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Chronic toxicity tests with Daphnia magna: the effects of different food and temperature regimes on survival, reproduction and growth
Environmental Pollution (Series A) 36 (1984) 95-107
Chronic Toxicity Tests with Daphnia magna: the Effects of Different Food and Temperature Regimes on Survival, Reproduction and Growth
R. R. Stephenson & S. A. Watts Shell Research Limited, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, Great Britain
ABSTRACT Chronic" toxicity tests with Daphnia magna are proposed in both the Toxic Substances Control Act in the USA and under the 6th Amendment of the 1967 Directive of the EEC on the classi[ication, packaging and labelling o f dangerous substances. Guidelines .[or such tests have been proposed by the US EPA and by the OECD. The experiments reported here examined the effects of l o o d and water temperature on the survival, reproductive performance and growth of individually held D. magna in a static water test system. Nine test regimes were compared, three temperatures and three rations, in a full 3 2 factorial experimental design replicated 25 times. The three temperatures were 17, 20 and 23°C and the three rations, Chlorella alone, Chlorella plus ),east extract, and a compound diet based on trout pellets. Under live of the nine regimes tested the D. magna survived and reproduced in a manner which fulfilled the current US EPA test guidelines .for a valid test. Only two regimes ful/illed the current OECD criteria. The most successful regimes were those in which the D. magna were.led Chlorella alone and held at 20 or 23 °C. The results reported here, along with published and unpublished results from other laboratories, suggest that the conditions under which valid tests might be carried out require further investigation, or that some relaxation of the criteria ,[or a valid test needs to be brought about, or both. 95
96
R. R. Stephenson, S. A. Watts
INTRODUCTION Daphnia magna Straus has long been recognised as a useful species for estimating the acute toxicity of substances to aquatic organisms (Anderson, 1944). More recently, toxicity tests based on reproductive performance and long-term survival of D. magna have been developed (Biesinger & Christensen, 1972: Winner & Farrell, 1976: Adema, 1978; Maki, 1979). These descriptions show no clear consensus as to the preferred conditions for chronic toxicity tests with D. magna, and Buikema et al. (1980) identified research on the nutrition and maintenance of healthy cultures as the highest-priority research need. Indeed over recent years it has become increasingly apparent that whilst some laboratories appear to have no problems in maintaining cultures of D. magna and carrying out chronic toxicity tests on a routine basis, others have significant difficulties (Blok, 1981). In our laboratory we have encountered problems with cyclical patterns of productivity in stock cultures and the abortion of eggs in cultures and tests, particularly during the first brood. In view of problems we have encountered and the lack of information in the literature, we have investigated the combined effects of ration and water temperature on survival and reproductive performance of D. magna in order to try to establish basic test conditions for these two parameters for D. magna from our laboratory culture. Two important guidelines for chronic toxicity tests with D. magna have recently been produced (OECD, 1981; EPA, 1982). Both of these simply define an acceptable feeding regime as one which meets the guidelines' criteria for acceptable survival and reproduction of control organisms. They do, however, agree that the test temperature should be around 20 °C (20_+ I°C in EPA guideline: 18-22°C controlled within _-L-0.5°C in OECD guideline). In the experiment reported here the three rations used have been previously advocated as suitable for chronic toxicity tests with D. magna. The test temperatures used ( 17-23 °C) extended beyond those specified in the EPA and OECD guidelines in order to see if relaxation or redefinition of the specified ranges would enhance the prospects of the routine achievement of the guidelines' criteria for survival and reproduction. The opportunity was also taken to see if young D. magna, produced under the various regimes, differed in either their susceptibility to a toxicant, potassium dichromate, or in their ability to withstand starvation.
Toxicity tests with Daphnia magna
97
MATERIALS A N D M E T H O D S
Reproductionexperiment The effects of water temperature and ration on the reproduction and survival of individually held D. magna were examined during a 21-day test. The D. magna used were obtained from the Institut National de Recherche Chimique Appliqu& France, some 2 years prior to the experiment. During this time they were maintained as an asexually reproducing clone. The test used a full 3 z factorial experimental design with three temperatures, 17, 20 and 23°C, and three rations: Ration I (R1) Ration 2 (R2) Ration 3 (R3)
Chlorella pyrenoidosa (0.4 x 106 cells mi- 1) Chlorella pyrenoidosa + yeast extract (0.4 × 106 cells ml - 1 + 10 #g ml - l) C o m p o u n d food ( 2 p l m l - 1 )
C. pyrenoidosa was collected daily from 20-1itre culture vessels and concentrated, and the volume needed to produce the rations was calculated. The yeast extract (Oxoid) was made up daily and used as a concentrated suspension in water. A single batch of the compound food was made up at the beginning of the experiment, following Biesinger & Christensen (I 972). It contained 20 g of trout pellets plus 1 g of dried grass in 500 ml of distilled water. The batch was divided into aliquots, frozen and defrosted daily for feeding. Twenty-five D. magna, less than 24-h old, were allocated to each of the nine test regimes. During the test the D. magna were held individually in 150-ml crystallising dishes containing 100ml of filtered (8#m) mains water, which had been passed through activated charcoal, aerated for 24 h and had the ration added. Twenty-five dishes from each of the ration regimes were allocated at random to positions in each of three temperature-controlled ( + I ° C ) water baths. The D. magna were transferred daily to clean dishes containing fresh water with the appropriate ration. The maximum and minimum temperatures of each water bath were recorded daily as was the total hardness as C a C O 3, concentration of dissolved oxygen and pH of the test media. The numbers of young produced and mortalities of the parent generation were recorded daily for individual D. magna. The young produced were discarded daily unless required for the toxicity and starvation experiments. At the end of the experiment the total length of
98
R. R. Stephenson, S. A. Watts
the surviving adults, which was taken as the distance from the anterior of the head to the base of the caudal spine, was measured.
Toxicity experiment On three ocasions during the reproduction experiment samples of young produced during the preceding 24 h were collected and their susceptibility to potassium dichromate was determined. The D. magna were exposed for 48 b to an approximately logarithmic series of concentrations of potassium dichromate made up in filtered (8/am) dechlorinated mains water with a total hardness 240 + 20 mg litre - 1as CaCO3, a pH value of 8.3 + 0.2, and a concentration of dissolved oxygen > 70 0% of the air-saturation value. The tests were carried out at 20 + 1 °C in 250-ml glass crystallising dishes containing 100 ml of test solution. Ten D. magna were exposed at each test concentration. At the end of 24 and 48 h the number of D. magna immobilised was recorded. Immobilisation is defined as those organisms unable to swim during a 10 s period of observation after the contents of the dish had been briefly stirred.
Starvation experiment On one occasion during the reproduction experiment, between days 14 and 18 depending on the treatment regime, young produced during a 24-h period were collected and a sample of 150 was taken from each test regime. The ability of these less than 24-h-old D. magna to withstand starvation at 17, 20 and 23 °C was determined. The 150 young from each regime were impartially allocated into groups of ten and placed in 250-ml glass crystallising dishes containing 100 ml of filtered (8/~m) dechlorinated mains water (total hardness 240 + 2 0 m g l i t r e - ' as C a C O 3 , pH 8.3 _+0.2, and concentration of dissolved oxygen > 70 OJ/oof the air-saturation value). Five of the 15 dishes from each test regime were introduced into each of the three constanttemperature ( + 1 °C) water baths held at 17, 20 and 23 °C. The number of D. magna immobilised in each dish was recorded at intervals during the period 24-120h after their introduction.
Statistical analysis When using analysis of variance two transformations were examined, log and square root. However, since neither transformation was consistently more appropriate, all analyses were performed on untransformed data.
Toxicity tests with Daphnia magna
99
Reproduction experiment Mortalities of the parent D. magna up to day 21 under the different regimes were compared using Fisher's exact test to determine significant differences between pairs of treatment means. The nominal level of the significance test was 5 II{,. However, since many tests were performed, the actual level was higher than this. Data on the daily production of young by each D. magna were used as a basis for the examination of treatment differences in reproductive performance. The following parameters were examined: The time o f production and size o f each of the /irst three broods. Where young were present on consecutive days they were assumed to come from a single brood unless moults were present on both days, when they were taken as coming from separate broods. If a brood was split over two days, the second day was taken as the brood day. For both time of production and size of broods there were no data for D. magna reared at 17 °C on ration 3, so a full two-way analysis of variance with an interaction term was not possible for either. An approximate analysis was therefore performed in each case by considering two separate analyses of variance, each with an interaction term, the first excluding D. magna at 17°C, the second excluding the D. magna fed ration 3. Although the main effects means were very different for the two analyses of variance, the pairs of "treatment' means (the interaction means) differed by less than I'!0. Using an ad hoc method the mid-value of these pairs was taken as an estimate of the means of the full analysis with ration 3 at 17°C excluded. Throughout these analyses the interaction term significantly reduced variability and so comparisons between main effects are not relevant. Comparisons between individual treatment means were based on the smallest estimate of error from the two analyses: these estimates did not differ greatly from each other. (2) The number o./young produced by D. magna under each treatment regime. The mean number of young produced by D. magna which survived to day 21 under each treatment regime were compared using a two-way analysis of variance with an interaction term. This analysis was only approximate since the number of parents for each treatment was different.
(I)
I00
R. R. Stephenson, S. A. Watts
Treatment-induced differences in the size of the D. magna alive at the end of the experiment were examined using a two-way analysis of variance. There was no significant interaction (50,o level) between temperature and ration and this term was therefore omitted from the analysis. Toxicitv experiment The data were analysed by probit analysis using log-transformed concentration values (Finney, 197 I) and the concentration immobilising 50 !!il of those exposed (ECso) for 24 and 48 h was calculated. These EC.~o values were subjected to a factorial analysis of variance, similar to that used to compare the time of production of the first broods and size of broods. Only one pair of means differed by more than 1 "',o, and that was by only 2 " O ' Starvation experiment The data were analysed by probit analysis using log-transformed time values (Finney, 1971) and the median time to immobilisation (ETs0) calculated. These ETso values were subjected to an analysis of variance. There were only limited data for young produced from parents held at 17 °C so a three-way analysis of variance incorporating the following was used: parent temperature -20 and 23 °C: parent ration - R I , R2 and R3, temperature young were starved at -17, 20 and 23°C. No interactions were included in the analysis as they were not significant (5 '!'0 level).
RESULTS Reproduction experiment
The test media had total hardnesses of 200-260 mg litre ~ and pH values of 7.5 -9-0; concentrations of dissolved oxygen were always in excess ot" 70"~, of the air-saturation value. There were significant differences in the survival of D. magna reared under the different.regimes (Table I). D. magna fed ration I had a low mortality when compared with those fed rations 2 and 3, and 23 °C was associated with higher mortalities than 17 or 20 ~C. During the 21-day experiment all of the D. magna which survived
T o x i c i t y tests with D a p h n i a m a g n a
101
TABLE I M o r t a l i t y o f D. m a g n a R e a r e d u n d e r Different R a t i o n a n d T e m p e r a t u r e Regimes ~ Temperature
N u m b e r d e a d after 21 d a y s b
(~C)
......... Ration 1
.
.
.
.
.
.
.
.
.
17 20 23
Total
.
.
.
Ration 2 .
.
.
.
.
.
.
1,
5,b ,
2,b 2,b
3,b ~ I (),
5
18
Ration 3 .
.
.
.
.
.
Total .
.
.
O,
6
3,h , 8b,
8 20
II
" T h o s e values with a subscript in c o m m o n are not significantly different ( P < 0.05, Fisher's exact test). Initial n u m b e r in each t r e a t m e n t g r o u p was 25.
produced at least one brood of young, except for those held at 17 °C and fed ration 3 of which only one produced a brood (Table 2). The mean time to the production of the first brood varied from 8-9 days for D. magna held at 23 °C and fed ration 1 to in excess of 15 days for those fed ration 3 and held at 17 and 20 °C. At all three temperatures the mean time to the production of each of the first three broods was shortest for D. magna fed ration i and longest for those fed ration 3. Whichever the ration, the mean time to brood production was shorter at 23 °C than at 17~C. TABLE 2 T h e Effect o f Different T e m p e r a t u r e a n d R a t i o n R e g i m e s on the Time o f B r o o d P r o d u c t i o n by D. m a g n a ° Temperatur,'
Mean time to production O! brood (days)
'('l First brood
17 20 23
Second brood
Third hrood
Ration I
Ration 2
Ration 3
Ration 1
Ration 2
Ration 3
Ration 1
lO.9t, 10"4h 8-9,
13.6a 12"2~ 10"8b
>211 16'2. 12"8¢
14.8,.a 13"4h I 1"6,
17"5 15"2d 14"0h,
>21 s 18"5t 14-9d
18"9a 16"5h 14"4,
Ration 2
196a 18-0~ 16'3 b
Ration 3
>21, 20.7 17"3
" Within each brood (i.e. first, second or third) those values with a subscript in common are not significantly different I P <_ I).05. SE I. I 1.41.
R. R. S t e p h e n s o n , S. A, W a t t s
102
The results of the analysis of variance of the times of production of the broods showed significant temperature, ration and interaction effects. D. magna fed ration 1 and held at 23 °C produced each of their first three broods significantly earlier than did those reared under other regimes. D. magna fed ration I at 20 and 17 °C and ration 2 at 23 °C also performed well, generally having a mean time to brood production significantly shorter than the remaining five regimes. First broods were generally smaller than subsequent ones, regardless of the treatment regimes; this tendency was particularly marked for D. magna fed ration i (Table 3). The mean sizes of the first three broods under the different regimes show the relatively poor performance of D. magna fed ration 3, none of which had mean brood sizes of greater than 25. This can be compared with the performances of the D. magna fed rations 1 and 2, where the mean sizes of the second and third broods all exceeded 30, and two exceeded 40. Table 4 gives the mean number of young produced by D. magna surviving to the end of the experiment. Again it is apparent that D. magna fed rations 1 and 2 and held at 23 or 20 °C performed best, all producing significantly more young than the other regimes. Both temperature and ration influenced the size of D. magna at the end of the experiment, with ration taking account of more variation than temperature (Table 5). The mean sizes of D. magna from the three ration regimes were significantly different from each other, with ration 1 producing the largest and ration 3 the smallest individuals. There was no TABLE 3 T h e Effect o f Different T e m p e r a t u r e a n d R a t i o n R e g i m e s o n the Size o f B r o o d s P r o d u c e d by D. m a g n a " Mean si--e o / b r o o d
Temperature ('C) Fir.~t brood Ration I
17 20 23
Second brood
I'hird brood
Ration 2
Ration .t
Ration I
Ration 2
Ration 3
1 I "4,1~ 23'3t,, 17.4,,j 33.6= 25.9 b 24-0 b
10"5. 9.8~.
35"0h 39.9, 34"6h
32. I h 31.8 h 33"2h
10.3, 15.2,
Ration I
Ration 2
43.()~ 35.8,,~ 4 1 . 2 ~ b 32.9,j 37.0h, j 38.2b,
Ration 3
8.7, 22.1
u Within each brood (i.e. first, second or third) those values with a subscript in common are not significantly different ( P < 0 . 0 5 , SE 7.6 11.6).
103
Toxicity t e s t s with Daphnia magna TABLE 4 The Effect of Different Temperature and Ration Regimes on the Mean Number of Young Produced by D. magna Surviving to the End of the Experiment" Temperature ('C)
17 20 23
Mean b number qf young .......... Ration I
Ration 2
Ration 3
88' 5d 159~b 171 a
68' 8~. 140, 150t,,
0 23"2~ 80"4.
" Those values with a subscript in c o m m o n are not significantly different ( P _< 0.05, SE 22.9). h Means are approximate since the replication (number of parents) in each regime was different (Table I ).
significant difference in the mean size of the D. magna reared at 20 and 23 °C but both of these groups were significantly larger than those reared at 17 °C. Toxicity experiment The susceptibility to potassium dichromate of the young D. magna from the different regimes was similar. Mean 24-h ECso values for D. magna from the different regimes ranged from 0-26 to 0.61 mg litre- 1 and 48-h ECso values from 0" I0 to 0.34 mg litre t (Table 6). Although there were a number of significant differences in the mean 24-h ECso values for the different regimes, there was only one in the 48-h ECso values. Neither the TABLE 5 Mean Length of Survivors at the End of the Experiment" l'emperaturc ((')
Length (mm)
17
3. ~a
20
4'1. 4"1 h
23
Ralioll
Length (ram)
43~ 2 3
" T h o s e values with a subscript in c o m m o n significantly different ( P _< 0 0 5 . SE 0 18).
4"1 h 3"2, are not
R. R. Stephenson, S. A. Watts
104
TABLE 6 Toxicity of Potassium Dichromate to D. magna Produced Under Different Food and Temperature Regimes"
Mean 24h ECso values (mg litre -I) Temperature
Ration
(~C)
Mean 48tl ECso values (nlglitru l) Temperature
- -
Ration
(~(')
/
:'
3
17
0"61~
0"47b,
-
17
0"25h
0"34 b
20 23
0"26~ 0"40.b
0"46b. 0"54.
0"30ab
20 23
0"23h 0"24~
0"31~, 0'31,,
0-26.
/
2
3
0"I0 0"29 b
" Those values with a subscript in c o m m o n are not significantly different (p < 0.05, SE 0.07 0.15).
24-h or 48-h ECso values indicated that particular ration or temperature regimes were consistently influencing the susceptibility of D. magna to potassium dichromate. Starvation experiment The temperature and ration regimes of the parents had no discernible effect on the ability of the young they produced to withstand starvation TABLE 7 Immobilisation of D. magna Produced from Different Food and Temperature Regimes when Starved at Different Temperatures Expressed as ET~. Values"
Ell'eel olparental temperature regitm" Temperature ("C') Number of tests Mean ET~o (h)
20 9 84,
23 9 88~
I 6 89
2 6 79~
3 6 91~
17 6 107,
20 6 7%,
23 6 73~,
E[]ect ol parental ration Ration Number of tests Mean ET 5 j(h)
E[]ect o/temperature at which young were starred Temperature (:'C) Number of tests Mean ET~o (h)
" Those values with a subscript in common are not significantly different (P < 0.05, SE 12).
T o x i c i t y tests with
Daphnia magna
105
(Table 7). The temperature at which the young were starved did affect the mean time taken for 50'~', to become immobilised, the young remaining mobile significantly longer at 17 °C than at 20 or 23 °C.
DISCUSSION In both the USA and Europe it has been proposed that reproduction/chronic toxicity tests with D. magna should play an important role in the testing, prior to notification, of new chemicals. The EPA and the OECD have published guidelines for such a test (OECD, 1981: EPA, 1982). The guideline requirements for an acceptable test can be compared with the results obtained in the present experiment. Two of the nine test regimes failed to fulfil the EPA guideline requirements for at least 80 0,, survival: mortality ofD. magna fed rations 2 and 3 and held at 23~'C exceeded 20"ii on day 21 (Table 1). The EPA criterion for acceptable reproductive performance, 60 or more young per Daphnia during the test, was achieved by D. magna fed rations i or 2 and by those fed ration 3 at 23 ~C, but not by those held at 17 or 20 °C and fed ration 3 (Table 4). Only D. magna fed ration 3 and those held at 23 ~C and fed ration 2 failed to fulfil both the survival and reproductive criteria of the EPA guideline. The OECD guideline requirement for survival of D. magna is the same as that in the EPA scheme, and it was under the same two test regimes-those in which D. magna were fed rations 2 and 3 and held at 23 °C--that the requirement was not achieved (Table 1). The first OECD criterion for an acceptable reproductive performance is that the first brood be produced within 9 days. Since in normal tests D. magna are held in groups and not individually, the data from Table 2 cannot be used directly. However, it can be inferred from the experimental data that D. magna fed ration 1 at 23 and 20°C and those fed ration 2 at 23°C achieved the required performance. In none of the test regimes did D. magna produce 3 broods within 14 days (Table 2). But all except those held at 17 °C and fed ration 3 had mean times for the production of the third brood of less than 21 days (Table 2), and hence fulfilled the second OECD reproductive criterion of three broods of young. Similarly the third OECD reproductive requirement, more than 20 young per Daphnia, was fulfilled by D. magna under all regimes, except for those held at 17 °C and fed
106
R. R. Stephenson. S. A. Watts
ration 3. Taking all three reproductive criteria together, only the D. magna held at 23 °C and fed rations I and 2 and those held at 20 °C and fed ration 1 produced a satisfactory result. Considering survival and reproduction together, only D. magna fed ration I at 23 and 20°C fulfilled the OECD criteria for a valid test. The food and temperature regimes used in the experiment reported here encompass many of those reported by others as giving satisfactory conditions for conducting chronic toxicity experiments with D. magna. Nevertheless, under only five of the nine test regimes did D. magna fulfil the current EPA requirements for a valid test and under only two the current OECD requirements. Furthermore, unreported studies in this laboratory have indicated that there are also significant differences in the survival and reproductive performance of D. magna when reared under the same test regimes on different occasions. Similar problems of reproducibility have been encountered by other laboratories conducting chronic toxicity tests with D. magna (Blok, 1981: Nebeker, 1982). In the light of these findings it would seem necessary that either the conditions under which a valid test might routinely be carried out require further investigation, or that some relaxation of the criteria for a valid test needs to be brought about, or both. Some relaxation would seem to be particularly desirable for the OECD requirement of first brood production within 9 days. The toxicity and starvation experiments showed that despite the significant effects of the different test regimes on the survival and reproductive performance of D. magna there was no effect on the susceptibility of the young produced to a toxicant, potassium dichromate, or on their ability to withstand starvation.
A C K N O W L E D G E M ENTS The technical assistance of N. Pearson and C. M. Sherwood is acknowledged, as is the help and comments ot" other colleagues at Sittingbourne Research Centre. REFERENCES Adema, D. M. M. (1978). Daphnia magna as a test animal in acute and chronic toxicity tests. H.vdrohiologia, 59. 125 34.
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Anderson, B. D. (1944). The toxicity thresholds of various substances found in industrial wastes as determined by the use of Daphnia magna. Sewage Works. J., 16, 1156-65. Biesinger, K. E. & Christensen, G. M. (1972). Effects of various metals on survival, growth, reproduction and metabolism of Daphnia magna. J. Fish. Res. Bd Can., 29, 1691 -700. Blok, J. (1981). Measuring the reproduction rate of Daphnia magna.
Proceedings o[ "'Colloque International d'Ecotoxicologie'" INSERM, Villeneuve, France, November 1981. Buikema, A. L., Jr.. Geiger, J. G. & Lee, D. R. (1980). Daphnia toxicity tests. In Aquatic invertebrate hioassays, ed. by A. L. Buikema Jr. and J. Cairns, Jr, A S T M Spec. Tech. Pubis, 715, 48 69. EPA (1982). EPA Environmental effects testing guidelines, EG-2 and E5-1. Chemical Regulation Reporter Supplement N-52. Bureau of National Affairs Inc. Washington DC. Finney, D. J. (1971). Prohit anal.vsis, 3rd edn. Cambridge, Cambridge University Press. Maki, A. (1979). Correlations between Daphnia magna and fathead minnow (Pimephales promelas) chronic toxicity values for several classes ot" test substances. J. Fish. Res. Bd Can., 36, 411 -21. Nebeker, A. V. (1982). Evaluation of a Daphnia magna renewal life-cycle test method with silver and endosulfan. Water Res., 16, 739 44. OECD (1981). Section 2, Effects on biotic systems 202. In OECD guidelines/or testing ~[chemicals. Paris, OECD. Winner, R. W. & Farrell, M. P. (1976). Acute and chronic toxicity of copper to four species of Daphnia. J. Fish. Res. Bd Can., 33, 1685 91.
Chronic Toxicity Tests with Daphnia magna: the Effects of Different Food and Temperature Regimes on Survival, Reproduction and Growth
R. R. Stephenson & S. A. Watts Shell Research Limited, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, Great Britain
ABSTRACT Chronic" toxicity tests with Daphnia magna are proposed in both the Toxic Substances Control Act in the USA and under the 6th Amendment of the 1967 Directive of the EEC on the classi[ication, packaging and labelling o f dangerous substances. Guidelines .[or such tests have been proposed by the US EPA and by the OECD. The experiments reported here examined the effects of l o o d and water temperature on the survival, reproductive performance and growth of individually held D. magna in a static water test system. Nine test regimes were compared, three temperatures and three rations, in a full 3 2 factorial experimental design replicated 25 times. The three temperatures were 17, 20 and 23°C and the three rations, Chlorella alone, Chlorella plus ),east extract, and a compound diet based on trout pellets. Under live of the nine regimes tested the D. magna survived and reproduced in a manner which fulfilled the current US EPA test guidelines .for a valid test. Only two regimes ful/illed the current OECD criteria. The most successful regimes were those in which the D. magna were.led Chlorella alone and held at 20 or 23 °C. The results reported here, along with published and unpublished results from other laboratories, suggest that the conditions under which valid tests might be carried out require further investigation, or that some relaxation of the criteria ,[or a valid test needs to be brought about, or both. 95
96
R. R. Stephenson, S. A. Watts
INTRODUCTION Daphnia magna Straus has long been recognised as a useful species for estimating the acute toxicity of substances to aquatic organisms (Anderson, 1944). More recently, toxicity tests based on reproductive performance and long-term survival of D. magna have been developed (Biesinger & Christensen, 1972: Winner & Farrell, 1976: Adema, 1978; Maki, 1979). These descriptions show no clear consensus as to the preferred conditions for chronic toxicity tests with D. magna, and Buikema et al. (1980) identified research on the nutrition and maintenance of healthy cultures as the highest-priority research need. Indeed over recent years it has become increasingly apparent that whilst some laboratories appear to have no problems in maintaining cultures of D. magna and carrying out chronic toxicity tests on a routine basis, others have significant difficulties (Blok, 1981). In our laboratory we have encountered problems with cyclical patterns of productivity in stock cultures and the abortion of eggs in cultures and tests, particularly during the first brood. In view of problems we have encountered and the lack of information in the literature, we have investigated the combined effects of ration and water temperature on survival and reproductive performance of D. magna in order to try to establish basic test conditions for these two parameters for D. magna from our laboratory culture. Two important guidelines for chronic toxicity tests with D. magna have recently been produced (OECD, 1981; EPA, 1982). Both of these simply define an acceptable feeding regime as one which meets the guidelines' criteria for acceptable survival and reproduction of control organisms. They do, however, agree that the test temperature should be around 20 °C (20_+ I°C in EPA guideline: 18-22°C controlled within _-L-0.5°C in OECD guideline). In the experiment reported here the three rations used have been previously advocated as suitable for chronic toxicity tests with D. magna. The test temperatures used ( 17-23 °C) extended beyond those specified in the EPA and OECD guidelines in order to see if relaxation or redefinition of the specified ranges would enhance the prospects of the routine achievement of the guidelines' criteria for survival and reproduction. The opportunity was also taken to see if young D. magna, produced under the various regimes, differed in either their susceptibility to a toxicant, potassium dichromate, or in their ability to withstand starvation.
Toxicity tests with Daphnia magna
97
MATERIALS A N D M E T H O D S
Reproductionexperiment The effects of water temperature and ration on the reproduction and survival of individually held D. magna were examined during a 21-day test. The D. magna used were obtained from the Institut National de Recherche Chimique Appliqu& France, some 2 years prior to the experiment. During this time they were maintained as an asexually reproducing clone. The test used a full 3 z factorial experimental design with three temperatures, 17, 20 and 23°C, and three rations: Ration I (R1) Ration 2 (R2) Ration 3 (R3)
Chlorella pyrenoidosa (0.4 x 106 cells mi- 1) Chlorella pyrenoidosa + yeast extract (0.4 × 106 cells ml - 1 + 10 #g ml - l) C o m p o u n d food ( 2 p l m l - 1 )
C. pyrenoidosa was collected daily from 20-1itre culture vessels and concentrated, and the volume needed to produce the rations was calculated. The yeast extract (Oxoid) was made up daily and used as a concentrated suspension in water. A single batch of the compound food was made up at the beginning of the experiment, following Biesinger & Christensen (I 972). It contained 20 g of trout pellets plus 1 g of dried grass in 500 ml of distilled water. The batch was divided into aliquots, frozen and defrosted daily for feeding. Twenty-five D. magna, less than 24-h old, were allocated to each of the nine test regimes. During the test the D. magna were held individually in 150-ml crystallising dishes containing 100ml of filtered (8#m) mains water, which had been passed through activated charcoal, aerated for 24 h and had the ration added. Twenty-five dishes from each of the ration regimes were allocated at random to positions in each of three temperature-controlled ( + I ° C ) water baths. The D. magna were transferred daily to clean dishes containing fresh water with the appropriate ration. The maximum and minimum temperatures of each water bath were recorded daily as was the total hardness as C a C O 3, concentration of dissolved oxygen and pH of the test media. The numbers of young produced and mortalities of the parent generation were recorded daily for individual D. magna. The young produced were discarded daily unless required for the toxicity and starvation experiments. At the end of the experiment the total length of
98
R. R. Stephenson, S. A. Watts
the surviving adults, which was taken as the distance from the anterior of the head to the base of the caudal spine, was measured.
Toxicity experiment On three ocasions during the reproduction experiment samples of young produced during the preceding 24 h were collected and their susceptibility to potassium dichromate was determined. The D. magna were exposed for 48 b to an approximately logarithmic series of concentrations of potassium dichromate made up in filtered (8/am) dechlorinated mains water with a total hardness 240 + 20 mg litre - 1as CaCO3, a pH value of 8.3 + 0.2, and a concentration of dissolved oxygen > 70 0% of the air-saturation value. The tests were carried out at 20 + 1 °C in 250-ml glass crystallising dishes containing 100 ml of test solution. Ten D. magna were exposed at each test concentration. At the end of 24 and 48 h the number of D. magna immobilised was recorded. Immobilisation is defined as those organisms unable to swim during a 10 s period of observation after the contents of the dish had been briefly stirred.
Starvation experiment On one occasion during the reproduction experiment, between days 14 and 18 depending on the treatment regime, young produced during a 24-h period were collected and a sample of 150 was taken from each test regime. The ability of these less than 24-h-old D. magna to withstand starvation at 17, 20 and 23 °C was determined. The 150 young from each regime were impartially allocated into groups of ten and placed in 250-ml glass crystallising dishes containing 100 ml of filtered (8/~m) dechlorinated mains water (total hardness 240 + 2 0 m g l i t r e - ' as C a C O 3 , pH 8.3 _+0.2, and concentration of dissolved oxygen > 70 OJ/oof the air-saturation value). Five of the 15 dishes from each test regime were introduced into each of the three constanttemperature ( + 1 °C) water baths held at 17, 20 and 23 °C. The number of D. magna immobilised in each dish was recorded at intervals during the period 24-120h after their introduction.
Statistical analysis When using analysis of variance two transformations were examined, log and square root. However, since neither transformation was consistently more appropriate, all analyses were performed on untransformed data.
Toxicity tests with Daphnia magna
99
Reproduction experiment Mortalities of the parent D. magna up to day 21 under the different regimes were compared using Fisher's exact test to determine significant differences between pairs of treatment means. The nominal level of the significance test was 5 II{,. However, since many tests were performed, the actual level was higher than this. Data on the daily production of young by each D. magna were used as a basis for the examination of treatment differences in reproductive performance. The following parameters were examined: The time o f production and size o f each of the /irst three broods. Where young were present on consecutive days they were assumed to come from a single brood unless moults were present on both days, when they were taken as coming from separate broods. If a brood was split over two days, the second day was taken as the brood day. For both time of production and size of broods there were no data for D. magna reared at 17 °C on ration 3, so a full two-way analysis of variance with an interaction term was not possible for either. An approximate analysis was therefore performed in each case by considering two separate analyses of variance, each with an interaction term, the first excluding D. magna at 17°C, the second excluding the D. magna fed ration 3. Although the main effects means were very different for the two analyses of variance, the pairs of "treatment' means (the interaction means) differed by less than I'!0. Using an ad hoc method the mid-value of these pairs was taken as an estimate of the means of the full analysis with ration 3 at 17°C excluded. Throughout these analyses the interaction term significantly reduced variability and so comparisons between main effects are not relevant. Comparisons between individual treatment means were based on the smallest estimate of error from the two analyses: these estimates did not differ greatly from each other. (2) The number o./young produced by D. magna under each treatment regime. The mean number of young produced by D. magna which survived to day 21 under each treatment regime were compared using a two-way analysis of variance with an interaction term. This analysis was only approximate since the number of parents for each treatment was different.
(I)
I00
R. R. Stephenson, S. A. Watts
Treatment-induced differences in the size of the D. magna alive at the end of the experiment were examined using a two-way analysis of variance. There was no significant interaction (50,o level) between temperature and ration and this term was therefore omitted from the analysis. Toxicitv experiment The data were analysed by probit analysis using log-transformed concentration values (Finney, 197 I) and the concentration immobilising 50 !!il of those exposed (ECso) for 24 and 48 h was calculated. These EC.~o values were subjected to a factorial analysis of variance, similar to that used to compare the time of production of the first broods and size of broods. Only one pair of means differed by more than 1 "',o, and that was by only 2 " O ' Starvation experiment The data were analysed by probit analysis using log-transformed time values (Finney, 1971) and the median time to immobilisation (ETs0) calculated. These ETso values were subjected to an analysis of variance. There were only limited data for young produced from parents held at 17 °C so a three-way analysis of variance incorporating the following was used: parent temperature -20 and 23 °C: parent ration - R I , R2 and R3, temperature young were starved at -17, 20 and 23°C. No interactions were included in the analysis as they were not significant (5 '!'0 level).
RESULTS Reproduction experiment
The test media had total hardnesses of 200-260 mg litre ~ and pH values of 7.5 -9-0; concentrations of dissolved oxygen were always in excess ot" 70"~, of the air-saturation value. There were significant differences in the survival of D. magna reared under the different.regimes (Table I). D. magna fed ration I had a low mortality when compared with those fed rations 2 and 3, and 23 °C was associated with higher mortalities than 17 or 20 ~C. During the 21-day experiment all of the D. magna which survived
T o x i c i t y tests with D a p h n i a m a g n a
101
TABLE I M o r t a l i t y o f D. m a g n a R e a r e d u n d e r Different R a t i o n a n d T e m p e r a t u r e Regimes ~ Temperature
N u m b e r d e a d after 21 d a y s b
(~C)
......... Ration 1
.
.
.
.
.
.
.
.
.
17 20 23
Total
.
.
.
Ration 2 .
.
.
.
.
.
.
1,
5,b ,
2,b 2,b
3,b ~ I (),
5
18
Ration 3 .
.
.
.
.
.
Total .
.
.
O,
6
3,h , 8b,
8 20
II
" T h o s e values with a subscript in c o m m o n are not significantly different ( P < 0.05, Fisher's exact test). Initial n u m b e r in each t r e a t m e n t g r o u p was 25.
produced at least one brood of young, except for those held at 17 °C and fed ration 3 of which only one produced a brood (Table 2). The mean time to the production of the first brood varied from 8-9 days for D. magna held at 23 °C and fed ration 1 to in excess of 15 days for those fed ration 3 and held at 17 and 20 °C. At all three temperatures the mean time to the production of each of the first three broods was shortest for D. magna fed ration i and longest for those fed ration 3. Whichever the ration, the mean time to brood production was shorter at 23 °C than at 17~C. TABLE 2 T h e Effect o f Different T e m p e r a t u r e a n d R a t i o n R e g i m e s on the Time o f B r o o d P r o d u c t i o n by D. m a g n a ° Temperatur,'
Mean time to production O! brood (days)
'('l First brood
17 20 23
Second brood
Third hrood
Ration I
Ration 2
Ration 3
Ration 1
Ration 2
Ration 3
Ration 1
lO.9t, 10"4h 8-9,
13.6a 12"2~ 10"8b
>211 16'2. 12"8¢
14.8,.a 13"4h I 1"6,
17"5 15"2d 14"0h,
>21 s 18"5t 14-9d
18"9a 16"5h 14"4,
Ration 2
196a 18-0~ 16'3 b
Ration 3
>21, 20.7 17"3
" Within each brood (i.e. first, second or third) those values with a subscript in common are not significantly different I P <_ I).05. SE I. I 1.41.
R. R. S t e p h e n s o n , S. A, W a t t s
102
The results of the analysis of variance of the times of production of the broods showed significant temperature, ration and interaction effects. D. magna fed ration 1 and held at 23 °C produced each of their first three broods significantly earlier than did those reared under other regimes. D. magna fed ration I at 20 and 17 °C and ration 2 at 23 °C also performed well, generally having a mean time to brood production significantly shorter than the remaining five regimes. First broods were generally smaller than subsequent ones, regardless of the treatment regimes; this tendency was particularly marked for D. magna fed ration i (Table 3). The mean sizes of the first three broods under the different regimes show the relatively poor performance of D. magna fed ration 3, none of which had mean brood sizes of greater than 25. This can be compared with the performances of the D. magna fed rations 1 and 2, where the mean sizes of the second and third broods all exceeded 30, and two exceeded 40. Table 4 gives the mean number of young produced by D. magna surviving to the end of the experiment. Again it is apparent that D. magna fed rations 1 and 2 and held at 23 or 20 °C performed best, all producing significantly more young than the other regimes. Both temperature and ration influenced the size of D. magna at the end of the experiment, with ration taking account of more variation than temperature (Table 5). The mean sizes of D. magna from the three ration regimes were significantly different from each other, with ration 1 producing the largest and ration 3 the smallest individuals. There was no TABLE 3 T h e Effect o f Different T e m p e r a t u r e a n d R a t i o n R e g i m e s o n the Size o f B r o o d s P r o d u c e d by D. m a g n a " Mean si--e o / b r o o d
Temperature ('C) Fir.~t brood Ration I
17 20 23
Second brood
I'hird brood
Ration 2
Ration .t
Ration I
Ration 2
Ration 3
1 I "4,1~ 23'3t,, 17.4,,j 33.6= 25.9 b 24-0 b
10"5. 9.8~.
35"0h 39.9, 34"6h
32. I h 31.8 h 33"2h
10.3, 15.2,
Ration I
Ration 2
43.()~ 35.8,,~ 4 1 . 2 ~ b 32.9,j 37.0h, j 38.2b,
Ration 3
8.7, 22.1
u Within each brood (i.e. first, second or third) those values with a subscript in common are not significantly different ( P < 0 . 0 5 , SE 7.6 11.6).
103
Toxicity t e s t s with Daphnia magna TABLE 4 The Effect of Different Temperature and Ration Regimes on the Mean Number of Young Produced by D. magna Surviving to the End of the Experiment" Temperature ('C)
17 20 23
Mean b number qf young .......... Ration I
Ration 2
Ration 3
88' 5d 159~b 171 a
68' 8~. 140, 150t,,
0 23"2~ 80"4.
" Those values with a subscript in c o m m o n are not significantly different ( P _< 0.05, SE 22.9). h Means are approximate since the replication (number of parents) in each regime was different (Table I ).
significant difference in the mean size of the D. magna reared at 20 and 23 °C but both of these groups were significantly larger than those reared at 17 °C. Toxicity experiment The susceptibility to potassium dichromate of the young D. magna from the different regimes was similar. Mean 24-h ECso values for D. magna from the different regimes ranged from 0-26 to 0.61 mg litre- 1 and 48-h ECso values from 0" I0 to 0.34 mg litre t (Table 6). Although there were a number of significant differences in the mean 24-h ECso values for the different regimes, there was only one in the 48-h ECso values. Neither the TABLE 5 Mean Length of Survivors at the End of the Experiment" l'emperaturc ((')
Length (mm)
17
3. ~a
20
4'1. 4"1 h
23
Ralioll
Length (ram)
43~ 2 3
" T h o s e values with a subscript in c o m m o n significantly different ( P _< 0 0 5 . SE 0 18).
4"1 h 3"2, are not
R. R. Stephenson, S. A. Watts
104
TABLE 6 Toxicity of Potassium Dichromate to D. magna Produced Under Different Food and Temperature Regimes"
Mean 24h ECso values (mg litre -I) Temperature
Ration
(~C)
Mean 48tl ECso values (nlglitru l) Temperature
- -
Ration
(~(')
/
:'
3
17
0"61~
0"47b,
-
17
0"25h
0"34 b
20 23
0"26~ 0"40.b
0"46b. 0"54.
0"30ab
20 23
0"23h 0"24~
0"31~, 0'31,,
0-26.
/
2
3
0"I0 0"29 b
" Those values with a subscript in c o m m o n are not significantly different (p < 0.05, SE 0.07 0.15).
24-h or 48-h ECso values indicated that particular ration or temperature regimes were consistently influencing the susceptibility of D. magna to potassium dichromate. Starvation experiment The temperature and ration regimes of the parents had no discernible effect on the ability of the young they produced to withstand starvation TABLE 7 Immobilisation of D. magna Produced from Different Food and Temperature Regimes when Starved at Different Temperatures Expressed as ET~. Values"
Ell'eel olparental temperature regitm" Temperature ("C') Number of tests Mean ET~o (h)
20 9 84,
23 9 88~
I 6 89
2 6 79~
3 6 91~
17 6 107,
20 6 7%,
23 6 73~,
E[]ect ol parental ration Ration Number of tests Mean ET 5 j(h)
E[]ect o/temperature at which young were starred Temperature (:'C) Number of tests Mean ET~o (h)
" Those values with a subscript in common are not significantly different (P < 0.05, SE 12).
T o x i c i t y tests with
Daphnia magna
105
(Table 7). The temperature at which the young were starved did affect the mean time taken for 50'~', to become immobilised, the young remaining mobile significantly longer at 17 °C than at 20 or 23 °C.
DISCUSSION In both the USA and Europe it has been proposed that reproduction/chronic toxicity tests with D. magna should play an important role in the testing, prior to notification, of new chemicals. The EPA and the OECD have published guidelines for such a test (OECD, 1981: EPA, 1982). The guideline requirements for an acceptable test can be compared with the results obtained in the present experiment. Two of the nine test regimes failed to fulfil the EPA guideline requirements for at least 80 0,, survival: mortality ofD. magna fed rations 2 and 3 and held at 23~'C exceeded 20"ii on day 21 (Table 1). The EPA criterion for acceptable reproductive performance, 60 or more young per Daphnia during the test, was achieved by D. magna fed rations i or 2 and by those fed ration 3 at 23 ~C, but not by those held at 17 or 20 °C and fed ration 3 (Table 4). Only D. magna fed ration 3 and those held at 23 ~C and fed ration 2 failed to fulfil both the survival and reproductive criteria of the EPA guideline. The OECD guideline requirement for survival of D. magna is the same as that in the EPA scheme, and it was under the same two test regimes-those in which D. magna were fed rations 2 and 3 and held at 23 °C--that the requirement was not achieved (Table 1). The first OECD criterion for an acceptable reproductive performance is that the first brood be produced within 9 days. Since in normal tests D. magna are held in groups and not individually, the data from Table 2 cannot be used directly. However, it can be inferred from the experimental data that D. magna fed ration 1 at 23 and 20°C and those fed ration 2 at 23°C achieved the required performance. In none of the test regimes did D. magna produce 3 broods within 14 days (Table 2). But all except those held at 17 °C and fed ration 3 had mean times for the production of the third brood of less than 21 days (Table 2), and hence fulfilled the second OECD reproductive criterion of three broods of young. Similarly the third OECD reproductive requirement, more than 20 young per Daphnia, was fulfilled by D. magna under all regimes, except for those held at 17 °C and fed
106
R. R. Stephenson. S. A. Watts
ration 3. Taking all three reproductive criteria together, only the D. magna held at 23 °C and fed rations I and 2 and those held at 20 °C and fed ration 1 produced a satisfactory result. Considering survival and reproduction together, only D. magna fed ration I at 23 and 20°C fulfilled the OECD criteria for a valid test. The food and temperature regimes used in the experiment reported here encompass many of those reported by others as giving satisfactory conditions for conducting chronic toxicity experiments with D. magna. Nevertheless, under only five of the nine test regimes did D. magna fulfil the current EPA requirements for a valid test and under only two the current OECD requirements. Furthermore, unreported studies in this laboratory have indicated that there are also significant differences in the survival and reproductive performance of D. magna when reared under the same test regimes on different occasions. Similar problems of reproducibility have been encountered by other laboratories conducting chronic toxicity tests with D. magna (Blok, 1981: Nebeker, 1982). In the light of these findings it would seem necessary that either the conditions under which a valid test might routinely be carried out require further investigation, or that some relaxation of the criteria for a valid test needs to be brought about, or both. Some relaxation would seem to be particularly desirable for the OECD requirement of first brood production within 9 days. The toxicity and starvation experiments showed that despite the significant effects of the different test regimes on the survival and reproductive performance of D. magna there was no effect on the susceptibility of the young produced to a toxicant, potassium dichromate, or on their ability to withstand starvation.
A C K N O W L E D G E M ENTS The technical assistance of N. Pearson and C. M. Sherwood is acknowledged, as is the help and comments ot" other colleagues at Sittingbourne Research Centre. REFERENCES Adema, D. M. M. (1978). Daphnia magna as a test animal in acute and chronic toxicity tests. H.vdrohiologia, 59. 125 34.
Toxicity tests with Daphnia magna
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Anderson, B. D. (1944). The toxicity thresholds of various substances found in industrial wastes as determined by the use of Daphnia magna. Sewage Works. J., 16, 1156-65. Biesinger, K. E. & Christensen, G. M. (1972). Effects of various metals on survival, growth, reproduction and metabolism of Daphnia magna. J. Fish. Res. Bd Can., 29, 1691 -700. Blok, J. (1981). Measuring the reproduction rate of Daphnia magna.
Proceedings o[ "'Colloque International d'Ecotoxicologie'" INSERM, Villeneuve, France, November 1981. Buikema, A. L., Jr.. Geiger, J. G. & Lee, D. R. (1980). Daphnia toxicity tests. In Aquatic invertebrate hioassays, ed. by A. L. Buikema Jr. and J. Cairns, Jr, A S T M Spec. Tech. Pubis, 715, 48 69. EPA (1982). EPA Environmental effects testing guidelines, EG-2 and E5-1. Chemical Regulation Reporter Supplement N-52. Bureau of National Affairs Inc. Washington DC. Finney, D. J. (1971). Prohit anal.vsis, 3rd edn. Cambridge, Cambridge University Press. Maki, A. (1979). Correlations between Daphnia magna and fathead minnow (Pimephales promelas) chronic toxicity values for several classes ot" test substances. J. Fish. Res. Bd Can., 36, 411 -21. Nebeker, A. V. (1982). Evaluation of a Daphnia magna renewal life-cycle test method with silver and endosulfan. Water Res., 16, 739 44. OECD (1981). Section 2, Effects on biotic systems 202. In OECD guidelines/or testing ~[chemicals. Paris, OECD. Winner, R. W. & Farrell, M. P. (1976). Acute and chronic toxicity of copper to four species of Daphnia. J. Fish. Res. Bd Can., 33, 1685 91.