Acclimation and tolerance of Artemia salina to copper salts

Marine Biology 38, 231-238 (1976) �9 by Springer-Verlag 1976

Acclimation and Tolerance of Artemia salina to Copper Salts

L. J. Saliba and R. M. Krzyz

Department of Biology, University of Malta; Msida, Malta

Abstract

The brine shrimp Artemia salina L. was acclimated in sea water with cupric chloride, acetate, carbonate, and sulphate, each at concentrations of O.1, 0.05 and 0.025 ppm Cu ++, together with sea water controls. Growth inhibition was observed in all four compounds, generally in direct relationship to the concentration. It was least in sulphate, and increased progressively in chloride, acetate and carbonate in that order. No inhibition however was observed in carbonate at 0.025 ppm Cu ++. In toxicity tests, 2-week old larvae from each solution were exposed to concentrations of 10, 7.5, 5, 2.5 and I ppm Cu ++ of the same compounds, together with unacclimated larvae of the same age. Similar tests were held with 6-week old adults acclimated (a) in O.1 ppm Cu ++ (chloride, acetate and sulphate) using the same concentrations and (b) in 0.5 ppm Cu++ (carbonate), using 150, 125, 100, 75, and 50 ppm Cu++. Toxicity to unacclimated larvae and adults differed with the compounds, carbonate being the least toxic, followed by sulphate, chloride and acetate in in- creasing order. Larvae acclimated in chloride (0.025 ppm Cu ++) and sulphate (O.1 and 0.5 ppm Cu++) showed an increased tolerance to I and 2.5 ppm Cu++ compared to untreated controls. Tolerance was not enhanced from 5 ppm Cu++ upwards. In both compounds, adults acclimated in 0.1 ppm Cu ++ showed an increased tolerance to concentrations between I and 7.5 ppm Cu++ compared to controls. Larval mortality in carbonate was below 50% in all test solutions. Adults acclimated at 0.5 ppm Cu ++ showed an increased tolerance to 50 ppm Cu ++ compared to controls. Considerable precipitation occurred with the high levels of this compound, thus effecting the "final" concentrations. No acclimation effect was observed in acetate for either larvae or adults. It is suggested that in A. salina, copper toxicity depends on the particular form of the metal, and that this difference is also evident in growth inhibition and in the potential acquisition of increased tolerance through exposure to low concentrations.

Introduction

At its normal concentration in sea water (0.005 ppm: Riley and Taylor, 1968), copper is an essential element for many liv- ing processes, forming part of cupropro- teins required for oxygen transport and electron transfer systems. It also forms part of many enzymes (e.g. tyrosinase) and acts as an activator for others (e.g. malate dehydrogenase). Its toxicity at higher levels has been extensively studied, results (as with several other heavy metals) showing a considerable variation. Apart from differences in methodology, factors influencing toxicity include the physical and chemical

nature of the toxicant (Portmann, 1970) and more specifically, the form of the metal itself (Bryan, 1971). The toxicity of copper sulphate to Artemia salina L. and Ophryotrocha labronica La Greca and Bacci, studied by Saliba and Ahsanullah (1973), was found to differ for both animals from that of copper sodium citrate, as recorded by Brown and Ahsanullah (1971), and the results also indicated that pre- exposure of A. salina to low levels of copper sulphate (0.025 to O.1 ppm Cu++) increased the tolerance of both larvae and adults to medium (I ppm Cu++), but not to high concentrations (10 ppm Cu ++ and above) of the same salt. This acclimation effect was not observed in o. labronica.

232 L.J. Saliba and R.M. Krzyz: Copper Tolerance in Artemia salina

Growth-rate measurements on larvae of phate) and 0.5 ppm Cu ++ (carbonate) for Artemia salina and Ophryotrocha labronica ex- 6 weeks, using the same procedures. posed to low concentrations of copper sodium citrate (Brown and Ahsanullah, 1971) and copper sulphate (Saliba and Ahsanullah, 1973) showed growth inhibition in both instances, the degree of such inhibition being generally in pro- portion to the concentration, but dif- fering in both animals between the two salts.

The purpose of the present study on the crustacean Artemia salina L. was to in- vestigate the differential effects of four copper salts on growth inhibition, acute toxicity, and on acclimation in-

These specimens were not measured. Toxicity tests were held on 2-week

old larvae and 6-week old adults. These were made in 250 ml beakers containing 200 ml solution. Concentrations used for larvae were 10, 7.5, 5, 2.5 and I ppm Cu ++. Those for adults were the same except for carbonate, which were 150, 125, 100, 75 and 50 ppm Cu ++. In both sets of experiments, 20 individuals from each "acclimating" concentration were placed in each test solution of the same salt, as were controls of the same age previ- ously kept in sea water. Each concentra-

duced by previous exposure to low concen- tion was replicated, and the whole ex- trations, periment repeated. Observations were

Materials and Methods

Artemia salina L. eggs were incubated in aerated sea water for 48 h in 1,OOO ml beakers held in a water bath at 24oc• O.5C o. The larvae were then transferred to 400 ml sea water in 500 ml beakers (approximately 150 larvae per beaker) containing the various test solutions, and fed 0.1 g "Mixed Cereal" (Cow and Gate Ltd) at 3-day intervals. Solutions were renewed weekly, larvae being re- tained on fine muslin during transfer. All containers were aerated throughout the experiments.

Experimental media were prepared by dissolving copper chloride, copper acetate, copper carbonate, and copper sulphate in sea water to give the required concentrations in terms of ppm (mg/l) copper ion, amounts being calculated by atomic weight. All salts were used in

made regularly at 4 h intervals, dead individuals being removed. The death point was considered to have been reached when an individual failed to respond to me- chanical stimulation. The cumulative number of dead at given times was plotted on semilog paper as percentage against time, giving sigmoid curves, from which the LT50 value was obtained. During toxicity tests, the solutions were not aerated, and the shrimp were not fed. Ex- periments were generally continued until 1OO% mortality was reached. Precipita- tion generally occurred above 5 ppm. This was not filtered out, and the ac- tual concentrations in the sea water, which were not measured, were therefore lower than those described. Precipita- tion was slight, except in the high levels of carbonate, and invariably occurred within I to 2 h of preparation of solutions. It was not observed to increase thereafter.

All cultures and toxicity tests were the cupric form. Sea water was collected held at 21 ~ to 22~ from Marsaxlokk Bay, in the Southeastern part of Malta (salinity 37.5~), sterilized at 7OoC for 2 h, topped up with dis- Results tilled water, and then passed through a 0.45 ~ Oxoid filter before use. Final Acclimation and Growl Rate

concentrations used for the growth-rate experiments were O.1, 0.05, and 0.025 ppm Under control conditions, Artemia salina Cu ++ for all four salts. Solutions were larvae survived in all concentrations, prepared fresh for every experiment or mortality being below 5%. Growth-rate medium-renewal. Two replicates for each measurements are shown in Fig. I a-d for concentration were used, including sea chloride, acetate, carbonate, and sul- water controls, and the whole experiment phate, respectively, compared with the was repeated twice. Measurements of Arte- mia salina were taken using an eyepiece micrometer in a binocular microscope, larvae being transferred to a slide for ease of handling. Means were taken of the measured length of 20 larvae from each concentration after I, 3, 5, 7, 10

sea water controls. The degree of inhibition recorded appeared to correspond directly with the concentration. It also differed to varying extents between the four salts.

Statistical analyses of the results were performed by Student's t-test. A

and 13 days. number of significant growth differences Separate cultures of Artemia salina were occurred in all four compounds, both be-

also maintained at concentrations of tween different levels of the same com- O.1 ppm Cu ++ (chloride, acetate and sul- pound, and between these and normal sea

L.J. Saliba and R.M. Krzyz: Copper Tolerance in Artemia salina 233

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(b) c o p p e r a c e t a t e , Fig. i. Artemia salina. Growth of larvae in (a) copper chloride,

carbonate, and (d) copper sulphate, acclim.: acclimation concentration

d

I I

11 13

(c) copper


Table i. Artemia salina. Statistical analysis of growth differences of larvae in varying concentrations of copper salts and in sea water controls

Salt Concentration Significance (P level) (ppm Cu ++) 5 days 7 days iO days 13 days

Chloride Control/O.O25 * * * 0.002 Control/O.O5 O.OO1 0.05 O.O1 0.025/0.05 * * 0.02 *

Acetate Control/O.O25 * O.O1 * 0.02 Control/O.O5 * 0.02 0.025/0.05 * * * O.O1

Carbonate Control/O.O25 * O.OO1 O.O1 * 0.025/0.05 O.O1 0.05 0.02 O.OO1

Sulphate Control/O.O5 * * O.OO1 O.OO1 0.025/0.05 O.O1 O.OO1 0.02

*: P >5% (0.05).

Table 2. Artemia salina. Statistical analysis of growth differences of larvae in equal concentrations of varying copper salts

Concentration Salts (ppm Cu ++)

Significance (P level) 7 days 10 days 13 days

0.025

0.05

O.O1

*P >5% (0.05).

Sulphate/chloride 0.02 O.01 O.O1 Chloride/carbonate * 0.02 *

Sulphate/acetate 0.002 * 0.05

Sulphate/chloride O.OO1 * * Sulphate/ace tate O. 05 O. 002 Ch lori de/ace tate * * O. 02 Chloride/carbonate * O. 002 O. 05

water (Table I), generally starting at 7 days, except in carbonate and chloride where they started at 5 days. In all four compounds, however, growth differences between 0.05 and 0.1 ppm Cu ++ were not statistically significant.

A comparison of the four compounds shows sulphate as giving the least de-

each salt, and also placed in (a) sea water, and (b) a fresh acclimation solution of the same concentration. Cumula- tive LT50 values for chloride, acetate and sulphate are shown in Fig. 2 a-c, respectively. The results for chloride (Fig. 2a) indicate a degree of increased tolerance for larvae acclimated in

gree of inhibition, followed by chloride, 0.025 ppm, compared to the sea water con- acetate, and carbonate in that order. At trols. These differences were signifi- 13 days, however, larvae in carbonate cant at the 2% level at 2.5 ppm and at increased their growth considerably. Dif- the 0.1% level at I ppm. The differences ferences between growth in the different between larvae acclimated at O.1 and compounds at the same concentration started to become statistically significant at 7 days (Table 2).

Acute Toxicity

Two-week larvae from each acclimation- solution were transferred to solutions of 10, 7.5, 5, 2.5 and I ppm Cu ++ of

0.05 ppm were not significant relative to the controls.

In acetate (Fig. 2b , there was no difference in toxicity between acclimated larvae and controls. The results for sulphate (Fig. 2c) showed no defi- nite increase in tolerance from 5 ppm upwards. At 2.5 ppm, differences between O.1 and 0.05 ppm acclimated larvae were


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Concentrat ion (ppm Cu ++)

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Fig. 2. Artemia salina. Tolerance of 2-week larvae to (a) copper chloride, (b) copper acetate, and (c) copper sulphate

significant at the 2% level. At I ppm, in O.1 ppm Cu ++ (chloride, acetate, and these were significant at the 0.1% level, sulphate), and in 0.5 ppm Cu ++ (carbon- and those between 0.05 and 0.025 ppm at the 0.2% level. There was no significant difference between 0.025 ppm and sea water.

Mortality in carbonate was very low and, in the concentrations used, did not reach the 50% level. Differences in mortality between control larvae in the other three salts were insignificant from 5 ppm upwards. In 2.5 ppm, comparative LT50 values for sulphate/acetate and sulphate/chloride showed significant differences at the 2% level, and in I ppm

ate). Test concentrations for the first three salts were 10, 7.5, 5, 2.5 and I ppm Cu ++. For carbonate, concentrations used were 150, 125, 100, 75 and 50 ppm Cu ++. LT50 values are shown in Fig. 3. Differences between acclimated and control adults in chloride (Fig. 3a) were significant at the 2% level (7.5 ppm), I% level (5 ppm), 2% level (2.5 ppm), and 2% level (I ppm). There was no significant difference in acetate (Fig. 3b). In sulphate (Fig. 3c), the differences between 7.5 and 1 ppm were all significant at the 2% level. In carbonate

at the I% level. There was no signifi- (Fig. 3d) although some increase in tol- cant difference between acetate and chloride. erance was evident, the differences be-

tween acclimated and control adults was Further tests were held with 6-week only significant (at the I% level) in

old adults. These were acclimated for 50 ppm. In this compound considerable the whole period, i.e., from hatching, precipitation occurred at all test lev-


50

40

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0

2O

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I I I I

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50

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Concent ra t ion (ppm Cu ++)

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200

150

v

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50

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d

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50 75 100 125 150

Concent ra t ion ( p p m C u ++ )

Fig. 3. Artemia salina. Tolerance of 6-week adults to (a) copper chloride, (b) copper acetate, (c) copper sulphate, and (d) copper carbonate

els, thus affecting the amount actually in solution. Adults both fed on the bottom precipitate and continually stirred it up by their movements.

Differences between LT50 values for control adults in chloride, acetate, and sulphate did not show any statistical significance at I ppm. Those between sulphate/chloride and sulphate/acetate were all significant at the 2% level between 2.5 and 10 ppm.

In all experiments, test individuals from all cultures held in sea water and in the acclimating concentrations under the same conditions suffered negligible (below 5%) or no mortality throughout the experimental period.

Discussion and Conclusions

(1973) in the same species. The earliest symptom was an increase in activity, followed by a quick decline, the shrimp ex- hibiting irregular spasmodic movements of the limbs which gradually diminished up to death point. In addition, in high concentrations of carbonate (50 to 150 ppm), a considerable amount of defaeca- tion occurred in practically all adults, the "strings" of faeces being several times as long as the shrimps themselves. This might have been partly due to the adults feeding on the bottom precipitate.

Copper Toxicity

The results obtained would indicate that one of the factors influencing copper toxicity is certainly the form of the metal itself. Carbonate was by far the

Toxicity symptoms for Artemia salina in all least toxic form. This may be due to the four salts were as described for copper fact that it precipitates more readily. sulphate earlier by Saliba andAhsanullah However, one should also consider the


facts that (a) larval mortality in the the purity of the sea water used. In ear- I to 10 ppm range, where precipitation ly experiments, reverse results were ob- was slight or absent, was very low; (b) tained, and an increase, rather than in- in the high levels (50 to 150 ppm) adults hibition of growth was evident, this in- continually moved through, and were ob- crease being directly proportional to served to graze upon the bottom precipi- the concentration. Microscopical examina- tate, this also to a lesser extent being tion of the filtered sea water indicated the case with larvae in 5 to 10 ppm; and the presence of several ciliates in the (c) mortality in both acclimated and con- control and 0.025 ppm concentrations, trol adults corresponded generally with these being less abundant in 0.05 ppm, the test concentrations prepared. This and totally absent in O.1 ppm. Competi- trend was more pronounced in the accli- tion for food therefore became an impor- mated individuals, tant factor. As a result, all sea water

Of the other three salts, sulphate was both sterilized and filtered before was the least toxic to 2-week old larvae, use in hatching and in growth experi- especially at I and 2.5 ppm. Acetate ap- ments. peared to be slightly less toxic than chloride, although the differences in LT50 values were not statistically significant. In the case of-6-week adults, sulphate again was the least toxic, and there were no apparent differences between acetate and chloride. One could postulate that age of the test organism is aDother contributing factor.

Aubert et al. (1972) subjected 25 to 50 Artemia salina larvae to various concentrations of copper sulphate in 20 ml of solution, recording no mortality up to 12.5 ppm after 9 days, and 60% mortality at 25 ppm in the same period. These results contrast strongly both with the present and with previous (Saliba and Ahsanullah, 1973) experiments on copper sulphate toxicity in this species. Although the fact that Aubert's larvae were fed with I ml algal inoculum might have contributed thereto, other factors, either arising out of the particular experimental procedure em- ployed, and/or possibly inherent in the larvae themselves, must have been respon- sible for the relatively high degree of tolerance recorded by Aubert et al.

Growth Rate

The data obtained support previous work, both on Artemia salina and on various other

Acclimation and Tolerance

Mortality tests with larvae and adults indicate that while Artemia salina can be confirmed in its ability to acquire a certain degree of tolerance to copper compounds after initial pre-exposure to low concentrations, this tolerance again appears to differ with the particular salt. In the case of sulphate, tolerance to I ppm increased according to the acclimating concentration. This was also so, although less distinct, at 2.5 ppm. Pre-exposure to acetate did not appear to produce any increase in tolerance, significant or apparent. In the case of chloride, while all pre-exposed larvae survived longer than the controls, the highest degree of tolerance was shown by those acclimated in 0.025 ppm. This would appear to indicate an optimum acclimation level in this instance.

Comparison of the mortality figures for 2-week larvae and 6-week adults indicate that a longer acclimation period does not necessarily result in increased tolerance. In sulphate, the difference in tolerance decreased somewhat with the longer period, although it extended over a wider range of test concentrations. In chloride, tolerance was generally more

species, that growth is inhibited by cop- pronounced at 6 weeks than at 2. In ace- per at concentrations above normal, and tate, there was no difference between that such inhibition corresponds direct- the two comparative values, and it ap- ly with the concentration. The growth pears that Artemia salina is unable to differences obtained with the four salts acclimate itself to this salt. also indicate that this sub-lethal ef- The results suggest that acclimation, feet of copper varies with the particu- with its resultant increase in tolerance, lar salts, and that these differences in- is subject to several factors, among crease with the concentration. In gener- which are age, the length of the pre- al, more significant differences were exposure period, and the concentrations recorded between the O.1 ppm concentra- at both pre-exposure and toxic levels. tions than between the 0.05 and 0.025 ppm The variations observed among these fae- among the various salts, tors would further suggest that in Artemia

One important factor affecting the salina, at least in the case of copper, growth rate of Artemia salina larvae in acclimation is very much dependent on copper concentrations was found to be the particular form of the metal.


Literature Cited

Aubert, M., R. Bittel, F. Laumond, M. Romeo, B.

Donnier et M. Barelli: Utilisation d'un chaine trophodynamique de type p~lagique pour l'~tude des transferts des pollutions m~tall-

iques. Revue int. oc~anogr. M&diterr. 28, 27-52 (1972)

Brown, B. and M. Ahsanullah: Effect of heavy

metals on mortality and growth. Mar. Pollut. Bull. 3, 182-188 (1971)

Bryan, G.W.: The effects of heavy metals (other than mercury) on marine and estuarine animals.

Proc. R. Soc. (Set. B) 177, 389-410 (1971) Portmann, J.E.: Results of acute toxicity tests

with marine organisms, using a standard meth-

od. In: Marine pollution and sea life, pp 212-217. Ed. by M. Ruivo. London: Fishing

News (Books) 1972

Riley, J.P. and D. Taylor: Chelating resins for the concentration of trace elements from sea water, and their use in conjunction with atomic absorption spectrophotometry. Analyti- ca chim. Acta 40, 479-485 (1968)

Saliba, L.J. and M. Ahsanullah: Acclimation and tolerance of Artemia salina and Ophryotrocha labronica to copper sulphate. Mar. Biol. 23, 297-302 (1973)

Dr. L.J. Saliba

Department of Biology University of Malta

Msida

Malta

Date of final manuscript acceptance: July 23, 1976. Communicated by J.H.S. Blaxter, Oban

Documents

Acclimation and tolerance of Artemia salina to copper salts