TOXICITY OF COPPER TO THE SHRIMP
METAPENAEUS ENSIS
by
Janet Kwai-yu Cheung
Thesis submitted as partial fu"lfillment for
the degree of Master of Philosophy
July, 1992
Marine Science Laboratory
Department of Biology
The Chinese University of Hong Kong 360103 ~ OL 4-!f'+ f/133 e Lf3 TOXICITY OF COPPER TO THE SHRIMP
METAPENAEUS ENSIS
by
Janet Kwai-yu Cheung
M. Phil. Thesis, Chinese University of Hong Kong,
July, 1992
ABSTRACT
Heavy metal pollution draws much public attention in recent years. Hong Kong waters are heavily polluted by heavy metals. The presence of heavy metals in water would affect marine life and pose the risk of being poisoned by seafood containing heavy metals. The present study aims at providing information on the impact of copper on the different life history stages of the local shrimp, Metapenaeus ensis.
Metapenaeus ensis is a common local shrimp of commercial importance. Its larval development consists of six naupliar stages, three protozoeal and three mysid stages. The myses then metamorphose to post larvae. Toxicity of copper was tested in five different life history stages of Metapenaeus ensis, including protozoea
I, mysis I, day-1 postlarva, day-4 postlarva and day 15- 20 postlarva.
Acute and chronic toxicity tests were conduct~d for
48 or 96 hand 8 or 10 days respectively. Acute toxicity tests showed that the presence of copper decreased the survival of the larvae and postlarvae of
Metapenaeus ensis. In addition, individuals from different spawners were shown to be different in their
sensitivity to copper. There was also a trend of increase in tolerance to copper through development. The rate of larval development retarded in the presence of
copper. Exposure to copper also resulted in retarded growth in day 15-20 postlarva. Decrease in developmental rate indicates an increase in the moult cycle duration of the larvae, which may adversely affect
the larval survival and thus the recruitment of this
species in the natural environment.
Sublethal effects of copper were assessed in terms
of three parameters, including oxygen consumption,
ammonia excretion and feeding rate. Upon sublethal exposure to copper, the respiratory rate and feeding
rate significantly increased and decreased, respectively, in some experiments. Ammonia excretion
showed no significant changes in most cases studied. An
2 imbalance in the energy budget may result from the increase in metabolism and the decrease in food intake. Both changes may act together in affecting the survival and growth of the shrimp upon exposure to copper.
3 ACKNOWLEDGEMENTS
I would like to thank Dr. K. H. Chu, my supervisor, for his invaluable support and advice. I,am also grateful to Dr. P. K. Wong and Dr. E. C. Ooi, for their comments on the thesis. Thanks are also given to all staffs in the Marine Science Laboratory, especially Mr. Y. C. Tam and Mr. M. K. Cheung for their technical assistance. I would like to express my sincere thanks to my family and my friends, especially Miss Yvette Y. M. Kan and Miss Merav Uziel, for their encouragement and support during these two years. Lastly, I thank the Biology Department , of the Chinese University of Hong Kong to provide me a chance to complete this study.
4 ( TABLE OF CONTENTS
.' Page
ABSTRACT 1
ACKNOWLEDGEMENTS 4
TABLE OF CONTENTS 5
LIST OF TABLES 7
LIST OF FIGURES 9
CHAPTER 1
GENERAL INTRODUCTION 11
CHAPTER 2
LITERATURE REVIEW
2.1 Toxicity of heavy metals to crustaceans 13
2.2 Heavy metal pollution in Hong Kong waters 20
2.3 Copper: its nature and occurrence 27
2.4 General biology of Metapenaeus ensis 32
5 CHAPTER 3
ACUTE AND CHRONIC TOXICITY OF COPPER
TO METAPENAEUS ENSIS
3.1 Introduction 39
3.2 Materials and Methods 39
3.3 Results 45
3.4 Discussion 68
CHAPTER 4
SUBLETHAL EFFECTS OF COPPER
TO METAPENAEUS ENSIS
4.1 Introduction 76
4.2 Materials and Methods 76
4.3 Results 82
4.4 Discussion 96
CHAPTER 5
GENERAL CONCLUSIONS 101
REFERENCES 103
6 LIST OF TABLES Page
3.1: LCso values estimated from the fitted regression lines for acute toxicity experiments 59
3.2: Body length of day-1 post larvae of Metapenaeus ensis upon chronic exposure to different copper concentrations 66
3.3: Toxicity of copper to some crust?cean species 67
4.1: Dry weight of selected life history stages of Metapenaeus ensis 86
4.2: Oxygen consumption and ammonia excretion of PZI from two spawners of Metapenaeus ensis 87
4.3: Oxygen consumption and ammonia excretion of
MI from two spawners of Metapenaeus ensis 88
4.4: Oxygen consumption and ammonia excretion of PL1 from two spawners of Metapenaeusensis 89
4.5: Oxygen consumption and ammonia excretion of
PL4from two spawners of Metapenaeus ensis 90
4.6: Oxygen consumption and ammonia excretion of PL15-20 from two spawners of Metapenaeus ensis 91
4 .7: Ingestion and clearance rates of PZI fed Chaetoceros gracilis 92
7 4.8: Ingestion and clearance rates of MI fed Chaetoceros gracilis 93
4.9: Predation rate of PL1 on Artemia nauplii 94
4.10: Predation rate of PL4 on Artemia nauplii 95
8 L~TOFllG~ Page
3.1: Percentage mortal~ty of protozoea I exposed to different concentrations of copper for 48 h. 49
3.2: Percentage mortality of mysis I exposed to different concentrations of copper for 48 h. 50
3.3: Percentage mortality of day-1 postlarvae exposed to different concentrations of copper for 48h. 51
3.4: Percentage mortality of day-4 post larvae exposed to different concentrations of copper for 48 h. 52
3.5: Percentage mortality of day 15-20 postlarvae exposed to different concentrations of copper for 48 and 96 h. 53
3.6: Relationship between mortality in probit scale and log copper concentration for protozoea I exposed to different concentrations of copper for 48 h. 54
3.7: Relationship between mortality in probit scale and log copper concentration for mysis I exposed to different concentrations of copper for 48 h. 55
3.8: Relationship between mortality in probit scale and log copper concentration for day-l postlarvae exposed to different concentrations of copper for 48 h. 56
9 3.9: Relationship between mortality in probit scale and log . copper concentration for day-4 postlarvaeexposed to different concentrations of copper for 48 h. 57
3.10: Relationship between mortality in probit scale and log copper concentration for day 15-20 postlarvae exposed to different concentrations of copper for 48 and 96.h. 58
3.11: Percentage of protozoea III among survivors for " protozoea I exposed to different concentrations of copper for 48 h. 60
3.12: Percentage of mysis 11 among survivors for mysis I exposed to different concentrations of copper for 48 h. 61
3.13: Percentage survival of larvae upon chronic exposure to different copper concentrations. 62
3.14: Growth index of larvae upon chronic exposure to different copper concentrations. 63
3.15: Percentage survival of day 15-20 postlarvae upon chronic exposure to different copper concentrations. 64
3.16: Percentage increase in body length of day 15-20 postlarvae upon chronic exposure to different copper concentrations. 65
10 CHAPTER 1
GENERAL INTRODUCTION
Metapenaeus ensis is a commercially important shrimp in Southeast Asia. It forms one of the most important species in the shrimp catches in Malaya, Singapore, Indonesia and Philippines (Holthuis, 1980). It is also extensively cultured in many Asian countries, including Taiwan, Philippines, Thailand and Malaysia. Local shrimp culture takes place at the Zhujiang River estuary that is often polluted with heavy metals. Thus, the growth of this shrimp is possibly confined to the coastal waters that are seriously polluted.
Among the heavy metals found in the coastal waters of Hong Kong, copper exhibits particularly high concentration both in the sediments and in the tissues of bio-indicators (see Chapter 2). In addition, copper is found to be very toxic to marine invertebrates at low concentrations (Mance, 1987) and thus has a high toxic potential.
Wong et al. (in press) reported that copper is the most toxic heavy metal among nickel, chromium and copper
11 to the larval and postlarval stages of Metapenaeus ensis. Studies on the effects of copper to different developmental stages Qf Metapenaeus ensis may thus confirm the toxicity to copper to this shrimp, and possibly shed light on the impact of this metal on the survival, growth and development of Metapenaeus ensis.
Pollutant concentrations in natural environment are generally much lower than the LCso values. Studies on the sublethal effects of copper to the larval and postlarval stages of this shrimp in respect to its respiration, ammonia excretion and feeding rate provide information on the sublethal toxic effects of copper to the metabolic activities of this shrimp. And thus possibly shed light on the mechanism of the toxic effect of copper to this shrimp in the metabolic level.
This thesis consists of 5 chapters. Chapter 1 is
a general introduction. Chapter 2 is a literature
review on topics that are relevant to this study.
Chapter 3 presents the toxicity of copper to different
life history stages of Metapenaeus ensis, including acute and chronic toxicity. Experiments on the sublethal effects of copper on respiration, ammonia excretion and feeding rate are presented in Chapter 4.
A brief conclusion is given in Chapter 5.
12 CHAPrER2
LITERATURE REVIEW
2.1 Toxicity of heavy metals to crustaceans
Crustaceans are extremely sensitive to heavy metals (Mance, 1987 , review). Both lethal and sublethal effects of heavy metals have been reported in the literature. Studies of lethal effects are usually adopted to determine the sensitivity of the organisms to the heavy metals concerned. The deleterious effects of heavy metals on crustaceans may result not only in death, but also in a variety of sublethal effects. In addition, the heavy metal concentrations in polluted environment are generally much lower than the median lethal concentrations (LCso values). Studies of the sublethal toxicity of heavy metals may therefore provide more realistic and meaningful information on the toxicity of heavy metals to organisms. Sublethal effects may include the reduction of an organism's capability to perform ,and adapt to its habitat and thus reduce its chances of survival, as well as its potential for growth and reproduction. In this review, the acute toxicity of heavy metals to crustaceans will be
13 introduced first, which is then followed by sublethal toxic effects.
2.2.1 Lethal effects
Al though numerous studies concerning the lethal effects of various metals to crustaceans have been carried out (Mance, 1987, review; Ahsanullah and Williams, 1991), the lack of a standard procedure in the experimental design or methods, reporting of results, and even the units of metal concentration used, makes it very difficult to make comparisons among the numerous experimental data. In acute toxicity tests, several heavy metals were commonly studied, which include copper (Conner, 1972; Johnson and Gentile, 1979; Ahsanullah et al., 1981a; Verriopoulos et al., 1987; Ismail et al., 1990; Liao and Hsieh, 1990), mercury (Conner, 1972; Glickstein, 1978; Johnson and Gentile, 1979; McClurg, 1984; Piyan et al., 1985), zinc (Conner, 1972; Ahsanullah et al., 1981a; Liao and Hsieh, 1990), cadmium (Johnson and Gentile, 1979; McClurg, 1984; Ahsanullah et al., 1981a; Giudici et al., 1987; Liao and Hsieh, 1990) and chromium (Verriopoulos et al., 1987).
Martin et al. (1981) tested the toxicity of ten heavy metals to Cancer magister larvae. Of the ten heavy metals, including arsenic, cadmium, chromium,
14 copper, lead, mercury, nickel, selenium, silver, and zinc, the most toxic metals expressed as 48-h LC~ are mercury ( 8 . 2 JJg 1-1), copper ( 49 JJg 1-1) and si 1 ver ( 55
JJg 1-1). Since mercury pollution does not commonly occur, the toxicity of other metals such as copper is of great concern. It has been suggested that copper is the second most toxic metal after mercury (Ahsanullah,
1982). Copper is also more toxic than zinc and cadmium to Callianassa australiensis (Ahsanullah and Arnott,
1978; Arnott and Ahsanullah, 1979; Ahsanullah et al.,
1981a). The toxicity of copper, cadmium and zinc to
A110rchestes compressa in their decreasing order is CU > Cd > Zn (Ahsanullah and Williams, 1991). Wong et al. (in press) also suggested that compared with copper, cadmium is relatively non-toxic to the larvae of
Metapenaeus ensis.
As mentioned above, studies of lethal toxicity are usually used to determine the sensitivity of organisms to the heavy metals concerned. From the results of acute toxicity tests, values of median lethal concentration (LCso ) can usually be estimated. These LCso values are commonly us~d to compare the sensitivities of different organisms or different life history stages of an organism, to the heavy metals concerned. It was well documented that young life history stages are more susceptible than later stages for the same species.
15 Conner (1972) reported that the larvae of sand shrimp,
Crangon crangon, the green crab, Carcinus maenas, and the European lobster, Homarus gammarus are 14 to 1000 times more sensitive to copper and zinc than the adults of the same species. Acute toxicity tests using , the marine amphipod, Al10rchestes compressa showed that the
96-h LCso values of copper are O. 11 and 0.5 mg 1-1 for the juveniles and the adults, respectively (Ahsanullah and
Florence, 1984). Juveniles are 4.5 times more sensitive than adults. Many other studies have also demonstrated that larval stages of marine invertebrates are more sensitive to metals than adults (Collier et al., 1973;
Saliba and Ahsanullah, 1973; Vernberg et al., 1973;
Brown and Ahsanullah, 1974; Ahsanullah and Arnott, 1978;
Arnott and Ahsanullah, 1979; McKenney and Neff, 1979).
A suggestion to explain the phenomenon is that the mechanisms of detoxification found in adults may not be well developed in their young forms (Bernard and Lane,
1961), so that the regulation of metals is less effective in young forms than in adults (Bryan and
Hummerstone, 1973).
2.1.2 Sublethal effects
The sublethal toxicity of heavy metals on growth and development, respiratory metabolism, swimming and
16 feeding behaviour as well as reproduction will be introduced below.
The developmental rate of Artemia salina was retarded upon exposure to zinc (Alayse-Danet et al., 1979) • The larval growth rates of Rhithropanopeus harrissii (Benijts-Claus and Benijts, 1975) and Daphnia magna (Winner, 1981) were also found to be retarded when the animals were exposed to sublethal doses of zinc. It has been suggested that zinc toxicity is related with the inhibition of enzymatic activities of crustaceans. According to Bitter et al. (1982), zinc stops the catalytic action of the cyclic AMPkinase on the sodium flux of the muscle of Balanus nubilis. Haya et al. (1983) also noticed a decrease in the activity of Na/K ATPase in the lobster, Homarus americanus, after exposure to zinc. Sublethal doses of cadmium at 10 ~g
1-1 can also cause a retardation in the developmental rate of Eurypanopeus depressus from the megalopa to juvenile crab (Mirkes et al., 1978). Both copper and cadmium were shown to affect the growth rate of nauplii of the copepod, Eurytemora arrinis (Sullivan et al., 1983). Cadmium can disturb the activity of enzymes in crustaceans (Amiard et al., 1982, cited in Gaudy et al., 1991). When the crab, Carcinus maenas was exposed to
2 .5 mg Cd 1-1 for 2 to 3 weeks, stimulation of protein
17 catabolism as indicated by the activities of trypsinases and aminopeptidases resulted.
Contradictory results are of common occurrence in the studies of the sublethal effects of heavy metal$ on respiratory metabolism. Copper was found to reduce respiration in Balanus amphitrite (Bernard and Lane,
1963), but activate respiration in Acartia clausi
(Apostolopoulou and Verriopoulos, 1979). Further, respiratory rates remained stable in copepod, Pseudocalanus sp. and Calanus sp. exposed to copper
(Reeve et al., 1977). According to Apostolopoulou and
Verriopoulos (1979), the oxygen consumption rate of
Acartia clausi showed a continuous increase for 10 days when the copepod exposed to copper ranged from 0.001 to
0.01 mg 1-1. It was also found that cadmium depresses metabolism in Pleuronectes americanus (Calabrese et al. " 1975) and increase metabolism in Acartia clausi
(Apostolopoulou et al. 1979). Recently, Gaudy et al.
(1991) also reported that sublethal doses of cadmium at concentrations between 0.05 and 0.10 mg 1-1 decrease respiratory metabolism in Leptomysis lingvura. These contradictory results ,were explained by the possibility that there are two phases in the physiological response of organisms to cadmium (Relexans et al., 1988, cited in
Gaudy et al., 1991). During the first phase, the organism responds by increased oxidative phosphorylation
18 which can be considered to be a physiologically adaptive-response to heavy metals. In the second phase, intoxication is characterized by a reduction in respiratory rate, during which the organism fails to
'meet the increased demand of ATP and finally died.
Barthalmus (1977), who studied the behavioural effects of mercury on the grass shrimp, Palaemonetes pugio, found that sublethal exposure of this shrimp to
O. 05 mg 1-1 mercury signif icantly impairs conditioned avoidance responses. Sublethal copper concentrations can cause reduction in swimming speed of the nauplii of the barnacle, Balanus improvisus (Lang et al., 1980). The swimming speed decreases progressively with increasing copper concentration. Both copper and cadmium were shown to reduce the swimming speed as well as the number of escape responses of Eurytemora affinis nauplii (Sullivan et al., 1983). Upon sublethal exposure to copper, the feeding activity was found to be reduced in the copepods, Pseudocalanus sp. and Calanus sp. (Reeve et al., 1977), Acartia clausi (Apostolopoulou and Verriopoulos, 1979), and Acartia tonsa (Sunda et al., 1987). Wong et al. (in press) studied the toxici ties of three heavy metals, including copper, chromium, and nickel, to the shrimp, Metapenaeus ensis. Both grazing and predation rates were reported to be lowered after exposure to heavy metals.
19 Nimmo et al. (1978) showed that cadmium delays reproduction it:l, Mysidopsis bahia. This result was further confirmed by Carr et al. (1985). Both zinc and copper affected the fecundity of the copepod, Acartia tonsa (Sunda et al., 1987). Cadmium also affects ovulation of the copepod, Acartia tonsa (Toudal and Riisgaard, 1987).
2.2 Heavy Metal Pollution in Hong Kong waters
Hong Kong is located on the southern coast of ' China. It has a long coastline in contrast to its limited land area (Morton, 1976). The coastal waters of Hong Kong can be divided into three zones, with the clear oceanic waters in the east and the turbid estuarine waters in the west. In between these 2 zones is a transition zone where victoria Harbour is located. Hong Kong is a city with the majority of its population and industrial activities along the coast. These situations particularly apply to the Kowloon and Hong Kong city centres bordering victoria Harbour. New towns such as Sha Tin, Tai Po and Tuen Mun are also located at the coast.
According to EPD (1991a), there are approximately six hundred streams, rivers and open nullahs which drain directly into the coastal waters of Hong Kong. These
20 , :
watercourses are useful for domestic supplies, irrigation, aquaculture, recreation, preservation of aquatic life, and as a stormwater passage in town areas.
On the other hand, the sea also plays an important role
in daily life. It has various beneficial uses ranging
from the provision of food resources to the disposal of wastes.
The waters of Hong Kong have been deteriorating in quality over the years as a result of population pressure and economic growth (EPD, 1991a). It was found that more than two-thirds of the watercourses were polluted by livestock waste, domestic sewage and
industrial effluents. It is worsened by the fact that
around 70% of the river baseflows are stored in the reservoirs for domestic uses. Hong Kong's coastal waters are also badly polluted (Morton, 1976; Wu, 1988; reviews). Pollutants in residential, agricultural and
industrial effluents from 6 million people enter into pipes or streams and finally to the sea (Wu, 1988;
Morton, 1988; Phillips, 1989). It was estimated that
Hong Kong produces 2 million tonnes of waste water every
day (EPD, 1991a).
The potential hazards of seafoods contaminated with heavy metals have lead to an increasing concern about heavy metals as pollutants in the coastal environment.
21 There have been many studies on heavy metal pollution in Hong Kong waters. The levels of heavy metals in Hong Kong waters were first reported by Chan et al. (1974). Water samples were collected from 54 areas around Hong Kong island, Kowloon, New Territories, and outlying islands. The concentrations of five heavy metals, including zinc, lead, cadmium, iron and copper, were found to be several hundred micrograms per litre. In
1 the case of iron, the concentration was several mg 1- • The concentrations of lead and cadmium were 160 and 180 times those found in the open ocean. An analysis of 12 drain samples showed a lead content on average 50 times higher, and in one case 1,000 times' higher, than that found in the open sea (Chan et al., 1973). However, the sampling methods and the analytical techniques involved in these two studies suggest that these data should be treated with caution (Phillips, 1989).
Due to the growing population around Victoria Harbour, its water quality has declined (Barnes, 1991). Phillips (1979) made a survey of the levels of heavy metals, including cadmium, copper, iron and zinc in the rock oyster, Saccostrea glomerata, from 54 sites in Hong Kong waters. The result showed elevated levels of metals in several areas. Moreover, the entire area of Victoria Harbour was found to be heavily contaminated by copper and zinc, the levels being some 5 and 2.5 times
22 higher than those found in other areas, concentrations being particularly high in the eastern portion of the
Harbour. Using Saccostrea glomerata as the biological indicator again, Phillips and Yim (1981) confirmed that there was a considerable contamination of Victo,ria
Harbour by copper and zinc. These results agreed well with the data on the heavy metal contents in sediments
(Yim and Fung, 1981; Phillips and Yim, 1981). Phillips
(1985) used the green-lipped mussel, Perna viridis, to indicate unusually large ranges in bioaccumulated concentrations of copper and lead (8.5-278 ~g ~1 and
1. 4-60 ~g g-1 dry weight respectively) . The concentration of zinc was found to be 77 to 164 ~g g~ dry weight, but mercury was below the detection limit of o. 11 ~g g-1 dry weight. These results indicated no major source of mercury pollution in Hong Kong waters.
Ho (1987) also found higher metal concentrations of copper, lead, nickel and zinc in three species of macroalgae, Enteromorpha compressa, Enteromorpha flexuosa and Gymnogongrus flabelliformis from victoria
Harbour than in algae from the south or east of Hong
Kong island.
Recently, the heavy metal pollution in victoria
Harbour was intensively studied by the EPD (1987-1991b).
Through these studies, high contents of heavy metals, including cadmium, chromium, nickel, zinc, lead, copper
23 and mercury, were found in the bottom sediments, particularly those in areas where on-shore industrial activities are intense, , including the waters off Tsuen Wan, North West Kowloon and Kowloon Bay. For example, there are more than 150 factories in the industrial areas of Tsuen Wan, Tsing Yi and Kwai Chung, so that it is not difficult to realize the severity of pollution arising from the large amount of industrial effluents produced. The contents of chromium, zinc and copper in sediments from Victoria Harbour were found to be 54-110
I-'g g-I, 150-240 I-'g g-1 and , 220-750 I-'g g-1 dry weight, respectively. These concentrations were higher than those found in some areas of the the united Kingdom where industrial pollution had been recognized. The general high levels of heavy metals may be due to the discharge of waste water arising from the textile, electroplating, electronic industries and the urban storm runoff which contains heavy metals from vehicle emissions. Copper content was particularly high. It could result mainly from the printed circuit board industry along the Harbour, but other industrial sources may also exist.
Tolo Harbour is another blackspot of heavy metal pollution. It connects to the open sea at Mirs Bay at the eastern side of Hong Kong. The rapid development of Shatin and Tai Po was identified to be the source of
24 deterioration of the water quality in Tolo Harbour (Wong et al., 1980). The effects of iron ore tailings on the coastal area of Tolo Harbour were studied by Wong et al. (1978, 1979, 1980). Higher heavy metal levels of copper, manganese and zinc were found in the sediments from the tailings area than those from two distant sites (Wong et al., 1978). In addition, the concentration of heavy metals found in the tissues of Scopimera intermedia was also high. Two marine algae, Chaetomorpha brychagona and Enteromorpha crinita from the iron ore tailings area also exhibited higher levels of heavy metals, including iron, manganese, lead and zinc, in their tissues than those from the distant sites (Wong et al., 1979). All these studies suggest that there was a local elevation in the bioavailable form of these metals . . These results were further supported by Wong et al. (1980), who showed that the Blue Snake River which passes through the iron ore tailings accumulated high contents of metal contaminants in the sediments as well as in the water. Chu et al. (1990) determined heavy metal concnetrations in the sediments, the mussel, Perna viridis and the rock oyster, Saccostrea cucullata, sampled from Tolo Harbour during March and May in 1986. Higher concentrations of metals, including iron and zinc, were found in sediments from inner Tolo Harbour than sediments from the Tolo Channel. Lower concentrations of iron, copper, and cadmium in Perna
25 viridis occurred in May suggesting seasonal variations of these heavy metals in this mussel. The EPD (1991b) also studied the heavy metal pollution in Tolo Harbour. Relatively high contents of heavy metals were found in the bottom sediments from inner Tolo Harbour wh.ere industry is active in the catchment. For other coastal waters on the eastern side of Hong Kong, including the Southern Waters, Port Shelter and outer Tolo Harbour, the contents of heavy metals in the bottom sediments were lower than those found in Victoria Harbour during 1986 to 1991 (EPD, 1987-1991b).
Located at the estuary of the Shenzen River, Deep Bay also receives large amount of effluents from the north-western New Territories, the towns of Yuen Long, Fanling, Sheung Shui and the surrounding areas. Rivers and streams in the catchment were heavily polluted the EPD (1991a). Levels of heavy metals were determined in the Pacif ic oysters, Crassostrea gigas, a common seafood cultured in Deep Bay (Wong et al., 1981). The results indicated a high content of cadmium in various parts of the oyster. Phillips (1982) also showed that cadmium and mercury in Crassoetrea gigas were clearly enriched, at concentrations of 0.14-5.4 and 0.01-1.26 p,g g-1 dry weight , respectively . Relatively high contents of heavy metals were also found in the bottom sediments of inner Deep Bay (EPD, 1987-1991b). Since Deep Bay is
26 commercially important for oyster productioni the high level of cadmium found there is considered to be a significant potential human health hazard in Hong Kong (Wu, 1988).
For other coastal areas of Hong Kong, the contents of heavy metals in the bottom sediments of the waters off various regions, including Sha Tin, Tai Po, Kwun Tong, Tsuen Wan, Sai Ying Pun and Pillar Point had increased in 1987 when compared to those in 1986 (EPD,
1987; 1988). The waters with the greatest percentage increase in heavy metal contents were those off Kwun
Tong, Tsuen Wan and Pillar Point, indicating the
increasing industrial activities around these areas.
Recently, Phillips (1989) claimed that the coastal waters of Hong Kong exhibit significantly elevated concentrations of heavy metals on both local and regional scales.
2.3 Copper: its nature and occurrence
Copper is a 3-d orbital filled heavy metal. It is
one of the members of the 3-d family which is made up of scandium, titanium, vanadium, chromium, manganese, iron,
cobalt, nickel and zinc (Partington, 1965; Moeller et
al., 1984). Copper has an electronic configuration of 3d104s1, with some important physical characters as
27 follows: atomic number, 29; atomic mass, 63.546; common
oxidation state ,. +1 and +2 i relati ve density, 8. 94 i
boiling point, 2310°Cj' and melting point, 10830 C
(Partington, 1965). As a typical metal, it is ductile, malleable, a good conductor of heat and electricity, ~nd has metallic lust er (Moeller et al., 1984).
Copper is widely distributed in nature. In the primary rocks of the earth's crust, copper is invariably restricted to sulphides (Minear and Keith, 1982). The commonest ores of copper are copper pyrites, CuFeS2 and bornite, Cu3FeS3 • Other forms of ores such as malachite,
CUCo3 ·Cu(OH)2' chrysocolla, CuSi03 .2H20 and atacamite, CuCl2.3Cu(OH)2' can also be found (Partington, 1965).
It is well known that copper is an essential element for normal growth and development in all organisms. According to Partington (1965} and Moeller et al. (1984), copper is an essential component of several enzymes, including catalase, peroxidase, tyrosinase, cytochrome oxidase, superoxide dismutase, amine oxidases and uricase. It also plays an important physiological role in . both animals and plants. For example, it can be found in the haemocyanin of most crustaceans and is important in the mobilization of iron and formation of haemoglobin in man. In addition, it is also essential for the biosynthesis of chlorophyll.
28 Lower organisms are very sensitive to copper salts and traces are . added to drinking water to destroy bacilli and algae (Hill, 1979). In surface waters, copper is toxic to algae at concentrations below 1.0 mg 14 and has frequently been used in the form of sulphate to control growth of algae in water supply reservoirs (Engel and Sunda, 1981). Medicinally, copper sulphate is used as an emetic. It has also been used for its astringent and caustic action and as an anthelmintic. Copper sulphate mixed with lime has been used as fungicide (Hill, 1979). Furthermore, copper is also of common use in our daily life, including in electrical applications, ornaments, jewellery, tableware, pipes, plumbing, gutters, industrial machinery and coinage (Moeller et al., 1984).
Copper, like other metals, enters aquatic environment from a variety of sources. The rocks and soils directly exposed to surface waters are usually the largest natural source. The amount of copper varies with the rock type and mineral content. Decomposing organic matter and the atmospheric fallout of particulates derived from volcanic eruption, erosion and forest fires also contribute small amounts of this metal to adjacent waters. Another principal source is anthropogenic in nature, including the direct discharge of various treated or untreated agricultural, municipal,
29 'residential or industrial effluents. Activities such as the combustion of fossil fuels, the processing of metals, construction, mining and lumbering can introduce large quantities of various heavy metals to the aquatic environment. It has been estimated that much of the copper in the aquatic environment is associated with anthropogenic inputs such as corrosion of copper tubing, algicides, insecticides, fungicides, fertilizers, textile, pigmentation, tanning, electroplating, smelting, engraving, mining, and many other industrial sources (Hill, 1979; Wong et al., 1980).
Heavy metals can exist in a variety of different chemical forms in natural waters including free ions, inorganic complexes, organic complexes, and metal adsorbed on or incorporated into particulate matter
(Engel and Sunda, 1981). According to Minear and Keith (1982), copper occurs in the +1,+2, and +3 oxidation states in aquatic systems. The stable oxidation state of copper is +2. Copper (I) readily disproportionates in water to form Cu++ and Cuo. Only small amounts of Cu+ can exist unless it forms stable complexes such as
Cu(NH3 )2+ and cu(CN)l-. Copper (Ill) is known to occur in K CuF and KCU02. cuFl- complex has also been reported 3 6 (Minear and Keith, 1982).
30 Due to the presence ·of the d orbital, copper(II) ion has a great . tendency to form both cationic and anionic complexes (Moeller et al., 1984). Copper (II) hydrolyses to form the mononuclear species CuOH+,
CU(OH)3-' CU(OH)42-, and the polynuclear complex CU2(OH)2~+.
It also forms soluble complexes with ammonia, disulphide, borate, carbonate, chloride, glycine, and histidine. other copper (II) complex ions are CuCI + ,
CU[B(OH)], CU(HS)33-, CUS(Hs)l-, Cu(glycine)2'
Cu(histidine)+, and Cu(histidine)2 (Minear and Keith,
1982).
According to Mance (1987), copper is toxic when its concentration is high. It is believed that the bioavailability and toxicity of copper to aquatic organisms are · dependent on the free metal ion concentration in water (Borgmann, 1983; Ahsanullah and
Florence, 1984; Blust, 1988). Free hydrated cupric ion has been shown to be the most toxic physico-chemical form of copper to aquatic organisms (Sunda and Lewis,
1978; Fowler et al., 1981; Borgmann, 1983; Ahsanullah and Florence, 1984; Stauber and Florence, 1987; Blust,
1988; Sunda, 1989). In addition, the concentration of the free cupric ion is determined by the total dissolved copper concentration as well as the concentration and nature of both organic and inorganic ligands present in solution (Sunda and Lewis, 1978; Engel and Sunda, 1981;
31 Borgmann, 1983; Ahsanullah and Florence, 1984; Blust, 1988). Associated with complex formation, chelation is of tremendous value in altering the properties of the metal ion by complex formation (Moeller et al., 1984). Experimental evidence showed that chelation lowers copper toxicity by lowering the concentration of the free ions (Lawrence et al., 1981; Ahsanullah and Florence, 1984). Naturally, copper is heavily chelated by organic ligands in seawater, and its biological availability and thus toxicity depend very much on the interactions of both biotic and abiotic parameters of the seawater (Sunda and Lewis, 1978; Blust, 1988; Sunda, 1989).
2.4 General bioloqy of Metapenaeus ensis
Metapenaeus ensis is a member of the family Penaeidae which is widely distributed throughout the world in tropical and subtropical seas. According to Yu and Chan (1986), Metapenaeus ensis is recorded from Sri Lanka, Malay Archipelago to south-east China, Hong Kong, Taiwan, Japan, to New Guinea and Australia. It is found at 8-64 m depth of sandy and muddy bottoms, and may penetrate to deeper waters up to 100 m or more. Adults are found in ocean and usually in shallow waters above 50 meters, while juveniles are found along the coast of
southern China. Metapenaeus ensis is a commercially
32 important shrimp in Southeast Asia. It is cultured extensively in many " Asian countries, including Taiwan,
Philippines, Thailand, Malaysia and Hong Kong.
According to Yu and Chan (1986), Metapenaeus ensis has a narrow rostrum with apical portion turned slightly upward. There are 6-10 dorsal rostral teeth. It is heterosexual. The petasma is large and the apical portion appears as a triangular process. The thelycum is rod like. Its body colour varies with size.
Individuals smaller than 6 cm are grey-green to dark green, whereas those larger than 6 cm are pale brown yellow and covered with dense minute pale dark brown dots. The antennal flagellum of the shrimp is red. The dorsal carina on abdomen is blackish or grey. Pleopods and tips of uropods are red. Adults exceeding 9 cm are pale grey blue, with pereiopods whitish with red bands, and tips of uropods blue and red.
The local life history of Metapenaeus ensis was first studied by Cheung (1964) in 1958-1960. Metapenaeus ensis occurred in all the areas sampled within the Hong Kong territorial waters. The spawning season is from March to October. Its distribution in
Hong Kong waters suggests that its migratory pattern is similar to other penaeid shrimp, i.e., the general off shore spawning and in-shore nursery habits.
33 According to the records from World Wide Fund For Nature (1991), various shrimp species, including Penaeus monodon, P. merguiensis" P. penicillatus, Metapenaeus ensis, M. affinis, M. burkenroadi, Ma crobranchi um nipponense and Palaemon orientalis, can be found in ~he Mai Po gei wais, which occupy the landward section of the mangrove marshes. All are grouped together as "gei wai ha". Of these, the most important one is Metapenaeus ensis. The fishmen call it "sand shrimp". Juveniles of Metapenaeus ensis are trapped as they come into the marshes to feed. During the day, they hide in the mud at the bottom of the pond. They are euryhaline (7-35°/00) and adapt to a wide range of temperature (10- 30°C). These shrimp mature to marketable size inside gei wais. The mature size is about 12-18 cm body length with body weight of 9-15 grams.
In Hong Kong, the maximum life span of Metapenaeus ensis was estimated to be 15-20 months in local waters (Cheung, 1964; Chan, 1984). From laboratory studies by Chan (1984), the spawning time lies between 2100 to 0300h in the morning. The temperature range of egg hatching is 14-36°C. The optimum salinity for spawning and hatching is 30°/00. Chu and So (1987) also showed that salinities above 20°/00 are required for hatching of eggs and early larval development of Metapenaeus ensis.
34 Further, there is an increase in low salinity tolerance as the larvae age.
Penaeid shrimp exhibit the most complicated larval development among decapod crustaceans (Williamson, 1982). Starting from non-feeding nauplius, the larvae develop to protozoeae and myses. Each of these larval stages, in turn, consists of a number of instars. Ong
(1969) gave the first description of the rearing procedures and the morphology of larval stages of Metapenaeus ensis. Four naupliar, three protozoeal and three mysid stages were reported. Later, Tseng et al.
(1979) made an account on the artificial propagation and cUltivation of Metapenaeus ensis from eggs, with records of the general morphology of the larval stages. According to Tseng et al. (1979), the larval stages of Metapenaeus ensis include six naupliar stages (NI-NVI), three protozoeal (PZI-PZIII) and three mysid (MI-MIII) stages. The myses then metamorphose to postlarvae (PL) which become benthic on the fifth day of development (PL5) . Larval development and morphological details of these larval stages up to postlarva I were further described by Leong (1991).
The diet of the larvae of most penaeid species in their natural habitat is unknown (Chan, 1984). General practice in the shrimp hatcheries is to use the
35 microalgae for the protozoeae, followed by the addition of Artemia nauplii for the late mysid and early postlarval stages (AQUACOP, 1983). Chu and Shing (1986) found that Metapenaeus ensis myses ingest little
Artemia nauplii possibly because of their smaller s~ze when compared to that of other penaeids. They also suggested that the shift from herbivorous to omnivorous or carnivorous diet in Metapenaeus ensis takes place at early postlarval stages instead of during mysid stage. Therefore the addition of Artemia nauplii is unnecessary for rearing the myses of Metapenaeus ensis. Leong (1991) studied the relationships between the morphological and the dietary changes during larval development of Metapenaeus ensis. Predation on Artemia nauplii increased rapidly during mysid stage. This result suggests that the presence of appropriate appendages for catching prey in mysid stage may be the critical factor in determining the diet rather than the size factor mentioned by Chu and Shing (1986). In addition, a gradual shift from herbivory to omnivory occur during mysid development rather than in early postlarval stages.
simon (1978) and Kuban et al. (1985) demonstrated that the diatom Chaetoceros gracilis, is an efficient diet for the protozoea of many penaeids. It was found that Chaetoceros gracilis is also an adequate diet for
36 rearing Metapenaeus ensis larvae (Chu, 1989; Chu and Lui, 1990). The use of two artificial diets alone, namely, the microencapsulated diet from the Centre Oceanologique du Pacifique, Tahiti, and Artificial Plankton B. P. (Nippai Shrimp Feed Inc., Japan), for rearing Metapenaeus ensis larvae resulted in a decrease in larval survival and retarded development (Chu and Lui, 1990; Chu, 1991). From these studies, they suggested that the use of artificial diets as the exclusive feed for rearing Metapenaeus ensis needs further investigations.
Ovsianico-Koulikowsky (1987) studied the physiological and biochemical changes during growth and development in Metapenaeus ensis. Analysis of the O:N ratio suggests that nauplius V/VI stages utilized protein in a great demand. Dietary carbohydrate or lipid were metabolized in later developmental stages. These results are in good agreement with the facts that nauplii live on the yolk while later stages feed on phytoplankton or zooplankton. An increasing weight specific rate of oxygen consumption also suggested an
increasing energy demand from protozoeal to early
postlarval stages. This agrees well with the change from herbivorous to omnivorous or carnivorous feeding habits through these stages. The reduction in oxygen . consumption in later postlarval stage was suggested to
37 be associated with the shift from planktonic to benthic mode of life.
In recent years, pollution and overexploitation have led to rapid decline of natural penaeid shrimp populations in many areas of Southeast Asia. According to the records from World Wide Fund For Nature (1991), the severe pollution in Deep Bay has reduced productivity of shrimp ponds in Mai Po marshes. Not only the pollutants kill the larval and juvenile shrimp in their natural Deep Bay habitats, but also cause a reduction in the number of days of harvest per spring tide cycle.
Wong et al. (in press) studied the toxicities of three heavy metals, including copper, chromium, and nickel, to the survival and feeding behaviour of the early developmental stages of Metapenaeus ensis. Copper was the most toxic among the three in reducing the larval survival. In addition, there was an increase in tolerance to the heavy metals with age. Both the grazing and predation rates were reported to be lowered after exposure to heavy metals.
38 CHAPrER3
ACUTE AND CHRONIC TOXICITY OF
COPPER TO METAPENAEUS ENSIS
3.1 Introduction
This Chapter presents the acute and chronic toxicity of copper to different developmental stages of Metapenaeus ensis, in respect to their survival, growth and development.
3.2 Materials and Methods
3.2.1 Spawners and larval rearing
Gravid Metapenaeus ensis females were obtained from local fish markets between April and October, 1991, and transported to the laboratory. The spawners were held individually in 500-1 indoor fiberglass tanks. These tanks were filled with 300 1 of seawater from Tolo Harbour. Disodium salt of ethylene-diamino tetra-acetic acid (EDTA) at 20 mg I-I was added to each tank for chelating heavy metals in the seawater. The seawater was well aerated to prevent the sinking of eggs. A
39 wooden board was used to cover the tank to avoid disturbance to the animals. Spawning usually occurred at early morning of the , following day. Spawners were removed from the tank in the next morning after spawning in order to prevent a second spawning so as to ensure that larvae were from a single spawning. Eggs usually hatched during the morning at about 0700 to 1100 h.
Larvae were inspected daily and the stages were identified according to Tseng et al. (1979).
Metapenaeus ensis has six naupliar (NI-VI), three protozoeal (PZI-III) and three mysid (MI-III) stages.
Larvae from PZI to MI were fed with cultured Chaetoceros
4 gracilis at a density of 5 x 10 to 1 x 105 cells ml-I , supplemented daily with 2 mg I-I of Artificial Plankton
B.P. (Nippai Shrimp Feed Inc., Japan). From MII onwards, freshly hatched Artemia nauplii were added at excess concentration together with Mixed Feed for
Penaeus monodon (Higashimaru Foods Inc., Japan) at 4 mg
1-1 daily. From MI onwards, about half of the water in the rearing tank was changed every 2 days. During the rearing period, the water temperature was 27-29°C and
salinity was 30-34°/00 •. Day-4 postlarvae (PL4) were transferred into outdoor tanks for further development.
Temperature in outdoor tanks was found to be 28-35°C during the day. The pH of seawater is quite stable and always found to be 7.9. 40 Under these culture conditions, the duration of the entire protozoeal. and mysid stages was 3-4 and 6-7 days, respectively. Larval development was completed to day-1 postlarvae (PL1) in 10 to 12 days from fertilized eggs. Postlarvae became benthic at about day 5-6. Larvae of PZI and MI, postlarvae of 1 and 4 days, as well as shrimp at about day 15-20 of postlarval development were taken for experiments.
3.2.2 Culturing of Chaetoceros gracilis
stock culture of Chaetoceros gracilis was obtained from the Centre Oceanologique du Pacifique, Tahiti, French Polynesia. It was maintained in Walne medium (Walne, 1966) as described in AQUACOP (1983). Algae for rearing shrimp were grown in 500-1 fiberglass tanks in seawater enriched with KN03 (100 mg 1-1), KH2P04 (10 mg 1-1), and Na2Si03 (5 mg 1-1). Algae grown in 5-1 conical flask at a cell concentration of about 6 x 106 cells ml~ was then added.
Chaetoceros gracilis used in laboratory experiments was grown in Walne medium in 0.5 to 5-1 conical flasks under fluorescent light. The seawater used was artificial seawater prepared from Instant Ocean Synthetic Sea Salts. Growth was monitored by making cell counts under the microscope with a haemocytometer.
41 cultures at active growth phase were used to feed the larvae in experiments.
3.2.3 Acute toxicity tests
The acute toxicity tests aimed at the determination of 48-h or 96-h LCso values for the stages PZI, MI, PL1,
PL4 and PL15-20. stock solution of copper of 500 mg l~ was prepared from hydrated salts of copper (II) sulphate
(CuS04 ·5H20) and stored at 4°C. Toxicity of copper was tested in 20-~m filtered artificial seawater prepared from Instant Ocean Synthetic Sea Salts. The experiment was carried out at 26-28°C with salinity of 30°/00 and pH
7.9-8.1. Individuals from a single spawning were used in each experiment. All individuals tested were preconditioned for at least 4 h in Instant Ocean with appropriate food (see below). After 4 h, fifty individuals were transferred to each experimental vessel. For experiments on PZI, MI, PL1 and PL4, 500-ml cone-shaped vessels were used. For experiments on PL15-
20, 500-ml rectangular plastic tanks were used. Five to
7 different copper concentrations, each in duplicate, were tested in each experiment. Shrimp in seawater without the addition of copper, in duplicate, served as control. The food provided for PZI and MI was
Artificial Plankton B.P. at 2 mg I-I and Chaetoceros gracilis at 104 cells ml-I daily. Mixed Feed for Penaeus
42 monodon at 3 mg 1-1 and 4 mg 1-1 daily were provided for
PL1, PL4 and PL15-20, respectively. Half of the seawater was renewed every 24 h. Copper and food were replenished. After 48 h of exposure, mortality was checked. Death was defined as no signs of life wnen stimulated with a copper wire under the microscope. Mortality of PL15-20 was also checked after 96 h. In experiments with PZI and MI, the stage of larval development of each survivor was also noted. The percentage mortality was then used in probit analysis
(Finney, 1971) for the estimation of the median lethal concentration (LC~). Experimental data with controls which had a mean percentage mortality of greater than 20% were excluded for analysis.
3.2.4 Chronic toxicity tests
Larval development from PZI to PL1 in the presence of copper was studied. After 4 h preconditioning in
Instant Ocean, 100 PZI were transferred to each 1000-ml cone-shaped vessels. Five different copper concentrations, each in duplicate, were tested in each experiment. Shrimp in seawater without the addition of copper, in duplicate, served as control. Food was provided as those used in acute toxicity tests except from MII onwards, Artificial Plankton B.P. at 2 mg I-I and fleshly hatched Artemia at 5 nauplii ml-I daily were
43 provided. Mortality was determined every two days. The stage of development in 10 randomly sampled larvae was determined and half of the seawater was renewed. Copper and food were replenished. Growth indexes of 1 to 7 were assigned to the developmental stages PZI, PZlI,
PZIII, MI, MII, MIll and PL1, respectively. An averaged growth index for the 10 larvae was then calculated
(Teshima and Kanazawa, 1983). No growth index was calculated in cases with less than 10 larvae left in a given time. The body length (from postorbital margin of the carapace to the end of telson) of PL1 was also measured.
The survival and growth of PL15-20 after chronic exposure to copper were also tested. The mean body length of the shr imp reared in outdoor tanks were determined in 30 randomly sampled individuals. One hundred individuals were transferred to each 1000-ml rectangular plastic tank. Three different copper concentrations, each in duplicate, were tested in each experiment. Shrimp in seawater without the addition of copper, in duplicate, served as control. Food as those used in acute toxicity test was provided. Mortality was monitored and half of the seawater was renewed every two days. Copper and food were replenished. After 8 days, survivors were preserved in 5% formaldehyde and their
44 body length were measured under microscope. The percentage increase in body length was calculated.
3.3 Results
3.3.1 Acute toxicity tests
Mortality of PZI, MI, PL1 and PL4 in different copper concentrations from three different spawners are
shown in Figs. 3.1, 3.2, 3.3 and 3.4, respectively.
Mortality of PL15-20 from two other spawners is shown in
Fig. 3.5. The mortality of control shrimp was also indicated.
Mortality of PZI at 0.24 mg 1-1 of copper was 100%
(Fig. 3.1). From MI onwards, great variability in the mortality occurred among individuals from different
spawners (Figs. 3.2-3.4). Copper concentration at or
above 2.4 mg 1-1 used in experiments on PL4 resulted in
the precipitation of copper salts during the
experimental period. These data were excluded from
subsequent analysis. The percentage mortality for PZI, MI, PL1 and PL4 was subjected to two-way analysis of variance after arc-sin transformation with spawners and copper concentrations as the main effects. The results suggested that both parameters significantly (p < 0.05)
45 affect mortality rate. Significant interaction (p < 0.05) of these two factors occurred in the cases of PL1 and PL4.
Mortality data were transformed into probit sc~le
(Finney, 1971) which may have linear relationship with the logarithm of concentration. It has been suggested that Goodness-of-fit Test is important to determine whether the log-probit line models the data before the line can be used to estimate the LCso value (Hubert, 1980; Buikema et al., 1982). The log-probit line was thus subjected to test for Goodness-of-fit. Data beyond the lowest copper concentration where 100% mortality was observed were not analysed. Data points that showed to be outliers (one-way ANOVA) were also excluded in the estimation of LCso • Exclusion of 1 experimental value occurred in two cases of MI and PL1 from one of the spawners. The fitted probit lines with equations for all stages tested were shown in Figs. 3.6-3.10. The estimated LC~ values were summarized in Table 3.1. The
48-h LCso values for stages PZI, MI, PL1, PL4 and PL15-20 varied among different spawners. Trends of increase in
LCso values during development were quite consistent for animals from a single spawner indicating an increase in the tolerance to copper during development. The 48-h
LC~ of PL15-20 were much higher than those of larvae and postlarvae. The higher 96-h LCso than 48-h LCso in the 46 juvenile shrimp indicated that toxicity increased with exposure time.
stages of development of each survivor were noted in experiments with PZI and MI. For experiment on PZI, larvae were either moult to PZII or PZIII, the percentage of PZIII among survivors was shown in Fig. 3.11. For experiment on MI, larvae were either moult to MII or remained as MI, the percentage of MII among survivors was shown in Fig. 3.12. Trends of progressive decrease in these percentages with increasing copper concentration was apparent in almost all cases. In PZI, percentage of PZIII among survivors dropped to below 50%
at concentrations above 0.12 mg 1-1 (Fig. 3.11).
3.3.2 Chronic toxicity tests
The percentage survival of shrimp under chronic exposure to copper is shown in Fig. 3.13. Only the data on two of the copper concentrations tested were shown for illustration purpose. compared with the control, shrimp exposed to copper, had a lower percentage survival during the experimental period. The growth index of the control shrimp increased from day 0 towards the end of the experiment (Fig. 3.14). Growth index of shrimp exposed to copper never exceeded that of the control
47 indicating a decrease in the developmental rate in larvae exposed to copper. The body length of PLl from two spawners was shown in Table 3.2. No significant differences from the control were found.
The result of chronic exposure of PL1S-20 to copper is shown in Fig. 3 .1S. The percentage survival of shrimp exposed to 0.4 mg eu 1-1 dropped to 20 to 30% at day 8, while that of control dropped to about 70%. The percentage increase in body length was shown in Fig.
3.16. One-way analysis of variance after arc-sin transformation indicated a significant difference (p < O.OS) in the percentage increase in body length among different groups. Further analysis with Tukey's test showed that the percentage increase in the body length of shrimp exposed to copper above 0.8 mg 1-1 was signif icantly different (p < o. OS) from that of the control except in one case at a copper concentration of
1.0 mg 1-1 where no significant difference was found.
48 Fig. 3.1: Percentage mortality of protozoea I exposed to different concentrations of copper for 48 h. Results for larvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
49 100 a 0 0 0 0
'80 0
60 0 § 40 0
20 § 0 § 0
0.00 0.08 0.16 0.24 0.32
100 b 8 0 0 0
80
60 0 0 40 § ...... -4• ?-"4 0 cd o 0 ~ 20 0 H 8 0 0 :;s 0 0.00 0.08 0.16 0 .24 0.32 ~
100 C © 0 0 0
80
60 0 0 40 0 0 20 o § © 0
0.00 0.08 0 . 16 0 .24 0.32 Copper concentration (mg 1-1) Fig. 3.2: Percentage mortality of mysis I exposed to different concentrations of copper for 48 h .. Results for larvae from spawners 1, 2 and 3 are presented in
(a), (b) and (c) respectively.
50
Fig. 3.3: Percentage mortality of day-1 postlarvae exposed to different concentrations of copper for 48 h. Results for postlarvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
51 100 a o 0 o 0 80 © 60 8 § 0 § 0 40 0 20 8 0
0
0.0 0.4 0.8 1 .2 1 .6
100 b 0 0 0 80 0 0 60 0 o 0 ~ ~ 40 .~ ~ © 0 ro 0 ~ 20 0 H 0 0 0 0 0 ~ 0.0 0.4 0.8 1 .2 1 .6 ~ 100 C 0 o 0 0 0 80 0 8 0 0 60 0 40
20 0
0 0 8 0.0 0.4 0.8 1.2 1.6 Copper concentration (rug 1-1) Fig. 3.4: Percentage mortality of day-4 postlarvae exposed to different concentrations of copper for 48 h. Results for postlarvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
52 100 a 0 80 0 0 0 0 0 0 0 60 0 8 0 40 0 0 20 0 8 0 b
0.0 0.8 1 .6 2.4 3.2
100 b 0 0 80 o 0 0 © 8 0 0 60 0 0 0 ~ ~ .~ ...-4 40 cd ~ 8 ~ 20 0 ~ 0 ~ 0.0 0.8 1 .6 2.4 3.2
100 C 0 0 80 0 0 0 60 0 © © 40 © © 0 20 0 0 8 0.0 0.8 1 .6 2.4 3.2 Copper concentration (mg I-I) Fig. 3.5: Percentage mortality of day 15-20 postlarvae exposed to different concentrations of copper for 48 and 96 h. Results for postlarvae from spawners
4 and 5 are presented in (a) and (b) respectively. 48 h (closed circle) and 96 h (open circle).
53 80 a 0 0 0 0 '@ o .8 60 0 0 0 I 40 o I I • • I 20 • 0 • • 0 0 I
~ 0.0 0.6 1.2 1 .8 2.4 ~ .~ ~ ~ ~ ~ 0 ~ 80 b o 0 • ~ 60 o i 0 • 0 0 40 0 • 0 • • • 20 • 0 • • • 0 I I
0.0 0.·6 1.2 1.8 2.4
Copper concelltration (mg 1-1) Fig. 3.6: Relationship between mortality in probit scale and log copper concentration for protozoea I exposed to different concentrations of copper for 48 h. Results for larvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
54 8 a 0
7 ' 0
6 - y== 11.4+6.57X 5 0 0 0 4
3
2
-1.4 -1.2 -1.0 -0.8 -0.6
~ ~ .~ 00 ~ 8 b ro ~ ~ 7 0
e 6 y== lO.3+5.26X ~ 0 5 Q) 0 0 0 ~ro 4 0 0 V'J 3 ~ .~ ..D -1.4 -1.2 -1.0 -0.8 -0.6 0 ~ ~ 8 C 0 7
6
5
A Lt ·
3 .
2
-1.4 -1.2 -1.0 -0.8 - -0.6
I Log copper concentration (mg 1-1) Fig. 3. 7: Relationship between mortality in probit scale and log copper concentration for mysis I exposed to different concentrations of copper for 48 h. Results for larvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
55 ·0 6.0 a o
5.5 Y==5.59+0.86X o 5.0
o 4.5 -0.9 -0.6 -0.3 0.0 0.3
~ 7.0 0 CD ~ b ...... c.~ 0 ro 6:5 0 ~ ~ Y==6.49+1.60X 0 6.0 S 0 ~ 5.5 0 0 0 Q) 5.0 ...... c ro 0 () 4.5 0 (JJ
~ .~ -0.9 -0.6 -0.3 0.0 0.3 ..0 0 ~ ~ 8 C 0
7
"6
5
4
-0.9 -0.6 -0.3 0.0 0.3
Log copper concentration (mg 1-1) Fig. 3.8: Relationship between mortality in probit scale and log copper concentration for day-1 postlarvae exposed to different concentrations of copper for 48 h. Results for postlarvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
56 0 6.5 a 0
6.0
5.5 Y=5.73+2.07X
5.0
4.5
4 .0
-0.8 -0.6 -0.4 -0.2 0.0 0.2
~ 0 ~ 6.0 b .~ 0 ...... c C\:$ ~;...... ,0 5.5 S ~ 0 5.0 (l) ...... c C\:$ 0 () en 4.5
~ .~ -0.8 -0.6 -0.4 -0.2 0.0 0.2 ..0 0 ;...... ~ 8 C 00
7 Y=B.81+4.47X 6 0 0 5 0
0 4 0 -0.8 -0.6 -0.4 -0.2 0.0 0.2
Log copper concentration (mg I-I) Fig. 3.9: Relationship between mortality in probit scale and log copper concentration for day-4 postlarvae exposed to different concentrations of copper for 48 h. Results for postlarvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
57 o 5.5 a o
5.0 Y=4.88+1.77X
4.5 :
4.0 , . I -0.4 -0.2 0.0 0.2 0 .4
6.5 0 ~ b ~ .~ ~ro 6.0 0 ~;..... Y =5.43+ 1.46X 0 0 5.5 0 S 0 5.0 ~ 0 Q) 4.5 0 ~ 0 ~ () 4.0 V) -0.4 -0.2 0.0 0.2 0.4 ~ .~ .,.0 0 ;..... 5.5 C ~ 0 0
5.0 Y=4.71 + 1.65X
4.5 8
4.0
-0.4 -0.2 0.0 0.2 0.4
Log copper concentration (mg 1-1) Fig. 3.10: Relationship between mortality in probit scale and log copper concentration for day 15-20 post larvae exposed to different concentrations of copper for 48 h (closed circle) and 96 h (open circle). Results for postlarvae from spawners 4 and 5 are presented in (a) and (b) respectively.
58 6.0 a 0 Y=5.34+0.65X 5.~ §
5.0 ~ I ,...... • ~.....c 0 C\$ 4.5 • ~ • ~ • 0 4.0 • Y=4.56+0.66X S ~ -0.6 -0.4 -0.2 0.0 0.2 0.4 0 ,...... (]) C\$ () 6 b V'J
~ • .....c 5 ..C; 0 ~ 4 ~ • 3 Y=4.17+3.14X 2 • -0.6 -0.4 -0.2 0.0 0.2 0.4
Log copper concentration (mg I-I) Table 3. 1: LCso values estimated from the fitted regression lines for acute toxicity experiments
Spawner Tested Fitted line Goodness of LCso value stage Fit mg 1-1
1 PZI y= 11.4+6.57X 0.9 >p >0.1 0.11+0.01 MI y= 5.59+0.86X P >0.995 0.21+0.12 PL1 y= 5.73+2.07X P >0.995 0.44+0.08 PL4 y= 4.88+1.77X P >0.995 1.17+0.25
2 PZI y= 10.3+5.26X 0.9 >p >0.1 0.10+0.01 MI y= 6.49+1.60X P > 0.995 0.12+0.05 PL1 y= 5.64+1.71X 0.9 >p >0.1 0.42+0.05 PL4 y= 5.43+1.46X P > 0.95 0.51+0.23
3 PZI y= 11.1+6.38X 0.9 >p >0.1 0.11+0.01 MI y= 6.79+4.79X P > 0.975 0.42+0.04 PL1 y= 6.81+4.47X P > 0.995 0.39+0.04 PL4 y= 4.71+1.65X P > 0.995 1.50+0.40
4 PL15-20 48 h y= 4.56+0.66X p > 0.995 4.64+0.76
96 h
y= 5.34+0.65X p > 0.995 0.30+0.14
5 PL15-20 48 h y= 4.17+3.14X P > 0.995 1.84+0.51
96 h
y= 5.31+1.66X P > 0.995 0.65+0.15
Y represents mortality in probit scale (mg 1-1) X represents logarithm of copper concentration confident interval LCso values are mean + 95 %
59 Fig. 3.11: Percentage of protozoea III among survivors protozoea I exposed to different concentrations of copper for 48 h. Results for larvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
60 Copper concentration (mg 1-1) Fig. 3.12: Percentage of mysis 11 among survivors for mysis I exposed to different concentrations of copper for 48 h. Resul ts for larvae from spawners 1, 2 and 3 are presented in (a), (b) and (c) respectively.
61 100 a
80 0 60 0
40 0 § 0 0 20 0 8 8 0 0 0 0 0
0.0 0.3 0.6 0.9 1.2 1.5
rJJ ~ 0 :> b .~ 100 00 0 0 0 0 :> 0 0 ~ 80 0 0 ::::3 0 rJJ 0 bl) 60 ~ 0 40 S 0 cd 20
~ ~ 0 ~ 0.0 0.3 0.6 0.9 1 .2 1.5 ~ 0 C ~ 100 §O 0 0 80 © 0 60 0 40 0 0 2.0 0
0 0 o 0 0.0 0.3 0.6 0.9 1.2 1.5
Copper concentration (mg I-I) Fig. 3.13: Percentage survival of larvae upon chronic exposure to different copper concentrations. Control
(square) , 0.04 mg 1-1 (circle), O. 06 mg 1-1 (diamond),
0.08 mg 1-1 (triangle) and O. 10 mg 1-1 (inverted triangle).
Results for larvae from three spawners are presented in (a),
(b) and (c). Error bars are mean + range of duplicate.
62 b 100
80
60
~ ~ 40 • >....c > 20 ~ en~ 0 ~ 0 2 4 6 8 10 12
C 100
80
60
40
20
0
0 2 4 6 8 10 12 Day Fig. 3.14: Growth index of larvae upon chronic exposure to different copper concentrations. Control (square),
0.04 mg 1-1 (circle), 0.06 mg 1-1 (diamond), 0.08 mg 1-1
(triangle) and 0.10 mg 1-1 (inverted triangle). Results for larvae from three spawners are presented in (a), (b) and
(c). Error bars are mean + range of duplicate.
63
"'~:#.~. ..-J\!;iP' , '~ J~"~ ,:).'_. : (\r ~"- .~ .:. ~ . " o 4 6 8 10
b
1 o o 2 4 8 10 12
c
1 o 6 Day Fig. 3.15: Percentage survival of day 15-20 postlarvae upon chronic exposure to different copper concentrations. control (square), 0.04 mg 1-1 (circle), 0.08 mg 1-1
(diamond) and 0.10 mg 1-1 (inverted triangle) . Results for larvae from spawners 4 and 5 are presented in (a) and (b) respectively. Error bars are mean + range of duplicate.
64 100
80
o 2 4 6 10
b 100
o Day Fig. 3. 16: Percentage increase in body length of day 15- 20 postlarvae upon chronic exposure to different copper concentrations. Results for larvae from spawners 4 and 5 are presented in (a) and (b) respectively. Values are means and error bars represent standard error (n = 10-70). Data with an asterisk are significantly different (p < 0.05) from the control.
65 14 a 12
10
8
6
4
2
~ 0 "---__'_--L...-----L-J.....-----L----1.----'----L-- ~ 0.0 0.4 0.8 1.0 o ..0 .S 16 b 14 , 12 10 8 6 4 2
o ~__'_~~~~~~-L- 0.0 0.4 0.8 1.0
Copper concentration (mg 1-1) Table 3.2: Body length of day-1 postlarvae of Metapenaeus ensis upon chronic exposure to two copper concentrations
Control 0.04 mgCu 1-1 0.06 mgCu 1-1
Body length (1) 3.09+0.02 3.01+0.09 3.05+0.02 (mm) (2) 3.00+0.04 3.03+0.12 2.89+0.09
Values are mean + standard error (n = 5-10). Rows (1) and (2) represent data from different spawners.
66 Table 3.3: Toxicity. of copper to some crustacean species
Crustacean Life Duration Reference species stage (day)
Acartia clausi adult 4 0.05 USEPA (1980) Acartia tonsa adult 4 0.02-0.06 Sosnowski & Gentile (1978) Tigriopus japonicus adult 4 0.48 USEPA (1980) Tisbe holothuriae adult 2 0.40 Verriopoulos & Dimas (1988)
Eurylemora affinis adult 4 0.53 USEPA (1980) Mysidopsis bigelowi adult 4 0.14 USEPA (1980)
Mysidopsis bahia adult 4 0.18 USEPA (1980) Euphausia pacifica adult 1 0.01-0.03 Reeve (1976) Palaeomonetes pugio adult 4 35.9 curtis et al. (1979)
Crangon crangon larva 2 0.33 Connor (1972) adult 2 30
Homarus gammarus larva 2 0.1-0.3 Connor (1972) Homarus americanus larva 4 0.05 Johnson & Gentile (1979)
adult 4 0.10 McLeese (1974)
Cancer magister zoea 4 0.05+0.02 Martin et al. ( 1981)
Carcinus maenas larva 2 0.6 Connor (1972) adult 2 109
67 3.4 Discussion
Mance (1987) reported that the 24 to 96-h LCso of copper to marine organisms varied considerably with the species tested and the test conditions. Table 3. ~ summarized the LCso values for some crustaceans. In the present study, the 48-h LCsofor PZI and MI larvae ranged from 0.10 to 0.42 mg 1-1, and those for PL1 and PL4 were
0.39 to 1.50 mg 1-1. Comparison of 48-h LCso values with those in Table 3. 3 showed that PZI and MI were as sensitive to copper as larvae of Homarus gammarus and Crangon crangon as well as adults of the copepod Tisbe _holothuriae, whereas PL1 and PL4 were as sensitive to copper as larvae of Carcinus maenas. The 96-h LCso for PL15-20 was O. 3 to O. 65 mg 1-1 in the present study.
Comparison of the 96-h LCso suggested that PL15-20 was as sensitive to copper as the adults of Tigriopus japonicus and Eurylemora affinis.
Verriopoulos and Dimas (1988) suggested that different experimental conditions, such as temperature, water quality or food supply, might cause differences in the estimated LC~ values . . For example, three different
48-h LCso values have been reported for ovigenous females of the copepod, Tisbe holothuriae upon acute exposure to copper. These reported values were 0.08 mg l~
68 I •
(Apostolopoulou and Verriopoulos, 1982), 0.42 mq I-I
(Verriopoulos et al., 1987) and 0.37 mq I-I (Verriopoulos
and Dimas, 1988). Wong et. al. (in press) reported that
the 48-h LCso for PZIII, MIll and day-3 postlarvae were
0.16, 1.58 and 4.76 mq cu I-I respectively. Hiqher LCso values compared with the present study were reported. In addition to the fact that different spawners were used, two other reasons could account for this
discrepancy. The first one was differences in experimental conditions. In the study by Wonq et al.
(in press), the test medium used was HW-Marinemix with Bioelements and the pH of the test medium was 8. 7 • Many . studies on toxicity of heavy metals to orqanisms have shown that the free (hydrated) metal ion is the most toxic form (Lawrence et al., 1981; Florence, 1983; Ahsanullah and Florence, 1984). Different test media with different composition may possibly affect the speciation of heavy metal and thus its toxicity.
Furthermore, Sunda and Hanson (1979) found that the complexation of copper by natural orqanic liqands increases with pH. Zuehlke and Kester (1983) reported that the chemical speciation of copper in salt solution is highly pH-dependent. At low pH, the cupric ion is the dominant inorganic species, but with increasing alkaline conditions its concentration declines quickly with carbonate and hydroxide species become the prevalent inorganic forms. Shaner and Knight (1985)
69 also concluded that carbonate complexation reduces copper toxicity. It was also suggested that pH not only alters the chemical speciation of heavy metals, but also charges the binding sites of heavy metals by protonation and thus affects the sensitivity of the organism. The second reason to account for the differences in LCso reported in the two studies was the method involved in estimating the LCso values. Wong et al. (in press) reported that the mortality in the control animals ranged from 12-31% for PZIII, 5-40% for MIll and 7-29% for PL3. The mortality in individuals exposed to metals was corrected for the mortality in control. In this way, the toxicity of copper might be under-estimated, giving rise to higher LCso values. In the present study, there was no adjustment of the mortality data for the control, so that LCso might be under-estimated, and consequently, the toxic effects of copper might be over estimated.
Buikema et al. (1982) stated that control was an integral part of any toxicity test. Controls were necessary in determining whether the death of organism was due to the toxicant or some other factors. Toxici ty test was suggested to be valid when the control mortality was less than 10%. However, why 10% mortality is chosen as the critical level is not clear. Large number of deaths in larvae was common under laboratory
70 rearing conditions (Chu, 1991). Preliminary tests with larvae and postlarvae of Metapenaeus ensis showed that 20% individuals might die .within 48 h for no apparent reason under the experimental conditions. Thus, in the present study a higher mortality of 20% was allowed for the control.
Results of two-way analysis of variance with copper concentration and spawner as the main factors indicated that mortality was a function of copper concentration. The test results also suggested that individuals of the same stage from different spawners showed significant differences in their sensitivities to copper toxicity. Spawners used in this study were acquired from local markets. The variability may stem from some inheritable factors of the spawners. Moreover, spawners from waters which are polluted with heavy metals might possibly gave birth to larvae more tolerant to the metals.
The present study also demonstrated that there was a trend of increase in tolerance to copper through development of Metapenaeus ensis. Wong et al. (in press) also reported that Metapenaeus ensis showed an increase in tolerance to heavy metals including copper, nickel and chromium during development from protozoeae to postlarvae. Increase in tolerance to copper with age in crustaceans has also been reported by many authors
71 (see Chapter 2). It has also been well documented that young life stages are considerably more sensitive than adults to pollutants other than heavy metals. For example, Penaeus monodon showed a progressive increase in tolerance to ammonia (Chin and Chen, 1987) and nitrate (Chen and Chin, 1988) as the shrimp developed from nauplii to postlarvae. This phenomenon might be explained by the suggestion that the mechanisms of detoxification present in adults may not be well developed in the earlier developmental stages (Bernard and Lane, 1961). Thus, the regulation of metals is less effective in the young stages (Bryan and Hummerstone, 1973).
In the present study, the decrease in the percentage of PZIII and MlI larvae in experiments on acute toxicity and the decrease in the growth index of larvae under chronic exposure showed that the developmental rate of larvae retarded in the presence of copper. Exposure to copper also resulted in retarded growth in PL15-20. Decrease in the rate of larval development, indicates that the planktonic phase of the life cycle is lengthened, which may adversely affects larval survival and thus the recruitment of the animal in the natural environment.
72 Buchanan et al. (1970) reported that the moult cycle duration of larval Cancer magister was lengthened
during exposure to the insecticide Sevin. Retarded growth has also been reported in the copepods,
Trigriopus sp. (D'Agostino and Finney, 1974) an~ Eurytemora affinise (Sullivan et al., 1983) and in the shrimp, Macrobrachium rosenbergii (Liao and Hsieh, 1990)
upon exposure to copper. Enzymatic disturbances caused
by heavy metals were reported in other crustaceans
(Gould, 1980; Amiard et al., 1982, cited in Gaudy et
al., 1991; Berglind, 1985). Further, sublethal metabolic disturbances would possibly be manifested by
a relative reduction in growth rate. From the results
of the present study, it was difficult to interpret the
results until the cellular mechanism of copper toxicity can be elucidated.
In the present study, all toxicity tests are static
in nature, but with renewal of media. This method has
the advantages of being simple, inexpensive, rapid and
requiring minimum space. However, the nature of the
test medium and thus the concentrations of copper may
change over the experimental period. Thus, the toxicity
values reported in this study were probably conservative
due to the nature of the test. In this study, the
background levels of the heavy metals in the test medium were unknown. Heavy metals may also be present in the
73 diets provided. In addition, the bioavailability and toxicity of copper are dependent on the free hydrated cupric ion concentration. which is affected by many factors present in seawater such as salinity and pH.
Due to the nature of being easily adsorbed ~y particulates or even to the glass, the concentration of copper in the test medium may be much lower than the nominal values. Therefore, the toxicity of copper may be under-estimated.
Metapenaeus ensis is one of the major species in the shrimp fishery of the Zhujiang River estuary. It is also cultured in Gei wais located in the Mai Po Marshes of Hong Kong (see Chapter 2). Due to the widespread use of copper, estuaries with industrial and municipal pollution may exhibit particularly high localized copper concentrations. In addition, particularly high copper concentration was observed in Hong Kong waters relative to other heavy metals (see Chapter 2) . The concentration of copper in the coastal area of Hong Kong has been reported in form of its concentration in seawater (Chan et al., 1974), in sediments (Ho et al.,
1991) and in organisms (Phillips, 1979; Phillips and
Yim, 1981; Ho, 1987). Most of these data were reported in form of copper concentrations in sediments and in organisms. It is diff icul t to relate the biological effects produced in the laboratory to the concentration
74 of copper in the natural habitat of Metapenaeus ensis. Moreover, in the natural aquatic environment, organisms are exposed not to a single metal but a mixture of different metals. Exposure to mixtures may result in toxicological interactions (0' Agostino and Finney, 1974;,
Ahsanullah et al., 1981b; 1988). Although about 10% of copper was reported to be in the chelated form in natural waters (Mantoura et al., 1977), and the concentrations of copper in natural seawater are expected to be below the reported LCso values. Chronic exposure to sublethal levels of copper, instead of acute lethal effect, may be one of the factors in reducing the productivity of the shrimp ponds along the Zhujiang
River estuary.
75 CHAPTER 4
SUBLETHAL EFFECTS OF
COPPER TO METAPENAEUS ENSIS
4.1 Introduction
This Chapter presents the sublethal effects of copper to oxygen consumption, ammonia excretion and feeding rate of the larval and postlarval stages of Metapenaeus ensis.
4.2 Materials and Methods
4.2.1 Spawners and rearing procedure
Procedures and experimental conditions for rearing larvae and post larvae of Metapenaeus ensis were the same as those described in sections 3.2.1-3.2.2.
4 • 2 . 2 Measurements of oxygen consumption and ammonia excretion
Rates of oxygen consumption and ammonia excretion of the stages PZI, MI, PL1, PL4 and PL15-20 were
76 , . determined in 20 ~m filtered artificial seawater
I (Instant Ocean). stock solution . of copper at 500 mg 1-1
was prepared from hydrated .salts of copper (II) sulphate
(CUS04 • 5H20) and stored at 4°C. Experiments were carried
out at 26-28°C with salinity of 30°/00 and pH 7.9-8.1. Individuals from a single spawning were used in each
experiment. All individuals tested were preconditioned
for at least 3 h in artificial seawater. Food for PZI
and MI consisted of Artificial Plankton B.P. at 0.7 mg
1-1 and Chaetoceros gracilis at 104 cells ml-I • Mixed
Feed for Penaeus monodon at 1 mg I-I and fleshly hatched
Artemia at 5 nauplii ml-I were provided for PL1 and PL4.
Mixed Feed for Penaeus monodon with 1.5 mg 1-1 was
provided for PL15-20.
After precohditioning, the shrimp were transferred
to well-aerated artificial seawater for 0.5-1.0 h. No
food was provided during this period and the subsequent
experimental period. Shrimp for experiments were then
transferred to stoppered glass bottles with volume
ranging from 43 to 47 ml. Based on results of
preliminary experiments, the number of shrimp and the
duration of the experiment were adjusted to ensure that
the oxygen tension of the seawater did not drop below
70% of its initial value at the end of the experiment.
The initial value of oxygen was within + 10% of
saturation. Thus, oxygen consumption measured was not 77 affected by oxygen tension. Experiments were conducted at about 1200 to 1800 h to minimize variation due to the diel changes in respiratory .and ammonia excretory rates.
The number of shrimp used in experiments on PZI, . MI, PL1, PL4 and PL15-20 were 150, 50, 20, 20 and 10, respectively. Two different copper concentrations, each in 6 replicates, were tested in each experiment. The two concentrations were close to the 48-h LC lO and LC2S of the stages based on the results of acute toxicity tests (see Chapter 3). Shrimp in uncontaminated seawater, in 6 replicates, served as control. Four bottles without shrimp, were used as blanks to account for changes in the amount of dissolved oxygen and ammonia in artificial seawater during the experimental period. There was no obvious change in the amount of dissolved oxygen and ammonia in bottles without shrimp. All glass bottles were tightly sealed and checked for air bubbles.
After 6 h of exposure, the amount of dissolved oxygen in half of the replicates in each test condition was determined using the Winkler method (Parsons et al. , 1984). Ammonia concentration in the other half of the replicates was determined with the oxidation method described in Parsons et al. (1984). Mortality of shrimp was checked in each glass bottle. Results excluded for analysis where mortality exceeded 5%.
78 Oxygen consumption and ammonia excretion were expressed in nmol individual-l h-1 and nmol I-£g-1 dry weight h~, respectively. Dry weights of PZI, MI, PL1, PL4 and PL15-20 were determined by using samples of 100 to 10000 individuals. Shrimp were washed thoroughfully with water to prevent crystallization of salts and then dried at 80°C to constant weight. Dry weight was determined to nearest 0.01 mg.
4.2.4 Measurement of feeding rates
Feeding rates of the stages PZI, MI, PL1 and PL4 were determined in the same conditions as in experiments on oxygen consumption and ammonia excretion. The number of shrimp used in experiments for PZI, MI, PL1 and PL4 were 100, 100, 20, and 20, respectively. After 3 h precondition with the same food item used in subsequent feeding experiment, shrimp were transferred to well aerated artificial seawater without food for 0.5-1.0 h. Shrimp were then transferred to capped bottles with a volume of 1175 ml for experiments.
For experiments on PZI and MI, the larvae were feed with Chaetoceros gracilis at an initial cell concentration of about 105 cells ml-!. Concentration of Chaetoceros gracilis before and after the experiment was determined with a Coulter counter (Model ZBI). Two 79 different copper concentrations, close to 48-h LC lO and
LC~ for the stage, each in duplicate, were tested in each experiment. Shrimp in seawater without the addition of copper, in duplicate, served as control. Bottles without shrimp, in duplicate, served to indicate the change in the density of Chaetoceros gracilis during the experimental period. Changes in the density of Chaetoceros gracilis were within 5% of its initial value. The bottles were mounted on a turning wheel rotating at 1.5 rpm. Experiments were carried out in darkness and lasted for 24 h to eliminate the variation due to the diel changes in feeding rate. Larvae may moult into next instar during the experimental period. Feeding rate data were assigned to the instar at which the experiments started. Mortality of shrimp was assayed after the experiment. Replicates with mortality greater than 5% were excluded from analysis. Clearance rates and ingestion rates were calculated according to Frost (1972). The growth constant for algae, k, and the grazing coefficient, g, were calculated as follows:
k(t2-tl) and C·= C·e(k-g)(t2-tl) C2 -C 1 e 2 . 1
1 ) where Cl and C2 are cell concentrations (cells ml- in the bottle without shrimp at the beginning (t}> and the
80 end ~t2) of an experiment, and Cl· and C2• are cell concentrations in the bottle with shrimp at time tl and t 2 •
The k and g were used to calculate the average cel,l concentration [Cl during the experimental period using the equation,
Cl • [e(k-g)(t2-tl) -1] [C) -
Thus, clearance rate is given by:
l F Cml individual-l h- ) = Vg/N where V is the volume (ml) of the bottle, and
N in the number of larvae in the bottle
Ingestion rate, I, is then calculated from:
1 I (cells individual-l h- ) = [Cl x F
For feeding experiments with PL1 and PL4, freshly hatched Artemia nauplii at a density of 200 nauplii 1-1 were used as prey. Two di.fferent copper concentrations, each in duplicate, were tested in each experiment. Shrimp in seawater without the addition of copper, in duplicate, served as control. After 4 h, the change in the number of nauplii was determined and used to
81 calculate the predation rate of the shrimp in preys
1 1 1 individual- h- and · preys 1-'9- dry weight h-1 • Mortality of shrimp was assayed after experiment. Results with mortality greater than 5% were excluded from analysis.
4.2.5 Analysis of results
All data was subjected to two-way ANOVA with different spawners and different copper concentrations
as main effects. Data where differences in animals
exposed to different copper concentrations were found were further subjected to Tukey's test. Significant
difference refers to the probability level of 0.05.
4.3 Results
4.3.1 Sublethal effects of copper on oxygen consumption
and ammonia excretion
The dry weights of PZI, MI, PL1, PL4 and PL15-20
are shown in Table 4.1. The dry weight increased from
several microgrammes in PZI to 100 microgrammes in PL15-
20, indicating a progressive increase in body weight
through development.
82 Two-way ANOVA with different spawners and different copper concentrations as main effects indicated that there was no interaction of these two main effects on oxygen consumption, ammonia excretion and feeding rate in all life history stages studied. Different spawner,s affected oxygen consumption of all stages tested, but did not affect ammonia excretion and feeding rate, except the feeding rate of PL1. Oxygen consumption, ammonia excretion and feeding rate were affected by the presence of copper dependent on the life stages.
Results of the effects of copper in seawater on the above three parameters are summarized in Tables 4.2-
4.10.
Results of oxygen consumption and ammonia excretion were presented in Tables 4.2-4.6. The oxygen consumption of PZI, MI, PL1, PL4 and PL15-20 in the control animals were about 1.5, 6.2, 15.8, 14.2 and 34.6
I I nmol O2 ind- h- respectively. And their weight-specific oxygen consumption ranged from 0.2 to 0.4 nmol O2 ~g~ dry weight h- I • General increase in oxygen consumption occurred in PZI-PL4 exposed to copper. significant increase (p < 0.05) in ox,ygen consumption occurred with
PZI at 0.05 mg I-I and PL4 at 0.30 mg I-I copper in shrimp from one of the spawners.
83 The ammonia excretion of PZI, MI, PL1, PL4 and PL15-20 in the control animals were about 0.3, 0.5, 0.9,
1. 6 and 5. 2 nmol ind-1 h ~ l respectively. And their weight-specif ic ammonia excretion ranged from o. 02 to
1 1 O. 07 nmol J.Lg- dry weight h- • No signif icant changes in ammonia excretion in all stages exposed to copper were found, except in one case where there was a decrease in
ammonia excretion of MI exposed to 0.10 mg 1-1 copper.
The feeding rates of PZI and MI are shown in Tables 4.7 and 4.8, respectively. Ingestion rate of PZI and MI on Chaetoceros gracilis was about 10-20 x 103 cells ind-1
h~ and the clearance rate was about 80-200 J.Ll ind~ h~. The ingestion and clearance rates expressed per unit dry weight were 2.0-3.0 x 103 cells J.Lg-1 h-1 and 20-35 J.Ll J.Lg-1
h~, respectively, in PZI, and 0.4-0.8 x 103 cells J.Lg~
1 1 and 6-8 JLl J.Lg- h- , respecti vely, in MI. No significant differences were found in the feeding rates of PZI and MI upon exposure to the two copper concentrations tested.
The predation rates of PL1 and PL4 on Artemia nauplii are shown in Tables 4.9 and 4.10 respectively. The predation rates of PL1 and PL4 were O. 3-0.4 and 0.4-
0.5 preys in~l ~1, respectively. Their predation rates
expressed in dry weight were 8-10 and 6-8 preys JL~l h~
respectively. There was a general decrease in the 84 predation rates of PLl and PL4 upon exposure to copper. Significant decrease in predation rate of PLl from one spawner was found when the shrimp were exposed to 0.10 and 0.20 mg 1-1 copper. No significant decrease in the predation rate of PL4 was found.
85 Table 4.1: Dry weight of selected life history stages of Metapenaeus ensis
Life stage Dry weight (J..£g) N
PZI 4.0 + 1.4 6 MI 24.9 + 5.3 6 PL1 36.0 + 6.7 3 PL4 64.7 + 3.2 3 PL15-20 100.8 + 8.6 6
Dry weights are expressed as mean + SE. N represents the number of measurements.
86 Table 4. 2 : Oxygen consumption and ammonia excretion of PZI from two spawners of Metapenaeus ensis
Control o. 03 mgCu l-t 0.05 mgCu l-t
Oxygen consumption nmol ind-t h-t (1) 1.62+0.14 1.88+0.06 2.42+0.15* (2) 1.29+0.04 1.32+0.15 1.71+0.17 nmol p,g-l h- l (1) 0.404+0.034 0.471+0.015 0.604+0.037* (2) 0.321+0.011 0.331+0.038 0.426+0.043 Ammonia excretion nmol ind-l h- l (1) 0.320+0.028 0.373+0.038 0.365+0.036 (2) 0.204+0.079 0.349+0.205 0.125+0.042 nmol p,g-l h-l (1) 0.080+0.007 0.094+0.010 0.092+0.009 (2) 0.051+0.020 0.088+0.052 0.031+0.011
Oxygen consumption and ammonia excretion are expressed as mean + SE from triplicate. Rows (1) and (2) represent data from different spawners. Data with an asterisk are significantly different (p < 0.05) from the control.
87 Table 4.3: Oxygen consumption and ammonia excretion of MI from two spawners of Metapenaeus ensis
Control 0.05 mgCu I-I 0.10 mgCu I-I
Oxygen consumption nmol ind-I h-1 (1) 7.27+0.29 7.39+0.09 8.06+0.28 (2) 5.10+1.10 3.54+0.50 6.79+0.45 nmol Jjg-l h-I (1) 0.292+0.011 0.297+0.004 0.323+0.011 (2) 0.205+0.044 0.142+0.020 0.273+0.018 Ammonia excretion nmol ind-I h-I (1) 0.416+0.042 0.396+0.136 0.323+0.066 (2) 0.645+0.292 0.315+0.029 0.097+0.024* nmol Jjg-l h-1 (1) 0.017+0.002 0.016+0.006 0.013+0.003 (2) 0.026+0.012 0.013+0.001 0.004+0.001*
Oxygen consumption and ammonia excretion are expressed as mean + SE from triplicate. Rows (1) and (2) represent data from different spawners. Data with an asterisk are significantly different (p < 0.05) from the control.
88 Table 4.4: Oxygen consumption and ammonia excretion of PL1 from two spawners of Metapenaeus ensis
Control o. 10 mgCu 1-1 0.20 mgCu 1-1
Oxygen consumption nmol ind-1 h-1 (1) 13.3+0.6 14.9+1.2 14.3+0.6 (2) 18.2+0.9 20.2+0.4 19.1+0.3 nmol JJg-1 h-1 (1) 0.371+0.016 0.415+0.033 0.397+0.017 (2) 0.506+0.024 0.561+0.012 0.531+0.009 Ammonia excretion nmol ind-1 h-1 (1) 0.611+0.023 0.744+0.057 0.837+0.295 (2) 1.24+0.36 0.609+0.205 1.18+0.13 nmol JJg-1 h-1 (1) 0.017+0.001 0.021+0.002 0.023+0.008 (2) 0.034+0.010 0.017+0.006 0.033+0.004
Oxygen consumption and ammonia excretion are expressed as mean + SE from triplicate. Rows (1) and (2) represent data from different spawners.
89 Table 4. 5: Oxygen consumption and ammonia excretion of PL4 from two spawners of Metapenaeus ensis
Control o. 15 mgCu 1-1 0.30 mgCu 1-1
Oxygen consumption nmol ind-1 h-1 (1) 12.9+0.4 14.6+0.4 15.2+0.5* (2) 15.5+0.9 18.9+1.2 19.1+0.9 nmol p,g-1 h-1 (1) 0.199+0.007 0.226+0.006 0.235+0.007* (2) 0.240+0.014 0.293+0.018 0.295+0.015 Ammonia excretion nmol ind-1 h-1 (1) 1.79+0.18 1.16+0.34 1.92+0.12 (2) 1.51+0.35 1.57+0.23 2.31+0.37 nmol p,g-1 h-1 (1) 0.028+0.003 0.018+0.005 0.029+0.002 (2) 0.023+0.005 0.024+0.004 0.036+0.006
Oxygen consumption and ammonia excretion are expressed as
mean + SE from triplicate. Rows (1) and (2) represent data from different spawners.
Data with an asterisk are significantly different (p <
0.05) from the control.
90 Table 4.6: Oxygen consumption and ammonia excretion of
PL15-20 from two spawners of Metapenaeus ensis
Control O. 50 mgCu 1-1 1.00 mgCu 1-1
Oxygen consumption nmol ind-1 h-1 (1) 41.6+1.8 40.8+2.2 36.5+1.7 (2) 27.6+1.0 32.0+4.5 32.8+1.7 nmol p,g-1 h-1 (1) 0.412+0.018 0.405+0.022 0.362+0.017 (2) 0.274+0.010 0.318+0.044 0.326+0.016 Ammonia excretion nmol ind-1 h-1 (1) 6.03+2.65 4.48+1.11 3.53+0.26 (2) 4.34+2.29 5.48+0.57 3.77+0.20 nmol p,g-1 h-1 (1) 0.060+0.026 0.044+0.011 0.035+0.003 (2) 0.043+0.023 0.054+0.006 0.037+0.002
Oxygen consumption and ammonia excretion are expressed as mean + SE from triplicate. Rows (1) and (2) represent data from different spawners.
91 Table 4.7: Ingestion and clearance rates of PZI fed Chaetoceros gracilis
Control o. 03 mgCu I-I 0.05 mgCu I-I
Ingestion rate (X103) cells ind-I h-I (1) 9.69/11.9 11.7/10.9 13.1/9.8 (2) 11.3/14.1 10.4/9.5 10.4/8.6 cells J,Lg-I h-l (1) 2.43/2.98 2.93/2.75 3.30/2.46 (2) 2.89/3.50 2.56/2.36 2.18/2.60 Clearance rate J,Ll ind-l h-l (1) 101/125 120/113 133/99 (2) 120/150 104/99 88/107 J,Ll J,Lg-I h-l (1) 25.4/31.3 30.1/28.5 33.4/24.9 (2) 30.2/37.5 26.9/24.6 22.3/26.9
Values in rows are results of duplicate. Rows (1) and (2) represent data from different spawners.
92 Table 4.8: Ingestion and clearance rates of MI fed Chaetoceros gracilis
Control o. 05 mgCu 1-1 0.10 mgCu 1-1
Ingestion rate (X103) cells ind-1 h-1 (1) 14.8/13.7 13.2/14.3 12.0/10.6 (2) 17.0/13.0 10.7/15.1 18.8/13.0 cells p,g-1 h-1 (1) 0.59/0.55 0.53/0.57 0.47/0.43 (2) 0.68/0.52 0.43/0.61 0.75/0.52 Clearance rate
~l ind-1 h-1 (1) 160/147 141/153 122/111 (2) 183/140 110/154 194/134
~l p,g-1 h-1 (1) 6.48/5.91 5.66/6.16 4.88/4.45 (2) 7.34/5.61 4.41/6.20 7.78/5.39
Values in rows are results of duplicate. Rows (1) and (2) represent data from different spawners.
93 Table 4.9: Predation rate of PL1 on Artemia nauplii
Control 0.10 mgCu I-I 0.20 mgCu I-I preys ind-I h- I (1) 0.38/0.40 0.23/0.39 0.31/0.23 (2) 0.31/0.26 0.013/0.038* 0.075/0.13* preys JJg-I h-I X 10-3 (1) 10.6/11.1 6.39/10.83 8.61/6.39 (2) 8.68/7.29 0.35/1.04* 2.08/3.47*
Values in rows are results of duplicate. Rows (1) and (2) represent data from different spawners. Data with an asterisk are significantly different (p < 0.05)
from the control.
94 Table 4.10: Predation rate of PL4 on Artemia nauplii
control O. 15 mgCu I-I 0.30 mgCu I-I
preys ind-I h-I
(1) 0.40/0.29 0.35/0.25 0.13/0.063 (2) 0.51/0.40 0.36/0.24 0.25/0.10 preys J1,g-1 h-1 X 10-3 (1) 6.19/4.45 5.41/3.87 1.93/0.97 (2) 7.89/6.19 5.57/3.71 3.87/1.55
Values in rows are results of duplicate.
Rows (1) and (2) represent data from different spawners.
95 4.4 Discussion
Weight-specific oxygen consumption of PZI to PL4 of Metapenaeus ensis was found to range from 0.2 to 0.4 nmol I-£g-1 dry weight h-1 in the present study. These
values were equivalent to about 4. 5 to 9. 0 1-£1 02 mg-1 dry
weight h~. Chan and Lawrence (1974) reported that the weight-specific oxygen consumption of Penaeus aztecus ranged from 4.7 to 9.8 JJl mg-1 dry weight h-1 in protozoea to day 7-8 post1arva at 28°C. Hightower and Lawrence (1975) also reported that the oxygen consumptions of larvae and post1arvae of P. setiferus and P. aztecus were 5.2 to 11.3 1-£1 mg-1 dry weight h-1 and 4.6 to 6.9 1-£1
1 mg-1 dry weight h- , respecti ve1y , at 28°C. Recently, Kurmaly et al. (1989) who studied the oxygen consumption of P. monodonand reported that the weight-specific oxygen consumption ranged from 6.3 to 6.9 1-£1 mg-1 dry
weight h~ in PZI to PL1 at 25 to 28°C. The values in - the present study are in consistent with the values reported in the above studies.
Mean oxygen consumption of PL15-20 in the present
study was about 0.34 nmo1 JJ~l dry weight ~1, equivalent
to about 7. 6 JJ1 mg- 1 dry weight h-1• This value was in good agreement with those reported for post1arva of P. setiferus and P. aztecus with oxygen consumption of 7.7
96 and 6. 9 ILl mg-1 dry weight h- 1 respectively (Hightower and Lawrence, 1975). .Gaudy and Sloane (1981) reported that oxygen consumption of day~32 postlarva of P. monodon and day-35 postlarvae of P. stylirostris were 3.5 and 3.9 ILl
1 1 mg- dry weight h- , respectively, at 35°/00 at 27°C. These values were about half the value of the present study, i.e. 7.6 ILl m~l dry weight ~l. This difference may be explained by the fact that weight-specific oxygen consumption decreased with increased body weight (Chan and Lawrence, 1974; Hightower and Lawrence, 1975; Kurmaly et al. 1989).
The rate of ammonia excretion per individual increased from O. 3 to 5. 2 nmol ind-1 h-1 in PZI to PL15. Weight-specific ammonia excretion in the present study ranged 0.02-0.08 nmol JL~l dry weight ~l. These values
1 1 were equivalent to about 0.34-1.36 JLg mg- dry weight h- • These values are consistent with those reported by Spaargaren et al. (1982) and Cockcroft and Mclachlan (1987). The ammonia excretion rates are 0.37-0.87 and
0.35 JLg mg-1 h-1 for juvenile Penaeus japonicus and adult
Macropetasma africanus respectively. The values are also at the same order of magnitude as those of 0.02-3.6 nmol ind-1 h-1 for NV-PL9 of Metapenaeus ensis reported by ovsianico-Koulikowsky (1987).
97 The present study showed that the oxygen consumption of different life history stages of
Metapenaeus ensis up~n exposure to sublethal concentrations of copper was either unaffected or significantly increased. Different results of the sublethal effect of copper on oxygen consumption of marine animals have been reported. Bernard and Lane (1963) reported that the oxygen consumption of the barnacle, Balanus amphitrite exposed to sublethal concentrations of copper reduced. However, the oxygen consumption of the copepod, Acartia clausi was reported to increase upon exposure to copper (Apostolopoulou and Verriopoulos, 1979). In this study, the oxygen consumption rate of Acartia clausi exhibited a continuous increase during 10 days of exposure to copper ranged .from 0.001 to 0.01 mg 1-1. On the other hand, Reeve et al. ( 1977 ) reported tha t the oxygen consumption remained stable in the copepods,
Pseudocalanus sp. and Calanus sp. in exposure to sublethal dosages of copper. Relexans et al. (1988, cited in Gaudy et al., 1991) suggested that there may be two phases in the physiological response of organisms to heavy metals. During the first phase, the organism responds by increased oxidative phosphorylation which can be considered to be a physiologically adaptive response to heavy metals. In the second phase, intoxication is characterized by a reduction in oxygen
98 consumption, during which the organism fails to meet the
increased demand .of ATP and finally died. The copper
concentrations used in this study were about the 48-h
LC10 and LC2S values and, oxygen consumption was measured in the first 6 h of exposure. Thus, the increase in
oxygen consumption in the present study possibly
represents the first phase of response as suggested by
Relexans et al. (1988).
Unlike oxygen consumption, ammonia excretion was
relatively insensitive· to the presence of copper. Gaudy
et al. (1991) also reported that the daily ammonia
excretion of the mysid, Leptomysis lingvura remain
unchanged at 10°C upon exposure to 0.1 mg 1-1 cadmium.
Ingestion and clearance rates of PZI and MI on
Chaetoceros gracilis in the present study were about 10-
1 1 20 x 103 cells ind-1 h- 1 and 80-200 JLl ind- h- • And the
. predation rates of PL1 and PL4 on Artemia nauplii were
0.3-0.4 and 0.4-0.5 preys ind~ ~1 respectively. These
values were comparable to those reported by Leong
(1991). Leong (1991) reported that in PZI and MI, the
ingestion and clearance rates were 6-15 x 103 cells ind-1
h-1 and 60-120 JLl ind-1 h-1 respectively. And in PL2-PL5,
1 1 the predation rate was o. 76-1. 09 preys ind- h- • Feeding rate of Metapenaeus ensis decreased in some cases. Wong
et al. (in press) also reported that the feeding rates 99 of different stages of Metapenaeus ensis reduced upon
exposure to heavy metals, including nickel, chromium and
copper. Upon sublethal exposure to copper, the feeding
activity also reduced in the copepods, Pseudocalanus sp.
and Calanus sp. (Reeve et al., 1977), Acartia clausi
(Apostolopoulou and Verriopoulos, 1979), and Acartia tonsa (Sunda et al., 1987).
Two-way ANOVA indicated that different spawners
affected oxygen consumption of all stages tested and
copper concentration affected oxygen consumption of some
life stages. The result suggested that oxygen
consumption was a sensitive parameter among the three
parameters studied, to both the effect of spawner and
copper concentration. The increase in oxygen
consumption of Metapenaeus ensis in the presence of
copper indicated an increase in the rate of metabolism.
Thus, an imbalance in the energy budget may result from
. the increased metabolism and the decrease in nutritional
energy intake from food. Both these two changes are
closely linked and act together to affect the survival
and growth of Metapenaeus ensis exposed to sublethal
concentration of copper.
100 CHAPTERS
GENERAL CONCLUSIONS
The conclusions of this study are listed as follows:
(a) The survival of the larvae and postlarvae of
Metapenaeus ensis decreases in the presence of copper.
(b) Individuals from different spawners are different
in their sensitivities to copper toxicity.
(c) Trends of increase in tolerance to copper through
development ,of Metapenaeus ensis is evident.
(d) The rate of larval development retards in the
presence of copper.
(e) Exposure to copper results in retarded growth in
day 15-20 postlarvae.
(f) Oxygen consumption increases and feeding rate decreases in the presence of copper, while ammonia excretion is relatively insensitive to copper.
101 / " (g) The increase in metabolism \~nd the decrease in
- nutritional intake may upset the energy balance of the animal, resulting in a reduction of the adaptive ability and eventually death of the
shrimp~ )
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