Critical analysis of factors affecting the sensitivity of zooplankton and the reproducibility of toxicity test results

Critical analysis of factors affecting the sensitivity of zooplankton and the reproducibility of toxicity test results

War. Res. Vol. 21, No. 12, pp. 1453-1462, 1987 Printed in Great Britain. All rights reserved 0043-1354/87 $3.00+0.00 Copyright © 1987 Pergamon Journa...

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War. Res. Vol. 21, No. 12, pp. 1453-1462, 1987 Printed in Great Britain. All rights reserved

0043-1354/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd

CRITICAL ANALYSIS OF FACTORS AFFECTING THE SENSITIVITY OF ZOOPLANKTON A N D THE REPRODUCIBILITY OF TOXICITY TEST RESULTS U. M. COWGILL Dow Chemical Co., Health and Environmental Sciences, 1702 Bldg, Midland, MI 48674, U.S.A.

(Received May 1986) Abstract--This paper concerns the variables that affect the sensitivity of test zooplankton and the reproducibility of results obtained from repeated toxicity tests. Among the variables considered are: (a) the nutrition of the test zooplankton, their health and diet; (b) culturing techniques, in particular, the effect of the ambient medium on demographic variables and testing results; (c) physical and chemical characteristics of the test compound; (d) purity of the test compound, and finally (e) the variation in results obtained with nominal and measured concentrations of the test compound. Critical factors addressed include the effects of inadequate cobalamine or selenium concentrations in the diet, adversely affecting fertility, which may result in misleading interpretation of short-term toxicity test results involving Ceriodaphnia or chronic studies using Daphnia. Other factors considered are, for example, the problems of poor health, as measured by fertility or by the reproducibility of static acute toxicity test results, and their effect on zooplankton sensitivity to toxicants. The observation that diets of several algae result in greater tolerance of zooplankton to toxicants than do diets based on synthetic food are discussed. It is also noted that organisms reared in clean, frequently replenished and renewed habitats and fed axenic cultures of algae, are healthier than animals maintained in static environments. The observation that the results of interlaboratory comparisons of zooplankton response to toxicants are more uniform and exhibit smaller standard deviations when the test animals are sustained on algal foods rather than on synthetic diets is emphasized. Variability due to test compound characteristics include the observation that test compounds of variable purity often are responsible for poor test results reproducibility, and that the reproducibility of the toxic response is often variable due to physical and chemical characteristics of the test chemical. Finally, poor interlaboratory comparisons may result when nominal concentrations arc the only ones considered. Data are selected from the published literature to illustrate the above discussion.

Key words--daphnid sensitivity, nutrition, diet, ambient medium, toxicity, bioassays, zooplankton

INTRODUCTION

Toxicity tests utilizing zooplankton as test organisms are used to establish water quality criteria; to monitor effluents and to ascertain their effect on the environment; to test the effect of new compounds for premanufacturing notification requirements and for registration of pesticides; to comply with U.S. Food and Drug Administration requirements and finally, to protect the waterways of the United States from the introduction of toxic substances. As a rule, such tests are developed as consensus documents under the aegis of some standards development organization. Shortly after their acceptance and subsequent publication, they are often embraced by some government agency which in turn publishes them in the U.S. Federal Register. At this point, the agency in question subjects the toxicity test to interlaboratory comparisons by involving a n u m b e r of laboratories to carry out the particular test as uniformly as possible, Such results are often published in the U.S. Federal Register. Due to their high variability, interlaboratory results involving toxicity tests using zoo-

plankton have been unacceptable (L. R. Williams, unpublished data). The variables that affect the sensitivity of test zooplankton and the reproducibility of results obtained from repeated toxicity tests include physiological characteristics of the test organism (nutrition, diet, health), the influence of culture and rearing techniques (ambient environment), the characteristics of the test compound (solubility, degradation rates, purity), and, finally, whether the a m o u n t of the test compound was estimated or measured throughout the test period. PHYSIOLOGY OF TEST ORGANISMS

Nutrition of zooplankton The chemical composition of Daphnia magna or any other aquatic organism, is governed by the composition of the food it consumes, as well as the water or ambient medium in which it lives. D. magna gathered from its natural habitat (Farkas and Herodek, 1964; Farkas et al., 1981) or reared in natural water and algal-fed (Cowgill, 1976) contains de-

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COWGILL

tectable quantities of 55 elements, a large variety of saturated and unsaturated fatty acids (Farkas and Herodek, 1964; Farkas et al., 1981; Cowgill et al., 1984) and 18 amino acids (Cowgill etal., 1986b). The carbohydrate and vitamin content of daphnid tissue is less well-defined. Scientists have presumed that this basic matrix is essential to the health of the organism, If these substances are absent for generations, often the case in laboratory cultures, deficiency symptoms may become apparent. Two such deficiency symptoms have been recently described (Keating and Dagbusan, 1984; Keating, 1985). If insufficient selenium (< 0.1/~g 1-1) is present in the ambient medium, daphnids exhibit premature cuticle deterioration. In addition, progressive loss of distal segments of the second antennae have been observed. Second generation progeny fail to attain reproductive maturity, Generally, selenium deficient animals exhibit a reduced lifespan. Addition of2/~g1-1 of selenium to the ambient medium alleviates these symptoms and the animals return to the behavior shown by normal healthy daphnids. Daphnia pulex deprived of vitamin B~2 in the ambient medium (<0.375#gl -~) were identical in color, size and activity to non-deprived animals, but failed to produce reproductively viable progency. These deprived organisms molted less frequently than non-deprived animals and produced their first brood at a later time. Addition of l#g 1-1 of vitamin B12 to the ambient medium produced a D. pulex population with a normal life span and an average of 17 broods during that time (Keating, 1985). Diet of zooplankton Diet affects the response to toxicity. Table 1 shows that variation in diet widely affects daphnid response to toxic substance. Generally, organisms that are algal-fed are more tolerant to the test substance than those maintained on trout chow and alfalfa or

Table 1. Variation in toxic response Diet T r o u t chow + alfalfa

cerophyl (CAS 100842-92-0: trade name for cereal leaves prepared from dehydrated leaves of wheat, oats, barley and rye while in the grass stage). In addition, variations in LCs0 results are caused by different algal diets when the same daphnid population is examined. It is clear that a diet of three algae produces daphnids with greater tolerance to sodium chloride than a diet of one alga. It has often been questioned as to why natural food such as algae provide the consuming daphnid with greater resistance to disease (Scymore et al., 1984) and toxicants than those maintained on synthetic food such as trout chow and alfalfa or cerophyl. Daphnids in nature derive the majority of their nutrition from the consumption of a variety of algae. Algae as noted above contain 55 detectable elements in their tissues, at least 18 amino acids and a substantial array of saturated and unsaturated fatty acids. Although feral daphnids have been collected and analyzed for their chemical composition (cf. Cowgili et al., 1984, 1986b; Farkas and Herodek, 1964; Farkas et al., 1981) no deficiency symptoms have been noted. Furthermore, the chemical composition of feral daphnids is similar to that of algal-reared daphnids but not to trout chow-reared daphnids (cf. Cowgill et al., 1986b). It is very easy to produce deficient daphnids in a laboratory. Once they are deficient in some nutrient requirement testing with toxicants will show them to be more sensitive. For example, Se deficient daphnids exhibit an LCs0 for NaCI of 1661 mg 1-~ while Se sufficient daphnids shows an LCs0 for NaCI of 4571 (unpublished data, The Dow Chemical Co.). Many investigators have postulated that high reproductive rate implies good health (Banta, 1921; Buikema et aL, 1980) and, hence, good health suggests resistance to toxic substances. Recently, Keating and Dagbusan (1984)have presented data that clearly indicate that high reproductive rate and

by Daphnia magna Substance

Acetone (mg 1- ~)

Chlorella pyrenoidosa Chick Trout chow + alfalfa

Selenastrum capricornutum Printz Chlamydomonas reinhardti Dangeard C. reinhardti + Ankistrodesmus convolutus Corda + Nitzschia frustulum Kutzing Trout chow + alfalfa

NaCI (rag I- i ) NaCI (mg 1- t )

Pentachlorophenol (/zg 1-a )

C. pyrenoidosa Trout chow + alfalfa

Picloram (rag 1- i )

S. capricornutum Trout chow + Cerophyl

CuSO4 (/~ g l -I )

C. reinhardti • Unpublished data, the D o w Chemical Co. tCanton and A d e m a (1978). ~(D. pulex) Keating and Dagbusan (1986). §Adema (1978). ¶ M a y e s and Dill (1985). IlWinner et al. (1977). • * M A T C - - M a x i m u m Acceptable Toxicant Concentration.

in relation to diet 48-h LCso

MATC**

6900* 12,657t 1661 * 4571" 2250:1:

------

3500:1: 260*

---

600~

--

34.4* 50.7¶ 83.3 II

S5.011

--20 40

Daphnids and the reproducibility of test results toxic response are diet dependent and not necessarily related. In addition, Keating and Dagbusan (1984) have shown that toxic response varies with the brood number. In support of these results (Keating and Dagbusan, 1984, 1986), other investigators have shown that broods one through three should not be used at all for toxicity testing (CowgiU et al., 1985c) since these broods are made up of underweight ( < 9 g per individual dry wt) individuals who do not contain sufficient lipids to survive (Cowgill et al., 1984) the 48 h static acute toxicity test requirement of no food for the length of the test. Data of this kind clearly indicate some of the reasons that interlaboratory results show poor concordance. Up to the present, there has been no stipulation as to which brood should be utilized in testing. Thus, if one laboratory utilizes brood 2 while another utilizes brood 5 in testing the results from the two laboratories will be too divergent to be acceptable, Diet has been shown to affect longevity, size and test data precision. D. magna sustained on the green alga Chlamydomonas reinhardti exhibited a mean longevity (101 + 32 days) greater than daphnids maintained on trout chow and cerophyl (56 + 22 days) (Winner et al., 1977). Longevity is important in life-cycle toxicity tests. Daphnids maintained on synthetic diets barely can survive the 48 h static acute tests let alone a 28 day test (Cowgill et al., 1984). A comparison of size based on three differing diets (Fig. 1) shows startling results. Lastly, interlaboratory comparisons utilizing D. magna exhibit smaller standard deviations for results obtained from algal-fed daphnids than trout chow maintained organisms (Canton and Adema, 1978; Cowgill et al., 1985b; Gersich et al., 1987). [Percent coefficient of variation 21 + 12 as compared to 56 4- 52 (Lewis and Weber, 1985; Nebeker, 1982; Cardwell et al., 1977).] In conclusion, it is clear that algal-fed daphnids exhibit a lower response to toxicants than trout chow-maintained organisms; that algal-fed organisms exhibit a longer lifespan than those fed trout chow and that high reproductive rate and high resistance, both thought to be components of good health, do not appear to be consistently related, Finally, algal-fed organisms utilized in repeated toxicity tests or interlaboratory comparisons provide precision and reproducibility of test results than trout chow-fed organisms,

Health o f zooplankton Before comparisons can be made between the standards of health of isolated laboratory cultures and those of more sophisticated aquatic communities, criteria for the assessment of health must be defined. Universal agreement on an adequate definition of the word health is paramount to developing criteria. Since one of the main objectives of aquatic toxicity testing is to simulate the behavior of a toxicant in the aquatic environment, being able to

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measure the health of laboratory cultures takes on renewed importance. This is especially true in view of the paucity of reports of disease in aquatic invertebrates under natural conditions. In the case of daphnids the major discussion is by Petersen 0910) who described an infection of daphnids in Danish lakes by what now appears to have been due to Aphanomyces daphniae Prowse. It is assumed therefore that most daphnid populations in nature are healthy. In addition, it should be noted that electrophoretic studies on laboratory and feral populations indicate that long-term maintenance of laboratory populations do not bring about genetic changes. The feral source of laboratory populations can, through such studies, be identified (C. E. Goulden and C. Lee, personal communication). Basic criteria of daphnid health must include survival, longevity, reproductive frequency, brood size, and resistance to disease and toxicants. Each of these criteria varies with the diet. Survival depends not only on the availability and size of food but also, in the case of neonate survival, on the nutrition of the reproducing female (Cowgill et aL, 1984). Longevity varies with the diet, (Cowgill et al., 1985a; Winner, 1976) as well as, with the ambient medium (Cowgill et al., 1985a, 1986a). It is important to recognize that, in the case of longevity, diet refers not only to the composition of the diet (synthetic vs natural) but also to life-sustaining properties of various diets. It may be postulated that the more varied the algal diet, the longer the lifespan. Reproductive frequency, mean brood size, the time of first brood, number of broods in a lifespan, as well as, brood interval are diet dependent (Cowgill et al., 1985a, c; 1986b; Keating and Dagbusan, 1984, 1986; Keating, 1985). Resistance to disease is lowered by stress. Stress in laboratory cultures may be induced by poor nutrition, starvation, sudden high temperatures or maintenance temperatures that exceed the usual one for an acclimatized population. Other stress-inducing conditions include sudden decline in dissolved oxygen, overcrowding, and dirty living conditions (Seymore et al., 1984). Dirty living conditions include infrequent habitat renewal, accumulation of discarded carapaces and dead daphnids in culture vessels, as well as the accumulation of daphnid fecal material. Research has demonstrated (Seymore et al., 1984) that such stressful conditions promote infection of D. magna by aquatic fungi. A number of investigators have addressed the subject of good culture health. Banta (1921) proposed a reproductive index which was the ratio of the mean brood size of the first brood and the mean age of the primiparous female. The higher the ratio the more vigorous the culture. This ratio varies with the brood number of which the primiparous female came (personal observation). More recently, good culture health has been defined as the absence of ephippial eggs in the cultivating vessels (U.S. EPA 1975). Buikema et al. (1980) summarize their view of the

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characteristics of good culture health which include a high reproductive rate, a large first brood, early appearance of the first brood, and more young than adults in a free breeding population. Goulden and Hornig (1980) showed that a high lipid content in nongravid females was necessary for survival. Later, Cowgill et al. (1984) demonstrated that the lipid content of reproducing females governed the length of survival of their young. Yet Keating (1985) observed high lipid concentrations in animals incapable of reproduction. Finally, dry weight in relation to daphnid age class should be included in any list of characteristics of good culture health, It is suggested that no single variable should be used as a guide to good culture health, but rather the principal criterion should be that of consistent demographic results achieved over time. Envisioned demographic results include mean brood size of brood No. 2-12, day of first brood, number of broods per lifespan, broods per female and finally, brood interval. In addition, consistent results should be attained by testing five to ten benchmark chemicals semiannually using the 48 h static acute toxicity test method. These tests should be carried out in triplicate but not simultaneously. The goal is to achieve a small percent coefficient of variation among results obtained with the same toxicant. These results should be reproducible within a small standard deviation of each other from year to year.

Culture and rearing techniques One of the reasons that interlaboratory comparisons of static acute toxicity tests involving vailous zooplankton species provide divergent results is that different laboratories carry out their tests in waters from many different sources. Generally, some natural waters encourage higher reproductive rate, reduced sensitivity to toxicants and encourage longer lifespan. Various factors which are affected by water quality include: reproductive capacity (Cowgill et al., 1985a, 1986a), longevity (Cowgill et al., 19875a; Winner, 1976), tolerance to toxicants (Cardwell et aL, 1977; Winner, 1976; Dowden and Bennett, 1965), day of first brood (Cowgill et al., 1985a, 1986a), percent mortality (Frear and Boyd, 1967) and number of young per female (Cowgill et al., 1985a, 1986a). It may be suggested that a defined animal medium (a "reconstituted water") not necessarily related to that of Marking and Dawson (1973) could be made acceptable were it to contain the appropriate and necessary minor elements (Cowgill, 1976; Cowgill et al., 1986b) in adequate concentrations. In particular, adequate amounts of selenium (Keating and Dagbusen, 1984) and vitamin B12 (Keating, 1985) should be included. In this way, another source for divergent results commonly achieved among testing laboratoties could be eliminated. It should be noted that as of this writing, no adequate and practical defined medium has been published,

CHARACTERISTICS OF TEST COMPOUNDS Physical and chemical characteristics o f the test cornpounds

Although the data are scant, it is possible to make some tentative suggestions as to the inherent characteristics of a test compound and the type of toxicity test results that may he obtained. This concept originated among workers of the last century, (Richet, 1893; Fuhner, 1904; Crum-Brown and Fraser, 1868-1869) however, the results of their studies have largely been forgotten. Within a given class of cornpounds (see Figs 2 and 3) there is a significant and negative relationship between LCs0 results of 48 h static acute tests using D. magna as a test organism and molecular weight and boiling point of the test compound. The results indicate that the higher the molecular weight or the boiling point of the test compound, the lower is the LCs0 result. Generally, infinitely soluble compounds such as n-propanol, acetone, allylamine and pyridine and very soluble compounds such as phenol exhibit a higher LCs0 than do slightly soluble or insoluble compounds. This is true for repeated tests within a laboratory and for interlaboratory comparisons. A detailed description of metal toxicity in relation to solution pressure has been published by Jones (1939). The relationship of solubility and toxicity has long been known. The more soluble the test compound the larger the LCs0 value or the more resistant is the test organism (Bobra et al., 1983). Moreover, the greater the molecular weight of the test compound the more likely the LCs0 value will be small and indicate high toxicity to the test organisms. The degree of chlorination also has an effect on toxicity response (Bobra et aL, 1983; Richter et al., 1983). For example, in the chlorinated ethanes and benzenes the toxicity increases with increasing chlorination. The kind of compound being tested determines to some extent the interlaboratory variation in test results. High precision in interlaboratory test results may originate from test compounds that are selected with the following characteristics: (1) infinitely soluble in water, (2) slightly or nontoxic, (3) low molecular weight, (4) no more than one chlorine and (5) low boiling point. On the other hand, poor precision and high percent coefficient of variation may be observed in interlaboratory test results if compounds selected have the following characteristics: (1) low water solubility to insoluble in water, (2) high toxicity, (3) high molecular weight, (4) several chlorine atoms and (5) high boiling point. It is clear that interlaboratory comparisons should address both types of cornpounds in order to ascertain the kind of variation that occurs under such conditions. The purity of test compounds is enormously important as mixtures often vary in the amount present of different components and these have different levels of toxicity to aquatic organisms. An example of

Fig. 1. Twenty-one day old Daphnia magna adults. From left to right--D, magna reared on trout chow and alfalfa; on Selenastrum cultured in an inorganic medium supplemented with vitamins; and on Selenastrum cultured in a largely organic medium.

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Daphnids and the reproducibility of test results

1

0~

0

0

1459

• Alkanes + cycloalkanes n = 12 r = -.0996 P< ,005 •

Monoaromatics n = 10 r = -0.888 P< 0.005

A Polynuclear aromatics n = 10 r = -0.70 P< 0,05 © Chlorinated aromatic hydrocarbons n = 8 r = -0.64 P< 0,10

100

E

10 b

©

o

©

0.1 50

100

150

200

250

300

350

400

Molecular Weight

Fig. 2. The relationship between static acute toxicity to Daphnia magna and the molecular weight of the test compound. Data are from Canton and Adema 0978), Bobra et al. (1983) and The Dow Chemical Co. (unpublished data).

this problem is illustrated in Table 2. Significant amounts of substances that are highly toxic in mixtures will affect the final LCs0 when the individual components are unknown. Toxicity test results of mixtures should be corrected and the quantity of the particular compound tested should be noted. N o m i n a l vs measured concentrations

Recently Hermens et al. (1984) published the resuits of 48 h static acute toxicity tests utilizing D. magna as the test organism and compared the results achieved with nominal and measured amounts of toxicants. The differences between LCs0 results achieved with nominal concentrations in contrast to measured concentrations was statistically significant

(x2test) beyond0.1% level for lindane, 1,3-dichlorobenzene, 2,4-dichloroaniline, 2,6-dimethylquinoline and di-isopropylamine. No statistically significant difference was noted for malathion, dinitro-o-cresol, phenol, sodium bromide and potassium dichromate. It is recommended therefore, that interlaboratory tests should use only measured concentrations of the compounds being tested, since serious discrepancies may occur in final LCs0 results when only nominal concentrations are employed. SUMMARY

It is clear that algal-fed daphnids exhibit more consistent toxicity test results, more consistent

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1000 [

~

[

• Alkanel + ¢ycloalkanesn " 11 r - -0.972 P< .00S • Monoaromltio=n = 13 r = -0.84 P< 0.006



A

Polynucleararomaticsn P<0.1

= 11 r = - 0 . 6 1

© Chlorinatedaromatic hydrocarbons n = 8 r = -0.882

\

°

~ 1o

P


©

d

--



©

O

0

. 25

75

1 125

~ 175 225 Boiling Point °C

275

325

375

Fig. 3. The relationship between static acute toxicity to Daphnia magna and the boiling point (°C) of the test compound. Data are from Canton and A d e m a (1978), Bobra et al. (1983) and The Dow Chemical Co. (unpublished data).

Table 2. Purity of test compounds Trichlorocthylene contaminants of technical grade (rag 1- i ) Daphnia magna Contaminants* % 48-h LCs0 Epichlorohyderin Epoxybutane Carbon tetrachloride Chloroform l,l,l-Trichloroethan¢ Di-isobutylene (2,2,4-trimethylpentene-l) ¢thylacctate Pentanol-2 Butanol-2 Trichloroethylene NA = not available. *Verscbucren (1983). ?The Dow Chemical Co. ~:La Blanc (1980). ~Canton and Adema (1978).

0.22 0.20 0.05 0.01 0.035 0.020

23.9t NA 35.0,~ 65.7? 11.27 NA

0.052 0.015 0.051 Technical grade

NA NA NA 60.9~

Daphnids and the reproducibility of test results demographic data, higher LCs0 results and greater precision in test results than do trout chow-fed organisms. Algal-fed organisms are cheaper to test' as a smaller loss of control organisms is experienced and therefore, there is less need to repeat tests, and tests carried out with algal-fed organisms are more reproducible with a smaller standard deviation than with trout chow-fed organisms. Such algal-fed animals have been referred to as inappropriate for testing because they are "super" daphnids. In fact, they are more likely, than are animals reared in cultures containing synthetic diets, to resemble ani-

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results: therefore, some time should be spent in trying to understand more about this problem. Finally, since even variation among strains of a single animal species can introduce disastrous effects on "RoundRobin" results, it is suggested that all animals eraployed in all interlaboratory studies should be of the same strain to minimize such variation.

REFERENCES Adema D. M. M. (1978) Daphnia magna as a test animal in acute and chronic toxicity tests. Hydrobiologia 59, 125-134. Banta A. M. (1921) Selection in Cladocera on the basis of mals collected from natural systems since they have a physiological character. Publication No. 305, Carnegie been reared in circumstances more closely resembling Institution of Washington. those in nature and have been fed a diet more closely BlanckH., Wallin G. and Wangberg S. A. (1984) Speciesresembling that available in nature (for further disdependent variation in algal sensitivity to chemical comcussion cf. Hutchinson, 1967). pounds. Ecotoxic. envir. Safety 8, 339-351. Bobra A. M., Shiu W. Y. and MacKay D. (1983) A Calculations based on published information indipredictivecorrelation for the acute toxicity of hydrocate that the obtained LCs0 may reflect the test carbons and chlorinated hydrocarbons to the water flea compound characteristics. Characteristics of the (Daphniamagna). Chemosphere 12, 1121-1129. compound in question responsible for this obser- Buikema A. L. Jr, Geiger J. G. and Lee D. R. (1980) Daphnia Toxicity Tests, pp. 46-69. STP 715, American vation include water solubility, molecular weight, Societyfor Testing and Materials, Philadelphia, Pa. LCs0 values, and boiling point. It is recommended Canton J. H. and Adema D. M. M. (1978) Reproducibility that all proposed interlaboratory studies address this of short-term and reproduction toxicity experiments with question prior to their initiation. Daphnia magna and comparison of the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata in short-term experiments. Hydrobiologia 59, 135-140. Cardwell R. D., Foreman D. G., Payne T. R. and Wilbur RECOMMENDATIONS D.J. (1977) Acute and chronic toxicity of chlordane to fish and invertebrates. EPA-600/3-77-019, U.S. EnvironTo achieve good precision and low percent mental Protection Agency, Duluth, Minn. coefficient of variation in all interlaboratory com- CowgillU. M. (1976) The chemical composition of two parisons, the following recommendations should be speciesof Daphnia, their algal food and their environconsidered: ment. Sci. Total Envir. 6, 79--102. Cowgill U. M., Williams D. M. and Esquivel J. B. (1984) (1) Initiate procedures that ascertain the health of Effectsof maternal nutrition on fat content and longethe test organism brood stock of each participating vity of neonates of Daphnia magna. J. Crust. Biol. 4, 173-190. laboratory prior to the initiation of any "Round- CowgillU. M., Keating K. I. and Takahashi I. T. (1985a) Robin" test procedure. Select a group of benchmark Fecundity and longevity of Ceriodaphnia dubia/affinis in chemicals and examine their toxicity in triplicate relation to diet at two different temperatures. J. Crust. semiannually. After several years the calculated Biol. 5, 420-429. coefficient of variation should be acceptably small. CowgillU. M., Takahashi I. T. and Applegath S. L. (1985b) A comparison of the effect of four benchmark chemicals Accumulate demographic data on the zooplankton of Daphnia magna and Ceriodaphnia dubia/affinis tested brood stock. Test data should be gathered quarterly, at two different temperatures. Envir. Toxic. Chem. 4, Consistency in demographic results, as well as, L C s 0 415-422. values derived from testing the benchmark chemicals CowgillU. M., Hopkins D. L., Applegath S. L., Takahashi I. T., Brooks S. D. and Milazzo D. P. (1985c) BroodSize should provide a measure of zooplankton health, and Neonate Weight of Daphnia magna Produced by Nine (2) Ascertain the purity of all compounds studied Diets, pp. 233-244. STP 891, American Society for Testprior to the initiation of the interlaboratory coming and Materials, Philadelphia, Pa. parison. Cowgill U. M., Emmel H. W., Hopkins D. L., Applegath S. L. and Takahashi I. T. (1986a) The influence of (3) Utilize measured rather than nominal concenwater on reproductive success and chemical composition trations for all test compounds and correct the of laboratory reared populations of Daphniamagna. Wat. resulting LC50 values accordingly. Res. 20, 317-323. (4) It is always possible that a particular strain of CowgillU. M., Emmel H. W., Hopkins D. L., Takahashi test organism may be inherently sensitive to a particI.T. and Parker W. M. (1986b) Variation in chemical composition, reproductive success and body weight of ular class of chemical compounds. Some research Daphnia magna in relation to diet. Int. Rev. ges. Hydroeffort should be devoted to investigating the problem biol. 71, 79-99. of species-dependent variation to particulargroupsor Crum-Brown A. and Fraser T. R. (1868-1869) On the classes of chemical compounds. Relatively little is connection between chemical constitution and physiological action. Part I. On the physiologicalaction of the known about this problem, though it does occur in salts of the ammonium bases derived from strychnia, algae (Blanck et al., 1984). Phenomena of this kind bucia, thebaia, codeia, morphia and nicotia. Part II. On could have disastrous effects on "Round-Robin" the physiological action of the ammonium bases derived WR

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B

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