Molecular structure—biological properties relationships in anionic surface-active agents

Waler Research Vol. 12. pp. 25 to 30. Pergamon Press 1978. Printed in Great Britain.

MOLECULAR STRUCTURE--BIOLOGICAL PROPERTIES RELATIONSHIPS IN ANIONIC SURFACE-ACTIVE AGENTS P. LUNDAHL a n d R. CABRIDENC Service Ecologie-Biochimie-Eaux, I.R.CH.A., B.P. No 1, 91710, Vert-le-Petit, France (Received 3 March 1977) Abstract--Molecular structure-toxicity relationships in anionic surface-active agents are studied, using aquatic organisms for the bioassays. In a series of homologs the toxicity increases sharply when the hydrophobic chain length increases and a simple logarithmic equation describes the relationship. This equation can be used to predict the toxicities. A simplified thermodynamic interpretation is proposed. Variations of the biodegradability with the chain length are tentatively explained.

INTRODUCTION Very little work has been done in the application of structure-activity relationships to environmental problems. However such studies could be very useful because the results could be used to predict the env i r o n m e n t a l impact of pollutants. A study of structure-toxicity relationships in anionic surface-active agents is presented here. A simplified t h e r m o d y n a m i c interpretation of some of the equations relating acute toxicity to a molecular parameter is given. The feasability of predicting the toxicity of a surfactant belonging to a given series of homologs, when the toxicities of two of the homologs are known, is demonstrated. Anionic surface-active agents were chosen because of their importance as pollutants and because a large series of homologs (products with the same hydrophilic group) were available. Toxicity toward aquatic organisms was chosen as a measure of the biological activity b e c a m e of its significance and accessibility. As a matter of convenience, acute toxicity tests o n D a p h n i a were used in all cases, often completed by short term tests of immobilization of algal cells, and acute toxicity tests toward fish. MATERIALS AND

METHODS

All concentrations are in mg ! - t Bioassays Acute toxicity on Daphnia maona Straus, 1820. The concentration immobilizing 50% of the animals in 24h (ICS0) is determined by the technique of the A.F.N.O.R. (1974). Acute toxicity on Chlamydomonas variabilis. Morano (1970) was the first to use the immobilization of algae to detect toxicity. He studied the immobilization of Dunaliella bioculata by tobacco smoke. Gayral (1971) and Lepailleur studied the structure of the flagella of zoids or non pheophycean chrysophyceae. The principle of the following bioassay in which the response is the immobilization of the cells was proposed by Lepailleur (personal communication). He participated in the development of the bioassay. The algae are grown in a medium (A.M.) containing: Ca (NO3)2 27.8, KNO3 100, MgSO4 14.7, KH2PO, 40, micronutrients solution A 0.5 ml l- t, solution B 0.5 ml l- t. 25

Solution A contains: CuSO4 44, ZnSO, 33.7, COCI2 32.8, Mn (NO3)2 42.8, H3BO3 60, citric acid 55. Solution B contains: ferric citrate 1625, Fe2 (SO,,) 3 625, FeCI 3 375. Double-distilled water is used. The medium is steam-sterilized at 120°C for 20min. A light--dark cycle of 6 h/6 h is used. Light is provided by GROLUX T 12 GRO tubes. The illumination at the level of the surface of the medium, on the outside of the flasks is 2000 Ix. Cultures are made in 600 ml flasks containing 180 ml of medium and 20 ml of inoculum. The inocolum is a similar culture with an optical density of 0.1 at 578 nm. Tests are made on an eight days old culture of which optical density is adjusted to 0.1 at 578 nm. Open test-tubes containing a total volume of liquid of 10ml are used. The toxic solution is added first, then the appropriate volume of A.M., and 5 ml of inoculum. The tubes are then gently agitated. There are three test-tubes at each concentration of toxic agent and the concentrations are in geometric progression with a ratio of 1 : 3. If the toxicity is completely unknown a preliminary test with a larger ratio and one test-tube at each concentration is done first. After four hours at 20°C, under usual laboratory light conditions, the test-tubes are agitated, a small quantity of liquid is taken from about 1 cm beneath the surface with a capillar glass tube, deposited on a microscope plate and observed uncovered at a magnification of 150. The percentage of motility of the cells is estimated (no count is taken). The average percentage of motility in each series of three test-tubes is plotted on a Iog-probit paper as a function of the concentration. The ICso is given by the point of the best fitting straight line corresponding to 50% of motility. It is then corrected by multiplying it by the ratio of the average ICso of a standard toxic agent to the ICso of this standard measured in the experiment. Sodium dodecylbenzenesulphonate cited in A.F.N.O.R. (1971) was used as standard. Its average ICs0 was 2.84 with a standard deviation of 0.39. Acute toxicity toward fish, The LCso 24h toward Phoxinus phoxinus is determined in natural spring water, pH 7.7, Ca t+. 8.55, Mg 2+. 3.34 (Temperature during acclimatization and assays is 13°C.) There are 10 fish per aquarium and 2 aquaria per concentration. Concentrations are in geometric progression; the ratio is adjusted so as to get 4 to 6 significant points on the dosage-mortality regression line. The line is estimated on a graph on probit paper. Fish are caught in streams and kept for a few weeks in large continuous flow tanks where they are fed with pellets of trout commercial food. They are transferred into the aquaria 48 h before the beginning of the test. No food is given during this period or during the test. Except during the first week in the tanks no mortality occured during acclimatization or in the blanks. In all cases, the concen-

Alkane disulphonates

15

14.6

Primary alkanesulphonates

35.6 11.8 1 t.6 8.6

. . . .

503 288 t08 50.3 28.8 11.5 10.1 21.5

. . . . . . . . . . . .

. . . . . . . . .

atoms

# M 1- 1

carbon

1000 1400

6900 3200 220 60

319 t33 111 34.2 30.1 12.3 3.31 6.30

35 17 12 . . . . .

3257 4460

43000 14800 810 200

1290 513 371 109 92 36 9 16

100 49 35

Daphnia m g 1- t #M I - 1 36.8 18.4 17.2 12.9

. . . .

. . . .

. . . .

. . . . . . . . . . . . . .

. . . .

----34.5 115 8.5 27 3.11 9.5 . . . . . ------

12.8 6.4 6.0 4.5

Phoxinus m g 1- ~ /~M 1-1

in t h e a l i p h a t i c p a r t o f t h e h y d r o p h o b i c

Sulfoacetate

l,n carboxylates

Primary alkylsuphates

Toxicant

part of the molecule

Chlamydomonas variabilis, Daphnia magna a n d Phoxinus phoxinus. D u r a t i o n

Algae

. . . . . . . .

. . . .

4 8 12 14

12.4 4.1 3.7 3.0

Secondary alkanesulphonates

12 12 12 12

125 74.9 32.4 15.8 9A2 3.93 3.7t 8.47

(1)

J L K P

mg I - l

10.3 11.2 14 15 16 17 18.9 20.7

Docecylbenzenesulphonates

2

1. IC~o o f s u r f a c e - a c t i v e a g e n t s t o w a r d

Toxicant

Table

2750 2510 1950 1800 1600 t050 900 135 65 29 12

1 3 4 5 6 7 8 9 10 It 12

809

8200 4350 2300 800 80 42

1

4, 24 a n d

5.480

33,600 22,800 15,700 13,100 10,500 6,320 5,000 695 315 t 31 51

46,900 18,700 9,150 3,280 278 139

Daphnia m g 1-1 /~M 1 - i

4 8 9 10 12 13

)[

of the bioassays:

2 = number

--

--

----106 --

.

.

.

.

. . . . . --. . . . -----. . . . . . .

. . . . .

--. . . . -. . . . .

---30.5 --

Phoxinus m g 1- l g M 1- l

24h;

of

rn "Z

>

,v r-'

rz

Molecular structure--biological properties relationships

27

trations of toxic agents which are used to draw the regression line are those introduced at zero time. Controls have shown that variations of the concentrations during the tests were not significant.

Biodegradability A die-away test is used. The composition of the medium is: NaCI: 1000, (NH4)2SO4: 1600, KH2PO4: 400, NaHPO4: 400; FeCI a 0.1, MgCI2 0.5. The initial concentrations of surfactant are 1.44.10-SM or 5.75.10-SM. Microorganisms come from 10 ml of water from a Daphnia rearing tank (see A.F.N.O.R. (1974)) which are added to the medium at the beginning of the test. 750 ml flasks containing a total volume of.200 ml of liquid are used. The temperature is 28°C. The flasks are placed on a rotary shaker. Analyses are done by the methylene blue active substance method which is described by the A.F.N.O.R. (1971).

iO~L

"-.+.

X

X ,, \',, X',

~o"

o u

,o-~

~0:

Surface-active agents and related products Analytical grade products were used except where it is I0' otherwise specified. Sodium acetate: Prolabo. Sodium propionate: Merck. Sodium 1, n carboxylates from butyrate to tridecanoate: Fluka. I I I I I I I I I I i I I I I Sulphoacetic acid. Eastman. Sodium 1, n alkylsulphates 2 3 4 5 6 7 El 9 I0 II 12 13 14 15 16 17 18 C4, Ca, C9, C1o, C13, C14- Merck, C12 Eastman. SecondX ary n alkanesulphonates, mixed position isomers, average chain lengths of narrow cuts: Ci0.3, CiL2, C~4, C~.~. C~o. Fig. 1. ICs0 of surfactants as functions of chain lengths: C~7, Cts.9, C2o.7. S.N.P.A. Sodium secondary alkanedisul--~-secondary-alkanesulphonates, algae; - - 0 - - 0 ~ phonates, mixed position isomers, average chain lengths secondary aikanesulphonates, daphniae; - - A - - / X - - priof narrow cuts: C~4.6 and Ct~: S.N.P.A. Sodium primary maryalkanesulphonates, daphniae: - • 0 - 0 . • primary n alkanesulphonates C4, Ca, C~4: Merck; C12: Schuchardt. alkylsulphates, daphniae; + - - + carboxylates, daphniae. Sodium tetrapropylbenzenesulphonate, (J) mixed isomers. industrial product. Sodium I, n dodecylbenzeneparasulphonate (P) made at the I.R.CH.A. by sulphonation of Table 3. The half-life time for a n initial concentration Eastman 1, n dodecylbenzene. Industrial soditim dodecylbenzenesulphonates composed of mixed isomers: a sample of 5 mg 1-1 is also given in Table 3. The half-life was made by Hills (K) and the other by Shell IL). The time has been estimated graphically from the data first one contained less branched isomers than the second. of the table. More detailed characteristics of the products are given by The percentages of biodegradation after 40, 72, 144 Lundahl (1974). and 216 h have been plotted as a function of ). on Fig. 2. The effect of the chain length on the biodegraRESU LTS dation rate is strong and complicated. A maximum The results of the IC.~0 experiments are summarized is observed for 2 = 16, and a m i n i m u m for 2 = 19. for the three species in Table l. In these experiments Daphnia is a little more sensitive to anionic surfaceDISCUSSION active agents than Chlamydomonas and Phoxinus. Toxicity Chlamydomonas and Phoxinus have nearly equal senThe relations between 2 and the IC5o are similar sitivities. The classilication of the products by order in different series of h o m o l o g surfactants and with of decreasing toxicity is the same with the three organisms. In all the series of products, the toxicity Table 2. Coefficients of the equation In IC5o = ~ + f12 and increases with the increase of the number, 2, of carbon corresponding correlation coefficients. ,i. = number of caratoms in the aliphatic part of the hydrophobic group bon atoms in the aliphatic part of the hydrobic part of the molecule of the surfactanl molecule. The IC.~o is multiplied by approximately 2 when the hydrophobic chain length Organism Product 2 ~ fl r2 increases by one carbon atom. For each series of homolog surfactants the IC50 Algae Secondary 10.3- 11.4 -0.50 0.97 alkanesulphonates 18.9 in molcs/I has been plotted as a function of 2 on semi logarithmic paper (Fig. I). For 5 <~ ~. < 19, Secondary 10.3- 13.2 -0.57 0.94 straighl lines are obtained. The coefficients ct and /~ alkanesulphonates 18.9 of the equations In IC~o = 7 + fl 2 and and the correPrimary lati,m coellicients r-' arc given in Table 2. For 2 < 5, alkanesulphonates 8-14 10.7 -0.72 1.00 only the carboxylates have been studied. A straight Daphnia Primary line is also obtained, but the slope is different from alkylsulphates 8-13 13.7 - 1.04 0.99 the slope obtained with longer carboxylates. Primary 6-12 11,0 -0.98 0.97 The values of the extent of the biodegradation are carboxylates 1-5 6,1 -0.25 0.97 expressed as percent of the initial concentration in

28

P. LUNDAHL and R. CABRIDENC at least partly, into the core. The reaction of D with R is assumed to be reversible:

Table 3. Biogradation of secondary alkanesulphonates Initial COllCentration 20 mg I- '

10.3 I 1.2 t4 15 16 17 18.9 20.7

6d

14d

99 100 100 99 100 0 0 0

100 100 100 100 100 15 24 15

D + R ~DR 5 mg I Time 40h 72h 1 4 7 17 21 16 0 0

37 68 92 86 96 35 0 0

surrounding R. If transport factors are not limiting, and as the total quantity of D is very large compared with the total quantity of R, (D) is equal to the introduced concentration of D. If transportation factors are limiting and if a steady state is attained, (D) in the biological medium is in a constant ratio with (Dt in the external water, and the calculations give the same final result. A given response percentage in a toxicity test is obtained for a constant ratio r = IDR)/(R), independent of the surfactant under consideration. If the chosen response percentage is 50'!.>. (D) is equal to the ICso. Considering theses assumptions equation (l) may be written as:

100 100 100 100 100 100 12 87

90 68 58 58 55 106 >216 116

different types of organisms lalgae, daphnia, fish). Therefore it is assumed that at the molecular level the mechanism of action is the same in all cases. This ~iew is supported by the fact that the toxicity of anionic surfactants on aquatic organisms probably results from the inhibition of enzymes or of the selectivity of the transportation of ions through membranes. It is also supported by the fact that anionic surfactant-proteins interactions increase when the hydrophobic tail gets longer. In the following proposed model. D is the surfactant, R is the biological receptor of D (enzyme or membrane) and DR the product of the reaction of D with R. DR is supposed to be unactive, so that the reaction of D with R produces (indirectly) the response observed in the bioassay. It is also assumed that R is composed of an external layer with many ionic groups and a hydrophobic core. When D reacts with R, the hydrophilic part of D is linked to the outer layer of R and its hydrophobic chain penetrates. - ~ - - -,,~--~

~--=~

/

/

L/ ~°l/

k = r/1C~o or In IC50 = In r - In k.

A similar equation was proposed by Pacheco (1973) to explain molecular structure-biological activity relationships in drugs. As a first approximation AGo can be broken down to AGh + AGi where AGh is the standard free energy of the hydrophobic interactions between D and R and AGi the standard free energy of ionic interactions including solvatation. If D has an aliphatic linear saturated hydrophobic tail with ). carbon atoms, AGh = 2Ag + A'y where Ag is the standard free energy of passage of a CH2 from the water into the hydrophobic part of R and A',q

.... ~---~..

/

',

/ i

~

|/ '

i

25 l -

,/

"

,," ,/

\'\

1

i

/

.~f"\\'

t

~ i /

• ......

\

/'/

'

',

\ ~ "

\\~' \\ \

//

x / Xd /

\\'~

/'

i IO

II

f2

15

14

15

(2)

If A Go is the standard free energy of the reaction of D with R, In k R T A Go and equation {2) gives In 1C50 = R T A Go + In r. (31

o/\~

I'

(1)

(D) is the concentration of the surfactant in the liquid

Half-life iime 144h 216h (hi 80 100 95 100 100 69 4 86

k = [(DR)/(D) x ( R ) ]

16

17

18

19

20

21

22

X t-ig. 2. Biodegradation of secondary alkanesutphonales b i o d e g r a d a l i o n after: - O - - O - 4 0 h : - - + - - + - - 7 2 h :

as function o f chain length. Percentages of -O ...... O - 1 4 4 h : -2~ A. 21611.

Molecular structure--biological properties relationships the standard free energy of passage of the rest of the hydrophobic part of D into the hydrophobic part of R. Equation (3~ gives: In ICso = ,;, R T A g + RTA'.q + R T A G i + lnr (4) This model shows that In IC~o is a linear function of 2 and is in agreement with the experimental results. The increase of the slope of In ICso = f C2) for small values of 2 which is observed in Fig. 1 can be interpreted by considering that the CH2 groups which are near the hydrophilic part of the molecule cannot penetrate deeply into the hydrophobic core of R, stay in a more external and ionic region of R, and have smaller absolute values of A# than the other CH2 groups. Therefore, it is not surprising that secondary alkanesulphonates are less toxic than their primary counterparts: they have more CH2 groups near the hydrophilic group. Similarly Hirsch (1963) has shown that n alkylbenzenenesulphonates of a given chain length are more toxic when the benzene ring is attached to one of the ends of the alkyl chain than when it is attached to an intermediate carbon atom. The very low toxicity of alkanedisulphonates when compared to monosulphonates of the same chain length could be due to the fact that in disulphonates many CH2 groups are not able to penetrate into the core of R because they are near and/or between hydrophilic groups. These hydrophilic groups are linked to the ionic external surface of R. Swisher (1964) gives another example of this phenomenon: sulphophenylundecanoic acid is much less toxic than undecylbenzenesulphonate. The toxicity of alkylbenzenesulphonates of the same molecular weight increases with the linearity of their aliphatic tails. This increase can be due to the deeper penetration of linear tails if compared to branched tails and to the fact that in branched tails many carbon atoms are hidden and unavailable for interactions with R. Alkylbenzenesulphonates (1) are less toxic than alkanesulphonates (II) having the same number of carbon atoms. This is probably due to the fact that the absolute value of the standard free energy IA'glI of transfer of a benzene ring from water to R is inferior to the absolute value of the standard free energy I6A#II[ of transfer of six C H 2 groups IA'gll < 16Aglll because more CH2 groups can penetrate the hydrophobic core of R in the case of (I1) than in the case of (I) and because the absolute value of the standard free energy of solvatation of a benzene ring is higher than that of six CH2 groups belonging to an aliphatic chain. The logarithm of the critical micelle concentration (CMC) of surface active agents is a linear function of the number of carbon atoms in the hydrophobic tail. This relation can be interpreted by calculations similar to the preceeding ones, if R is the micelle, and AM# the standard free energy of transfer of a CH2 group from the water into the hydrophobic core of the micelle. The relation can be written as: In C M C = 2 R T AM# + constant.

(51

29

If In ICso is plotted as a function of In CMC in a series of homolog surfactants a straight line is obtained. From equations (4) and (5) it can be seen that In IC5o = Ag/Au,q In CMC + constant. With Daphnia the observed slopes are: primary alkanesulphonates: 1 . 0 4 ; secondary alkanesulphonates: 1.16, carboxylates: 1.44, alkylsulphates: 1.64. With Chlamydomonas the slope for secondary alkanesulphonates is 1.42. As AM.q characterizes hydrophobic interactions in the core of the micelle, the fact that the values of Ag/Au.q are not very far from 1 supports the assumption that variations in hydrophobic interactions are the explanation of the observed correlation between the hydrophobic chain length and the toxicity. A micelle is grossly similar in its structure (hydrophobic core, ionic surfacel to a globular protein or a biological membrane. The study of the toxicity of secondary alkanesulphonates shows that if 2 > 19, equation (4) does not apply and furthermore, that the toxicity decreases when 2 increases. It was also shown that the toxicity of long chain linear primary alkylsulphates and linear primary alkanesulphonates decreases when 2 increases. This phenomenon seems to be general: Galbraith (1971) observed it in studying the bacteriostatic activity of fatty acids, Schott (1973) in studying the toxicity of alkylsulphates toward mice and the irritant action of aikysulphates and fatty acids on the skin. Marchetti (1965) and Janicke (1973) in studying the toxicity of linearalkylbenzenesulphonates on fish. The decrease of the activity of homolog drugs with increasing molecular weight is frequently attributed to the slow down of their transportation through the biological membranes. In the case of surfactants this explanation is not satisifactory because Czok (1969) has shown that the inhibition thresholds of alkylsulphates on in vitro enzyme activities also increase with increasing chain length for 2 > 17. Another reason to abandon the "transportation slow down" explanation is that the toxicity decrease was observed in the Chlamydomonas bioassay, where the surfactant, very probably, inhibits the flagellum without going into the cell. Some biophysical properties of surfactants having very long hydrophobic tails could be due to the folding of the chain (Dupeyrat, personal communication). Perhaps it is the case here. The effect of the hydrophilic group on the toxicity is very significant. Figure 1 shows that when equal chain lengths are considered, and if ,~ > 9, carboxylates are more toxic than sulphates, which are more toxic than sulphonates. As the number of different hydrophilic groups which were studied is small, no attempt to find correlations between their structure parameters and the toxicity was done. Biodegradability

The results of Swhisher 0971) concerning biodegradability as a function of chain length were confirmed. The following qualitative interpretation is proposed. Measured biodegradability results from the combina-

P. LUNDAHL and R. CABRIDENC

30

tion of intrinsic biodegradability and inhibition of the biodegradability by the studied product itself. Intrinsic biodegradability is a theoretical property: it is the biodegradability in a system exactly similar to the real one except that it is a theoretical system where there is no toxic effect. Intrinsic biodegradability can be regarded as a measure of the biological activity. As one of the determining factors of intrinsic biodegradability is the affinity of the surfactant for the enzymes, intrinsic biodegradability increases with increasing chain length, except for very long chains. But the toxicity increases concurrently. Below the toxicity threshold the observed biodegradability is equal to the intrinsic biodegradability. At the toxicity threshold the observed biodegradability decreases sharply, in spite of the increase in the intrinsic biodegradability which is overcome by the self-inhibitory effect. When the chain becomes very long the toxicity decreases, and so does the intrinsic biodegradability, but because toxicity decreases faster than the intrinsic biodegradability, the observed biodegradability increases again. The toxicity decreases faster than the intrinsic biodegradability because the inhibition of the biodegradation prevents the growth of the bacterial population (no other source of carbon than the surfactant is present), and a lower bacterial concentration brings a lower toxicity threshold.

the detrimental effects of the products toward the water fauna, in the optimization process. Prediction of the relative biodegradation speeds seems much more difficult. REFERENCES

A.F.N.O.R. (1971) D~termination de la biod~gradabilit~ des d~tergents contenant des agents de surface anioniques,

Norme exp6rimentale T.73260. A.F.N.O.R. (1974) D~termination de l'inhibition de la mobilit~ de Daphnia magna Straus (Crustac~,s, Cladocl, re),

CONCLUSION

Norme exp6rimentale T.90301. Czok R., Kaiser G. & T~iuber G. (1969) Interaction of anionic surfactants with enzpmes. Chimie, Physique et Application Pratiques des Agents de Surface, 4 volumes Ediciones Unidas Barcelona. Proceedings of the fifth international congress of surface activity (Barcelona 1968). Galbraith H., Miller T. B., Paton A. M. & Thompson J. K. (1971) Antibacterial activity of long chain fatty acids and the reversal with calcium, magnesium, ergocalciferol and cholesterol. J. appl. Bact. 34, 803--813. Gayral P. and Lepailleur H. (1971) Etude de deux Chrysophyc6es filamenteuses: Nematochrysopis roscoffensis Chadefaud, Mematochrysis hieroglyphica Waern. Rerue. g~n. Bot. 78, 61-74. Janicke W. (1973) Schadworkungen yon tensiden unter wasserwirtschafllichem gesichtpunkt, Eine Ubersicht. Bundesgesundheits BI. 16, 242-246. Kopperman H. L., Carlson R. M. and Capte R. (1974) Aqueous Chlorination and Ozonation studies. 1. Structure toxicity correlations of phenolic compounds to Daphnia magna. Chem. Biol. Interactions 9, 245--251. Lundahl P. (1974) Contribution ?t I'~tude de la pollution des

Equations relating toxicity to structural parameters (hydrophobic chain length and standard free energy of transfer of the hydrophobic group from water to a hydrophobic medium) have been developed. As those of Kopperman (1974) who studied phenols they show the feasability of using this kind of work to predict the toxicity of pollutants when the toxicity of a few homologs can be measured. If a choice between products of a given family has to be done before industrial scale production, and if the products are potential toxic water pollutants, this kind of equation could be used to take into account quantitatively

teur-ing6nieur, Paris VI University, 200 p. Marano F. & Izard C. (1970) Le Dunaliella bioculata: ultrastructure et modifications exp6rimentales de l'ultrastructure. Bull. Soc. phycol. Ft. 15, 70. Marchetti R. (1965) Revue critique des effets des d(~tergents synth&iques sur la vie aquatique. Stud. Rer. gen. Fish. Coun. Mediterr. 26, 1-35. Pacheco H. (1973) La Pharmacologie MolFculaire. 222 p. Presses Universitaires de France. Schott H. (1973) Effect of chain length in homologous series of anionic surfactants on irritant action and toxicity. J. Pharm. Sci. 62, 34t-343. Swisher R. D. (1971) Surfactant Biogradation. Marcel Dekker. New York.

eaux par les substances toxiques; propri~tFs biologiques de quelques agents de surface anioniques. Thbse de doc-