- Email: [email protected]
The toxicity of some detergents tested on Aëdes aegypti L., Lebistes reticulatus peters, and Biomphalaria glabrata (Say)
THE TOXICITY OF SOME DETERGENTS TESTED ON AEDES AEG YPTI L., LEBISTES RETICULA TUS PETERS, AND BIOMPHALARIA GLABRA TA (SAY)
H. M. van EMDEN, C. C. M. KROON, E. N. Sc,OEMAN & H. A. VAN SEVENTER
Institute o / Tropical Hygiene, Department of the Royal Tropical Institute, Laboratory of Parasitology, University of Amsterdam, Amsterdam, The Netherlands"
ABSTRACT
Afides aegypti L., the urban vector mosquito of yellow fever, the 'guppy' Lebistes reticulatus Peters (a small tropical fish) and the snail Biomphalaria glabrata (Say), vector of the disease Bilharziasis, were exposed to different concentrations of anionic and non-ionic commercial detergents. Biodegradation products of these detergents were also tested for possible toxic action on these in water-living animals. Afides aegypti larvae are more sensitive to anionic than to non-anionic detergents. The most sensitive stage during development in the ecdysis is from 4th stage larvae to pupae. The oviposition was most frequent in the lowest available concentration. The detergent A P H - - 1 1 EO (branched nonylphenol--11 EO), with a rather long biodegradation time, has two different modes of action: it is toxic at low concentrations and causes drowning of the larvae at higher concentrations because of the lowering of the surface tension. Results with tests on Lebistes reticulatus show irreversible damage of the epithelium of the gills by anionic, as well as long chained non-ionics, and a reversible paralysing effect with short (and branched) non-ionic detergents. Adaptation to the paralysing effect is possible by slowly increasing concentrations far above the LC ~oo 24 h. Biomphalaria glabrata is equally susceptible to anionic and non-ionic detergents. Survival is possible after exposure in the LC~ oo 24 h concentration if the snails are placed in fresh water after 20 h. Biodegradation products of 1000 ppm detergent have no influence on the mortality of the three tested animals.
INTRODUCTION
The development of synthetic surface-active products has taken place primarily since the First World War. The research was intended to develop washing prep297 Era,iron. Pollut. (6) (1974)--© Applied Science Publishers Ltd, England, 1974 Printed in Great Britain
298
H . M . VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A. VAN SEVENTER
arations less sensitive to water-containing calcium and magnesium salts (hard water) than the fatty acid soaps. We differentiate between four surface-active materials: anion active, cation active, non-ionic substances and ampholytes. The most important group are the anion active compounds, which include fatty acid soaps. Other examples are primary and secondary alkyl sulphates and alkyl benzene sulphonates. The non-anionic compounds, which are generally based on ethene- and propene-oxide, have recently been produced in rapidly increasing numbers. The cation active compounds have primarily a bactericide action, while the ampholytes are rather expensive specialities (for instance shampoos). This study concerns the toxicity of some anionic active or non-ionic surface-active substances and their biodegradation products tested on three different groups of animals living in water: a mosquito, a fish and a snail: 1 : A~des aegypti L; the yellow fever transmitting mosquito of urban areas. The strain used in the tests originally came from India. 2: Lebistes reticulatus Peters, 'Guppies', a fish used to try to control mosquitoes. 3: Biomphalaria glabrata (Say) a snail and vector of the tropical disease Bilharziasis. All these animals have been cultured in the laboratory for many years.
MATERIALS
Anionic active detergents LAS 345: A linear alkyl benzene sulphate with 10-13 carbon atoms in a straightchain and an average molecule weight of 345; 2 6 . 8 ~ A M (Active Material). LAS 358: A linear alkyl benzene sulphate with 10-15 carbon atoms in a straightchain and an average molecule weight of 358; 16.4 ~ AM. PAS 311: A sulphated primary alcohol with 12-15 carbon atoms in a straightchain and an average molecule weight of 311 ; 23-0~ AM. PAES : A sulphated polyglycol ether of a primary alcohol with 3 EO groups on each molecule, 59.5 ~o AM.
Non-ionic detergents PA - - 9EO:
A polyglycol ether of a primary alcohol with 12-15 carbon atoms in a straight-chain and 9 EO groups on each molecule, 100%AM. APH - - ! lEO: A polyglycol ether of an alkylphenol with a branched chain of 9 carbon atoms and 11 EO groups on each molecule, 100 %AM. 'TEEPOL CH - - 53': A mixture of a linear alkylbenzene sulphate and an alkylphenol ethene-oxide condensate, 30 % AM.
TOXICITY OF D E T E R G E N T S TO MOSQUITOES A N D O T H E R O R G A N I S M S
299
The ppm concentrations used in our tests are related to the Active Materials (AM) in the different substances.
ANALYTICAL METHODS
The anion active detergents were tested by the methods of Longwell & Maniece (1955). Standard curves were drawn for concentrations between 0 and 25 ppm. No standard curves could be drawn for non-ionic detergents by the method described by Huddleston & Allred (1965) because of the laborious method and its inaccuracy. The tests with the animals were conducted at a temperature of 25°C and with 12 h TL light/day. 1. R e s u l t s with A~des aegypti, 24 h e x p o s u r e
The LCloo in 24 h, and LCso in 24 h were determined for 2nd and 3rd stage larvae (Table 1.) TABLE 1 24 h OF 40 2ND AND 3RD STAGE A~des aegypti IN DIFFERENTDETERGENTS
LCs0 AND L C I 0 0 - -
Detergent
LAS 345 LAS 358 PAS 311 PAES PA - - 9 EO APH -- I1 EO TEEPOL CH53
LARVAE OF
LCso - - 24 h in ppm - - A M
LCI oo - - 24 h in ppm - - A M
6 2 4 11 nd 500 nd
8 3 5 12 200 1000 170
nd = notdone. Tests lasting a period of more than 24 h could be performed with the detergent APH - - I l EO, because it has a rather slow biodegradation. 2. R e s u l t s with A~des aegypti e x p o s u r e to A P H -
11 E O f o r longer p e r i o d s
Those concentrations were examined at which: (A) (B) (C) (D) (E)
50 ~ 50~ "5 0 ~ 50~ 50 ~
of of of of of
the the the the the
eggs were deposited. eggs hatched. 2nd and 3rd stage larvae died (LCs0). pupae died (LCso). mosquitoes hatched from the pupae.
All compared with the controls.
300
H. M. VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A. VAN SEVENTER TABLE 2 EGGSOF A~des aegypti 1N APH
ppm--AM
First experiment Second experiment 100~7 103S3' 5 3 ~ 24d'~ No. of eggs laid No. of eggs laid
210 nd nd 0 0 0 22 nd nd
Control 5 20 25 50 75 100 150 200
225 183 nd 79 nd 85 51 45 0
-
-
11
EO
Third experiment Fourth experiment 4 3 ~ 12d'd' 29~ lld'~ No. of eggs laid No. of eggs laid
nd nd 130 nd 0 0 0 nd nd
nd nd nd 59 nd 35 38 13 nd
n d = not done.
(A) The n u m b e r of eggs deposited at different c o n c e n t r a t i o n s of A P H - - 11 E O were determined (Table 2). This experiment was conducted with 100 newly-hatched female mosquitoes and 103 newly-hatched male mosquitoes placed in a m o s q u i t o cage and provided with sugar-water. A b l o o d m e a l from a rabbit was given. Five days 'after this bloodmeal the eggs deposited in petri-dishes with different concentrations of A P H - - 1 1 E O were counted. Two days after the first c o u n t a fresh bloodmeal was provided. Again, after 5 days, the n u m b e r of eggs was counted. The third a n d fourth experiments were conducted in a similar fashion and each time the deposited eggs were counted after a lapse of 5 days. Discussion o f Table 2: F r o m the results it seems that the female mosquitoes prefer to deposit their eggs at the lowest c o n c e n t r a t i o n of detergents. I n the first experiment, the majority of eggs were deposited in the control, in the second experiment in the control and at the 5 p p m concentration. I n experiments three a n d four, in which n o control was provided, most eggs were deposited at the lowest available c o n c e n t r a t i o n s of 20 a n d 25 ppm.
TABLE 3 OUT OF 60 DEPOSITEGGS IN APH (ppm -- AM) ATDIFFERENTCONCENTRATIONS
NUMBER OF EGGS HATCHED
--
11
EO
ppm
Hatched e g g s
Hatched eggs in %
Control 50 100 150 200 375 600 1000
45 46 42 42 36 25 20 12
75 77 70 70 60 42 33 20
TOXICITY OF DETERGENTSTO MOSQUITOESAND OTHER ORGANISMS
301
(B) 5 0 ~ of the deposited eggs hatch: In each c o n c e n t r a t i o n of A P H - - 11 EO were placed two batches o f 30 eggs. After 10 days the n u m b e r of live and dead larvae were counted. [nterpolation gives a c o n c e n t r a t i o n of + 500 ppm A P H - - 11 EO at which 50 ~o of the total n u m b e r of eggs hatch, compared with the hatch of the control eggs (Table 3). (C) LCs o .for larvae of 2nd and 3rd stage exposed to concentrations of A P H - - 11 EO: The d u r a t i o n of the tests at which the 2nd a n d 3rd stage larvae of A~desaegypti were exposed to different c o n c e n t r a t i o n s of A P H - - 11 EO was 12 days. This was only possible with this detergent because the biodegradation is rather slow. Table 4 shows the n u m b e r of dead a n d living larvae at the different c o n c e n t r a t i o n s of this particular detergent. P u p a e were n o t included in the table. TABLE 4 LC50 OF 2ND AND 3RD STAGELARV-AEOF A~des aegypti AT DIFFERENTCONCENTRATIONS OF A P N - - 11 EO (PPM- - AM)
Hours
ppm detergent Control 32 50 100 150 200 250 275 500 750 1000
0*
24
48
72
96
20 20 10 10 10 10 10 120 80 80 40
20 20 10 91 10 10 10 8139 4040 2555 040
20 20 10 9 10 10 91 4041 2515 223
20 20 10 9 10 82 63 319 205 11
20 20 10 81 10 71 33 1813 163 1
120 144 168 192 216 240 264 288 20 19 10 8 9j 52 21 153 12 4 1
20 18 16 19 19 19 10 91 7 6 6 6 9 63 41 32 21 lJ 11 1 Ol 123 66 0 6 4 12 0 1 0
15 14 14 14 19 18 16 16 51 3 0 5 5 3 1 3 3 3 I 1
1
1
0
* Number of original larvae. 'missing' numbers indicate dead or alive pupae. exponents indicate the number of dead larvae. A graph was m a d e showing the LCso for different c o n c e n t r a t i o n s of A P H - - 11 EO. T h e n u m b e r o f exposed larvae in each c o n c e n t r a t i o n was called 100K, the dead larvae were c o u n t e d as a percentage a n d deducted from the original 1 0 0 ~ . Dead or live pupae were n o t counted. Discussion of Fig. l : It seems that this graph is composed of two different straight lines. This gives the impression that at higher c o n c e n t r a t i o n s two different processes are at w o r k : I. T h e lowering o f the surface tension. 2. The toxicity of the detergent. If the larvae are n o longer able to adhere to the surface to exchange CO2 and 0 2 they drown. Russell & R a o (1941) gave as critical surface tension for Culex sp.
302
H . M . VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A, VAN SEVENTER
ppun 1000 •
I
|
900. l l 800
l I I
700
600
500
400
300 I 200
100
days Fig. 1.
L C s 0 of 2nd and 3rd stage larvae of A(des aegypti by different concentrations of A P H - - 11 E O (in p p m - - AM).
larvae 26-8 dynes (the normal surface tension of water/cm varies from 75-64 at 0°C to 58.85 at 100°C). Below the concentration of 285 ppm the surface tension is so high that the death of the larvae is not caused by drowning but can only be accounted for as being due to the toxicity of the detergent. Above this concentration both processes have their influence. (D) Determination o f the LCso on AEdes aegypti pupae with A P H - - 1 1 EO: Many dead pupae were found in experiment (C). Tests with 2 × 20 larvae in the 4th stage of development with rising concentrations of APH - - 11 EO showed that 50 ~o of the pupae stayed alive for 10 days at 50 ppm (Fig. 2). This test was repeated
TOXICITY OF DETERGENTS TO MOSQUITOES AND OTHER ORGANISMS
[]
larvae
~-~ Mosquitoes
Pupae
~
303
Pupae t
40
36
32 i,.
0 _m
28
IE 8
m
24
20
16
12
1
Fig. 2.
2
3
4
5
6
7
8
9 10 days
11
L C 50 o f Ak'des aegypti pupae, w h e n 40 4th stage larvae are exposed to 50 ppm - - A M . A P H -- 11 EO.
with 95 larvae in 3rd and 4th stage provided with newly-made solutions after 10 days because of a possible effect of biodegradation. After 14 days a total of 50 live mosquitoes and 45 dead pupae was counted. The sex ratio, according to Christophers (1960), is 45 ~ female. In our tests about 75 ~o live females developed. It seems that the smaller male pupae are more susceptible to the toxic detergent than the much larger female pupae, hence this different sex ratio in our experiments. The death of
304
H. M. V A N E M D E N , C. C. M. K R O O N , E. N . S C H O E M A N , H. A. V A N SEVENTER
the pupae occurs directly after ecdysis of the 4th stage larvae. The newly-developed cuticula of the fresh pupae is much more susceptible to the toxic action of the detergent. (E) From the surviving pupae live mosquitoes developed without any difficulty. The slight lowering of the surface tension at this concentration of 50 ppm has no influence on this process. Experiments on the effect of insecticides on pupae as performed by LiJdemann & Neumann (1960) showed that the pupae are much less susceptible to insecticides. A similar situation apparently exists with the toxic influence of detergents on pupae. 3. R e s u l t s with Lebistes reticulatus b y 24 h e x p o s u r e
The guppies used in the experiments were females with a weight between 0-12 and 0.38 g and a length of approximately 2 cm. Male fishes weighed between 0.05 g and 0.08 g and had a length of 2 cm. Young, 14-days-old fishes of both sexes were also used in the experiments; they weighed +0.007 g and were 0-7 cm long. To establish a curve of the LCso and LCloo at different concentrations of detergent is hardly possible for two reasons. 1. There is a very small margin in the detergent concentrations at which the fishes are all alive or are all dead. 2. Very low detergent concentrations are necessary to kill the fishes. Therefore the error in the amount of detergent is more critical than in the preparations of higher concentrations. Preliminary tests were performed at different concentrations in which 3 females, 3 males and 5 young fishes were tested. To establish the LC 1o o 24 h, new tests were carried out and repeated at intermediate concentrations (Table 5). The LCloo 24 h was established by interpolation from the different results at varying concentrations. It turned out that the LCso 24 h is very close to the concentration used for the LC1 oo 24 h.
TABLE 5 INFLUENCE OF DIFFERENT DETERGENTS IN PPM - - AM TESTED ON FEMALES~ MALES AND YOUNG OF Lebistes reticulatus
Detergent Tested on
LAS 345 LAS 358 PAS 311 PAES PA -- 9 EO APH - - 11 EO TEEPOL CH53 nd = not done.
LC10o -- 24 h in ppm - - A M ~ dd Young
2 1 2 5 9 57 4
2 1 2 8 11 64 4
1 0.5 0'5 4 6 52 nd
0 42 42 44 46 48 50 52
ppm
3-3 2-2 2-2 2-2 2-2 2-2 2-2 2-2
3-3 2-2 2-2 2-2 2-2 2-2 2-2 2-2
~-~ ?-~
Days 7-14 3-3 2-2 2-2 2-1 1-2 2-2 2-2 1-2
~-~ ?-~
Days 14-21
0 3-3 52 2-2 52 2-2 54 2-1 56 1-2 58 2-2 60 2-2 62 1-2
ppm 3-3 2-2 1-2~ 2-1 1-2l 2-2 2-2l 1-2
~-~ ?-~
Days 21-28
0 3-3 62 2-2 62 1-2 64 2-1 66 1-2 68 2-2 70 2-2 72 1-2
ppm
3-4
*82
92
3--4 88
3-4
*78 3-3
¢-~ ~-¢
Days 35-42
1-1
0-2
1-1
0-2
0 3-3 3-3 82 0-2 0-2 84 0-2 0--2
ppm
3-3 I-2 2-3
?-~ ~-~
Days 28-35
0 3-3 72 2-2 *74 3-3
ppm
* After the death of some fishes combinations of batches were made to test higher concentrations.
3-3 2-2 2-2 2-2 2-2 2-2 2-2 2-2
?-~ ~-~
Days 0-7
Control3-3 32 2-2 32 2-2 34 2-2 36 2-2 38 2-2 40 2-2 42 2-2
ppm
102
98
0 92 94
ppm
0-0
0-2
3-3 0-2 0-0
0-2
3-3 0-2
~-~ ~-~
Days 42-49
TABLE 6 ADAPTATION OF Lebistes reticulatus TO DIFFERENTCONCENTRATIONSOF APH m l 1 EO (PPM - - AM)
108
0 102
ppm
0-0
3-3 04)
~-~
Day 49
E~
,/
©
M
© -I
~7
.q O t-M
-] O
Z
tr~ ..] rn
-] O
306
H . M . VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A. VAN SEVENTER
Adaptation tests in A P H - - 11 EO with Lebistes reticulatus: With guppies it was possible to adapt them from sublethal dose concentrations to concentrations far above the original LCloo 24 h. For this purpose the guppies were placed in 1 litre beakers in a sublethal concentration of A P H - 11 EO. After seven days each batch of fishes was placed in a new concentration 10 p p m higher than the last one (Table 6). Discussion of results with Lebistes reticulatus: Lang (1963) and Liebmann (1967) explained the two different actions of detergents on fishes. (a) A permanent damage of the gills occurs by anionic detergent and non-ionic detergents with a long C chain and/or an ethylene oxide chain, through the change of surface tension from which the fish dies. When the fishes had been replaced in fresh water no recovery took place. Lang's and Liebmann's descriptions of the struggle before the fish dies were the same as that observed in our experiments, with the exception of A P H - - 11 EO which has, however, a non-ionic branched short C chain. Liebmann claims that the toxicity of the detergent increases when the C chain is longer and when the EO chain is shorter and that no damage was observed by the detergent in the alimentary tract. (b) Short-chained (branched) non-ionic detergents cause little damage to the gills. Histological examination shows that this damage cannot be the cause of death. The above-mentioned workers explain the death of the fishes by action on the central nervous system which paralyses the respiration. In a number of cases spontaneous recovery occurred in the same solution in which the fishes are tested. In all our experiments the fishes recovered when they were placed in fresh water after a short exposure. This is totally different from the results as described under (a) above. These paralysing effects were only seen with A P H - - 1 1 EO detergent. The lethal concentration of the detergent with this typical paralysing effect can be raised to a much higher concentration when initially the fishes are brought in a sublethal dose and the concentration is slowly increased. 4. Results with Biomphalaria glabrata by 24 h exposure The size of the snails varied from 1.5 cm to 2 cm. The criteria for death was that the foot of the snail, when pinched by blunt tweezers, showed no reaction at all. The water in our tests was copper-free tap water. In each experiment two batches of 3 snails in 250 cc water were tested, together with controls (Table 7).
hTfluence of biodegradation products o[ the detergents The prepared solutions were put in one-litre glass bottles and continuously aerated.
T O X I C I T Y OF DETERGENTS TO MOSQUITOES A N D O T H E R ORGANISMS
307
TABLE 7 LCj00 -- 24 h OF Biomphalariaglabrata IN DIFFERENT DETERGENTS (PPM - - AM)
Detergent
LC~ oo -- 24 h
LAS 345 LAS 358 PAS 311 PAES PA - - 9 EO APH - - 11 EO TEEPOL CH 53
3 1 4 12 11 23 7
At intervals the biodegradation of anionic active detergents was measured. Total degradation takes place by all tested detergents with this method in about a month for concentrations of 32 ppm. If activated sludge from the Sewage Works at Amsterdam was added as a bacterial source it took about one week to break down 32 ppm and about 4 weeks to break down a concentration of 1000 ppm. The method of Longwell & Maniece (1955) shows errors because of the developing H/S and SO2 which interfere with the test. It is possible that after four weeks not all the detergent is broken down, but is absorbed by the sludge (Borstlap & Kooyman, 1963) and therefore seems to have disappeared from the solution. The animals in our tests were exposed after filtration of the sludge.
5. Results with A~des aegypti larvae on biodegradation products of detergents Two batches of ten 4th stage larvae were exposed to biodegradation products of original 1000 pprr] solution. LAS 345 showed 3 dead pupae, T E E P O L CH - - 53 one dead larva and 2 dead pupae and APH - - 11 EO only one dead larva. None of the controls showed any dead larvae or dead pupae. All the other tested detergents after biodegradation showed no mortality in the tests.
6. Results of the hatching of eggs of A~des aegypti in biodegradation products of L A S 358 In our tests and in the controls 8 batches of 20 eggs were exposed to biodegradation products of originally 1000 ppm LAS 358. Four batches of 20 eggs were submerged. This was done to test possible surface tension action of the biodegradation products. Hatched larvae were counted after 23 days. The results were:
Surface eggs Control LAS 358
51 larvae 63.75 ~o 53 larvae 66.25 ~
Submerged eggs 61 larvae 76.25 61 larvae 76.25
Conclusion: The biodegradation products have no influence on the hatching of eggs.
308
H.M. VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A. VAN SEVENTER
Note." Gillett (1955) states that submerged eggs hatch better t h a n floating eggs; this also occurred in our tests. Influence o f biodegradation products o f detergents on fishes and snails The influence of these biodegradation products of the various detergents was tested on the fishes a n d the snails. After 9 days n o difference was f o u n d in mortality between the exposed animals and the controls in fresh water.
ACKNOWLEDGEMENT The authors would like to express their gratitude to the research workers of the Royal D u t c h Shell L a b o r a t o r y in A m s t e r d a m for their valuable advice and the supplies of the detergents.
REFERENCES
BORSTLAP, C. d~. KOOYMAN,P. L. (1963). A study of the biodegradation of anionic synthetic detergents. J. Am. Oil Chem. Soc., 40, 78-80. CHRISTOPHERS, S. R. (1960). A~des aegypti L., the yellow fever mosquito. Cambridge, University Press. GILLETT,J. D. 0955). Variation in the hatching-response of A~des eggs (Diptera: Culicidae). Bull. ent. Res., 46, 241-54. HUDDLESTON,R. L. • ALLRED,R. C. (1965). Determination of non-ionic surfactants, biodegradability physical properties v.s. colorimetry. J. Am. Oil Chem. Soc., 46, 983-6. LANG, W. (1963). Untersuchungen zur Wirkungsweise yon grenzfldchenaktiven Stoffen auf histoIogische Besehaffenheit und Funktion verschiedener Organe bei Fischen. Ph.D. Thesis, University of Cologne. LIEBMANN, H. (1967). Detergentien und Ole im Wasser und Abwasser. Munich and Vienna, Oldenbourg. LONGWELL,J. & MAN1ECE,W. D. (1955). Determination of anionic detergents in sewage, sewage effluence and river waters. Analyst, Lond., 80, 167-71. LODEMANN,D. & NEUMANN,H. Z. (1960). Versuche fiber die akute toxische Wirkung neuzeitlicher Kontaktinsektizide auf Siisswassertiere. Z. angew. Zool., 47, 303-21,493-505. RUSSELL, P. F. • RAMACHANDRARAO, T. (1941). On surface tension of water in relation to behavior of Anopheles larvae. Am. J. trop. Med. Hyg., 21, 767-77.
H. M. van EMDEN, C. C. M. KROON, E. N. Sc,OEMAN & H. A. VAN SEVENTER
Institute o / Tropical Hygiene, Department of the Royal Tropical Institute, Laboratory of Parasitology, University of Amsterdam, Amsterdam, The Netherlands"
ABSTRACT
Afides aegypti L., the urban vector mosquito of yellow fever, the 'guppy' Lebistes reticulatus Peters (a small tropical fish) and the snail Biomphalaria glabrata (Say), vector of the disease Bilharziasis, were exposed to different concentrations of anionic and non-ionic commercial detergents. Biodegradation products of these detergents were also tested for possible toxic action on these in water-living animals. Afides aegypti larvae are more sensitive to anionic than to non-anionic detergents. The most sensitive stage during development in the ecdysis is from 4th stage larvae to pupae. The oviposition was most frequent in the lowest available concentration. The detergent A P H - - 1 1 EO (branched nonylphenol--11 EO), with a rather long biodegradation time, has two different modes of action: it is toxic at low concentrations and causes drowning of the larvae at higher concentrations because of the lowering of the surface tension. Results with tests on Lebistes reticulatus show irreversible damage of the epithelium of the gills by anionic, as well as long chained non-ionics, and a reversible paralysing effect with short (and branched) non-ionic detergents. Adaptation to the paralysing effect is possible by slowly increasing concentrations far above the LC ~oo 24 h. Biomphalaria glabrata is equally susceptible to anionic and non-ionic detergents. Survival is possible after exposure in the LC~ oo 24 h concentration if the snails are placed in fresh water after 20 h. Biodegradation products of 1000 ppm detergent have no influence on the mortality of the three tested animals.
INTRODUCTION
The development of synthetic surface-active products has taken place primarily since the First World War. The research was intended to develop washing prep297 Era,iron. Pollut. (6) (1974)--© Applied Science Publishers Ltd, England, 1974 Printed in Great Britain
298
H . M . VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A. VAN SEVENTER
arations less sensitive to water-containing calcium and magnesium salts (hard water) than the fatty acid soaps. We differentiate between four surface-active materials: anion active, cation active, non-ionic substances and ampholytes. The most important group are the anion active compounds, which include fatty acid soaps. Other examples are primary and secondary alkyl sulphates and alkyl benzene sulphonates. The non-anionic compounds, which are generally based on ethene- and propene-oxide, have recently been produced in rapidly increasing numbers. The cation active compounds have primarily a bactericide action, while the ampholytes are rather expensive specialities (for instance shampoos). This study concerns the toxicity of some anionic active or non-ionic surface-active substances and their biodegradation products tested on three different groups of animals living in water: a mosquito, a fish and a snail: 1 : A~des aegypti L; the yellow fever transmitting mosquito of urban areas. The strain used in the tests originally came from India. 2: Lebistes reticulatus Peters, 'Guppies', a fish used to try to control mosquitoes. 3: Biomphalaria glabrata (Say) a snail and vector of the tropical disease Bilharziasis. All these animals have been cultured in the laboratory for many years.
MATERIALS
Anionic active detergents LAS 345: A linear alkyl benzene sulphate with 10-13 carbon atoms in a straightchain and an average molecule weight of 345; 2 6 . 8 ~ A M (Active Material). LAS 358: A linear alkyl benzene sulphate with 10-15 carbon atoms in a straightchain and an average molecule weight of 358; 16.4 ~ AM. PAS 311: A sulphated primary alcohol with 12-15 carbon atoms in a straightchain and an average molecule weight of 311 ; 23-0~ AM. PAES : A sulphated polyglycol ether of a primary alcohol with 3 EO groups on each molecule, 59.5 ~o AM.
Non-ionic detergents PA - - 9EO:
A polyglycol ether of a primary alcohol with 12-15 carbon atoms in a straight-chain and 9 EO groups on each molecule, 100%AM. APH - - ! lEO: A polyglycol ether of an alkylphenol with a branched chain of 9 carbon atoms and 11 EO groups on each molecule, 100 %AM. 'TEEPOL CH - - 53': A mixture of a linear alkylbenzene sulphate and an alkylphenol ethene-oxide condensate, 30 % AM.
TOXICITY OF D E T E R G E N T S TO MOSQUITOES A N D O T H E R O R G A N I S M S
299
The ppm concentrations used in our tests are related to the Active Materials (AM) in the different substances.
ANALYTICAL METHODS
The anion active detergents were tested by the methods of Longwell & Maniece (1955). Standard curves were drawn for concentrations between 0 and 25 ppm. No standard curves could be drawn for non-ionic detergents by the method described by Huddleston & Allred (1965) because of the laborious method and its inaccuracy. The tests with the animals were conducted at a temperature of 25°C and with 12 h TL light/day. 1. R e s u l t s with A~des aegypti, 24 h e x p o s u r e
The LCloo in 24 h, and LCso in 24 h were determined for 2nd and 3rd stage larvae (Table 1.) TABLE 1 24 h OF 40 2ND AND 3RD STAGE A~des aegypti IN DIFFERENTDETERGENTS
LCs0 AND L C I 0 0 - -
Detergent
LAS 345 LAS 358 PAS 311 PAES PA - - 9 EO APH -- I1 EO TEEPOL CH53
LARVAE OF
LCso - - 24 h in ppm - - A M
LCI oo - - 24 h in ppm - - A M
6 2 4 11 nd 500 nd
8 3 5 12 200 1000 170
nd = notdone. Tests lasting a period of more than 24 h could be performed with the detergent APH - - I l EO, because it has a rather slow biodegradation. 2. R e s u l t s with A~des aegypti e x p o s u r e to A P H -
11 E O f o r longer p e r i o d s
Those concentrations were examined at which: (A) (B) (C) (D) (E)
50 ~ 50~ "5 0 ~ 50~ 50 ~
of of of of of
the the the the the
eggs were deposited. eggs hatched. 2nd and 3rd stage larvae died (LCs0). pupae died (LCso). mosquitoes hatched from the pupae.
All compared with the controls.
300
H. M. VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A. VAN SEVENTER TABLE 2 EGGSOF A~des aegypti 1N APH
ppm--AM
First experiment Second experiment 100~7 103S3' 5 3 ~ 24d'~ No. of eggs laid No. of eggs laid
210 nd nd 0 0 0 22 nd nd
Control 5 20 25 50 75 100 150 200
225 183 nd 79 nd 85 51 45 0
-
-
11
EO
Third experiment Fourth experiment 4 3 ~ 12d'd' 29~ lld'~ No. of eggs laid No. of eggs laid
nd nd 130 nd 0 0 0 nd nd
nd nd nd 59 nd 35 38 13 nd
n d = not done.
(A) The n u m b e r of eggs deposited at different c o n c e n t r a t i o n s of A P H - - 11 E O were determined (Table 2). This experiment was conducted with 100 newly-hatched female mosquitoes and 103 newly-hatched male mosquitoes placed in a m o s q u i t o cage and provided with sugar-water. A b l o o d m e a l from a rabbit was given. Five days 'after this bloodmeal the eggs deposited in petri-dishes with different concentrations of A P H - - 1 1 E O were counted. Two days after the first c o u n t a fresh bloodmeal was provided. Again, after 5 days, the n u m b e r of eggs was counted. The third a n d fourth experiments were conducted in a similar fashion and each time the deposited eggs were counted after a lapse of 5 days. Discussion o f Table 2: F r o m the results it seems that the female mosquitoes prefer to deposit their eggs at the lowest c o n c e n t r a t i o n of detergents. I n the first experiment, the majority of eggs were deposited in the control, in the second experiment in the control and at the 5 p p m concentration. I n experiments three a n d four, in which n o control was provided, most eggs were deposited at the lowest available c o n c e n t r a t i o n s of 20 a n d 25 ppm.
TABLE 3 OUT OF 60 DEPOSITEGGS IN APH (ppm -- AM) ATDIFFERENTCONCENTRATIONS
NUMBER OF EGGS HATCHED
--
11
EO
ppm
Hatched e g g s
Hatched eggs in %
Control 50 100 150 200 375 600 1000
45 46 42 42 36 25 20 12
75 77 70 70 60 42 33 20
TOXICITY OF DETERGENTSTO MOSQUITOESAND OTHER ORGANISMS
301
(B) 5 0 ~ of the deposited eggs hatch: In each c o n c e n t r a t i o n of A P H - - 11 EO were placed two batches o f 30 eggs. After 10 days the n u m b e r of live and dead larvae were counted. [nterpolation gives a c o n c e n t r a t i o n of + 500 ppm A P H - - 11 EO at which 50 ~o of the total n u m b e r of eggs hatch, compared with the hatch of the control eggs (Table 3). (C) LCs o .for larvae of 2nd and 3rd stage exposed to concentrations of A P H - - 11 EO: The d u r a t i o n of the tests at which the 2nd a n d 3rd stage larvae of A~desaegypti were exposed to different c o n c e n t r a t i o n s of A P H - - 11 EO was 12 days. This was only possible with this detergent because the biodegradation is rather slow. Table 4 shows the n u m b e r of dead a n d living larvae at the different c o n c e n t r a t i o n s of this particular detergent. P u p a e were n o t included in the table. TABLE 4 LC50 OF 2ND AND 3RD STAGELARV-AEOF A~des aegypti AT DIFFERENTCONCENTRATIONS OF A P N - - 11 EO (PPM- - AM)
Hours
ppm detergent Control 32 50 100 150 200 250 275 500 750 1000
0*
24
48
72
96
20 20 10 10 10 10 10 120 80 80 40
20 20 10 91 10 10 10 8139 4040 2555 040
20 20 10 9 10 10 91 4041 2515 223
20 20 10 9 10 82 63 319 205 11
20 20 10 81 10 71 33 1813 163 1
120 144 168 192 216 240 264 288 20 19 10 8 9j 52 21 153 12 4 1
20 18 16 19 19 19 10 91 7 6 6 6 9 63 41 32 21 lJ 11 1 Ol 123 66 0 6 4 12 0 1 0
15 14 14 14 19 18 16 16 51 3 0 5 5 3 1 3 3 3 I 1
1
1
0
* Number of original larvae. 'missing' numbers indicate dead or alive pupae. exponents indicate the number of dead larvae. A graph was m a d e showing the LCso for different c o n c e n t r a t i o n s of A P H - - 11 EO. T h e n u m b e r o f exposed larvae in each c o n c e n t r a t i o n was called 100K, the dead larvae were c o u n t e d as a percentage a n d deducted from the original 1 0 0 ~ . Dead or live pupae were n o t counted. Discussion of Fig. l : It seems that this graph is composed of two different straight lines. This gives the impression that at higher c o n c e n t r a t i o n s two different processes are at w o r k : I. T h e lowering o f the surface tension. 2. The toxicity of the detergent. If the larvae are n o longer able to adhere to the surface to exchange CO2 and 0 2 they drown. Russell & R a o (1941) gave as critical surface tension for Culex sp.
302
H . M . VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A, VAN SEVENTER
ppun 1000 •
I
|
900. l l 800
l I I
700
600
500
400
300 I 200
100
days Fig. 1.
L C s 0 of 2nd and 3rd stage larvae of A(des aegypti by different concentrations of A P H - - 11 E O (in p p m - - AM).
larvae 26-8 dynes (the normal surface tension of water/cm varies from 75-64 at 0°C to 58.85 at 100°C). Below the concentration of 285 ppm the surface tension is so high that the death of the larvae is not caused by drowning but can only be accounted for as being due to the toxicity of the detergent. Above this concentration both processes have their influence. (D) Determination o f the LCso on AEdes aegypti pupae with A P H - - 1 1 EO: Many dead pupae were found in experiment (C). Tests with 2 × 20 larvae in the 4th stage of development with rising concentrations of APH - - 11 EO showed that 50 ~o of the pupae stayed alive for 10 days at 50 ppm (Fig. 2). This test was repeated
TOXICITY OF DETERGENTS TO MOSQUITOES AND OTHER ORGANISMS
[]
larvae
~-~ Mosquitoes
Pupae
~
303
Pupae t
40
36
32 i,.
0 _m
28
IE 8
m
24
20
16
12
1
Fig. 2.
2
3
4
5
6
7
8
9 10 days
11
L C 50 o f Ak'des aegypti pupae, w h e n 40 4th stage larvae are exposed to 50 ppm - - A M . A P H -- 11 EO.
with 95 larvae in 3rd and 4th stage provided with newly-made solutions after 10 days because of a possible effect of biodegradation. After 14 days a total of 50 live mosquitoes and 45 dead pupae was counted. The sex ratio, according to Christophers (1960), is 45 ~ female. In our tests about 75 ~o live females developed. It seems that the smaller male pupae are more susceptible to the toxic detergent than the much larger female pupae, hence this different sex ratio in our experiments. The death of
304
H. M. V A N E M D E N , C. C. M. K R O O N , E. N . S C H O E M A N , H. A. V A N SEVENTER
the pupae occurs directly after ecdysis of the 4th stage larvae. The newly-developed cuticula of the fresh pupae is much more susceptible to the toxic action of the detergent. (E) From the surviving pupae live mosquitoes developed without any difficulty. The slight lowering of the surface tension at this concentration of 50 ppm has no influence on this process. Experiments on the effect of insecticides on pupae as performed by LiJdemann & Neumann (1960) showed that the pupae are much less susceptible to insecticides. A similar situation apparently exists with the toxic influence of detergents on pupae. 3. R e s u l t s with Lebistes reticulatus b y 24 h e x p o s u r e
The guppies used in the experiments were females with a weight between 0-12 and 0.38 g and a length of approximately 2 cm. Male fishes weighed between 0.05 g and 0.08 g and had a length of 2 cm. Young, 14-days-old fishes of both sexes were also used in the experiments; they weighed +0.007 g and were 0-7 cm long. To establish a curve of the LCso and LCloo at different concentrations of detergent is hardly possible for two reasons. 1. There is a very small margin in the detergent concentrations at which the fishes are all alive or are all dead. 2. Very low detergent concentrations are necessary to kill the fishes. Therefore the error in the amount of detergent is more critical than in the preparations of higher concentrations. Preliminary tests were performed at different concentrations in which 3 females, 3 males and 5 young fishes were tested. To establish the LC 1o o 24 h, new tests were carried out and repeated at intermediate concentrations (Table 5). The LCloo 24 h was established by interpolation from the different results at varying concentrations. It turned out that the LCso 24 h is very close to the concentration used for the LC1 oo 24 h.
TABLE 5 INFLUENCE OF DIFFERENT DETERGENTS IN PPM - - AM TESTED ON FEMALES~ MALES AND YOUNG OF Lebistes reticulatus
Detergent Tested on
LAS 345 LAS 358 PAS 311 PAES PA -- 9 EO APH - - 11 EO TEEPOL CH53 nd = not done.
LC10o -- 24 h in ppm - - A M ~ dd Young
2 1 2 5 9 57 4
2 1 2 8 11 64 4
1 0.5 0'5 4 6 52 nd
0 42 42 44 46 48 50 52
ppm
3-3 2-2 2-2 2-2 2-2 2-2 2-2 2-2
3-3 2-2 2-2 2-2 2-2 2-2 2-2 2-2
~-~ ?-~
Days 7-14 3-3 2-2 2-2 2-1 1-2 2-2 2-2 1-2
~-~ ?-~
Days 14-21
0 3-3 52 2-2 52 2-2 54 2-1 56 1-2 58 2-2 60 2-2 62 1-2
ppm 3-3 2-2 1-2~ 2-1 1-2l 2-2 2-2l 1-2
~-~ ?-~
Days 21-28
0 3-3 62 2-2 62 1-2 64 2-1 66 1-2 68 2-2 70 2-2 72 1-2
ppm
3-4
*82
92
3--4 88
3-4
*78 3-3
¢-~ ~-¢
Days 35-42
1-1
0-2
1-1
0-2
0 3-3 3-3 82 0-2 0-2 84 0-2 0--2
ppm
3-3 I-2 2-3
?-~ ~-~
Days 28-35
0 3-3 72 2-2 *74 3-3
ppm
* After the death of some fishes combinations of batches were made to test higher concentrations.
3-3 2-2 2-2 2-2 2-2 2-2 2-2 2-2
?-~ ~-~
Days 0-7
Control3-3 32 2-2 32 2-2 34 2-2 36 2-2 38 2-2 40 2-2 42 2-2
ppm
102
98
0 92 94
ppm
0-0
0-2
3-3 0-2 0-0
0-2
3-3 0-2
~-~ ~-~
Days 42-49
TABLE 6 ADAPTATION OF Lebistes reticulatus TO DIFFERENTCONCENTRATIONSOF APH m l 1 EO (PPM - - AM)
108
0 102
ppm
0-0
3-3 04)
~-~
Day 49
E~
,/
©
M
© -I
~7
.q O t-M
-] O
Z
tr~ ..] rn
-] O
306
H . M . VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A. VAN SEVENTER
Adaptation tests in A P H - - 11 EO with Lebistes reticulatus: With guppies it was possible to adapt them from sublethal dose concentrations to concentrations far above the original LCloo 24 h. For this purpose the guppies were placed in 1 litre beakers in a sublethal concentration of A P H - 11 EO. After seven days each batch of fishes was placed in a new concentration 10 p p m higher than the last one (Table 6). Discussion of results with Lebistes reticulatus: Lang (1963) and Liebmann (1967) explained the two different actions of detergents on fishes. (a) A permanent damage of the gills occurs by anionic detergent and non-ionic detergents with a long C chain and/or an ethylene oxide chain, through the change of surface tension from which the fish dies. When the fishes had been replaced in fresh water no recovery took place. Lang's and Liebmann's descriptions of the struggle before the fish dies were the same as that observed in our experiments, with the exception of A P H - - 11 EO which has, however, a non-ionic branched short C chain. Liebmann claims that the toxicity of the detergent increases when the C chain is longer and when the EO chain is shorter and that no damage was observed by the detergent in the alimentary tract. (b) Short-chained (branched) non-ionic detergents cause little damage to the gills. Histological examination shows that this damage cannot be the cause of death. The above-mentioned workers explain the death of the fishes by action on the central nervous system which paralyses the respiration. In a number of cases spontaneous recovery occurred in the same solution in which the fishes are tested. In all our experiments the fishes recovered when they were placed in fresh water after a short exposure. This is totally different from the results as described under (a) above. These paralysing effects were only seen with A P H - - 1 1 EO detergent. The lethal concentration of the detergent with this typical paralysing effect can be raised to a much higher concentration when initially the fishes are brought in a sublethal dose and the concentration is slowly increased. 4. Results with Biomphalaria glabrata by 24 h exposure The size of the snails varied from 1.5 cm to 2 cm. The criteria for death was that the foot of the snail, when pinched by blunt tweezers, showed no reaction at all. The water in our tests was copper-free tap water. In each experiment two batches of 3 snails in 250 cc water were tested, together with controls (Table 7).
hTfluence of biodegradation products o[ the detergents The prepared solutions were put in one-litre glass bottles and continuously aerated.
T O X I C I T Y OF DETERGENTS TO MOSQUITOES A N D O T H E R ORGANISMS
307
TABLE 7 LCj00 -- 24 h OF Biomphalariaglabrata IN DIFFERENT DETERGENTS (PPM - - AM)
Detergent
LC~ oo -- 24 h
LAS 345 LAS 358 PAS 311 PAES PA - - 9 EO APH - - 11 EO TEEPOL CH 53
3 1 4 12 11 23 7
At intervals the biodegradation of anionic active detergents was measured. Total degradation takes place by all tested detergents with this method in about a month for concentrations of 32 ppm. If activated sludge from the Sewage Works at Amsterdam was added as a bacterial source it took about one week to break down 32 ppm and about 4 weeks to break down a concentration of 1000 ppm. The method of Longwell & Maniece (1955) shows errors because of the developing H/S and SO2 which interfere with the test. It is possible that after four weeks not all the detergent is broken down, but is absorbed by the sludge (Borstlap & Kooyman, 1963) and therefore seems to have disappeared from the solution. The animals in our tests were exposed after filtration of the sludge.
5. Results with A~des aegypti larvae on biodegradation products of detergents Two batches of ten 4th stage larvae were exposed to biodegradation products of original 1000 pprr] solution. LAS 345 showed 3 dead pupae, T E E P O L CH - - 53 one dead larva and 2 dead pupae and APH - - 11 EO only one dead larva. None of the controls showed any dead larvae or dead pupae. All the other tested detergents after biodegradation showed no mortality in the tests.
6. Results of the hatching of eggs of A~des aegypti in biodegradation products of L A S 358 In our tests and in the controls 8 batches of 20 eggs were exposed to biodegradation products of originally 1000 ppm LAS 358. Four batches of 20 eggs were submerged. This was done to test possible surface tension action of the biodegradation products. Hatched larvae were counted after 23 days. The results were:
Surface eggs Control LAS 358
51 larvae 63.75 ~o 53 larvae 66.25 ~
Submerged eggs 61 larvae 76.25 61 larvae 76.25
Conclusion: The biodegradation products have no influence on the hatching of eggs.
308
H.M. VAN EMDEN, C. C. M. KROON, E. N. SCHOEMAN, H. A. VAN SEVENTER
Note." Gillett (1955) states that submerged eggs hatch better t h a n floating eggs; this also occurred in our tests. Influence o f biodegradation products o f detergents on fishes and snails The influence of these biodegradation products of the various detergents was tested on the fishes a n d the snails. After 9 days n o difference was f o u n d in mortality between the exposed animals and the controls in fresh water.
ACKNOWLEDGEMENT The authors would like to express their gratitude to the research workers of the Royal D u t c h Shell L a b o r a t o r y in A m s t e r d a m for their valuable advice and the supplies of the detergents.
REFERENCES
BORSTLAP, C. d~. KOOYMAN,P. L. (1963). A study of the biodegradation of anionic synthetic detergents. J. Am. Oil Chem. Soc., 40, 78-80. CHRISTOPHERS, S. R. (1960). A~des aegypti L., the yellow fever mosquito. Cambridge, University Press. GILLETT,J. D. 0955). Variation in the hatching-response of A~des eggs (Diptera: Culicidae). Bull. ent. Res., 46, 241-54. HUDDLESTON,R. L. • ALLRED,R. C. (1965). Determination of non-ionic surfactants, biodegradability physical properties v.s. colorimetry. J. Am. Oil Chem. Soc., 46, 983-6. LANG, W. (1963). Untersuchungen zur Wirkungsweise yon grenzfldchenaktiven Stoffen auf histoIogische Besehaffenheit und Funktion verschiedener Organe bei Fischen. Ph.D. Thesis, University of Cologne. LIEBMANN, H. (1967). Detergentien und Ole im Wasser und Abwasser. Munich and Vienna, Oldenbourg. LONGWELL,J. & MAN1ECE,W. D. (1955). Determination of anionic detergents in sewage, sewage effluence and river waters. Analyst, Lond., 80, 167-71. LODEMANN,D. & NEUMANN,H. Z. (1960). Versuche fiber die akute toxische Wirkung neuzeitlicher Kontaktinsektizide auf Siisswassertiere. Z. angew. Zool., 47, 303-21,493-505. RUSSELL, P. F. • RAMACHANDRARAO, T. (1941). On surface tension of water in relation to behavior of Anopheles larvae. Am. J. trop. Med. Hyg., 21, 767-77.