with a probability of over 90%. These ®ndings were produced with group-reared animals in a 16-to-8 hour light/dark photoperiod, {``Most authors agree that there is no true pairing of homologous chromosomes in Daphnia and so there is no chance of gene recombination'' (Zaagnini, 1987)
and the same maintenance conditions did not lead to any other eect in the case of dichromate. In¯uence of the medium on the sensivity towards potassium dichromate Those who have gained experience over many years using the daphnid test and the reference substance potassium dichromate will view our laboratory with understandable scepticism, as the high degree of sensitivity with EC50 values around 0.2 mg lÿ1 documented in our experiments has never been observed elsewhere. Nonetheless, our values are correct and reproducible. They have come about on the basis of the premise, falsely stated in the German standard DIN 38412 Teil 31 (1989), that the dilution water for the ®sh test is identical to that of the daphnid test according to DIN 38412 Teil 30 (1989) and thereby to ISO 6341. In fact the ®sh water only contains 13% of the amount of NaHCO3 described in ISO 6341 (and in addition only 33% of the amount of KCl, but 110% CaCl2). This dierence alone causes the higher toxicity of potassium dichromate as documented in Fig. 4; it does not aect the general conclusions to be drawn from the age dependent sensitivity and the steep gradient of the dosage-eect correlation curve. DISCUSSION
The results presented here explain beyond doubt why under the premises of the various national and international norms concerning the acute daphnid test it has remained impossible to introduce a positive control with potassium dichromate into the test. The aim of a positive control would consist in the demonstration of uniform sensitivity, and aim of a positive control would consist in the demonstration of uniform sensitivity, and this is not the
Age-dependent sensitivity of Daphnia magna
case in the age range of 0±24 h with Daphnia magna. Curiosly enough Fig. 3 suggests that with test groups of unknown age structure it is rather possible to estimate the average age after the test than to predict the test result in advance. For this reason the EC50 determination for potassium dichromate as prescribed by the ISO 6341 cannot undertake the function of a validity criterion. The steep gradient of the dosage-eect correlation curve for potassium dichromate (Fig. 4) obliges us to recognise that EC50 values in the wide range of 0.6±1.7 mg lÿ1 allow no more exact conclusions than that the daphnids used in the determination were between 0±24 h old. As this is required by the standard anyway, the inference is trivial and provides no more information than that an EC50 value of 0.6 would show that mainly older daphnids were involved, whereas a value of 1.7 indicates a population with a large proportion of daphnids only a few hours old. But in a concrete test situation this is irrelevant; the point at issue is to show that the test animals are uniformly sensitive. This can only be achieved with reference substances in de®ned concentrations. Potassium dichromate exhibits a number of advantages as a reference substance: for instance very good solubility in water, stability and commercial availability as titration solution. Its only disadvantage in the acute daphnid test is normally seen in the steep dosage-eect correlation curve. Small errors in producing the reference solution can result in the tolerance range required by the validity criterion being failed. On the other hand, the steep correlation curve de®nes two very proximate concentration bounds above which nothing and below which everything occurs. Thus, in the same way that Enserink et al. (1990), following on from Stephan (1977), used these two bounds as 99% con®dence limits for LC50 values in case no partial mortality occured, two de®ned concentrations of potassium dichromate could function as positive control and enable collectives with comparable sensitivity to be established. The choice of a reference substance with a less steep dosage-eect correlation curve would not solve the problem of the missing positive control. The validity criterion could be too easily satis®ed and would lose the necessary property of demonstrating uniform sensitivity. And as in addition uniform sensitivity can be more eectively demonstrated in the range of highest sensitivity than in a range of less sensitivity, in using dichromate as reference substance the age group 20±24 h would be the most suitable. The consequence of demanding a uniformly sensitive test animal collective is of course that the test can no longer be established relatively easily in a laboratory, as appeared to be the case up to now. If the age of the test animals has to be taken in account because of its signi®cance for the results,
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only those born during normal working hours will be suitable, or in other words two thirds of the progeny from a daphnid culture will be unusable. Nonetheless, the new situation will have to be taken into account, as a biotest with individuals of demonstrably variable sensitivity can only be used for screening purposes. In any attempt to employ the method in comparative tests on chemicals (ranking), and certainly in formulating legal requirements for waste water evaluation, the lack of a positive control, of a proof of uniform sensitivity, will preclude a plausible and legally watertight test result. The very dierent degrees of sensitivity in the test animals, unobserved up to now even though the variation occurs within a few hours, probably constitutes the main reason for the unacceptably wide distribution of the test results using de®ned concentrations of reference substances. They are certainly sucient to explain the divergence documented in Fig. 1. Nevertheless, this is no guarantee that there may not be other in¯uences which have not yet been taken into sucient consideration and which must be investigated and incorporated into the standard, so that a bona ®de positive control can function as validity criterion. In this connection we mention the in¯uence of quantity and quality of the feed used in the culture, often mentioned in papers (Belanger et al. 1989; Enserink et al. 1990; Goser 1997; Vigano 1993) but not yet addressed in concrete standard recommendations, as well as the in¯uence of the quantity of feed available to the juvenile daphnids prior to the test, which recent trials suggest should not be left out of account (Klein, in preparation). However, it is not a matter for speculation, but a de®nite necessity as con®rmed by Fig. 4, that the test medium mentioned in ISO 6341 can no longer be considered as merely exemplary, but must be exactly de®ned if potassium dichromate is to continue to be used as a reference substance. For the test medium which we used also ful®lled the generalised requirements of the ISO 6341 concerning the Ca/Mg ratio and the resulting hardness. As the German standard DIN 38412 Teil 30 contains no mention of the pH-value of the dilution water, using the ®sh water of DIN 38412 Teil 31 was quite legitimate according to the German norm, even if it were erroneously equated to the daphnid water. The signi®cant dierence compared with the daphnid water according ISO 6341 consists in the lower degree of carbonate hardness, which lowers the pHvalue by circa 0.5 units, thereby shifting the dissociation balance chromate/dichromate in favor of dichromate, which appears to be the more toxic constituent. AcknowledgementsÐThis paper is dedicated to Prof. Dr. Horst Neumann, who instituted the biotest laboratory in the State Agency, on the occasion of his 70th birthday.
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Berthold Klein REFERENCES
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