Bull. Environm. ISontam. foxlcol. 20, I-B (1918)

Effects of Chlordane and Heptachlor on the Marine Dinoflagellate, Exuviella baltica, Lohmann Barbarajean Magnani, C. Donald Powers, Charles F. Wurster,* and Harold B. O'Connors, Jr. Marine Sciences Research Center, State University of New York at Stony Brook,

Stony Brook, N.Y. 11794

Chlordane and heptachlor are widely used chlorinated cyclodiene insecticides. While the effects of both compounds on certain higher organisms have been studied (EISLER 1970; HARBISON 1973; MEHRLE et al. 1974; MIRANDA and WEBB 1972), little is known of their effects on phytoplankton, the base of marine and freshwater food webs. The present study examined growth, productivity, chlorophyll a content and cell size distribution of a marine dinoflagellate treated with chlordane or heptachlor. MATERIALS AND METHODS The marine dinoflagellate Exuviella b a l t i c a Lohmann was cultured according to POWERS et al. (1975). To prepare experimental cultures, 150 ml of exponentially growing cells were introduced aseptically to a sterile aspirator bottle containing 2850 ml of m~dium, yielding a final cell concentration of 6.4 x i0 J cells/ml. After mixing by magnetic st 8 cell suspensions were transferred to each of six 500ml Erlenmeyer flasks fitted with Teflon-lined screw caps (two replicate flasks for each treatment). Cultures were treated with 50 ~g/l (ppb) of chlordane or heptachlor dissolved in 50 ~i of methanol (POWERS et al. 1975); control cultures recelved an equal volume of methanol. Experimental cultures were grown as previously described (POWERS et al. 1975). Technical h e p t a c h l o r (74% 1 , 4 , 5 , 6 , 7 , 8 , 8 a - h e p t a c h l o r o - 3 a , 4 , 7 a - t e t r a h y d r o - 4 , 7 - m e t h a n o i n d e n e and 26% related insecticidal compounds) and technical chlordane (60% o c t a c h l o r o - 4 , 7 - m e t h a n o t e t r a h y d r o i n d a n e and 40% related insecticidal compounds) were p r o v i d e d by Velsicol Chemical Corporation, Chicago, lllinois. Cultures were sampled shortly after starting the experiment and every 24 to 72 hours thereafter for seven days. Cell numbers were determined optically

y y171 Center,

requests should be sent to this author. 199 of the Marine Sciences Research State Univ. of N.Y. at Stony Brook

0007-4861/78/0020-0001 $01.60 O 1978 Springer-Verlag New York, Inc.

using a Fuchs-Rosenthal counting chamber. Chlorophyll a was determined f l u o r o m e t r i c a l l y (LOFTUS and CARPENTER 1971) using a Turner F l u o r o m e t e r (Turner Designs, Palo Alto, Calif.). For d e t e r m i n a t i o n of photosynthetic activity, 6 ml of sample were pipetted into each of three vials (two light and one dark) and 0.i ml of NaHI4CO 3 (New England Nuclear stock diluted in sterile f/2 medium) was added. Vials were capped and incubated for two hours with gentle shaking every 30 minutes. Photosynthesis was terminated with the addition of 1.0 ml of 0.5 N HCI to each vial. Samples were filtered (0o45-~m Millipore), and filters were dried and placed in s c i n t i l l a t i o n vials containing 5 ml of toluene-based fluor (toluene, 0.3% PPO and 0.01% POPOP). Radioactivity was determined using a Mark II Liquid Scintillation System (Searle Radiographics, Des Plains, Iii.). Cell size distribution was determined w i t h an E l e c t r o Z o n e / C e l l o s c o p e | (Particle Data, Inc., Elmhurst, Iii.) particle counting and sizing system equipped with a calibrated 120-~m orifice tube. Fiveto 50-ml volumes of E. baltica suspension were diluted with filtered seawater to produce particle concentrations below those at which coincident particle passage through the orifice would occur (SHELDON and PARSONS 1968). This system was adjusted to enumerate particles per ml in i00 channels, ranging in size from 2.4 to 23.4 ~m equivalent spherical diameters. RESULTS AND DISCUSSION Cell densities in treated cultures were lower than controls throughout the experiment (Fig. I), resulting in reductions in chlorophyll a concentrations per liter (Table i). Levels of chlorophyll a per cell were not significantly different, however, in treated and untreated cultures, suggesting that inhibition of 14C uptake per treated cell (Table I) was due to interference with chlorophyll function rather than its synthesis. Consequently, considerably less carbon was fixed per unit of chlorophyll a in treated cells than in controls. These results generally agree with those of MACFARLANE et al. (1972), who described reduced photosynthesis per cell and per unit of chlorophyll a in DDT-treated cultures of N i t z s c h i a delicatissima. They also observed a reduction in chlorophyll a per cell, however.

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or chlordane. E a c h point r e p r e s e n t s the m e a n of cell counts from two r e p l i c a t e c u l t u r e s , the a c t u a l values i n d i c a t e d by bars above and b e l o w the point. Growth rates (~), e x p r e s s e d as d i v i s i o n s per day b e t w e e n days I and 7, were c a l c u l a t e d a c c o r d i n g to the e x p r e s s i o n = (35)(in n t - in n t 0 ) / ( t - t0) w h e r e t - t 0 r e p r e sents e l a p s e d time in hours and-n t and nt0 are the p o p u l a t i o n d e n s i t i e s at times t and t0, r e s p e c t i v e l y (EPPLEY and S T R I C K L A N D 1968).

Chlordane

Heptachlor

Control

155.32

237.51

4

7

7

17.34 14.33 19.60

2 4

177.19

7

12.67

101.79

4

0

13.5o 29.41

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2

12.89 41.47

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Day

Samples and Time of sampling

45.24

12.06

2

1.64 1.52 1.94

1.87 1.43 1.96

14.33 27.14

0.97

1.04

16.36

1.82

2.12

1.87

1.70 1.68

1.07

1.89

2.11 1.84

2.22

1.97

1.89

2

1.97

2.07

2.02

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1.98

11.99

79.92 135.72

26.92

10.86

177.19 214.89

~g/l

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0.75 1.46 1.02

0.36

3.99 7.08

1.74

3.12 7.60 ll.30

2

0.67 1.40

5.73 9.10

3.02

3.02 8.83 11.40

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Cpm/cell (x io-~)

0.40 0.89 0.67

0.75 0.25

3.90

4.29

0.34

2.45

3.12

5.10

5.80

1.03

1.66 3.86

1.50 4.26

1.43

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Biomass and photosynthesis in E. baltica exposed to 50 Dg/l of heptachlor or chlordane

TABLE i

Recent e x p e r i m e n t s u t ~ 9 natural phytoolankton isolates e x p o s e d to p o i y c h l o r i n a t e d b i p h e n y l s (PCB), a n o t h e r group of c h l o r i n a t e d h y d r o c a r b o n s , r e v e a l e d a response somewhat d i f f e r e n t from that r e p o r t e d in this study. S u p p r e s s e d 14C uptake per t r e a t e d cell was a t t r i b u t e d to less c h l o r o p h y l l a per cell w i t h no loss of c a r b o n - f i x i n g e f f i c i e n c y per unit of e x i s t i n g c h l o r o p h y l l a (POWERS et al. 1978). I n h i b i t i o n per cell was ~ot d e t e c t e d by F I S H E R (1975) in three p h y t o p l a n k t o n species e x p o s e d to DDT or PCB. No a t t e m p t was made to define the m e c h a n i s m of i n h i b i t i o n of p h o t o s y n t h e s i s in this study. Interference w i t h e l e c t r o n t r a n s p o r t may have been involved 87 as in algae t r e a t e d w i t h other c h l o r i n a t e d h y d r o c a r bons (BOWES and GEE 1971; BOUGES 1972), since both h e p t a c h l o r and c h l o r d a n e i n h i b i t e d m i t o c h o n d r i a ! electron t r a n s p o r t in m a m m a l i a n ce!Is (PARDINI et al. 1971). The effect of c h l o r d a n e on cell si ze d i s t r i b u t i o n may be seen in Figs. 2 and 3. Since h e p t a c h l o r t r e a t e d cells d i f f e r e d only s ! i g h t l y from controls, the data are not included. As c o r r o b o r a t e d by microscope m e a s u r e m e n t s , the peak in p a r t i c l e n u m b e r (Fig. 2) and p a r t i c l e volume (Fig. 3) b e t w e e n 8 and 16 Mm c o n s i s t e d of E. b a l t i c a cells. Both p a r a m e t e r s were s u p p r e s s e d by chlordane in that size range. Concomitantly, both were h i g h e r than controls b e t w e e n 4 and 8 Mm, p a r t i c u l a r l y on day 4. Microscope examination r e v e a l e d c o n s i d e r a b l e c e l l u l a r debris in t r e a t e d cultures in this size range, s u g g e s t i n g c h l o r d a n e i n d u c e d d i s r u p t i o n of cells. A s i m i l a r effect on this species by dieldrin, another c h l o r i n a t e d cyclodiene, was r e c e n t l y r e p o r t e d (POWERS et al. 1977). The i n h i b i t i o n of E. b a l t i c a growth and p h o t o s y n thesis o b s e r v e d in this study, c o u p l e d w i t h the shift in cell size d i s t r i b u t i o n , might have i m p o r t a n t ecological implications. In nature, such d i s r u D t i o n s could i n f l u e n c e the a v a i l a b i l i t y of food for s e l e c t i v e l y - f e e d i n g h e r b i v o r e s (PARSONS and L e B R A S S E U R 1970). SUMMARY C h l o r d a n e and h e p t a c h l o r at 50 Mg/l r e d u c e d cell density, c h l o r o p h y l l a per unit volume of culture, 14C uptake per cell and carbon f i x a t i o n per unit of chlorophyll a in the marine d i n o f l a g e l l a t e E x u v i e l l a b a l t i c a Lohman~. The c o n c e n t r a t i o n of c h l o r o p h y l l a

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per cell was not reduced, however, by treatment with either compound. Chlordane was more toxic than heptachlor at this concentration, and caused the disintegration of many cells, thus affecting particle size distribution in the cultures. In nature, such an inhibition and shift in size class distribution could affect the availability of food for p a r t i c l e - f e e d i n g herbivores. ACKNOWLEDGEMENTS We thank Ralph G. Rowland for technical assistance and pertinent suggestions. This work was supported by grants from the National Science F o u n d a t i o n (BMS 7419665), The Rockefeller Foundation (GA NES 7505), the MESA New York Bight Project (ERL-NOAA) and New York Sea Grant Institute. REFERENCES BOWES, G . W . : Plant Physiol. 49, 172 (1972). BOWES, G. W. and R. W. GEE: Bioenergetics ~, 47 (1971). EISLER, R.: Tech. Papers Bur. Sport Fish. Wildl. no. 46 (1970). EPPLEY, R. W. and J. D. H. STRICKLAND: Kinetics of marine phytoplankton growth, In Advances in Microbiology of the Sea, vol. i. London and New York: Academic Press 1968. FISHER, N. S.: Science 189, 463 (1975). HARBISON, R. D.: Toxicol. Appl. Pharmacol. 25, 472 (1973). LOFTUS, M. E. and J. H. CARPENTER: J. Mar. Res. 29, 319 (1971). MACFARLANE, R. B., W. A. GLOOSCHENK0 and R. C. HARRISS: Hydrobiologia 39, 373 (1972). MEHRLE, P. M., W. W. JOHNSON and F. L. MAYER, Jr.: Bull. Environ. Contam. Toxicol. 12, 513 (1974). MIRANDA, C. L. and R. E. WEBB: Fed. Proc. Amer. Soc. E x p . Biol. 31, 726 (1972). PARDINI, R. S., 7.. C. HEIDKER and B. PAYNE: Bull. Environ. Contam. Toxicol. ~, 436 (1971). PARSONS, T. R. and R. J. LeBRASSEUR. The availability of food to different trophic levels in the marine food chain, In Marine Food Chains. I ed. Edinburgh: Oliver and Boyd (1970). POWERS, C. D., R. G. ROWLAND, R. A. MICHAELS, N. S. FISHER and C. F. WURSTER: Environ. Pollut. 9, 253 (1975). POWERS, C. D., R. G. ROWLAND, H. B. O'CONNORS, Jr. and C. F. WURSTER: Appl. Environ. Microbiol. 34, 760 (1977a).

POWERS, C. D., R. G. ROWLA:i0, H. B. O'CONNORS, Jr. and C. F. WURSTER: Appl. Snviron. Microbiol. 3__5, no. I (1978). SHELDON, R. W. and T. R. PARSONS: A P r a c t i c a l Manual on the Use of the C o u l t e r C o u n t e r in Marine Science. Ottawa, Canada: Coulter Counter E l e c t r o n i c Sales Co. 1968.