Culture of the Freshwater Cladoceran, Daphnia Pulex F\
CULTURE OF THE FRESHWATER CLADOCERAN, DAPHNIA PULEX F\
UTILIZING SCENEDESMUS OBLIQUUS GROWN IN DAIRY WASTE MEDIUM
by
NELSON M. CASTILLO
B. Sc., University of the Philippines, Quezon City Philippines, 1975
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES
(Department of Agricultural Mechanics)
We accept this thesis as conforming
to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA October 1981
©Nelson M. Castillo, 1981 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library, shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of Q^O^!Sn^ QUx&qV\flg*>
The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5
Date SAW \&j R?\ J ii
ABSTRACT
Scenedesmus obliquus was grown in batch cultures at varying concentrations of digested dairy medium ranging from 200 to
2,000 ug-at • N-l1. Higher growth rates were observed at low N- concentrations while higher cell yields were observed at high N- concentrations. Aeration enhanced both algal growth rates and biomass yields. Results show an advantage in adjusting the nitrogen to phosphorus atomic ratio of the medium. More biomass was produced in cultures with higher N:P ratios.
The algal biomass produced was used as food for the freshwater cladoceran, Daphnia pulex F. Three feeding levels -1 were used: 50,000, 100,000 and 150,000 cells-ml . However, no significant differences were observed in both Daphnia biomass yields and biomass conversion efficiencies. The tendency of
Scenedesmus cells to settle down in the bottom and cling to the sides of the tank presented a major problem in the study.-
Intensive feeding did not increase the biomass production of
Daphnia, although larger-sized adults with larger brood sizes were produced. Animals in culture reached a density of 1.24 animals-mfAand obtained conversion efficiencies as high as 40-
50%.
Dr. J,W. Zahradnik
Thesis Supervisor iii
TABLE OF CONTENTS
Page
ABSTRACT ii
TABLE OF CONTENTS . iii
LIST OF TABLES v
LIST OF FIGURES . vi
ACKNOWLEDGEMENTS ix
INTRODUCTION 1
LITERATURE REVIEW '.. 3
Recycling of Wastes through Aquaculture 3
Algal Production from Waste 5
Daphnia as a Potential Fish Food in Aquaculture 8
Present State-of-the-Art on Daphnia Culture 12
MATERIALS AND METHODS 14
Algae 14
Daphnia pulex 14
Media Preparation 15
Algal Culture Medium 15
Daphnia Culture Medium 15
Description of Culture Units 16
Algal Culture Unit 16
Daphnia Culture Unit 18
Culture Methods 18
Algal-Culture Method 18
Daphnia Culture Method 19
Chemical Analysis 21
Kheldahl Nitrogen 21
Ammonia/Nitrate/Nitrite 21 iv
Total Phosphate 22
. Ortho-Phosphate 22
Algal Cell Weight 22
Growth Rate Experiments ( Daphnia ) 23
Length-Weight Relationship ( Daphnia ) 23
RESULTS 25.
Dairy Waste Analysis .25
Algal Experiments 26
Daphnia Experiments 37
Algal Cell Weight 68
Growth Rate Experiments 68
Length-Weight Relationship 71
Brood Size in Relation to Daphnia length 71
DISCUSSION 77
Livestock Waste as a Nutrient Source in
Algal Production 77
Algal Growth on Manure Medium 78
Daphnia Biomass Production 81
Population Growth and Size Structure 82
Growth Rate Experiments 84
Length-Weight Relationship 85
Nutritional Inadequacy of Certain Algae
Daphnia Culture 85
Toxicity in Daphnia Culture 87
Scale-up Considerations 89
SUMMARY AND CONCLUSIONS 91
REFERENCES 94
APPENDICES 103 V
LIST OF TABLES
Table Title Page
1 Summary of chemical composition of each media and the algal experiment in which they were used 25
2 Summary of the algal experiments, the different- treatments and the results 27
3 Chemical composition of media before and after algal growth .33
4 Amount of algae consumed by Daphnia at 3-day intervals .42
,5 Fecundity of Daphnia cultures in all five experiments 69
6 Ammonia, pH and DO levels measured daily in Daphnia culture tanks in D^ Expts. #4 #5 70
7 Daphnia brood size in relation to total length 75 vi
LIST OF FIGURES
Figure Title Page
1 A generalized view of the Algal Culture Unit 17
2 Daily cell density of S.obiiquus grown in varying N-concentrations of dairy waste medium (Algal Expt.#1) 28
3 Daily cell density of S.obiiquus grown in varying N-concentrations of.dairy waste medium (Algal Expt.#2) 30
4 Daily cell density of S.obiiquus grown in varying N-concentrations of dairy waste medium (Algal Expt.#3) 31
5 Daily cell density of S.obliquus grown in varying . N-concentrations of dairy waste medium (Algal Expt.#4). .32
6 Daily cell density of S.obiiquus grown in varying N-concentrations of dairy waste medium (Algal Expt.#5) • 35
7 Daily cell density of S.obiiquus grown in varying N-concentrations of dairy waste medium (Algal Expt.#6) 36
8 Daily cell density of S.obliquus grown in dairy waste medium with Nitrogen to Phosphorus atomic ratios of 17 and 64 (Algal Expt.#7) 38
9 Daily cell density of S.obliquus grown in dairy waste medium with Nitrogen to Phosphorus atomic ratios of 4, 22 and 69 (Algal Expt.#9) 39
10 Daily cell density of S.obiiquus grown in dairy waste medium with and without aeration (Algal Expt.#8) 40
11 N-consumed vs. Algal yield 41
12 Daphnia biomass at the three feeding levels at 3-day intervals ( D^ Expt.#l) 45'
13 Biomass conversion efficiency of Scenedesmus to Daphnia at the three feeding levels at 3-day intervals ( EL Expt.#l) 45
14 Daphnia biomass at the three feeding levels at 3-day intervals ( D^ Expt.#2) 46
15 Biomass conversion efficiency of Scenedesmus to Daphnia at the three feeding levels at 3-day •VI1
intervals ( IK Expt'.#2) 46
16 Daphnia biomass at the three feeding levels..at 3-day intervals (-'IK Expt. #3)' 47
17 Biomass conversion efficieny of Scenedesmus to Daphnia at the three feeding levels at 3-day . intervals (. IK" Expt. #3) 47
18 Daphnia biomass at 3-day intervals fed with 100,000 Scenedesmus cells ml 2 to 3 times daily (IK. Expt.#4 and #5) ; 48
19 Biomass conversion efficiency of Scenedesmus to Daphnia in EK_ Expts.#4 and #5 at.intervals 48.
20 Proportion of juvenile, young adult and adult Daphnia in terms of number and biomass in cultures at the three feeding levels at 3-day intervals ( EL Expt.#1) 49
21 Proportion of juvenile, young adult and adult Daphnia in terms of number and biomass in cultures at the three feeding levels at 3-day intervals ( IK Expt.=2) 50
22 Proportion of juvenile, young adult and adult Daphnia in terms of number and biomass in cultures at the three feeding levels at 3-day intervals ( IK Expt.#3) 51
23 Proportion of juvenile, young adult, and adult Daphnia in terms of number and biomass at 3-day intervals in EK_ Expt.#4 and #5 52
24 Daphnia size-frequency structure at 50,000 cells ml feeding level at 3-day intervals ( IK Expt.#1) 53
25 Daphnia size-frequency structure at 100,000 cells ml feeding level at 3-day intervals ( IK Expt.#1) 54
26 Daphnia size-frequency structure at 150,000 cells ml feeding level at 3-day intervals ( IK Expt.#1) 55
27 Daphnia size-frequency structure at 50,000 cells ml feeding level at 3-day intervals ( IK Expt.#2) 56
28 Daphnia size-frequency structure at 100,000 cells ml feeding level at 3-day intervals ( IK Expt.#2) 57
29 Daphnia size-frequency structure at 150,000 VI11
cells ml feeding level at 3-day intervals ( EL Expt.#2) 58
30 Daphnia size-frequency structure at 50,000 cells ml feeding level at 3-day intervals ( EL Expt.#3) 59
31 Daphnia size-frequency structure at 100,000 cells ml feeding level at 3-day intervals ( EL_ Expt.#3) 60
32 Daphnia size-frequency structure at 150,000 cells ml feeding level at 3-day intervals ( EL Expt.#3) .61
33 Daphnia size-frequency structure at 3-day intervals fed. with 100,000 cells ml 2 to 3 times daily ( IK Expt.#4- Repl ."l) 62
34 Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml 2 to 3 times daily ( IL Expt.#4- Repl.2) 63
35 Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml 2 to 3 times daily ( IL Expt.#4- Repl.3) 64
36 Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml 2 to 3 times daily ( Expt.#5- Repl.1) 65
37 Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml 2 to 3 times daily ( EL Expt.#5- Repl.2) 66
38 Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml 2 to 3 times daily ( EL Expt.#5- Repl.3) 67
39 Daily total length of D.pulex at the three food concentrations (Growth Rate Expt.#1) 72
40 Daily total length of D.pulex at the three food concentrations (Growth Rate Expt.#2) 73
41 Length-weight relationship of D.pulex 74 ACKNOWLEDGEMENT
My sincerest gratitude to Dr. J.W. Zahradnik, my advisor,
Department of Bio-Resource Engineering. I am deeply indebted to all members of my committee: Dr. P.J. Harrison, Department of
Oceanography and Dr. W.E. Neill, Institute of Animal Resource
Ecology, for their help at the initial stages of the experiments and their constructive criticisms in the preparation of the manuscript; Dr. V. Lo for his comments.
My sincerest appreciation to the following: Dr. P. Liao,
Department of Bio-Resource Engineering, for conducting most of the chemical analysis; J. Pehlke and N. Jackson, Department of
Bio-Resource Engineering, for the technical assistance; to the rest of the Bio-E staff and graduate students for their moral support.
Financial support from the International Development
Research Centre (I.D.R.C.) is very sincerely acknowledged. INTRODUCTION
Aquaculturists world-wide have expressed the need for nutrionally adequate diets in animal husbandry. The search for suitable feeds is stimulated by the costs and unpredictable supply of Artemia. sp. Cysts and manufactured diets have only been partially successful due to uncontrolled leaching of vitamins and minerals as soon as the food touches the water
(Norman et al., 1979). The possibility of using cladocerans
(such as Daphnia and Moina ) which can easily be grown especially in wastewater have already been investigated by a number of researchers (Bogatova and Askerov, 1958; Dewitt and
Candland, 1971; Dinges, 1974, 1976; Norman et al.,. 1979; Rees and Oldfather, 1980). Daphnia is an unexploited potential
source of animal protein and can be a protein supplement in fish
food pellets used by hatcheries and commercial fish-farmers.
Daphnia (Cladocera:Daphniidae) are filter-feeders and feed upon bacteria, certain unicellular green algae, and a mixture of various protozoa and protophytes from the sediments of ponds
(Banta, 1921). In past studies, the green algae such as,
Chlorella, Chlamydomonas and Scenedesmus have been used as food
for the culture of Daphnia (Watanabe et al., 1955; Richman,
1958; Sasa et al., 1960; Schindler, 1968; Gordon, 1975).
Agricultural wastes have been used successfully for the culture
of microorganisms such as bacteria and phytoplankton (Hephner,
1972; Schroeder, 1974). The utilization of livestock wastes as
a nutrient source for phytoplankton culture and subsequently, as
food for Daphnia has not been investigated yet. 2
The general objective of the present . research was to evaluate the culture potential of the freshwater cladoceran,
Daphnia pulex fed with the green alga, Scenedesmus obiiquus grown in dairy manure medium.
The experiments were conducted in two phases:
Phase I - Culture studies of Scenedesmus obliquus grown in
dairy manure medium;
Phase II - Culture studies of Daphnia pulex fed with waste-grown
algae. 3
LITERATURE REVIEW
Recycling of Wastes through Aquaculture
For hundreds of years people have been using wastes of household and farm animals as fertilizers in fishponds to stimulate production of acceptable food organisms for fish and other aquatic animals (Brown and Nash, 1979). The integration of agricultural wastes with fish culture is being practiced especially in lesser developed countries. On the other hand, aquaculture as a means for wastewater treatment is being
investigated in more developed countries. Aquaculture systems have been integrated with wastewater stabilization ponds to benefit from large quantities of aquatic food material produced as a by-product of microbial degradation, and to assist in the stabilization of the pond.
In most cases, natural foods present in ponds and other aquatic containments are not sufficient to support the fish especially when grown in great densities. Supplemental feeding with artificial diets should be given, which is often the
largest single operating expense in fish-farming (Hepher and
Schroeder, 1974). One way of reducing the need for this is by
increasing the amount of natural food in the pond.
Investigators have demonstrated many times to have increased plankton production of small ponds as much as 50 to 200% by the addition of commercial and natural fertilizers, and it is well
known that sewage effluents greatly increase the plankton of
lakes and rivers (Pennak, 1946; Schroeder, 1974). 4
Animal and domestic wastes are ideal as a growth medium for the production of algae since they are rich in all macro- and micro-nutrients necessary for algal growth; Effluents from secondary and tertiary sewage treatments, mixed with seawater had been used as a source of nutrients to grow single-celled marine algae in mass cultures (Songer et al., 1974; Ryther,
1975; Schroeder and Hepher, 1976; Trief et al., 1976). Animal wastes such as swine and cow-shed manure were also effective in growing algae (Schroeder, 1977; Maddox et al., 1978).
Numerous investigators have applied agricultural wastes directly in fishponds and reported increased fish yields and decreased supplemental feeding (Buck et al., 1976; Moav et al.,
1977; Schroeder, 1977; Maddox et al., 1979). The effectiveness of these wastes in fish culture maybe based on a food chain that
starts with bacteria and protozoa active in the decomposition of
the organic matter in the manure.
There is a big potential of incorporating wastes in fish culture. However, we are still faced with a number . of
restraints for its wider application. Allen and Hepher (1976)
enumerates them:
(1) unsatisfactory dissolved oxygen levels in ponds
(2) presence of toxic materials in wastewaters
(3) presence of unacceptable tastes and odours in fish
(4) presence of parasites and diseases
(5) various problems of public health
(6) difficulties of satisfying pond effluent standards
(7) meeting public acceptance of the practice. 5
Algal Product ion From Waste
Chlorella, Scenedesmus and Chlamydomonas have been reported to be the principal constituents of sewage oxidation ponds and suffice as a diet for Daphnia and related cladoceran Crustacea
(Hintz et al., 1966; Hintz and Heitman, Jr., 1975; Gordon,
1975) . Proliferation of blue-green algae has also been noted in waters receiving domestic outfalls. However, these algae serve as inferior food to cladocerans compared to green algae. Arnold
(1971) in his study on seven different species of blue-green algae found that ingestion, assimilation, survivorship and reproduction of Daphnia pulex fed with blue-green algae • were
lower than those fed with green algae.
The micro-algae Chlorella, Scenedesmus and Chlamydomonas are excellent feeds for Daphnia (Watanabe et al., 1955; Richman,
1958; Schindler, 1968). Listed below are the caloric values of
these algae obtaned by Richman (1958):
Chlamydomonas reinhardi 5,269 cal/g
Chlorella pyrenoidosa 5,444 cal/g
Scenedesmus obliquus 5,507 cal/g.
Of the three algal species mentioned above, only Scenedesmus can
be grown successfully in the laboratory using cow-shed manure.
Scenedesmus is commonly found in freshwaters and soils but
it can also withstand domestic sewage pollution (Trainor et al.,
1976) . Because of its simple nutritional requirements and rapid
growth rates, it can be easily maintained in the laboratory.
Members of this genus are coenobic and occur in the plankton,
among benthic algae in quiet bodies of water. The cylindrical •6.
cells, with rounded or pointed ends are laterally joined in groups of 4, 8 or 16. The terminal cells and some of the other cells, in some species have spines. Reproduction is by autocolony formation in which each parental cell forms a miniature colony, that is liberated through a tear in the parental wall (Bold and Wynee, 1978).
The genus Scenedesmus is composed of two groups, the
"obliquus" or non-spiny and the spiny group. The "obliquus" group is characterized by the following:
• (1) cells fusiform
(2) no ornamental pectic layer surrounding colony
(3) unicells form by fragmentation of colonies as they age
(4) cells may be joined end.to end in a Dactylococcus stage
(5) may reproduce sexually by fusion of biflagellated
segments (Trainor et al., 1976).
Dimentman et al.(l975) observed that Scenedesmus . obliquus was the absolute dominant sewage alga in the shallow treatment ponds near Jerusalem and Haifa (Israel). This sewage grown alga allowed a considerable growth rate of five species of fairy shrimps anostracans studied and hastened maturation and egg production. Different species of Scenedesmus were also shown to be compatible in growing daphnids (D'Agostino and Provasoli,
1970; Lampert, 1976; Rees and Oldfather, 1980).
Provasoli and Pintner (19 ) list the pre-requisites and versatile media for photo-autotrophic algae:
(a) total-solids concentrations
(b) concentration of major elements to suit the
prevalent ions required 7
(c) adequate sources of N, and growth factors
(d) sources of P, and avoidance of precipitates
in alkaline pH's
(e) pH buffering
(f) trace metal buffering.
Phosphates often become toxic above 5-20 mg% except for organisms living in polluted waters and ammonia tends to become toxic above 3-5 mg% in alkaline media except for Eurobionts living in polluted waters. Most algae utilize nitrates although
Scenedesmus tends to . show a s,light preference for NH^over NO^
(Krauss, 1958 ) .
In the 1960's, researchers began exploring the possibility of culturing algae on a mass scale (Davis et. al., 1961; Casey et al., 1963; Loosanoff and Davis, 1963; Ukeles, 1965). These were directed most especially for aquacultural purposes. Three types of mass culture were identified: batch, semi-continuous and continuous (Wisely and Purday, 1961; Monod, 1950; Herbert, 1958,
1961). Goldman (1978) has identified the problems relating
specifically to mass culture of algae:
(1) culture mixing
(2) nutrient availability and addition
(3) species control
(4) CO^addition
(5) water supply and evaporation
(6) algal separation and harvesting. 8
Daphnia as a Potential Fish-food in Aquaculture
Daphnia pulex has a caloric content of 5,350 cal gm dry weight (Richman, 1958). Of the total calories, about 21 - 27% is carbohydrate, 4 - 20% is fat. and 47% is protein. A comparison'is made between the composition of a typical dry trout ration and Daphnia:
Composition Proximate Composition Dry Trout (%Dry Matter) of Daphnia pulex Ration (Yurokowski and (Cho et al., Tobachek, 1978) 1 974)
Protein 47.7 49.7
Crude Fat 10.3 16.3
Fiber 2.3 6.9
Gross Energy 5.0 3.6
(Utilizable energy)
The figures above clearly suggest that a Daphnia diet can meet
the requirements of a fish ration.
Daphnia inhabit temporary pools, small ponds and lakes.
They are common aquatic crustaceans which feed upon bacteria, certain unicellular green algae, and a mixture of various protozoa and protophytes from the sediments of ponds (Banta,
1921). In some major lakes, the major portion of their food consists of detritus and bacteria, rather than living algae
(Pennak, 1946). 9
Daphnia exhibit both sexual, and parthenogenic (diploid) reproduction. During favourable conditions Daphnia reproduces by parthenogenesis. The eggs are liberated a few hours before the mother moults. The young produced in this way are all females which mature and reproduce in the same manner. The. number of young produced increases to a peak, thereafter declines with age (Anderson et al., 1937; Anderson and Jenkins,
1942; Frank et al., 1957; Richman, 1958). A parthenogenic female produce as high as 30 - 40 new Daphnia every two days when conditions are optimum (Dinges, 1974). A maximum of 70 parthenogenic eggs per brood was even reported by Daborn et al.(l978) of very large female Daphnia (4.4mm TL) in aerated, sewage treatment ponds (Nova Scotia, Canada). However, when conditions become unfavourable males appear among the offspring and sexual eggs are produced which require fertilization. Two eggs are enclosed in a dense, durable structure called the ephippium (Green, 1955). Females which produce ephippia may return to parthenogenetic reproduction when conditions return to normal. The environmental stimuli associated with the reversal of parthenogenesis to sexual reproduction are the following: density of culture, evaporation of habitat, starvation, high and low temperatures, diet, metabolic depresants and photoperiodism
(Stross, 1965).
A newly-born Daphnia is about 0.667 mm in total length.
The number of pre-adult instars, the number of instars elapsing between the time of release of the individual female from the brood chamber of her mother and the appearance of eggs in its own brood chamber, is usually from 4-6 depending on 10 environmental conditions. Adult females range from 1.5 - 3.5 mm in total length.
The rate of growth of Daphnia, the age at sexual maturity, the size of the broods, the interval between broods and the age at death are influenced by food concentration, temperature, crowding and other external conditions. Food is necessary for the growth of Daphnia. Semi-starved animals do not grow as quickly as well-fed animals. Starvation decreases growth in two ways: it increases the duration of the instars and reduces the increment at each moult (Ingle et al., 1937; Dunham, 1938).
Daphnia are filter-feeders. Food particles collected on the filtering setae are swept into the food groove by tufts of laterally inclined setules located on the second, third, and fourth thoracic appendages. In the food groove it moves forward to the maxillules and mandibles which pass it into the oral cavity where it collects until a peristaltic wave of the oesophagus carries it into the midgut (McMahon and Rigler,
1963).
Temperature affects the frequency of moulting and hence the frequency with which young are produced. Reproduction occurs every 2.0 days at 25°C; every 2.6 days at 20°C; and every 8.0 days at 11°C (Hall, 1964). An increase in temperature, up to a sub-lethal level, increases the initial growth rate by shortening the duration of instars (MacArthur and Baillie, 1929;
Green, 1955). The longevity in Daphnia magna varied as an inverse function of temperature between 8°C and 28°C (MacArthur and Baillie, 1929). Hall (1964) reported that the median lifespan of D.galeata mendotae is 30 days at 25°C; 60 - 80 days 11 at 20 C; and about 150 days at 11°C. Temperature also affects the filtering and metabolic rates of Daphnia. Filtering rate and maximum feeding rate of D.magna both continued to increase as temperature increased to 24°C, but above this temperature, the rate rapidly decreased (McMahon, 1965). Increase in temperature increased metabolic rates such as heartbeat rate and
C02production (MacArthur and Baillie, 1929). Brown and Crozier
(1927) reported better Daphnia cultures in terms of survival rate at room temperature (21°C) than in any other temperatures.
All of these findings suggest that the best operating conditions for the culture of D.pulex is in the range 20 - 24°C.
Studies show that photoperiodism affects the growth of
Daphnia. Stross (1965) found that short day photoperiods
(12:12, 13:11, 14:10 vs. 16:8LD) induced sexual reproduction.
On the other hand, Gordon (1975) using two photoperiods (18:6 and 16:8LD) found photoperiod to have a little effect on Daphnia biomass production with 16:8LD achieving slightly more biomass.
There is a limit to how many Daphnia a given volume of water can hold depending on the environmental conditions.
Increased density is accompanied, over a wide range, by decreased birth rate and lowered growth rate (Frank et al.,
1957; Frank, 1960; Smith, 1963; Mace, 1975). Increased crowding of young lengthens the immature period, whereas decreased crowding of adults increases the brood size.
The amount of 0^present in the water is also important in
the culture of Daphnia. The rate of Ogconsumption per animal
increases with body size but on a unit weight basis, the rate of
0^uptake is higher in the smaller animals. Animals larger than 12
1.00 mm show a relatively constant rate of consumption per unit weight, the mean being 7.21 ul«mg hr (Richman, 1958). 0 -1 levels of 3 mg-1 is considered to be the lower limit in D.pulex cultures (Richman, 1958; Ivleva, 1973; Kring and O'Brien, 1976).
Below these levels the filtering rates of Daphnia are Og _ dependent.
D.pulex often possesses considerable haemoglobin which is an adaption to enable it to withstand low Ogconcentration (Fox,
1948, 1951; Kring and O'Brien, 1976). Animals that have prolonged, exposure to low 0^ concentrations respond by facultatively increasing their haemoglobin levels. This enhances such functions as filtering rates, egg production, general activity and survivorship of Daphnia at low Og concentration.
D.magna can survive at a pH range of 5.4 to 9.5 (Klugh and
Miller, 1926). However, most investigators working on Daphnia culture found that these animals generally do very well in a slightly alkaline medium with a pH of somewhere between 7.6 to
8.6 (Klugh and Miller, 1926; Viehoever, 1935; Ivleva, 1973;
Conklin and Provasoli, 1978).
Present State-of-the-Art on Daphnia Culture
For years, cladocerans have shown considerable merit as test animals for studies in population behaviour (Pratt, 1943;
Frank, 1952, 1957; Slobodkin, 1954). They are easy to culture, their life cycle is short, they do not have free egg or larval
stages and, they produce homogeneous populations through 13 parthenogenic reproduction.
General laboratory methods for cultivating daphnids have evolved from the "stable tea" of Banta (1939) to the defined media of Murphy (1970) and D'Agostino and Provasoli (1970). In the laboratory (Bio-Resource Engineering Department, U.B.C.), stock cultures of Daphnia have been maintained using reconditioned pond and distilled water containing dairy manure/ and some green algal species as food.
Most of the studies done so far on Daphnia were basic experiments. To date, only a few studies have been focussed on the mass culture of Daphnia for its possible application in aquaculture (Bogatova and Askerov, 1958; Dewitt and Candland,
1971; Dinges, 1974, 1976; Rees and Oldfather, 1980). All large scale Daphnia studies were based on the utilization of Daphnia for the improvement of wastewater effluent. Dewitt and Candland
(1971) reported a commercial Daphnia harvest of 40 tons in
California stabilization ponds, with one pond yielding 25 tons -z -1 at a rate of 1.5 tons per acre per day(0.0406 kg. m day ).
Bogatova and Askerov (1958), mass culturing Daphnia in concrete tanks on media including yeast and fertilizer, achieved a substantial yield of 76.7 lbs per acre foot per•day(0.0854 kg-m" day"1) . Ik
MATERIALS AND METHODS
Algae
Scenedesmus obiiquus, a major contaminant of algal cultures in the laboratory (Bio-Resource Eng. Dept., U.B.C), was isolated in June, 1980. The alga was grown and maintained under noh-axenic conditions in 25-ml Erlenmeyer flasks in alternating light and darkness (16:8LD) at 20° C. The alga was then transferred to 20.0-ml Erlenmeyer flasks where they were maintained as "starter" cultures. These were used for inoculating'cultures in the experiments.
Daphnia pulex
The freshwater cladoceran, Daphnia pulex F., was obtained from the Civil Engineering Laboratory (U.B.C), and was originally collected from Deer Lake (Burnaby, B.C.) in July,
1978.
Stock cultures of Daphnia pulex were maintained in 20-1 aquaria. The culture medium was reconditioned pond water mixed with distilled water. Digested dairy manure and some green algal species were added as food from time to time. These cultures were kept at laboratory conditions at 20i 2°C and a photoperiod of 16:8LD. Slight aeration was provided to keep the -X dissolved oxygen above the minimum limit ( 3.0 mg-O^l ). 15
Media Preparation
Algal Culture Medium:
Fresh dairy manure was collected from the University of
British Columbia Dairy Barn. Raw wastes were collected in 25-1 plastic pails and immediately taken to the laboratory. Two kilograms of manure was placed in each of three 50-1 plastic garbage buckets containing 20 liters of dechlorinated water.
They were then aerated vigorously with the use of airstones to promote aerobic digestion of the wastes. Lids were provided to prevent any growth of photo-autotrophic organisms. After 10 days, the supernatant was filtered through 9-cm glass fiber filters (Reeve Angel). The liquid was then placed in plastic bags and stored in the freezer at -10°C.
The samples were analyzed for total Kjeldahl nitrogen
(TKN), ammonia-nitrogen, nitrate-nitrogen, nitrite-nitrogen, ortho-phosphate and total phosphates by colorimetric methods after two days of freezing. The ratio between digested dairy waste and distilled water volume in the medium was varied depending on the nitrogen concentration, but on the average the ratio was ca 10 - 15% wastes and 85 - 90% distilled water. The -1 nitrogen concentration used varied from 200 to 2,000 ug-at-N«l .
Daphnia Culture Medium:
Water was collected from a U.B.C pond located infront of the Physics Department. This was filtered using 9-cm glass fiber filters. This was then mixed with distilled water and used for growing Daphnia.
In the Daphnia experiments, reconditioned water from the 16 stock cultures.was used. This was also filtered using glass fiber filters. The green alga, Scenedesmus obiiquus, was supplied as food for Daphnia.
. Description of Culture Units
Algal Culture Unit:
To provide a uniform temperature to the algal cultures, a big water bath container (102cm x 64cm x 10cm) was constructed out of Plexiglas. A Temporite Package water chiller (TR4-20
3/4HP 20GPH) maintained the water temperature at 20°C. A liquid circulating pump (Model D-6 Eastern, 3 1/2GPM at 0 pressure) recirculated the water from the cultures to the chiller and back to the cultures. A generalized view of the whole system is shown in Figure 1. All cultures were arranged in a blocked randomized design fashion.
The algal experiments were conducted using 1-liter flat bottom boiling flasks (Experiments #1 - #6, #9) and 1-gal jars
(Experiments #7 and #8). Six VHO (Daylight 48" Sylvannia) fluorescent lamps provided illumination for the growth of algae. -1 -2 -1 An initial light intensity of ca 0.03 ly-min (113 uE.m sec ) was used. On the sixth day of growth when the cultures got thicker, -1 the light intensity was increased to ca 0.05 ly^min (161 uE-m sec~L) by lowering the light source. Illumination was measured by Quantum/Radiometer/Photometer Sensors (Lambda LI-185). A timer regulated the day and night cycle (16:8LD) of the cultures.
No aeration was used in Experiments #1 thru #3. The flasks were fitted with Neoprene rubber stoppers (No.8). Aeration was
18
incorporated beginning with Experiment #4. Air was first passed through 1N H ^ SO^. solution to dry it and to get rid of contaminants {e.g.NHo, in air) and washed with distilled water before being distributed into the cultures. Plastic tubing
(Tygon R-3'603 0.476cm x 0.635cm x 0.140cm) fitted to glass tubing (0.5cm OD) connected the culture flasks, to the aeration system. Glass tubing outlets were also provided to prevent air pressure build-up. Neoprene rubber stoppers (No.8) supported the glass tubing.
Daphnia Culture Unit:
Daphnia pulex was cultured in 50cm x 30cm x 15cm aquaria of
20-1 capacity. The containers were made shallow because shallow cultures tend to favour Daphnia growth (Viehover, 1935). This tendency was also observed in the laboratory. The experiments were conducted inside a Controlled Environment (CONVIRON) chamber. Temperature was made constant at 20° C and a photoperiod of 16:8LD was maintained throughout the experiments.
Light intensity was ca 0.01 ly-miri"i(50 uE-m^sec"1). No aeration was provided. However, the dissolved oxygen and pH were checked regularly using an Oxygen meter (Model 54 YSI) and a pH/Ion meter (Fisher Model 420 Digital).
Culture Methods
Algal Culture Methods:
None of the culture units remained bacteria-free, however, there was no contamination by other algae.
The density of the inoculum was determined before it was 19 introduced to the culture units. Initial inoculations averaged ca 5,000 - 10,000 cells«ml . Two to three generations were allowed for the algae to acclimatize to the new culture conditions. The algal cells were then counted daily using a haemacytometer (American Optical Corporation). Five squares on the grid of each chamber were counted and the average count, Q,
4 . was multiplied by 10 . Thus, given the average Q, the.density
(cells ml..-), d, of the suspension in the haemacytometer was calculated from the expression,
d = I04x Q.
Four replicate counts were made and the mean value, calculated.
When the cultures reached their peak concentration and started to level-off, the experiment . was terminated. For
Experiment #5, #6 and #8, the culture medium was filtered twice using glass fiber filters and then anlalyzed for nutrients remaining or nutrients not consumed by the algae.
Daphnia Culture Method:
Before the experiments were conducted, stock cultures of
Daphnia were maintained for several generations at the experimental conditions to account for effects of acclimation.
In Experiment #1, 1,000 Daphnia pulex were randomly taken from a homogeneously mixed stock culture and placed in each of three aquaria containing 10 liters of reconditioned pond water.
The initial density was one Daphnia per ml of water. The green alga, Scenedesmus obliquus, grown from dairy manure medium, was used for feeding the Daphnia in the experiments. Cell -1 concentrations of 50,000, 100,000 and 150,000 cells«ml were 20
maintained once a day in each aquarium, respectively. This was done by first making cell counts of the algal feed and the. culture tanks before feeding, and then calculating the volume of feed needed. Only algal cells in their , logarithmic phase of growth were used as was suggested by Ryther (1954). Bacteria and detritus also developed in the tanks and undoubtedly contributed to the food supply, but at all times the algae represented the major food source.
The initial Daphnia biomass was determined and the increase in biomass and total number of Daphnia were noted every three days until peak levels were reached. Fifty Daphn ia were randomly collected from each aquarium. Each animal was placed on a slide and its total length measured with an ocular micrometer. They were then divided into three size classes:
0.60 - 1.70 mm, 1.71 - 2.30 mm and>2.30 mm. They were placed on weighed 4-cm aluminum foil strips, dried for 24 hours at 60 C, cooled in a desiccator and weighed immediately with a CAHN-21
Automatic Electrobalance. Gravid females were noted recording the number of eggs and embryos.
The procedure above was repeated for Experiments #2 and #3.
However, in Experiments #4 and #5, only one feeding level was -1 used, 100,000 cells* ml . This time food was monitored two to three times a day. More attention was given to the physico- chemical conditions of the water. Dissolved oxygen and pH levels were monitored daily. Accumulation of ammonia was also determmined daily with the use of a Technicon Auto-Analyzer II. 21
Chemical Analysis
Kjeldahl Nitrogen:
The method followed was an adaption of Wall et al. (1974) and that in the Technicon Auto-Analyzer Manual (1971b). Five mis of the sample (dairy manure medium) were introduced into a
50 ml digestion tube. To enhance the oxidation-reduction, 0.5 g of a digestion catalyst (composed of 960g K^SO^, 35g CuSO^and 5g
SeOg) were added to each tube. The tubes were then placed in a digestor rack at 3600 C for 6 - 12 hours. Boiling chips were added to prevent excessive bumping during digestion and a small glass funnel was placed on the open end of the tubes to prevent spillage and excessive evaporation. After digestion, the tubes were removed from the digestor and allowed to cool for one to two hours. Each tube was diluted to 50 ml/' with distilled water. Approximately 10 ml of each diluted sample were placed on a rotating sampler of the Auto-Analyzer for Total Kjeldahl
Nitrogen determination. Whenever the auto-analysis graphical recorder went off-scale, the sample was rediluted and again run on the Auto-Analyzer.
Ammonia/Nitrate/Nitrite:
The automated procedure for the determination of the above nitrogeneous compounds was done through colorimetric methods and adapted to that of Technicon Auto-Analyzer II Manual (1969,
1971a). 22
Total Phosphate:
Samples were digested following the same procedure as with the determination of Total Kjeldahl Nitrogen. The automated procedure for the determination of total phosphate was done through colorimetric methods and adapted to that of Technicon
Auto-Analyzer II Manual (1972).
Ortho-Phospahte:
The automated procedure for the determination of ortho- phosphate was done through colorimetric methods and adapted to that of Technicon Auto-Analyzer II Manual (1973).,
Algal Cell Weight
The weight per algal cell was determined by drying and weighing a known volume of algal suspension of known cell concentration. Two hundred ml of algal suspension was filtered on a pre-weighed glass fiber filter which was then placed in a cruicible, dried at 60°C for 30 minutes and weighed on a Mettler
H6 Digital Balance. This was done in triplicates. The weight
per algal cell was calculated from the following equation:
. , (Dry wt. Filter+ algae)-(Dry wt. Filter)(mg) algal cell weight = — (mg.cell-:r) (200 ml) • (Cell cone, cells.ml )
Four additional trials were conducted using different algal
cultures. 23
Growth Rate Experiments (Daphnia)
The growth rate of Daphnia was determined at three different food levels: 50,000, 100,000 and 150,000 Scenedesmus -1 cells- ml . Gravid female Daphnia were placed in a 2-1
Erlenmeyer flask and supplied with excess food. The following day, the newly-born young were collected and used in the experiment. Two hundred-fifty Daphnia each were placed in three
1-1 Erlenmeyer flasks containing 500 mlf1 of reconditioned pond water from previous Daphnia cultures. Food was adjusted to the desired concentration and monitored once a day. The experiment was conducted inside the CONVIRON at 20°C, a photoperiod of -1 2 -1
16:8LD, and a light intensity of ca 0.01 ly.min (50 uE«m sec ).
The initial length (TL) was measured using an ocular micrometer.
Ten Daphnia were sampled from each flask and measured daily until Day 10. The appearance of the first brood and the number of progeny were noted down.
A second trial was repeated. This time, food was monitored twice a day.
Length-Weight Relationship
A length-weight relationship on Daphnia was performed.
Live animals were removed from the experimental flasks by means of a 10-ml pipette, placed on a slide and measured with an ocular micrometer. They were then placed on weighed 4-cm' aluminum foil strips, dried for 24 hours at 60°C, cooled in a desiccator and weighed immediately. Newly-borns, juveniles and adults with and without eggs and embryos were measured. 25
RESULTS
Dairy Waste Analysis
Six dairy manure media were used during the course of the. experiments. Table 1 below summarizes the various chemical
composition (TKN, NH^ -N, NOs-N, NOg-N, total PCfy-and PO^-P) of each medium and the algal experiments in which they were used.
Table 1. Summary of chemical composition of each medium and the algal experiment in which they used.
Media* TKN NH^-N N03-N N02-N PO^-P Total PO^ Algal ug-at.N-l"1 Expt.#
1 3 1 3 I 9. 5x1 0 1 .7x10 2. 4x 1 0 1 . 9x 1 0 1 . 6x1 0 - 1
a 1 II 2. 6x1 1 .2x10* 3. 4x1 0* 2 .7xl0 5 .1x10 - 2 % 3 3 III 3. 1x1 0 1 .3x1 0 2. 1X10 1 .5x1 0^ 3 .6x10 - 3, 4 Z 3 Z Z 2 IV 4 .8x 1 0* 1 .2X10 3. 4x1 0 4 .3x1 0 5 . 0x1 0 1 . 2x1 0 5, 6 z 1 2 8 V 6. 1x10 2 . 6x 1 0^ 3. 3x1 0 1 . 3x1 0 1 .7x1 0 2 .2x10 7
VI 7. 0x1 Q 3 . 1 x 1 03 4. 2x1 01 1 .4x1 0* 1 .5x1 0Z 1 .6X102 8, 9
Note that the chemical composition varied from one medium to the other. Although the dairy wastes were collected from the same source, the rate of manure digestion could have been different each time. 26
Algal Experiments
A summary of all the algal experiments, the different treatments and the results is shown in Table 2.
Algal Experiment #1 was conducted to determine the growth of Scenedesmus obiiquus with increasing nitrogen concentrations.
Statistical analysis using analysis of variance (ANOVA) showed significant differences between treatments (Appendix 11a). The statistical analysis was done through the U.B.C. Computer
System using a General Least Squares Analysis of Variance
Programme known as GENLIN (Greig and Bjerring, 1977). Maximum cell yields increased as nitrogen concentration in the medium increased. Statistical analysis showed significant differences between treatments (Appendix 11b). Daily cell densities are plotted in Figure 2.
In Algal Experiments- #2, #3 and #4, the growth of
Scenedesmus was 'again determined at five different nitrogen concentrations (400, 800, 1,200., 1,600 and 2,000 ug-at • N- l"1 ) .
Statistical analysis showed significant differences between treatments for both growth rates and cell yields in Experiment
#2 (Appendices 12a and 12b). Experiment #3 showed similar results. Statistical analysis showed significant differences between treatments for both growth rates and cell yields
(Appendices 13a and 13b).
Aeration enhanced both growth rates and cell yields in
Experiment #4. Statistical analysis showed significant differences between treatments for both growth rates and cell yields (Appendices 14a and 14b). I
Table 2. Summary of the algal experiments, the different treatments and the results.
Algal TKN NH4-N N03-N N02-N , P04-P Total P04 Aeration pH K Cell Yield Expt. ug-at'W"1 ug-at'W ug-at'Ml uj-at'W" ug-at-P-l ug-at-P-r-1 (dlv.day") (maximum)
2.0XI02 3.5x10 S.OxlO"1 3.6X10"1 3.5 - 7.72 0.88±0.99 0.99x10* 4.0 7.0 , 1.0x10° 7.2 7.0 - 8.35 1.18+0.03 1.42 #1 8.0 ^ 1.4x10* 2.0 1.4x10° 1.4x10 - without 8.51 1.31+0.14 1.44 1.6X10 2.8 4.0 2.9 2.8 - 8.95 0.94+0.01 1.72 3.2 5.6 8.0 5.8 5.6 - 9.20 0.61+0.01 2.07
4.0x1O& 1.8x10 5.2x10 4.1x10 7.7 - 8.65 1.21+0.03 0.82X106 8.0 , 3.6 I.OxlO2 8.2 1.5x10 - 8.71 1.10+0.03 1.36 #2 1.2x10* 5.4 1.6 1.2x10 2.3 - without 8.69 1.00+0.05 1.59 t.6 7.2 2.1 1.6 3.1 - 8.65 0.95+0.03 2.61 2.0 9.0 2.6 2.0 3.9 - 8.63 0.8810.06(1.35+0.07) 2.75
4.0x10* 1.6x10 2.6x10Z 1.9x10 2.1x10 - 8.84 1.22*0.04 1.38X106 8.0 3.2 5.2 3.8 4.2 - 8.83 1.13+0.04 1.33 #3 1.2x10* 4.8 7.8 5.7 6.3 - without 8.84 0.9910.07 1.13 3 1.6 6.4 I.OxlO 7.6 8.4 4 - 8.79 0.9710.04 1.23 2.0 8.0 1.3 9.5 1.1x10 - 8.84 0.8310.04(1.18+0.03) 2.00
4.0x10* 1.6x10 2.6xl02 1.9x10 2.1x10 - 9.12 1.90+0.09 S.01X106 8.0 3.2 5.2 3.8 4.2 - 9.16 1.27+0.03 7.91 #4 1.2x10* 4.8 7.8 5.7 6.3 - with 9.10 0.98+0.02(1.14+0.03 11.11 1.6 6.4 1.0x10' 7.6 8.4 , - 9.04 0.84+0.02(0.93+0.04) 12.79 2.0 8.0 1.3 9.5 1.1x10 - 9.O0 0.75+0.03(0.92+0.03) 15.28
2.0X102 4.9x10 I.SxIO2 1.8x10 1.9 5.2 7.66 0.98+0.09 1.80x106 *S 4.0 9.8 „ 2.6 3.6 3.8 1.0x10 with 8.27 0.98+0.04(1.24+0.04) 4.42 8.0 2.0x10 5.2 , 7.2 7.6 2.1 8.81 0.9810.04(1.15+0.09) 7.15 1.6x10* 3.9 1.0x10* l^xlO"* 1.5X1CT 4.2 8.87 1.01*0.04 9.69
2.0x10* 3.8x10^ 1.3x102 1.8x10 2.9 5.2 7.55 1.75+0.07 2.30X106 *6 4.0 7.6 2.6 3.6 5.8 1.0x10 with 8.32 1.7410.07 4.89 8.0 „ 1.5x10° 5.2 7.2 , 1.2x10 2.1 8.86 1.7410.02 7.47 1.6x10 3.0 1.0x10' 1.4x10 2.3 4.2 9.00 1.30+0.06 11.68
#7 80xt02 3.5xlOZ 4.3 . 1.7x10 2 3x10 2.8x10 with 8.80 1.3410.02 8.01x10fe 8.0 3.5 1.1x10* 1.7 2.3 2.8 8.80 1.30+0.00 9.17
*8 8.0x102 3.5x10Z 4.8 1.7x10 1.7x10 1.8x10 with 8.83 1.90+0.15 3.98x10* 8.0 3.5 4.8 1.7 1.7 1.8 without 8.83 1.60+0.12 1.38
8.0x10Z 3.Sx10Z 4.8 1.6x10 9.3x10 9 5x10 8.92 1.4610.04 4.50x1O6 *9 8.0 3.5 4.B 1.6 1.7 1.8 with 8.92 1.4910.06 4.59 8.0 3.5 8.0x10* 1.6 1.7 1.8 8.92 1.41+0.09 6.41 28
TIME ( days)
Figure 2. Daily cell, density of S_. obliquus grown in varying N-concentrations of dairy manure medium (Algal Expt. #1). The values-are means and ranges of replicates per treatment (n=3K (200 ug-at Nl"1*; H-OOo • 800 * ; 1,600* ; 3,200 •) 29
When the light intensity was increased from 0.03 to 0.05 ly. min"-4-, growth rates dramatically increased in cultures with high nitrogen concentrations. In Experiments #2 and #3, growth rates -1 ~i at 2,000 ug-at-N-1 increased from 0.88 to 1.35 div.day and from
0.83 to 1.18 div.day"'*". In Experiment #4, growth rates at 1,200,
1,600 and 2,000 ug-at•N•l^were increased to 1.14, 0.93 and 0.92 -1 div.day , respectively.
Daily cell densities for Experiments #2, #3 and #4 are plotted in Figures 3, 4 and 5.
Experiment #5 was conducted mainly to determine the amount of nutrients consumed by the algae. Nitrogen concentrations of
200, 400, 800 and 1,600 ug-at-N • l^were used. No significant differences were found between growth rates (Appendix 15a).
However, significant differences were found between cell yields (Appendix 15b). When the light intensity was increased to 0.05 -1 ly.min , higher growth rates were attained at concentrations 400 -l and 800 ug-at-N-1 . Some discrepancies were obtained during the determination of TKN at the end of the experiment since the samples were not 100% free of algae. Thus, higher TKN values were obtained.at the end than initially.
Experiment #6 was conducted with improved means of separating the algae from the culture medium. Although the same nitrogen concentrations were used as in Experiment #5, higher growth rates were achieved. Significant differences were found- between treatments (Appendix 16a). Maximum cell yields were also found to be significantly different (Appendix 16b). Table
3 shows the amount of nutrients consumed by the algae during the
- course of the experiment. All of the NO3-N, N02 N and PO^-P was TIME (days) Figure 3« Daily cell density of S_. obliquus grown in varying N-concentrations of dairy waste medium (Algal Expt. #2). The values are means and ranges of replicates per treatment (n=3). (^00 ug-at Nl-1*; 800 o; 1,200A ; 1,600 A; 2,000B) 31
TIME (days)
Figure Daily cell density of S. obliquus grown in varying N-concentrations of dairy waste medium (Algal Expt. #3). The values are means and ranges of replicates per treatment (n=3). (^00 ug-atN 1_1• ; 800o ; 1,200Aj 1.600A; 2,000a) 32
TIME (days) Figure 5» Daily cell density of S. obliquus growuin varying N-concentrations of dairy waste medium (Algal Expt. #k) \ with aeration. The values are means and ranges of replicates per treatment (n=3). (^00 ug-atN ; 800 o; 1,200 A; 1,600 A; 2,000 •) Table 3. Chemical' composition of media before and after algal growth.
TKN NH4-N N03-N N02-N. P04-P ^ Total Pfl4 ug-at-N-l"1 ug-at-N-l" ug-at-N-l"1- ug-at«N-l ug-at-P. f ug-at.P-1
in out In out . 1n out In out in out in out
2.0x10 1.7x10 3.BX10 -?*0 1.3x10 0 1.8x10 O 3.0 0 5.0
Algal 4.0 2.1 7.6 4.0 2.G O 3.6 O 6.0 O 1.0x10 -*
Expt. + /C6 8.0 2.3 1.5x10 1.1x10 5.2 0 7.2 O 1.2x10 0 2.0 - 1.6x103 2.5 3.0 1.8 1.0x10* O 1.4x108 O 2.4 O 4.0
a 2 2 * 8.0x10 4.4x10 3.5x10 4.0 5.0 O 1.6x10 O 1.7x10 O 1.8x10 -T Algal (w/alr) Expt. ^ HS 8.0 5.1 3.5 7.6x10 5.0 3.0 1.6 2.7 1.7 1.0x10 1.8 (w/o air)
'HfHad difficulty measuring very dilute concentrations In -AutoAnalyzer. 3^ assimilated by all cultures. Traces of NH^-N were found in the cultures containing the lowest nitrogen concentration (200 ug-at-
N • 1 ) and about 5 - 7% of it were left in the rest of the
cultures. Total Kjeldahl Nitrogen (organic + ammonia) was
partly consumed by the algae. Appreciable amounts of TKN -1
ranging from 170 to 250 ug-at'N-1 were left in the cultures.
Plots of daily cell densities for Experiments #5 and #6 are
shown in Figures 6 and 7.
In Experiment #7, the nitrogen to phosphorus atomic ratio
of the medium was adjusted to determine any effects on the cell
yields of the algae. KNO^was added to make a N:P ratio of 64.
This was tested against the original medium (N:P ratio of 17).
As expected, there were no significant differences on the growth
rates (Appendix 17a). A slightly higher maximum cell yield was
achieved with N:P ratio of 64. However, statistical analysis
showed no significant differences between the two values
(Appendix 17b). Note that when the experiment was terminated,
the algal cultures with N:P ratio of 64 have not reached their
peak concentrations yet. Had they been given more time to grow,
their yield could have been significantly different from those
with N:P ratio of 17.
Experiment #9 was conducted to check again, the effect of
varying N:P ratios on the cell yields of the algae. The ratios
investigated were 4, 22 (original medium) and 69. As expected,
growth rates were not affected by varying the N:P ratio
(Appendix 18a). This time, significant differences were
obtained between cell yields (Appendix 18b). Plots of the daily cell densities for Experiments #7 and #9 35
TIME (days) Figure 6. Daily cell density of S. obliquus grown in varying N-concentrations of dairy waste medium (Algal Expt. #5){ with aeration. The values are means and ranges of replicates per treatment (n=3). (200 ug-at N l--*- • ; 400 o ; 800 A; 1,600 A) 36
TIME (days) Figure•?• Daily cell density of S. obliquus grown in varying N-concentrations of dairy waste medium (Algal Expt. #6); with aeration. The values are means and ranges of replicates per treatment (n=3). (200 ug-at N 1 ; 400 o ; 800 A; 1,600 A) 37 are shown in Figures 8 and 9.
Aeration had a pronounced effect on algal growth as shown by the results of Experiment #8. Both higher growth rates and cell yields were obtained by aerated cultures. Statistical analysis showed significant differences between these values
(Appendices 19a and 19b). All of the NO3-N, NOg-N and PO^-P, most of the NH^-N and about half of the TKN were consumed by
aerated cultures (See Table 3). On the other hand, only partial
amounts of nutrients were consumed by non-aerated cultures.
Daily cell densities for Experiment #8 are plotted in
Figure 1.0.
Scenedesmus seemed to have utilized some of the inorganic
nitrogen present in the medium. There was a high correlation (r*
=0.9729) found between the N consumed and the algal yield.
Figure 11 shows this relationship.
Daphnia Experiments
Table 4 shows the amount of algae consumed by Daphnia in
all five experiments. As expected, more algae were consumed at
higher feeding levels.
In general, Daphnia biomass increased from Day 0 to a peak
level (usually at Day 9) after which it started to drop off. In
both Experiments #1 and #2, feeding levels of 100,000 and -1 150,000 cellS'ml achieved the highest biomass values. A maximum -l yield of 126.4 mg for 100,000 cells-ml and 108.8 mg for 150,000 -i
cells, ml were attained in Experiment #1. Higher yields were
reached in Experiment #2: 264.2 and 228.7 mg. At the lowest 38
TIME (days)
Figure 8. Daily cell density of S. obliquus grown in dairy waste medium with nitrogen to phosphorus atomic ratios of 17 and 6k (Algal Expt. #7); with aeration. The values are. means and ranges of replicates per treatment (n=3). (NiP of 17 o j N:P of 6k • ) 39
TIME (days) Figure 9' Daily cell density of S. obliquus grown in dairy waste medium with nitrogen to phosphorus atomic ratios of 4, 22 and 69"' (Algal Expt. #9); with aeration. The values are means and ranges of replicates per treatment (n=3). (N:P of 4»;N:P of 22o;N:P of 69 *•) 40
TIME (days )
Figure 10. Daily cell density of S. ooliquus grown in dairy waste medium with and without aeration (Algal Expt. #8). The values are means and ranges of replicates per treatment (n=3)• (w/air • ; w/o air o) Figure 11. N-consumed vs. algal yield. k2
Table 4.. Amount of algae consumed by Daphnia at 3-day intervals.
(in mg.)
Expt. cells-ml1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12
50,000 - 56.2 114.5. 177.6
£1 100,000. - 124.5 253.2 386.4 .'
150,000 - 195.1 398.2 599.8
50,000 - 60.9 109.6 173.5 242.3
#2 100,000 - 123.5 244.4 366.7 492.6
150,000 - .175.6 361.3 546.4 737.7
50,000 - 57.7 116.3 175.9 244.7
#3 100,000 - 117.6 246.7 370.9 494.7
150.000 - 173.8 364.3 532.2
#4 100,000 - 216.4+ 437.5± 746.3± 1118.5+ 11.9 27.9 26.4 29.9
#5 100,000 - 191.7± 377.8+ 715.0* 1081.8+ 14.7 41.4 41.4 41.4 43
feeding level (50,000 cells«ml ), minimum biomass values were
recorded: 69.0 mg in Experiment #1 and 102.1 mg in Experiment
#2. However, these findings were reversed in Experiment #3.
The highest biomass was attained at the lowest feeding level.
Biomass values were 138.7, 99.1 and 76.2mg at 50,000, 100,000
and 150,000 cellS'inl* feeding levels, respectively. Statistical
analysis showed no significant differences between the three
treatments due to the wide range of values obtained in the three
experiments (Appendix 20a).
The maximum number of Daphnia in the cultures was in the
range 7,000 and 8,000, attaining densities of 0.7 - 0.8 Daphnia'
ml\ These numbers were obtained at all three feeding levels.
Maximum biomass conversion efficiencies were in the range 40 - -1 50%. At 50,000 cells • ml feeding level, biomass conversion
efficienies of 45.3, 47.2 and 49.3% were obtained. At 100,000
• -A ' . cells.ml feeding level, values were 43.2, 49.5 and 21.8%. At -1 .
150,000 cells • ml feeding level, values were 36.2, 38.2 and
15.9%. However, statistical analysis showed no significant
differences between treatments due to the wide range of values
obtained (Appendix 20b).
Intensive feeding did not increase the amount of Daphnia
produced in Experiment #4 amd #5. Maximum yields of only 143.4
and 234.6 mg were obtained. Statistical analysis showed
significant differences at 5% level of significance between the
two trials conducted (Appendix 21a). A much higher frequency of
12,400 Daphnia or a density of 1.24 Daphnia .ml*' was attained by
cultures in Experiment #5. This was due to the increased
reproduction rates with more food available. As expected, biomass conversion efficiencies were much lower due intensive feeding. Maximum efficiencies recorded were 17.7 and 21.0%.
Statistical analysis showed no significant differences between the two values (Appendix 21b).
To get a clearer view of the biomass and biomass conversion efficiencies obtained at each feeding treatment, data were plotted in graphs (Raw 3). Increase in Daphnia:biomass for Experiments #1. thru #5 are shown in Figures 12, 14, 16 and 18, respectively. Conversion efficiencies on the other hand, are shown in Figures 13, 15, 17 and 19. The proportion of juveniles (0.60 - 1.70 mm), young adults (1.71 - 2.30 mm) and adults ( >2.30 mm) in the cultures are depicted in Figures 20 thru 23. The proportion in terms of biomass is also included. The increase in Daphnia frequency coincided with the increase in biomass except for Experiment #2 -1 (100,000 cells«ml feeding level) and Experiment #5, where at Day 12, the number of adults still increased despite the decrease in the total number of animals in the culture. Generally, most of the biomass was accounted for by the adult animals which weigh so many times more than the juveniles. A more detailed size-frequency structure of Daphnia in the cultures was investigated. The number of Daphnia was noted in increments of 0.20 mm at all feeding levels. Figures 24 thru 38 depict these results (Raw data is tabulated in Appendices 4 thru 8). Generally, the Daphnia population increased from Day 0 to a peak level after which huge mortalities in young occurred resulting to a decline in population. There was a big increase 45 150 0 3 6 9 TIME ( days) Figure 12. Daphnia biomass at the three feeding levels at 3-day intervals (D. Expt. #1). (50,000 cells ml-1* 100,0000. 150,0004-) TIME (days) Figure 13• Biomass conversion efficiency of Scenedesmus to Daphnia at the three feeding levels at 3-day--, intervals (D. Expt. #1). (50,000 cells ml-l#) 100,0000; 150,000,4.) 6 9 12 TIME days Figure 14. Daphnia biomass at the three feeding- levels at 3-day intervals (D. Expt. #2). (50,000 cells ml-1^! ioo,ooo05 150,0004) TIME (days) Figure 15. Biomass conversion efficiency of Scenedesmus to Daphnia at the three feeding levels at 3-day intervals (D. Expt. #2). (50,000 cells ml-1^; ioo,oooO; 150,0004) 47 TIME (days) Figure 16. Daptola biomass at the three feeding levels at 3-day intervals (D. Expt. #3). (50,000 cells ml"1^; IOO.OOOO; 150,OOOA) TIME (days) Figure 17. Biomass conversion efficiency of Scenedesmus to Daphnia at the three feeding levels at 3-day _1 intervals (D. Expt. #3). (50,000 cells ml 0i 100,0000; 150,000,4) 200H o E < 2 O CO iooh TIME Figure 18. Daphnia hiomass at 3-day intervals fed with 100,000 Scenedesmus cells ml :2 to 3 times daily (D. Expts. #4 and #5)• The values are means and ranges of replicates v(n=3). (Expt.#4g; Expt.#5D) ;25 T 1 O ffl 6 "9" TIME (days) 12 Figure 19 Biomass conversion efficiency of Scenedesmus to Daphnia in D. Expts. #4 and #5 at 3-day intervals. The values are means and ranges'of replicates (n=3)« (Expt.#4>; Expt.#5d) 49 200t 10000fr D 0.61-1.70 mm Hi.71-2.30 mm E cc • > 2.30 mm CO UJ co 5000 «21001 3 o CD 3 6 3 6 TIME (days) TIME (days) ( A ) 10000I cc HI CQ 5000h 2 z> z 3 6 3 6 TIME (days) ( C ) TIME (days) Figure 20. The proportion of juvenile, young adult and adult Daphnia in terms of number and biomass in cultures at 3-day intervals (D. Expt. #1). (A-50,000 cells ml"1 B- 100,000 cells ml-1, C- 150,000 cells ml-1) lOOOOf D 0.61-1.70 mm 50 I 1.71-2.30 mm DC • >2.30 mm UJ m 5000 3 3 6 3 6 9 TIME (days) TIME (days) 6 TIME (days) TIME TIME (days) The proportion of juvenile;,young adult and adult Daphnia in terms of number and biomass in cultures at the three feeding levels at 3-day intervals (D. Expt. #2). (A- 50,000, B- 100,000, C- 150,000 cellls ml-1) Figure 22. The proportion of juvenile, young adult and adult Daphnia in terms of number and "biomass in cultures at the three feeding levels at 3-day intervals (D. Expt. #3). (Avo50,000f B- 100,000, C- 150,000 cells ml-1) 52 300h —200 10000H E DC CO 111 CO m < 5 100 03 TIME The proportion of juvenile, young adult and adult Figure 23. Daphnia in terms of number and "biomass at 3-day intervals in D. Expts. #k (B)'-arid #5 (A). Figure 2k. Daphnia size-frequency structure at 5°»000 cells ml feeding level at 3-day intervals (D. Expt. #i). 0.60 0L80 1J0O 1.20 1.40 1.60 1.80 2X>0 2.20 2.40 2.60 2.80 3.00 3.20 3.40 TOTAL LENGTH (mm) Figure 25. Daphnia size-frequency structure at 100,000 cells feeding level at 3-day intervals (D. Expt. #1). 55 1,0001 DAY 0 2,000|« DAY 9 1,000 060 0 80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.8 3.00 3.20 3.40 3£0 TOTAL LENGTH (mm) Figure 26. Daphnia size-frequency structure at 150,000 cells ml""1 feeding level at 3-day intervals (D. Expt. #1). 1,000 DAY O 3,0O01- 2000 1,0001- DAY 12 1,000h 0.60 0 80 1.00 1.20 1.40 1.60 1.80 2X)0 2.20 2.40 2.60 2.80 3 00 3 20 TOTAL LENGTH(mm) 000 Figure 27. Daphnia size-frequency structure at 5°» cells feeding level at 3-day intervals (D. Expt. #2). DAY O 57 i.oooh 1,000 DAY 3 DAY 6 2,000 2,000K DAY 12 1,000 a60 0 80 1.00 1.20 140 1.60 180 2.00 2.20 2.40 2.60 2.80 3.00 TOTAL LENGTH(mm) _ -j Figure 28. Daphnia size-frequency structure at 100,000 cells ml feeding level at 3-day intervals (D. Expt. #2). 58 1,000* DAY O DAY 6 i,oooK > o z LU o LU DAY 9 cc 2,000 1,000 0.60 0-80 1.00 1.20 1.40 1.60 180 2.00 2.20 2.40 2.60 2.80 3.00 3.20 TOTAL LENGTH(mm) Figure 29. Daphnia size-frequency structure at 150,000 cells ml -1 feeding level at 3-day intervals (D. Expt. #2). 59 1,000 DAY O 000 Figure 30. Daphnia size-frequency structure at 5°» cells ml" feeding level at 3-day intervals (D. Expt. #3). 60 1, 1,000* DAY 3 DAY 6 1,0001- DAY 9 1,0004" DAY 12 0.60 0.80 tOO 1.20 140 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 TOTAL LENGTH (mm) Figure 31. Daphnia size-frequency structure at 100,000 cells ml-1 feeding level at 3-day intervals (D. Expt. #3). LOOCM- DAY O TOTAL LENGTH(mm) Figure 32. Daphnia size-frequency structure at 150,000 cells ml feeding level at 3-day intervals (D. Expt. #3). 62 D A Y O 1,00(* 1,0001- DAY 3 DAY 6 1,000f >- 5,000+ o z 4,000 3,000}- 2£00t 1,000- 1,0001 0.60 0.80 1.00 1.20 140 160 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3A0 3.60 3.8—40o" TOTAL LENGTH{mm) Figure 33- Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml-1 2 to 3 times daily (D. Expt. #4- Repl. 1). 63 DAY O i^oor 0.60 0.80 1.00 WO 140 1.60 1,80 2JOO 2.20 2.40 2.60 2.80 3.00 3.2 3.40 3.60 3.80 TOTAL LENGTH (mm) Figure 34. Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml-1 2 to 3 times daily(D. Expt. #4- Repl. 2). 64 1,000* DAY 0 O 1,OOOf DAY 3 DAY 6 2,000 > 1,000 oz LU 3 o LU O DAY 9 ipooj- DAY 12 ipoof 0.60 0.80 1.00 1.20 1A0 1.60 1.80 2X>0 2.20 2 AO 2.60 2.80 3.00 3.20 3.40 3.60 3.80 TOTAL LENGTH (mm) Figure 35• Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml"1 2 to 3 times daily (D. Expt. #4- Repl. 3). TOTAL LENGTH [mm) Figure 36. Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml-1 2 to 3"times daily (D. Expt. #5- Repl. 1). 66 Figure 37. Daphnia size-frequency structure at 3-day intervals fe'd with 100,000 cells ml-1 2 to 3 times daily (D. Expt. #5r Repl. 2). 67 1,000 L DAY 0 1,000 k DAY 3 DAY 6 1,000 DAY 9 2,000 1,000 DAY 12 1,000 0.60 0.80 1.00 1.20 140 1.60 1.80 2.00 2.20 240 2.60 2.80 3.00 3.20 3.40 3.60 3.80 TOTAL LENGTH (mm) Figure 38. Daphnia size-frequency structure at 3-day intervals fed with 100,000 cells ml-1 2 to 3 times daily (D. Expt. #5- Repl. 3). 68 in the number of juvenile Daphnia due to the high reproductive rate of the few adults. There was also a steady increase in the number of adults up to the peak level until food became limiting and greatly reduced their reproductive rate and survival. The total number of eggs and embryos at each sampling day was recorded (Table 5). Epphipial eggs were also . noted when cultures aged. In Experiments #4 and #5, water quality was monitored daily. Table 6 shows the fluctuations in pH and Og levels and the accumulation of NH^-N as cultures aged. Algal Cell Weight The weight of Scenedesmus was determined on cell concentrations ranging from 4.14 to 9.20 x 10 per 200 ml of sample. The mean weight was 4.5846 x 10 mg per Scenedesmus cell ± a standard deviation of 1.0818 x 10 . The coefficient of variation was 23.6%. Growth Rate Experiments Growth rates were essentially the same at the three feeding levels up to the third or fourth day after which growth rapidly levelled-off with the average maximum length at the end of the experimental period being dependent on the food level. Statistical analysis shows significant differences between the lengths of Daphnia in both experiments (Appendices 22a and 22b). 69 Table 5. Fecundity of Daphnia cultures in all five experiments Total # of eggs and embryos Expt. cells-ml"1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 50,000 1 ,456 2, 180 636 * (145) (706) # 1 100,000 960 4,130 1,784 793 % (614) 150,000 5,302 1,899 1,110 - (392)* 50,000 3, 168 3,280 1 ,768 1,176 (219) (295)* (392) #2 100,000 500 2,720 4,864 3,080 1,254 (608) (308)* (2787)* 150,000 3,009 1 ,560 3,329 (260) ( 303) (460)* 50,000 2,146 1 ,064 2,198 1,855 j. (419)* (1427* #3 . 100,000 829 2,736 3,479 807 218 M (161 r (851 V 150,000 2,407 1,444 0 * (61 r 2,830 8,722 5,130 1,628 - (88)* #4 100,000 180 2,836 11,344 3,502 9,384 (109) 2,520 9,792 12,799 7,543 2,127 7,191 11,704 7,400 #5 100,000 280 2,210 6,048 8,976 6,533 1,632 5,274 13,696 10,122 iff ( ) epphipial eggs Table 6. Ammonia, pH and DO levels measured in Daphnia culture tanks in Experiment #4 and #5. NH4-N (ug-at' N< 1 ) Expt DAY 0 DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 DAY 1 1 DAY 12 DAY 13 MA 14 . 3 14.3 15.0 15.7 16.4 19.3 24 . 3 26 . 4 28 . 6 32 .9 38.6 38 . 6 43.6 67.9 #5 28 . 6 18.6 2 1.4 10.7 17.9 19.3 17.9 19.3 23 . 6 45.0 60. 7 63.6 75 . 7 101.4 pH Expt DAY 0 DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 DAY 1 1 DAY 12 DAY 13 #4 8 . 38 7 . 83 8.11 8.47 8 . 49 8 . 30 8.19 8.11 7 . 88 7 .65 7 . 48 7 . 36 7 . 60 7 . 74 8 . 06 8 .09 8.61 8 . 80 8 .92 8 . 87 8 .60 8 . 28 7 . 98 7 .93 7.65 7 . 53 7 . 42 7.71 DO (ug-a t' Expt DAY 0 DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 DAY 1 1 DAY 12 DAY 13 #4 240.6 206 . 3 231 . 3 262 . 5 259.4 237 . 2 253 . 1 243.8 265.6 200.0 206 . 3 23 1 . 3 240.6 200.0 #5 231 . 3 265 .6 265.6 268.8 268 . 8 268 . 8 268 .8 268.8 253 . 1 22 1 . 9 209.4 212.5 178 . 1 187.5 o 71 When fed twice a day, Daphnia attained larger sizes. At Day 10, the average total lengths of Daphnia at 50,000, 100,000 -1 and 150,000 cells.ml. feeding level were 2.91, 2.96 and 3.01 mm, respectively. In contrast, total lengths of 2.36, 2.44 and 2.52 mm were attained by Daphnia when fed only once a day. Figures 39 and 40 depict these results (Raw data is tabulated in Appendix 9) . Length-Weight Relationship The relation between the length and dry weight of parthenogenic female Daphnia with and without eggs and embryos in the brood chamber is shown in Figure 41 (Raw data is, tabulated in Appendix 10). These values fit two lines, one during juvenile stage and the other one during adult stage. The regression equation was Y = 14.88X - 10.57 (r = 0.7268) with animals between 0.80 and 1.82 mm in total length and Y = 80.28X 146.18 (r3 = 0.9355) with animals larger than 1.8.2 mm. The length-weight relation was the same regardless of fecundity. Brood Size in Relation to Length Daphnia brood size was investigated during the course of the growth and feeding rate experiments. Table 7 shows the number of eggs and embryos in relation to Daphnia size. The average number of young per brood increased for the first few broods followed by an irregular but relatively high average 72 TIME (days) Figure 39. Daily total length of D. pulex at the three food con• centrations (Growth Rate Expt. #1). The values are means and ranges of replicates per treatment (n=10). (50,000 cells ml"1*; lOO.OOOB; 150,0000) 73 0.8h TIME (days) Figure 40. Daily total length of D. pulex at the three food con• centrations (Growth Rate Expt. #2). The values are means and ranges of replicates per treatment (n=10). (50,000 cells ml-l#; 100,000 150,000Q) 74 0.8 1.0 2.0 3.0 TOTAL LENGTH (mm) Figure 41. Length-weight relationship of D. pulex. (w/eggs and embryostf-'jv.w/o eggs and embryos#) Table 7. Daphnia brood size in relation to total length. (number of eggs and embryos) Fed Fed Fed Fed Length(mm.) 50,000 100,000 1 50 , 000 _± 100,000 ^ cells «ml cells - ml" cells-ml cells.ml 1x a day 1x a day 1x a day 2-3x a day 1.80-2.00 2-4 - '- . 2.01-2.20 3-5* 3-4 , 3-4 , - 2.21-2.40 3-6 3-6 3-6 3-6 2.41-2.60 3-9 4-9 5-9 4-9 2.61-2.80 4-9 4-9 4-13 6-16 2.81-3.00 6-12 6-12 6-10 6-17 3.01-3.20 6-12 6-7 8-12 9-15 3.21-3.40 6- 10-14 1 3- • ; 8-21 3.41-3.60 12-33 3.61-3.80 20-35 3.81-4.00 21- 76 production of young. A maximum of 35 young per brood was recorded. 77 DISCUSSION Livestock Waste as a Nutrient Source in Algal Culture Although the dairy manure was pre-digested for 10 days prior to its use, organic and ammonia-nitrogen still comprised most of the nitrogen content except for Medium III in which large amounts of nitrate- and nitrite-nitrogen were observed. The composition of each medium varied from one batch to another and this might be attributed to the difference in the digestion rates of the manure. Nitrogen appears to be the major nutrient limiting primary production in world's, oceans as well as in certain freshwater systems (Owens and Osaias, 1976). Among the nitrogen forms, ammonium appears to be the primary inorganic N form which enters into synthetic reactions; both N03and NO^must first be reduced to NH^prior to assimilation into proteins. Ammonium is the most energetically favourable source of inorganic N because of its reduced state, and both laboratory and field studies demonstrate that NH4" is taken up in preference to NO^and N02when all forms are present (Owens and Osaias, 1976). The green alga, Scenedesmus obiiquus, is thought to have a slight preference for NH4 over NOj and N02~ (Krauss, 1958). However, in the present study, no preference was found. Both NO3" and NOg were always completely assimilated by the algae but substantial amounts of NH^were always left in the medium after growth. Animal wastes may contain toxic substances from residues and growth stimulants added to the food ration of the animals. 78 However, in the present study, the manure medium did not show any toxic effects to the algae even at higher concentrations. The toxicants may have been reduced to insignificant levels through the digestion process. Algal growth was mainly affected by the light intensity which was much reduced at higher manure concentrations. Algal Growth on Manure Medium Most of the Scenedesmus obiiquus grown in manure medium were composed of unicells. The media contained a high mineral content (e.g.Na+ , K+ , Ca* ) including a high phosphate concentration favouring unicell formation over coenobia. This agrees with the observation of Shubert and Taylor (1974) that Scenedesmus may be found only in areas where there is sufficient phosphate, e.g.near sewage outfalls or run-offs from agricultural areas. Among the nitrogen concentrations used, the growth constant k was highest at lower concentrations while cell yields was highest at higher concentrations. Cell division was affected by the light intensity rather than by the amount of nitrogen present in the medium. The light penetrating the cultures decreased as N-concentration increased. This accounted for the low cell division rates in N-concentrated cultures. However, when the light intensity was increased from 0.03 to 0.05 ly.min^ cell division in N-concentrated cultures also increased to normal rates. High light intensity was not used initially since photo-inhibition might occur in less N-concentrated cultures. 79 Only when the. algae had become dense was the light increased. As the algal concentration increases, penetration of light in the water decreases. A limit is reached at which further production of algae ceases even though an excess of chemical nutrients may be present (Hepher, 1962). Sorokin and Krauss (1958) observed that the light saturation for Scenedesmus obiiquus was 0.02 ly-min . At this intensity, the growth constant k was 2.2 div.day while at.half- saturation 1.intensity it was 1.5 div.day . The growth rates obtained in the present study were lower than these values and this might be because the actual light intensities penetrating the cultures were very much reduced below saturation levels by the medium used. However, the Scenedesmus used here might be of a different clone, a slower grower, than that used by Sorokin and Krauss. The environmental conditions might have been different, too. The first three algal experiments were conducted without aeration. Carbon limitation might have caused the low cell yields achieved by the cultures. These yields were increased five-fold or more when aeration was introduced into the system. Agitation might have reduced the light limitation to the cultures. Cell division rates also increased significantly. Various studies show that the addition of CO^through aeration and agitation enhances algal growth (Gates and Borchardt, 1963; Trainor, 1964; Trainor and Shubert, 1973; Perez et al., 1977). It provides better light utilization, prevents algae from settling or accumulating at the surface where the light intensity is sufficient to deactivate the cells, and 80 provides a more homogeneous environment for the algae. In the experiments, aeration may.have also enhanced bacterial activity transforming some of the organic nitrogen into more usable forms of nitrogen.and producing vitamins for algal growth. Investigators in the past have also showed that the total algal biomass produced on liquid wastes was. determined by the limiting nutrient, usually carbon . (Goldman et al., 1971; Foree et al.-., 1973; Dor et al., 1974; Oswald and Beneham, 1977). The amount of algae may be increased by injecting COginto the ponds to give added carbon. Inorganic nitrogen may be a limiting factor with regards to the amount of *growth which can be produced (Mead et al., 1945). The ratio of nitrogen to phosphorus (N:P) must therefore be given due consideration. In the experiments conducted, the ratios used showed significant difference in algal growth. Growth constants were the same as expected. A higher biomass was produced at N:P of 69 than at 4 or 2.2. Using much higher ratios might result to more convincing results. Rhee (1974, 1978) showed that the optimal yield of Scenedesmus occurred at N:P of 30. Below this optimal ratio, yield was determined solely by N-limitation and above it, by P- limitation. Cell-N remained constant up to the optimal ratio and increased linearly with N:P above it. The level of cell-P was high at low N:P (N-limited state) but decreased rapidly until the ratio approached the optimal and remained constant at a low level at high N:P (P-limited state). No residual inorganic or dissolved N or P was detected in the medium at any N:P examined, indicating excess accumulation of both nutrients. 81 This excess accumulation of N and P was also observed in the present study. Cell division rates varied from time to time despite uniformity in medium and culture conditions. This observation is also true for other algal species (Harrison, personal communication). Daphnia Biomass Production Statistical analysis showed no significant differences between the three feeding levels .used in terms of biomass production and biomass conversion efficiencies. This was due to the wide range in replicates obtained in each treatment. However, slightly bigger biomass values were obtained at the higher feeding levels. The amount of food made available to Daphnia may have been the same despite the feeding level. Scenedesmus cells always settled in the bottom and dinged to the sides of the tanks and therefore, were never made very available to Daphnia. In the higher feeding levels, algal cells settled faster and so the actual concentration of suspended food could have been the same in each treatment. No aeration nor mixing in tanks was employed since Daphnia was very much affected by even slight water agitation. Intensive feeding did not seem to increase the biomass production of Daphnia. When feeding was conducted two to three -1 times daily at 100,000 cells«ml , low biomass values were still obtained. Again, there was the problem of algae settling to the bottom of the tanks. The increased algal feed might have also 82 increased the toxic effects of the residues found in the manure medium. Although low biomass values were produced, Daphnia showed high biomass conversion efficiencies. Maximum efficiencies were in the range 40 - 50%. These values are comparable to the conversion efficiency of 38 - 42% that Sasa et al. (i960) obtained when Chlorella was fed to Daphnia. Population Growth and Size Structure Generally, populations follow a sigmoid population curve. A small population of organisms introduced into a suitable container will increase slowly at first, then its increase will accelerate and finally the increase in population will become very slow and approach a level of upper asymptote (Smith, 1952). This assumes that organisms in the population can be considered identical with each other. This assumption has been demonstrated to be false to Daphnia (Slobodkin,. 1954). Daphnia populations are characterized by intrinsic oscillations due to physiological differences between individual Daphnia and do not follow a sigmoid population curve. This seems to be the case in the present study. Knowledge of the age and structure of metazoan populations is necessary to analyze growth properly (Slobodkin, 1954). Daphnia exhibits no specific characters, making it difficult to describe the age structure of the population. Therefore, the population size structure was considered in this study, instead. In general, the peaks in population number coincided with 83 the maximum proportion of small animals due to the high birth rates. On the other hand, the troughs coincided with the maximum proportion of large animals due to the high juvenile death rate after peak level was reached. Similar results were observed for D.obtusa (Slobodkin, 1954), D.magna (Pratt, 1943) and D.pulicaria (Frank, 1952). In the early stages of population growth the few adults had a high reproductive rate due to the abundance of food resulting in a size-frequency distribution that was skewed towards the small end. The population increased until it was at its numerical peak. At this point, the food supply became limiting. In the competition for food, the small animals do not exert a strong a competitive pressure as large animals and so huge mortalities in young occurred. The reproduction rates of the adults was also greatly .reduced. The size frequency distribution of the population shifted towards,the large animals with low reproduction. The increased severity of competition for food increased the ratio of deaths to births so that the total number of animals in the population curve grew smaller while the size frequency distribution moved towards a larger mean size. Adult animals started to produce epphipial eggs. The same observation was found by Slodbokin (1954) on D.obtusa. When intensive feeding was conducted, Daphnia attained great sizes and high fecundities. More young was produced due to the high reproduction rates. As a result, cultures attained -1 higher frequencies. A density of 1.24 Daphnia.ml was reached, -1 much higher than the usual 0.7 to 0.8 Daphnia » ml in previous experiments. The production of epphipial eggs was negligible or 84 very much reduced. Maximum length and brood size of Daphnia is apparently determined by the abundance of food (Hall, 1964; Daborn et al., 1978). Growth Rate Experiments Daphnia exhibited different growth rates at the three feeding levels when fed once a day with Scenedesmus obliquus. Growth rates were essentially the same up to the third or fourth day after which growth levelled-off with the average maximum length at the end of the experimental period being dependent on the food level. Adult stages are affected more by different food levels than are juvenile stages specifically the growth per instar, the duration of instar and the maximum carapace length (Ingle et al., 1937; Hall, 1964; Richman, 1958). Higher growth rates were observed at the higher food concentrations. Furthermore, the duration of the pre-adult instars was one day shorter at the highest food concentration. Similar results were obtained when Daphnia was fed twice a day at the same food concentrations. However, larger-sized Daphnia were produced. This again shows the relationship between the food level and the maximum size attained by Daphnia. 85 Length-Weight Relationship A strong correlation was found between the length.and weight of Daphnia. The values fit . two lines, one during juvenile stage and the other during adult stage. The same trend was observed in earlier studies (Edmonson, 1956; Richman, 1958; Schindler, 1968; Burns, 1969; Kring and O'Brien, 1976). Daphnia with and without eggs and embryos in the brood chamber showed the same relationship. . This finding does not conform with Richman's observation that Daphnia with eggs and embryos weighed twice as much as animals of the same length with empty brood chambers. Nutritional Inadequacy of Certain Algae to Daphnia Culture Investigators show contradictory results on the adequacy of certain green algae as food for Daphnia. Taub and Dollar (1969) in their studies found that Daphnia failed to reproduce normally when fed with either Chlorella pyrenoidosa or Chlamydomonas reinhardi cultured and suspended in defined medium. Life span was reported shorter and ovulation reduced, and a large percentage of ovulated eggs failed to complete embryonic development. The same algal culture suspended in "biologically conditioned water" supported a normal life span and ample but not maximal reproduction. Taub and Dollar suspected the algae to be deficient in meeting the nutritional requirements of D.pulex, especially with respect to reproduction. They further 86 suggested that proper utilization of the algae seemed to depend on factors supplied by other organisms, probably bacteria, and probably provided in the water from natural sources used in most experiments. On the other hand, Watanabe et al.(l955) reported that the micro-algae Chlorella, Chlamydomonas and Scenedesmus were excellent feeds for Daphnia. Furthermore, Sasa et al. (1960) in their studies were successful in feeding Chlorella to Daphnia obtaining 38 - 42% conversion efficiencies. Gordon (1975) also reported that Daphnia tend to prefer mixed diets. More biomass was achieved when Daphnia was fed with both Chlorella and Scenedesmus than with either algae alone. It must be pointed out that the algal cultures used in the above studies were non- axenic. In the present study, success was achieved in growing Daphnia on Scenedesmus cells. Growth was normal and high reproduction rates were achieved expecially when more food was given. Biomass conversion efficiencies were also high. It has to be pointed out that the cultures were grown in re-conditioned pond water. Detritus and bacteria growing in it may have contributed to the diet of the Daphnia. Ryther (1954) reported that the filtering rate of Daphnia was inhibited by substances produced by the three algal species ( Chlorella, Scenedesmus and Navicula ) he tested. The inhibition was mediated partially by substances which diffused from the cells and accumulated in the water. A much more pronounced effect appeared to be produced by the release of inhibitory products ingested within the animal. Minimum 87 inhibition was produced by actively growing algae from cultures which were in thier log phase of growth. Maximum inhibition was produced by scenescent, non-dividing algae. Ryther further noted that the filtration rate of D.magna decreased as the concentration of food particles increased which he interpreted as an increase in inhibitory factor as food concentration increased. This was contested by O'Brien and de Noyelles (1972) who have shown that the mortality factor which Ryther observed was probably high pH. Other investigators have since demonstrated that the reduction in feeding rate is an accommodation of the animals to increased food concentration (Rigler, 1961; McMahon and Rigler, 1963). Toxicity in Daphnia Culture Rees and Oldfather (1980) suggest that there is a direct relationship between pH and the number of Daphnia ultimately appearing in the cultures. Rise in pH in natural and laboratory conditions correlate with photosynthetic activity as removal of CO^by phytoplankton results in decreased buffering activity of the system. This results to decreased feeding of Daphnia and eventually death. Although cladocerans have an upper limit of pH tolerance between 10.5 and 11.0 (Bogatova, 1952), the greatest filtration rate of D.pulex occurs at pH range 6.0 - 8.0 (Ivanova, 1969). Furthermore, Davis and Ozburn (1969) suggest that D.pulex will not thrive in a pH outside the range 7.0 - 8.7. In the present study, pH range was within safe limits(5.4- 9.5). A slight increase in pH was observed when the cultures 88 reached their peak numbers and started declining. The toxicity of ammonia to aquatic biota is greatly affected by the chemistry of the water in which it is dissolved. Although ammonia toxicity is influenced by alkalinity, temperature, free-Co^ and dissolved (Brown, 1968), the factor of primary importance is pH (McKee and Wolf,- 1968) because it- controls the dissociation of ammonia in solution. Ammonia is not toxic in its undissociated form. The higher the pH, the greater the proportion of ammonia that will be undissociated. Ammonia was already shown to be toxic to Daphnia at elevated pH (Dinges, 1974). In one toxicity study, Parkhurst et al. (1979) found that ammonia was toxic to D.magna at a very -1 . high concentration of 25 mg»l . D.magna thrive in lakes where high ammonia concentrations are found and this explains their very high threshold limit. On the other hand, D.pulex is a pond species and is not ordinarilly exposed to high ammonia concentrations. Only D.magna was reported to grow in stabilization and effluent ponds. In the present study, ammonia levels were very low. There was an accumulation of NH^from the first day of culture until Daphnia reached peak levels and started to drop. This does not necessarily mean that NH^caused the mortalities. At present, the toxicity of ammonia to D.pulex has not been investigated yet, so no conclusions can be made. In addition, the 0^levels were very high, near saturation level. Susceptibility of animals to NHj, poisoning is reduced when 0S levels are maintained near saturation levels (Kinne, 1976). Other toxicities to Daphnia have been investigated. Taub and Dollar (1964) reported the toxicity of salt solutions like 89 KNG^and NaNO^to D.pulex. They also reported the possibility of volatile contaminants in distilled water and to other toxicities that may be introduced through non-inert containers. Hydrogen sulfide was also reported to be toxic to Daphnia in stabilization ponds (Dinges, 1974). Toxic materials might have been present in the dairy manure medium used which could have caused the mortalities in the present study. Scale-up Considerations The study was conducted with the intension of applying it in the Philippines at SEAFDEC (Southeast Asian Fisheries Development Center) Aquaculture Department. Success was achieved in growing Scenedesmus obiiquus on dairy waste medium and this could possibly be expanded into large-scale operations for aquacultural purposes. Agricultural wastes are readily available in the Philippines and the presence of solar energy eliminates the problem of providing artificial light to the cultures. The algae can be either grown in big outdoor tanks or earthen ponds. However, there are certain problems that must be overcome. Proper mixing should be employed to reduce algal settling. Another problem is harvesting the algae. In tanks, this is no problem as water can be pumped directly into the zooplankton cultures. With -earthen ponds, this can not be done as water becomes muddy when disturbed. One solution to this problem is to have the bottom lined with concrete. Another solution is to grow Daphnia with Scenedesmus simultaneously, but this has to be investigated first if it is feasible. 90 Culturing Daphnia under the present method.can not still be applied to large-scale operations. Other strains of Daphnia as well as some other cladocerans (e.g. Moina ) should be tried and hopefully, a much more robust species can be cultured to withstand water agitation. 91 SUMMARY AND CONCLUSIONS The green alga Scenedesmus obliquus was successfully grown in a medium using digested dairy manure as the nutrient source. Best growth rates were achieved by algal cultures containing -1 nitrogen concentrations of 400 and 800 ug-at'N-1 . Decreasing light intensities caused by high biomass at increasing nitrogen concentrations accounted for the lower growth.rates attained by algal cultures with high N-concentrations. On the other hand, cultures with high N-concentrations produced more biomass than those with low N-concentrations. There was a high correlation found between the N-concentration consumed and the algal biomass produced. Evidence showed that Scenedesmus can also utilize organic nitrogen aside from the inorganic nitrogen forms. Aeration enhanced both algal growth rates and biomass yields. There was evidence of COg -limitation in cultures without aeration. Nutrients were readily made available to the algae due to bubbling. The tendency of Scenedesmus cells to settle down was also reduced by aeration. Nitrogen to phosphorus atomic ratio in digested dairy manure was observed to fall in the range 15 to 25. Altering this ratio showed significant difference on algal growth. The medium with a N:P ratio of 69 produced a higher biomass than the two other media (N:P's of 4 and 22). The results show that there is an advantage of adjusting the N:P ratio. The three feeding levels used did not show any significant differences in both Daphnia biomass yields and biomass conversion efficiencies. The algae always settled down in the 92 tanks before being consumed since no aeration was provided. The amount of food available to Daphnia may have been the same despite the feeding level with faster algal settling rate observed in higher feeding levels. Intensive feeding did not increase the biomass production of Daphnia. However, larger-sized Daphnia were produced. Brood size was increased to a maximal of 30 - 40 young per animal. A . -1 . higher density of 1.24 Daphnia-ml was obtained as a result of the increased production of young. Daphnia seem to reach a peak density in culture and decline afterwards. The drop is suspected to be either caused by a shortage of • food supply as cultures get denser or by the degeneration of the water quality. There is the problem of algae settling down even with intensive feeding. Dissolved oxygen and pH levels were always within safe limits. Only the ammonia levels tended to accumulate as cultures aged but in very small amounts. No conclusions can be made regarding NH^toxicity since the toxicity of NH3 to IK Pulex is not known at the present time. This merits investigation. The batch and semi-continuous culture systems appear to be the best methods of growing Dahpnia especially in large-scale operations. Cultures should be totally harvested at their peak level or partially harvested with a sufficient portion allowed to regenerate and so forth. Scenedesmus obliquus though grown successfully in dairy manure does not appear to be a very good food for Daphnia. The cells may be too small and its tendency to settle down is a major problem. 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Expt. cells-ml1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 50,000 36.2 69.0 61.5 — #1 100,000 17.0 67.9 126.4 78.5 - 150,000 87.6 • 108.8 65.6 - 50,000 36.7 60.2 1 02. 1 72.0 #2 100,000 20.2 39.0 112.9 191.9 264.2 150,000 43.9 8.1.6 228.7 100.5 50,000 31.7 38.6 77.3 138.7 #3 100,000 18.1 38.8 66. 1 99. 1 66.6 150,000 31.3 76.2 42.7 - #4 100,000 11.4 52.8+ 88.31 143.4+ 76.11 4.5 13.9 35.3 25.4 #5 100,000 20.5 53.81 80.01 170.2+ 234.6+ 4.8 14.7 4.4 46.6 Appendix 2 Daphnia frequency in cultures at 3-day. intervals. Expt. cells-ml1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 50,000 2,800 3,633 3,533 #1 100,000 1,000 3,500 7,433 3,966 150,000 3,083 8,633 3,266 50,000 1,800 5,466 7,366 3,266 #2 100,000 1,000 2,566 7,600 7,700 6,966 150,000 2,033 6,500 7,566 2,300 50,000 2,333 2,533 5,233 7,133 #3 100,000 1,000 2,533 3,866 4,033 1,933 150,000 2,866 3,800. 1,533 #4 100,000 1,000 1,100+ 5,233+ 7,655+ 3,178± 1 27 869 1918 1260 #5 100,000 1,000 1,233+ 3,511+ 12,400± 8,822± 88 407 1473 1248 105 Appendix 3 Biomass conversion efficiencies of Daphnia cultures at 3-day intervals. (in %) Expt. cells-ml1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 50,000 - 34.2 45.3 . 25.0 # 1 100,000 40.9 43.2 15.9 — 150,000 36.2 23. 1 8.1 50,000 27. 1 36.5 47.2 21.4 #2 100,000 15.2 38.0 46.8 49.5 150,000 13.5 17.0 38.2 10.9 50,000 23.6 17.6 33.7 49.3 #3 100,000 17.6 19.5. 21.8 9.8 150,000 7.5 15.9 4.6 #4 100,000 19.2±2.6 17.5±2.4 17.7+4.9 5.7+2.1 #5 100,000 17.311.2 15.6 + 2.3 21 . 0±1.8 19.9 + 5.0 106 Appendix 4 S1ze-frequency distribution of Dnphnia in cultures at 3-day Intervals (Expt.#1). 50,000 cells-ml" 100,000 cells-ml"-1 -150.000 cells •ml" Class Interval, DAY 0 DAY 3 DAY 6 DAY 9 DAY 0 DAY 3 DAY 6 DAY 9 DAY 0 DAY 3 DAY 6 DAY 9 0 .60-0 .80 120 112 73 0 120 140 149 0 120 185 345 131 0 .81-1 .00 220 728 436 1 , 201 220 840 1 , 487 1 , 348 220 493 2 , 763 1 , 764 1 .01-1 . 20 100 280 944 7 1 100 140 1 ,932 79 100 431 1 ,381 131 1 .21-1 . 40 160 728 872 2 12 160 630 743 0 160 678 1 ,036 0 1.,41- 1 . 60 40 336 73 495 40 630 595 397 40 431 690 0 1 .61-1 . 80 40 56 73 7 1 40 70 297 159 40 0 5 18 65 1 .81-2 .00 80 1 12 145 424 80 140 595 476 80 62 863 196 2 .01-2 . 20 140 26 2 18 14 1 140 4 90 149 7 14 140 62 173 326 2 .2 1-2 . 40 20 224 363 353 20 2 10 1 ,040 397 20 432 518 196 2 ,41-. 2 . 60 • 20 168 0 353 20 0 297 159 20 0 173 131 2 6 1-2 .80 0 0 2 18 353 0 70 0 79 0 62 173 196 2 .8 1-3 .00 60 0 2 18 0 60 70 0 79 GO 123 0 85 3 01-3 . 20 0 0 0 0 0 0 149 0 0 62 0 0 3 .2 1-3 . 40 0 0 O 0 0 0 0 79 0 0 0 65 3 .41- 3 . 60 0 0 0 0 0 0 0 0 0 62 0 0 3 .61- 3 . 80 0 0 0 0 0 0 0 0 0 0 0 0 3 .8 1-4 .00 0 0 0 0 O 0 0 0 0 0 0 0 Tota 1 1 ,000 2.800 3,,63 3 3 , 533 1 ,000 3,500 7 ,433 3,966 1I .000 3,08 3 8.633 3.266 Size-frequency distribution of Daphnia In cultures at 3-day 1nterva1s(Expt.H7 ) 50.000 eel IS' ml"1 1OO,000 eel 1 s • m 1 "A 150,OOO eel 1s.ml Class Interva1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 0 . 6 1- 0 . 80 40 36 109 0 0 40 5 1 152 0 75 40 0 0 0 0 0 . 8 1- 1 .00 100 432 1 . 858 3, 388 1 , 306 100 975 1 ,, 368 770 139 100 69 1 1 ,040 15 1 0 1 .01 - 1 . 20 120 180 1,093 1,.47 3 587 120 257 2,, 280 308 557 120 285 1 ,430 303 0 > 1 .21 - 1 . 40 120 144 438 442 0 1.20 308 456 1 , 232 4 18 120 203 650 908 - 0 1 . 4 1- 1 .60 80 180 656 590 O 80 103 912 914 557 80 122 1 , 170 908 138 CD 1 .61 - 1 . 80 80 144 2 19 295 131 80 205 456 1 , 848 557 80 122 260 1 ,815 230 1 .81 -2 .00 80 144 109 442 131 80 0 304 154 697 80 81 650 454 46 Ch H' 2 .0. 1 -2 . 20 120 72 219 0 131 120 205 304 462 557 120 4 1 520 303 4 14 2 . 2 1-2 . 40 140 252 109 147 131 140 154 456 924 697 140 122 520 2 ,118 460 X 2 . 4 -12 .60 40 72 438 44 2 26 1 40 308 456 462 1,811 40 244 0 303 644 2 .6. 1 -2 .80 60 36 109 0 457 60 0 304 462 697 60 81 0 303 368 2 . 8 1-3 .00 20 72 109 147 131 20 0 152 154 279 20 4 1 130 O 0 3 .0. 1 -3 . 20 0 36 0 0 0 0 0 0 0 0 0 0 130 0 0 3 . 2 -13 . 40 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 . 4 1-3 . 60 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 .6. 1 -3 . 80 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 3 . 8 1-4 .00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 1 ,000 1 ,800 5,466 7. 36G 3,266 1 ,ooo 2 , 566 7 ,60 0 7 , 600 6.966 1 .000 2,,03 3 6,500 7 , 566 2 , 300 I-1 O -v3 Size-frequency distribution of Daphnia 1n cultures at 3-day 1 nterva 1 s ( E xpt .//3 ) 50,000 cells-mr1 100.000 cells-ml"-1 150,OOO cells-ml*-1 Class Interval DAY 0 DAY 3 DAY G DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 0 .61-0.80 20 0 0 105 0 20 101 0 0 0 20 0 0 0 0 . 81 - 1.00 340 980 405 2 , 302 2 ,568 340 507 773 887 0 340 688 304 61 1 .01- 1 .20 80 140 203 1 . 150 856 80 405 155 8 1 39 80 745 228 61 1 .2 1 - 1 .40 80 280 557 209 428 80 304 6 18 823 155 80 516 532 0 1 .4 1 - 1 . 60 60 93 658 105 142 60 253 464 0 155 60 0 836 123 1 .61-1 .80 40 140 152 105 428 40 _152 464 7 16 348 40 57 380 307 1 .81 -2.00 60 47 203 105 7 14 60 203 - 309 403 385 60 1 15 684 276 1 .01-2.20 40 233 101 3 14 0 40 O 232 484 232 40 286 304 582 2 .21-2.4. 0 120 93 5 1 105 7 14 120 101 309 484 309 120 1 15 228 92 2 . 4 1 -2 .60 140 47 101 3 14 570 140 203 155 403 155 140 1 15 152 31 2 61-2.80 20 186 51 105 428 20 152 155 81 155 20 229 76 0 2 .8 1 - 3.00 0 47 5 1 209 285 0 101 232 161 0 O 0 76 0 3 ..01-3.2 0 0 0 0 105 0 0 51 0 0 0 O 0 0 3 .2 1 -3.40 0 47 0 0 0 0 0 0 0 0 O 0 0 O 3 .41-3.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 .61-3.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 3 .8 1 - 4.00 0 0 0 0 0 0 0 0 0 0 O 0 0 • 0 \ Tota 1 1.000 2,,33 3 2,,53 3 5. 233 7. 133 1 .000 2.,53 3 3.,86 6 4 .033 1 .933 1.000 2 ,866 3 .800 1 . 533 Size-frequency distribution of Oaphnla In cultures at 3-day 1nterva1s(Expt . #4 ) Replicate 1 Replicate 2 Replicate 3 Class Interval DAY O DAY 3 DAY 6 DAY 9 DAY 12 DAY O DAY 3 DAY 6 DAY 9 DAY 12 DAY O DAY 3 DAY 6 DAY 9 DAY 12 0. 6 1-0.8 0 o 21 187 592 44 0 0 249 0 368 0 0 192 133 55 0. 81-1 ..0 0 320 248 1 ,213 5 . 130 880 320 354 1,994 2 ,959 2 , 300 320 240 2 , 208 1,466 1 ,476 1. 01-1 . 20 320 83 840 395 0 320 101 1 ,496 257 276 320 100 672 267 109 1..21-1 .. 40 160 21 654 395 0 160 51 748 257 92 160 0 576 800 109 1..41-1 ..6 0 20 0 373 1 , 579 44 20 76 623 257 - 92 20 40 192 667 0 1..61- 1 . 80 40 21 747 197 132 40 101 125 643 O 40 40 384 400 55 1 .81-2 .00 40 41 0 987 176 40 127 249 386 0 40 140 0 1 ,066 0 2 .01-2 . 20 0 4 1 93 197 220 0 76 125 386 184 O 20 96 267 109 2 .21-2 . 40 O 4 1 93 0 440 O 0 0 901 368 0 60 0 800 164 2 .41-2 .60 40 83 93 197 132 40 0 125 129 552 40 0 96 133 273 2 .61-2 .80 0 103 93 197 44 0 25 0 129 92 0 0 0 267 109 2 .81-3 .00 60 83 0 0 0 60 51 125 0 92 60 180 0 0 55 3 .01-3 . 20 0 123 0 0 44 0 51 0 0 0 0 60 0 0 55 3 .21-3 .40 0 103 0 0 0 0 177 0 0 0 0 120 96 0 0 3 .41-3 .60 0 2 1 93 0 0 0 76 125 0 92 0 0 192 133 164 3 .61-3 .80 0 0 187 0 0 0 0 249 129 92 0 0 96 267 0 3 .81-4 ,oo 0 0 0 0 44 0 0 O O 0 0 0 0 0 0 Total 1.000 1.033 4.666 9.866 2,200 1,000 1,266 6,233 6,433 4,600 1.000 1.000 4.800 6,666 2,733 Size-frequency distribution of Daphnia In cultures at 3-day 1nterva1s(Expt.US ) . Replicate 1 Replicate 2 Replicate 3 Z1 ass Interva1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 0 .61 -0 . 80 0 0 310 0 0 0 26 2 16 528 0 0 23 184 O 0 0 . 8 1- 1 .00 160 76 2 , 165 3., 458 1 , 294 160 78 2 ,088 4 . 224 2 ,053 160 204 1 .657 2 , 354 1 . 363 1 .01 - 1. 20 140 5 1 155 2 , 926 296 140 52 72 2 .640 747 140 O 61 2 , 140 779 1 . 2 1- 1. 40 40 101 465 3 .45, 8 148 40 26 288 1 . 848 560 40 0 368 1 . 926 389 1 . 4 1- 1 .60 260 101 77 1 ., 330 296 260 104 0 2 .112 747 260 45 184 1 , 926 1 , 168 1 .61 - 1 . 80 60 76 77 0 888 60 78 72 0 933 60 0 0 856 973 1 ,81 -2 .00 60 152 0 798 592 60 130 0 264 560 60 63 61 0 1 , 363 2 .01 -2 . 20 60 177 77 532 1 .036 60 182 72 264 560 60 , 226 184 2 14 779 2 . 2 1- 2 . 40 20 177 77 0 1 , 332 20 260 144 264 1 , 680 20 204 61 214 1 , 168 2 . 4 1-2 .60 100 51 0 0 , 148 100 130 288 0 933 100 136 61 0 584 2 61 -2 . 80 40 152 77 266 148 40 52 72 792 0 40 91 0 642 194 2 .8 1 -3 .0 0 20 127 77 0 148 20 104 144 0 0 20 136 184 214 0 3 .0 1 -3 .2 0 0 0 155 266 296 0 0 72 0 373 0 0 0 0 584 3 .2 1 -3 .4 0 20 0 77 0 148 20 52 72 264 187 20 0 0 0 389 3 .4 1 -2 .6 0 20 25 77 266 0 20 26 0 0 0 20 0 0 2 14 0 3 .6 1 -3 .B O 0 0 0 0 0 0 0 0 0 0 0 0 6 1 0 0 3 .8 1 -4 .O O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 1.00O 1.266 3.866 13000 7.400 1.000 1.300 3,600 13200 9.333 1.OOO 1,133 3.066 10700 9,733 Dally measurements of Daphnia total length and size range In Growth Rate Experiment #1. Total Length (mm.) Feeding level DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 50.000 cells-mf* 0.85+0.06 1.08+0.07 1.42+0.07 1.53*0.15 1.66*0.08 1.91*0.09 1.96*0.05 2*22*0.16 2.16+0.03 2.36+0.0 100.000 eel Is-ml"^ 0.85*0.06 1.12+0.07 1.44*0.08 1.76*0.08 1.82+0.21 2.68+0.09 2.42*0.08 2*4*5+0.12 2.43+0.06 2.44+0.0 150.OOO cells.ml' 0.85+0,06 1.12+0.05 1.45+0.08 1.74+0.09 1?88+0.20 2.15+0.13 2*^20+0.08 2.57+0.16 2.47+0.12 2.52+0.0 *f1rst adult instar observed •••first youngs observed Size Range (mm.) Feeding level DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 " DAY 7 DAY 8~ DAY 9 DAY 10 50.OOO eel Is-ml"^ 0.83/0.89 0.99/1. 19 1.29/1.52 1.35/1.78 1.45/1.75 1.75/2.08 1.91/2.01 2.11/2.31 2.14/2.21 2.24/2.4 100.000 eel Is. ml'+ 0.83/0.89 1 .06/1 .19 1 . 29/1 . 55 1 .62/1 .88 1 .58/2. 15 '1.91/2.18 2.31/2.54 2.28/2.61 2.34/2.51 2 . 38/2 . 6 150.OOO cells-ml"1 O.83/0,89 1.06/1.19 1.39/1.52 1.58/1.88 1.72/2.21 1.88/2.28 2.18/2.31 2.41/2.84 2.31/2.64 2.38/2.6 Dally measurement of Daphnia total length and size range in Growth Rate Experiment til . -y Total Length (mm.) Feeding level DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 50.000 cells-mf* 0.92+0.11 1.14*0.09 1.42*0.15 1.77+0.132.06+0.21 2*34*0.21 2.52+0.102*59+0.192.92*0.072.91*0.0 100.000 eel Is.ml"* 0.92*0. 1 1 1.14+0.11 1.45+0.16 1.78+0.15 2 . 15+0 . 09 2 ?*44+0. 17 2.62t0.22 2**33+0.13 2.94+0.04 2.96+0.0 150.000 cells-ml"1 0.92+0.11 1.13+0.07 1.49±0.14 1.81+0.16 2.*32+0.10 2.51+0.14 2**61+0.16 2.71+0.10 3.00+0.11 3.01+0.0 *ftrst adult instar observed **first youngs observed S1 ze Range (mm. ) Feeding level DAY 1 DAY 2 DAY 3 DAY 4 DAY* 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 50.OOO eel Is.mf* 0.76/1 . 12 1.02/1.32 1.16/1.55 1.58/1.95 1.75/2.31 2.1 1/2.67 2.31/2.64 2.08/2.74 2.81/2.97 2.87/2.9 100.OOO eel 1s-ml 0.76/1.12 1.02/1.09 1.19/1.78 1.55/2.01 2.05/2.28 2.24/2.67 2.28/2.94 2.44/2.81 2.87/2.97 2.90/3.0 150.OOO cells-ml 0.76/1.12 1.06/1.25 1.19/1.68 1.62/1.98 2.18/2.44 2.34/2.77 2.31/2.81 2.57/2.94 2.94/3.14 2.94/3.1 112 Appendix 10 Length-weight relationship of Daphnia with and without eggs and embryos. Daphnia w/o eggs & embryos Daphnia with eggs & embryos T.Length(mm.) Weight(l0 mg.) T.Length(mm.) WeightOO mg.) 0.84 2.1 1.93 20.5 0.92 5.3 1 .96 26.4 1.08 4.7 2.15 28.0 1.13 7.8 2.16 35.8 1.14 6.6 2.17 28.7 1 .32 7.5 2.20 20.8 1 .35 9.6 2.22 31.4 1 .42 7.5 2.22 33.2 1 .45 10.0 2.36 33.0 1 .49 10.8 2.41 37.3 1 .53 9.1 2.42 46.6 1 .66 17.3 2.44 36.0 1 .74 12.5 2.45 48.8 1 .75 23.1 2.51 51 .2 1 .76 11.7 2.51 57.0 1 .77 19.3 2.54 55.5 1 .78 17.0 2.56 56.9 1 .80 12.5 2.56 61.5 1.81 14.7 2.61 62.3 1 .82 19.2 2.63 69.6 2.06 27.0 2.64 78.0 2.07 31.2 2.66 56.7 2.08 25.0 2.66 63.5 2.15 29. 0 2.69 67. 1 2.19 38.2 2.86 69.0 2.27 24.0 2.91 82.2 2.31 44.0 2.92 89.4 2.31 50.0 2.94 76.6 2.33 31.5 3.02 84.4 2.38 45.5 3.06 114.8 2.43 32.0 3.07 96.2 2.43 47.0 3.08 97.8 2.45 59.0 3.14 108.0 2.54 66.0 3.27 124.0 2.61 57.0 3.37 144.0 2.67 68.0 3.50 146.0 2.74 55.0 3.63 150.0 2.77 90.0 3.70 141.0 2.84 78.0 3.86 173.0 2.97 91 .0 3.10 76.0 3.17 125.0 3.54 143.0 1 Appendix 11a ALG EXPT #1 Analysis for K Analysis of variance table Sum of Mean F-rat1o Probability Test term Source squares DF square 46.477 0.00131 RESIDUAL TREAT 0.S9S76 4 . 0.14919 0.16810E-01 5.2368 0.08401 RESIDUAL REP 0.1681OE-01 1 . 0.3210OE-O2 Residual 0.12840E-01 4. Total 0.62641 9. Overal1 Overal1 mean standard deviation O.9830O O.26382 Frequencies, means, standard deviations for TREAT 1. 4. 2 2 2 2 0.61000 0.87500 1 . 18O0 1 .3100 0.940O0 O MEAN 1 . 1800 1 .3100 0.94000 0.61000 OSTDV 0.8750o1l9^E-00 1 OMSEE-01 O'MM o! 14,4^-01 0.14142E-01 S ERR M OAwlil-Ol 0.4O062E-01 0.40O62E-01 0.40062E-01 0.40062E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probability of freedom Ch1-square Probability warn TREAT 5.1516 0.27209 4 9.8718 0.04264 < 10 Time for homogeneity of variance test was 0.18229E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 4 homogeneous subsets which are listed as follows: ) . 4. ) . 2. ) , 3. ) Duncan test at 5% probability level There are 3 homogeneous subsets which are 1tsted as follows: ( 5. ) ( 1.. 4. ) ( 2., 3. ) Time for multiple range test was 0.58724E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 5 5 0 MEAN 0.94200 1.0240 P MEAN 0.94200 1.0240 0 STDV 0.25193 0.29821 S ERR M 0.25338E-01 O.25338E-01 Homogeneity of variance test Size Bartlett Degrees Layard Probability warn Factors Chl-square Probability of freedom Chi-square 0.65428 < 10 REP 0.10064 0.75106 1 0.20054 Time for homogeneity of variance test was 0.16667E-02 seconds. Multiple range tests F-ratlo is not significant at probability 0.08401 STOP 114 Appendix lib- ALG EXPT #1 Analysis for CELLY Analysis of variance table Sum of Mean Source squares DF square F-rat lo Probability Test term TREAT 0.12786E+13 4. 0.31966E+12 18.S82 0.00748 RESIDUAL REP 0.84100E+10 1 . 0.8410OE+10 0.49153 0.52189 RESIDUAL Residual O.68440E+1 1 4. 0.17110E+11 Total 0.135S5E+13 9. Overal1 Overall mean standard deviation 0.15286E+07 0.38808E+06 Frequencies, means, standard deviations for TREAT 1 . 2. 3. 4. 5. 2 2 2 2 2 0 MEAN 0.99350E+06 0.14155E+07 0.14400E+07 0.17225E+07 0.20715E+07 P MEAN 0.993S0E+0S O.14155E+07 0.14400E+07 0.17225E+07 0.20715E+07 0 STDV 2121.3 31820. 53740. 19092. 0.26941E+06 S ERR M 92493. 92493. 92493. 92493. 92493. Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probability of freedom Cht-square Probability warn TREAT 10.512 0.03263 4 18.098 0.00118 < 10 Time for homogeneity of variance test was 0.16797E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 3 homogeneous subsets which are listed as follows: ( 1.. 2., 3. ) ( 2.. 3.. 4. ) ( 4.. 5. ) Duncan test at 5% probability level There are 3 homogeneous subsets which are listed as follows: ( 1. ) ( 2.. 3., 4. ) ( 4.5.) Time for multiple range test was 0.56380E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 5 S 0 MEAN 0.15576E+07 0. 14996E+07 P MEAN 0.15576E+07 0.14996E+07 0 STOV 0.47324E+OG 0. 33587E»06 S ERR M 58498. 58498. Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probability warn REP 0.41008 0.52193 1 0.61398 0.43329 < 10 Time for homogeneity of'variance test was 0.17579E-02 seconds. Multiple range tests F-rat1o Is not significant at probability 0.52189 STOP Appendix 1-2 a ALG EXPT #2 Analysis for K Analysis of variance tablt Sum of Mean Source squares DF square F-rat 1o Probability Test term TREAT 0.201 16 4. 0.50290E-01 20.781 0.00028 RESIDUAL REP 0.39996E-04 2 . 0.19998E-04 0.82636E-02 0.99178 RESIDUAL Residual 0.19360E-01 8. 0.24200E-02 Total 0.22056 14 . Overal1 Overall mean standard deviation 1.0260 O.12552 Frequencies, means, standard deviations for TREAT t. 2. 3. 4. 5. 3 3 3 3 3 O MEAN 1.2067 1.1000 0.99667 0.95000 0.87667 P MEAN 1.2067 I.10OO 0.99667 0.95000 0.87667 0 STDV 0.30551E-01 0.34641E-01 0.50332E-01 0.300OOE-01 0.64291E-01 S ERR M 0.28402E-01 0.28402E-01 0.28402E-01 0.28402E-01 0.28402E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom Chl-square Probability warn TREAT 1.5947 0.80974 4 2.5675 0.63259 < 10 Time for homogeneity of variance test was 0.17188E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 3 homogeneous subsets which are listed as follows: ( 5.. 4.. 3. ) ( 3.2.) ( 2.1.) Duncan test at 5% probability level There are 4 homogeneous subsets which are listed as follows: ( 5.. 4. ) ( 4.3.) ( 1. ) Time for multiple range test was 0.61719E-02 seconds. Frequencies, means, standard deviations for REP 5 5 5 0 MEAN 1.0240 1.0260 1 .0280 P MEAN 1.0240 1.0260 1.0280 0 STDV 0.14011 0.13278 0. 13368 S ERR M 0.22OO0E-01 0.22OO0E-01 0.220OOE-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probability warn REP 0.12459E-01 0.99379 2 0.2348IE-01 0.98833 < 10 for homogeneity of variance test was 0.16146E-02 seconds. Multiple range tests F-ratlo is not significant at probability 0.99178 STOP lie-' Appendix 12b ALG EXPT HI Analysis for CELLY Analysis of variance table Sum of Mean Source squares DF square F-rat 1o Probability Test term TREAT 0.82405E+13 4. 0.20601E+13 91.81.5 0.00000 RESIDUAL REP ' 0.34830E+11 2. 0. 174 15E+ 1 1 0.77614 0.49196 RESIDUAL Residual 0.17950E+12 8. 0.22438E+11 Total 0.84548E+13 14 . Overa11 Overa11 mean standard deviation CELLY 0. 18270E+07 0. 77712E+06 Frequencies, means, standard deviations for TREAT 1 . 2 . 3 . 4 . 5 . 3 3 3 3 0 MEAN 0.82300E+06 0. 13583E+07 0.15943E+07 0.26057E+07 0.27537E+07 P MEAN 0.82300E+06 0. 13583E+07 0.15943E+07 0.26057E+07 0.27537E+07 0 STDV 22000. 51326 . 0.14889E+06 88008. 0.27227E+06 S ERR M 86483. 86483. 86483. 86483. 86483. Homogeneity of variance test Bartlett Degrees Layard Size Factors Cht-square Probability of freedom Chi-square Probability warn TREAT 9.3835 0.05220 4 11.382 0.02259 < 10 Time for homogeneity of variance test was 0.16667E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 3 homogeneous subsets which are listed as follows: ( 1. ) ( 2., 3. ) ( 4.. 5. ) Duncan test at 5% probability level There are 3 homogeneous subsets which are listed as follows: ( 1. ) ( 2.. 3. ) ( 4.. 5. ) Time for multiple range test was 0.56511E-02 seconds. Frequehc i es, means, standard deviations for REP . 1 .2 .3 5 0 MEAN 0.18180E+07 0.18900E+07 0.17730E+07 P MEAN 0. 18180E+07 0.18900E+07 0.17730E+07 0 STDV 0.87 18 1E+06 0.90660E+06 0.72321E+06 S ERR M 66989. 66989. 66989. Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch i-square Probability of freedom Chi-square Probability warn REP 0. 20056 0.90458 2 0.58680 0.74572 < 10 Time for homogeneity of variance test was 0.15755E-02 seconds. STOP Appendix 13a ALG EXPT #3 Analysis for K Analysis of variance table Sum of Mean F-ratlo Probability Test term Source squares DF square TREAT 0.27183 4 . 0 G7957E-01 24.607 0.00015 RESIDUAL REP 0.17334E-03 2. 0.86670E-04 0.31383E-01 0.96922 RESIDUAL Residual 0.22093E-01 8. 0.27617E-02 Total 0.29409 14 . Overa11 Overall mean standard deviation 1.0273 0.14494 Frequencies, means, standard deviations for TREAT 1. 2. 3. 4. 5. 3 3 3 3 3 0 MEAN 1.2200 1.1267 0.99000 O.97000 0.83000 P MEAN 1.2200 1.1267 0.99000 0.970O0 O.8300O 0 STDV 0.40O00E-01 0.41633E-01 0.65574E-01 0.43589E-01 0.40O00E-01 S ERR M 0.30341E-01 0.3O341E-01 0.30341E-01 0.30341E-01 0.30341E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probability warn TREAT 0.68668 0.95296 4 1.1573 0.88507 < 10 Time for homogeneity of variance test was 0.17058E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 4 homogeneous subsets which are listed as follows: ( 5., 4. ) ( 4.. 3. ) ( 3.2.) (2.1.) Duncan test at 5% probability level There are 3 homogeneous subsets which are listed as follows: ( 5. ) ( 4.. 3. ) ( 2.1.) Time for multiple range test was 0.66536E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 .3 5 5 5 0 MEAN 1.0260 1.0240 1.0320 P MEAN 1.0260 1.0240 1.0320 0 STDV 0.13957 0.14223 O. 18377 S ERR M 0.23502E-01 0.23502E-01 O.23502E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square ProbabiIity warn REP 0.35667 0.83666 2 O.67542 0.71340 < 10 Time for homogeneity of variance test was 0. 16016E-02 seconds. Multiple range tests F-ratio is not significant at probability 0.96922 STOP 118 Appendix 13b ALG EXPT #3 Analysis for CELLY Analysis of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 0. 13804E+13 4 . 0.34509E+12 15.462 0.00078 RESIDUAL REP 0.31712E+11 2 . 0. 1585GE+1 1 0.71042 0. 52000 RESIDUAL Res1dual 0. 17855E+12 8 . 0. 22319E+11 Total 0. 15906E+13 14 . Overal1 Overall mean standard deviation CELLY 0. 14130E+07 0.33707E+06 Frequencies, means, standard deviations for TREAT 1 2 . 5. 3 3 3 3 3 0 MEAN 0.13757E+07 0.13278E+07 0.11325E+07 0.12327E+07 0.19963E+07 P MEAN 0.13757E+07 0.13278E+07 0.11325E+07 0.12327E+07 0.19963E+07 0 STDV 0.24254E+06 0.17719E+06 0.11504E+06 30587. 27186.. S ERR M 86253. 86253. 86253. 86253. 86253. Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom Ch1-square Probability warn TREAT 9.1561 0.05731 4 15.583 0.00363 < 10 Time for homogeneity of variance test was 0.16667E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 2 homogeneous subsets which are listed as follows: ( 3..4..2..1.) / ( 5. ), Duncan test at 5% probability level There are 2 homogeneous subsets which are listed as follows: ( 3..4..2..1. ) ( 5. ) Time for multiple range test was 0.51302E-02 seconds. Frequencies, means, standard deviations for REP 0 MEAN 0. 14700E+07 0.13574E+07 0.14 116E+07 P MEAN 0. 14700E+07 0.13574E+07 0. 14116E+07 0 STDV 0.37611E+06 0.34411E+06 0.36036E+06 S ERR M 66812. 66812. 66812. Homogeneity of variance test Bartlett Degrees Layard' Size Factors Ch1-square Probability of freedom Chl-square Probability warn REP 0.28412E-01 0.98589 2 0.40041E-01 0.98018 < 10 Time for homogeneity of variance test was 0.16016E-02 seconds. Multiple range tests F-ratio is not significant at probability 0.52000 STOP 11 Appendix ika. ALG EXPT #4 Analysis for K Analysis of variance table Sum of Mean Source squares DF square F-ratio Probability Test term TREAT 2.5603 4. 0.64007 241.23 O.OOOOO RESIDUAL REP 0.37334E-03 2. 0.18667E-03 0.70352E-01 0.93264 RESIDUAL Residual 0.21227E-01 8. 0.26533E-02 Total 2.5819 14. Overal1 Overall mean standard deviation 1.1473 0.42944 Frequencies^'means. standard deviations for TREAT 3 3 3 3.J — 0 MEAN 1.8967 1.2667 0.98000 0.84000 0.75333 P MEAN 1.8967 1.2667 O.98000 0.84000 0.75333 0 STDV 0.92916E-01 0.25166E-01 0.17320E-01 0.20O00E-01 0.28867E-01 S ERR M 0.29740E-01 0.29740E-01 0.2974OE-01 0.29740E-01 0.29740E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom Chl-square Probability warn TREAT 7.4238 0.11512 4 4.3685 0.35842 < 10 Time for homogeneity of variance test was 0.16927E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 4 homogeneous subsets which are listed as follows: ( 5.. 4. ) ( 4.3.) ( 2. ) ( 1. ) Duncan test at 5% probability level There are 4 homogeneous subsets which are listed as follows: ( 5.4.) ( 3. ) ( 2. ) ( 1. ) Time for multiple range test was 0.66667E-02 seconds. Frequencies~~means. standard deviations for REP . 1 5 5 S n ucAM 1 1420 1•154° 1 1460 2 M1£N 1420 L1S40 1-1460 0 STDV O 4 22° 0.48066 0.49435 S ERR M 0.23O36E-01 O.23036E-O1 O.23036E-01 Homogeneity of variance test D Bartlett . «?'1!HL Chl-square Probability warn Factors Chl-square Probability of freedom Ch square Q 90B02 < ,0 REP 0.13243 0.93593 •< Time for homogeneity of variance test -as 0. 16406E-02 seconds. Multiple range tests F-ratio is not significant at probability 0.93264 120 Appendix 14b ALG EXPT #4 Analysis for CELLY Analysis of variance table Sum of Mean Source squares DF square Probability Test term TREAT O.19568E+15 4. 0.48920E+14 375.50 0.00000 RESIDUAL REP O.47982E+12 2 . O.23991E+12 1 .8415 O.21985 RESIDUAL Residual O.10422E+13 8. O.13028E+12 Total O.19720E+15 14. Overa11 Overall mean standard deviation 0.10418E+08 0.37531E+07 Frequencies, standard deviations for TREAT 1 . 2. 3. 4. 0 MEAN O. 50108E+07 0.79088E+07 0.11 108E+08 12790E+08 O.15275E+OB P MEAN 0.50108E+07 0.79088E+07 0.11 108E+08 12790E+08 0. 15275E+08 0 STDV O.46429E+06 0.29175E+06 0.5G896E+06 1450OE+0G 0.340OOE+06 S ERR M 0.20839E+06 0.20839E+0G 0.20839E+06 20839E+06 0.20839E+06 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probability of freedom Ch1-square Probability warn TREAT 2.8885 0.576G5 4 5.9449 0.20330 < 10 Time for homogeneity of variance test was 0. 16927E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 5 homogeneous subsets which are listed as follows: ( 1. ) ( 2. ) ( 3. ) ( 4. ) ( 5. ) Duncan test at 5% probability level There are 5 homogeneous subsets which are listed as follows: ( 1. ) ( 2. ) ( 3. ) ( 4. ) ( S. ) Time for multiple range test was 0.71094E-02 seconds. Frequencies, means, standard deviations for REP . 1 5 5 0 MEAN 0.10355E+08 O.10238E+08 O.10662E+0B P MEAN 0.10355E+08 O. 10238E+08 0. 10662E*08 0 STDV 0.43557E+07 O. 39274E*07 0.38449E*07 S ERR M O. 16142E+06 0. 16142E+06 0.16142E+OG Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probabl11ty of freedom Chi-square Probabl1ity warn REP 0.G5492E-01 0.9G778 2 0.14154 0.93168 < 10 for homogeneity of variance test was 0.1G01GE-02 seconds. Multiple range tests F-rat1o is not significant at probability 0.21985 121 Appendix 15a ALG EXPT 05 Analysis for K Analysis of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 0.23583E-02 3. 0.78611E-03 0. 39915 0.75886 RESIDUAL REP 0.11667E-03 2. 0.58334E-04 0.29619E 0.97096 RESIDUAL Res idual 0.11817E-01 s 6. 0.19695E-02 Total 0.14292E-01 11. Overa11 Overa11 mean -standard deviation K 0.98917 0.3G045E-01 Frequencies, means, standard deviations for TREAT 1 . 2 . 3. 4. 3 3 3 3 0 MEAN 0. 98000 0.98333 0.98000 1 .0133 P MEAN 0. 98000 0.98333 0.98000 „,,<•- 1 .0133 0 STDV 0. 36056E -01 0.40415E -01 0.36056E-01 0. 41633E-01 S ERR M 0. 25622E -01 0.25622E -01 0.25622E-01 0. 25622E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probability warn TREAT 0.56569E-01 0.99648 3 0.13433 0.98742 < 10 Time for homogeneity of variance test-was 0.17058E-02 seconds. Multiple range tests F-rat1o is not significant at probability 0.75886 Frequencies, means, standard deviations for REP .1 .2 .3 444 0 MEAN 0.990*00 0.98500 0 99250 P MEAN 0.990O0 0.98500 0^99250 0 STDV 0.21603E-01 0.54467E-01 0.35940E-01 S 'ERR M 0.22189E-01 0.22189E-01 0.22189E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probability warn REP 2.0421 0.36022 2 2.8100 0.24536 < 10 Time for homogeneity of variance test was 0.15625E-02 seconds. Multiple range tests F-ratio Is not significant at probability 0.97096 . - - STOP 122 Appendix .15b ALG EXPT #5 Analysis for CELLY Analysis of variance table Sum of Mean F-ratlo Probabl11ty Test term Source squares DF square 33.055 0.00040 RESIDUAL TREAT 0.10474E+15 3. 0.34913E+14 1 .2059 0.36289 RESIDUAL REP 0.25474E+13 2. 0.12737E+13 Residual 0.63371E+13 S. 0. 10562E-M3 Total 0.11362E+15 1 1 . Overal1 Overall mean standard deviation 0.32139E+07 CELLY 0.57648E+07 Frequencies, means, standard deviations for TREAT 1. 2. 3. 4. 3 3 3 3 96933E+07 • MEAN 0.17985E+07 0.44150E+07 0.71525E+07 0. 9G933E+07 P MEAN 0.179B5E+07 0.44150E+07 0.71525E+07 0. 19581E+07 0 STDV 0 15242E+06 0.46808E+06 O.60486E+06 0. 59335E+06 S ERR M O.S933SE+06 0.59335E+06 0.59335E+06 0. Homogeneity of variance test Bartlett Degrees Layard Size warn Factors Chl-square Probability of freedom Ch1-square Probabl1ity < 10 TREAT 8.8741 0.03101 3 8.7317 0.03308 Time for homogeneity of variance test was 0.18229E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 3 homogeneous subsets which are listed as follows: ( 1.. 2. ) ( 2-. 3. ) ( 3.. 4. ) Duncan test at 5% probability level There are 4 homogeneous subsets which are listed as follows: ( 1. ) < 2. ) ( 3. ) ( 4. ) Time for multiple range test was 0.55469E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 .3 4 4 4 0 MEAN 0.52563E*07 0.566S4E+07 0.63719E+07 P MEAN 0.525S3E*07 0.566G4E+07 0.S3719E+07 0 STDV 0.26736E*07 0.34297E+07 0.42560E+07 S ERR M 0.51385E+O6 0.51385E+O6 0.51385E+OG Homogeneity of variance test Size Bartlett Degrees Layard Probability warn Factors Chl-square Probability of freedom Chl-square 0.56345 < 10 REP 0.34990 0.75961 2 1.1474 Time for homogeneity of variance test was 0.15755E-02 seconds. Multiple range tests F-ratlo 1s not significant at probability 0.36289 STOP Appendix 15c ALG EXPT #5 Ana'lysls for K Analysis of variance table Sum of Mean Source squares DF square F-rat 1o Probab111ty Test term TREAT 0.82755E-01 2 . 0.41378E-01 29.323 0.00408 RESIDUAL REP 0. 18289E-01 2 . 0.91445E-02 6.4803 0.05562 RESIDUAL Res 1dua1 0.56445E-02 4 . 0.14 111E-02 Total 0. 10G69 8 . Overal1 Overal1 mean standard deviation 1.1311 0.11548 Frequencies, means, standard deviations for TREAT . 1 . 2 . 3. 3 3 3 0 MEAN. 1.2400 1.1467 1.0067 P MEAN 1.2400 1.1467 1:0067 0 STDV 0.40000E-01 0.94517E-01 0.37859E-01 S ERR M. 0.21688E-01 0.21688E-01 0.21688E-01 Homogeneity of, variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Ch1-square Probability warn TREAT 1.8503 0.39647 2 2.3816 0.30398 < 10 Time for homogeneity of variance test was 0.15885E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 2 homogeneous subsets which are listed as follows: (. 3.. 2. ) ( 2. , 1 . ) Duncan test at 5% probability level There are 3 homogeneous subsets which are listed as follows: ( 3. ) ( 2. ) ( 1. ) Time for multiple range test was 0.42708E-02 seconds. Frequencies, means, standard deviations for REP . 1 .2 .3 3 3 3 0 MEAN 1 .0733 1 . 1367 1 . 1833 P MEAN 1 .0733 1 . 1367 1.1833 0 STDV 0. 1 1372 0. 13051 0. 1 1930 S ERR M 0.21688E-01 0.21688E-01 0. 21688E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probability of freedom CM-square Probability warn REP 0.32352E-01 0.98395 2 0.77128E-01 0.96217 < 10 Time for homogeneity of variance test was 0.15755E-02 seconds. Multiple range tests F-rat1o Is not significant at probability 0.05562 STOP 12k Appendix 16a ALG EXPT 06 Analysis for K Analysis of variance table Sum of Mean Source squares DF square F-rat 1 o Probability Test term TREAT 0.44242 3. 0.14747 36.792 0.00029 RESIDUAL REP O.G1G66E-03 2. 0.30833E-03 0.76922E-01 0.92686 RESIDUAL Res 1dua1 O.24050E-01 6 O.40083E-02 Total 0. 46709 1 1 Overa11 Overall mean standard deviation 1.6358 0.20607 Frequencies, means, standard deviations for TREAT 1 2 . 3 3 3 3 0 MEAN 1.7533 1 . 7433 1 . 7433 1 .3033 P MEAN 1 .7533 1 . 7433 1 . 7433 1 .3033 0 STDV 0.6G583E-01 0.66583E-01 0.15276E-01 0.56863E-01 S ERR M 0.36553E-01 0.36553E-01 0.36553E-01 0.36553E-01 Homogeneity of variance test Bartlett >. Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probability warn TREAT 2.9917 0.39291 3 . 9.3312 0.02520 < 10 Time for homogeneity of variance test was 0.17447E-02 seconds. Multiple range tests ' Duncan test at 1% probability level There are 2 homogeneous subsets which are listed as follows: ( 4. ) ( 2. . 3. , 1 . ) Duncan test at 5% probability level There are 2 homogeneous subsets which are listed as follows: ( 4. ) ( 2. . 3. . 1 . ) Time for multiple range test was 0.45182E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 .3 4 4 4 0 MEAN 1.6450 1.6350 1.6275 P MEAN 1.6450 1.6350 1.6275 0 STDV 0.22189 0.19122 0.26399 S ERR M 0.31656E-01 0.31656E-01 0.31656E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probability warn REP 0.27214 0.87278 2 0.40349 0.81730 < 10 Time for homogeneity of variance test was 0.17839E-02 seconds. Multiple range tests F-rat1o Is not significant at probability 0.92686 STOP 1 Appendix 16b ALG EXPT #6 Analysis for CELLY Analysis of variance table Mean Sum of Test term DF square F-ratio Probabt11ty Source squares RESIDUAL 3. 0.479G1E+14 143.88 0.00001 TREAT 0.14388E+15 RESIDUAL 2. 0.34003E+12 1.0201 0.41557 REP 0.68007E+12 6. 0.33333E+12 Residual 0.20000E+13 11. Total 0.14656E+15 Overal1 Overal1 mean standard deviation 0.36502E+07 CELLY 0.65858E+07 Frequencies, means, standard deviations for TREAT 1 . 2. 3. 4. 3 3 3 3 0 MEAN 0.23008E+07 0.48925E+07 0.74700E+07 0.11680E+08 P MEAN O.23OO8E*07 O.48925E+07 0. 74700E+07 0.11680E+08 0 STDV 0.22500E+06 0.410O2E+06 0.10252E+07 0.26520E+06 S ERR M 0.33333E+06. 0.33333E+06 0.33333E+06 0.33333E+06 Homogeneity of variance test Size Bartlett Degrees Layard Probability warn Factors Chl-square Probability of freedom Chl-square 0.22458 < 10 TREAT 4.9G02 0.17474 3 4.3658 Time for homogeneity of variance test was 0.18359E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 4 homogeneous subsets which are listed as follows: ( 3. ) ( 4. ) Duncan test at 5% probability level There are 4 homogeneous subsets which are listed as follows: ( 1 ) ( 2. ) ( 3. ) ( 4. ) Time for multiple range test was 0.56641E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 .3 4 4 -4 0 MEAN 0.65856E+O7 0.62944E+07 0.68775E+07 P MEAN 0.65B56E+07 0.62944E+07 0.68775E+07 0 STDV 0.40507E+07 0.41259E+07 0.38983E+07 S ERR M 0.28867E+06 0.28867E+O6 0. 2B867E+06 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chl-square Probability warn REP 0.87032E-02 0.99566 2 0.18446E-01 0.99082 < 10 Time for homogeneity of variance test was 0.15755E-02 seconds. Multiple range tests F-ratio Is not significant at probability 0.415S7 STOP Appendix 17a ALG EXPT tn Analysis for K Analysis of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 0. 12250E-02 1 . 0.12250E-02 5.4441 0. 25777 RESIDUAL REP 0.22501E-03 1 . 0.22501E-03 1.0000 0. 50000 RESIDUAL Residual 0.22501E-03 1 . 0.22501E-03 Total 0.16750E-02 3. Overal1 Overal1 mean standard deviation 1 . 3175 0.23629E-01 Frequencies, means, standard deviations for TREAT 1 . 2 . 2 2 0 MEAN 1.3000 1 .3350 P MEAN 1.3000 1 .3350 0 STDV 0.0 0.21214E-01 N S ERR M 0.10607E-01 0.10607E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom Chi-square Probability warn TREAT Uncalculable due to standard deviation of zero. Time for homogeneity of variance test was 0.13281E-02 seconds. Multiple range tests F-rat1o is not significant at probability 0.25777 Frequencies, means, standard deviations for REP .1 .2 2 2 0 MEAN 1.3100 1 :3250 P MEAN 1 .3 100 1 .3250 0 STDV 0.14142E-01 0.35355E-01 S ERR M 0.10607E-01 0.10607E-O1 Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom Chl-square Probability warn REP 0.49545 0.48151 1 1.3304 0.24873 < 10 Time for homogeneity of variance test was 0.1G927E-02 seconds. Multiple range tests F-ratlo Is not significant at probability 0.50000 STOP 127 Appendix 17b ALG EXPT HI Analysis for CELLY Analysis of variance table Sum of Mean Source squares DF square F-rat 1o Probability Test term TREAT 0. 13340E-M3 1 . 0.13340E+13 1.0540 0. 49162 RESIDUAL REP 0.53592E+13 1 . 0.53592E+13 4.2344 0. 28798 RESIDUAL Res 1dua1 O. 12656E+13 1 . 0. 12656E+13 Total 0.79589E+ 13 3 . Overa11 Overal1 mean standard deviation CELLY 0. 85875E+07 0. 16288E+07 Frequencies, means, standard deviations for TREAT 1 . 2 . 2 2 o 0 MEAN 0.91650E+07 0.80100E+07 P MEAN 0.91650E+07 0.80100E+07 0 STDV 0.24324E+07 0.84146E+06 S ERR M 0.79550E+06 0.79550E+06 Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom Ch1-square Probabl11ty warn TREAT 0.64187 0.42303 1 1.7215 0. 18950 < 10 Time for homogeneity of variance test was 0.16276E-02 seconds. Multiple range tests F-ratio 1s not significant at probability 0.49162 Frequencies, means, standard deviations for REP .1 .2 2 2 0 MEAN 0.97450E+07 O.74300E+07 P MEAN 0.97450E+07 0.74300E+07 0 STDV 0.16122E+07 21213. S ERR M 0.79550E+06 0.79550E+06 Homogeneity of variance test Bartlett DegreesA Layard Size Factors Chl-square Probability of freedom" Chl-square Probability warn REP 4.8503 0.02764 1 25.013 O.O0000 < 10 Time for homogeneity of variance test was 0.17188E-02 seconds. Multiple range tests F-ratlo Is not significant at probability 0.28798 STOP 1(28 Appendix 18a ALG EXPT #9 Analysis for K Analysis of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 0.10689E-01 2 . 0.53444E-02 2 . 2 166 0. 22498 RESIDUAL REP 0.15089E-01 2 . 0.75445E-02 3. 1290 0. 15205 RESIDUAL Res 1dua t 0.96445E-02 4 . ,0.24111E-02 Total 0.35422E-01 8 . Overa11 Overal1 mean standard deviation 1 . 4556 0.G6542E-01 Frequencies, means, standard deviations for TREAT 1 . 2. 3. 3 3 3 0 MEAN 1.4633 1.4933 . 1.4100 P MEAN 1.4633 1.4933 1.4100 0 STDV 0.37859E-01 0.55076E-01 0.88882E-01 S ERR M 0.28350E-01 0.28350E-01 0.28350E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom Chl-square Probability warn TREAT 1.1662 0.55817 2 2.0261 0.36311 < 10 Time for homogeneity of variance test was 0.16016E-02 seconds. Multiple range tests F-ratlo 1s not significant at probability 0.22498 Frequencies, means, standard deviations for REP .1 .2 .3 3 3 3 0 MEAN 1 .4233 1.4 300 1.5133 P MEAN 1 .4233 1 .4 300 1.5133 0 STDV 0.76376E-01 0.55677E-01 0.35119E-01 S ERR M 0.28350E-01 0.28350E-01' 0.28350E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probab111ty warn REP 0.91109 0.63410 2 1 . 8634 0.39389 < 10 Time for homogeneity of variance test was 0.16146E-02 seconds. Multiple range tests F-ratlo 1s not significant at probability 0.15205 STOP 129 Appendix 18b ALG EXPT #9 Analysis for CELLY Analysis of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 0.69562E+13 2 . 0.34781E+13 21 .920 0.00G99 RESIDUAL REP 0. 14574E+12 2 . 0.728G9E+11 0.45925 0.66138 RESIDUAL Res 1dua1 0.63468E+12 4 . 0. 158G7E+ 12 Total 0.773G6E+13 8 . Overal1 Overa11 mean standard deviation CELLY " 0.51661E+07 0. 98340E+06 Frequencies, means, standard deviations for TREAT 1 . 2. 3 3 3 0 MEAN 0.45000E+07 0.45900E+07 0.64083E+07 P MEAN 0.45000E+07 0.45900E+07 0.64083E+07 0 STDV • 0.39752E+06 0.34868E+06 0.33258E+06 S ERR M 0.22998E+06 0.22998E+06 0.22998E+06 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Ch1-square Probability warn TREAT 0.57151E-01 0.97183 2 0.13341 0.93547 < 10 Time for homogeneity of variance test was 0.16146E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 2 homogeneous subsets which are listed as follows: ( 1 . . 2. ) Duncan test at 5% probability level There are 2 homogeneous subsets which are listed as follows: ( 1 . , 2. ) ( 3. ) Time for multiple range test was 0.38672E-02 seconds. Frequenc i es, means, standard deviations for REP . 1 .2 " .3 0 MEAN . 53233E+07 0.50117E+07 0. 5 1633E+07 P MEAN . 53233E+07 0.50117E+07 0.51633E+07 0 STDV . 12359E+07 0 . 99925E + 06 0.11267E+07 S ERR M 22998E+06 0.22998E+06 0. 22998E+06 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probabl11ty of freedom Ch t-square Probab11 1 ty warn REP 0.73380E-01 0.96398 2 0. 17655 0.91551 < 10 Time for homogeneity of variance test was 0.16276E-02 seconds Multiple range tests F-rat1o is not significant at probability 0.66138 STOP 100 Appendix 19a ALG EXPT.us Analysis for K Analysis of variance table Sum of Mean Source squares DF square F-ratio Probability Test term TREAT 0. 13500 1. 0.13500 385.70 0.00258 RESIDUAL REP 0.74100E-01 2. 0.37050E-01 105.85 0.00936 RESIDUAL Res i dua1 0.70003E-03 2. 0.35002E-03 Total 0. 20980 5 . Overal1 Overa11 mean standard deviation 1 . 7500 0.20484 Frequencies, means, standard deviations for TREAT 1 . 2 . 3 3 0 MEAN 1.9000 1.6000 P MEAN 1.9000 1.6000 0 STDV 0. 14933 0.12288 S ERR M 0.10801E-01 0.10801E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probability of freedom Chi-square Probability warn TREAT 0.60428E-01 0.80582 1 0.14659 0.70182 < 10 Time for homogeneity of variance test was 0.17058E-02 seconds. Multiple range tests Duncan test at 1% probability level There are 2 homogeneous subsets which are listed as follows: ( 2. ) ( -1 . ) Duncan test at 5% probability level There are 2 homogeneous subsets which are listed as follows: ( 2. ) ( 1 . ) Time for multiple range test was 0.35287E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 .3 2 2 2 0 MEAN 1.7 100 1.8050 1.7350 P MEAN 1.7 100 1.8050 1.7350 0 STDV 0.'28284E-01 0.21920 0.38891 S ERR M O.12757 • 0.12757 0.12757 Homogeneity of variance test Bartlett Degrees Layard Factors Ch1-square Probability of freedom Chi-square Probability REP 2.7226 O.25633 2 10.522 0.00519 Time for homogeneity of variance test was 0.18880E-02 seconds. Multiple range tests F-ratio is not significant at probability 0.87032 STOP . . •131 ALG EXPT #8 Appendix 19b Analysis for CELLY Analysts of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 0.10101E+14 1 . 0.10101E+14 181 .47 0.00547 RESIDUAL REP 0. 1232GE+12 2 . 0.61G29E+11 1 . 1072 0.47456 RESIDUAL Residual 0.11 132E+12 2 . 0.55G62E+11 Total 0. 10336E+14 5 . Overa11 Overa11 mean standard deviation CELLY 0.26808E+07 . 0.14377E+07 Frequencies, means, standard deviations for TREAT 1 • 2 • 3 3 • 0 MEAN 0.39783E+07 0.13833E+07 P MEAN 0.39783E+07 0.13833E+07 , 0 STDV . 0.31262E+06 0.13985E+06 S ERR M 0.13621E+06 0.13621E+06 Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom Ch1-square Probability warn TREAT 0.93982 0.33232 1 1.7922 0.18066 < 10 Time for homogeneity of variance test was 0.16276E-02 seconds. .Multiple range tests Duncan test at 1% probability level There are 2 homogeneous subsets which are listed as follows: ( 2. ) ( 1. ) Duncan test at 5% probability level There are 2 homogeneous subsets, which are listed as follows: ( 2. ) ( 1. ) Time for multiple range test was 0.33594E-02 seconds. Frequencies, means, standard dev1 at ions.for REP .1 .2.3 2 2 2 0 MEAN 0.28775E+07 0.2G250E+07 0.25400E+07 P MEAN 0.28775E+07 0.26250E+07 0.25400E+07 0 STDV 0.19622E+07 0.19799E+07 0.15G27E+07 S ERR M 0.16683E+06 0.16683E+06 0.16683E+06 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Ch1-square Probability warn REP 0.46991E-01 0.97678 2 0.14105 0.93191 < 10 Time for homogeneity of variance test was 0.15885E-02 seconds. Multiple range tests F-ratlo Is not significant at probability 0.47456 STOP . 1-32 Appendix 20a DAP EXPT/M .2,3 Analysis for- BIOM Analysis of variance table Sum of Mean Source squares DF square F-rat 1o Probability Test term TREAT 5437.2 2 . 2718.6 0.84869 0.49291 RESIDUAL REP 18180. 2 . 9090.1 2.8377 0. 17092 RESIDUAL Res1dual 12813. 4 . 3203.3 Total 36431. 8 . Overal1 Overa11 mean standard deviation BIOM 134.80 67.482 Frequencies, means, standard deviations for TREAT 1 . 2. 3. 3 3 3 0 MEAN 103.27 163 . 23 137.90 P MEAN 103.27 163 . 23 137.90 0 STDV 34.865 88.499 80.307 S ERR -'M 32.677 32 .677 32.677 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chi-square Probabl11ty warn TREAT 1.3233 0.51600 2 3.2167 0. 20022 < 10 Time for homogeneity of variance test was 0.17968E-02 seconds. Multiple range tests F-ratio 1s not significant at probability 0.49291 Frequencies, means, standard deviations for REP .1 .2 .3 3 3 3 0 MEAN 101.40 198.33 104.67 P MEAN , 101.40 198.33 104.67 0 STDV 29.407 85.210 31.620 S ERR M 32.677 32.677 32.677 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probability of freedom Chi-square Probability warn REP 2.4551 0.29301 2 2.8809 0.23682 < 10 Time for homogeneity of variance test was 0.15885E-02 seconds. Multiple range tests F-ratio is not significant at probability 0.17092 STOP Appendix 20b DAP EXPT#1,2,3 Analysis for BCEFF Analysis of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 442.58 2 . 22 1 . 29 2.8581 0. 16948 RESIDUAL REP 424.42 2 . 212.21 2.7408 0. 17797 RESIDUAL Residual 309.70 4 . 77.424 Total 1 176 . 7 8 . Overa11 Overal1 mean standard deviation BCEFF 38.51 1 12. 128 Frequencies, means, standard deviations for TREAT 1 . 2 . 3 3 3 0 MEAN 47.267 38 . 167 30.100 P MEAN 47 . 267 38. 167 30.100 0 STDV 2.0008 14 .520 12.338 S ERR M 5.0802 5.0802 5.0802 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Ch1-square Probability warn TREAT 4.3481 0.11372 2 12.862 0.00161 < 10 Time for homogeneity of variance test was 0.16016E-02 seconds. Multiple range tests F-ratio is not significant at probability 0.16948 Frpauendes, means, standard deviations for REP .1 -2 -3 3 3 3 0 MEAN 4 1.567 44.967 29.000 P MEAN 4 1.567 44.967 29.000 0 STDV 4.7648 5.9719 17.826 J S ERR M 5.0802 5.0802 5.0802 Homogeneity of variance test Bartlett Oegrees Layard Size Factors Chl-square Probability of freedom Ch1-square • Probability warn REP 3.3317 0.18903 2 3.6563 0.16071 < 10 Time for homogeneity of variance test was 0.15885E-02 seconds. Multiple range tests F-ratio Is not significanv t at probab111ty 0.17797 STOP 134- Appendix 21a DAP EXPT#4,5 Analysis for BIOM Analysis of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 12485. 1 . 12485. 21 .829 0.04289 RESIDUAL REP 5698 . 1 2 . 2849 . 1 4.9812 0. 16719 RESIDUAL Res 1dua1 1143.9 2 . 571.96 Total 19327. 5 . Overa11 Overal1 mean standard deviation BIOM 189.02 62 . 173 Frequencies, means, standard deviations for TREAT 1 . 2. 3 3 0 MEAN ' 143.40 234.63 P MEAN 143.40 234.63 0 STDV 35.286 46.647 S ERR M 13.808 13.808 Homogeneity of variance test Bartlett Degrees Layard Size Factors Ch1-square Probability of freedom, Ch1-square Probability warn TREAT O.12305 0.72575 1 0.29012 0.59015 < 10 Time for homogeneity of variance test was 0.16406E-02 seconds. Multiple range tests Duncan test at 5% probab111ty level There are 2 homogeneous subsets which are listed as follows: ( 1 . ) ( 2. ) Time for multiple range test was 0.20703E-02 seconds. 1 Frequencies, means, standard deviations for REP 2 2 2 0 MEAN 158.30 177.60 231 . 15 P MEAN 158.30 177.60 231 . 15 0 STDV 39. 174 86.691 67 .670 S ERR M 16.911 16.911 16.911 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Chl-square Probability warn REP 0.39743 0.81978 2 1.1548 0.56137 < 10 Time for homogeneity of variance test was 0.16927E-02 seconds. Multiple range tests F-ratio 1s not significant at probabil*ty O.J.6J7J9 — STOP 1-3^ Appendix 21b DAP EXP#4,5 Analysis for BCEFF Analysis of variance table Sum of Mean Source squares DF square F-rat1o Probability Test term TREAT 16.007 1 . 16.007 3. 1222 0.21927 RESIDUAL REP 43 . 773 2 . 21 .887 4 . 2692 0. 18978 RESIDUAL Res1dual 10.253 2. 5. 1267 Total 70.033 5. Overa11 Overal1 mean standard deviation BCEFF 19.367 3 . 7426 Frequencies, means, standard deviations for TREAT 1.2. 3 3 0 MEAN 21.000 17.733 P MEAN 21.000 17.733 0 STDV 1.8193 4.8686 S ERR M 1.3072 1.3072 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probability of freedom Ch1-square Probability warn TREAT 1.3501 0.24526 1 2.4687 0.11614 < 10 Time for homogeneity of variance test was 0.17318E-02 seconds. Multiple range tests \ F-rat1o Is not significant at probability O.21927 Frequencies, means, standard deviations for REP 1 .2 .3 2 2 0 MEAN 18.200 16.800 23.100 P MEAN 18.200 16.800 23.100 0 STDV 2.4042 4.5255 0.0 S ERR M 1.6010 1 .6010 1 .6010 Homogeneity of variance test Bartlett Degrees ' Layard Size Factors Chi-square Probability of freedom Chl-square Probability warn REP Uncalculable due to standard deviation of zero. Time for .homogenelty of variance test was 0.13412E-02 seconds. Multiple range tests F-rat1o 1s not significant at probability 0.18978 STOP --• 136 Appendix 22a GROWTH R/M Analysis for TL Analysis of variance table Sum of Mean Source squares DF square F-rat lo Probability Test term TREAT 0.64013E-01 2. 0.32007E-01 6.8488 0.01848 RESIDUAL REP 0. 51693E-01 4. 0.12923E-01 2 . 7653 0. 10305 RESIDUAL Res 1dua1 0.37387E-01 8. 0.4G733E-02 Total 0. 15309 14 . Overal1 Overal1 mean standard deviation TL 2 . 4427 0.10457 Frequencies, means, standard deviations for TREAT 1 . 2. 3. 5 , 5 5 0 MEAN . 2.3620 2.4440 2 . 5220 P MEAN 2.3620 2.4440 2.5220 0 STOV 0.74632E-01 0.51283E-01 0. 1 1862 S ERR M 0.30572E-01 0.30572E-01 0.30572E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probability of freedom Chi-square Probability warn TREAT 2.4677. 0.29117 2 3.4273 0.18021 < 10 Time for homogeneity of variance test was 0.16276E-02 seconds. Multiple range tests , Duncan test at 5% probability level There are 2 homogeneous subsets which are listed as follows: ( 1., 2. ) . ( 2. . 3. ) Time for multiple range test was 0.26302E-02 seconds. Frequencies, means, standard deviations for REP 1 • -2 .3 .4 . 5 3 3 3 3 0 MEAN 2.4567 2.4333 2.3400 2 . 4633 2 . 5200 P MEAN 2.4567 2 .4333 2 .3400 2 .4633 2 . 5200 0 STDV 0.4X3414E-01 0. 80829E-01 0.88882E-01 0. 12858 0. 13454 S ERR M 0.39469E-01 0. 39469E-01 0. 39469E-01 0. 39469E-01 O. 39469E;-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chi-square Probabl11ty of freedom Chi-square Probability warn REP 2.4113 0.66059 4 5.4338 0.24561 < 10 Time for homogeneity of variance test was 0.17448E-02 seconds. Multiple range tests F-ratio is not significant at probability 0.10305 STOP 137 Appendix 22b GROWTH R#2 Analysis for TL Analysis of variance table Sum of Mean Source squares DF square F-rat 1o Probability Test term TREAT 0. 24040E-01 2 . 0. 12020E-01 6 . 37 10 0.02213 RESIDUAL REP 0.93067E-02 4. 0.23267E-02 1 . 2332 0. 3G980 RESIDUAL Residual 0. 15093E-01 8. 0.18867E-02 Total 0.48440E-01 14 . Overal1 Overa11 mean standard deviation TL 2.9580 0.58822E-01 Frequencies, means, standard deviations for TREAT 1 3. 5 5 5 0 MEAN . 2.9100 2.95G0 3 .0080 P MEAN 2.9100 2.9560 3 .0080 0 STDV 0.30000E-01 0.43359E-01 0.57620E-01 S ERR M 0.19425E-01 0.19425E-01 0.19425E-01 Homogeneity of variance test Bartlett Degrees , Layard Size Factors Chi-square Probability of freedom Chi-square Probability warn TREAT 1.4514 0.48399 2 1.7924 0.40812 < 10 Time for homogeneity of variance test was 0.166G7E-02 seconds. Multiple range tests Duncan test at 5% probability level There are 2 homogeneous subsets which are listed as follows: ( 1 . . 2. ) ( 2.. 3. ) Time for multiple range test was 0.26433E-02 seconds. Frequencies, means, standard deviations for REP 1 • -2 .3 ,a 3 3 3 0 MEAN 2.9600 2 .9467 3.0000 2 .9233 2.9600 P MEAN 2.9600 2 . 9467 3.OOOO 2.9233 2 .9600 0 STDV 0.34641E-01 0. 50332E-01 0. lOOOO i 0.68069E-01 0. 346.4 IE-01 S ERR M 0.25078E-01 0.25078E-01 0.25078E-01 0.25078E-01 0. 25078E-01 Homogeneity of variance test Bartlett Degrees Layard Size Factors Chl-square Probab111ty of freedom Ch1-square Probability warn REP 2.8198 0.58841 4 3.8880 0.42138 < 10 Time for homogeneity of variance test was 0.17578E-02 seconds. Multiple range tests F-ratio is not signif1 cant.at probability 0.36980