Effects of cadmium on consumption, assimilation and biochemical parameters of Daphnia magna: possible implications for reproduction

0306~4492/88$3.00 + 0.00 0 1988 Pergamon Press plc

Camp. Biochem. Physiol. Vol. 9OC, No. 2, pp. 341-346, 1988

Printed in Great Britain

EFFECTS OF CADMIUM ON CONSUMPTION, ASSIMILATION AND BIOCHEMICAL PARAMETERS DAPHNIA MAGNA: POSSIBLE IMPLICATIONS FOR REPRODUCTION C. W. M. BODAR, I.

VAN

OF

DER SLUIS, P. A. VOOCT*

and D. I. ZANDEE Research Group for Aquatic Toxicology, and *Research Group for Reproduction Physiology of Invertebrates, State University of Utrecht, P.O. Box 80058, 3508 TB Utrecht, The Netherlands (Received 26 June 1987)

Abstract-l.

The effects of cadmium on consumption, assimilation rates and biochemical oarameters of

Daphnia magna were determined.

2. The consumption and assimilation rates of 14 days 1.0 DDbCd treated animals tended to decrease slightly, the decline of these rates at 5.0 ppb Cd (14 days), h&ever, was highly significant (P < 0.001). 3. The assimilation efficiencies of daphnids exposed to cadmium did not significantly differ from control. 4. No notable changes in the biochemical composition of daphnids could be noticed after 7, 14 and 21 days of cadmium exposure. 5. It seems as if not one metabolic process in particular was depressed due to cadmium, but metabolic activities seemed to be inhibited on the whole. 6. Results are discussed in relation with data of a previous study on the reproduction of D. magna under cadmium stress.

INTRODUCTION For many years daphnid reproduction tests have been used in aquatic toxicology for the establishment

of water quality and aquatic safety assessment. The toxic effects of heavy metals on the reproduction of daphnids have been reported in several studies (Van Leeuwen et al., 1985; Biesinger and Christensen, 1972). The factors causing deleterious effects of these metals on the reproduction of aquatic organisms, however, are still poorly understood. Toxic agents might interfere with the normal reproduction in three different ways, viz. directly, indirectly or a combination of both. Effects of toxicants on processes like oogenesis, embryogenesis and regulation (hormones) express themselves directly on reproduction, while malfunctioning of e.g. feeding, digestion and resorption caused by toxic agents, finally results in a lack of essential reproduction components, thus affecting reproduction indirectly. Sangalang and Freeman (1974) showed that very low concentrations of cadmium (1 .Oppb) affect the levels of 11-ketotesterone and testosterone in the rainbow trout. Recently Voogt et al. (1987) reported the effects of cadmium and zinc on steroid metabolism and steroid levels in the starfish. There is no information about direct toxic effects of heavy metals on the reproduction of daphnids. Little is known about the effects of toxicants on feeding rates of daphnids. Gliwicz and Sieniawska (1986) found that the pesticide lindane at very low concentrations reduced the filtering rates of Duphnia pulex. Flickinger et al. (1982) reported that filtering

rate might be used as an index of chronic copper stress in D. magna. A first attempt to establish a relationship between toxicant related changes in biochemical parameters and survival and reproduction was made by McKee and Knowles (1986a; 1986b). They studied the effects of fenvalerate and chlordecone, respectively, on reproduction and biochemical parameters of D. magna.

In this study indirect toxic effects of the heavy metal cadmium on the reproduction of D. magna will be reported. For this, consumption and assimilation rates were measured according to the modified 14C-technique (Gulati et al., 1982) at different sublethal concentrations of cadmium. In addition, the effects of cadmium exposure on the levels of glycogen, protein and various lipid-classes were studied. Results are discussed in relation with data of a previous study on the reproduction of D. magna under cadmium stress (Bodar et a1.,1988). MATERIALS AND METHODS Daphnia magna The laboratory strain of Daphnia magna used in our tests was a gift from Dr H. Canton (National Institute of Public Health and Environmental Hygiene, Bilthoven). The medium for rearing the daphnids was prepared according to Dutch Standard NPR 6503 (1980): pH 8.4 + 0.2; and hardness, (as CaCO,) about 150 mg/l. The animals were fed daily with Chlorella pyrenoidosa. Chlorella was harvested from axenic cultures, washed in Daphnia medium and resuspended to a concentration of 109cells/ml. D. magna cultures were kept in an environmental chamber at 20 f 1°C with a 1ight:dark regime of 14 hr: 10 hr.

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C. W. M. BODARet al.

Cadmium Cadmium was added to the culture medium as CdCl,. Hz0 (Merck, Darmstadt). Cadmium concentrations tested: control, 1.O and 5.0 ppb bg/l). Experiments I. Consumption and assimilation experiment. For the experiment about 150 young daphnids ( < 24 hr old) were transferred to plastic aquaria containing 8 1 test solution each for control, and for 1.O and 5.0 ppb Cd. Each experiment was carried out in duplicate. Thrice a week the juveniles were collected and half of the medium was refreshed. The medium was aerated gently. The amount of Chlorella added to the animals was restricted in order to prevent loss of cadmium because of uptake by or adherance to Chlorella. The Chlorella densities used were: 5.0 x lo6 (cells/day.animal) (day 1); 12.5 x lo6 (day 2); 2.0 x 10’ (days 3 and 4) and 4.0 x IO’ (days 514), respectively. Prior to the consumption and assimilation experiment 10 groups of 2 animals each were collected at each concentration and the animals were dried at 110°C in an oven. Dry weights were measured using a microbalance (Mettler Me30). Consumption and assimilation rates were measured using the V-technique (Gulati et al., 1982). The tracer food was prepared by adding 100 PCi NaH14C0, (Amersham, 4&60 mCi/mmol) to 1 1 Chlorella suspension. The suspension was incubated for 48 hr at 20°C and a light intensity of about 30 W/m*. Again 100 /ICYNaH’%O, was added and after 24 hr of inoculation the cells were centrifuged (4000 rpm) and resuspended in daphnid culture medium to a volume of 100 ml. Prior to the use of the tracer food, six times 100 ~1 of the “C-Chlorella suspension was filtered on a 0.045 pm membrane filter (Schleicher and Schuell, BA 85) and washed with 0.05 n HCl. The filters were dried and transferred to scintilation vials and after addition of the toluene-based scintillation cocktail (Packard Toluene Scintillator) the radioactivity was counted. On day 15 the animals were transferred to 1 1Chlorella-free daphnid medium (control, 1.0 and 5.0 ppb Cd). After acclimation for 30 min, 7.5 ml of the tracer food was added to the beakers, mixing gently while adding. Consumption Ten minutes after adding of the tracer food 30 animals were sieved out of each vessel, rinsed and killed with hot (90°C) water and sorted into scintillation vials (5 animals per vessel). For further procedure see below. Assimilation Sixty minutes after the tracer addition the rest of the animals were transferred to non-active medium, and allowed about 45 minutes to clear their guts of the ingested labelled food. Subsequently, the animals were filtered on a sieve, rinsed and killed with hot water and transferred into scintillation vials (5 animals per vial). Both groups of animals (consumption and assimilation) were solubilized in 0.25 ml of Soluene 350 (Packard Instruments) added to each vial, and kept closed in an oven (55°C) for about 12 h. Thereafter, 10ml of scintillation cocktail was added and radioactivity was counted. The consumption rates (C) and assimilation rates (A) are calculated as follows: C,A=R,xR;‘xn-‘x24xt-’ where C and A are clearance rates (ml/animal. day), expressed as consumption and assimilation respectively; R, and R are the radioactivities of the animals (DPM) and the tracer food (DPM/ml), respectively; n is the number of animals per vial and t is the-feeding time in tracer food (in hours). Specific filtering clearance rates (SFR) as volume equivalents from which the food was consumed or assimilated, expressed as number of ml of medium filtered per day per

mg dry weight, can be calculated by substituting dry weight values per animal for n in the equation above. The assimilation efficiency (%) is computed from (A/C) x 100. II. Biochemical experiments. Three experiments were carried out, in which animals were exposed to cadmium for 7, 14 and 21 days respectively. On day one 150 young daphnids ( < 24 hr old) were placed in plastic aquaria each containing 8 1 test medium (control, 1.0 and 5.0 ppb Cd). Both the Cd concentrations and control consisted of four replicates. Three times a week the juveniles were collected and 50% of the medium was refreshed. The medium was aerated gently. Food concentration in the experiments was the same as for the consumption and assimilation experiments (day H--21:4.0 x 10’ cells/day. animal). At the end of the experiment the animals were collected, counted and lyophilized. Biochemical analysis The four replicates per concentration were pooled pairwise so as to get two groups of daphnids for each concentration. This was necessary for obtaining the minimum amount of dry weight for carrying out all analyses. The samples were weighed and pulverised. Glycogen. The glycogen content was determined according to Keppler and Decker (1970) using 10mg of the pulverized daphnids. The remainder of the sample was homogenized in 4ml distilled water employing an Ultra Turrax homogenizer and used for the other estimations. Protein. For analyzing the protein content (Coomassie Brilliant Blue method, Bradford (1976) 200 ~1 of the homogenate was used. Bovine serum albumin (Fluka AG, Buchs SG) was used as a standard. Lipids. Total lipid fractions were extracted from the rest of the homogenate with chloroform-methanol according to Bligh and Dyer (1959). Lipid fractions were separated into a polar lipid (PL) fraction and a neutral lipid (NL) fraction by column chromatography (Silicic acid (SillicAR CC,, 100 mesh, Malinckrodt) in chloroform). NL-fractions were eluted with chloroform and subsequently PL-fractions with chloroform-methanol 1: 1 (v/v). The fractions were evaporated under nitrogen and weighed. NL-fractions were seuarated bv thin laver chromatoaraphv (silicagel 60F 254, Merck, Da&tadt)-using the F&e&an and West (1966) solvent system. Levels of the various NL-classes were determined by photodensitometry using a TLC densitometer (Vitatron Densitometer TLD-100, equipped with a Vitatron Integrator). Statistics Differences in mean body weights of daphnids and their consumption and assimilation rates, and assimilation efficiencies between the Cd treated animals and the control animals, were tested by performing t-test (a = 0.05).

RESULTS Consumption eficiency.

and assimilation

rates;

assimilation

Body weights, consumption and assimilation rates (SFR) and AE-values of daphnids exposed to 0, 1.0 and 5.0ppb Cd for 2 weeks are presented in Table 1. Body weights of animals reared at both 1.0 and 5.0ppb Cd were significantly lower compared with control animals. The consumption and assimilation rates of the 1.0 ppb Cd treated daphnids tended to decrease slightly. The decline of these rates at 5.0 ppb Cd was highly significant. The assimilation efficiencies of the Cd-treated animals did not significantly differ from controls, there is even a

Effects of cadmium

343

on Duphniu magna

Table 1. Body weights (mg), consumption and assimilation rates (SFR) and assimilation efficiencies (%) of Daphnia magna exposed to 0, 1.O and 5.0 ppb Cd for 14 days. SD are given in parentheses. *P ~0.05, when compared with control Body weight Consumption Assimilation Ass. efficiency

control

1.O.DDb . Cd

5.0 Dub . . Cd

0.43 (0.09) 23.0 (3.7) 15.5 (2.1) 67.9 (16.2)

0.19 (0.06). 18.9 (6.3) 13.7 (3.7) 75.1 (29.2)

0.21 (0.09). 9.2 (4.4). 5.3 (2.4). 81.0 (58.9)

tendency for an increase due to cadmium. Apparently the smaller amount of food consumed by the Cd treated animals is still being assimilated quite efficiently. The coefficient of variation of the assimilation efficiencies of the Cd stressed animals, however, is higher than those of the controls. Biochemical components

Mean body weights of animals and the mean values of the major biochemical components of daphnids exposed to 0, 1.O and 5.0 ppb Cd for 7, 14 and 21 days respectively, are shown in Table 2. At each exposure time a decrease of the animal’s body weight due to cadmium was found, the difference between the Cd-treated animals and control animals was much more evident after 14, and 21 days, however. The biochemical components in Table 2 are expressed in relative proportions. The protein content of daphnids treated with cadmium did not obviously differ from controls. Protein levels of 7-day old daphnids reared at 5.0 ppb Cd and 21-day old animals exposed to 1.0 and 5.0 ppb Cd decreased slightly. Glycogen levels of Cd-exposed animals decreased slightly at all exposure times. Striking, however, was the halved glycogen content of the 21-day old animals. Total lipid levels of 7-day animals exposed to 1.0 ppb Cd were slightly increased compared with controls, in general total lipid levels were rather constant. No obvious changes in the ratio neutral lipids/polar lipids could be noticed either. The main neutral-lipid classes in Cd-stressed Daphnia magna are shown in Table 3. Approximately 50% of the neutral lipids consisted of triacylglycerols. The triacylglycerols constitute a pool of stored energy, which apparently is not influenced by Cd stress. Free sterols, especially cholesterol, form an important part of the structural material of cell membranes in all tissues. On the average the contribution of sterols to the NL-fraction is ll-12%, although in 21-day old daphnids their part seemed to be reduced slightly. The components No. 7 and No. 8 will probably belong to the class of ether lipids. Because of their low levels, the increase in 7 and 1Cday old animals should be considered cautiously when it is brought in relation to cadmium. Diacylglycerols and monocylglycerols in most instances function as intermediates of the triacylglycerol metabolism. These classes of neutral lipids are present in daphnids in only small amounts. Both mono- and diacylglycerols seemed to be reduced slightly due to cadmium at each of the exposure times. The group of free fatty acids tended to reach a higher level in 21-day old animals. A slight increase of free fatty acid levels might be noticed with increasing cadmium concentrations in 14 and 21-day animals.

DISCUSSION

The consumption and assimilation experiments indicated a drastic effect of cadmium on the consumption rates of D. magna. At 5.0 ppbCd the consumption rates of 1Cday old daphnids were only 40% of controls. Assimilation rates of cadmiumexposed animals were depressed as well, but this might be due only to the reduced consumption. The constant assimilation efficiencies confirm this assumption. So cadmium-stressed animals are not capable of maintaining the filtering or food collecting mechanism at control levels. Once ingested however, digestion and absorption of the food by intestine epithelium cells seemed to be less affected. Gulati et al. (1988) studied the effects of short-term cadmium exposure (20 and 48 hr) on the consumption and assimilation rates of daphnid species, other than D. magna, in lake water. Much higher concentrations of cadmium (10,25,50 and 100 ppb) were tested, and in their study in particular the assimilation rates were found to be inhibited. Kersting and Van der Honing (1981) pointed out that filtering rates of daphnids might be used as very sensitive toxicity parameters. A concentration dichlobenil of a tenth of the 48-hr LC~~, depressed the feeding rate of Daphnia by half. Gliwicz and Sieniawska (1986) found that a concentration of the pesticide lindane of 0.05 mg/l, much lower than the 48-hr LC~, caused a 25% depression of the frequency of movements of filtering limbs and mandibles of D. pulex.

In the subsequent experiments the effects of the reduced feeding rates on the biochemical composition of Daphnia were investigated. Daphnids were exposed to 1.0 and 5.0ppb Cd for 7, 14 and 21 days respectively. As in the previous experiment, body weights of the animals declined due to cadmium. Growth inhibition due to cadmium was found to be low after 7 days. Daphnid neonates initially do not feed (Cowgill et al., 1984) and they mainly live on their yolk content. So, in the early life of daphnids the contact with the toxicant might be limited. On the other hand the uptake of water will be relatively high during the first growth period of the animal and might have raised the cadmium level in the animal. If so this cadmium uptake did not reveal any harmful effects after 7 days of exposure. After 14 and 21 days of exposure to cadmium body weights declined approximately 40% compared with controls. No obvious changes in the biochemical composition of daphnids could be noticed after cadmium exposure (Table 2). It seems as if not one metabolic process in particular was depressed due to the toxic agent, but metabolic activities seemed to be inhibited on the whole.

Body weight Protein Glycogen Total lipid Neutral lipid Polar lipid

Monoacylglycerols Diacylglycerols Free sterols Free fatty acids Triacylglycerols No.7 No.8 Steryl esters Hvdrocarbons

Table 3. Neutral

0.32 (0.04) 183.2 (6.0) 101.2(21.2) 153.6 (3.0) 102.4 (3.4) 36.0 (0.2)

Control

0.30 (0.01) 184.0(17.6) 101.4(7.1) 172.6 (5.8) 106.7(13.9) N.D.

7 days 1.O ppb Cd

components

(0.01) (2.5) (6.4) (5.8) (5.1) (3.9) 0.51 (0.03) 192.5 (15.6) 103.6 (1.2) 157.2(1.8) 96.4(1.3) 39.2(1.6)

Control 0.47 (0.01) 180.2 (7.8) 90.2 (0.4) 158.2(3.3) 96.8 (0.8) 33.8 (0.5)

14 days 1.O ppb Cd 0.32 (0.03) 182.8 (38.0) 81.8(12.9) 163.2(1.9) 101.0(0.9) 30.6 (3.2)

5.0 ppb Cd 0.68 205.6 52.2 147.8 88.9 36.0

(0.09) (8.6) (7.1) (1.7) (3.8) (0.0)

Control 0.54 (0.05) 163.4 (19.7) 51.2(8.8) 150.5 (6.4) 87.3 (0.8) 37.8 (14.3)

21 days 1.0 ppb Cd

3.8 (0.5) 5.6 (0.9) 12.2(1.1) 6.0 (0.5) 50.6 (3.0) 1.6(0.1) 3.2 (0.1) 2.7 (0.1) 14.4(1.5)

Control

5.0 (0.5) 6.6 (0.1) 11.5(0.1) 5.8 (0.3) 49.0 (1.5) 1.O (0.6) 2.2 (0.5) 3.0 (0.3) 16.0 (2.1)

Control 7.6 (0.6) 8.4 (0.4) 12.7 (0.1) 5.2 (0.1) 46.2 (0.2) 1.2 (0.4) 2.0 (0.2) 2.7 (0.1) 14.0 (1.6)

5.0 ppb Cd 4.2 (0.2) 5.2(1.1) 11.9 (0.8) 5.6 (0.4) 50.6 (4.5) 1.7(0.1) 4.3 (0.4) 3.0 (0.1) 13.6(1.X) 6.6 (0.1) 8.0 (0.3) 11.1 (0.6) 6.2 (0.4) 49.8(1.6) 1.4(0.1) 2.8 (0.1) 2.7 (0.3) 11.4(2.1)

14 davs 1.O ppbCd

4.7 (0.4) 5.6 (0.3) 9.0 (0.6) 6.6 (1.3) 50.7 (1.3) 1.8 (0.2) 4.1 (0.1) 3.5 (0.7) 14.0{1.7j

5.0 ppb Cd

11.8jl.Oj

5.5 (0.1) 7.8 (0.1) 8.4 (0. I) 8.4 (0.6) 50.3 (0.0) 1.4 (0.6) 2.5 (0.1) 2.4 (0.4)

Control

5.4 (0.8) 7.0 (0.6) 7.7 (0.4) 9.6(1.9) 50.6 (0.4) 0.8 (0.1) 2.5 (0.3) 2.2 (0.2) 13.1(1.8j

21 days 1.O ppb-Cd

.I

5.2 (0.8) 6.8 (0.6) 8.0 (0.1) 10.7 (0.9) 48.3 (0 41 1.3 (0.1) 2.4 (0.1) 2.0 (0 . .-IA’ 14.6 (2.3)

5.0 ppb Cd

N.D. = not

0.47 (0.01) 174.0 (23.8) 46.4 (3.5) 146.1 (1.8) 78.8 (4.9) 31.2(4.6)

5.0 ppb Cd

exposed to 0, 1.0 and 5.0 ppb Cd for 7, 14 and 21 days. SD are given in parentheses. detected

magna

(%) of Dapnia magna exposed to 0, 1.0 and 5.0 ppb Cd for 7, 14 and 21 days. SD are given in parentheses

0.26 159.0 86.7 158.5 105.8 39.8

5.0 ppb Cd

(mg per g dry wt) of Daphia

1 davs 1.0 ppb Cd

lipid composition

Table 2. Body weights (mg) and biochemical

Effects of cadmium on Daphnia magna

The amount of lipids has been used as a measure of feeding success for various cladoceran species (Tessier and Goulden, 1982). They store large quantities of lipid, predominantly triacylglycerols although glycogen may be stored as well. Smith (1915) on the other hand stated that parthenogenetic females of daphnids store up reserve material almost exclusively in the form of glycogen. In the present study it was found that 50% of the neutral lipid fractions consisted of triacylglycerols (Table 3) in contrast with marine zooplankton in which wax esters are the common storage lipids (Lee, 1975). Under normal conditions lipid is stored in adult cladocerans whenever an animal achieves a positive net energy balance (metabolic costs < food assimilated). In addition, Tessier and Goulden (1982) suggested that lipid reserves in cladocerans predict the sensitivity of that individual to environmental stress, including toxicity and starvation. Blazka (1966) found that non-protein substrates are preferred for obtaining energy, and proteins are rather emergency fuel used during lack of other nutrients in daphnids. In this study glycogen was the only component that decreased constantly with increasing cadmium concentrations at all exposure times, although very slightly. Lipid and protein levels remained at more constant levels. Depletion of glycogen has previously been suggested as a bio-indicator of chronic stress in aquatic organisms (Carr and Neff, 1981). Studies of McKee and Knowles (1986) have shown that the glycogen content was significantly reduced relative to lipid content at several concentration of fenvalerate. Effects of cadmium on the lipid synthesis in aquatic organisms have been reported as well. High cadmium concentrations in molluscs led to considerable changes in the phospholipid composition in their organs (Evthushenko et al., 1986). Cadmium is also known to inhibit lipid synthesis in the liver and kidney of rats fed cadmium-enriched food (Rana et al., 1980). Due to cadmium the food uptake rate of D. magna was reduced, resulting in lower body weights. The stressed animal starts to deplete glycogen reserves first of all, probably followed by the lipid and finally the protein supplies. These results are in accordance with studies of Lemcke and Lampert (1975) in which the effects of starvation on biochemical components in Duphniu pulex were investigated. An important question is where does this inhibition process start? The toxicity of cadmium might exert its effect very first by a direct inhibition of the muscle activities of the feeding apparatus. Indeed, neurotoxicological effects of heavy metals have been reported in several studies. For example, cadmium was shown to affect the release of neurotransmitters in the frog at concentrations of lo-’ M or less (Cooper et al., 1984). Because of the reduced amounts of nutrients, the energy available for the movements of the feeding appendages will be further reduced. Moreover cadmium is known to inhibit the activity of ATP-ase enzyme (Evtushenko et al., 1986). Inevitably, a lack of the ATP-energy will account for decreased muscle cell activities. In a previous study the effects of sublethal cadmium concentrations on the reproduction of D. magna were investigated (Bodar et al., 1988). After 25 days of exposure to 1.0 and 5.0 ppb Cd total quan-

345

tities of neonate-biomass per female increased, at the first Cd concentration but decreased at the higher one. Due to cadmium the reproductive biomass was divided up differently from controls: the number of progeny per female increased with increasing cadmium concentrations, however, the size of those neonates was reduced. Both in the consumption, assimilation, and biochemical experiments of the present study adult body weights were reduced under Cd-stress. So apparently at the cost of their own health and viability females of D. magna are capable of increasing or at least limiting the loss of the reproductive biomass. At concentrations above 5.0 ppb Cd daphnids will fail to cope with the lack of nutrients and reproduction will be affected negatively as well. Beside these indirect effects of cadmium on daphnid reproduction, direct effects of this heavy metal might have accelerated the damaging process. In further studies on the toxicity of cadmium on daphnid reproduction, the effects on processes like embryogenesis and regulation will be investigated.

Acknowledgements-The authors wish to thank Dr R. D. Gulati, Dr J. H. Kluytmans and Dr C. J. van Leeuwen for their critical comments; Mr. G. Postema for technical assitance and Miss M. van Hattum for typing the manuscript. This investigation was supported financially by the Institute for Inland Water Management and Waste Water Treatment. REFERENCES

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