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.H.S. Santos et al. J. Exp. Mar. Biol. Ecol. 247 2000 233 –242
1. Introduction
Industrial activity along coastal regions has significantly contributed to increases of natural levels of heavy metals in aquatic systems. In the Patos Lagoon estuary Southern
Brazil, areas in which copper and zinc concentrations exceed the estimated natural levels have been reported Baisch et al., 1988; Niencheski et al., 1994. However, this
region is a natural hatchery for the pink shrimp Farfantepenaeus paulensis, which is the most important fishing resource in Southern Brazil Valentini et al., 1991. The life cycle
includes both, marine and estuarine phases. They penetrate the Patos Lagoon estuary as postlarvae 6th substage at approximately 3 weeks of age Iwai, 1978, and develop
until a pre-adult stage 7–8 cm total length when they return to sea D’Incao, 1991. During their residence in the estuary, postlarvae are susceptible to all contaminants
available in the environment, and as mentioned below, the effects of excessive copper and zinc concentrations on postlarvae physiology must be considered.
Copper and zinc are essential metals to normal physiology of crustaceans. However, they become toxic if high environmental concentrations are attained. Some general
effects of heavy metals on crustacean physiology have been reported Thurberg et al., 1973; Spicer and Weber, 1991; Wong et al., 1993. These effects also include growth
and reproduction disturbances Nimmo and Hamaker, 1982. Some of these effects may be due to an energetic imbalance carried out by the action of heavy metals on enzymatic
systems, which in turn lead to metabolic changes. Considering food ingestion as the major input of energy in crustaceans, and oxygen consumption as a measure of
metabolism, the effects of sub-lethal copper and zinc concentrations on these parameters were analysed in F
. paulensis postlarvae. Their effects on feeding response were also considered. Chronic effects of sub-lethal concentrations of copper and zinc on PL
17
growth was studied for 35 days. All studies were performed using copper and zinc singly, and in a mixture of equipotent concentrations.
2. Material and methods
Farfantepenaeus paulensis postlarvae PL were reared in the Marine Aquaculture ˜
Station of the ‘‘Fundac¸ao Universidade Federal do Rio Grande’’ Rio Grande, RS, Southern Brazil following the method described by Marchiori 1996. Two day old PL
PL were previously acclimated to aerated saline water at 2561.5‰; 2561.08C and
2
photoperiod 12L:12D, for 15 days. Saline water employed was pumped from Cassino Beach Rio Grande, RS, Southern Brazil and filtered 1 mm mesh. The acclimated
PL total length 5 9.860.4 mm; wet weight 5 3.760.4 mg were then employed for
17
the experiments. Before tests, they were starved for 24 h in plastic flasks containing 500 ml of saline water at the same conditions of acclimation.
In order to determine the 96h-LC , twenty PL were exposed to each of the following
50
nominal concentrations of copper or zinc: 0 control; 0.5; 1.0; 2.0; 4.0 and 8.0 ppm of
21 21
Cu as CuSO .5H O or Zn
as ZnSO .7H O. After 96 h of exposure, LC for
4 2
4 2
50
copper and zinc were determined by means of probit analysis and compared using the respective 95 confidence intervals.
M .H.S. Santos et al. J. Exp. Mar. Biol. Ecol. 247 2000 233 –242
235
Based on the 96h-LC values, PL were exposed to the following nominal con-
50 21
centrations of copper and zinc: 0 control; 17; 43; 85 and 212 ppb of Cu as
21
CuSO .5H O; and 0 control; 41; 106; 212 and 525 ppb of Zn as ZnSO .7H O. It
4 2
4 2
must be pointed out that both concentration sequences present the same lethal toxicity and correspond to approximately 0; 1.2; 3; 6 and 15 of the 96h-LC
see Results.
50
Cooper and zinc were tested singly or in mixture. Copper–zinc mixtures approximately 1:2.5 were obtained adding the equipotent concentrations of these metals, e.g. 17 ppb
21 21
Cu 1 41 ppb of Zn
. All experimental media were prepared employing the same water used for postlarvae acclimation. Experiments were performed under the same
conditions temperature, salinity and photoperiod.
2.1. Growth experiments Acclimated PL
were exposed to the different concentrations of copper and zinc,
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singly or in mixture, in plastic trays containing 10 l of the experimental medium, for 35 days. Every 2 days, the experimental media were totally renewed. During the exposure
period, PL were fed ad libitum with nauplii first 15 days or adults of Artemia sp. At the beginning of the experiment N 5 43 and after 35 days of exposure N 5 13–129;
Table 1, PL were captured, measured total length and weighed wet weight and dry weight. Experiments were performed in duplicate. Results were submitted to Kruskal-
Wallis analysis followed by a posteriori test described in Conover 1980.
2.2. Feeding experiments Acclimated PL
N 5 10–13; Table 2 non pre-exposed to the metals were in-
17
dividually exposed to the different concentrations of copper and zinc, singly or in mixture, for 30 min. They were exposed in separated plastic flasks containing 10 ml of
experimental medium and 10 nauplii of Artemia sp., in darkness because F . paulensis
PL is more active and feeds at night Iwai, 1978. PL was then isolated and weighed wet weight. The nauplii remaining in the flask were counted and the results were
expressed in terms of number of nauplii predated 30 min. In the control group, a possible effect of PL weight on the food consumption was analysed by means of linear
regression. Data of food consumption were submitted to Kruskal-Wallis analysis followed by a posteriori test described in Conover 1980.
In order to study the effect of copper and zinc on the postlarvae feeding response, we
o ]
employed the method described by Santos F 1983. Acclimated PL N 5 10 non
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pre-exposed to the metals were individually exposed to the highest concentrations of copper and zinc tested singly or in mixture, for 4 min. They were exposed in separated
flasks containing 10 ml of experimental medium and a piece of filter paper Whatman No. 1; 2.5 3 2.5 mm, previously immersed in a saturated sea water solution of
L-isoleucine and dried at 378C. This aminoacid was used since it was demonstrated to be
o ]
the major inducer of feeding response in F . paulensis juveniles Santos F , 1983.
Feeding response was classified as either positive or negative. According to the same author, a positive feeding response could be defined as: ‘‘holding and putting the piece
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.H.S. Santos et al. J. Exp. Mar. Biol. Ecol. 247 2000 233 –242
of filter paper in their mouths within 2 min after presentation and swallowing it or, rejecting it only after, at least, 4 min in their mouths’’. Data were submitted to the
Kolmogorov-Smirnov two-sample test.
2.3. Oxygen consumption experiments Acclimated PL
N 5 5–8; Table 3 non pre-exposed to the metals were exposed to
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the different concentrations of copper and zinc, singly or in mixture, in a static respirometer containing 7.5 ml of experimental medium, for 15 min. In this case, saline
water was previously filtered 1 mm mesh and autoclaved 20 min at 1208C. The respirometer was mounted on a magnetic stirrer and provided with an O
electrode
2
connected to an oxymeter Digimed, Brazil. Each 5 min, dissolved O was measured
2
and the oxygen consumption calculated, taking into account the respirometer volume. Previous experiments indicated that by 15 min constant consumption rates were
obtained. So, after 15 min the PL oxygen consumption was measured, in duplicate.
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PL were then isolated and weighed wet weight. Results were expressed in mg
