Toxicology, 4 (1975) 17--29 Q Elsevier/North-Holland, Amsterdam -- Printed in the Netherlands
ABSORPTION, METABOLISM AND EXCRETION BY GOLDFISH OF THE ANIONIC DETERGENT SODIUM L A U R Y L SULPHATE
P.W.A. TOVELL, D. HOWES and C.S. NEWSOME Environmental Safety Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford (Great Britain)
(Received September 7th, 1974)
SUMMARY The route of absorption, tissue distribution, metabolism, route of excretion, and excretion rate of the anionic detergent sodium lauryl sulphate (SLS) in goldfish was investigated using [35 S] or [1-14 C] SLS. It has been shown t h a t goldfish absorb SLS from solution principally through the gills, and t h a t SLS is rapidly distributed t h r o u g h o u t the body tissues, the highest concentration being found in the gall bladder. SLS was metabolised by the fish to butyric acid-4-sulphate, and was excreted by the kidney. Over a 24-h period 68% and 38% of a prescribed dose was excreted from freely fed and unfed fish, respectively.
INTRODUCTION There have been several studies on the toxicity of synthetic detergents to freshwater fish [4,9,11,12,18,21,23] and aquatic organisms in general [20]. Other work on the reaction of freshwater fish to detergent toxicity has included reports of changes induced in certain tissues such as the gill [1,16, 19,22], behavioural changes [10,13], the effect of environmental parameters [3,14], and the influence of other pollutants such as metals [2,5]. No a t t e m p t has been made to determine whether or not detergent is absorbed by the fish, whether or n o t it is concentrated in any particular tissue, and to what e x t e n t the detergent is metabolised and excreted. This paper describes experiments which provide answers to these questions, and give an indication of the route of entry of an anionic detergent and the route of excretion of its metabolites. The anionic detergent SLS was used t h r o u g h o u t this study since it is available as a labelled c o m p o u n d (14C or 35S), and has been used by m a n y Abbreviation: R.T., reproductive tissue; SLS, sodium lauryl sulphate.
17
workers as a model detergent. It is anticipated that the techniques estabfished using SLS can be applied routinely to most pollutants, provided that they are defined compounds and can be satisfactorily labelled. MATERIALS AND METHODS Goldfish (Carassius auratus, Linnaeus) of total length 2.5 or 6 in. (average weight 3.8 and 105 g) were obtained from L. Cura and Sons, Hemel Hempstead, and were held in continuously flowing local Bedford tap water (total hardness 300 ppm as calcium carbonate, pH 6.8--7.2, temperature 12 + 1 °) in 3000-1 polythene tanks. Fish were fed daily with grade III t r o u t food pellets, Cooper Nutrition Products Ltd., Witham, Essex. Water hardness (total hardness equivalent to ppm calcium carbonate) was found by measuring the total concentration of bivalent cations using BDH total hardness pellets and titrating against EDTA. Local water had a hardness of 290--320 ppm and contained 100 ppm Ca 2+, 20 ppm Na +, 5 ppm K + and 8 ppm Mg2+. Except where otherwise stated, fish under test were n o t fed and were held in glass tanks containing continuously aerated treatment solution at 18-+ 1 ° under static conditions; the water used for these tests was glassdistilled [24,25]. Fish were acclimatised in the respective treatment solution hardness for 7 days. Rats were 8-week-old 150-g females of the Colworth/Wistar strain. [1-14C] SLS was prepared by a similar m e t h o d to that of Denner et al. [7], using the reaction between [1-14 C]-lauryl alcohol and sulphuric acid at --15 ° in 40--60 petroleum ether. The specific activity was 1.97 pCi/mg. Radiochemical purity was 98.5% by TLC, and 99.0% by isotope dilution analysis. [35S] SLS was purchased from the Radiochemical Centre, Amersham. The specific activity was 54.8 pCi/mg on date of purchase. Radiochemical purity was found to be 96% by TLC, the sample contained some free 35 SO~. Unlabelled SLS was obtained in specially pure form from BDH Chemicals Ltd., Poole. Preliminary experiments showed that goldfish could tolerate a m a x i m u m concentration of 50 ppm SLS in the treatment solution for 24 h. Similarly, a t r e a t m e n t solution volume of 250 ml per 2.5 in. fish was found to be adequate for the tests described below: To assess the tissue distribution of absorbed SLS, fish were treated in solutions containing 50 ppm of either [14C]SLS (4 fish) or [aSS]SLS (2 fish). After 24 h the fish were removed and individually dissected into 14 separate fractions as follows: gut, kidney, R.T., brain, eye, swim bladder, gall bladder, spleen, liver, heart, gill, scales, surface mucus and remainder of body tissue (body). Scale and mucus fractions were obtained from lightly blotted whole fish from which all the scales were removed by scraping with a scalpel, the scrapings were suspended in 10 ml of water and left to settle, the supernatant being taken as the mucus fraction and the sediment as the scale fraction. Each sample (except the surface mucus sample) was weighed, dissolved in
18
Soluene (Packard Instruments Ltd., Chicago, Ill.), and counted in a Packard 4322 liquid scintillation spectrometer following the m e t h o d suggested by the manufacturers. The surface mucus samples and all other aqueous samples were counted in a Triton X-100 toluene liquid scintillator. The counting efficiency of each sample was determined by internal standardisation or a channels ratio technique. The equivalent concentration of SLS in each sample in ppm was then calculated from the weight of tissue and the known specific activity of the labelled material. The use of t r e a t m e n t solutions containing SLS makes subsequent analysis of excreted metabolites difficult since the water contains an excessive proportion of unabsorbed SLS. The feasibility of injecting the fish with suitable concentrations of SLS was investigated. It was found by experiment that an i.p. injection of 0.5--1.0 mg SLS per 2.5 in. goldfish corresponded to the m a x i m u m tolerable 24-h dose (over a 24-h period 2.5 in. goldfish accumulate approximately 250 gg SLS from a t r e a t m e n t solution containing 50 ppm SLS). To assess the tissue distribution of SLS following i.p. injection of [35S] SLS, 8 fish were each injected i.p. with 0.5 mg of labelled SLS in 0.05 ml of water. Immediately after injection each fish was placed in 150 ml of distilled water for 5 min (to check that there was no leakage from the site of injection) before being placed in 500 ml of continuously aerated distilled water for 24 h. Each fish was then dissected into 13 tissues and the radioactivity counted (as described above). The most likely surface through which detergent absorption takes place is the gill epithelium, however, two other possible routes include cutaneous and alimentary absorption. Three experiments were performed to investigate the route of entry of SLS. (i) To establish whether or not ingested SLS could be absorbed from the gastro-intestinal tract 50 pg of [35 S] SLS in 0.05 ml water was administered intragastrically to each of 7 goldfish (2.5 in.) using Portex PP30 nylon cannula. The fish were then placed in a 130-1 continuous flow holding tank. After 18 h the fish were killed and the gall bladders and livers removed so t h a t the radioactivity in each tissue could be counted. (ii) An indication of the extent of ingestion and alimentary and gill absorption of detergent from a t r e a t m e n t solution was investigated by placing 50 goldfish (2.5 in.) in 12 l of 50 ppm [3~]SLS; random groups of 5 fish were removed from the treatment solution after 5, 15, 30 and 45 min and 1, 2, 3, 4, 11 and 24 h, and the liver, gut and gall bladder were removed from each fish so t h a t the accumulated radioactivity in these tissues could be counted. In this way the build-up of labelled material in the three tissues was monitored. (iii) Cutaneous absorption as a proportion of total absorption was assessed by exposing the posterior two-thirds of 8 goldfish to a solution of [3 ~ S] SLS in distilled water, whilst the anterior third of each fish (including mouth, buccal cavity and gills) was exposed to tap water containing no SLS: 8 goldfish (6 in.) were lightly anaesthetised with 20 ppm tricaine methane sulphonate (MS 222) and placed in tailored polythene bags up to their pectoral fins as shown in Fig. 1. The bagged fish were then put individually into separate holding tanks each containing 6 1 of tap water. Each bag con-
19
Fig. 1. The position of a tailored polythene bag on a 6 in. goldfish. The bag is held in position with coarse thread, and the rear end of the fish is bathed in the contents of the bag, whilst the front end and the outside of the bag are free within the 51 of water in the holding tank. The bags did not restrict movement of the fish, but did impair their ability to swim. The fish were apparently able to control swimming depth in the normal way. There was found to be negligible leakage from the bag following tests with methylene blue and radiotracers. tained a p p r o x i m a t e l y 2 0 0 ml o f 50 p p m [a SS] SLS, a n d was held securely in place with coarse t h r e a d . T h e fish w e r e k e p t in the h o l d i n g t a n k s f o r 4 h. The h o l d i n g w a t e r in each t a n k was m o n i t o r e d every h o u r f o r t h e p r e s e n c e of r a d i o l a b e l l e d m a t e r i a l to e n s u r e t h a t t h e r e was n o leakage f r o m t h e bags. At t h e e n d o f t h e 4-h e x p o s u r e p e r i o d t h e liver and gall b l a d d e r w e r e rem o v e d f r o m each fish a n d t h e r a d i o a c t i v i t y in these tissues c o u n t e d . A f u r t h e r 5 goldfish (6 in.) w e r e placed ( u n b a g g e d ) in a t r e a t m e n t s o l u t i o n c o n t a i n i n g 50 p p m [ a S S ] SLS for 4 h, t h e livers a n d gall b l a d d e r s w e r e t h e n r e m o v e d and t h e i r r a d i o a c t i v i t y c o u n t e d . T h e p r o p o r t i o n o f c u t a n e o u s abs o r p t i o n was assessed b y c o m p a r i n g t h e average c o u n t s f r o m bagged and u n b a g g e d fish; c a l c u l a t i o n s were based on an e x p o s u r e o f 75% o f t h e t o t a l skin surface area t o 50 p p m SLS w i t h i n e a c h bag. T h e m o s t likely r o u t e o f e x c r e t i o n o f SLS or its m e t a b o l i t e s is via t h e k i d n e y , h o w e v e r , e x c r e t i o n c o u l d also o c c u r t h r o u g h t h e skin or the respirat o r y m e m b r a n e s o f the gill. T h e r o u t e o f e x c r e t i o n was investigated b y the f o l l o w i n g e x p e r i m e n t . Five 6 in. goldfish w e r e each given an i.p. i n j e c t i o n o f [35S] SLS ( t h e i n j e c t i o n s o l u t i o n c o n s i s t e d o f 16 m g o f labelled SLS dissolved in 1.6 m l o f distilled w a t e r giving a c o n c e n t r a t i o n o f 10 m g / m l ) ; 0.1 ml o f this s o l u t i o n was i n j e c t e d i n t o each fish e q u i v a l e n t to a dose o f 1 mg. T h e fish were a c c l i m a t i s e d to tap (300 p p m ) water, a n d t h e e x p e r i m e n t was r u n in t a p water. A f t e r b e i n g i n j e c t e d t h e fish w e r e placed in c o n t i n u o u s f l o w h o l d i n g t a n k s f o r 15 m i n so t h a t a n y free SLS at t h e i n j e c t i o n site was w a s h e d a w a y . T h e fish were t h e n lightly a n a e s t h e t i s e d (20 p p m MS 2 2 2 ) a n d p l a c e d in t a i l o r e d p o l y t h e n e bags u p to their p e c t o r a l fins as s h o w n in Fig. 1 b e f o r e b e i n g p u t individually i n t o s e p a r a t e h o l d i n g t a n k s each c o n t a i n i n g 5 1 o f water. T h e bags each c o n t a i n e d a p p r o x i m a t e l y 175 ml o f water. A n y r a d i o l a b e l l e d m a t e r i a l e x c r e t e d f r o m t h e k i d n e y was t h e r e f o r e c o l l e c t e d in the bag, a n d a n y r a d i o l a b e l l e d m a t e r i a l e x c r e t e d f r o m t h e gill w o u l d a c c u m u -
20
late in the 5 1 of water in each holding tank. Material excreted from the skin would be found in both the bag and the holding water. Samples (2.0 ml) of the holding water were taken at the beginning and end of the collection time (300 min), and a 2-ml sample was taken from the measured bag volume at the end of the experiment. The radioactivity of each 2-ml sample was counted. In order to assess the excretion rate of absorbed SLS, fish were first placed in a t r e a t m e n t solution containing labelled SLS (uptake phase), and then placed in a large volume of continuously flowing water from which the fish were removed in groups of five after set time intervals and the level of radioactivity remaining in their tissues assayed (excretory phase). Thirty five 2.5 in. goldfish were placed in 4 1 of treatment solution containing 50 ppm [35S] SLS for 5 h; the fish were then transferred into tap water in a continuous flow 130-1 glass fibre tank (7 1/min). Five fish were withdrawn after 5 min (time 0), each of the 5 fish was homogenised and the tissue level of radioactivity counted. Further groups of 5 fish were withdrawn after 1, 3, 6, 9, 19 and 24 h, and the tissue radioactivity was counted. Fish were not fed during this experiment. The effect of feeding was investigated by repeating the experiment (using 20 fish) with food given ad lib. during the excretory phase. Groups of 5 fish were taken for assay after 0, 7, 19 and 24 h. A TLC examination was made of the excretion products and bile from [14 C] SLS-treated goldfish, and of the urine from similarly treated rats. Five samples were run on a TLC plate: (A) excretion products from goldfish given [14 C] SLS i.p.; (B) urine from rats given [14C]SLS i.p.; (C) bile from goldfish given [14C]SLS i.p.; (D) pure [14C] SLS; (E) a mixture of A, B and D. The excretion products from goldfish were obtained from 1 1 of distilled water in which 4 [ l a c ] SLS-injected 2.5 in. goldfish (0.1 ml of a 10 mg/ml solution) had been held for 24 h. The bathing solution was filtered once through No. 1 Whatman filter paper, freeze-dried immediately and taken up in 1 ml of water. Rat urine (collected over the first 6 h following i.p. injection of 0.5 ml of a 10 mg/ml solution of [14C] SLS) was filtered through a bed o f Celite 545 (Johns-Manville Co. Ltd.) and freeze-dried, 1(} mg of solid was taken up in 1 ml of water. The gall bladders were removed from the 4 fish given i.p. [14 C] SLS injections for the collection of excretion products in the bathing solution as described above; each gall bladder was punctured and the bile collected. Aliquots (10 pl) of each sample (A to E) were spotted as bands onto Silica gel G (Merck Ltd., Darmstadt) coated TLC plates (250 p) and developed in n-butanol--acetic acid--water ( 4 : 1 : 1 by volume). Each sample had previously been diluted such that the activity in the 10-pl aliquot was approximately 20 nCi. Plates were air-dried at room temperature and exposed to Kodirex X-ray film (Kodak Ltd., London) for 24 h. To provide a clearer definition between individual bands an aliquot of goldfish excretion products was also run on Silica gel H (250 p) and developed in isobutyric acid--0.2 N ammonia ( 5 : 3 by volume)against pure [14C] SLS as a marker.
21
The molecular structure of the principal metabolite of SLS excreted by fish and rat was investigated using GLC and mass spectral analysis. Freezedried solids from rat urine collected over 6 h after i.p. injection of 35S_ or 14 C-labelled SLS, and from water in which fish had been held for 24 h after i.p. injection of [14C]SLS, were used as the source of metabolites. The sulphate group was hydrolysed from 35 S:labelled metabolites by refluxing in 4 N HC1 (approx. 2 ml) for 16 h. The hydrolysate was neutralised with 1 N NaOH to pH 6--7, and 1.0 ml of 5% sodium sulphate solution added. Excess 5% barium chloride solution was added and the resulting barium sulphate precipitate was centrifuged down. The supernatant was monitored for 35 S. The precipitate was washed with ethanol and then with diethyl ether and these washings monitored for 35S. The washed precipitate was monitored for 3 5 S by suspending in a liquid scintillator. The 14 C-labelled metabolites were hydrolysed in a similar manner to the 3 5 S metabolites above. The hydrolysates were extracted with benzene in a similar manner to that described by Denner et al. [7], and evaporated down to a small volume (approx. 1 ml). Aliquots of the benzene extracts were run on a Pye 104 Gas Liquid Chromatograph System fitted with a 9' × 1/8" column packed with 10% FFAP (free f a t t y acid phase) on 80--100 mesh G-cel. The column was fitted with a stream splitter which directed approximately half the column effluent through a flame ionisation detector, and half through a Panax radiochromatogram attachment. The column was run isothermally at 130 ° and the carrier gas was argon containing 5% CO2 flowing at 50 ml/min. Mass spectroscopy of the peaks containing 14 C was performed using a similar gas liquid chromatograph system coupled to an A.E.I. MS 12 for identification of the rat metabolite. Positive identification of the fish metabolite had to be performed on a high resolution mass spectrometer (A.E.I. MS 903A), since only a small quantity of material was available. RESULTS
Tissue distribution of externally administered SLS The distribution of radioactivity (expressed as equivalent ppm SLS) in the tissues of the 6 fish exposed for 24 h to a treatment solution of distilled water containing 50 ppm labelled SLS is displayed graphically in Fig. 2. The pattern of tissue accumulation was found to be the same following exposure to either [14C]SLS or [3~S]SLS, indicating that the results were not affected by the inclusion of any labelled impurities in either preparation, and that the sulphate group is not removed from the alkyl chain. It is clear t h a t the radioactivity is concentrated primarily in the gall bladder, and to a lesser extent in the gut and liver (Fig. 2). The gill, kidney and scales did not contain an appreciable a m o u n t of labelled material; and the body fraction (muscle and bone) and some other tissues had not reached equilibration with the level of SLS in the treatment solution; there was, however, an overall rise above equilibration in the combined total body tissue level (76 +- 11.8 ppm SLS).
22
5000
3OOO I 5O
IOO0
200
,oo 50
j
i
i
iiiJ
i
i
i
Jl
!0
in z I i .il
Fig. 2. The fate of SLS in goldfish tissues. Combined results from two separate experiments showing the tissue distribution of radioactivity (expressed as ppm SLS) following external administration of 50 ppm [ 1-14C ] SLS and [ 35 S ] SLS for 24 h. Fig. 3. The radioactivity in individual tissues following exposure to [1-14C]SLS or [35S]SLS in the treatment solution. Individual results are expressed as a percentage of the total body activity rather than as a concentration as in Fig. 2. T h e greatest p r o p o r t i o n (or p e r c e n t a g e ) o f SLS was p r e s e n t in t h e " b o d y " a n d gut, f o l l o w e d b y t h e liver and gall b l a d d e r (Fig. 3). T h e gall bladder c o n t a i n s a relatively small p e r c e n t a g e o f the t o t a l b o d y activity due t o its small size. T h e high p r o p o r t i o n in t h e m u c u s f r a c t i o n w o u l d in part be due t o w e t t i n g w i t h t r e a t m e n t solution.
Tissue distribution of internally administered SLS T h e tissue d i s t r i b u t i o n p a t t e r n was similar t o t h a t following e x t e r n a l l y a d m i n i s t e r e d [ 3 5 S ] S L S (Fig. 4); t h e r e was, h o w e v e r , a very high level of r a d i o a c t i v i t y in t h e gall bladder. Figures f o r the scales and m u c u s were n o t d e t e r m i n e d s e p a r a t e l y as t h e s e were e x p e c t e d t o be very low following internal a d m i n i s t r a t i o n . T h e p e r c e n t a g e d i s t r i b u t i o n o f r a d i o a c t i v i t y w i t h i n the tissues s h o w e d t h a t t h e gall b l a d d e r c o n t a i n e d a l m o s t half o f the t o t a l activity (Fig. 5); o t h e r tissues c o n t a i n i n g high p r o p o r t i o n s i n c l u d e d b o d y , gut and liver, as was t h e case f o l l o w i n g e x t e r n a l l y a d m i n i s t e r e d SLS.
Route of absorption of SLS A p p r o x i m a t e l y 22% o f t h e 50 #g SLS a d m i n i s t e r e d intragastrically to each o f 7 goldfish was r e c o v e r e d in t h e liver and gall b l a d d e r a f t e r 18 h, showing t h a t SLS can be a b s o r b e d f r o m t h e intestine. Fig. 6 shows t h e d i s t r i b u t i o n o f r a d i o a c t i v i t y (expressed as pg SLS) in t h e liver, gall b l a d d e r and intestine o f t h e 50 goldfish e x p o s e d for the t i m e s s h o w n to a t r e a t m e n t s o l u t i o n c o n t a i n i n g 50 p p m [ 3 5 S ] S L S . During the
23
5000 3000 O l OO
400
':'00
150
i
tO0
•
i,
i . . i
i
I ill
0
~
i
Fig. 4. The tissue distribution o f radioactivity (expressed in ppm SLS) 24 h after internal administration (i.p.) of 0.5 mg of [35SISLS.
Fig. 5. The percentage of total body radioactivity in individual tissues following internal administration of 0.5 mg of [35S] SLS. first h o u r t h e intestine c o n t a i n e d the g r e a t e s t q u a n t i t y o f d e t e r g e n t , foll o w e d b y t h e liver a f t e r 2 h e x p o s u r e a n d t h e gall b l a d d e r f r o m 11 to 24 h exposure. T h e c u t a n e o u s a b s o r p t i o n e x p e r i m e n t s h o w e d t h a t SLS was a b s o r b e d across t h e skin surface. A f t e r 4 h e x p o s u r e t h e liver a n d gall b l a d d e r o f each fish c o n t a i n e d an average o f 26.8 '-+ 2.9 p g SLS, a f t e r a c c o u n t i n g f o r t h e t o t a l surface a r e a o f skin. In c o m p a r i s o n , t h e livers a n d gall bladders f r o m goldfish p l a c e d free in 50 p p m SLS a c c u m u l a t e d an average o f 128.6 -+ 24.4 pg SLS o v e r t h e s a m e t i m e . This r e p r e s e n t s a c u t a n e o u s a b s o r p t i o n o f a p p r o x i m a t e l y 20% o f t o t a l a b s o r p t i o n b y all r o u t e s .
R o u t e o f excretion T h e r a d i o a c t i v i t y o f t h e 1 m g d o s e o f SLS injected into each fish was 38 891 0 0 0 d p m / m g . T a b l e I s h o w s t h e d i s t r i b u t i o n o f r a d i o a c t i v i t y in the 5 1 o f h o l d i n g w a t e r and in t h e bag w a t e r a f t e r t h e 3 0 0 - m i n c o l l e c t i o n t i m e . I t is clear t h a t labelled m a t e r i a l was e x c r e t e d o n l y i n t o t h e bag, a n d t h a t the r o u t e o f e x c r e t i o n was t h e r e f o r e n o t via the skin o r gills. Rate o f excretion O v e r t h e 24-h e x c r e t o r y p h a s e t h e level o f r a d i o a c t i v i t y in t h e tissues o f u n f e d fish fell 38%, c o m p a r e d w i t h a fall o f 68% in t h e fish fed ad lib. o v e r
24
60
Liver _~S£ ~g i
Gall Bladder "S.E.
~LS
G u t _~S.E.
30
I-C
~g SLS
o.,,j u $
15
30
45
60//
2
3 4
I1
rain
24 hr
Fig. 6. T h e d i s t r i b u t i o n o f r a d i o a c t i v i t y (expressed as ~g SLS) in t h e liver, gall b l a d d e r a n d i n t e s t i n e o f goldfish e x p o s e d for t h e t i m e s s h o w n to a t r e a t m e n t s o l u t i o n c o n t a i n i n g 50 p p m [ 3 5 S ] S L S .
the same period (Fig. 7). Thus, feeding causes a considerable increase in the excretion rate of externally absorbed SLS.
TLC autoradiographic analysis of excretion products Fig. 8 shows a TLC autoradiogram of fish metabolite (A), rat metabolite TABLE I ROUTE OF EXCRETION D i s t r i b u t i o n o f labelled m a t e r i a l in h o l d i n g w a t e r a n d bag a f t e r 3 0 0 min. Fish
Holding water 0 min (dpm/ml)
Holding water 300 min (dpm/ ml)
Bag w a t e r 300 m i n (dpm/ml)
Volume o f bag (ml)
T o t a l activity in b a g ( d p m )
A B C D E
54 57 55 50 43
56 55 51 57 53
18 17 25 27 17
181 210 173 152 162
3 3 4 4 2
957 351 747 343 329
431 644 454 156 807
000 000 000 000 000
25
5C
"L Excreted 3(
1
3
6
7
9
19 Time
24
(h }
Fig. 7. The rate o f e x c r e t i o n over 24 h o f radiolabelled material from fed (©) and u n f e d ( e ) fish f o l l o w i n g 5 h e x p o s u r e to a t r e a t m e n t s o l u t i o n c o n t a i n i n g 50 ppm [ 3 5 S ] S L S .
(B), fish bile (C), pure SLS (D) and a mixture of fish and rat metabolite with SLS (E); these samples were obtained from animals dosed i.p. with [14C] SLS. The principal metabolites excreted from fish and rat had identical RF values. Fish bile contained a minor band corresponding to the excreted metabolite, and also larger proportions of other breakdown products in
Fig. 8. TLC autoradiogram o f e x c r e t e d m e t a b o l i t e s f r o m goldfish ( A ) and rat (B). Also s h o w n are bile from goldfish (C), pure SLS (D), and a m i x t u r e o f fish and rat m e t a b o l i t e w i t h pure SLS (E). T h e m e t a b o l i t e s were 14C-labelled f o l l o w i n g i.p. administration o f [ 1-14C ] SLS. S i l i c a gel G d e v e l o p e d in n - b u t a n o l - - a c e t i c a c i d - - w a t e r (4 : 1 : 1 v/v).
26
S o l v e n t f r o n t ~,-
C12
lO 8 6
Origin
~,-
Excreted
SLS
m e t a bol ites
Fig. 9. TLC autoradiogram of 14C_labelled metabolites excreted from goldfish following i.p. administration o f [14C]SLS; and pure [14C]SLS. The postulated structure of each band is marked as C12 , ClO , CS, C6 and C 4. Silica gel H developed in isobutyric acid-0.2 N NH 3 (5 : 3 v/v).
addition to a considerable a m o u n t of non-metabolised SLS. Further examination of the metabolites of SLS excreted from goldfish shows the presence of four individual bands in addition to the principal metabolite (Fig. 9); one band corresponds to SLS, whilst the other three may represent successive products of ~-oxidation of the alkyl chain.
Identification of the metabolite The acid hydrolysis of the 35S.labelled metabolites from both species yielded a barium salt which was not soluble in water or in the organic solvents used to wash the precipitate. This indicated that the 3 5 s was present in the hydrolysate as 35 SO7,. The gas liquid chromatographs of the extractable 14 C-labelled hydrolysis product from both species were similar. There was, however, 50 times more material in the rat urine extract than in the fish extract. Mass spectra showed that the 14 C-labelled peaks from both fish and rat were similar. A peak of mass 86.0363 was found, compared with a peak of 86.0368 for butyrolactone, which is consistent with the hydrolised metabolic product being butyrolactone and the untreated metabolite being butyric acid-4-sulphate. DISCUSSION
SLS entered the b o d y tissues of goldfish when this substance was present in the t r e a t m e n t solution. The principal route of entry was across the oral or respiratory membranes, though cutaneous absorption and the drinking rate
27
accounted for 20% and 8% of the total, respectively. Drinking rates were calculated on an intestinal uptake of I pg SLS/h from a 50 ppm solution of SLS (Fig. 6) which gave a drinking rate of 526 pl/100 g/h, compared with 646 #l/100 g/h for Carassiops compressus (Krefft) [6]. Absorbed SLS entered the circulation and was distributed freely throughout the body tissues. The higher than average levels of radioactivity found in the liver and gall bladder suggest that the detergent was actively absorbed and metabolised by the liver, and the metabolites were subsequently returned to the circulation or secreted into the gall bladder. The large proportion of SLS which was also present in the bile did not seem out of place since the major constituents of bile have detergent properties. Circulatory SLS metabolite was excreted by the kidney; there was, however, no evidence to exclude alimentary excretion, although cannulation of the urinary tract of [35 S] SLS-treated goldfish yielded very high concentrations of labelled metabolite in the urine (Tovell, unpublished observation). It was significant that no labelled metabolite was excreted from the gill as this organ has been suggested to have an excretory role [15]. Similarly, the kidney appeared unable to clear SLS from the circulation with the same efficiency as it was able to clear the short chain metabolite (Fig. 9). The increased excretion rate of radiolabelled metabolite from freely fed fish would be in part due to the clearance of residual material in the intestinal lumen, and to the release of radiolabelled material stored in the gall bladder [8]; this "biliary isolation" in unfed fish accounts for 13% of the total body radiolabelled material, which on feeding becomes available for re-absorption from the intestine. Lech et al. [17] have reported marked biliary accumulation by rainbow trout of three pesticides when exposed to low concentrations of these substances, and have suggested using fish bile analysis as an aid to the detection of very low concentrations of contaminants in the aquatic environment. The results from this study indicate that fish bile analysis could also be used to monitor for th~ presence of low concentrations of water-borne detergents. Evidence from the TLC studies suggested that the end metabolite of SLS was butyric acid-4-sulphate (Fig. 9). Denner et al. [7] have claimed that in rat the principal metabolite of SLS was butyric acid-4-sulphate, which indicates that the degradation of SLS is by co-oxidation and subsequent ~-oxidation of the alkyl chain. In the present study evidence from TLC of urine from rats injected i.p. with [14C] SLS and from fish similarly treated showed that the metabolites from both animals had similar Rv values. The purification of these metabolites was achieved, and their subsequent analysis has shown them to be butyric acid-4-sulphate. ACKNOWLEDGEMENTS We are grateful to Mr. C. James for preparing the [ l f l 4 C ] SLS, to Mr. D. Welti and Mr. H. Hadaway for performing the mass spectral analysis, to Mrs. A. Cordell for technical assistance, and to Dr. L.J. Morris and Dr. A.J. Collings for their advice and encouragement.
28
REFERENCES 1 J.E. Bardach, M. Fujiya and A. Holl, Sci. N.Y., 148 (1965) 1605. 2 V.M. Brown, V.V. Mitrovic and G.T.C. Stark, Water Res., 2 (1968) 255. 3 J. Cairns and A. Scheier, Proc. 17th Ind. Waste Conf., Purdue Univ. Engng. Ext. Ser., 112 (1962) 14. 4 J. Cairns and A. Scheier, Notul. Nat., 367 (1964) 1. 5 D. Calamari and R. Marchetti, Water Res., 7 (1973) 1453. 6 W. Dall and N.E. Milward, Comp. Biochem. Physiol., 30 (1969) 247. 7 W.H.B. Denner, A.H. Olavesen, G.M. Powell and K.S. Dodgson, Biochem. J., 111 (1969) 43. 8 R.W. Denton, L.L. Gershbein and A.C. Ivy, J. Appl. Physiol., 2 (1950) 671. 9 T.P. Dooley and J. Cavil, Texas J. Sci., 16 (1964) 202. 10 N.R. Foster, A. Scheier and J. Cairns, Trans. Am. Fish. Soc., 95 (1966) 109. 11 C. Henderson, Q.H. Pickering and J.M. Cohen, Sew. Ind. Wastes, 31 (1959) 295. 12 D.W.M. Herbert, G.H.J. Elkins, H.T. Mann and J. Hemens, War. Waste Treat. J., 6 (1957) 394. 13 E. Hirsch, Vom. Wasser, 30 (1963) 249. 14 K.E.F. Hokanson and L.L. Smith, Trans. Am. Fish. Soc., 100 (1971) 1. 15 A.B. Keys, Z. Vergl. Physiol., 15 (1931) 364. 16 W. Lang, Arch. Fisch Wiss., 18 (1967) 25. 17 J.J. Lech, S.K. Pepple and C.N. Statham, Toxicol. Appl. Pharmacol., 25 (1973) 430. 18 E. Leclerc and F. Devlaminck, Bull. Cent. Belge Etud. Docum. Eaux, 17 (1952) 165. 19 A.E. Lemke and D.I. Mount, Trans. Am. Fish. Soc., 92 (1963) 372. 20 R. Marchetti, Stud. Rev. Gen. Fish. Counc. Mediterr., 26 (1965) 1. 21 Q.H. Pickering and T.O. Thatcher, J. Water Pollut. Control Fed., 42 (1970) 243. 22 O.J. Schmid and H. Mann, Arch. Fisch Wiss., 13 (1962) 41. 23 T.O. Thatcher and J.F. Santner, Proc. 21st Ind. Waste Conf., Purdue Univ. Engng. Ext. Ser., 121 (1966) 966. 24 P.W.A. Tovell, C. Newsome and D. Howes, Water Res., 8 (1974) 291. 25 P.W.A. Tovell, C. Newsome and D. Howes, (1974) in preparation.
29
ABSORPTION, METABOLISM AND EXCRETION BY GOLDFISH OF THE ANIONIC DETERGENT SODIUM L A U R Y L SULPHATE
P.W.A. TOVELL, D. HOWES and C.S. NEWSOME Environmental Safety Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford (Great Britain)
(Received September 7th, 1974)
SUMMARY The route of absorption, tissue distribution, metabolism, route of excretion, and excretion rate of the anionic detergent sodium lauryl sulphate (SLS) in goldfish was investigated using [35 S] or [1-14 C] SLS. It has been shown t h a t goldfish absorb SLS from solution principally through the gills, and t h a t SLS is rapidly distributed t h r o u g h o u t the body tissues, the highest concentration being found in the gall bladder. SLS was metabolised by the fish to butyric acid-4-sulphate, and was excreted by the kidney. Over a 24-h period 68% and 38% of a prescribed dose was excreted from freely fed and unfed fish, respectively.
INTRODUCTION There have been several studies on the toxicity of synthetic detergents to freshwater fish [4,9,11,12,18,21,23] and aquatic organisms in general [20]. Other work on the reaction of freshwater fish to detergent toxicity has included reports of changes induced in certain tissues such as the gill [1,16, 19,22], behavioural changes [10,13], the effect of environmental parameters [3,14], and the influence of other pollutants such as metals [2,5]. No a t t e m p t has been made to determine whether or not detergent is absorbed by the fish, whether or n o t it is concentrated in any particular tissue, and to what e x t e n t the detergent is metabolised and excreted. This paper describes experiments which provide answers to these questions, and give an indication of the route of entry of an anionic detergent and the route of excretion of its metabolites. The anionic detergent SLS was used t h r o u g h o u t this study since it is available as a labelled c o m p o u n d (14C or 35S), and has been used by m a n y Abbreviation: R.T., reproductive tissue; SLS, sodium lauryl sulphate.
17
workers as a model detergent. It is anticipated that the techniques estabfished using SLS can be applied routinely to most pollutants, provided that they are defined compounds and can be satisfactorily labelled. MATERIALS AND METHODS Goldfish (Carassius auratus, Linnaeus) of total length 2.5 or 6 in. (average weight 3.8 and 105 g) were obtained from L. Cura and Sons, Hemel Hempstead, and were held in continuously flowing local Bedford tap water (total hardness 300 ppm as calcium carbonate, pH 6.8--7.2, temperature 12 + 1 °) in 3000-1 polythene tanks. Fish were fed daily with grade III t r o u t food pellets, Cooper Nutrition Products Ltd., Witham, Essex. Water hardness (total hardness equivalent to ppm calcium carbonate) was found by measuring the total concentration of bivalent cations using BDH total hardness pellets and titrating against EDTA. Local water had a hardness of 290--320 ppm and contained 100 ppm Ca 2+, 20 ppm Na +, 5 ppm K + and 8 ppm Mg2+. Except where otherwise stated, fish under test were n o t fed and were held in glass tanks containing continuously aerated treatment solution at 18-+ 1 ° under static conditions; the water used for these tests was glassdistilled [24,25]. Fish were acclimatised in the respective treatment solution hardness for 7 days. Rats were 8-week-old 150-g females of the Colworth/Wistar strain. [1-14C] SLS was prepared by a similar m e t h o d to that of Denner et al. [7], using the reaction between [1-14 C]-lauryl alcohol and sulphuric acid at --15 ° in 40--60 petroleum ether. The specific activity was 1.97 pCi/mg. Radiochemical purity was 98.5% by TLC, and 99.0% by isotope dilution analysis. [35S] SLS was purchased from the Radiochemical Centre, Amersham. The specific activity was 54.8 pCi/mg on date of purchase. Radiochemical purity was found to be 96% by TLC, the sample contained some free 35 SO~. Unlabelled SLS was obtained in specially pure form from BDH Chemicals Ltd., Poole. Preliminary experiments showed that goldfish could tolerate a m a x i m u m concentration of 50 ppm SLS in the treatment solution for 24 h. Similarly, a t r e a t m e n t solution volume of 250 ml per 2.5 in. fish was found to be adequate for the tests described below: To assess the tissue distribution of absorbed SLS, fish were treated in solutions containing 50 ppm of either [14C]SLS (4 fish) or [aSS]SLS (2 fish). After 24 h the fish were removed and individually dissected into 14 separate fractions as follows: gut, kidney, R.T., brain, eye, swim bladder, gall bladder, spleen, liver, heart, gill, scales, surface mucus and remainder of body tissue (body). Scale and mucus fractions were obtained from lightly blotted whole fish from which all the scales were removed by scraping with a scalpel, the scrapings were suspended in 10 ml of water and left to settle, the supernatant being taken as the mucus fraction and the sediment as the scale fraction. Each sample (except the surface mucus sample) was weighed, dissolved in
18
Soluene (Packard Instruments Ltd., Chicago, Ill.), and counted in a Packard 4322 liquid scintillation spectrometer following the m e t h o d suggested by the manufacturers. The surface mucus samples and all other aqueous samples were counted in a Triton X-100 toluene liquid scintillator. The counting efficiency of each sample was determined by internal standardisation or a channels ratio technique. The equivalent concentration of SLS in each sample in ppm was then calculated from the weight of tissue and the known specific activity of the labelled material. The use of t r e a t m e n t solutions containing SLS makes subsequent analysis of excreted metabolites difficult since the water contains an excessive proportion of unabsorbed SLS. The feasibility of injecting the fish with suitable concentrations of SLS was investigated. It was found by experiment that an i.p. injection of 0.5--1.0 mg SLS per 2.5 in. goldfish corresponded to the m a x i m u m tolerable 24-h dose (over a 24-h period 2.5 in. goldfish accumulate approximately 250 gg SLS from a t r e a t m e n t solution containing 50 ppm SLS). To assess the tissue distribution of SLS following i.p. injection of [35S] SLS, 8 fish were each injected i.p. with 0.5 mg of labelled SLS in 0.05 ml of water. Immediately after injection each fish was placed in 150 ml of distilled water for 5 min (to check that there was no leakage from the site of injection) before being placed in 500 ml of continuously aerated distilled water for 24 h. Each fish was then dissected into 13 tissues and the radioactivity counted (as described above). The most likely surface through which detergent absorption takes place is the gill epithelium, however, two other possible routes include cutaneous and alimentary absorption. Three experiments were performed to investigate the route of entry of SLS. (i) To establish whether or not ingested SLS could be absorbed from the gastro-intestinal tract 50 pg of [35 S] SLS in 0.05 ml water was administered intragastrically to each of 7 goldfish (2.5 in.) using Portex PP30 nylon cannula. The fish were then placed in a 130-1 continuous flow holding tank. After 18 h the fish were killed and the gall bladders and livers removed so t h a t the radioactivity in each tissue could be counted. (ii) An indication of the extent of ingestion and alimentary and gill absorption of detergent from a t r e a t m e n t solution was investigated by placing 50 goldfish (2.5 in.) in 12 l of 50 ppm [3~]SLS; random groups of 5 fish were removed from the treatment solution after 5, 15, 30 and 45 min and 1, 2, 3, 4, 11 and 24 h, and the liver, gut and gall bladder were removed from each fish so t h a t the accumulated radioactivity in these tissues could be counted. In this way the build-up of labelled material in the three tissues was monitored. (iii) Cutaneous absorption as a proportion of total absorption was assessed by exposing the posterior two-thirds of 8 goldfish to a solution of [3 ~ S] SLS in distilled water, whilst the anterior third of each fish (including mouth, buccal cavity and gills) was exposed to tap water containing no SLS: 8 goldfish (6 in.) were lightly anaesthetised with 20 ppm tricaine methane sulphonate (MS 222) and placed in tailored polythene bags up to their pectoral fins as shown in Fig. 1. The bagged fish were then put individually into separate holding tanks each containing 6 1 of tap water. Each bag con-
19
Fig. 1. The position of a tailored polythene bag on a 6 in. goldfish. The bag is held in position with coarse thread, and the rear end of the fish is bathed in the contents of the bag, whilst the front end and the outside of the bag are free within the 51 of water in the holding tank. The bags did not restrict movement of the fish, but did impair their ability to swim. The fish were apparently able to control swimming depth in the normal way. There was found to be negligible leakage from the bag following tests with methylene blue and radiotracers. tained a p p r o x i m a t e l y 2 0 0 ml o f 50 p p m [a SS] SLS, a n d was held securely in place with coarse t h r e a d . T h e fish w e r e k e p t in the h o l d i n g t a n k s f o r 4 h. The h o l d i n g w a t e r in each t a n k was m o n i t o r e d every h o u r f o r t h e p r e s e n c e of r a d i o l a b e l l e d m a t e r i a l to e n s u r e t h a t t h e r e was n o leakage f r o m t h e bags. At t h e e n d o f t h e 4-h e x p o s u r e p e r i o d t h e liver and gall b l a d d e r w e r e rem o v e d f r o m each fish a n d t h e r a d i o a c t i v i t y in these tissues c o u n t e d . A f u r t h e r 5 goldfish (6 in.) w e r e placed ( u n b a g g e d ) in a t r e a t m e n t s o l u t i o n c o n t a i n i n g 50 p p m [ a S S ] SLS for 4 h, t h e livers a n d gall b l a d d e r s w e r e t h e n r e m o v e d and t h e i r r a d i o a c t i v i t y c o u n t e d . T h e p r o p o r t i o n o f c u t a n e o u s abs o r p t i o n was assessed b y c o m p a r i n g t h e average c o u n t s f r o m bagged and u n b a g g e d fish; c a l c u l a t i o n s were based on an e x p o s u r e o f 75% o f t h e t o t a l skin surface area t o 50 p p m SLS w i t h i n e a c h bag. T h e m o s t likely r o u t e o f e x c r e t i o n o f SLS or its m e t a b o l i t e s is via t h e k i d n e y , h o w e v e r , e x c r e t i o n c o u l d also o c c u r t h r o u g h t h e skin or the respirat o r y m e m b r a n e s o f the gill. T h e r o u t e o f e x c r e t i o n was investigated b y the f o l l o w i n g e x p e r i m e n t . Five 6 in. goldfish w e r e each given an i.p. i n j e c t i o n o f [35S] SLS ( t h e i n j e c t i o n s o l u t i o n c o n s i s t e d o f 16 m g o f labelled SLS dissolved in 1.6 m l o f distilled w a t e r giving a c o n c e n t r a t i o n o f 10 m g / m l ) ; 0.1 ml o f this s o l u t i o n was i n j e c t e d i n t o each fish e q u i v a l e n t to a dose o f 1 mg. T h e fish were a c c l i m a t i s e d to tap (300 p p m ) water, a n d t h e e x p e r i m e n t was r u n in t a p water. A f t e r b e i n g i n j e c t e d t h e fish w e r e placed in c o n t i n u o u s f l o w h o l d i n g t a n k s f o r 15 m i n so t h a t a n y free SLS at t h e i n j e c t i o n site was w a s h e d a w a y . T h e fish were t h e n lightly a n a e s t h e t i s e d (20 p p m MS 2 2 2 ) a n d p l a c e d in t a i l o r e d p o l y t h e n e bags u p to their p e c t o r a l fins as s h o w n in Fig. 1 b e f o r e b e i n g p u t individually i n t o s e p a r a t e h o l d i n g t a n k s each c o n t a i n i n g 5 1 o f water. T h e bags each c o n t a i n e d a p p r o x i m a t e l y 175 ml o f water. A n y r a d i o l a b e l l e d m a t e r i a l e x c r e t e d f r o m t h e k i d n e y was t h e r e f o r e c o l l e c t e d in the bag, a n d a n y r a d i o l a b e l l e d m a t e r i a l e x c r e t e d f r o m t h e gill w o u l d a c c u m u -
20
late in the 5 1 of water in each holding tank. Material excreted from the skin would be found in both the bag and the holding water. Samples (2.0 ml) of the holding water were taken at the beginning and end of the collection time (300 min), and a 2-ml sample was taken from the measured bag volume at the end of the experiment. The radioactivity of each 2-ml sample was counted. In order to assess the excretion rate of absorbed SLS, fish were first placed in a t r e a t m e n t solution containing labelled SLS (uptake phase), and then placed in a large volume of continuously flowing water from which the fish were removed in groups of five after set time intervals and the level of radioactivity remaining in their tissues assayed (excretory phase). Thirty five 2.5 in. goldfish were placed in 4 1 of treatment solution containing 50 ppm [35S] SLS for 5 h; the fish were then transferred into tap water in a continuous flow 130-1 glass fibre tank (7 1/min). Five fish were withdrawn after 5 min (time 0), each of the 5 fish was homogenised and the tissue level of radioactivity counted. Further groups of 5 fish were withdrawn after 1, 3, 6, 9, 19 and 24 h, and the tissue radioactivity was counted. Fish were not fed during this experiment. The effect of feeding was investigated by repeating the experiment (using 20 fish) with food given ad lib. during the excretory phase. Groups of 5 fish were taken for assay after 0, 7, 19 and 24 h. A TLC examination was made of the excretion products and bile from [14 C] SLS-treated goldfish, and of the urine from similarly treated rats. Five samples were run on a TLC plate: (A) excretion products from goldfish given [14 C] SLS i.p.; (B) urine from rats given [14C]SLS i.p.; (C) bile from goldfish given [14C]SLS i.p.; (D) pure [14C] SLS; (E) a mixture of A, B and D. The excretion products from goldfish were obtained from 1 1 of distilled water in which 4 [ l a c ] SLS-injected 2.5 in. goldfish (0.1 ml of a 10 mg/ml solution) had been held for 24 h. The bathing solution was filtered once through No. 1 Whatman filter paper, freeze-dried immediately and taken up in 1 ml of water. Rat urine (collected over the first 6 h following i.p. injection of 0.5 ml of a 10 mg/ml solution of [14C] SLS) was filtered through a bed o f Celite 545 (Johns-Manville Co. Ltd.) and freeze-dried, 1(} mg of solid was taken up in 1 ml of water. The gall bladders were removed from the 4 fish given i.p. [14 C] SLS injections for the collection of excretion products in the bathing solution as described above; each gall bladder was punctured and the bile collected. Aliquots (10 pl) of each sample (A to E) were spotted as bands onto Silica gel G (Merck Ltd., Darmstadt) coated TLC plates (250 p) and developed in n-butanol--acetic acid--water ( 4 : 1 : 1 by volume). Each sample had previously been diluted such that the activity in the 10-pl aliquot was approximately 20 nCi. Plates were air-dried at room temperature and exposed to Kodirex X-ray film (Kodak Ltd., London) for 24 h. To provide a clearer definition between individual bands an aliquot of goldfish excretion products was also run on Silica gel H (250 p) and developed in isobutyric acid--0.2 N ammonia ( 5 : 3 by volume)against pure [14C] SLS as a marker.
21
The molecular structure of the principal metabolite of SLS excreted by fish and rat was investigated using GLC and mass spectral analysis. Freezedried solids from rat urine collected over 6 h after i.p. injection of 35S_ or 14 C-labelled SLS, and from water in which fish had been held for 24 h after i.p. injection of [14C]SLS, were used as the source of metabolites. The sulphate group was hydrolysed from 35 S:labelled metabolites by refluxing in 4 N HC1 (approx. 2 ml) for 16 h. The hydrolysate was neutralised with 1 N NaOH to pH 6--7, and 1.0 ml of 5% sodium sulphate solution added. Excess 5% barium chloride solution was added and the resulting barium sulphate precipitate was centrifuged down. The supernatant was monitored for 35 S. The precipitate was washed with ethanol and then with diethyl ether and these washings monitored for 35S. The washed precipitate was monitored for 3 5 S by suspending in a liquid scintillator. The 14 C-labelled metabolites were hydrolysed in a similar manner to the 3 5 S metabolites above. The hydrolysates were extracted with benzene in a similar manner to that described by Denner et al. [7], and evaporated down to a small volume (approx. 1 ml). Aliquots of the benzene extracts were run on a Pye 104 Gas Liquid Chromatograph System fitted with a 9' × 1/8" column packed with 10% FFAP (free f a t t y acid phase) on 80--100 mesh G-cel. The column was fitted with a stream splitter which directed approximately half the column effluent through a flame ionisation detector, and half through a Panax radiochromatogram attachment. The column was run isothermally at 130 ° and the carrier gas was argon containing 5% CO2 flowing at 50 ml/min. Mass spectroscopy of the peaks containing 14 C was performed using a similar gas liquid chromatograph system coupled to an A.E.I. MS 12 for identification of the rat metabolite. Positive identification of the fish metabolite had to be performed on a high resolution mass spectrometer (A.E.I. MS 903A), since only a small quantity of material was available. RESULTS
Tissue distribution of externally administered SLS The distribution of radioactivity (expressed as equivalent ppm SLS) in the tissues of the 6 fish exposed for 24 h to a treatment solution of distilled water containing 50 ppm labelled SLS is displayed graphically in Fig. 2. The pattern of tissue accumulation was found to be the same following exposure to either [14C]SLS or [3~S]SLS, indicating that the results were not affected by the inclusion of any labelled impurities in either preparation, and that the sulphate group is not removed from the alkyl chain. It is clear t h a t the radioactivity is concentrated primarily in the gall bladder, and to a lesser extent in the gut and liver (Fig. 2). The gill, kidney and scales did not contain an appreciable a m o u n t of labelled material; and the body fraction (muscle and bone) and some other tissues had not reached equilibration with the level of SLS in the treatment solution; there was, however, an overall rise above equilibration in the combined total body tissue level (76 +- 11.8 ppm SLS).
22
5000
3OOO I 5O
IOO0
200
,oo 50
j
i
i
iiiJ
i
i
i
Jl
!0
in z I i .il
Fig. 2. The fate of SLS in goldfish tissues. Combined results from two separate experiments showing the tissue distribution of radioactivity (expressed as ppm SLS) following external administration of 50 ppm [ 1-14C ] SLS and [ 35 S ] SLS for 24 h. Fig. 3. The radioactivity in individual tissues following exposure to [1-14C]SLS or [35S]SLS in the treatment solution. Individual results are expressed as a percentage of the total body activity rather than as a concentration as in Fig. 2. T h e greatest p r o p o r t i o n (or p e r c e n t a g e ) o f SLS was p r e s e n t in t h e " b o d y " a n d gut, f o l l o w e d b y t h e liver and gall b l a d d e r (Fig. 3). T h e gall bladder c o n t a i n s a relatively small p e r c e n t a g e o f the t o t a l b o d y activity due t o its small size. T h e high p r o p o r t i o n in t h e m u c u s f r a c t i o n w o u l d in part be due t o w e t t i n g w i t h t r e a t m e n t solution.
Tissue distribution of internally administered SLS T h e tissue d i s t r i b u t i o n p a t t e r n was similar t o t h a t following e x t e r n a l l y a d m i n i s t e r e d [ 3 5 S ] S L S (Fig. 4); t h e r e was, h o w e v e r , a very high level of r a d i o a c t i v i t y in t h e gall bladder. Figures f o r the scales and m u c u s were n o t d e t e r m i n e d s e p a r a t e l y as t h e s e were e x p e c t e d t o be very low following internal a d m i n i s t r a t i o n . T h e p e r c e n t a g e d i s t r i b u t i o n o f r a d i o a c t i v i t y w i t h i n the tissues s h o w e d t h a t t h e gall b l a d d e r c o n t a i n e d a l m o s t half o f the t o t a l activity (Fig. 5); o t h e r tissues c o n t a i n i n g high p r o p o r t i o n s i n c l u d e d b o d y , gut and liver, as was t h e case f o l l o w i n g e x t e r n a l l y a d m i n i s t e r e d SLS.
Route of absorption of SLS A p p r o x i m a t e l y 22% o f t h e 50 #g SLS a d m i n i s t e r e d intragastrically to each o f 7 goldfish was r e c o v e r e d in t h e liver and gall b l a d d e r a f t e r 18 h, showing t h a t SLS can be a b s o r b e d f r o m t h e intestine. Fig. 6 shows t h e d i s t r i b u t i o n o f r a d i o a c t i v i t y (expressed as pg SLS) in t h e liver, gall b l a d d e r and intestine o f t h e 50 goldfish e x p o s e d for the t i m e s s h o w n to a t r e a t m e n t s o l u t i o n c o n t a i n i n g 50 p p m [ 3 5 S ] S L S . During the
23
5000 3000 O l OO
400
':'00
150
i
tO0
•
i,
i . . i
i
I ill
0
~
i
Fig. 4. The tissue distribution o f radioactivity (expressed in ppm SLS) 24 h after internal administration (i.p.) of 0.5 mg of [35SISLS.
Fig. 5. The percentage of total body radioactivity in individual tissues following internal administration of 0.5 mg of [35S] SLS. first h o u r t h e intestine c o n t a i n e d the g r e a t e s t q u a n t i t y o f d e t e r g e n t , foll o w e d b y t h e liver a f t e r 2 h e x p o s u r e a n d t h e gall b l a d d e r f r o m 11 to 24 h exposure. T h e c u t a n e o u s a b s o r p t i o n e x p e r i m e n t s h o w e d t h a t SLS was a b s o r b e d across t h e skin surface. A f t e r 4 h e x p o s u r e t h e liver a n d gall b l a d d e r o f each fish c o n t a i n e d an average o f 26.8 '-+ 2.9 p g SLS, a f t e r a c c o u n t i n g f o r t h e t o t a l surface a r e a o f skin. In c o m p a r i s o n , t h e livers a n d gall bladders f r o m goldfish p l a c e d free in 50 p p m SLS a c c u m u l a t e d an average o f 128.6 -+ 24.4 pg SLS o v e r t h e s a m e t i m e . This r e p r e s e n t s a c u t a n e o u s a b s o r p t i o n o f a p p r o x i m a t e l y 20% o f t o t a l a b s o r p t i o n b y all r o u t e s .
R o u t e o f excretion T h e r a d i o a c t i v i t y o f t h e 1 m g d o s e o f SLS injected into each fish was 38 891 0 0 0 d p m / m g . T a b l e I s h o w s t h e d i s t r i b u t i o n o f r a d i o a c t i v i t y in the 5 1 o f h o l d i n g w a t e r and in t h e bag w a t e r a f t e r t h e 3 0 0 - m i n c o l l e c t i o n t i m e . I t is clear t h a t labelled m a t e r i a l was e x c r e t e d o n l y i n t o t h e bag, a n d t h a t the r o u t e o f e x c r e t i o n was t h e r e f o r e n o t via the skin o r gills. Rate o f excretion O v e r t h e 24-h e x c r e t o r y p h a s e t h e level o f r a d i o a c t i v i t y in t h e tissues o f u n f e d fish fell 38%, c o m p a r e d w i t h a fall o f 68% in t h e fish fed ad lib. o v e r
24
60
Liver _~S£ ~g i
Gall Bladder "S.E.
~LS
G u t _~S.E.
30
I-C
~g SLS
o.,,j u $
15
30
45
60//
2
3 4
I1
rain
24 hr
Fig. 6. T h e d i s t r i b u t i o n o f r a d i o a c t i v i t y (expressed as ~g SLS) in t h e liver, gall b l a d d e r a n d i n t e s t i n e o f goldfish e x p o s e d for t h e t i m e s s h o w n to a t r e a t m e n t s o l u t i o n c o n t a i n i n g 50 p p m [ 3 5 S ] S L S .
the same period (Fig. 7). Thus, feeding causes a considerable increase in the excretion rate of externally absorbed SLS.
TLC autoradiographic analysis of excretion products Fig. 8 shows a TLC autoradiogram of fish metabolite (A), rat metabolite TABLE I ROUTE OF EXCRETION D i s t r i b u t i o n o f labelled m a t e r i a l in h o l d i n g w a t e r a n d bag a f t e r 3 0 0 min. Fish
Holding water 0 min (dpm/ml)
Holding water 300 min (dpm/ ml)
Bag w a t e r 300 m i n (dpm/ml)
Volume o f bag (ml)
T o t a l activity in b a g ( d p m )
A B C D E
54 57 55 50 43
56 55 51 57 53
18 17 25 27 17
181 210 173 152 162
3 3 4 4 2
957 351 747 343 329
431 644 454 156 807
000 000 000 000 000
25
5C
"L Excreted 3(
1
3
6
7
9
19 Time
24
(h }
Fig. 7. The rate o f e x c r e t i o n over 24 h o f radiolabelled material from fed (©) and u n f e d ( e ) fish f o l l o w i n g 5 h e x p o s u r e to a t r e a t m e n t s o l u t i o n c o n t a i n i n g 50 ppm [ 3 5 S ] S L S .
(B), fish bile (C), pure SLS (D) and a mixture of fish and rat metabolite with SLS (E); these samples were obtained from animals dosed i.p. with [14C] SLS. The principal metabolites excreted from fish and rat had identical RF values. Fish bile contained a minor band corresponding to the excreted metabolite, and also larger proportions of other breakdown products in
Fig. 8. TLC autoradiogram o f e x c r e t e d m e t a b o l i t e s f r o m goldfish ( A ) and rat (B). Also s h o w n are bile from goldfish (C), pure SLS (D), and a m i x t u r e o f fish and rat m e t a b o l i t e w i t h pure SLS (E). T h e m e t a b o l i t e s were 14C-labelled f o l l o w i n g i.p. administration o f [ 1-14C ] SLS. S i l i c a gel G d e v e l o p e d in n - b u t a n o l - - a c e t i c a c i d - - w a t e r (4 : 1 : 1 v/v).
26
S o l v e n t f r o n t ~,-
C12
lO 8 6
Origin
~,-
Excreted
SLS
m e t a bol ites
Fig. 9. TLC autoradiogram of 14C_labelled metabolites excreted from goldfish following i.p. administration o f [14C]SLS; and pure [14C]SLS. The postulated structure of each band is marked as C12 , ClO , CS, C6 and C 4. Silica gel H developed in isobutyric acid-0.2 N NH 3 (5 : 3 v/v).
addition to a considerable a m o u n t of non-metabolised SLS. Further examination of the metabolites of SLS excreted from goldfish shows the presence of four individual bands in addition to the principal metabolite (Fig. 9); one band corresponds to SLS, whilst the other three may represent successive products of ~-oxidation of the alkyl chain.
Identification of the metabolite The acid hydrolysis of the 35S.labelled metabolites from both species yielded a barium salt which was not soluble in water or in the organic solvents used to wash the precipitate. This indicated that the 3 5 s was present in the hydrolysate as 35 SO7,. The gas liquid chromatographs of the extractable 14 C-labelled hydrolysis product from both species were similar. There was, however, 50 times more material in the rat urine extract than in the fish extract. Mass spectra showed that the 14 C-labelled peaks from both fish and rat were similar. A peak of mass 86.0363 was found, compared with a peak of 86.0368 for butyrolactone, which is consistent with the hydrolised metabolic product being butyrolactone and the untreated metabolite being butyric acid-4-sulphate. DISCUSSION
SLS entered the b o d y tissues of goldfish when this substance was present in the t r e a t m e n t solution. The principal route of entry was across the oral or respiratory membranes, though cutaneous absorption and the drinking rate
27
accounted for 20% and 8% of the total, respectively. Drinking rates were calculated on an intestinal uptake of I pg SLS/h from a 50 ppm solution of SLS (Fig. 6) which gave a drinking rate of 526 pl/100 g/h, compared with 646 #l/100 g/h for Carassiops compressus (Krefft) [6]. Absorbed SLS entered the circulation and was distributed freely throughout the body tissues. The higher than average levels of radioactivity found in the liver and gall bladder suggest that the detergent was actively absorbed and metabolised by the liver, and the metabolites were subsequently returned to the circulation or secreted into the gall bladder. The large proportion of SLS which was also present in the bile did not seem out of place since the major constituents of bile have detergent properties. Circulatory SLS metabolite was excreted by the kidney; there was, however, no evidence to exclude alimentary excretion, although cannulation of the urinary tract of [35 S] SLS-treated goldfish yielded very high concentrations of labelled metabolite in the urine (Tovell, unpublished observation). It was significant that no labelled metabolite was excreted from the gill as this organ has been suggested to have an excretory role [15]. Similarly, the kidney appeared unable to clear SLS from the circulation with the same efficiency as it was able to clear the short chain metabolite (Fig. 9). The increased excretion rate of radiolabelled metabolite from freely fed fish would be in part due to the clearance of residual material in the intestinal lumen, and to the release of radiolabelled material stored in the gall bladder [8]; this "biliary isolation" in unfed fish accounts for 13% of the total body radiolabelled material, which on feeding becomes available for re-absorption from the intestine. Lech et al. [17] have reported marked biliary accumulation by rainbow trout of three pesticides when exposed to low concentrations of these substances, and have suggested using fish bile analysis as an aid to the detection of very low concentrations of contaminants in the aquatic environment. The results from this study indicate that fish bile analysis could also be used to monitor for th~ presence of low concentrations of water-borne detergents. Evidence from the TLC studies suggested that the end metabolite of SLS was butyric acid-4-sulphate (Fig. 9). Denner et al. [7] have claimed that in rat the principal metabolite of SLS was butyric acid-4-sulphate, which indicates that the degradation of SLS is by co-oxidation and subsequent ~-oxidation of the alkyl chain. In the present study evidence from TLC of urine from rats injected i.p. with [14C] SLS and from fish similarly treated showed that the metabolites from both animals had similar Rv values. The purification of these metabolites was achieved, and their subsequent analysis has shown them to be butyric acid-4-sulphate. ACKNOWLEDGEMENTS We are grateful to Mr. C. James for preparing the [ l f l 4 C ] SLS, to Mr. D. Welti and Mr. H. Hadaway for performing the mass spectral analysis, to Mrs. A. Cordell for technical assistance, and to Dr. L.J. Morris and Dr. A.J. Collings for their advice and encouragement.
28
REFERENCES 1 J.E. Bardach, M. Fujiya and A. Holl, Sci. N.Y., 148 (1965) 1605. 2 V.M. Brown, V.V. Mitrovic and G.T.C. Stark, Water Res., 2 (1968) 255. 3 J. Cairns and A. Scheier, Proc. 17th Ind. Waste Conf., Purdue Univ. Engng. Ext. Ser., 112 (1962) 14. 4 J. Cairns and A. Scheier, Notul. Nat., 367 (1964) 1. 5 D. Calamari and R. Marchetti, Water Res., 7 (1973) 1453. 6 W. Dall and N.E. Milward, Comp. Biochem. Physiol., 30 (1969) 247. 7 W.H.B. Denner, A.H. Olavesen, G.M. Powell and K.S. Dodgson, Biochem. J., 111 (1969) 43. 8 R.W. Denton, L.L. Gershbein and A.C. Ivy, J. Appl. Physiol., 2 (1950) 671. 9 T.P. Dooley and J. Cavil, Texas J. Sci., 16 (1964) 202. 10 N.R. Foster, A. Scheier and J. Cairns, Trans. Am. Fish. Soc., 95 (1966) 109. 11 C. Henderson, Q.H. Pickering and J.M. Cohen, Sew. Ind. Wastes, 31 (1959) 295. 12 D.W.M. Herbert, G.H.J. Elkins, H.T. Mann and J. Hemens, War. Waste Treat. J., 6 (1957) 394. 13 E. Hirsch, Vom. Wasser, 30 (1963) 249. 14 K.E.F. Hokanson and L.L. Smith, Trans. Am. Fish. Soc., 100 (1971) 1. 15 A.B. Keys, Z. Vergl. Physiol., 15 (1931) 364. 16 W. Lang, Arch. Fisch Wiss., 18 (1967) 25. 17 J.J. Lech, S.K. Pepple and C.N. Statham, Toxicol. Appl. Pharmacol., 25 (1973) 430. 18 E. Leclerc and F. Devlaminck, Bull. Cent. Belge Etud. Docum. Eaux, 17 (1952) 165. 19 A.E. Lemke and D.I. Mount, Trans. Am. Fish. Soc., 92 (1963) 372. 20 R. Marchetti, Stud. Rev. Gen. Fish. Counc. Mediterr., 26 (1965) 1. 21 Q.H. Pickering and T.O. Thatcher, J. Water Pollut. Control Fed., 42 (1970) 243. 22 O.J. Schmid and H. Mann, Arch. Fisch Wiss., 13 (1962) 41. 23 T.O. Thatcher and J.F. Santner, Proc. 21st Ind. Waste Conf., Purdue Univ. Engng. Ext. Ser., 121 (1966) 966. 24 P.W.A. Tovell, C. Newsome and D. Howes, Water Res., 8 (1974) 291. 25 P.W.A. Tovell, C. Newsome and D. Howes, (1974) in preparation.
29
