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The toxicity of the mothproofing chemical eulan WA new to frog Rana Temporaria tadpoles
tz)l, tronmentu/ Pollulu,n I.~'rte~ .4 ) 24 ( 1981 ) I I "t 123
THE TOXICITY OF THE MOTHPROOFING CHEMICAL EULAN WA NEW TO FROG RANA TEMPORARIA TADPOLES D. OsHOrN & M. C French ,~v'~llurtl[ l-m'ironment Research Council, 11"1.~111111~~ oJ 7~,rre.stria/ Eco/og.l', ,.~onk,s Wood lc~xperimental Station..4bhot.~ Ripton, Huntingdon, PEI 7 2[S. Great Britain
ABSIRA('I
Eu/an WA New is rephwing dieMr& as a mothproqfing agent. Its to.vicitv to./i'og tadpoles has been assessed in a .static hioassav sv.~tem. Eu[an, at t'ariou.~ concentrations, killed tadpo&s or rethwed.[eeding- and suhsequentl.v the weight o[ the tadpoles. It is suegested that these eff~,cts c,uhl occur in wihl animals accumulating &'tween ] and ]Omgkg t o/Euhm.
INTRODU(']I()N
Euhm WA New is used as an alternative to dieldrin in certain mothproofing treatments in the UK and elsewhere. It is likely that some Eulan will enter waterways (Westo6 & Noren, 1977) and possible that it will accumulate in (Wells, 1979), and have undesirable effects on, wildlife. Accordingly, the toxicity of Eulan WA New to Rana temporaria tadpoles was tested in a static bioassay system which had previously been used to provide information on the toxicity of DDT. dieldrin, 2,4-D. cyanatryn, copper and cadmium to amphibian larvae (Cooke, 1972, Scorgie & Cooke, 1979: Osborn, details to be published later).
MA1ERIAL AND METIIOI)S
7",dpo]e, Frog spawn was collected from a site in Cambridgeshire. U K, and hatched in aged tap-water. (Aged tap-water is tap-water left in the open for at least 3 days.) When tadpoles were at stage 24 (Witschi, ]956), twenty individuals (weight/tadpole = 2 ] - 0 _ 1 . 0 m g ; n = ]0) were placed in one litre of aged tap-water that contained the following additions ofthe technical preparation of Eulan WA N e w : l - 0 mg/litre (1 ppm), 0. I mg/litre, 0.0] mg/litre and 0mg/litre as the contro]. There were four 117 Enriron. Pollut. Ser. A. 0143-1471 ,,'81/0024-0117/$02.50 .C Applied Science Publishers Ltd, England, 198 I Printed in Great Britain
I 18
D. OSBORN, M. ('. FRENCH
replicates of each treatment. Tadpoles were weighed in groups of five to improve reliability and the mean was taken as the weight of a 'typical' tadpole. The values obtained for movement were taken as the mean from four observations of the tadpoles in each dish made on each occasion movement was recorded. In accordance with earlier work, the animals were not fed during the first 48 h of exposure. Thereafter they were fed spinach leaf daily. The water, with suitable additions of Eulan, was changed three times a week and, over the course of the experiment, its temperature ranged between 1!.5 and 15°C. The number of tadpoles moving, the number hanging at the side of the dish, the stage of development and the number of various abnormalities were recorded at various times during the experiment. The experiment was ended when the tadpoles began tail resorption at stages 32 and 33. Eulan WA New was supplied by Bayer UK Ltd in a liquid technical form which had the following composition. 20'70 N-Chloromethylsulpho-pentachloro-2aminodiphenylether, sodium salt (the active ingredient), 2 % monosulphonated long chain hydrocarbon, 74,.0 1,2 propanediol, 3.7 ~, water, 0-3 % sodium hydroxide.
Chemical analysis The analysis used was amended from that of Wells (1979). Tadpoles were taken from the experiment, killed and frozen. Subsequently specimens were removed from the freezer and ground immediately with sand and anhydrous sodium sulphate. The friable mix was then extracted with n-hexane, and the volume reduced to 5 ml on a hot-water bath. This extract was analysed without further clean-up (a procedure not required in this instance) on a Perkin Elma Sigma 3 gas chromatograph equipped with an EC detector. The 1.85m column was packed with 1% Dexsii 300 on Chromabsorb W AW DMCS and run at a temperature of 250°C. Samples were compared with a sample of pure Eulan WA New. Statistics Results were analysed by a standard analysis of variance technique, after first transforming the observed proportions into angles using the variance stabilising arcsine transformation. After the analysis of variance, the various treatment means were compared via Student's t-statistic at the 5 % level of significance.
RESULTS
Movement Tadpole movements were recorded during the first 96 h of the experiment. There was a significant decrease in the movement of tadpoles in the 1.0 mg/litre groups in the first 48 h, the effect being greatest during the second 24 h (Table 1). The effect did
TOXICITY OF EULAN TO R a n a t e m p o r a r i a
119
not persist beyond this time, i.e. once animals had been fed, no difference in movement was observed. Hanging
H a n g i n g was r e c o r d e d d u r i n g the first 96 h o f the e x p e r i m e n t . D u r i n g the 5 h f o l l o w i n g e x p o s u r e h a n g i n g in b o t h thc 1.0 m g / l i t r e a n d 0.1 m g / l i t r c dishcs was TABLE 1 taDPOLe MOVI-:MEYl a. Tadpole movements during first 48 h of experiment during which time tadpoles were observd on eleven txzcasions Replicate
Control Number Total t o o l i n g a obser~'ation.~
A B C D Group total
49.5 53.5 54.00 55.75 212.75
220 220 220 220 880(24)
Eu/an 1.0 mg; litrv Number Total
O-1 mg. lim Number Total
0.01 ntg, htre Number Total
30.25 220 31.75 210 25.75 208 205 27.25 115-00" 843(13)
56.75 220 53.75 220 58"00 220 6400 220 232"50 880(26)
47.00 220 53"25 220 52-50 220 51 "50 220 204"25 880(23)
b. Tadpole movements during second 24 h of the experiment when tadpoles were observed on four occasions. (See also Table la.) R~Tlicate
A B C D
Group total
Control Number Total 1"0 t~lgllhlrt ' mol'ing" ob.gerl'ations Number Total
2(7.00 21.25 22-75 21.75 85.75
80 80 80 80 24(7(35)
7.25 80 7.25 71 5.50 68 6.25 65 26"25* 284(9)
Eu/an ll'l mghtrc Numher Total
24.75 22.75 23.75 2875 100
80 80 80 80 240(42)
0"01 rag. litrc Numher Total
18.75 23.50 23.50 24.25 90
80 8(7 80 8O 240(38)
"These values are the mean of four observations of each dish (.',co"Material and Methods'). |:igures in parentheses are group totals expressed as percentages. * Results so marked are significantly different from the controls (p <- ().05). see text. signiticantly less t h a n that in the c o n t r o l s a n d a p p a r e n t l y d o s e - r e l a t e d , but t h e r e a f t e r differences, a l t h o u g h persisting, were small a n d not signiticant {Table 2). Feeding
F e e d i n g on s p i n a c h leaf was r e c o r d e d d u r i n g the first 48 h in w h i c h s p i n a c h was present. T a d p o l e s in the 1.0 m g / l i t r e dishes w e r e o b s e r v e d feeding a b o u t t h r e e t i m e s less o f t e n t h a n the c o n t r o l s ( T a b l e 3). W h i l e a n i m a l s in the 0.1 mg,:litre a n d 0.01 m g / l i t r e dishes fed less t h a n the c o n t r o l s , the differences w e r e not significant. but their persistence s u g g e s t e d that a d o s e - r e l a t e d i n h i b i t i o n o f f e e d i n g m i g h t be o c c u r r i n g T h e cfl'ect on f e e d i n g persisted t h r o u g h o u t the e x p e r i m e n t .
120
D. OSBORN, M. C. FRENCH
TABLE 2 TADPOLE HANGING
a. Tadpole hanging during first 48 h of experiment. Tadpoles were observed on eleven occasions. (See also Table la.)
Replicate
A B C D Group total
Control Number Total 1.0 mg/litre hanging observations Number Total 92 87 71 64 314
220 220 220 220 880(36)
85 61 61 44 251
220 210 208 205 843(30)
Eulan 0"1 mg/litre Number Total 73 82 52 61 268
0"01 mg/litre Number Total
220 220 220 220 880(30)
84 77 74 78 313
220 220 220 220 880(36)
b. Tadpole hanging during first 5 h of experiment. Tadpoles were observed on three occasions. (See also Table la.)
Replicate
A B
C D Group total
Control Number Total 1"0 mg/litre hanging observations Number Total 21 21 19
60 60 60
7 6 10
60 60 60
Eulan 0"1 mg/litre Number Total 12 9 8
18
60
3
60
11
79
240(33)
26*
240( I 1)
40*
0"01 mg/litre Number Total
60 60 60 60 240(17)
15 18 15 24 72
60 60 60 60 240(30)
* Results so marked are significantly different from the controls (p < 0.05), see text.
TABLE 3 TADPOLES FEEDING OVER FIRST 48 h THAT FOOD WAS AVAILABLE (DAYS 3 AND 4 OF THE EXPERIMENT) DURING WHICH TIME TADPOI.FS WERE OBSERVED ON ELEVEN OCCASIONS. (SEE ALSO TABLE l a )
Replicate
A B
C D Group total
Control Number Total 1.0 rng;litre feeding observations Number Total 25 27 18 23 93
220 220 220 220 880( 11)
14 3 6 4 27*
220 166 171 135 692(4)
Eulan 0"1 mg,;litre Number Total 14 19 25 16 74
0.01 mg/litre Number Total
220 220 220 220 880(8)
26 13 22 19 80
220 220 220 220 880(9)
* Results so marked are significantly different from the controls (p < 0.05), sec text.
Mortalit)" Over the whole course of the experiment the mortality of tadpoles 1.0 m g / l i t r e d i s h e s w a s m u c h g r e a t e r t h a n t h a t o f t h e c o n t r o l s , o n l y six o u t o f t a d p o l e s s u r v i v i n g at t h i s d o s e c o m p a r e d w i t h s e v e n t y - e i g h t o u t o f e i g h t y c o n t r o l s , s e v e n t y - n i n e o u t o f e i g h t y in 0-01 m g / l i t r e d i s h e s a n d all e i g h t y 0. I m g / l i t r e g r o u p .
in t h e eighty in t h e in t h e
TOXICITY OF EULAN TO
Rana temporaria
121
Development The development of treated tadpoles was slower than that of the controls. This was most apparent with the 1.0 mg/litre group, none of which reached stage 30. Furthermore, in the 0. I mg/litre and 0.01 mg/litre dishes, development was slightly behind that of the controls. For example, on the 28th day of exposure twenty out of seventy-eight surviving tadpoles had reached stages 29 or 30 in the control groups, whereas only seven out ofeighty and three out of seventy-nine had reached stage 29 in the 0.1 mg/litre and 0.01 mg/litre groups. This effect was emphasised by the observation that 2 3 days after ten stage 31 tadpoles had been removed for chemical and biochemical analysis from each of the control, 0.1 mg/litre and 0.01 mg/litre dishes, 24°,0 (9,"38) of the remaining control animals were in stage 32, whilst only 15 ~, (6/39) of the 0.01 mg/litre and 5 01,(2/40) ofthc 0. ! mg/litre groups had reached this stage of development.
Abnormalities There was a slight suggestion that Eulan increased the occurrence of the type of abnormalities that occurred naturally in the tadpoles, for whereas there were two tail kinks in the control groups there were seven such kinks in the 1.0 mg/litre group. One such kink occurred in the 0.01 mg/litre group and none in the 0.1 mg/litrc group. The kinks in the 1.0 mg/litrc groups were much more severe than those in any other group.
Weight As can be seen in Table 4, 1-0 mg/litre tadpoles were lighter than the controls from day 3 onwards, and those in the 0" 1 mg/litre group lighter from day 24 until they reached stage 31 on days 35-37.0-01 mg/litrc tadpoles were lighter at stage 31 than thc controls, but the difference was not statistically significant. As expected, weight decreased between days 29 and 36. This was more pronounced in treated than in control tadpoles.
Eulan reshtues Table 5 shows the residues of Eulan found in the pooled sample ofwhole tadpoles, each made up of five individuals. The residues are of a similar order to those of D D T accumulated by R. temporaria tadpoles which had been exposed to comparable concentrations of D D T and which were suffering from the effects of D D T (Cooke, 1972). DISCUSSION
The results suggest that some effect on feeding leads to a reduction in weight during the later stages of metamorphosis of tadpoles, even when the effect on feeding was
122
D. OSBORN, M. C. FRENCH
TABLE 4 WEIGHTS OF TADPOLES(mg_+ I SE)
Day 3* 5 12 17 22 24 29 36b
Stage of de~'elopment 25/24' Not recorded in detail Not recorded in detail Not recorded indetail Not recorded indetail 28,'26-27' 29-30/26 28' 31/26 29~
Control
0"01 mg/litre
Eulan 0"1 mg/litre
22+0.7 (4)
23_+0.6 (4)
23_+0"3 (4)
19_+ 1"0 (4)*
36 _+0.5 (4)
36 +1.5 (4)
37 _+0.9 (4)
22 _+1.3 (4)*
53 + 2.8 (4)
53 _+2.8 (4)
53 + 1.3 (4)
29 + 1.9 (4)*
215+7
(4)
213+11 (4)
213-+8
(4)
373-+4 440_+ 16 590+6 541+12
(4) (4) (4) (8)
368-+25 452+ 15 588-+11 523_+9
369+9 409-+6 567_+33 493_+8
(4) (4) (4) (8)*
(4) (4) (4) (8)
I 0 mg/litre
79+8.0(3)* 146-+19 171 -+24 306_+31 382
(3)* (3)* (3)* (I)
Figures in parentheses are values of n, * = p < 0.05, Student's t-test, such results are significantly different from controls. "This weight was obtained before food was given. bTwo groups of tadpoles were weighed here. One group was used for chemical analyses and the other retained for future studies on body composition. For this reason n = 8 rather than 4. ' These figures relate to stage of development of the 1-0 mg/litre group, the others to the controls and the 0-1 and 0.01 mg/litre groups. TABLE 5 ELt.AN ('ON('EN'IRATIONS (mg k g
Control 1.0 mg/litre 0.1 mg,litre 0.01 mg/litre
~ WET WEIGHT} AFIER
35-36
DAYS OF EXPOSURE
Eulan
n
Stage of tadpole det,elopment
ND 24. I 7.71 + 1.60 0.53 ±0.11
4 1" 4 4
31 26-29 31 31
"Only one determination was possible owing to mortality.
not statistically signilicant. This conclusion would be supported by (i) the finding that hanging is also affected, since this is frequently associated with grazing on the glass surface at the side of the dish, and (ii) that weight declined more rapidly between days 29 and 36 in the treated animals than in the controls, i.e. body reserves were used more rapidly by treated animals. The effect of movement in the period when no food was present could indicate a decrease in the ability, or desire, of the animals to search for food. Thus it seems possible that the main effect of Eulan on tadpoles is to reduce weight by reducing feeding or food-secking activity. Rcduced feeding is said to be thc manner in which Eulan kills moth larvae. We would stress that in attempting to assess the environmental hazard Eulan presents, extrapolations from the nominal water concentrations quoted here should
TOXICITY OF I-.ULAN TO
Rana temporaria
123
not be made since it is possible that (i) the true water concentrations are far below the nominal values and (ii) much Eulan could be accumulated through food contamination. It would seem more appropriate in hazard assessments to use the tissue levels accumulated by these animals and the effects they brought about. Thus it is likely that an effect on feeding which could have serious implications for animals is to be expected in animals accumulating whole body concentrations of Eulan between 1 and 10 mg k g - ' wet weight, since reduced feeding could reduce individual survival and reduced body weight at metamorphosis could influence future survival, or breeding success. Similarly, mortality is likely to occur in individual members of groups of animals accumulating whole body residues in excess of 25 mg k g - ~ wet weight. It should be noted that this experiment was performed at a time when the tadpoles had passed one of their most sensitive periods to many other compounds, e.g. D D T and dieldrin, and that estimates of toxicity extrapolated from these data may underestimate the true toxicity to R. temporaria tadpoles. From these data it may be tentatively concluded that Eulan WA New seems about halfto one-fifth as toxic to tadpoles as dieldrin or D D T under similar conditions (Cooke, 1972). However, since about five times as much Eulan as dieldrin is used in mothproofing, Eulan could, at least locally, be as great an environmental hazard as dieldrin if large quantities of factory effluent containing Eulan reach waterways. However. such simple extrapolations may be naive.
ACKNOWLEDGEMENTS
We thank Drs I. Newton, J. P. Dempster and A. S. Cooke for comments on the manuscript and are indebted to Mr K. H. Lakhani for his statistical advice and other remarks. Bayer UK kindly supplied the Eulan preparation used in this study.
REFERENCES COOKE,A. S. (1972). The effectsof DDT, dieldrin and 2,4-D on amphibian spawn and tadpoles. Environ. Pollut., 3, 5l-8. SCORGIE,H. R. A. & COOKr,A. S. (1979). Effectsof the triazine herbicidecyanatryn on aquatic animals. Bull. environ. Contain. & Toxicol., 22, 135-42. WELLS, D. E. (1979). The isolation and identification of polychloro-2 (chloromethylsulphonamide) diphenyl ether isomers and their metabolites from Eulan WA New and fish tissue by gas chromatography-mass spectrometry. Analytica Ckim. Acta, 104, 253--66. W~'roO, G. & NOREr~,K. (1977). Polychlorinated 2-amidodiphenyl ethers in fish. Ambio, 6, 232--4. Wrrscm, E. (1956). Development of vertebrates. Philadelphia, Saunders.
THE TOXICITY OF THE MOTHPROOFING CHEMICAL EULAN WA NEW TO FROG RANA TEMPORARIA TADPOLES D. OsHOrN & M. C French ,~v'~llurtl[ l-m'ironment Research Council, 11"1.~111111~~ oJ 7~,rre.stria/ Eco/og.l', ,.~onk,s Wood lc~xperimental Station..4bhot.~ Ripton, Huntingdon, PEI 7 2[S. Great Britain
ABSIRA('I
Eu/an WA New is rephwing dieMr& as a mothproqfing agent. Its to.vicitv to./i'og tadpoles has been assessed in a .static hioassav sv.~tem. Eu[an, at t'ariou.~ concentrations, killed tadpo&s or rethwed.[eeding- and suhsequentl.v the weight o[ the tadpoles. It is suegested that these eff~,cts c,uhl occur in wihl animals accumulating &'tween ] and ]Omgkg t o/Euhm.
INTRODU(']I()N
Euhm WA New is used as an alternative to dieldrin in certain mothproofing treatments in the UK and elsewhere. It is likely that some Eulan will enter waterways (Westo6 & Noren, 1977) and possible that it will accumulate in (Wells, 1979), and have undesirable effects on, wildlife. Accordingly, the toxicity of Eulan WA New to Rana temporaria tadpoles was tested in a static bioassay system which had previously been used to provide information on the toxicity of DDT. dieldrin, 2,4-D. cyanatryn, copper and cadmium to amphibian larvae (Cooke, 1972, Scorgie & Cooke, 1979: Osborn, details to be published later).
MA1ERIAL AND METIIOI)S
7",dpo]e, Frog spawn was collected from a site in Cambridgeshire. U K, and hatched in aged tap-water. (Aged tap-water is tap-water left in the open for at least 3 days.) When tadpoles were at stage 24 (Witschi, ]956), twenty individuals (weight/tadpole = 2 ] - 0 _ 1 . 0 m g ; n = ]0) were placed in one litre of aged tap-water that contained the following additions ofthe technical preparation of Eulan WA N e w : l - 0 mg/litre (1 ppm), 0. I mg/litre, 0.0] mg/litre and 0mg/litre as the contro]. There were four 117 Enriron. Pollut. Ser. A. 0143-1471 ,,'81/0024-0117/$02.50 .C Applied Science Publishers Ltd, England, 198 I Printed in Great Britain
I 18
D. OSBORN, M. ('. FRENCH
replicates of each treatment. Tadpoles were weighed in groups of five to improve reliability and the mean was taken as the weight of a 'typical' tadpole. The values obtained for movement were taken as the mean from four observations of the tadpoles in each dish made on each occasion movement was recorded. In accordance with earlier work, the animals were not fed during the first 48 h of exposure. Thereafter they were fed spinach leaf daily. The water, with suitable additions of Eulan, was changed three times a week and, over the course of the experiment, its temperature ranged between 1!.5 and 15°C. The number of tadpoles moving, the number hanging at the side of the dish, the stage of development and the number of various abnormalities were recorded at various times during the experiment. The experiment was ended when the tadpoles began tail resorption at stages 32 and 33. Eulan WA New was supplied by Bayer UK Ltd in a liquid technical form which had the following composition. 20'70 N-Chloromethylsulpho-pentachloro-2aminodiphenylether, sodium salt (the active ingredient), 2 % monosulphonated long chain hydrocarbon, 74,.0 1,2 propanediol, 3.7 ~, water, 0-3 % sodium hydroxide.
Chemical analysis The analysis used was amended from that of Wells (1979). Tadpoles were taken from the experiment, killed and frozen. Subsequently specimens were removed from the freezer and ground immediately with sand and anhydrous sodium sulphate. The friable mix was then extracted with n-hexane, and the volume reduced to 5 ml on a hot-water bath. This extract was analysed without further clean-up (a procedure not required in this instance) on a Perkin Elma Sigma 3 gas chromatograph equipped with an EC detector. The 1.85m column was packed with 1% Dexsii 300 on Chromabsorb W AW DMCS and run at a temperature of 250°C. Samples were compared with a sample of pure Eulan WA New. Statistics Results were analysed by a standard analysis of variance technique, after first transforming the observed proportions into angles using the variance stabilising arcsine transformation. After the analysis of variance, the various treatment means were compared via Student's t-statistic at the 5 % level of significance.
RESULTS
Movement Tadpole movements were recorded during the first 96 h of the experiment. There was a significant decrease in the movement of tadpoles in the 1.0 mg/litre groups in the first 48 h, the effect being greatest during the second 24 h (Table 1). The effect did
TOXICITY OF EULAN TO R a n a t e m p o r a r i a
119
not persist beyond this time, i.e. once animals had been fed, no difference in movement was observed. Hanging
H a n g i n g was r e c o r d e d d u r i n g the first 96 h o f the e x p e r i m e n t . D u r i n g the 5 h f o l l o w i n g e x p o s u r e h a n g i n g in b o t h thc 1.0 m g / l i t r e a n d 0.1 m g / l i t r c dishcs was TABLE 1 taDPOLe MOVI-:MEYl a. Tadpole movements during first 48 h of experiment during which time tadpoles were observd on eleven txzcasions Replicate
Control Number Total t o o l i n g a obser~'ation.~
A B C D Group total
49.5 53.5 54.00 55.75 212.75
220 220 220 220 880(24)
Eu/an 1.0 mg; litrv Number Total
O-1 mg. lim Number Total
0.01 ntg, htre Number Total
30.25 220 31.75 210 25.75 208 205 27.25 115-00" 843(13)
56.75 220 53.75 220 58"00 220 6400 220 232"50 880(26)
47.00 220 53"25 220 52-50 220 51 "50 220 204"25 880(23)
b. Tadpole movements during second 24 h of the experiment when tadpoles were observed on four occasions. (See also Table la.) R~Tlicate
A B C D
Group total
Control Number Total 1"0 t~lgllhlrt ' mol'ing" ob.gerl'ations Number Total
2(7.00 21.25 22-75 21.75 85.75
80 80 80 80 24(7(35)
7.25 80 7.25 71 5.50 68 6.25 65 26"25* 284(9)
Eu/an ll'l mghtrc Numher Total
24.75 22.75 23.75 2875 100
80 80 80 80 240(42)
0"01 rag. litrc Numher Total
18.75 23.50 23.50 24.25 90
80 8(7 80 8O 240(38)
"These values are the mean of four observations of each dish (.',co"Material and Methods'). |:igures in parentheses are group totals expressed as percentages. * Results so marked are significantly different from the controls (p <- ().05). see text. signiticantly less t h a n that in the c o n t r o l s a n d a p p a r e n t l y d o s e - r e l a t e d , but t h e r e a f t e r differences, a l t h o u g h persisting, were small a n d not signiticant {Table 2). Feeding
F e e d i n g on s p i n a c h leaf was r e c o r d e d d u r i n g the first 48 h in w h i c h s p i n a c h was present. T a d p o l e s in the 1.0 m g / l i t r e dishes w e r e o b s e r v e d feeding a b o u t t h r e e t i m e s less o f t e n t h a n the c o n t r o l s ( T a b l e 3). W h i l e a n i m a l s in the 0.1 mg,:litre a n d 0.01 m g / l i t r e dishes fed less t h a n the c o n t r o l s , the differences w e r e not significant. but their persistence s u g g e s t e d that a d o s e - r e l a t e d i n h i b i t i o n o f f e e d i n g m i g h t be o c c u r r i n g T h e cfl'ect on f e e d i n g persisted t h r o u g h o u t the e x p e r i m e n t .
120
D. OSBORN, M. C. FRENCH
TABLE 2 TADPOLE HANGING
a. Tadpole hanging during first 48 h of experiment. Tadpoles were observed on eleven occasions. (See also Table la.)
Replicate
A B C D Group total
Control Number Total 1.0 mg/litre hanging observations Number Total 92 87 71 64 314
220 220 220 220 880(36)
85 61 61 44 251
220 210 208 205 843(30)
Eulan 0"1 mg/litre Number Total 73 82 52 61 268
0"01 mg/litre Number Total
220 220 220 220 880(30)
84 77 74 78 313
220 220 220 220 880(36)
b. Tadpole hanging during first 5 h of experiment. Tadpoles were observed on three occasions. (See also Table la.)
Replicate
A B
C D Group total
Control Number Total 1"0 mg/litre hanging observations Number Total 21 21 19
60 60 60
7 6 10
60 60 60
Eulan 0"1 mg/litre Number Total 12 9 8
18
60
3
60
11
79
240(33)
26*
240( I 1)
40*
0"01 mg/litre Number Total
60 60 60 60 240(17)
15 18 15 24 72
60 60 60 60 240(30)
* Results so marked are significantly different from the controls (p < 0.05), see text.
TABLE 3 TADPOLES FEEDING OVER FIRST 48 h THAT FOOD WAS AVAILABLE (DAYS 3 AND 4 OF THE EXPERIMENT) DURING WHICH TIME TADPOI.FS WERE OBSERVED ON ELEVEN OCCASIONS. (SEE ALSO TABLE l a )
Replicate
A B
C D Group total
Control Number Total 1.0 rng;litre feeding observations Number Total 25 27 18 23 93
220 220 220 220 880( 11)
14 3 6 4 27*
220 166 171 135 692(4)
Eulan 0"1 mg,;litre Number Total 14 19 25 16 74
0.01 mg/litre Number Total
220 220 220 220 880(8)
26 13 22 19 80
220 220 220 220 880(9)
* Results so marked are significantly different from the controls (p < 0.05), sec text.
Mortalit)" Over the whole course of the experiment the mortality of tadpoles 1.0 m g / l i t r e d i s h e s w a s m u c h g r e a t e r t h a n t h a t o f t h e c o n t r o l s , o n l y six o u t o f t a d p o l e s s u r v i v i n g at t h i s d o s e c o m p a r e d w i t h s e v e n t y - e i g h t o u t o f e i g h t y c o n t r o l s , s e v e n t y - n i n e o u t o f e i g h t y in 0-01 m g / l i t r e d i s h e s a n d all e i g h t y 0. I m g / l i t r e g r o u p .
in t h e eighty in t h e in t h e
TOXICITY OF EULAN TO
Rana temporaria
121
Development The development of treated tadpoles was slower than that of the controls. This was most apparent with the 1.0 mg/litre group, none of which reached stage 30. Furthermore, in the 0. I mg/litre and 0.01 mg/litre dishes, development was slightly behind that of the controls. For example, on the 28th day of exposure twenty out of seventy-eight surviving tadpoles had reached stages 29 or 30 in the control groups, whereas only seven out ofeighty and three out of seventy-nine had reached stage 29 in the 0.1 mg/litre and 0.01 mg/litre groups. This effect was emphasised by the observation that 2 3 days after ten stage 31 tadpoles had been removed for chemical and biochemical analysis from each of the control, 0.1 mg/litre and 0.01 mg/litre dishes, 24°,0 (9,"38) of the remaining control animals were in stage 32, whilst only 15 ~, (6/39) of the 0.01 mg/litre and 5 01,(2/40) ofthc 0. ! mg/litre groups had reached this stage of development.
Abnormalities There was a slight suggestion that Eulan increased the occurrence of the type of abnormalities that occurred naturally in the tadpoles, for whereas there were two tail kinks in the control groups there were seven such kinks in the 1.0 mg/litre group. One such kink occurred in the 0.01 mg/litre group and none in the 0.1 mg/litrc group. The kinks in the 1.0 mg/litrc groups were much more severe than those in any other group.
Weight As can be seen in Table 4, 1-0 mg/litre tadpoles were lighter than the controls from day 3 onwards, and those in the 0" 1 mg/litre group lighter from day 24 until they reached stage 31 on days 35-37.0-01 mg/litrc tadpoles were lighter at stage 31 than thc controls, but the difference was not statistically significant. As expected, weight decreased between days 29 and 36. This was more pronounced in treated than in control tadpoles.
Eulan reshtues Table 5 shows the residues of Eulan found in the pooled sample ofwhole tadpoles, each made up of five individuals. The residues are of a similar order to those of D D T accumulated by R. temporaria tadpoles which had been exposed to comparable concentrations of D D T and which were suffering from the effects of D D T (Cooke, 1972). DISCUSSION
The results suggest that some effect on feeding leads to a reduction in weight during the later stages of metamorphosis of tadpoles, even when the effect on feeding was
122
D. OSBORN, M. C. FRENCH
TABLE 4 WEIGHTS OF TADPOLES(mg_+ I SE)
Day 3* 5 12 17 22 24 29 36b
Stage of de~'elopment 25/24' Not recorded in detail Not recorded in detail Not recorded indetail Not recorded indetail 28,'26-27' 29-30/26 28' 31/26 29~
Control
0"01 mg/litre
Eulan 0"1 mg/litre
22+0.7 (4)
23_+0.6 (4)
23_+0"3 (4)
19_+ 1"0 (4)*
36 _+0.5 (4)
36 +1.5 (4)
37 _+0.9 (4)
22 _+1.3 (4)*
53 + 2.8 (4)
53 _+2.8 (4)
53 + 1.3 (4)
29 + 1.9 (4)*
215+7
(4)
213+11 (4)
213-+8
(4)
373-+4 440_+ 16 590+6 541+12
(4) (4) (4) (8)
368-+25 452+ 15 588-+11 523_+9
369+9 409-+6 567_+33 493_+8
(4) (4) (4) (8)*
(4) (4) (4) (8)
I 0 mg/litre
79+8.0(3)* 146-+19 171 -+24 306_+31 382
(3)* (3)* (3)* (I)
Figures in parentheses are values of n, * = p < 0.05, Student's t-test, such results are significantly different from controls. "This weight was obtained before food was given. bTwo groups of tadpoles were weighed here. One group was used for chemical analyses and the other retained for future studies on body composition. For this reason n = 8 rather than 4. ' These figures relate to stage of development of the 1-0 mg/litre group, the others to the controls and the 0-1 and 0.01 mg/litre groups. TABLE 5 ELt.AN ('ON('EN'IRATIONS (mg k g
Control 1.0 mg/litre 0.1 mg,litre 0.01 mg/litre
~ WET WEIGHT} AFIER
35-36
DAYS OF EXPOSURE
Eulan
n
Stage of tadpole det,elopment
ND 24. I 7.71 + 1.60 0.53 ±0.11
4 1" 4 4
31 26-29 31 31
"Only one determination was possible owing to mortality.
not statistically signilicant. This conclusion would be supported by (i) the finding that hanging is also affected, since this is frequently associated with grazing on the glass surface at the side of the dish, and (ii) that weight declined more rapidly between days 29 and 36 in the treated animals than in the controls, i.e. body reserves were used more rapidly by treated animals. The effect of movement in the period when no food was present could indicate a decrease in the ability, or desire, of the animals to search for food. Thus it seems possible that the main effect of Eulan on tadpoles is to reduce weight by reducing feeding or food-secking activity. Rcduced feeding is said to be thc manner in which Eulan kills moth larvae. We would stress that in attempting to assess the environmental hazard Eulan presents, extrapolations from the nominal water concentrations quoted here should
TOXICITY OF I-.ULAN TO
Rana temporaria
123
not be made since it is possible that (i) the true water concentrations are far below the nominal values and (ii) much Eulan could be accumulated through food contamination. It would seem more appropriate in hazard assessments to use the tissue levels accumulated by these animals and the effects they brought about. Thus it is likely that an effect on feeding which could have serious implications for animals is to be expected in animals accumulating whole body concentrations of Eulan between 1 and 10 mg k g - ' wet weight, since reduced feeding could reduce individual survival and reduced body weight at metamorphosis could influence future survival, or breeding success. Similarly, mortality is likely to occur in individual members of groups of animals accumulating whole body residues in excess of 25 mg k g - ~ wet weight. It should be noted that this experiment was performed at a time when the tadpoles had passed one of their most sensitive periods to many other compounds, e.g. D D T and dieldrin, and that estimates of toxicity extrapolated from these data may underestimate the true toxicity to R. temporaria tadpoles. From these data it may be tentatively concluded that Eulan WA New seems about halfto one-fifth as toxic to tadpoles as dieldrin or D D T under similar conditions (Cooke, 1972). However, since about five times as much Eulan as dieldrin is used in mothproofing, Eulan could, at least locally, be as great an environmental hazard as dieldrin if large quantities of factory effluent containing Eulan reach waterways. However. such simple extrapolations may be naive.
ACKNOWLEDGEMENTS
We thank Drs I. Newton, J. P. Dempster and A. S. Cooke for comments on the manuscript and are indebted to Mr K. H. Lakhani for his statistical advice and other remarks. Bayer UK kindly supplied the Eulan preparation used in this study.
REFERENCES COOKE,A. S. (1972). The effectsof DDT, dieldrin and 2,4-D on amphibian spawn and tadpoles. Environ. Pollut., 3, 5l-8. SCORGIE,H. R. A. & COOKr,A. S. (1979). Effectsof the triazine herbicidecyanatryn on aquatic animals. Bull. environ. Contain. & Toxicol., 22, 135-42. WELLS, D. E. (1979). The isolation and identification of polychloro-2 (chloromethylsulphonamide) diphenyl ether isomers and their metabolites from Eulan WA New and fish tissue by gas chromatography-mass spectrometry. Analytica Ckim. Acta, 104, 253--66. W~'roO, G. & NOREr~,K. (1977). Polychlorinated 2-amidodiphenyl ethers in fish. Ambio, 6, 232--4. Wrrscm, E. (1956). Development of vertebrates. Philadelphia, Saunders.