- Email: [email protected]
Effect of water hardness on the toxicity of a nonionic detergent to fish
It.lt~,~ ges~.:r.h Vol 9 pp 31 to 36 Pergamon Press ',u-5 P:'int~ad n Great Britain.
EFFECT OF WATER HARDNESS ON THE TOXICITY OF A NONIONIC DETERGENT TO FISH P. W. A. TOVELL. C. N E w s o x u : a n d D. HOWES Environmentat Safety Division. Unilever Research Laboratory Coh~orth Wel~? n . Colworth House. Sharnbrook. Bedtbrd. England
{Received 18 March 19741 Abstract--Fish (trout and goldfishl were placed in treatment solutions of different water hardnesses containing ethoxylate {EO} detergents. Survival times were recorded, zaC-labelled EO was used to assess absorption. The toxicity of ethoxylates to fish acclimatised and treated in different water hardnesses, and the effect of cations on ethoxylate toxicity were also investigated. EO is slightly less toxic in hard water than in soft water. The hardness of the treatment solution has no marked influence on the amount of EO absorbed by the fish. Evidence suggests that there is little relationship between the composition of cations constituting a particular hardness and the toxicity of EO. Acclimatisation m ditt~zrent water hardnesses does not affect the susceptibility of fish to nonionic ethoxylate detcrgents.
INTRODUCTION
pH 7.2-7.8, temperature 12 + I C ) in 200-1. glass fibre tanks. Goldfish (Carassius auratus, Linnaeus) of total length 2.5 in. and average weight 2.8 g were obtained from L. Cura and Sons. Hemel Hempstead, Hertfordshire, and were hem tinder similar conditions. Water hardness (total hardness equivalent to ppm calcium carbonate) was found by measuring the total concentration of bivalent cations using BDH total hardness pellets (B.D.H. Chemicals Ltd., Poole) and titrating against EDTA. Local water had an average hardness of 300 :__ 20ppm and contained 100ppm Ca "-~, 20ppm N a - , 5 p p m K ~ and 8 p p m M g - " . Single glass distilled water was taken as 0 ppm hardness and contained 0.4ppm C a - " . [-7ppm N a - . 0"3 ppm K + and <0.1 ppm M g - " . Water of 200 and 60 ppm hardness was obtained by mixing distilled and tap water. Batches offish from the holding tanks were acclimatised for at least seven days in water of 200, 60 and 0 ppm hardness by placing them in 45 I. of water of the appropriate hardness (continuously aerated, changed daily. 18 + 1 Clcontained in 60-1. glass fibre tanks. A one week acclimatisation period was regarded as st, fficient for the purposes of this study. In order to reduce stress during acclimatisation to 60 and 0 p p m hardness, fish were first held for three consecutive weeks in water of 200, 130 and 60 ppm hardness respectively. Attempts to acclimatise trout to distilled water were unsuccessful; the fish showed signs of stress throughout the distilled water acclimatisation period, and oxygenation rather than aeration was found to be necessary in order to reduce a spontaneous daily mortality of 10 per cent. However. goldfish held continuously in
The toxicity of detergents has been reported to increase, decrease or be unaffected by changes in water hardness. Henderson, Piekering and Cohen (1959) found alkyl benzene sulphonate (ABS) more toxic to the fathead minnow in hard water, sodit, m alkyl sulphate more toxic in soft water, and a nonionic ethoxylate unaffected by water hardness. Cairns and Scheier (1962) found that the toxicity of ABS to the bluegill was not significantly affected by ~'ater hardness, and Leclerc and Devlaminck (1952) found that water hardness did not affect the toxicity of ABS or sodium lauryl sulphate (SLS). However, Hokanson and Smith (1971) reported that linear alkylate sulphonate (LAS) was significantly less toxic to the bluegill in soft water compared to hard, whilst Lang (1967) showed that ABS and LAS were more toxic to carp in hard water. In comparison, heavy metals have been found to be uniIbrmly less toxic as the hardness of the water is increased (Brown, 1968). As a follow-up to previous work on the efl:,:ct of water hardness on anionic detergents (Tovell et al., 1974) this study describes a series of investigations on the effect of water hardness on the toxicity of a nonionic detergent. MATERIALS AND METHODS Rainbow trout (&thno gairdncri, Richardson) of total length 2..5 in. and average weight 2.8 g were obtained from Bibury Trout Farm, Bibury, Gloucestershire, and were held in continuously flowing local Bedford tap water (total hardness 300 ppm as calcium carbonate. 31 w.~. 9 I - - C
32
P. w A. TOVELL,C. NEWSO~,IEand D. HowEs
8 ppm Ct, 3 EO. Each fish ~as weighed, dissolved in Soluene (Packard Instruments Ltd.. lllinois), and the radioactivity counted in a toluene-based scintillator in a Packard 4000 liquid scintillation spectrometer. This experiment was repeated using goldfish with one additional group of four fish acctimatised and treated in water ofO ppm hardness. CH~ (CH.,).~+ CHz(O---CH,--CH_,),, OH To establish whether or not acclimatisation has any where n refers to the number of ethoxylate groups in effect on the susceptibility of trout to EO. fish from a pure compound, and the average number of ethox?- each hardness of acclimatisation were treated with EO in water of 300. 200 and 60 ppm hardness. Nine treatlate groups in a commercial compound. Commercial Alfol 12 ethoxylate, having a weighted ment tanks were set ttp such that there were three average ethoxylation (n) of 3.25 (designated C~: 3'25 tanks containing 21. of water at each of the three hardEO for brevity), was used for all tests except the uptake nesses chosen for acclimatisation (300. 200 and study in which pure n-alkyl triethoxylate with the CI 60ppm). The water in all treatment tanks contained carbon on the alkyl chain labelled t+C (designated I[-1- 5 ppm C~: 3"25 EO. Eight trout were placed in each t~C]--C.: 3 EO) was used together with pure unla- treatment tank such that eight fish from each level of belled CL: 3 EO as carrier. Ct: 3'25 EO was obtained acclimatisation (300, 200 and 60 ppm) were exposed to from Unilever Research Laboratory, Port St, nlight, water containing EO in each of the hardnesses investiCheshire. C~_, 3 EO was prepared in this laboratory. gated (300. 200 and 60 ppm, see Table 41. The effect of The radiochemical purity of the [l-t*C]--Ct_, 3 EO acclimatisation on the susceptibility of goldfish to EO was found to be 98"~, plus by TLC. The specific activity was similarly investigated, except that six fish were was 4.8 and 6.2/~Ci rag- ' in the two batches of labelled used for each treatment and an additional hardness of acclimatisation and treatment (0ppm) was included EO. To demonstrate the difference in toxicity of EO to (see Table 5). The survival time of each fish was noted. fish in hard and soft water, eight trout from the holding It had already been established that both trout and tanks were exposed to 5 ppm C,, 3'25 EO made t,p in goldfish were able to withstand the maximum change hard (300ppm) water, and a further eight trout from in hardness for considerably longer than the survival the holding tanks were exposed to 5 ppm C,_, 3"25 EO times following exposure to 5 ppm Ct: 3.25 EO. The role played by specific cations in the mechanism made up in soft (60 ppm) water. Similarly. two groups of soft (60 ppm) water acclimatised trout were treated of EO toxicity was investigated by placing goldfish in in the same way. The survival times in these treatment solutions of sodium, potassium, magnesium and calsolutions were noted;death being assumed at the stage cium chloride each containing 5ppm Ct_, 3-25 EO. ofopercular immobilisation, and when the fish gave no Eight batches of sixteen goldfish acclimatised to hard resistance to being removed from the water by hand. (300 ppm) water were set up in eight 4-1. tanks each This experiment was repeated using four groups of containing 4 1. ofdistilled (0 ppm) water and 5 ppm Ct_, eight goldfish, except that distilled (0 plum) water was 3-25 EO. In addition, each of the eight treatment solutions contained either 1 or 3 mxn of one of the followvsed in place of soft (60 ppm) water. The amount of EO absorbed by fish in different ing four salts: sodium chloride, potassium chloride, water hardnesses was determined by the use of ~'~C- magnesium chloride or calcium chloride (300ppm labelled EO. Four trout from the holding tanks were CaCI, is eqt, ivalent to a 3 mM solution. The survival placed in 1 1. of hard (300ppm)water: similarly, two time of each fish was recorded. A further eight batches batches of four trout acclimatised in water of 200 ppm of 16 goldfish acclimatised in distilled (0ppm) water and 60ppm hardness were each placed in 1 1. of were similarly treated, and their survival times 200ppm and 60ppm water respectively. 8 ml of recorded. taC--labelled C~_, 3 EO solution containing 1 mg EOml- ~ (active and carrier) were added to each tank to R ES U LTS give a final concentration of 8 ppm Ct : 3 EO. When the 1. Surt'ival times o]'fish ili EO solution.~ made up in hard first fish died the exposure time was noted and fish from all treatment solutions were removed and killed and soft water
distilled ~ater for three months showed no signs of stress. Fish under test were not fed and were held in glass tanks containing continuously aerated treatment solution at 18 _+ l:C under static conditions. The detergents used in this study were non-ionic C~ : alkyl ethoxylates with the formula:
so that the amount of labelled material absorbed by each of them could be counted. 2 ml samples of the treatment solutions were taken at the start of the experiment to assess the radioactivity equivalent to
The survival times of fish acclimatised in hard or soft water and exposed to 5ppm C~_, 3-25 EO made up in hard (300 ppm) or soft (60 ppm trout. 0 ppm goldfish) water are shown in Table I. It is clear that both trout
33
Effect of water hardness Table 1. Survival times of fish placed in 5 ppm EO solutions made up in v,ater of different hardnesses
Trout (Accl. in hard water) Trout (Accl. in soft water) Goldfish [Accl. in hard watcrl Goldfish (Accl. in soft water)
Hard water
Average survival time (min ± SE) Soft water
129 ± 22
66 ± 3
P < 0.05
152 ± 8
107 + 10
P < 0.0l
252 ± 44
161 ± 23
P < 0.1
267 ___39
170 ± 23
P < 0.05
and goldfish are less susceptible to EO made up in hard water.
2. Uptake of labelled EO hy trout in water of different hard nesse s The specific activity of the [l-t~C]-Ci2 3 EO was 6.2,uCi rag- ~. The radioactivitv of a standard 8 ppm solution was 27.693 dis min-~ ml- x (8'1 mg active + 24 mg carrier in 32.1 ml water. 8 ml of this solution being added to each litre of treatment solution). Fish were killed after 63 min (this being the shortest individual survival time) and their total radioactivity counted. Results are shown in Table 2. Table 2. Tissue levels of EO in trout placed in 8 ppm EO for 63 rain Treatment solution hardness (ppm)
Radioactivity in fish -(dismin-lg-l)
= lEO] (ltgg -I ± S E )
300 200 60
874,243 903,460 919.920
253 ± 17 261 ± I1 266 ± 1
These results show that the hardness of the treatment solution has no influence on the amount of EO accumulated by trout.
3. Uptake of labelled EO by 9ohlfish in water of'different har (hzesse s The specific activity of the second batch of [l-t'~C] Ct_, 3 EO was 4-8#Ci mg -~. The radioactivity of a standard 8 ppm solution was 21.386 dis m i n - t m l - t (8.7 mg active + 26 mg carrier in 34.7 ml water. 8 ml of this solution being added to each litre of treatment solution). Fish were killed after 50 rain (this being the shortest individual survival time) and their total radioactivity counted. Results are shown in Table 3. These results show that the hardness of the treatment solution has no marked influence on the amount of EO accumulated by goldfish.
Table 3. Tissue levels of EO in goldfish placed in 8 ppm EO for 50 min Treatment solution hardness (ppm)
Radioactivity in fish dis m i n - l g - t )
300 200 60 0
569.148 664,148 557.520 472.390
- lEO] i~tgg-t ± SE) 164 ± 192 ± 161 ± 137 ±
8 13 t0 14
4. The survit'al offish in EO followin 9 acclimatisation m water of diffi.,rent hardnesses Table 4 shows the survival times of trout acclimatised in water of 300, 200 and 60 ppm hardness and then exposed to 5 ppm Ct_, 3"25 EO made tip in water of thc hardnesses listed in the third column. Table 5 shows the survival times of goldfish acclimatised and treated in the same way as described for trout. An additional group of goldfish acclimatised in distilled (0ppm) water were also treated with EO in each hardness. It is apparent from these results that under the conditions described acclimatisation does not condition the susceptibility of trout or goldfish to C~_, 3.25 EO, but that they remain most susceptible to the effects of the detergent in soft water (60 ppm for trout and 0 ppm for goldfish).
5. Role of specific catiotzs in toxicity olEO Table 6 shows the survival times of hard (300 ppm) water acclimatiscd goldfish exposed to 5 ppm C~_, 3.25 EO made up in 1 mxt or 3 mM sodium, potassium. magnesium or calcium chloride. Table 7 shows the survival times of distilled water acclimatised goldfish exposed to 5 p p m C~_, 3"25 EO made up in 1 mM or 3 mM sodium, potassium, magnesiru~ or calcium chloride. These resuhs show that the cation composition (whether di- or mono-valent) has little effect on the toxicity of EO to goldfish. Also. the concentration of these ions appears to have little effect on toxicity.
Trout 5ppmCi23.25 EO
50C
03
E
0
5(3
IOC
60 60 60
8 8 8
E
200 200 2110
8 8 8
I
t
60 200 Hordness of t r e o t m e n t solution
I
300
152 + 8.0 173 ± 8.9 107 + 9.9
138 -I- 10-3 192 ± 19.7 74 -± 12.6 :
129 ± 21"7 143 ± 22"0 66 ± 3"4
Survival time (min ± SE)
• 3001 A200~ Hordness of o c c l i m o t i s a t i o n • 60 ]
300 200 60
300 200 61)
301) 200 60
300 300 300
8 8 8
Treatment h-lrdllt2ss (ppm)
Acclilnatisation hardness (ppnl)
No. o f tish
TaMe 4. Survival of trout in waters of 31)0, 200 and 60 ppm hardness containing 5 ppna C~_, 3-25 EO after previous acclimatisation in these hardncsscs
E
0 0 0 0
8 8 8 8
50
IO(
500
60 61) 60 60
8 8 8 8
300 2110 6(1 0
300 21~) 60 0
3IX) 3110 60 0
300 200 61) I)
Treatment hardness (ppm)
± ± ± ±
:~ :!: ± ±
I
200
Hordness of o c c l i m o t i s o t i o n
267 ± 315 ± 235 ± 171) ±
272 279 223 185
241) 267 256 209
252 4 233 ± 20(~ ± 161 ±
I
300
39 4() 48 23
19 17 25 23
18 12 18 34
44 21) 22 23
Survival lime (rain ± SE)
Hordness of l " r e o t m e n t s o l u t i o n
I
60
.6oO/
• 3001 A200~
Goldfish 5ppmCi23-25 EO
21)1) 21)1) 200 200
300 300 300 300
8 8 8 8 8 8 8 8
Acelimalisalion h:irdness (ppm)
No. o f lish
Table 5. Survival of goldfish in walcrs of 3110, 2/10, 60 and () ppm hardness containing 5 ppm C',_, 3"25 1'i0 ;l[lcr ]'}l'CViOtlS, acclimatisalion in these hardncsscs
©
F~
t.r-
-H o
"0
~C
Effect of water hardness Table 6. Survival times of hard 1300 ppm) water acclima-~.~tised goldfish exposed to 5 ppm C~ : _'_) EO made up in distilled water containing the salts listed
Table 7. Survival times of distilled water acclimatised goldfish exposed to 5 ppm EO made up in distilled water containing the salts listed
Survi,al time (min ± SE) I mr,t 3 m.,4
Salt NaCI KCI MgCI2 CaCI:
168 ± 127 ~ 176 ± 184 ±
16 17 13 10
214 ± 162 ± 175 ± 185 ±
Survival time (min _.- SE) 1 m.~t 3 mxl
Salt NaCI KCI MgCI: CaCI:
21 7 20 12
200 + 13(1 ± 217 ± I,";5 ±
16 19 14 II
15S ± 136 ± 175 z 134 _.
Goldfish
Goldfish 5pore 3.25 E Q Role of specfic cotions in toxicity of 3"25 EO
500
35
14
Ill 17 15
5pprn 3.25 E O
Role of specific co-ions in
500
toxicity
of 3'25 EO
E
E
i IS,E E 4-
ImM 3mM
>
I mM 3 mM
> >
I00
I00
50
.50 No
K
Mg
No
Ca
DISCUSSION
This work has shown that EO is less toxic to trout and goldfish in hard water than in soft water: however, its toxicity is much less affected by the treatment solution hardness than was found to be the case with the anionic detergent sodium lauryl sulphate (SLS). Similarly, it was found that the cation composition of the treatment solution did not affect the toxicity of E O to goldfish, and previous acclimatisation or trout and goldfish to water of different hardnesses did not modify the susceptibility of the fish to EO. The uptake experiment showed that the hardness of the treatment solution did not affect the amount of detergent accumulated by either trout or goldfish, and that trout accumulated the detergent to a greater extent than goldfish. The comparatively small difference in the amount of EO accumulated throughout the range of hardnesses investigated compared with the difference in accumu-
K
Mq
Ce
lation of SLS throughout the same range (Tovell et al., 1974) is shown in Table 8. This emphasises that water hardness has a much less marked cfli:ct on EO toxicity than on SLS toxicity. These findings could be explained in terms of the ionic characteristics of the two detergents, a nonionic being less likely to be affected by the presence of ions in solut i o n - a s was shown in the cation experiment. The implication from this and the previous study (Tovell et al., 19741 is that the presence of C a - " (or other diwtlent cations) does not affect the toxic response to detergents by some action on the diffusion membranes of the gill. instead, the toxic response relies on an interaction between the ions in solution and the detergent, which in turn modifies the solubility and/or diffusion characteristics of the detergent itself. On the other hand. the fact that prior acclimatisation to different water hardnesses affects the subsequent toxicity of an anionic
Table 8. The difference between the amount of detergent accumulated by fish in hard and soft water expressed as a ratio of that accumulated in soft water Detergent SLS SLS EO EO
Fish Trout Goldfish Trout Goldfish
Range of hardness (ppm)
Accumulation ratio (hard: softl
60-300 0-300 60-300 0-300
6-I 8.93 0.95 1-20
36
P.W.A. TOVELL. C. NEWSOMEand D. HowEs
detergent (Tovell et al., 1974) but does not affect the toxicit? of a nonionic detergent indicates that water hardness (in the absence of an~ toxinJ can elicit some form of selective a d a p t a t i o n of diffusion m e m b r a n e permeability. An investigation of the effect water hardness has on the toxicit? of a wider range of anionic and nonionie detergents and of some cationic detergents is necessary before a more complete evaluation can be made of the role a n d mechanism of water hardness in detergent toxicity. .4cknowledyemep~ts--We are grateful to Mr. C. T. James for preparing the labelled Ct: 3 EO. Mrs. A. Cordell for her technical assistance, and Dr. L. J. Morris and Dr. A. J. Collings tbr their help in preparing this manuscript. REFERENCES
Brown V. M. (1968) The calculation of the acute toxicity of mixtures of poisons to rainbow trout, ttater Rex. 2, 723733.
Cairns J. and Scheier A. (1962) The acute and chronic effects of standard sodium alkyl benzene sulphonate upon the pulapkinseed sunfish, Lepomis ,qibhosus (Linn.) and the bluegill sunfish, Lepomis mucrochirus(Raf.). Proc. 17th Ind. W~lsre C o ~ , Purdue Univ. Engng. Exten. Sr. 112, 14-28. Henderson C., Pickering Q. H. and Cohen J. M. (1959) The toxicity of synthetic detergents and soaps to fish. Sewage Iml. ~t~,~tes 31,295-306. Hokanson K. E. F. and Smith L. L. (197I) Some factors influencing toxicity of linear alkylate sulphonate (LAS) to the bluegill. Trans. ,qm. Fish. Soc. 100, t-12. Lang W. (1967) Investigations on the mode of action of anionic surl:actants on the histology and function of various organs of Carassius auratus. Arch. Fisch Wiss. 18, 25-,,t5. Leclerc E. and Devlaminck F. (1952) Natural or synthetic detergents and fish. B~tll. Ct, tlt. bt,l~lc Etttd. Docum. Eatt.v.. (17). 165-171. Tovell P. W. A.. Newsome C. and Howes D. (1974) Effect of water hardness on the toxicity of an anionic detergent to fish. Water Res. 8, 291-296.
EFFECT OF WATER HARDNESS ON THE TOXICITY OF A NONIONIC DETERGENT TO FISH P. W. A. TOVELL. C. N E w s o x u : a n d D. HOWES Environmentat Safety Division. Unilever Research Laboratory Coh~orth Wel~? n . Colworth House. Sharnbrook. Bedtbrd. England
{Received 18 March 19741 Abstract--Fish (trout and goldfishl were placed in treatment solutions of different water hardnesses containing ethoxylate {EO} detergents. Survival times were recorded, zaC-labelled EO was used to assess absorption. The toxicity of ethoxylates to fish acclimatised and treated in different water hardnesses, and the effect of cations on ethoxylate toxicity were also investigated. EO is slightly less toxic in hard water than in soft water. The hardness of the treatment solution has no marked influence on the amount of EO absorbed by the fish. Evidence suggests that there is little relationship between the composition of cations constituting a particular hardness and the toxicity of EO. Acclimatisation m ditt~zrent water hardnesses does not affect the susceptibility of fish to nonionic ethoxylate detcrgents.
INTRODUCTION
pH 7.2-7.8, temperature 12 + I C ) in 200-1. glass fibre tanks. Goldfish (Carassius auratus, Linnaeus) of total length 2.5 in. and average weight 2.8 g were obtained from L. Cura and Sons. Hemel Hempstead, Hertfordshire, and were hem tinder similar conditions. Water hardness (total hardness equivalent to ppm calcium carbonate) was found by measuring the total concentration of bivalent cations using BDH total hardness pellets (B.D.H. Chemicals Ltd., Poole) and titrating against EDTA. Local water had an average hardness of 300 :__ 20ppm and contained 100ppm Ca "-~, 20ppm N a - , 5 p p m K ~ and 8 p p m M g - " . Single glass distilled water was taken as 0 ppm hardness and contained 0.4ppm C a - " . [-7ppm N a - . 0"3 ppm K + and <0.1 ppm M g - " . Water of 200 and 60 ppm hardness was obtained by mixing distilled and tap water. Batches offish from the holding tanks were acclimatised for at least seven days in water of 200, 60 and 0 ppm hardness by placing them in 45 I. of water of the appropriate hardness (continuously aerated, changed daily. 18 + 1 Clcontained in 60-1. glass fibre tanks. A one week acclimatisation period was regarded as st, fficient for the purposes of this study. In order to reduce stress during acclimatisation to 60 and 0 p p m hardness, fish were first held for three consecutive weeks in water of 200, 130 and 60 ppm hardness respectively. Attempts to acclimatise trout to distilled water were unsuccessful; the fish showed signs of stress throughout the distilled water acclimatisation period, and oxygenation rather than aeration was found to be necessary in order to reduce a spontaneous daily mortality of 10 per cent. However. goldfish held continuously in
The toxicity of detergents has been reported to increase, decrease or be unaffected by changes in water hardness. Henderson, Piekering and Cohen (1959) found alkyl benzene sulphonate (ABS) more toxic to the fathead minnow in hard water, sodit, m alkyl sulphate more toxic in soft water, and a nonionic ethoxylate unaffected by water hardness. Cairns and Scheier (1962) found that the toxicity of ABS to the bluegill was not significantly affected by ~'ater hardness, and Leclerc and Devlaminck (1952) found that water hardness did not affect the toxicity of ABS or sodium lauryl sulphate (SLS). However, Hokanson and Smith (1971) reported that linear alkylate sulphonate (LAS) was significantly less toxic to the bluegill in soft water compared to hard, whilst Lang (1967) showed that ABS and LAS were more toxic to carp in hard water. In comparison, heavy metals have been found to be uniIbrmly less toxic as the hardness of the water is increased (Brown, 1968). As a follow-up to previous work on the efl:,:ct of water hardness on anionic detergents (Tovell et al., 1974) this study describes a series of investigations on the effect of water hardness on the toxicity of a nonionic detergent. MATERIALS AND METHODS Rainbow trout (&thno gairdncri, Richardson) of total length 2..5 in. and average weight 2.8 g were obtained from Bibury Trout Farm, Bibury, Gloucestershire, and were held in continuously flowing local Bedford tap water (total hardness 300 ppm as calcium carbonate. 31 w.~. 9 I - - C
32
P. w A. TOVELL,C. NEWSO~,IEand D. HowEs
8 ppm Ct, 3 EO. Each fish ~as weighed, dissolved in Soluene (Packard Instruments Ltd.. lllinois), and the radioactivity counted in a toluene-based scintillator in a Packard 4000 liquid scintillation spectrometer. This experiment was repeated using goldfish with one additional group of four fish acctimatised and treated in water ofO ppm hardness. CH~ (CH.,).~+ CHz(O---CH,--CH_,),, OH To establish whether or not acclimatisation has any where n refers to the number of ethoxylate groups in effect on the susceptibility of trout to EO. fish from a pure compound, and the average number of ethox?- each hardness of acclimatisation were treated with EO in water of 300. 200 and 60 ppm hardness. Nine treatlate groups in a commercial compound. Commercial Alfol 12 ethoxylate, having a weighted ment tanks were set ttp such that there were three average ethoxylation (n) of 3.25 (designated C~: 3'25 tanks containing 21. of water at each of the three hardEO for brevity), was used for all tests except the uptake nesses chosen for acclimatisation (300. 200 and study in which pure n-alkyl triethoxylate with the CI 60ppm). The water in all treatment tanks contained carbon on the alkyl chain labelled t+C (designated I[-1- 5 ppm C~: 3"25 EO. Eight trout were placed in each t~C]--C.: 3 EO) was used together with pure unla- treatment tank such that eight fish from each level of belled CL: 3 EO as carrier. Ct: 3'25 EO was obtained acclimatisation (300, 200 and 60 ppm) were exposed to from Unilever Research Laboratory, Port St, nlight, water containing EO in each of the hardnesses investiCheshire. C~_, 3 EO was prepared in this laboratory. gated (300. 200 and 60 ppm, see Table 41. The effect of The radiochemical purity of the [l-t*C]--Ct_, 3 EO acclimatisation on the susceptibility of goldfish to EO was found to be 98"~, plus by TLC. The specific activity was similarly investigated, except that six fish were was 4.8 and 6.2/~Ci rag- ' in the two batches of labelled used for each treatment and an additional hardness of acclimatisation and treatment (0ppm) was included EO. To demonstrate the difference in toxicity of EO to (see Table 5). The survival time of each fish was noted. fish in hard and soft water, eight trout from the holding It had already been established that both trout and tanks were exposed to 5 ppm C,, 3'25 EO made t,p in goldfish were able to withstand the maximum change hard (300ppm) water, and a further eight trout from in hardness for considerably longer than the survival the holding tanks were exposed to 5 ppm C,_, 3"25 EO times following exposure to 5 ppm Ct: 3.25 EO. The role played by specific cations in the mechanism made up in soft (60 ppm) water. Similarly. two groups of soft (60 ppm) water acclimatised trout were treated of EO toxicity was investigated by placing goldfish in in the same way. The survival times in these treatment solutions of sodium, potassium, magnesium and calsolutions were noted;death being assumed at the stage cium chloride each containing 5ppm Ct_, 3-25 EO. ofopercular immobilisation, and when the fish gave no Eight batches of sixteen goldfish acclimatised to hard resistance to being removed from the water by hand. (300 ppm) water were set up in eight 4-1. tanks each This experiment was repeated using four groups of containing 4 1. ofdistilled (0 ppm) water and 5 ppm Ct_, eight goldfish, except that distilled (0 plum) water was 3-25 EO. In addition, each of the eight treatment solutions contained either 1 or 3 mxn of one of the followvsed in place of soft (60 ppm) water. The amount of EO absorbed by fish in different ing four salts: sodium chloride, potassium chloride, water hardnesses was determined by the use of ~'~C- magnesium chloride or calcium chloride (300ppm labelled EO. Four trout from the holding tanks were CaCI, is eqt, ivalent to a 3 mM solution. The survival placed in 1 1. of hard (300ppm)water: similarly, two time of each fish was recorded. A further eight batches batches of four trout acclimatised in water of 200 ppm of 16 goldfish acclimatised in distilled (0ppm) water and 60ppm hardness were each placed in 1 1. of were similarly treated, and their survival times 200ppm and 60ppm water respectively. 8 ml of recorded. taC--labelled C~_, 3 EO solution containing 1 mg EOml- ~ (active and carrier) were added to each tank to R ES U LTS give a final concentration of 8 ppm Ct : 3 EO. When the 1. Surt'ival times o]'fish ili EO solution.~ made up in hard first fish died the exposure time was noted and fish from all treatment solutions were removed and killed and soft water
distilled ~ater for three months showed no signs of stress. Fish under test were not fed and were held in glass tanks containing continuously aerated treatment solution at 18 _+ l:C under static conditions. The detergents used in this study were non-ionic C~ : alkyl ethoxylates with the formula:
so that the amount of labelled material absorbed by each of them could be counted. 2 ml samples of the treatment solutions were taken at the start of the experiment to assess the radioactivity equivalent to
The survival times of fish acclimatised in hard or soft water and exposed to 5ppm C~_, 3-25 EO made up in hard (300 ppm) or soft (60 ppm trout. 0 ppm goldfish) water are shown in Table I. It is clear that both trout
33
Effect of water hardness Table 1. Survival times of fish placed in 5 ppm EO solutions made up in v,ater of different hardnesses
Trout (Accl. in hard water) Trout (Accl. in soft water) Goldfish [Accl. in hard watcrl Goldfish (Accl. in soft water)
Hard water
Average survival time (min ± SE) Soft water
129 ± 22
66 ± 3
P < 0.05
152 ± 8
107 + 10
P < 0.0l
252 ± 44
161 ± 23
P < 0.1
267 ___39
170 ± 23
P < 0.05
and goldfish are less susceptible to EO made up in hard water.
2. Uptake of labelled EO hy trout in water of different hard nesse s The specific activity of the [l-t~C]-Ci2 3 EO was 6.2,uCi rag- ~. The radioactivitv of a standard 8 ppm solution was 27.693 dis min-~ ml- x (8'1 mg active + 24 mg carrier in 32.1 ml water. 8 ml of this solution being added to each litre of treatment solution). Fish were killed after 63 min (this being the shortest individual survival time) and their total radioactivity counted. Results are shown in Table 2. Table 2. Tissue levels of EO in trout placed in 8 ppm EO for 63 rain Treatment solution hardness (ppm)
Radioactivity in fish -(dismin-lg-l)
= lEO] (ltgg -I ± S E )
300 200 60
874,243 903,460 919.920
253 ± 17 261 ± I1 266 ± 1
These results show that the hardness of the treatment solution has no influence on the amount of EO accumulated by trout.
3. Uptake of labelled EO by 9ohlfish in water of'different har (hzesse s The specific activity of the second batch of [l-t'~C] Ct_, 3 EO was 4-8#Ci mg -~. The radioactivity of a standard 8 ppm solution was 21.386 dis m i n - t m l - t (8.7 mg active + 26 mg carrier in 34.7 ml water. 8 ml of this solution being added to each litre of treatment solution). Fish were killed after 50 rain (this being the shortest individual survival time) and their total radioactivity counted. Results are shown in Table 3. These results show that the hardness of the treatment solution has no marked influence on the amount of EO accumulated by goldfish.
Table 3. Tissue levels of EO in goldfish placed in 8 ppm EO for 50 min Treatment solution hardness (ppm)
Radioactivity in fish dis m i n - l g - t )
300 200 60 0
569.148 664,148 557.520 472.390
- lEO] i~tgg-t ± SE) 164 ± 192 ± 161 ± 137 ±
8 13 t0 14
4. The survit'al offish in EO followin 9 acclimatisation m water of diffi.,rent hardnesses Table 4 shows the survival times of trout acclimatised in water of 300, 200 and 60 ppm hardness and then exposed to 5 ppm Ct_, 3"25 EO made tip in water of thc hardnesses listed in the third column. Table 5 shows the survival times of goldfish acclimatised and treated in the same way as described for trout. An additional group of goldfish acclimatised in distilled (0ppm) water were also treated with EO in each hardness. It is apparent from these results that under the conditions described acclimatisation does not condition the susceptibility of trout or goldfish to C~_, 3.25 EO, but that they remain most susceptible to the effects of the detergent in soft water (60 ppm for trout and 0 ppm for goldfish).
5. Role of specific catiotzs in toxicity olEO Table 6 shows the survival times of hard (300 ppm) water acclimatiscd goldfish exposed to 5 ppm C~_, 3.25 EO made up in 1 mxt or 3 mM sodium, potassium. magnesium or calcium chloride. Table 7 shows the survival times of distilled water acclimatised goldfish exposed to 5 p p m C~_, 3"25 EO made up in 1 mM or 3 mM sodium, potassium, magnesiru~ or calcium chloride. These resuhs show that the cation composition (whether di- or mono-valent) has little effect on the toxicity of EO to goldfish. Also. the concentration of these ions appears to have little effect on toxicity.
Trout 5ppmCi23.25 EO
50C
03
E
0
5(3
IOC
60 60 60
8 8 8
E
200 200 2110
8 8 8
I
t
60 200 Hordness of t r e o t m e n t solution
I
300
152 + 8.0 173 ± 8.9 107 + 9.9
138 -I- 10-3 192 ± 19.7 74 -± 12.6 :
129 ± 21"7 143 ± 22"0 66 ± 3"4
Survival time (min ± SE)
• 3001 A200~ Hordness of o c c l i m o t i s a t i o n • 60 ]
300 200 60
300 200 61)
301) 200 60
300 300 300
8 8 8
Treatment h-lrdllt2ss (ppm)
Acclilnatisation hardness (ppnl)
No. o f tish
TaMe 4. Survival of trout in waters of 31)0, 200 and 60 ppm hardness containing 5 ppna C~_, 3-25 EO after previous acclimatisation in these hardncsscs
E
0 0 0 0
8 8 8 8
50
IO(
500
60 61) 60 60
8 8 8 8
300 2110 6(1 0
300 21~) 60 0
3IX) 3110 60 0
300 200 61) I)
Treatment hardness (ppm)
± ± ± ±
:~ :!: ± ±
I
200
Hordness of o c c l i m o t i s o t i o n
267 ± 315 ± 235 ± 171) ±
272 279 223 185
241) 267 256 209
252 4 233 ± 20(~ ± 161 ±
I
300
39 4() 48 23
19 17 25 23
18 12 18 34
44 21) 22 23
Survival lime (rain ± SE)
Hordness of l " r e o t m e n t s o l u t i o n
I
60
.6oO/
• 3001 A200~
Goldfish 5ppmCi23-25 EO
21)1) 21)1) 200 200
300 300 300 300
8 8 8 8 8 8 8 8
Acelimalisalion h:irdness (ppm)
No. o f lish
Table 5. Survival of goldfish in walcrs of 3110, 2/10, 60 and () ppm hardness containing 5 ppm C',_, 3"25 1'i0 ;l[lcr ]'}l'CViOtlS, acclimatisalion in these hardncsscs
©
F~
t.r-
-H o
"0
~C
Effect of water hardness Table 6. Survival times of hard 1300 ppm) water acclima-~.~tised goldfish exposed to 5 ppm C~ : _'_) EO made up in distilled water containing the salts listed
Table 7. Survival times of distilled water acclimatised goldfish exposed to 5 ppm EO made up in distilled water containing the salts listed
Survi,al time (min ± SE) I mr,t 3 m.,4
Salt NaCI KCI MgCI2 CaCI:
168 ± 127 ~ 176 ± 184 ±
16 17 13 10
214 ± 162 ± 175 ± 185 ±
Survival time (min _.- SE) 1 m.~t 3 mxl
Salt NaCI KCI MgCI: CaCI:
21 7 20 12
200 + 13(1 ± 217 ± I,";5 ±
16 19 14 II
15S ± 136 ± 175 z 134 _.
Goldfish
Goldfish 5pore 3.25 E Q Role of specfic cotions in toxicity of 3"25 EO
500
35
14
Ill 17 15
5pprn 3.25 E O
Role of specific co-ions in
500
toxicity
of 3'25 EO
E
E
i IS,E E 4-
ImM 3mM
>
I mM 3 mM
> >
I00
I00
50
.50 No
K
Mg
No
Ca
DISCUSSION
This work has shown that EO is less toxic to trout and goldfish in hard water than in soft water: however, its toxicity is much less affected by the treatment solution hardness than was found to be the case with the anionic detergent sodium lauryl sulphate (SLS). Similarly, it was found that the cation composition of the treatment solution did not affect the toxicity of E O to goldfish, and previous acclimatisation or trout and goldfish to water of different hardnesses did not modify the susceptibility of the fish to EO. The uptake experiment showed that the hardness of the treatment solution did not affect the amount of detergent accumulated by either trout or goldfish, and that trout accumulated the detergent to a greater extent than goldfish. The comparatively small difference in the amount of EO accumulated throughout the range of hardnesses investigated compared with the difference in accumu-
K
Mq
Ce
lation of SLS throughout the same range (Tovell et al., 1974) is shown in Table 8. This emphasises that water hardness has a much less marked cfli:ct on EO toxicity than on SLS toxicity. These findings could be explained in terms of the ionic characteristics of the two detergents, a nonionic being less likely to be affected by the presence of ions in solut i o n - a s was shown in the cation experiment. The implication from this and the previous study (Tovell et al., 19741 is that the presence of C a - " (or other diwtlent cations) does not affect the toxic response to detergents by some action on the diffusion membranes of the gill. instead, the toxic response relies on an interaction between the ions in solution and the detergent, which in turn modifies the solubility and/or diffusion characteristics of the detergent itself. On the other hand. the fact that prior acclimatisation to different water hardnesses affects the subsequent toxicity of an anionic
Table 8. The difference between the amount of detergent accumulated by fish in hard and soft water expressed as a ratio of that accumulated in soft water Detergent SLS SLS EO EO
Fish Trout Goldfish Trout Goldfish
Range of hardness (ppm)
Accumulation ratio (hard: softl
60-300 0-300 60-300 0-300
6-I 8.93 0.95 1-20
36
P.W.A. TOVELL. C. NEWSOMEand D. HowEs
detergent (Tovell et al., 1974) but does not affect the toxicit? of a nonionic detergent indicates that water hardness (in the absence of an~ toxinJ can elicit some form of selective a d a p t a t i o n of diffusion m e m b r a n e permeability. An investigation of the effect water hardness has on the toxicit? of a wider range of anionic and nonionie detergents and of some cationic detergents is necessary before a more complete evaluation can be made of the role a n d mechanism of water hardness in detergent toxicity. .4cknowledyemep~ts--We are grateful to Mr. C. T. James for preparing the labelled Ct: 3 EO. Mrs. A. Cordell for her technical assistance, and Dr. L. J. Morris and Dr. A. J. Collings tbr their help in preparing this manuscript. REFERENCES
Brown V. M. (1968) The calculation of the acute toxicity of mixtures of poisons to rainbow trout, ttater Rex. 2, 723733.
Cairns J. and Scheier A. (1962) The acute and chronic effects of standard sodium alkyl benzene sulphonate upon the pulapkinseed sunfish, Lepomis ,qibhosus (Linn.) and the bluegill sunfish, Lepomis mucrochirus(Raf.). Proc. 17th Ind. W~lsre C o ~ , Purdue Univ. Engng. Exten. Sr. 112, 14-28. Henderson C., Pickering Q. H. and Cohen J. M. (1959) The toxicity of synthetic detergents and soaps to fish. Sewage Iml. ~t~,~tes 31,295-306. Hokanson K. E. F. and Smith L. L. (197I) Some factors influencing toxicity of linear alkylate sulphonate (LAS) to the bluegill. Trans. ,qm. Fish. Soc. 100, t-12. Lang W. (1967) Investigations on the mode of action of anionic surl:actants on the histology and function of various organs of Carassius auratus. Arch. Fisch Wiss. 18, 25-,,t5. Leclerc E. and Devlaminck F. (1952) Natural or synthetic detergents and fish. B~tll. Ct, tlt. bt,l~lc Etttd. Docum. Eatt.v.. (17). 165-171. Tovell P. W. A.. Newsome C. and Howes D. (1974) Effect of water hardness on the toxicity of an anionic detergent to fish. Water Res. 8, 291-296.