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Interaction of permethrin with Daphnia magna in the presence and absence of particulate material
I'.hrtronmental P, Ilution {Serte~A) 24 {19811135 144
INTERACTION OF PERMETHRIN WITH DAPHNIA MAGNA IN T H E P R E S E N C E A N D A B S E N C E O F PARTICULATE MATERIAL
GI.ENN W. STRAI'I-ON & CI.tARI.ES T. ('ORKE
Department oJ Em'ironmental Bioh~gv. University o.1 Guelph, Guelph, Ontario, ('anada N I G 214"1
ABSTRA('T
The 48-h LCso olpermethrin towards'juvenile and adult Daphnia magna rangedj}'om 0.2 to 0.6 tlg litre- '. Permethrin also induced the adhesion of particulate material. including algae, bacteria and silica powder, onto various setate appendages of D. magna. This led to immobilisation of the daphnids and sign(licant O"earlier mortalitv in some O'stems containing algae. The adhesion phenomenon resulted from some ~:l.]'eet o[permethrin on D. magna, as opposed to an interaction with the particulate materials used. Algal adhesion to D. magna was also imh,'ed, to vatting degrees. with diazinon, cypermethrin, methoxyehlor and carharvl.
I N TRODU(7"I"ION
There is great concern about the effects of pesticides on non-target aquatic organisms, since the aquatic environment is the final reservoir for many xenobiotics (Livingston, 1977). One pesticide being evaluated for its impact on aquatic ecosystems is the pyrethroid insecticide, permethrin (3-phenoxybenzyl (IRS)-cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-carboxylate). Synthetic pyrethroids are among the most potent pesticides known and are being evaluated for many applications (Elliott & Janes, 1978). Permethrin is already used to control various agricultural pests and has been under investigation as a mosquito larvicide (Mulla et al., 1978a,b). Data for the effects of permethrin on aquatic organisms are few and deal primarily with fish, where the LCso values range from I to 61 pglitre-1 (Zitko et al., 1977, 1979; Jolly et al., 1978; Mulla et al., 1978b; Coats & O'Donnell-Jeffry, 1979). Permethrin is toxic to crayfish, lobster and mayfly larvae at concentrations less than 10pglitre-~ (Jolly et al., 1978; Zitko et al., 1979) and inhibits growth of the 135 Environ. Pollut. Ser. A. 0143-1471/81/0024-0135/$02"50 ((5 Applied Science Publishers Ltd, England, 1981 Printed in Great Britain
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GLENN W. STRATTON, CHARLES T. CORKE
blue-green alga Anabaena above 500 #g litre- ~(Stratton et al., 1980). Growth of the marine diatom Skeletonema costatum is more sensitive to permethrin, with an LC5o of 68 to 72/lg litre- ~ (Walsh & Alexander, 1980), The only sublethal effect reported for permethrin is that it induces downstream drift with some invertebrates, such as Gammarus and Simulium (Muirhead-Thompson, 1978). This paper describes the effect of permethrin on the freshwater crustacean Daphnia magna--one of the more sensitive crustaceans used in pesticide toxicity studies (Sanders, 1970). Standard bioassay techniques showed that permethrin was extremely toxic to D. magna and induced the clumping of algae, bacteria and silica onto the crustacean's appendages.
MATERIALS AND METttODS
Rearing o[" Daphnia magna Daphnia magna was isolated from a small pond and cultured in 10-1itre glass aquaria containing filtered river water. D. magna was fed daily with a mixture of Scenedesmus quadricauda and Chlorella pyrenoidosa (approximately 108 cells per aquarium), that had been cultured in modified BGI 1 medium (Stratton & Corke, 1979) containing 1.5 g of N a N O 3 litre- ~. The algae and D. magna were grown at a temperature of 20°C and a light intensity of 7 klux, on a 12-h light- dark cycle. Acute toxicity study D. magna was exposed to permethrin in 250-ml Erlenmeyer flasks which contained 200 ml of filtered river water. To each flask were added ten juvenile (1-2 mm long) or adult (3-5 mm long) D. magna and 0.1 ml of permethrin (technical grade, Chipman Chemicals, Stoney Creek, Ontario) dissolved in acetone, to give a final permethrin concentration ranging from 0 to 10#glitre '~. in these and all subsequent experiments, flasks not containing permethrin had 0.05 '7o acetone (v/v) added. D. magna was added to each test system after the addition of permethrin and all treatments were carried out in triplicate. The flasks were incubated at 20 _+ 2 °C for 48 h and percent mortality was calculated. Death was defined as the inability to show any motion when prodded with a glass probe. Effect of particulate materials In some experiments, various particulate materials were added to the treatment flasks. These were the alga Chlorella pyrenoidosa (107 cells a day per flask), the bacterium Escherichia coli (109 cells a day per flask), and silica powder (0.1 g per flask; 240 mesh, Fisher Scientific Co.). For these experiments the permethrin concentration was 0.5/aglitre -~, which was chosen because it was within the calculated LCs0 range. D. magna was examined microscopically for treatment effects after 24 hours" and 48 hours' incubation. In one series of experi-
INTERACTION OF PERMETHRIN WITH Daphnia magna
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ments involving C. pyrenoidosa, p e r m e t h r i n was replaced with 1.0,ug l i t r e diazinon (O,O-diethyl O-2-isopropyl-6 methylpyrimidin-4-yl phosphorothioate), 0-5 #g l i t r e - t c y p e r m e t h r i n ((RS)-ct-cyano-3-phenoxybenzyl (IRS)-cis, trans-3-(2,2d i c h l o r o v i n y l ) - 2 , 2 - d i m e t h y l - c y c l o p r o p a n e c a r b o x y l a t e ) , 4/ag l i t r e - t m e t h o x y c h l o r (1,1, l - t r i c h l o r o - 2 , 2 - b i s ( 4 - m e t h o x y p h e n y l ) ethane) and 10/ag l i t r e - t c a r b a r y l ( ln a p h t h y l m e t h y l c a r b a m a t e (I)).
Statistics LCso values were c a l c u l a t e d from d o s a g e - m o r t a l i t y d a t a using probit analysis ( F i n n e y , 1971 ). Significant differences in m o r t a l i t y a m o n g systems with a n d without p a r t i c u l a t e m a t e r i a l were d e t e r m i n e d using D u n c a n ' s multiple range test at ct = 0.05, as outlined by Bliss (1967).
RESULTS
Acute toxicity study Daphnia magna was very sensitive to permethrin. The 48 h LCso for both juvenile a n d a d u l t D. magna ranged from 0.2 to 0"6/~glitre-~ and 1 . 0 l i g l i t r e - ~ usually caused 1 0 0 ~o mortality. Effect o f particulate materials C o m p a r a t i v e studies were carried out on the effect o f a d d i n g the alga Chlorella pyrenoidosa to test flasks d u r i n g p e r m e t h r i n toxicity experiments. W h e n D. magna a d u l t s were exposed to p e r m e t h r i n in the presence o f the alga, there was a significant increase in m o r t a l i t y d u r i n g the first 24 h, c o m p a r e d with the same system without algae (Table 1). After 48 h therc was no difference in m o r t a l i t y between the two systems. The experiment was r e p e a t e d using a bacterial food source, Escherichia TABLE I FTFE('T OF P A R T I C U L A T E MATERIAL ON THE T O X I C I T Y OF P E R M E T H R I N T O
Particulate component added NONE C. pyrenoidosa E. coil silica powder
Observed mortality 2
D. lrtlagntg I
24 h
48 h
Degree of adhesion~
0.0 (0)* 2.0 (20)b 0.7 (7)* 0.3 (3)*
6.3 (63)' 7.0 (70)' 7.0 (70)' 8.0 (80)'
++ + +
All flasks contained 0.5 ~uglitre- ~ permethrin and ten adult D. magna, plus the additional component listed. All components were added at time zero and each treatment was carried out in triplicate. 2 Mortality data are presented as the mean number of dead D. magna per flask. The number in parentheses is the calculated percent mortality. Those values followed by the same letter are not significantly different at ct = 0.05. based upon Duncan's multiple range test. 3( _ ) no adhesion. ( + ) moderate adhesion. ( + + ) extensive adhesion, as determined microscopically on antennae 2.
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GLENN W. STRATTON, CHARLES T. CORKE
coli, and an inert component, silica powder. Only the presence of the alga induced significantly higher mortality within 24 h (Table 1). After 48 h the level of mortality was the same in all systems regardless of the component added. In all cases, the particulate material was added at time zero, following permethrin addition. Adhesion of particulate material to D. magna All three particulate materials adhered in large masses to the swimming antennae of adult, permethrin-treated D. magna. This impeded daphnid mobility, causing them to sink to the bottom of the flask where they showed erratic, unco-ordinated movement and eventually died. The bottoms of the flasks which contained algae as the added component were also littered with large clumps of algae which had fallen from the antennae olD. magna. These masses retained their integrity upon shaking. Photomicrographs of the clumping phenomenon are shown in Figs 1 and 2. The biramous swimming antennae (antennae 2) of an organism exposed only to permethrin are depicted in Fig. I A. There are nine plumose setae on these antennae and each seta contains numerous fine setules (Fig. 1B). The antennae of D. magna exposed to both permethrin and C. pyrenoidosa had numerous algal cells adhering
Fig. I. Accumulation of particulate material onto the swimming appendages of D. magna treated with 0-5/zglitre-t permethrin. (A) Antennae 2 of an organism from a system containing D. magna and permethrin only. The arrows indicate the nine plumose setae. (B) Fine structure of one of the plumose setae from antennae 2 showing the distribution of setules. (C) Algal cells adhered to the setae and setules of antennae 2. (D) Silica powder adhered to antennae 2. The bar represents 0-1 mm.
INTERACTION OF PERMETHRIN WITH Daphnia magna
C
139
• I
Fig. 2. Accumulation ofparticulate material onto D. magna treated with 0.5 pg litre - n permethrin. (A) Spine of an organism from a system containing D. magna and permethrin only. (B) Spine with adhered algal cclls. (C) Spine with adhered bacterial cells. (D) Post-abdomen with adhered silica powder: F furca, S-. post-abdomenal spine, SB--post-abdomenal setae. The bar represents 0.1 ram.
to the setae and setules (Fig. I C). A similar pattern of adhesion was noted tbr both E. colt and silica powder (Fig. 1D). Another prominent area of algal cell adhesion was the posterior portion of the spine (Fig. 2B), which is bordered by a number of short, thick setae (Fig. 2A). Bacterial cells (Fig. 2C) and silica also adhered to this structure. The short, inconspicuous antennae 1 also accumulated particulate material, particularly at the tip, where a number of very fine setac arc present. Silica powder also clumped onto the filtering setae of the thoracic appendages and on the post-abdomen (Fig. 2D). The furca, tile post-abdomenal plumose setac and the post-abdomenal spines werc also covered with particulate matter.
Evtent of adhesion The amount of adhesion varied, depending on the particulate material present (Table I). This response was most pronounced when algal cells were the added component. Algal adhesion also occurred at permethrin concentrations of 0-05 and 0-005 l~g litre-~. However, mortality was not significantly affected in these two systems. The extent of algal cell accumulation on antennae 2 increased with increasing levels of permethrin. Sctal adhesion was only observed in those systems containing both permethrin, D.
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GLENN W. STRATTON, CHARLES T. CORKE
magna, and either algae, bacteria, or silica. When any one of the above components was omitted from the test system, no accumulation of particulates was observed. All systems not containing permethrin still contained acetone to ensure that the solvent was not a factor in the adhesion response. When dead D. magna were used, no accumulation of algal cells occurred, even when the test flasks were gently shaken to provide better contact between the various components in the flasks. When algae were added to systems containing permethrin and live D. magna after 24 h, rather than at time zero, adhesion was still observed after 2 hours' incubation. In such a system, much of the permethrin (0.5 pg litre- 1) would probably have been adsorbed to the glass flasks and D. magna during the first 24 h. When D. magna was treated with 0'5/ag litre- 1 permethrin for 24 h and then removed, washed and placed into fresh permethrin-free water containing algae, cell adhesion was noted within 2 h. Response of juvenile D. magna Juvenile D. magna treated with 0-5 pglitre-1 permethrin responded differently from adult organisms. With the former, there was no significant difference in mortality between those systems with and without algae added. Many algal cells adhered to the swimming antennae of juvenile D. magna but the extent of clumping and immobilisation was not as pronounced as that noted for adult organisms. There were numerous shed carapaces present in the flasks containing juvenile D. magna and these were heavily laden with attached algal cells. Insecticides other than permethrin For comparative purposes, several other insecticides were screened for their ability to induce algal cell adhesion onto adult D. magna. Diazinon (1/~glitre-~) induced extensive clumping of C. pyrenoidosa onto antennae 2 within 24 h. The affected daphnids were immobilised and settled out onto the bottom of the flasks. Less pronounced algal adhesion was also observed with cypermethrin (0" 5 pg litre- 1), methoxychlor (4/ag litre- 1) and carbaryl ( 10 pg litre - 1). However. the affected daphnids in these three systems were not immobilised. DISCUSSION
D. magnawas very sensitive to permethrin. The 48 h LCso of 0.2 to 0-6/~glitre- i is significant because permethrin has been added to water, experimentally, as a mosquito larvicide at surface rates of 11 to 28 g ha-~ (Mulla et al., 1978a). These rates translate into concentrations of 1 to 3/ag litre- 1, assuming a uniform water depth of one metre. The LCg0 of permethrin towards some mosquito larvae is 2-5/ag litre- ~ (Mulla et al., 1978b). Consequently, permethrin could have profound effects on natural Daphnia populations if used as a mosquito larvicide.
INTERACTION OF PERMETHRIN WITH
Daphnia magna
141
The pyrethroid insecticides are being considered as possible replacements for some of the organophosphatc, carbamate and organochlorine insecticides now considered unacceptable (Elliott & Janes, 1978). Therefore, it is important to know how permethrin compares with other insecticides in its toxicity towards non-target organisms, such as Daphnia. Permethrin and organophosphate insecticides have similar toxicity towards Daphnia. Forty-eight hour ICso values of 0.8 and 0.9/~g litre- ~ have been recorded for parathion and malathion, respectively, with D. magna (Frear & Boyd, 1967). Diazinon causes 50 0.,, mortality at a concentration ot" 0.91tglitre- ~ in D. puh, x (Sanders & Cope, 1966). Permcthrin is more toxic to D. magna than D D T or methoxychlor, which produce LCso values from 1.1 to 4.4/tg litre-1 (Frear & Boyd, 1967: Randall et al., 1979). Permethrin is also more toxic than the carbamate and cyclodiene insecticides, which have LC~ 0 values from 8 to 250~uglitre-1 (Sanders & Cope, 1966: Parker et al., 1970: Randall et al., 1979). Adult D. magna treated with permethrin and exposed to ('. p.l'renoMosa were more sensitive to the toxicant during the first 24 h of a 48-h incubation than were their unfed counterparts. This is in contrast to previously reported data. Adema (1978) reported that feeding has no effect on the 48h LC5o of some pesticides towards D. magna. Bicsinger & Christenscn (1972) found that D. magna that are fed during 48 h experiments arc more resistant to metals. Earlier mortality only in those systems containing algae could have resulted tor several reasons. Permethrin could have been adsorbed to the ingested algal cells, thereby providing another route of toxicant entry into D. magna and a more rapid uptake of the pesticide. However, this does not explain why the addition of bacterial cells did not give the same response, since permethrin could have been adsorbed to the bacteria which would also have been used as a source of food during incubation. The mortality response outlined in Table 1 could also have been related to the phenomenon of clumping of masses of algal cells, bacterial cells and silica onto various setate appendages of D. magna (Figs 1 and 2). This induced the immobilisation of the daphnids and could have affected mortality. Algae-coated D. magna had a greater extent of adhesion, relative to bacteria- and silica-coated animals, and became immobilised more rapidly. This variation in the extent of adhesion -and its subsequent effect on m o b i l i t y - c o u l d be responsible for the different patterns of mortality observed for the various particulate materials used (Table I ). In any case, this phenomenon and its resultant immobilisation appeared to bc an important sublethal effect of permethrin on D. magna, lmmobilisation would effectively remove such organisms from the water column, thereby affecting the entire aquatic food chain. Daphnia and other zooplankton are important sources of food for larger organisms. The adhesion phenomenon resulted from some effect ofpermethrin on D. magna, as opposed to an interaction with the particulate materials used. Evidence for this was provided by the fact that dead D. magna accumulated no algae, in addition, D.
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GLENN W. STRATTON, CHARLES T. CORKE
magna which had been pre-treated with permethrin and then washed and placed in an untreated system with algae, became coated with cells. If the sticking phenomenon was due to an effect of permethrin on the algal cells, then adhesion would still be observed with dead D. magna, unless mobility of the appendages was also required. If permethrin was only acting on the algae, then sticking would not have been observed in permethrin-free medium containing pre-treated D. magna and untreated algae. Also, the algal cells would probably clump together, even in those systems containing only algae and permethrin. This was not observed with algae or any of the other three particulate components tested, even when the flasks were gently shaken to provide better surface contact. All systems not containing permethrin were still treated with acetone, consequently the adhesion response was not due to the solvent. The above results and the fact that silica powder acted in the same manner as algae and bacteria eliminate any biological effects of permethrin on the added particulate materials as an answer for the adhesion phenomenon. Juvenile D. magna, unlike adults, did not evidence earlier mortality when they were treated with permethrin and then exposed to algal cells. The difference in response between adult and juvenile D. magna may be related to the fact that young Daphnia moult frequently, while adults do so only once every several days. Moulting could reduce the detrimental effects of algal clumping, since the affected carapaces would be discarded frequently. The cause of particulate matter adhesion is open to speculation. Permethrin is apparently affecting D. magna in such a way that its setae are becoming sticky. Daphnia is covered by a chitinous integument composed of an epidermis, a thick procuticle and a thin epicuticle (Schultz & Kennedy, 1977). The epicuticle is laminated and has a layer of loose, flaky material on the outside. The entire integument may be affected by permethrin and the atfinity of algae, bacteria and silica for various setate appendages on D. magna may be related to their greater surface area. This phenomenon could also be the result of some physiological difference induced in these structures. Daphnia physiology and biochemistry have not been studied in great detail but the setules on antenna 2 are known to contain only procuticular and epicuticular material (Schultz & Kennedy, 1977). Whether permethrin actually affects the setae themselves, or some other property, is unknown. More detailed physiological studies would be required to elucidate this problem. The fact that screening experiments with diazinon, cypermethrin, methoxychlor and carbaryl indicated that they could also induce algal cell adhesion implies that this response could be a general sublethal effect of insecticides. All the materials used are neurotoxins and this may, or may not, be a significant fact. In summary, permethrin was very toxic to D. magna and concentrations which have been used experimentally to control mosquito larvae could severely affect natural Daphnia populations. Crustaceans and insects share important physiological properties (Waterman, 1961) and since permethrin affects the nervous system in
INTERACTION OF PERMETHRIN WITH
Daphnia magna
143
insects (Wouters & van den Bercken, 1978), it could have a similar mode of action in Daphnia. However, the permethrin-induced adhesion of particulate materials, such as algae, bacteria and silica, to D. magna is apparently related to some other property. All natural aquatic ecosystems contain copious amounts of particulate matter and the above responsc may have important cnvironmental significance, cspecially since it occurs at permethrin concentrations well below the LCso for D. magna. Further study is required to determine the mode of action resulting in surface adhesion and its environmental significancc. Since other insecticides also induce a similar response, it could bc an important sublethal effect of insecticides on aquatic invertebrates. A( "KNOW LED( ;E MEN'IS
The authors wish to thank M. L. Kurp for technical assistance. This research was supportcd by the Natural Sciences and Engineering Research Council of Canada. REFERENCES ADEMA, D. M. M. (1978). Daphnia magna as a test animal in acute and chronic toxicity tests. H.l'drohiologia, 59, 125- 34. 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. BLISS, C. I. (1967). Statistics in biology, Vol. I. New York, McGraw-Hill. COATS, J. R. & O'DONNELL-JI~FFRY,N. L. (1979). Toxicity of four synthetic pyrethroid insecticides to rainbow trout. Bull. environ. Contain. & Toxicol., 23, 250 5. ELLIOTT, M. & JANES,N. F. (1978). Synthetic pyrethroids A new class of insecticide. ('hem. Soe. Rev., 7, 473. 505. FINNEY, D. J. (1971). Probit analysis, 3rd edn. Cambridge, Cambridge University Press. FREAR, D. E. H. & BOYD. J. E. (1967). Use of Daphnia rnagna for the microbioassay of pesticides, 1. Development of standardized techniques for rearing Daphnia and preparation of dosage-mortality curves for pesticides. J. econ. Ent., 60, 1228-36. JOLLY, A. L., AVAULT,J. W., KOONCE, K. L. & GRAVF.S,J. B. (1978). Acute toxicity of permethrin to several aquatic animals. Trans. Am. Fish. Soc.. 107, 825-7. L~VlNGSTON, R. J. (1977). Review of current literature concerning the acute and chronic effects of pesticides on aquatic organisms. CRC Crit. Rev. Environ. Control, 7, 325 51. MUIRHEAD-THOMPSON, R. C. (1978). Lethal and behavioural impact of permethrin (NRDC 143) on selected stream macroinvertebrates. Mosquito News, 38, 185-90. MULL^, M. S., NAVVAB-GOJRATI,H. A. & DARWAZEH,H. A. (1978a). Biological activity and longevity of new synthetic pyrethroids against mosquitos and some non-target insects. Mosquito News, 38, 90-6. MULLA, M. S., NAVV^B-GOJRATI,H. A. & DARWAZEH,H. A. (1978b). Toxicity of mosquito larvicidal pyrethroids to four species of fresh-water fishes. Environ. Ent., 7, 428- 30. PARKER,B. L.. DEWEY,J. E. & BACHE,C. A. (1970). Carbamate bioass,ay using Daphnia magna. J. econ. Ent., 63. 710-14. RANDALL, W. F., DENNIS, W. H. & WARNER, M. C. (1979). Acute toxicity of dechlorinated DDT, chlordane, and lindane to bluegill (Lepomis macrochirus) and Daphnia magna. Bull. environ. Contain. & Toxicol., 21,849-54. S^NDE~S, H. O. (1970). Toxicities of some herbicides to six species of fresh-water crustaceans. J. 14/at. Pollut. Control Fed., 42, 1544-50. S^NDERS,H. O. & COPE, O. B. (1966). Toxicities of several pesticides to two species ofcladocerans. Trans. Am. Fish. Soc., 95, 165-9. SCHULTZ,T. W. & KErn,ruby, J. R. (1977). Analysis of the integument and muscle attachment in Daphnia pulex (Cladocera: Crustacea). J. submieroscop. Cytol., 9, 37-51.
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STRATTO.~, G. W. & CORKE, C. T. (1979). The effect of nickel on the growth, photosynthesis, and nitrogenase activity of Anabaena inaequalis. Can. J. Microbiol., 25, 1094-9. STR^TION, G. W., HUBER, A. L. & CORKE, C. T. (1980). The effect of pesticides and their metabolites, alone and in combination, on algal processes. Can. Tech. Rep. Fish. Aquat. Sci., in press. WALSH, G. E. & ALEXANDER,S. V. (1980). A marine algal bioassay method: Results with pesticides and industrial wastes. Water, Air, Soil Pollut., 13, 45-55. W^rERM^N, T. H. ( 1961). Comparative physiology, in The physiology ofCrustacea, Vol. 2, ed. by T. H. Waterman, 521-3. New York, Academic Press. WOUrERS, W. & w,r~ DEN BERCKE.'q,J. (1978). Action of pyrethroids. Gen. PharrnacoL, 9, 387 98. ZITKO, V., CARSON,W. G. & MErCALVE,C. D. (1977). Toxicity of pyrethroids to juvenile Atlantic salmon. Bull. environ. Contain. & Tox&ol., 18, 35-41. Zt'rKo, V., McLEESE, D. W., MErC^LVL C. D. & C^RsOrq, W. G. (1979). Toxicity of permethrin, decamethrin, and related pyrethroids to salmon and lobster. Bull. environ. Contain. & Toxicol., 21, 338-43.
INTERACTION OF PERMETHRIN WITH DAPHNIA MAGNA IN T H E P R E S E N C E A N D A B S E N C E O F PARTICULATE MATERIAL
GI.ENN W. STRAI'I-ON & CI.tARI.ES T. ('ORKE
Department oJ Em'ironmental Bioh~gv. University o.1 Guelph, Guelph, Ontario, ('anada N I G 214"1
ABSTRA('T
The 48-h LCso olpermethrin towards'juvenile and adult Daphnia magna rangedj}'om 0.2 to 0.6 tlg litre- '. Permethrin also induced the adhesion of particulate material. including algae, bacteria and silica powder, onto various setate appendages of D. magna. This led to immobilisation of the daphnids and sign(licant O"earlier mortalitv in some O'stems containing algae. The adhesion phenomenon resulted from some ~:l.]'eet o[permethrin on D. magna, as opposed to an interaction with the particulate materials used. Algal adhesion to D. magna was also imh,'ed, to vatting degrees. with diazinon, cypermethrin, methoxyehlor and carharvl.
I N TRODU(7"I"ION
There is great concern about the effects of pesticides on non-target aquatic organisms, since the aquatic environment is the final reservoir for many xenobiotics (Livingston, 1977). One pesticide being evaluated for its impact on aquatic ecosystems is the pyrethroid insecticide, permethrin (3-phenoxybenzyl (IRS)-cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-carboxylate). Synthetic pyrethroids are among the most potent pesticides known and are being evaluated for many applications (Elliott & Janes, 1978). Permethrin is already used to control various agricultural pests and has been under investigation as a mosquito larvicide (Mulla et al., 1978a,b). Data for the effects of permethrin on aquatic organisms are few and deal primarily with fish, where the LCso values range from I to 61 pglitre-1 (Zitko et al., 1977, 1979; Jolly et al., 1978; Mulla et al., 1978b; Coats & O'Donnell-Jeffry, 1979). Permethrin is toxic to crayfish, lobster and mayfly larvae at concentrations less than 10pglitre-~ (Jolly et al., 1978; Zitko et al., 1979) and inhibits growth of the 135 Environ. Pollut. Ser. A. 0143-1471/81/0024-0135/$02"50 ((5 Applied Science Publishers Ltd, England, 1981 Printed in Great Britain
136
GLENN W. STRATTON, CHARLES T. CORKE
blue-green alga Anabaena above 500 #g litre- ~(Stratton et al., 1980). Growth of the marine diatom Skeletonema costatum is more sensitive to permethrin, with an LC5o of 68 to 72/lg litre- ~ (Walsh & Alexander, 1980), The only sublethal effect reported for permethrin is that it induces downstream drift with some invertebrates, such as Gammarus and Simulium (Muirhead-Thompson, 1978). This paper describes the effect of permethrin on the freshwater crustacean Daphnia magna--one of the more sensitive crustaceans used in pesticide toxicity studies (Sanders, 1970). Standard bioassay techniques showed that permethrin was extremely toxic to D. magna and induced the clumping of algae, bacteria and silica onto the crustacean's appendages.
MATERIALS AND METttODS
Rearing o[" Daphnia magna Daphnia magna was isolated from a small pond and cultured in 10-1itre glass aquaria containing filtered river water. D. magna was fed daily with a mixture of Scenedesmus quadricauda and Chlorella pyrenoidosa (approximately 108 cells per aquarium), that had been cultured in modified BGI 1 medium (Stratton & Corke, 1979) containing 1.5 g of N a N O 3 litre- ~. The algae and D. magna were grown at a temperature of 20°C and a light intensity of 7 klux, on a 12-h light- dark cycle. Acute toxicity study D. magna was exposed to permethrin in 250-ml Erlenmeyer flasks which contained 200 ml of filtered river water. To each flask were added ten juvenile (1-2 mm long) or adult (3-5 mm long) D. magna and 0.1 ml of permethrin (technical grade, Chipman Chemicals, Stoney Creek, Ontario) dissolved in acetone, to give a final permethrin concentration ranging from 0 to 10#glitre '~. in these and all subsequent experiments, flasks not containing permethrin had 0.05 '7o acetone (v/v) added. D. magna was added to each test system after the addition of permethrin and all treatments were carried out in triplicate. The flasks were incubated at 20 _+ 2 °C for 48 h and percent mortality was calculated. Death was defined as the inability to show any motion when prodded with a glass probe. Effect of particulate materials In some experiments, various particulate materials were added to the treatment flasks. These were the alga Chlorella pyrenoidosa (107 cells a day per flask), the bacterium Escherichia coli (109 cells a day per flask), and silica powder (0.1 g per flask; 240 mesh, Fisher Scientific Co.). For these experiments the permethrin concentration was 0.5/aglitre -~, which was chosen because it was within the calculated LCs0 range. D. magna was examined microscopically for treatment effects after 24 hours" and 48 hours' incubation. In one series of experi-
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ments involving C. pyrenoidosa, p e r m e t h r i n was replaced with 1.0,ug l i t r e diazinon (O,O-diethyl O-2-isopropyl-6 methylpyrimidin-4-yl phosphorothioate), 0-5 #g l i t r e - t c y p e r m e t h r i n ((RS)-ct-cyano-3-phenoxybenzyl (IRS)-cis, trans-3-(2,2d i c h l o r o v i n y l ) - 2 , 2 - d i m e t h y l - c y c l o p r o p a n e c a r b o x y l a t e ) , 4/ag l i t r e - t m e t h o x y c h l o r (1,1, l - t r i c h l o r o - 2 , 2 - b i s ( 4 - m e t h o x y p h e n y l ) ethane) and 10/ag l i t r e - t c a r b a r y l ( ln a p h t h y l m e t h y l c a r b a m a t e (I)).
Statistics LCso values were c a l c u l a t e d from d o s a g e - m o r t a l i t y d a t a using probit analysis ( F i n n e y , 1971 ). Significant differences in m o r t a l i t y a m o n g systems with a n d without p a r t i c u l a t e m a t e r i a l were d e t e r m i n e d using D u n c a n ' s multiple range test at ct = 0.05, as outlined by Bliss (1967).
RESULTS
Acute toxicity study Daphnia magna was very sensitive to permethrin. The 48 h LCso for both juvenile a n d a d u l t D. magna ranged from 0.2 to 0"6/~glitre-~ and 1 . 0 l i g l i t r e - ~ usually caused 1 0 0 ~o mortality. Effect o f particulate materials C o m p a r a t i v e studies were carried out on the effect o f a d d i n g the alga Chlorella pyrenoidosa to test flasks d u r i n g p e r m e t h r i n toxicity experiments. W h e n D. magna a d u l t s were exposed to p e r m e t h r i n in the presence o f the alga, there was a significant increase in m o r t a l i t y d u r i n g the first 24 h, c o m p a r e d with the same system without algae (Table 1). After 48 h therc was no difference in m o r t a l i t y between the two systems. The experiment was r e p e a t e d using a bacterial food source, Escherichia TABLE I FTFE('T OF P A R T I C U L A T E MATERIAL ON THE T O X I C I T Y OF P E R M E T H R I N T O
Particulate component added NONE C. pyrenoidosa E. coil silica powder
Observed mortality 2
D. lrtlagntg I
24 h
48 h
Degree of adhesion~
0.0 (0)* 2.0 (20)b 0.7 (7)* 0.3 (3)*
6.3 (63)' 7.0 (70)' 7.0 (70)' 8.0 (80)'
++ + +
All flasks contained 0.5 ~uglitre- ~ permethrin and ten adult D. magna, plus the additional component listed. All components were added at time zero and each treatment was carried out in triplicate. 2 Mortality data are presented as the mean number of dead D. magna per flask. The number in parentheses is the calculated percent mortality. Those values followed by the same letter are not significantly different at ct = 0.05. based upon Duncan's multiple range test. 3( _ ) no adhesion. ( + ) moderate adhesion. ( + + ) extensive adhesion, as determined microscopically on antennae 2.
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GLENN W. STRATTON, CHARLES T. CORKE
coli, and an inert component, silica powder. Only the presence of the alga induced significantly higher mortality within 24 h (Table 1). After 48 h the level of mortality was the same in all systems regardless of the component added. In all cases, the particulate material was added at time zero, following permethrin addition. Adhesion of particulate material to D. magna All three particulate materials adhered in large masses to the swimming antennae of adult, permethrin-treated D. magna. This impeded daphnid mobility, causing them to sink to the bottom of the flask where they showed erratic, unco-ordinated movement and eventually died. The bottoms of the flasks which contained algae as the added component were also littered with large clumps of algae which had fallen from the antennae olD. magna. These masses retained their integrity upon shaking. Photomicrographs of the clumping phenomenon are shown in Figs 1 and 2. The biramous swimming antennae (antennae 2) of an organism exposed only to permethrin are depicted in Fig. I A. There are nine plumose setae on these antennae and each seta contains numerous fine setules (Fig. 1B). The antennae of D. magna exposed to both permethrin and C. pyrenoidosa had numerous algal cells adhering
Fig. I. Accumulation of particulate material onto the swimming appendages of D. magna treated with 0-5/zglitre-t permethrin. (A) Antennae 2 of an organism from a system containing D. magna and permethrin only. The arrows indicate the nine plumose setae. (B) Fine structure of one of the plumose setae from antennae 2 showing the distribution of setules. (C) Algal cells adhered to the setae and setules of antennae 2. (D) Silica powder adhered to antennae 2. The bar represents 0-1 mm.
INTERACTION OF PERMETHRIN WITH Daphnia magna
C
139
• I
Fig. 2. Accumulation ofparticulate material onto D. magna treated with 0.5 pg litre - n permethrin. (A) Spine of an organism from a system containing D. magna and permethrin only. (B) Spine with adhered algal cclls. (C) Spine with adhered bacterial cells. (D) Post-abdomen with adhered silica powder: F furca, S-. post-abdomenal spine, SB--post-abdomenal setae. The bar represents 0.1 ram.
to the setae and setules (Fig. I C). A similar pattern of adhesion was noted tbr both E. colt and silica powder (Fig. 1D). Another prominent area of algal cell adhesion was the posterior portion of the spine (Fig. 2B), which is bordered by a number of short, thick setae (Fig. 2A). Bacterial cells (Fig. 2C) and silica also adhered to this structure. The short, inconspicuous antennae 1 also accumulated particulate material, particularly at the tip, where a number of very fine setac arc present. Silica powder also clumped onto the filtering setae of the thoracic appendages and on the post-abdomen (Fig. 2D). The furca, tile post-abdomenal plumose setac and the post-abdomenal spines werc also covered with particulate matter.
Evtent of adhesion The amount of adhesion varied, depending on the particulate material present (Table I). This response was most pronounced when algal cells were the added component. Algal adhesion also occurred at permethrin concentrations of 0-05 and 0-005 l~g litre-~. However, mortality was not significantly affected in these two systems. The extent of algal cell accumulation on antennae 2 increased with increasing levels of permethrin. Sctal adhesion was only observed in those systems containing both permethrin, D.
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GLENN W. STRATTON, CHARLES T. CORKE
magna, and either algae, bacteria, or silica. When any one of the above components was omitted from the test system, no accumulation of particulates was observed. All systems not containing permethrin still contained acetone to ensure that the solvent was not a factor in the adhesion response. When dead D. magna were used, no accumulation of algal cells occurred, even when the test flasks were gently shaken to provide better contact between the various components in the flasks. When algae were added to systems containing permethrin and live D. magna after 24 h, rather than at time zero, adhesion was still observed after 2 hours' incubation. In such a system, much of the permethrin (0.5 pg litre- 1) would probably have been adsorbed to the glass flasks and D. magna during the first 24 h. When D. magna was treated with 0'5/ag litre- 1 permethrin for 24 h and then removed, washed and placed into fresh permethrin-free water containing algae, cell adhesion was noted within 2 h. Response of juvenile D. magna Juvenile D. magna treated with 0-5 pglitre-1 permethrin responded differently from adult organisms. With the former, there was no significant difference in mortality between those systems with and without algae added. Many algal cells adhered to the swimming antennae of juvenile D. magna but the extent of clumping and immobilisation was not as pronounced as that noted for adult organisms. There were numerous shed carapaces present in the flasks containing juvenile D. magna and these were heavily laden with attached algal cells. Insecticides other than permethrin For comparative purposes, several other insecticides were screened for their ability to induce algal cell adhesion onto adult D. magna. Diazinon (1/~glitre-~) induced extensive clumping of C. pyrenoidosa onto antennae 2 within 24 h. The affected daphnids were immobilised and settled out onto the bottom of the flasks. Less pronounced algal adhesion was also observed with cypermethrin (0" 5 pg litre- 1), methoxychlor (4/ag litre- 1) and carbaryl ( 10 pg litre - 1). However. the affected daphnids in these three systems were not immobilised. DISCUSSION
D. magnawas very sensitive to permethrin. The 48 h LCso of 0.2 to 0-6/~glitre- i is significant because permethrin has been added to water, experimentally, as a mosquito larvicide at surface rates of 11 to 28 g ha-~ (Mulla et al., 1978a). These rates translate into concentrations of 1 to 3/ag litre- 1, assuming a uniform water depth of one metre. The LCg0 of permethrin towards some mosquito larvae is 2-5/ag litre- ~ (Mulla et al., 1978b). Consequently, permethrin could have profound effects on natural Daphnia populations if used as a mosquito larvicide.
INTERACTION OF PERMETHRIN WITH
Daphnia magna
141
The pyrethroid insecticides are being considered as possible replacements for some of the organophosphatc, carbamate and organochlorine insecticides now considered unacceptable (Elliott & Janes, 1978). Therefore, it is important to know how permethrin compares with other insecticides in its toxicity towards non-target organisms, such as Daphnia. Permethrin and organophosphate insecticides have similar toxicity towards Daphnia. Forty-eight hour ICso values of 0.8 and 0.9/~g litre- ~ have been recorded for parathion and malathion, respectively, with D. magna (Frear & Boyd, 1967). Diazinon causes 50 0.,, mortality at a concentration ot" 0.91tglitre- ~ in D. puh, x (Sanders & Cope, 1966). Permcthrin is more toxic to D. magna than D D T or methoxychlor, which produce LCso values from 1.1 to 4.4/tg litre-1 (Frear & Boyd, 1967: Randall et al., 1979). Permethrin is also more toxic than the carbamate and cyclodiene insecticides, which have LC~ 0 values from 8 to 250~uglitre-1 (Sanders & Cope, 1966: Parker et al., 1970: Randall et al., 1979). Adult D. magna treated with permethrin and exposed to ('. p.l'renoMosa were more sensitive to the toxicant during the first 24 h of a 48-h incubation than were their unfed counterparts. This is in contrast to previously reported data. Adema (1978) reported that feeding has no effect on the 48h LC5o of some pesticides towards D. magna. Bicsinger & Christenscn (1972) found that D. magna that are fed during 48 h experiments arc more resistant to metals. Earlier mortality only in those systems containing algae could have resulted tor several reasons. Permethrin could have been adsorbed to the ingested algal cells, thereby providing another route of toxicant entry into D. magna and a more rapid uptake of the pesticide. However, this does not explain why the addition of bacterial cells did not give the same response, since permethrin could have been adsorbed to the bacteria which would also have been used as a source of food during incubation. The mortality response outlined in Table 1 could also have been related to the phenomenon of clumping of masses of algal cells, bacterial cells and silica onto various setate appendages of D. magna (Figs 1 and 2). This induced the immobilisation of the daphnids and could have affected mortality. Algae-coated D. magna had a greater extent of adhesion, relative to bacteria- and silica-coated animals, and became immobilised more rapidly. This variation in the extent of adhesion -and its subsequent effect on m o b i l i t y - c o u l d be responsible for the different patterns of mortality observed for the various particulate materials used (Table I ). In any case, this phenomenon and its resultant immobilisation appeared to bc an important sublethal effect of permethrin on D. magna, lmmobilisation would effectively remove such organisms from the water column, thereby affecting the entire aquatic food chain. Daphnia and other zooplankton are important sources of food for larger organisms. The adhesion phenomenon resulted from some effect ofpermethrin on D. magna, as opposed to an interaction with the particulate materials used. Evidence for this was provided by the fact that dead D. magna accumulated no algae, in addition, D.
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GLENN W. STRATTON, CHARLES T. CORKE
magna which had been pre-treated with permethrin and then washed and placed in an untreated system with algae, became coated with cells. If the sticking phenomenon was due to an effect of permethrin on the algal cells, then adhesion would still be observed with dead D. magna, unless mobility of the appendages was also required. If permethrin was only acting on the algae, then sticking would not have been observed in permethrin-free medium containing pre-treated D. magna and untreated algae. Also, the algal cells would probably clump together, even in those systems containing only algae and permethrin. This was not observed with algae or any of the other three particulate components tested, even when the flasks were gently shaken to provide better surface contact. All systems not containing permethrin were still treated with acetone, consequently the adhesion response was not due to the solvent. The above results and the fact that silica powder acted in the same manner as algae and bacteria eliminate any biological effects of permethrin on the added particulate materials as an answer for the adhesion phenomenon. Juvenile D. magna, unlike adults, did not evidence earlier mortality when they were treated with permethrin and then exposed to algal cells. The difference in response between adult and juvenile D. magna may be related to the fact that young Daphnia moult frequently, while adults do so only once every several days. Moulting could reduce the detrimental effects of algal clumping, since the affected carapaces would be discarded frequently. The cause of particulate matter adhesion is open to speculation. Permethrin is apparently affecting D. magna in such a way that its setae are becoming sticky. Daphnia is covered by a chitinous integument composed of an epidermis, a thick procuticle and a thin epicuticle (Schultz & Kennedy, 1977). The epicuticle is laminated and has a layer of loose, flaky material on the outside. The entire integument may be affected by permethrin and the atfinity of algae, bacteria and silica for various setate appendages on D. magna may be related to their greater surface area. This phenomenon could also be the result of some physiological difference induced in these structures. Daphnia physiology and biochemistry have not been studied in great detail but the setules on antenna 2 are known to contain only procuticular and epicuticular material (Schultz & Kennedy, 1977). Whether permethrin actually affects the setae themselves, or some other property, is unknown. More detailed physiological studies would be required to elucidate this problem. The fact that screening experiments with diazinon, cypermethrin, methoxychlor and carbaryl indicated that they could also induce algal cell adhesion implies that this response could be a general sublethal effect of insecticides. All the materials used are neurotoxins and this may, or may not, be a significant fact. In summary, permethrin was very toxic to D. magna and concentrations which have been used experimentally to control mosquito larvae could severely affect natural Daphnia populations. Crustaceans and insects share important physiological properties (Waterman, 1961) and since permethrin affects the nervous system in
INTERACTION OF PERMETHRIN WITH
Daphnia magna
143
insects (Wouters & van den Bercken, 1978), it could have a similar mode of action in Daphnia. However, the permethrin-induced adhesion of particulate materials, such as algae, bacteria and silica, to D. magna is apparently related to some other property. All natural aquatic ecosystems contain copious amounts of particulate matter and the above responsc may have important cnvironmental significance, cspecially since it occurs at permethrin concentrations well below the LCso for D. magna. Further study is required to determine the mode of action resulting in surface adhesion and its environmental significancc. Since other insecticides also induce a similar response, it could bc an important sublethal effect of insecticides on aquatic invertebrates. A( "KNOW LED( ;E MEN'IS
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STRATTO.~, G. W. & CORKE, C. T. (1979). The effect of nickel on the growth, photosynthesis, and nitrogenase activity of Anabaena inaequalis. Can. J. Microbiol., 25, 1094-9. STR^TION, G. W., HUBER, A. L. & CORKE, C. T. (1980). The effect of pesticides and their metabolites, alone and in combination, on algal processes. Can. Tech. Rep. Fish. Aquat. Sci., in press. WALSH, G. E. & ALEXANDER,S. V. (1980). A marine algal bioassay method: Results with pesticides and industrial wastes. Water, Air, Soil Pollut., 13, 45-55. W^rERM^N, T. H. ( 1961). Comparative physiology, in The physiology ofCrustacea, Vol. 2, ed. by T. H. Waterman, 521-3. New York, Academic Press. WOUrERS, W. & w,r~ DEN BERCKE.'q,J. (1978). Action of pyrethroids. Gen. PharrnacoL, 9, 387 98. ZITKO, V., CARSON,W. G. & MErCALVE,C. D. (1977). Toxicity of pyrethroids to juvenile Atlantic salmon. Bull. environ. Contain. & Tox&ol., 18, 35-41. Zt'rKo, V., McLEESE, D. W., MErC^LVL C. D. & C^RsOrq, W. G. (1979). Toxicity of permethrin, decamethrin, and related pyrethroids to salmon and lobster. Bull. environ. Contain. & Toxicol., 21, 338-43.