Uptake pathways and elimination of a nonionic surfactant in cod (Gadus morrhua L.)

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UPTAKE PATHWAYS AND ELIMINATION OF A NONIONIC SURFACTANT IN COD (GADUS MORRHUA L.) A. GRANMO and S. KOLLBERG Kristineberg Marine Biological Station, 450 34 Fiskeb:,ickskil, Sweden (Receired 11 June 19751

A~tract--The uptake and elimination of a labelled surfactant, the nonionic nonylphenol ethoxylate. was studied in cod IGadus morrhua L.) exposed to a concentration of 5 ppm. The amount of labelled surfactant was analyzed by the scintillation counting method in various tissues from the fish. A penetration especially through the gills, but also some intestinal resorption was found. Eight hours from start a steady state condition was obtained. High concentrations were found especially in gall bladder and liver. The elimination process in clean sea water was quite rapid and after 24h the residues

INTRODUCTION

MATERIAL AND METHODS

The toxicity of surface active agents to fish has been Cod (Gadus morrhua L.) in the size range of 22 + 5 crn established by many studies {Henderson et al., 1969; were caught in the GuLlmar Fjord and acclimatized to Cairns and Sheir, 1962; Lemke and Mount, 1963; laboratory conditions and were not fed for 1 week preceeding the experiments. Thatcher, 1966; Thatcher and Santner, 1965; MarThe nonionic surfactant studied, nonylphenol ethoxylate, chetti, 1965; Pickering, 1966: Schmid and Mann, 1966 NP 10 EO, the formula of which is given in Fig. l, is and others) and for marine fishes and invertebrates frequently used in the manufacturing of synthetic washing powders and oil dispersants. For the present investigation particulary by Bellan et al. (1969);. Eisler, (1965) and the ethyLene oxide chain was uniformly labelled with 14C Swedmark et al. (1971). and three different batches with specific activities of 14.5, There is a strong indication that the principal site 114 and 132 ,uCi.g- t respectively were used. of effect is the respiratory organs (Bock, 1966; Lemke All experiments were performed in stagnant water in and Mount, 1963; Schmid and Mann, 1962 and aquaria of 25--401. volume at a temperature of It + 2°C others), as is shown by damage to the gills such as with the exception of one experiment at 18:C. Aeration kept the oxygen tension in the water at a satisfactory level swelling and thickening of gill filaments and finally (70~<~saturation). destruction of the epithelium, but neither liver, kidney After exposure to the surfactant, the fish were dissected and various organs removed and weighed. The following nor gut showed histological damages. The property of surfactants to be adsorbed on sur- samples were taken for analysis: gill filaments, blood from the dorsal aorta, liver (central part), kidney, gall bladder, faces and interfaces makes a physical effect on the urine (only 18~C test). gill epithelium with its enlarged area of tissue reasonThe tissue samples, weighing between 50 and 400mg able to presume. Hirsch (19631 also underlines the were washed in clean sea water to remove an) adsorbed importance of the chemical structure of the surfactant surfactant, dried on filter paper and transferred to counting vials. The tissues were then solubilized through addition to toxicity. of a sample-solubilizer (Packard TMt°°). After this proGloxhuber and Fischer (1968) and Swedmark et al. cess, generally completed within 48 h, liquid scintillator (197l) demonstrated sublethal effects of surfactants (Packard Instagel TM) was added and the samples were such as avoidance reactions and equilibrium distur- measured in a liquid scintillation counter (Unilux III, Nucbances in fish without simultaneous effects on gill tis- lear Chicago) for 10rain. Samples with high quenching were diluted with liquid scintillator to a defined fraction sues. These results indicate a different penetration rate of their initial concentration. into the gills and the body for different types of surThe investigation was performed in five parts. factants. Recently Calamari and Marchetti (1973) sug1. Immersion of the fish in 5ppm of N P 10 EO gest that nonionic surfactants penetrate into the fish II321~Ci.g -t) at II:C and thereby produce toxic effects similar to anaesteIn a previous work (Swedmark et al., 1971) this concentics. tration has proved not to be acutely toxic for cod up to The purpose of the present investigation has been to contribute to the explanation of the toxicity of a surfactant by studying a possible penetration, the acC9HI9 - ~ (OCH2CH2)IO-OH cumulation in tissues and organs and the final eliminFig. 1. Chemical formula of NP 10 EO. ation in clean sea water. 189

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Fig. 2. Uptake and elimination of NP 10 EO in various tissues of cod after exposure to 5ppm. The uptake is expressed in concentration, elimination in per cent. Values obtained in fish dead in 25 ppm ( x ) and 20 ppm (+). a period of 48 h. The exposure times were: 5 and 30 min, 2, 8, 24 and 48 h. A total of 30 fish were used for this experiment.

2. Exposure to 5ppm of NP 10 EO at 18°C (ll41zCi.g -~) This test was performed to evaluate the effect on the uptake of surfactant caused by a rapid increase of the temperature. Eight fish were taken from a storage tank (8°C) and kept for l h at the test temperature in clean sea water before the start of exposure, Exposure times were: 5, 30 and 50 rain and 4 h.

3. Rate of elimination of NP 10 EO (ll4#Ci.g -1) at ll°C After immersion during 8 h in 5 ppm of the surfactant, 9 cod were kept in clean sea water for 30 rain, 2, 8 and 24 h before samples were taken.

4. Intragastric injection (132 #CL g-l) at l l°C The purpose here was to find out the degree of resorption of NP l0 EO through the gut. A micropipette was inserted through the mouth and pharynx in order to avoid all contact with other tissues, especially the gills. Prior testing had shown that by using this technique no liquid was lost by regurgitation. The amount of injected surfactant was calculated according to a known drinking rate of 2 ml sea water per h (Larsson, 1970). The doses were chosen to correspond to 2 and 8 h drinking respectively of a solution of 5 ppm, and were dissolved in 0-5 ml sea water. After injection the fishes were stored in tanks with clean sea water at a temperature of 1VC. The technique used means that the fishes are at once exposed to the total amount of surfactant as compared to normal drinking. Therefore, the time in clean sea water was reduced by 't'--1 and 4 h respectively, before preparing the samples. In all, 4 fish were used for each period of time.

5. lramersion in 20 and 25 ppm of NP 10 EO (14-5 I~Ci.g-t) Both concentrations are sufficient to cause mortality of cod within 8 h. Immediately upon death, samples were taken for analysis. A total of 4 fish were exposed to each concentration. During each experiment, water samples were drawn shortly before and after sampling.

To evaluate the accuracy of the method of analysis, known concentrations of the surfactant were added to blood and liver samples taken from unexposed animals and analyzed by the liquid scintillation counter. The analyzed values of the contaminated tissues have been expressed as #g NP 10 EO per g of sample (ppm). The radioassays were based on a linear relation between activity and N P 10 EO over the range used.

RESULTS

The rate of uptake on NP 10 EO in the different tissues and organs studied after exposure to 5 ppm at 11°C is illustrated in Figs. 2 and 3(a), where the concentrations obtained are plotted against exposure time. A rapid uptake takes place and within 5 min all tissues except the gall bladder contain appreciable amounts of NP 10 EO. Thus, the highest concentration (10 times that of the ambient water) was found in the gills. After 30rain exposure, NP 10 EO starts to show up in the gall bladder (Fig. 2). The uptake is then extremely rapid and 1½h later the accumulated amount of surfactant exceeds that found in other tissues at the same time [Fig. 3(a)]. The values for the gall bladder show a much greater variation than other tissues, Furthermore the lowest values are found in the largest bladders. After 8 h of exposure, stable conditions are reached in all the tissues with the lowest values obtained in gills and blood (100 ppm), values around 500ppm in liver and kidney and a concentration in the gall bladder that amounts to 4000 ppm. This steady state condition lasts for the rest of the exposure time. If the samples from the different tissues are compared [Fig. 3(a)], the uptake of the surfactant between 5 min, and 8 h of exposure shows a 1000-fold increase

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Fig. 3. Uptake of NP 10 EO in various tissues of cod after exposure to 5 ppm. (a) at I I'C, (b) at 18C. x gills, [] blood. + liver, • gall bladder, © kidney. • urine. in the gall bladder, while the increase for the liver is 10-fold and the value for the gills only doubles. Samples taken during each experiment from the ambient water reveal a decrease in content during the longer exposure times (Fig. 4). The effect of a rapid rise of temperature on the uptake is shown in Fig. 3(b), where the accumulation from exposure in 18°C is given. Though not directly comparable to the II°C test due to seasonal variations, it is evident that the increased temperature causes a faster rate of uptake in all the tissues, particularly in the gills, compared with 11°C [Fig. 3(a)]. Blood and liver reach their maxima within 2 h at the higher temperature, but only for the gills does the amount of N P I0 EO clearly exceed that obtained at the lower temperature. Urine samples taken in this experiment show a rapidly increasing concentration

of surfactant which indicated an excretion process [Fig. 3(b)]. After 4 h the exposure was interrupted as the fishes were severely affected. When the fishes are exposed to lethal concentrations 20 and 25 ppm (Fig. 2; indicated with crosses), the gills are the only tissue where the concentrations obtained exceed those found at the exposure in 5 ppm. The values found in the different tissues are of the same size except for the gall bladder, where the longer exposure time for the fishes in 20 ppm gives a higher uptake on N P I0 EO than in 25 ppm. The values obtained in the different tissues after oral injection of the surfactant are low (Table 1) compared to those found when fishes are immersed in the surfactant. The results show a certain intestinal resorption of the surfactant. The concentrations obtained at 4 h exposure are about the same as those at 1 h for the gills and blood, but slightly higher for Table 1. Concentration of NP 10 EO (ppm) in various organs of cod after intragastric injection

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Elimination of a nonionic surfactant in cod Table 2. Concentrations obtained in blood and liver of cod (Gadus raorrhua L.) after addition of known amounts of NP 10 EO Concentration of NP 10 EO Blood Liver Added

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2,7 1,9 8.3 5.8 18.3 21.6

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5.9 6.9 12.0 11.4 37.2 36-3

the other organs. As in the immersion tests, the gall bladder accumulates the highest amounts of surthctant. The rate of elimination in the various tissues after the transfer to clean sea water is presented in Fig. 2. The rate is slow and irregular for all tissues except the gall-bladder during the first 8 h, after which time a maximum is reached, but after that the elimination is rapid. In the gall bladder, on the other hand, there is a maximum after 2 h. After that the values remain stable. The amount of surfactant at the end of the test is reduced by more than 60~o in the gills, blood and kidney, while the gall bladder shows an increase of about 60%. Also in this experiment the variation is small for all the tissues but the gall bladder. The test series performed in order to evaluate the accuracy of the methods used for analysis of all samples gives results (Table 2/ which coincide with those theoretically expected within the limits of the analytical methods, and no attempt was made to adjust the previous results.

DISCUSSION The results confirm that penetration and uptake of the nonionic NP 10 EO takes place in tissues and organs of cod and that elimination occurs during the recovery period in clean water.

(a) Penetration and uptake pathways The results give a clear indication that the site of penetration of the surfactant is the gill tissues, and also that only small amounts are resorbed by the intestine during drinking. The uptake is found to be a rapid process with high concentrations in the gills after only 5 min exposure. Further, the high amount of NP 10 EO found in blood after 5 min (4-5 times the concentration in the ambient water), shows that the blood is the chief vehicle of transportation to the various tissues/organs where deposition occurs. The blood values obtained upon exposure to lethal concentrations where all values are below those found at immersion in 5 ppm, indicate that no acutely physiological effects such as for example haemolyses, are present in the blood.

193

The liver rapidly takes up large amounts of the surfactant and forwards it to the gall bladder, where the highest amount of the surfactant was found. Only slight amounts of bile products were secreted during the tests as the fish were starving. Therefore the results indicate the total amount of surfactant accumulated in this organ. The high concentration obtained, and the lack of elimination, indicate that there is no further transport or turnover taking place. The great variations in the values must then be related to individual differences in bladder size and bile volume at the start of the tests. As a consequence of this. the highest amounts on NP 10 EO were found in the smallest gall bladders. For example bladders taken after a 24 h exposure weighing 195, 294, 360 and 424mg contained respectively 5728. 2970, 2412 and 1238 ppm surfactant. The decrease of the concentration in the ambient water observed during the longer exposure-times may be explained by various factors. 1. As shown in the immersion tests, some of the surfactant is taken up by the fish. A rough estimate of the amounts taken up in the tissues examined gives a result which would correspond to a decrease of the concentration in the tank in the range of 0-5 ppm at 24 h exposure. 2. Surface films may be formed at the water surface and along the walls of the tank in accordance with the properties of the surfactant and account for some decrease of the surfactant available in the water volume. As the surfactant tested has a branched ethyleneoxide chain, the rate of biodegradation is slow (Prat and Giraud, 1964) and therefore of minor importance in the tests. During the short exposure times any degradation of the surfactant within the fish is not likely to occur as the molecular bindings are stable and quite resistant to biodegradation (Eriksson, personal communication).

(b) Elimination Gills and blood are the organs found to be most important for the penetration and distribution of the surfactant. They also show the highest rate of elimination after transfer to clean sea water. After 24 h in clean water the elimination is evident and the increasing amounts in the gall bladder indicate that some of the surfactant leaving the tissues has accumulated here. The high values of the urine samples at the 18°C test show that an excretion process occurs within the kidney.

(c) Action of the surfactant In the present investigation the high levels of surfactant found in gills when fish were exposed to lethal concentrations, indicate that this organ is a possible site of toxic action. The observed swelling of the gill-lamellae (Swedmark et al., 1971) is likely to reduce the oxygen utilization. Such results were

194

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also obtained for anionic surfactants by Schmid and Mann (1962) using trout. A penetration through the gills may be started by the formation of surface films on the lipophilic gill epithelium, which is a consequence of the properties of the surfactant (K61bel and Kurzend6rfer, 1969). At h i ~ e r concentrations a more specific cell damage, as for example a protein denaturation in the cells as found by Manwell and Baker is supposed to occur. Such detergent-protein interactions were also found by Putnam (1948) and by Swisher et aL (1964). It is also suggested (Prat and Giraud, 1964) that a depressed surface tension is the principal cause of the toxic action of surfactants in vie,,,,' of the relationship between toxicity and order of surface tension depression. However, opinions against this theory have recently been given by Abel (1974) who suggests that the surfactants at lower concentrations alter the membrane permeability which is also supported by studies on common mussels (Braaten et al., 1972). It seems therefore reasonable that swelling of the gill lamellae and changes of the membrane permeability bring on asphyxiation as a main cause of acute poisoning. Acknowledgements--The authors are greatly indebted to

the Director of Kristineberg Marine Biological Station Prof. B. Swedmark for working facilities and generous support. Thanks are due to Dr. M. Swedmark who critically read the manuscript. We are most grateful to Drs. M. Hellsten, and G. Karlsson for fruitful discussions and all work involved with the analysis of. the samples, and to Bero[ Kemi AB, Stenungsund for supplying the labelled surfactant. This work was financially supported by the National Swedish Environment Protection Board, project leader Dr. M. Swedmark.

REFERENCES

Abel P. D. (1974) Toxicity of synthetic detergents to fish and aquatic invertebrates. J. Fish Biol. 6, 279-298. Bellan G., Caruelle F., Foret-Montardo P., Kaim-Malka R. A. & Leung Tack K. (1966) Contribution a l'6tude de diff~rents facteurs physico-chimiques polluants sur les organismes marins. I. Action des d&ergents sur la polychete Scolelepis fuliginosa (note preliminaire). Tethys 1, 367-374. Bock K. J. (1966) iJber die Wirkung von Waschrohstoffen auf Fisehe. Arch. Fisch 14~ss. 17, 68--77. Braaten B., Granmo A. & Lange R. (1972) Tissue-swelling in Mytilus edulis L. induced by exposure to a nonionic surface active agent. Norw. J. Zool. 20, 137-140.

Cairns J. & Scheir -\. qi902/ q'he acute and chrome cffect~ of standard sodit, m alk)Ibcnzcnc aulphonate upor~ the pumpkinseed suntish. Le~,,,mi., m,cr,chir,~* IRaf.~. Pro~. l';rir ind. Iti.,,r¢. (_',roll. t)urJ:~e Uni:. E~zqnq E.v:. Ser. 112, 14-28. Calamari D. & Marchetti R. 11973) The toxicit~ of mixtures of metals and surfactants to rainbov, trout (S,[mo g,irdneri Richmond). W~t,*erRes. 7. 1453-1464. Eisler R. (19651 Some effects of at synthetic detergent on estuarine fishes. Trans..4m. Fish. S,w. 94, 26-31. Gloxhuber C. & Fischer W. K. 119681 Untersuchungen f:ber die Wirkungen ~on Alkylpolyglykol'a.thern in hohen Koncentrationen auf Fische. Fd Cosmet. Toxicol. 6, 469-477. Henderson C.. Pickering Q. H. & Cohen J. M. 1t9591 The toxicity of synthetic detergents and soaps to fish. Sewa~te imt. }Ii~stes 31, 295-306. Hirsch E. 119631 Strukturelemente ~on Alkylbenzolsulfonaten und ihr Einttuss auf das Verhalten yon Fischen. Vom W , ss. 30, 249-259. K~Slbel H. & Kurzendtirfer P. (19691 Konstitution und Eigenschaffen ,,on tensiden. Forrsehr. Chem. Forsch. 12, 252-348. Larsson J. E. 11970) The drinki, 9 rate ~g Fish in the Ska9erak aml the Baltic. Rep. AE-383 from Aktiebolaget Atomenergi Studsvik, Nyk6ping Sweden. Lemke A. E. & Mount D. 1. (1963) Some effects of alkylbenzenesulfonate on the bluegill. Trans. Am. Fish. Soc. 92, 372-378. Manwell C. & Baker C. M. A. (1967) A study of detergent pollution by molecular methods: Starch gel e[ectrophoresis of a variety of enzymes and other proteins. J. mar. biol. Ass. 47, 659-675. Marchetti R. (1965) A critical review of the effects of synthetic detergents on aquatic life. Stud. Rev. fen. Fish. Coun. Medit. 26. 1-32. Pickering Q. H. {1966) Acute toxicity of alkyl benzene sulfonate and linear alkytate sulfonate to the eggs of the fathead minnow Pinwphules promelas. Int. J. Air Wat. Pollut. 10. 385-391. Prat J. & Giraud A. (1964) The Polh, tion of Water Deter9ents, 86 pp. Paris, O.E.C.D. Putnam F. W. (1948) The interactions of protein and synthetic detergents. Adr. Protein Chem. 4, 79-122. Schmid J. O. & Mann H. (1962) Die Einwirkung yon Dodecylbenzolsulfonat auf die Kiemen yon Forellen. Arch. Fisch Wiss. 13.41--51. Swedmark M., Braaten B., Emanuelsson E. & Granmo A. (1971) Biological effects of surface active agents on marine animals. Mar. Biol. 9, 183-201. Swisher R. D.. O'Rourke J. T. & Tomlinson H. D. (1964) Fish bioassays of linear alkylate sulfonates (LAS) and intermediate biodegradation products. J. Am. Oil Chem. Soe. 41, 746-752. Thatcher T. O. (1966) The comparative lethal toxicity of a mixture of hard ABS detergent products to eleven species of fishes. Int. J. Air War. Pollat. 10, 585-590. Thatcher T. O. & Santner A. A. (1967) Acute toxicity of LAS to various fish species. Proc. 21st Purdue Ind. Waste Conf. Purdue Univ. Enqn 9. Ext. Part 2, 50, 996.--1002. Ext. Bull. Purdue Univ. (Engng) Ser. No. 121.