Nutrient Removal by Algae Grown in CO2-Enriched Wastewater over a
Utilization of wastewater-grown zooplankton: Nutritional quality of zooplankton and performance of...
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Utilization of wastewater-grown zooplankton: Nutritionalquality of zooplankton and performance of silver perchBidyanus bidyanus (Mitchell 1838) (Teraponidae) fedon wastewater-grown zooplankton
G. KIBRIA & D. NUGEGODA Department of Environmental Management, Victoria University of Technology,
MCMC, Melbourne, Australia
R. FAIRCLOUGH Department of Biomedical Sciences, Victoria University of Technology, Melbourne, Australia
P. LAM Chemistry and Biology Department, City University of Hong Kong, Kowloon, Hong Kong
A. BRADLEY Zootech Research Group, Werribee, Victoria, Australia
Abstract
Zooplankton grow in the last stage of water puri®cation at
the Werribee Sewage Treatment Lagoons (WSTL) and the
resource is huge and unutilized. Daphnia carinata and Moina
australiensis are the dominant species at the WSTL. The
protein content of D. carinata and M. australiensis was
54.80% and 64.80%, respectively. Analysis of zooplankton
showed that both essential and nonessential amino acids were
present at a level that was higher than control diet. Silver
perch fed on D. carinata exhibited better growth, better food
conversion ratio (FCR), protein e�ciency ratio (PER) and
apparent net protein utilization (ANPU), which were not
signi®cantly di�erent from the control diet (P > 0.05).
Heavy metals concentrations were at very low levels in ®sh
fed on wastewater-grown zooplankton.
KEYKEY WORDSWORDS: amino acids, heavy metals, performance, prox-
imate composition, silver perch, wastewater reuse, zooplank-
ton
Received 10 February 1997, accepted 14 May 1998
Correspondence: Dr Golam Kibria, 45 Lowson Street, Fawkner, VIC 3060,
Australia. E-mail: [email protected]
Introduction
Zooplankton are the natural food item of many marine and
freshwater ®sh and crustaceans. They have been used
extensively to rear larvae and fry (De Pauw et al. 1981;
Tay et al. 1991). Both live (Dabrowski 1984; Alam et al.
1993) and frozen zooplankton (Brett 1971; Sargent et al.
1979) have been used to rear ®n®sh and crustaceans. Studies
have shown that fry performed better when fed zooplankton
than when fed with arti®cial dry diets (Dabrowski 1984;
Dave 1989). Frozen and freeze-dried zooplankton represent
an excellent food source for hatchlings of white ®sh Coreg-
onus sp., carp Cyprinus carpio, lake char Salvelinus sp. and
species which normally require live plankton for growth
(Bryant & Matty 1980).
Zooplankton are a valuable source of protein, amino
acids, lipid, fatty acids and enzymes (Ogino 1963; Millamena
et al. 1990; Munilla-Moran et al. 1990; Pillay 1990).
Yurkowski & Tabachek (1979) reported that essential amino
acid levels in the cladoceran Daphnia pulex and the copepod
Diaptomus sp. were equal to or greater than ®sh require-
ments. Studies on the nutrient composition of natural
zooplankton are signi®cant since they would aid in deter-
mining the suitability of the organisms as feed ingredients for
aquaculture (Yurkowski & Tabachek 1979). The nutritional
quality of living organisms can be evaluated by determining
their proximate and amino acid composition, and by protein
e�ciency ratio and net protein utilization (Watanabe et al.
1978).
Zooplankton grow abundantly in the nutrient-rich Werri-
bee Sewage Treatment Lagoons (WSTL), Melbourne, Aus-
tralia but the resource is unutilized. To date, there has been
no research on the utilization of this resource for aquaculture
in Australia. Fish meal is the highest quality protein source
for ®sh feeds (Lovell 1989). However, it is becoming
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Aquaculture Nutrition 1999 5;221^227. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Ó 1999 Blackwell Science Ltd
expensive owing to high demand and reduced supply (Barlow
1989). Therefore, it is essential to evaluate other ingredients
as alternative protein sources for rearing ®sh. Silver perch
Bidyanus bidyanus is an Australian native ®sh of high
aquaculture potential and represents the main freshwater
native aquaculture industry in Australia. This paper exam-
ines the proximate and amino acid composition of wastewa-
ter grown caldocerans (Daphnia carinata and Moina
australiensis) and the preliminary performance of silver perch
fed on wastewater grown zooplankton.
Materials and methods
Zooplankton source and production
Zooplankton grow abundantly in the last stage of the
wastewater treatment process of the WSTL, which are
located 35 km south-west of Melbourne, Australia. They
graze on algae and remove nitrogen and phosphorus (pol-
lutants) from the wastewater. A simpli®ed sequence of
zooplankton production at the WSTL is given in Fig. 1.
During 1995±96 (summer), Zootech (a private company
researching utilization of wastewater zooplankton) used a
proprietary zooplankton harvester called `Baleen' and har-
vested 40±84 kg h)1 of zooplankton from the lagoons.
Zooplankton was harvested every day during the summer
period (January±March). Zootech used carefully chosen
mesh for selective harvesting of D. carinata or M. austral-
iensis from the ponds because there is a distinct size variation
between the two populations (Lai 1994). Harvested zoo-
plankton were kept frozen at )20 C in blocks until used. The
dominant zooplankton at the WSTL were D. carinata King
and M. australiensis Sars (Lai 1994). These natural and
invaluable resources are normally drained into Port Philip
Bay by the Melbourne Water Authority.
Fish husbandry and experimental procedures
Preliminary rearing experiments were conducted at the
Zootech multipurpose hatchery located at the Werribee
Treatment Complex. Silver perch ®ngerlings (612 mg) were
bought from a local native ®sh hatchery. Fish were acclima-
tized in the hatchery environment for 3 weeks and were
subjected to a short salt bath (10 g L)1) for 60 min prior to
the start of feeding trials (Rowland & Ingram 1991).
Rectangular, dark-coloured PVC tanks (41 ´ 84 ´ 27 cm)
were selected randomly for each replicate of the seven feeding
treatments (Table 1). Each tank was stocked with 15 ®sh
with three replicates per treatment. Fish were reared in static
water. Water temperature was kept at 25 °C by a thermo-
statically controlled heater. Water quality was maintained at
pH 7.5±8.0, hardness at 80±100 mg L)1, dissolved oxygen
levels at > 6 mg L)1 and a cycle of 12 h light and 12 h dark
Figure 1 Simpli®ed sequence of zooplankton production at the Werribee Treatment Lagoons. 1. Wastewater (raw sewage) enters the aerobic
ponds. 2. Bacteria digest the organic material. 3. Sludge containing the heavy metals and other chemicals settle out. 4. Wastewater moves into
the aerobic pond. 5. Algae grow in the nutrient-rich water. 6. Bacteria and algae remove nitrogen from the water. 7. Zooplankton grow and feed
on algae and bacteria. 8. Treated wastewater, along with zooplankton, ¯ows into the Port Phillip Bay.
G. Kibria et al.
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was used. Fish were fed frozen zooplankton and dry diet
(control), twice per day at 3% of their body weight, 5 days
per week for 4 weeks (dry matter basis).
Biochemical analysis
Fish and zooplankton samples were stored at )20°C until
analysed. Triplicate samples of zooplankton (D. carinata and
M. australiensis), ®sh and feed were subjected to chemical
analysis. Samples were weighed using an analytical balance
(Mettler AE 200 USA). At the completion of the experiment,
pooled ®sh samples were dried and ground, using mortar and
pestle for crude protein, fat, ash and phosphorus determina-
tion. Dry matter was determined by weight loss after drying
in an oven at 100°C for 24 h (AOAC 1990). Ash was
determined after ignition at 600°C for 12 h (AOAC 1990),
crude protein by digesting samples with Kjeldahl tablets and
5 mL concentrated sulphuric acid (N ´ 6.25) (AOAC 1990),
carbohydrate by the di�erence between the dry matter of the
sample and the sum of the determined crude protein, fat and
ash, lipid by ether extract in a Soxhlet apparatus (New 1987)
and total phosphorus following AOAC (1990). Protein
retention was determined by subtracting the initial protein
content of ®sh from the ®nal protein content. Amino acid
composition of zooplankton and dry feed was analysed in the
State Chemistry Laboratory, Melbourne by high-pressure
liquid chromatography (HPLC).
Heavy metals analysis
Fish carcasses were analysed for heavy metals (zinc,
cadmium and lead). Samples were dried in an oven and
crushed gently in a mortar and pestle. Dried samples were
digested in analytical nitric acid (HNO3) at 100°C in a block
heater for 30 min (Corp & Morgan 1991). Metals were
analysed on a Varian spectra AA-400 Flame Absorption
Spectrophotometer with background correction using pro-
cedural blanks.
Statistical procedures
Mean, standard deviation and standard error of the mean of
speci®c growth rate (SGR), food conversion ratio (FCR),
protein e�ciency ratio (PER) and apparent net protein
utilization (ANPU) were calculated following Zar (1984). All
percentage data were transformed to arcsin values prior to
analysis. One-way analysis of variance (ANOVAANOVA) was used to
compare the means of SGR, FCR, PER and ANPU. All
ANOVAANOVAs were calculated using an IBM compatible Microsoft
EXCELEXCEL programme setting the signi®cance level at 0.05.
Results and discussion
Proximate composition of zooplankton
D. carinata and M. australiensis possessed an attractive
biochemical composition (Table 2). The protein content in
D. carinata and M. australiensis was 54.34% and 64.80%,
respectively, higher than the control commercial silver perch
diet. The protein content of Daphnia sp. has been reported to
be 49.70±70% (Blazka 1966; Yurkowski & Tabachek 1979;
Watanabe et al. 1983) whereas for Moina sp. it varies
between 59.00 and 77.85% (Tay et al. 1991; Creswell 1993).
It appears that D. carinata and M. australiensis are a
potentially valuable protein source.
The lipid content in the two zooplankton studied was in
the range 7.29±7.73% (Table 2). Watanabe et al. (1983)
analysed various zooplankton, with Daphnia containing 13%
and Moina 12±27% lipids. The lipid content in freshwater
zooplankton can be extremely variable (Vijverberg & Frank
1976) and may be in¯uenced by seasonal succession of
Table 1 Di�erent feeding regimes used in the silver perch perfor-
mance study
Feeding regimes Code
1 Control (artificial silver perch diet;100%) C2 Daphnia carinata (100%) D3 Moina australiensis (100%) M4 D. carinata (50%) and M. australiensis (50%) D + M5 D. carinata (33.33%) + M. australiensis (33.33%) +arti¢cial diet (33.33%)
D + M + C
6 D. carinata (50%) + control (50%) D + C7 M. australiensis (50%) + control (50%) M + C
Table 2 Proximate analysis of control feed and zooplankton
(Daphnia carinata and Moina australiensis) harvested from the
Werribee Sewage Treatment Lagoons
Dry diet(control) D. carinata
Moina
australiensis
Carbohydrate (g kg)1) 366.6 þ 10.4 271.0 þ 15.6 206.5 þ 9.7Crude ash (g kg)1) 98.4 þ 0.2 112.6 þ 0.1 68.2 þ 0.7Crude fat (g kg)1) 85.0 þ 4.9 72.9 þ 0.3 77.3 þ 0.6Crude protein (g kg)1) 450.0 þ 15.02 543.4 þ 28.3 648.0 þ 14.8Moisture (g kg)1) 98.8 þ 1.0 928.7 þ 0.9 937.2 þ 1.1Nitrogen (g kg)1) 72.0 þ 5.0 86.9 þ 3.1 103.7 þ 1.7Phosphorus (g kg)1) 11.6 þ 0.8 11.4 þ 0.2 11.1 þ 0.1Digestible energy
(DE, kcal kg)1)15273 5250 5700
DE/P (g kcal)1) 117 97 88
1Gross energy was calculated using kilocaloric values of 5.5 g)1 protein,9.1 g)1 fat and 4.1 g)1 carbohydrate (New 1987).2 Each value represents a mean þ SE; n = 3.
Nutritional quality of wastewater-grown zooplankton and performance of silver perch
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phytoplankton species (Je�ries 1970) or source of food fed to
zooplankton (Proulx & de la Noue 1985). Although the fatty
acid composition of D. carinata and M. australiensis was not
analysed, a study by Tucker (1992) noted that Daphnia sp.
may be de®cient in docosahexaenoic acid (DHA) and Moina
sp. in both DHA and eicosapentaenoic acid (EPA).
Amino acid content in zooplankton
Amino acid analysis revealed that both D. carinata and
M. australiensis contained appreciable levels of both essen-
tial and nonessential amino acids (Table 3). Compared
with the commercial silver perch diet (control), both
zooplankton appeared slightly superior. However, values
of some of the essential amino acids in D. carinata and M.
australiensis were lower than those previously cited for
Daphnia sp. (Yurkowski & Tabachek 1979; Hepher 1988)
and Moina sp. (Hepher 1988; Creswell 1993). The lower
values could have been due to seasonal variation in amino
acid content (Holm & Walther 1988), foods consumed by
zooplankton (Watanabe et al. 1983) or culture medium
(Allen & Allen 1981). The essential amino acid content of
zooplankton can meet the requirements of ®sh, for example
the essential amino acid content of Daphnia pulex was
found to be equal to or greater than the requirement of
Chinook salmon (Yurkowski & Tabachek 1979) (see also
Table 3). Both D. carinata and M. australiensis had a
relatively similar essential and nonessential amino acids
composition (average 51.5% essential amino acids). Holm
& Walther (1988) reported that 50.6% of amino acids in
daphnids were essential. The cladocerans, D. carinata and
M. australiensis, contained relatively similar proportions of
polar to nonpolar amino acids, (0.89 for D. carinata and
0.80 for M. australiensis). A higher proportion (1.57) was
calculated for daphnids by Holm & Walther (1988), which
may indicate a better nutritional status of those species.
Performance of silver perch fed on zooplankton
The control silver perch diet (treatment 1) resulted the best
growth among the seven feeding regimes, followed by ®sh fed
D. carinata (treatment 2) (Fig. 2). Gain in weight was not
signi®cantly di�erent between treatments 1 and 2 (P > 0.05).
Among the zooplankton-based treatments, ®sh fed
D. carinata (treatment 2) and a combination treatment of
D. carinata plus control diet (treatment 6) showed better
growth (P < 0.05). Silver perch fed M. australiensis showed
comparatively less growth. The results may indicate that
silver perch have a preference for D. carinata over
M. australiensis. Confer & Lake (1987) established that
yellow perch Perca ¯avescens has a greater a�nity for
Diaptomus sicilis than for other species of zooplankton.
Minimum essentialamino acid requirementsfor finfish1
Dry diet(control) D. carinata M. australiensis Omnivorous Carnivorous
Essential amino acidsArginine (Arg) 2.42 3.37 3.01 1.81 2.24Histidine (His) 1.29 1.65 1.37 0.76 0.95Isoleucine (Iso) 1.66 2.60 2.67 1.18 1.46Leucine (Leu) 3.25 5.22 4.94 2.15 2.66Lysine (Lys) 2.65 3.35 3.34 2.48 3.08Methionine (Met) 0.80 1.46 1.13 0.81 1.00Phenylalanine (Phe) 1.86 2.52 2.67 1.22 1.51Threonine (Thr) 1.68 2.86 2.79 1.35 1.67Tryptophan (Try) 0.43 0.71 0.76 0.25 0.31Valine (Val) 2.36 3.27 3.56 1.40 1.73
Nonessential amino acidsAlanine (Ala) 2.27 3.23 3.77Aspartic acid (Asp) 2.42 4.70 5.12Cystine 0.44 0.71 0.60Glutamic acid (Glu) 6.36 6.39 6.57Glycine (Gly) 2.03 2.48 2.59Proline (Pro) 2.03 2.20 2.36Serine (Ser) 1.88 2.82 2.62Tyrosine (Tyr) 1.17 2.14 2.38
1Tacon (1990).
Table 3 Essential and nonessential
amino acid composition of dry diet
(control) and zooplankton (Daphnia
carinata and Moina australiensis)
harvested from the Sewage Treatment
Lagoons and used in feeding trials
(g 100 g)1). Essential amino acid
requirements of ®sh (omnivorous and
carnivorous fry, 0.05 g) are also given
for comparison
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Similarly, Atlantic salmon Salmo salar showed a preference
for daphnids (Holm & Walther 1988). Furthermore, the
species and their origin (Watanabe et al. 1978), and seasonal
changes in the biochemical composition in zooplankton can
in¯uence their nutritional value to ®sh (Sadykhov et al. 1975;
Je�ries 1979). SGR, FCR, PER and ANPU were signi®-
cantly better with treatments 1 and 2 (Table 4). D. carinata
again gave the best FCR, PER and ANPU values compared
with M. australiensis. The maximum PER values obtained
with D. carinata were 1.57±1.64 (treatments 6 and 2). Trout
fed on zooplankton-krill (Euphsusia sp.) had a PER of 1.92
(Koops et al. 1979), whereas channel cat®sh had a PER of
1.03 (Hilge 1979). However, Watanabe et al. (1983) reported
a PER of 3.9 with Daphnia sp. and 2.6 with Moina sp. fed to
rainbow trout. Those zooplankton were raised indoors and
supplied with nutritionally enriched foods which might have
led to the better PER in ®sh. Nevertheless, D. carinata did
show comparable results for SGR, FCR, PER, ANPU in
comparison with the control commercial silver perch diet.
Effect of different feeding regimes on the chemicalcomposition of fish
The carcass composition of ®sh is shown in Table 5. The
moisture level decreased whereas protein, lipid and ash
content increased from initial values. Fish fed the control diet
had more lipids whereas ®sh fed on zooplankton had higher
ash and phosphorus contents. Rainbow trout fed on the
zooplankton Euphsusia paci®ca showed higher ash and lower
fat content (Spenelli 1979). Koops et al. (1979) also found a
higher phosphorus content in ®sh fed on krillmeal than on
®shmeal.
Heavy metals in fish carcass
Zinc, cadmium and lead were present at very low levels in ®sh
carcasses (Table 6). Fish fed mixed zooplankton (treatment
Figure 2 Gain in weight (%) of silver perch fed di�erent combina-
tions of zooplankton-based diets. Fish were fed at 3% body weight,
twice per day, 5 days per week for 4 weeks. The control diet (1) was a
commercial silver perch feed (mean � SE; n � 3); C � control,
D � Daphnia; M � Moina.
Table 4 Mean performance of silver perch juveniles fed either single zooplankton or a mixed zooplankton, or a combination of zooplankton
plus arti®cial diet. The ®sh were reared at 25°C for 4 weeks and fed at 3% body weight, twice per day, 5 days per week
C1 D M D + M D + M + C D + C M + C1 2 3 4 5 6 7
IAW2 610 609 611 613 608 617 619FAW3 1105 1104 901 863 904 1012 892SGR4,5 2.99 þ 0.04a 2.97 þ 0.16a 1.94 þ 0.09b 1.70 þ 0.05bc 1.99þ 0.11b 2.50 þ 0.04bdef 1.85 þ 0.02bcg
SGR as % of C 100.0 99.30 64.90 56.86 66.55 83.61 61.87FCR6 1.02 þ 0.07a 1.18 þ 0.12a 1.38 þ 0.04b 1.94 þ 0.47bcd 1.68þ 0.09b 1.23 þ 0.12ae 1.71 þ 0.13b
PER7 2.17 þ 0.14a 1.57 þ 0.11a 1.12 þ 0.09b 0.86 þ 0.11bc 1.10 þ 0.06b 1.64 þ 0.02adef 1.06 þ 0.05 bg
ANPU8 58.07 þ 2.1a 47.48 þ 4.0a 37.68 þ 3.10b 23.38 þ 2.13bcd 38.16 þ 0.48b 33.86 þ 1.77bd 23.37 þ 0.91bcdef
1SeeTable1for codes.2 IAW = Initial average weight (mg).3 FAW = Final average weight (mg).4 Each value represents a mean � SE; n = 3; numbers within a row not sharing the same superscript (a^g) are signi¢cantly di¡erent (P < 0.05).5 Speci¢c growth rate (SGR) = ln ¢nal body weight ) ln initial body weight/time (days) ´ 100 (Haiqing & Xiqin1994).6 Feed conversion ratio (FCR) = dry weight fed/wet weight gain (Haiqing & Xiqin1994).7 Protein e¤ciency ratio (PER) = wet weight gain/protein consumed (Haiqing & Xiqin1994).8 Apparent net protein utilization (ANPU) = body nitrogen at the end of the test ) body nitrogen at the beginning of the test/amount of nitrogenconsumed.(Jauncey1982).
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5) had signi®cantly higher levels of zinc than those of other
treatments (P < 0.05). Cadmium levels were negligible, with
no signi®cant di�erences among the treatments (P > 0.05).
Heavy metals were within the safe levels as set out by the
National Health and Medical Research Council (1987) for
human consumption. These preliminary data indicate that
zooplankton from the wastewater treatment lagoons were
not contaminated with the metals tested.
Conclusions
It is concluded that zooplankton from the Werribee waste-
water treatment facilities are rich in protein, essential amino
acids, lipid and phosphorus. Moreover, silver perch per-
formed signi®cantly better with D. carinata than with
M. australiensis (P < 0.05). SGR, FCR and PER were not
signi®cantly di�erent for ®sh fed a control commercial silver
perch diet or D. carinata. Further studies are needed on the
proximate, amino acid and fatty acid composition to
establish any seasonal variations in nutrient composition in
wastewater zooplankton. Research is also needed on the
possible loss of nutrients from frozen and freeze-dried
zooplankton, and on appropriate ways to reduce such losses.
Analysis of a wide range of contaminants, such as other
heavy metals and pesticides, would provide pertinent infor-
mation on the suitability of wastewater-grown zooplankton
for use in aquaculture.
Acknowledgements
The authors are indebted to the Department of Environ-
mental Management, Victoria University of Technology for
allowing the use of the equipment and chemicals required to
carry out the analysis. The Zootech Research Group
provided zooplankton and hatchery space to Golam Kibria.
We are grateful to two anonymous referees whose sugges-
tions helped to improve the manuscript.
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Table 5 E�ect of zooplankton-based feeding regimes on the chemical composition1 of silver perch juveniles. The ®sh were reared at 25°C for
4 weeks and fed at 3% body weight, twice per day, 5 days per week
Feeding regime2 Moisture (g kg)1) Crude protein (g kg)1) Crude lipid (g kg)1) Ash (g kg)1) Phosphorus (g kg)1)
1. C 759.7 þ 6.13 648.1 þ 1.8 116.2 þ 0.9 185.0 þ 0.9 25.6 þ 1.62. D 763.3 þ 1.9 647.5 þ 1.9 99.2 þ 0.3 200.5 þ 0.3 30.4 þ 1.23. M 781.2 þ 2.4 650.3 þ 2.8 93.6 þ 1.2 214.0 þ 0.6 29.5 þ 0.54. D + M 790.2 þ 3.4 634.3 þ 0.3 90.6 þ 1.4 243.4 þ 0.3 34.5 þ 2.75. D + M+ C 780.2 þ 0.9 652.2 þ 0.3 98.3 þ 0.8 225.6 þ 0.5 30.5 þ 0.16. D + C 775.4 þ 2.5 626.2 þ 0.4 106.3 þ 1.1 176.2 þ 0.4 24.9 þ 0.47. M + C 785.0 þ 2.9 624.0 þ 0.2 95.7 þ 1.1 188.6 þ 0.1 28.0 þ 1.1
1Body composition at the beginning of the experiment was: moisture 79.52%, protein 61.95%, lipid 9.07%, ash17.63%, phosphorus 2.51%.2 SeeTable1for codes.3 Each value represents a mean þ SE; n = 3.
Table 6 Metal concentrations (lg g)1, dry weight basis) in the
carcass of silver perch juveniles reared for 4 weeks and fed on
zooplankton and zooplankton plus arti®cial diet. The zooplankton
was harvested from the Werribee Sewage Treatment Lagoons
Feeding regimes1 Zinc2 Cadmium Lead
1C 207.6 þ 15.43a 3.77 þ 0.06a ND3
2 D 226.37 þ 8.05a 3.91 þ 0.03a ND3 M 214.84 þ 15.96a 3.91 þ 0.02a ND4 D + M 287.27 þ 8.10bc 3.96 þ 0.06a ND5 D + M+ C 278.54 þ 8.09a 3.84 þ 0.79a ND6 D + C 233.69 þ 0.00ad 3.85 þ 0.02a ND7 M + C 238.69 þ 7.64ae 3.82 þ 0.76a NDMaximum permitted conc. 75000.00 1000.00 1200.5
in fish as food (lg g)1)4
1SeeTable1for codes.2 Each value represents a mean þ SE; n = 3; numbers within the samecolumn not sharing the same superscript (a^e) are signi¢cantly di¡erent(P < 0.05).3 ND = not detected.4 National Health & Medical Research Council (1987).
G. Kibria et al.
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