IIIIIIIIIIIIIIIIIIIIIIIIIII llliNHflHHIIT“Willi!“lilliillfllfllflmi ; This is to certify that the thesis entitled A COMPARISON OF TWO ALGAL BIOASSAY PROCEDURES AND THEIR APPLICATION IN ASSESSING THE EFFECTS OF CUPRIC ION AND P-CRESOL 0N ALGAL GROWTH KINETICS presented by Michael John Toohiii has been accepted towards fulfillment of the requirements for flS degree in Fisheries & Wi'ldiife Major professor Date Juiy 1983 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES RETURNING MATERIALS: P1ace in book drop to remove this checkout from m your record. FINES wi'l] be charged if book is returned after the date stamped below. ;AW 3327 A COMPARISON OF TWO ALGAL BIOASSAY PROCEDURES AND THEIR APPLICATION IN ASSESSING THE EFFECTS OF CUPRIC ION AND P-CRESOL ON ALGAL GROWTH KINETICS BY Michael John Toohill A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1983 ABSTRACT A COMPARISON OF TWO ALGAL BIOASSAY PROCEDURES AND THEIR APPLICATION IN ASSESSING THE EFFECTS OF CUPRIC ION AND P-CRESOL ON ALGAL GROWTH KINETICS BY Michael John Toohill The purpose of this study was to evaluate the effective- ness of two types of static algal bioassays in assessing toxicant effects on algal growth. Batch cultures of Chlorella vulgaris Beijerinck were assayed for changes in growth kinetics caused by cupric ion and p-cresol. Cupric ion was found to be acutely toxic to Chlorella vulgaris cultures at a concentration of 0.15 mg/l. A con- centration of 0.05 mg Cu2+/l was found to have little effect on the algal cultures. Cupric ion levels of 0.075. 0.1, and 0.125 mg/l had increasing effects on algal growth. The sublethal effect was a graded decrease in photosynthesis with increasing cupric ion levels, prolonging the lag phase of growth and decreasing the maximum specific growth rate. This caused a prolongation of the growth cycle of the Chlorella vulgaris cultures. The maximum standing crop level was not affected. The organic toxicant, p-cresol, was found to be rapidly degraded under nonaxenic algal culture conditions. Four gnassible causes of loss from the media were tested (photoly- ssis, volatilization, algal sorbtion/degradation, bacterial degradation) and the results indicated that bacterial degra- dation of the compound was the major cause of p—cresol loss. A comparison of the two bioassay methods used showed that the pH method (Young and King, 1973) was more sensitive in assessing the toxic effects of cupric ion than was the Algal Assay Procedure (Miller et a1, 1978). In the p-cresol test, the pH method was adversely affected by the breakdown of the p-cresol. The pH method was found to be a more time-efficient procedure. ACKNOWLEDGEMENTS I wish to thank my major professor, Dr. Niles R. Kevern, for his guidance throughout my degree program at Michigan State University. I also wish to thank the other members of my guidance committee, Drs. D. L. King and J. A. Eastman. for their assistance. And to Dr. B. J. Mathis of Bradley Univer- sity, thank you for the inspiration you gave to a young college student. Finally, to my parents. thank you for raising me in an atmosphere conducive to thought and learning. I thank you and my brothers and sisters for asking ”why" and encouraging me to seek the answers. Nothing that I have accomplished would have been possible without you. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION LITERATURE REVI EW Cupric Ion p-Cresol Chlorella vulgaris ALGAL CULTURE METHODS Physical Conditions Algal Culture Medium Experimental Algae AAP Sampling, Enumeration, and Calculation Methods pH Method Sampling, Measurement, and Calculations Statistical Analyses and Computer Software PRELIMINARY EXPERIMENTS Experiment 1 - Cell Counting Procedure Error Experiment 2 - The Effects of Culture Concentration on the Cell Counting Procedure Experiment 3 - Trial Run of the AAP and pH Methods TOXICOLOGY EXPERIMENTS . Experiment 4 - The Effects of Cupric Ion on Chlorella vulgaris Culture Growth Experiment 5 - Further Study into the Effects of Cupric Ion on Chlorella vulgaris Culture Growth Experiment 6 - The Effects of p-Cresol on Chlorella vul aris Culture Growth Experiment 7 - Analysrs of the Degradation of p-Cresol Under Algal Bioassay Conditions iii 10 13 15 15 17 19 20 22 29 31 34 38 52 .53 80 105 116 SUMMARY APPENDIX - AAP and pH Method Data from the Experiments LITERATURE CITED iv LIST OF TABLES Table , Page 1. Predictions based on a nine-compartment environmental exposure model for p-cresol (0.8. Environmental Protection Agency, 1978)....... 12 2. Composition of synthetic algal nutrient medium..... 18 3. Nested analysis of variance studying the variability in the sampling, subsampling, and counting procedures used for the AAP............... 33 4. Experimental setup and results from a test of the modified McNabb (1960) method of enumerating algal 08118000....0.0.0000...0.000000000000000...OO 35 S. The statistical analysis of Experiment 3........... 45 6. Specific growth rates (u) of Chlorella vulgaris cultures in Experiment 3, as calculated by the AAP. 46 7. Statistical analysis of the AAP results from Experiment 40.0.000000000000000000000000IOIOOOOOOOO 59 8.‘ Specific growth rates (u) of Chlorella vulgaris cultures in Experiment 4, as determined by the AAP. 60 9. Statistical analysis of the pH method results firm Experiment 4.0000000000000000000000.0.0.000... 64 10. Kinetic constants for the Chlorella vulgaris cultures in Experiment 4, as determined by the pH mthOdOOOOOOQ0.00.000...OOOOOOOOOOOOOOOOOOOOOOO. 69 11. Statistical analysis of the AAP results from the first eight sampling times of Experiment 5..... 86 12. Statistical analysis of the AAP results from the last four sampling times of experiment 5....... 87 Table Page 13. Specific growth rates (u) of Chlorella vulgaris cultures in Experiment 5, as determined by the AAP. 88 14. Statistical analysis of the pH method results from the first six sampling times of Experiment 5....... 92 15. Statistical analysis of the pH method results from the last seven sampling times of Experiment 5...... 93 16. Kinetic constants for the Chlorella vulgaris cultures in Experiment 5, as determined by the pa mthOdOOOOOOOO0.00......OOOOOOOOOOOOOOOOOOOOOOO. 99 17. Statistical analysis of the AAP results of Exmrimnt 6......OOOOOOOOOOOOOOO0.000000000000000. 111 18. Specific growth rates (u) of Chlorella vulgaris cultures in Expermient 6, as determined by the AAPOOOOOIOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOOO 113 19. Results of the gas chromatographic analysis for p-cresol in Experiment 7........................... 125 Al. Cell counts as cells/ml from Experiment l.......... 131 A2. Cell counts as cells/m1 from Experiment 3.......... 132 A3. pH values for cultures of Chlorella vulgaris in experiment 3.0.0.0000000000000000000000000000000000 133 A4. Cell counts as cells/m1 from Experiment 4.......... 134 A5. pH values for cultures of Chlorella vulgaris in merimnt 4.00000000000000000000000.0.0....00...OO 13S A6. Cell counts as cells/m1 from Experiment 5.......... 137 A7. pH values for cultures of Chlorella vulgaris in Experiment 5.COO0.0.0000000000000000000000.0...O... 138 A8. Cell counts as cells/ml from Experiment 6.......... 140 A9. pH values for cultures of Chlorella vulgaris in Experimnt 6.0.0.00000000000000000000000000.00....O 141 vi LIST OF FIGURES Figure Page 1.. The relationship between the expected and observed biomass estimates using the modified McNabb (1960) method of algal cell enumeration..... 37 2. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 3...... 39 3. Contour map of the photosynthetically active radiation (uE/m2 /sec) falling on the assay platform in Experiment 3........................... 40 4. pH fluctuation of AAP cultures of Chlorella vulgaris from Experiments 3, 4, and 5.............. 42 5. AAP growth curves for Chlorella vulgaris cultures in Experiment 3.0....O..0.0000000000000000000000000 43 6. pH method growth curves for Chlorella vulgaris cultures in Experiment 3........................... 47 7. Specific growth rates of cultures of Chlorella vulgaris in Experiment 3 as a function of the corresponding free C02 concentrations of the medi‘mOOOOOOOOOOOOOOOOOOOOIOOO0.00000000000000.0... 49 8. Reciprocal plot of the specific growth rates of Chlorella vulgaris cultures in Experiment 3 versus the corresponding free C02 concentrations of the mediumOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 50 9. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 4...... 54 10. Contour map of the photosynthetically active radiation (uE/m2 /sec) falling on the assay platform in Experiment 4........................... 55 11. AAP growth curves for Chlorella vulgaris cultures in Experiment 4.0.0.0.000...OOOOOOOOOOOOOOOOOOOOOOO 58 vii Figure Page 12. pH method growth curves for Chlorella vulgaris CUltures in Experiment 40...OOOOOOOOOOOOOOOOOOOOOOO 63 13. Specific growth rates of the control Chlorella vulgaris cultures in Experiment 4 as a function of the C02fo concentration of the medium........... 66 14. Specific growth rates of the 0.05 mg Cu2+/1 treatment Chlorella vulgaris cultures in Experiment 4 as a function of the COsz concentration of the medium........................ 67 15. Specific growth rates of the 0.10 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiment 4 as a function of the COZfo concentration of the medium........................ 68 16. Reciprocal plot of the specific growth rates of the control Chlorella vulgaris cultures in Experiment 4 as a function of the COzfA concentration of the medium........................ 70 17. Reciprocal plot of the specific growth rates of the 0.05 mg Cu2+/1 treatment Chlorella vulgaris cultures in Experiment 4 as a function of the COzfA concentration of the medium.................. 71 18. Reciprocal plot of the specific growth rates of the 0.10 mg Cu2+/1 treatment Chlorella vulgaris cultures in Experiment 4 as a function of the COZfA concentration of the medium.................. 72 l9. Eadie-Hofstee plot of the control Chlorella vulgaris cultures in Experiment 4.................. 74 20. Eadie-Hofstee plot of the 0.10 mg Cu2+/1 treatment Chlorella vulgaris cultures in Experiment 4........ 7S 21. COZfA/U vs COZfA transformation of the control Chlorella vulgaris cultures in Experiment 4........ 76 22. coagA/u vs cozfA transformation of the 0.10 mg Cu /1 treatment Chlorella vulgaris cultures in Experiment 4.0.0.0....I.OOOOOOOOOOOOCOOOOOO0.00.... 77 23. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 5...... 81 24. Contour map of the photosynthetically active radiation (uE/mZ/sec) falling on the assay platform in Experiment 5...C......................O...O..... 82 viii Figure Page 25. AAP growth curves for Chlorella vulgaris onltures in Experiment SOOOOOOOOOOOOOOOOOOOOOO0..O. 85 26. pH method growth curves for Chlorella vulgaris CUltures in Experiment 5....OOOOOOOOOOOOOOOOOOOOOO. 91 27. Specific growth rates of the control cultures of Chlorella vulgaris in Experiment 5 as a function of the cozfo concentration of the mediumOOOOCOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 95 28. Specific growth rates of the 0.075 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiment 5 as a function of the COZfo concentration of the medium........................ 96 29. Specific growth rates of the 0.10 mg Cu2+/l treatment Chlorella vul aris cultures in Experiment 5 as a function of the COsz concentration of the medium........................ 97 30. Specific growth rates of the 0.125 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiment 5 as a function of the COZfo concentration of the medium........................ 98 31. Reciprocal plot of the specific growth rates of the control Chlorella vulgaris cultures in Experiment 5 as a function of the C02fA concentration of the medium........................ 100 32. Reciprocal plot of the specific growth rates of the 0.075 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiment 5 as a function of’the COZfA concentration of the medium.................. 101 33. Reciprocal plot of the specific growth rates of the 0.10 mg Cu2+/1 treatment Chlorella vulgaris cultures in Experiment 5 as a function of the CngA concentration of the medium.................. 102 34. Reciprocal plot of the specific growth rates of the 0.125 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiment 5 as a function of the COZfA concentration of the medium.................. 103 35. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 6...... 106 ix Figure Page 36. Contour map of the photosynthetically active radiation (uE/mZ/sec) falling on the assay platform in Experiment 6........................... 107 37. AAP growth curves for Chlorella vulgaris CUltures in Experiment 6....OOOIOOOOOOIOOOOOOOOOOOO 110 38. Flowchart of the experimental design of the p-cresol degradation test, Experiment 7............ 117 39. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 7...... 120 40. Contour map of the photosynthetically active radiation (uE/mZ/sec) falling on the assay platform in Experiment 7........................... 121 INTRODUCTION In recent years much research has been carried out on the effects of pollutants in the environment. In general, however, the bulk of this effort has been focused on their effects on higher organisms. All too often the primary pro- ducers have been neglected or, at best, considered second- arily. While the importance of primary producers is stressed in the classrooms and in the literature, toxicological research generally centers on the heterotrophic life forms in natural systems. A more concerted effort needs to be established in the area of defining toxicant effects on the primary producers in the aquatic ecosystem. As with researchers in any area of investigation, aquatic biologists have developed certain techniques useful in the determination of the effects of chemicals on organ- isms. Bioassays have been utilized for some time in these tests as technical tools. In this study, where the actions of two toxicants on Chlorella vulgaris Beijerinck were assessed, two of these bioassay methods were utilized. The first of these is the Algal Assay Procedure--Bottle Test (AAP-8T), currently listed by the United States Environmental Protection Agency as the accepted algal bioassay procedure. This method was outlined by the U.S. Environmental Protection 2 Agency (1971) and further defined by Miller et a1 (1978). In this procedure, an algal seed is introduced into a growth medium containing nutrients, and the variable under investi- gation and algal growth kinetics are studied for the effects of the variable. Quantification of the effects is via the change in cell counts (expressed as cells/ml) over time. The medium contains sufficient amounts of algal macro- and micro- nutrients to maintain the culture throughout the test period. Temperature and light conditions are held constant, and the pH and C02 supply are maintained by bubbling air through the medium, or by other means (Miller et a1, 1978). The theory behind this procedure is that if the variable under study has an inhibitory effect on the algal culture, it will be exhib- ited by a reduced growth of the culture, as compared to con- trols. Conversely, any substance that augments the growth of the algae will exhibit an increase in cell counts as compared to the controls. While this method does give information on the effect of the variable under study, it gives no informa- tion on the interaction of the variable with the pH, inor- ganic carbon supply, and other growth factors in the system (Young and King, 1973). Also the method of quantification (cell counts) tends to be tedious and may introduce a large source of error into the results (Forsythe, 1973). Many researchers have used the change in pH caused by algal uptake of C02 in a closed system as a measure of pro- duction (Verduin, 1951, Beyers and Odum, 1959; Beyers, 1963; Beyers et a1, 1963; Young and King, 1973). The procedure of 3 Young and King (1973) will be followed in this study. As with the AAP-8T, this method (hereinafter called the pH method) supplies all of the nutrients needed by the algae but provides a limited source of inorganic carbon. The basis of the method is that if the amount of inorganic carbon available to the algae is known (the carbonatefbicarbonate alkalinity of the system) and the carbon present in the ini— tial algal seed is known, the total amount of carbon in the closed system at any point in time is known. When, during growth, the algae fix some of the free C02 of the medium into algal biomass, the change in the free C02 concentration in the system may be measured as a change in the pH of the sys- tem. This effect may be seen in the first and second dis- sociations of carbonic acid (H2C03) in water: nzco3 (acoz-HZO)=Hco3‘ + a+=co32' + 23* (1) or as it relates to the algal uptake of C02 (adapted from Wetzel, 1975): 211(203"-->co2 + c032“ + 303 (2) The rise in pH as the algae ”pull" on the free C02 of the system also may be predicted with the following equation: K2 = (H+)(CO§2-) (Hco3‘) (3) where K2 is the temperature dependent second dissociation constant of carbonic acid. Rearranging this equation yields: (4) (c032') and by taking the logarithm of both sides of the equation: pH = pK2 - log (Hc03‘) (5) (c032‘) As the C02 is removed from the system by the algae, the C032’ concentration rises, the HC03' concentration drops, and con- sequently the pH rises. In this system, a variable which has a deleterious effect on algal growth will express itself in a decreased pH rise as compared to the controls. Con- versely, any variable which stimulates growth will cause a faster rise in pH of the cultures than is seen in the con- trols. A basic assumption in this method is that an increase in the pH of the system reflects an increase in algal bio- mass. This assumption was tested by Young and King (1973) and they found a correlation (r2) of 0.986 between the calcu- lated biomass estimates from the pH method and direct mea- surements of organic carbon. This pH method has two advantages over the AAP-ET. First, the change in pH is more easily monitored than the change in the quantity of cells. Secondly, interactions between the variable under investigation and inorganic car- bon (or other nutrient) availability may be seen in the pH method, but not in the AAP-BT. A The purposes of this study were twofold. The first was to examine the two bioassay methods discussed above and attempt to assess the relative sensitivity of each pertaining to changes in algal growth kinetics. By doing this, compari- sons between the two methods and their relative sensitivities were made. Secondly, an attempt was made to assess the 5 inhibitory effects of cupric ion and p-cresol on Chlorella vulgaris. The choice of these two toxicants is discussed in the following paragraphs. LITERATURE REVIEW Cupric Ion COpper is a trace nutrient in plant and animal metabo- lism and is found in many naturally occurring compounds (Lehniger, 1970). In plants, copper is an important con- stituent of cytochrome c oxidase, which catalyzes energy transformations during electron transport (Lehniger, 1970). Chloroplasts contain the blue copper protein plastocyanin, which may be the direct electron donor to P700+ in Photo- system I (weier et a1, 1974). The form of copper of interest in this study is cupric ion (Cu2+). This has been determined to be the active toxic form of the metal (den Dooren De Jong, 1965: Sunda and Guillard, 1976). Cupric ion is the active ingredient in one of the first known fungicides, Bordeaux mixture, which is a mixture of lime, water, and copper sulfate (Bullard, 1972). Copper sulfate also has been extensively used as an algicide, having been applied to lakes for that purpose as early as 1918 (Bullard, 1972). Besides its input into the environment as an algicide, cupric ion is found as a significant con- stituent in metal plating, pickling, and rayon manufacture wastes (Klein, 1962) as well as in mine tailings. While much of the copper which is mined is not lost to the environment, the indiscriminant use of copper sulfate has 6 7 caused problems. In cases like Lake Monona, Wisconsin, where over two million pounds of copper sulfate had been added over a 26-year period for the control of algae (MacKenthun and Cooley, 1952) or the San Francisco water storage reservoirs where, in the period 1912 to 1962, 800,000 pounds of copper sulfate were added annually (Palmer, 1964), the problem of copper persisting in the aquatic environment is acute. Symmes (1975) reported that of all the copper added to a lake, only 2 percent left the system: 2 percent was in the water and the rest had accumulated in the sediments and biota. Wagner and Bohl's (1978) study showed that upon the addition of copper sulfate or cutrine to the aquatic system, the c0pper content of the fish in a pond was raised twofold, but the content of the plants and sediments was raised 18 to 80 fold. Cutrine (copper triethanolamine complex) was chosen as the form of copper to be used in this study because of its present use as an algicide and because of the reactions of copper in the aqueous environment. Under slightly acidic (pH = 6) conditions, cupric ion precipitates as a hydrox- ide (Britton, 1927). Under alkaline conditions, cupric ion forms soluble and insoluble carbonate complexes (Stiff, 1971a and 1971b). Depending on the pH and alkalinity of the system, these forms include CuCO3, Cu(CO3)22' and CuCO3(OH)22- (Stiff, 1971a). The solubility of these com- plexes is pH dependent and Stiff (1971a) gives equilibrium constants for theSe complexes. Cutrine was chosen because 8 this complex is less susceptible to precipitation as a car- bonate, oxide, or sulfide than other c0pper compounds, and thus is more likely to remain actively toxic. Stiff (1971b) states that c0pper complexes with amino acids, polypepotides, and humic substances are the most soluble forms of copper. Much work has been done on assessing the toxicity of cupric ion towards aquatic life. Copper has been found to be toxic to fish in concentrations ranging from 0.05 ppm (Sprague, 1963) to 1.2 ppm (Todd, 1970), depending on the species tested and the test conditions. As previously stated, copper, applied in various forms, has been used for some time as an algicide. Young and Lisk (1972) reported complete inhibition of growth in Anabaena variablis by 0.30 ppm cupric ion and stated that green algae appear to be less sensitive to copper than are blue-green algae. This finding was supported by Kallqvist and Meadows (1978) when they reported that cupric ion applied at a concentration of 24 ppb prevented a blue-green algae bloom, but that the green algae developed normally. They also found that photosyn- thetic production of a natural assemblage was reduced to 80 percent of the control with the addition of 0.10 ppm cupric ion and to 50 percent of the control with 0.15 to 0.20 ppm cupric ion. They further report that the addition of 0.05 ppm cupric ion reduced the growth rate of the algae to 40 percent of the control within a few days (Kallqvist and Meadows, 1978). Bartlett et a1 (1974) found that 0.05 ppm cupric ion initiated growth inhibition in Selenastrum 9 capricornutum and that 0.09 ppm cupric ion was completely inhibitory towards growth. McBrien and Hassal (1967) found that a concentration of luM CuSO4 (0.255 ppm CuSO4) caused an increase in the lag phase of Chlorella culture growth and resulted in decreased cell counts after four days. They noted that a concentration of 2uM CuSO4 (0.51ppm CuSO4) com- pletely inhibited culture growth. Sunda and Guillard (1976) found that growth rate inhibition and copper toxicity are related to cupric ion activity and not to the total copper concentration. Various modes of action of cupric ion toxicity have been reported. Several researchers (McBrien and Hassal, 1967: Cedeno-Maldonado and Swader, 1976) reported that cupric ion reacts with protein sulfhydryl groups and in this way inhib- its algal growth. Cedeno-Maldonado et a1 (1972) also reported that cupric ion inhibits electron transport in chloroplasts and, in this way, cuts down on the photosyn- thetic production. Gross et a1 (1970) noted that at a con- centration ratio of 1.27ug Cu2+/106 Chlorella cells, there was a significant drop in cell chlorophyll content. Further- more, McBrien and Hassal (1965) found that cupric ion caused a release of potassium from Chlorella cells in amounts exceeding the number of equivalents of cupric ion. This indicates an increase in cell permeability. In the course of this study, the exact mode of action of copper toxicity will not be assessed. Because of the exten- 'sive amounts of research done on the element and because of 10 its known algicidal properties, it is useful as a test of the sensitivities of the two algal assay procedures chosen. p-Cresol p-Cresol (4,methyl-phenol or 4,hydroxy-toluene) is a phenolic compound with the chemical formula C7H30. p-Cresol is a weak acid with a pKa of 10.2 at 25°C. It melts at 34.8°C (Chemical Rubber Co., 1974). p-Cresol has a solu- bility in water of 1.8 mg/ml at 25°C (Lang, 1973) and has a low vapor pressure (Chemical Rubber Co., 1974). It is a constituent of creosote, which is widely used as a wood pre- servative and for a variety of other uses (U.S. Environmental Protection Agency, 1978). This phenolic compound is also a significant by-product of coal gasification, coal liquifica- tion, and petroleum refining processes (U.S. Environmental Protection Agency, 1978: Boling, personal communication). With the increased production of these fuel products, water contamination with p-cresol is a distinct possibility. Campbell et a1 (1979) have shown very clearly how underground coal gasification projects can lead to groundwater contamina- tion with p-cresol. They also proved that there may be a delayed release of this chemical due to the disruption of water movements in the area surrounding these coal gasifica- tion projects. In the aqueous environment, p-cresol has been shown to react in several ways. p-Cresol may be chemically oxidized 11 to yield Pummerer's ketone, 2,2‘—dihydroxy-4,4'-dimethyl- biphenol and the trimer of this compound (U.S. Environmental Protection Agency, 1978). Furthermore, p-cresol may undergo photolysis to yield a variety of organic compounds (Joschek and Miller, 1966). Both of these processes occur at a very slow rate (U.S. Environmental Protection Agency, 1978). The most significant degradation pathway of p-cresol in the aquatic environment is via biodegradation (U.S. Environmental Protection Agency, 1978). This is considered virtually the only breakdown pathway in eutrophic systems and photolysis becomes a significant factor only under oligotrophic condi- tions. Adsorption of p-cresol onto suspended particles and into the sediments is considered negligible (U.S. Environ- mental Protection Agency, 1978). These relationships can be seen in Table 1. Several genera of bacteria have been found to be biodegrade p-cresol. These include Escherichia coli (Landa et a1, 1953), Aerobacter aerogenes (Phillips and Hinschelwood, 1953), Pseudomonas sp. (Dagley and Patel, 1957: Claus, 1964: Bayly et a1, 1966), and Achromobacter s2. (Claus, 1964). Bunch and Chambers (1967) found that greater than 99.5 percent of the p-cresol initially in their samples was degraded in seven days in their biodegradability tests. A representative breakdown pathway for p-cresol in Pseudomonas is: p-cresol-—:p-hydroxymethylphenol-—:p-hydroxybenzaldehyde p-hydroxybenzoic ac id -9 protocatechnic ac id—: ring cleavage (U.S. Environmental Protection Agency, 1978). ‘12 Table 1. Predictions based on a nine-compartment environmental exposure model for p-cresol (U.S. Environmental Protection Agency, 1978) Half Suspended life Solution Solids Sediments Percent Environment (hr) (glml) (gig) (gig) sorbed River 0.55 0.980 9.80 9.65 0.1 Pond 12.0 0.017 0.17 0.17 0.3 Eutrophic 12.0 0.037 0.37 0.10 0.05 Lake OligotrOphic 2400 0.0221 2.20 0.94 0.05 Lake 13 p-Cresol may indeed be biodegraded in less than one hour (Boling, personal communication); however, little work has been done on this chemical in relation to its effect on aquatic life. LDso's ranging from 5 to 15 ppm p-cresol have been reported for fish and invertebrates (McKee and Wolf, 1963). Boling and CoOper (1980) found a significant shift in the nocturnal drift patterns of invertebrates upon the addition of 8 ppm p-cresol to their experimental stream. They also noted that bluegills exhibited abnormal behavior when exposed to 2.5 ppm p-cresol and a cessation of feeding when exposed to 8 ppm p-cresol. No literature was found pertaining to the effects of this chemical on algal growth. Chlorella vulgaris The experimental algae, Chlorella vulgaris Beijerinck, is a planktonic, unicellular green algae with large cup- shaped chloroplasts (Trainor, 1978). Its planktonic char- acter makes it suitable for a batch culture testing proce- dure, minimizing the loss of algae cells due to adhesion to the walls of the test vessel. Its unicellular habit makes it easy to enumerate, and its large cup-shaped chloroplast is an easily identifiable characteristic of this genus (Trainor, 1978). Chlorella is fairly ubiquitous in nature (Smith, 1933) and divides about as rapidly as any green plant, making it ideal for phycological studies (Trainor, 1978). It is a relatively small alga (4 to 5u), necessitating the use of a l4 high-power, high-resolution microscope (Smith, 1933). Its nearly perfect spherical shape makes it an ideal specimen for enumeration by electronic particle counters. This was one of the primary reasons why Chlorella vulgaris was chosen for this study. However, mechanical problems with the available particle counter precluded its use in this study. ALGAL CULTURE METHODS One of the aims of this study was to assess the effec- tiveness of two algal bioassay procedures in detecting the effects of toxicants on algal culture growth. The two methods chosen, the Alagal Assay Procedure--Bott1e Test (Miller et a1, 1978) and the method of Young and King (1973), differ in several respects. In this study, an attempt was made to adhere as closely as possible to the standards put forth by the United States Environmental Protection Agency (Miller et a1, 1978) for algal bioassays. The following sections briefly describe these standards and any modifica- tions which were made in order to make the two methods more comparable. For brevity, the modification of the Algal Assay Procedure--Bottle Test used in this study is referred to as the AAP. The method of Young and King (1973) is referred to as the pH method. Physical Conditions An assay platform, similar to that described by Miller et a1 (1978), was constructed and divided into 64 equally sized and spaced sections, each of sufficient size to accom- modate one culture vessel. Miller et a1 (1978) recommend that the algal cultures be grown under 400 *10 percent 15 16 footcandles of constant fluorescent illumination. The lighting for this study was supplied by eight 48-inch-long General Electric fluorescent plant-grow light bulbs mounted on a scaffolding above the assay platform. Light intensity measurements (footcandles) were taken prior to and following the conclusion of each experiment with a Model 756 weston Illmmination Meter equipped with a quartz filter Number 2403. Those spaces falling in the 360 to 440 footcandle range were used in the test. The Photosynthetically Active Radiation (PAR) also was measured because recent literature suggests that this is a more realistic measurement of the light avail- able to plants for photosynthesis (Trainor, 1978). A LiCor Integrating Photometer, Model Li 18813, equipped with a Li 1925B underwater sensor was used for this purpose. Fig- ures showing the variation of both footcandles and PAR across the assay platform surface are presented in the text. Glassware was cleaned with a laboratory soap solution, rinsed with distilled water, and acid washed with chromic acid. Following these steps, the glassware was soaked in a 30 percent hydrochloric acid solution for six hours and rinsed with distilled water. These steps were taken to mini- mize contamination of the Chlorella vulgaris cultures. Miller et a1 (1978) recommend that the pH of the cul- tures be maintained by continuous mixing on a shaker table at 100 rpm. Efforts were made to fulfill this requirement: however, the heat generated by the shaker table raised the temperature of the cultures to 32°C, far above the 17 recommended temperature of 25°C. An alternative method of pH control was devised. Line air was delivered through a filter and water satu- rator (glass wool in a 250 ml flask containing approximately 100 m1 of distilled water) and a series of brass aquarium gang valves to the culture vessels. The valves allowed regu- lation of the airflow to each flask. A pasteur pipette inserted through a Number 9 two-hole rubber stopper deliv- ered the air to the bottom of the culture vessel. A second pasteur pipette, inserted tip up and capped with a foam plug, provided a pressure release while minimizing culture evaporation and contamination. This closure was used for all AAP culture vessels and for the stock culture vessels. The closure for the pH method test vessels was a modi- fication of those described by Young and King (1973). A Number 9 two-hole rubber stopper was fitted with a Number 12 serum cap and a pressure-release apparatus. The pressure release consisted of a piece of tygon tubing inserted through the open hole of the stopper. The opposite end of the tubing was placed underwater in a beaker. This apparatus allowed for the release of evolved oxygen from the cultures while minimizing atmospheric recarbonation of the medium. Algal Culture Medium The components of the algal culture medium are listed in Table 2. This medium is a modification of that developed by Kevern (personal communication) and is higher in nutrient 18 Table 2. Composition of synthetic algal nutrient medium. The components listed are given as final concen- trations in milligrams per liter. MACRONUTRIENTS Concentration Compgund (3911) NaHC03 168.0 KNO3 114.0 CaC12'2H20 13.2 MgSO4'7H20 20.0 K32P04 3.0 MICRONUTRIENTS Concentration Compgund (fig/1) H3803 2.86 MnC12'4H20 1.81 ZnSO4‘7HZO 0.22 (NH4)5M07024 0.18 CuSO4 0.05 CoC12'6H20 0.40 FeC13'6H20 1.20 NazEDTA 3.00 19 concentrations than the medium recommended by Miller et a1 (1978). The intent of raising the nutrient concentrations was to ensure that all components were in nonlimiting quanti- ties. The exception to this is carbon in the case of the pH method, but not in the AAP where the medium is constantly recarbonated. Fitzgerald (1975) states that whereas the maximum yield of an algal culture depends on the concentra- tion of nutrients in the medium, the rate of growth of cultures in different media remains the same up to the point of nutrient deficiency. The medium chosen for these experi- ments should have alleviated any problems of nutrient defi- ciency which would have complicated the analyses. The medium was prepared with reagent grade chemicals and distilled-deionized water on a regular basis. Macronutrients were added directly to 18 1 of distilled-deionized water in a S-gallon glass carboy. This mixture was aerated for 48 hours to ensure carbonation of the medium. The micronutrients, with the exceptions of FeCl3'6H20 and NaZEDTA, were combined in a stock solution at 1,000 times the final medium concentra- tion. The FeCl3°6H20 and NaZEDTA were combined in the same manner. Eighteen m1 of each stock were added to the aerated macronutrient mixture. Experimental Algae The experimental algae, Chlorella vulgaris Beijerinck, were obtained from the University of Texas Algal Collection. 20 Upon receipt, the culture was transferred into freshly pre- pared nutrient medium. Weekly inoculations of fresh medium with 1 ml of 6- to 8-day-old culture kept the algae in an actively growing condition. AAP Sampling, Enumeration, and Calculation Methods The USEPA (1971) lists various methods of estimating the biomass of algal cultures. Although gravimetric methods may be preferable, these researchers state that indirect mea- surements of biomass, suCh as cell counts, normally will be required because of the difficulty in making accurate gravi- metric measurements at low cell densities. Therefore, the preferred method is indirect enumeration via an electronic particle counter. This was the method chosen for this study: however, as previously noted, an alternative method had to be found. This other method, found to be reliable by Miller et a1 (1978), is direct microscopic enumeration. The direct counting method chosen was that of McNabb (1960). A twenty- five-milliliter sample was withdrawn from each culture with a pipette. Portions of this sample were then filtered at 8 cm pressure through a 0.45u Millipore filter. The filters were air dried for l to 3 hours prior to mounting them on glass microscope slides with Type A, microscope objective immersion oil. This process had the effect of making the filters transparent while preserving the algae in their natural form. 21 After enumeration, the raw cell counts were converted to biomass estimates (cells/ml) by the following formula: cells/ml =3 (_N__C_) (ME) (RE) (6) F V where NC is the number of cells counted, F is the number of fields counted, NF is the number of filters counted, V is the volume of sample filtered (m1), and MP is the magnification factor. This factor is determined by the equation MF = effective filteringarea (7) (microscope field width)2 2 Using these cell number estimates, the specific growth rate, u, for the cultures can be determined by the equation u = N/At (8) n where N is the change in cells/m1 over the time increment At and n is the average number of cells/ml during this time increment. The USEPA (1971) states that the maximum specific growth rate (“max) for an individual flask is the largest u obtained at any time during incubation. Since this is an observed value, as opposed to a calculated “max: this parameter was referred to as the maximum u observed (observed “max, in this study. As discussed in the following section, the pH method allows for the calculation of “max rather than taking the observed “max: To avoid confusion, the distinction between these two parameters is noted. The recorded biomass estimates (cell counts), u values, and observed umax values for each treatment were compared in 22 order to detect changes in culture condition due to the toxi- cant being studied. pH Method Sampling, Measurement, and Calculations The basis for the estimation of algal growth response in the pH method is the change of culture pH over time. The starting point for the calculations in this method is the. initial carbonate-bicarbonate alkalinity (a) of the medium, the initial pH of the medium, and the organic carbon content of the algal seed inoculum. The alkalinity of the medium was measured prior to the start of a test by the potentio- metric method (American Public Health Assoc., 1965). The potentiometric method yields the total alkalinity of the system. To determine the noncarbonate portion of the alka- linity, a replicate sample of the medium was acidified to pH 3 with 1N H2804 and sparged with nitrogen gas for 5 min- utes to remove C02. The COz-free sample was back titrated to the initial pH of the medium with 0.02N NaOH to determine the noncarbonate alkalinity. Subtraction of the noncarbonate alkalinity from the total alkalinity yields the alkalinity due to the carbonate-bicarbonate buffering system (a). The total organic carbon content of the algal seed inoculum was determined with a Beckman Model 915A carbon analyzer. Replicate samples of the cultures were acidified to pH 3 with 1N HCl and sparged with nitrogen gas for 5 min- utes to rid the samples of inorganic carbon. Fifty ul sam- Ples of the cultures were injected into the 900°C. oven of 23 the carbon analyzer. The results were compared to standard curves of known organic carbon concentrations prepared by analysis of potassium acid phtlalate standards. Samples from the pH method test vessels were taken with 25-ml syringes fitted with 18-gauge hypodermic needles. The pressure release tubes were sealed with a cork stopper prior to sampling. The contents of the test vessels were mixed by swirling and inverting the vessels for sampling. The sam- pling procedure, penetration of the septum of the serum cap with the hypodermic needle, and withdrawal of the sample, created a slight vacuum in the test vessel. This was equal- ized by injecting 25 cc of nitrogen gas into the test vessel with a hypodermic needle. The samples were then injected into a 50 m1 beaker. This was done by placing the tip of the needle at the bottom corner of the beaker and slowly injecting the sample into the beaker so as to minimize recarbonation. The pH of the sample was measured with a Beckman Zeromatic pH meter equipped with an Orion Research Needle Tip combination electrode. Prior to each use, this instrument was calibrated with standard buffer solutions for the culture pH anticipated. In the first few days of growth, buffer solutions in the range of pH 7 to 10 were used. After the culture exceeded pH 10, the meter was calibrated with buffer solutions in the pH range 9 to 11 in an attempt to minimize interferences usually encountered in solutions at high pH (American Public Health Assoc., 24 1965). The accuracy of the pH meter and electrode was determined to be approximately 0.1 pH unit. Young and King (1973) recommend taking the pH of the sample in a nitrogen gas-filled apparatus, so as to avoid recarbonation of the sample. Testing of their method versus the one mentioned above revealed no appreciable difference in pH between replicate samples. Even at high pH, the recare bonation of the sample occurred at a slow enough rate as to allow accurate determination of the sample pH. Using the pH measurements, initial medium alkalinity (a) and the organic carbon content of the algal inoculum, the calculation method of Young and King (1973) was used to esti- mate the growth kinetics of the cultures. The pH measure- ments were transformed into values of carbon fixed by the algae by the equation Mt = C02 = ( C02t0 - C02t) (9) where Mt is the change in biomass (mM organic carbon/1) over the time increment t and C02 is determined by the equation Hz/Kl + H + K2 C02 = a( ) (10) H + 2K2 where C02 is the total molar sum of the inorganic carbon species in the medium (mM/l): a is the carbonate-bicarbonate alkalinity of the medium (meq/l) which has been corrected for the hydroxide ion concentration of the medium; H is the hydrogen ion activity as determined by a pH meter; and K1 and K2 are the first and second dissociation constants of 25 carbonic acid. Values of K1 and K2 are temperature- dependent and, for the purposes of this study (temperature = 27°C.), the values used were K1 2 4.3x10"7 and K2 = 4.9 x 10-11. Likewise, the free carbon dioxide concentration of the medium was determined from the pH and initial alkalinity of the medium by the equation “2 COzf = a( ) (11) K1 (H +2K2) where C02f is the free carbon dioxide concentration of the medium (mM/1). The specific growth rate, u, is calculated over the time interval t by the equation u =- 31/."- (12) m where M is the change in biomass (mM carbon fixed by the algae/l) over the time increment t, and m is the average standing crop biomass (mM C/l) during the time increment. Models predicting u as a function of the concentration of a limiting substrate have been used for some time. One such model is the Monod Equation (after Lehniger, 1970): U 3 “max S (13) KS + s where S is the concentration of a substrate, ”max is the maximum specific growth rate, and K3 is the saturation con- stant (substrate concentration at which u = l/2 “max- Equa- tion (13) is also known as the Michaelis-Menten Equation (Lehniger, 1970). 26 If an assumption is made that there exists a lower threshold concentration of the substrate in question; that is, a concentration below which the algae cannot assimilate the substrate, equation (13) can be shown as: S - Sq u = umax ( _ ) (14) (K3 - Sq) + (S - Sq) where Sq is the lower threshold concentration of the sub- strate. Therefore, when S = Sq, u = O. In the pH method, where C02f is the limiting substrate, equation (14) can be shown as: CO2fo ‘ KCO 3f° ) (15) u = umax ( (Kco * COzfq) + (C02fo ’ COzfq) 2fo where COZfo is the observed C02f concentration of the medium and COqu is the lower threshold concentration of C02f. The substrate concentration represented by COZfo - COqu is the actual concentration of C02f in the medium available to the algae for photosynthisis (COZfA). To obtain CngA, one must find Cngq. Two methods of obtaining COqu were assessed. The first is to estimate COzfq by observation. That is, when during the course of the experiment the calculated u for a replicate = O, the COzfq concentration at that point can be taken as COzfq. Averaging the COqu values for the replicates then gives an estimate of COzfq for that treatment. A second method is to estimate C02fq graphically from the experimental data. Since growth curves for algae are logrithmic in nature (up to the stationary growth phase), one 27 should expect that in a closed system that nutrient uptake also would be logrithmic. Since C02f is the limiting nutrient, a ”mirror image” of C02f uptake vs growth (biomass) is anticipated. Therefore, plots of u vs C02fo were used to estimate the C02f concentration at which u = O, the COzfq value. These curves are of the form u a a + b (IDCOZfo) (16) The second method of estimating C02fq was chosen for use in this study because it utilizes all of the data from the active growth phases of each replicate, as opposed to one value per replicate. Although this method led to some extrapolation of the data, the COqu estimates yielded by this method were several orders of magnitude below the C02f values obtained and did not influence the determination of Umax or Kcozfo The value for COqu obtained by this method was then subtracted from COZfo to yield COzfA (the actual C02f concen- tration available to the algae for photosynthesis at time t). Rearranging equation (15) and substituting C02fA for C02fo - COqu yields Kco2fo ' C02£q 1/u = l/umax + (l/COzfA) (17) umax which is a linear regression equation of the form Y = a + bx. This transformation is known as the Lineweaver-Burk Equation (Lehniger, 1970). 28 Curves of the linear transform l/u vs 1/C02fA were therefore used to determine the kinetic constants “max and - CO . The latter kinetic constant, as determined 2fo 2fo by the curve-fitting procedure, is the corrected C02f con- Kco centration where u equals l/Zumax, or KcozfA. The specific growth rates and corresponding COZfA con- centrations which occurred during the active growth phase of the cultures were used in this curve-fitting process. Attempts were made to use the Monod Equation as a mathematical model in this study. The three kinetic con- stants, derived from algal cultures exposed to various toxicant concentrations, were examined for changes due to the toxicant. When either COzfq, Kcozf, or “max varied as a function of the toxicant concentration, they were substi- tuted into the Monod equation as follows: C02f - f3(Z) u = f1(Z) ( ) (18) [f2(2) ' 53(2)] + [(C02f - f3(Z)] where f1(Z) equals umax as a function of the toxicant con- centration, f2(Z) equals KCOzf as a function of the toxicant concentration, and f3(Z) equals COzfq as a function of the toxicant concentration. Such models would have been useful in predicting the sublethal effects of a toxicant on the specific growth rates of Chlorella vulgaris cultures at various toxicant and medium C02f concentrations. However, as discussed in Experiments 4 and 5, the growth of the algal cultures did not follow the Michaelis-Menton growth kinetics 29 equations closely enough to warrant the extrapolation of the data in further modeling. Statistical Analyses and Computer Software The statistical methods employed varied with the setup of the experiments. Complete randomization was used wherever possible. Because of the volume of the data, analyses of variance, t-tests, multiple-range procedures, and the other statistics used were calculated using the Statistical Package for the Social Sciences (SPSS) computer software package (Nie et a1, 1975). Assay platform light level contour maps were produced by using the Surface II Graphics Software package (Sampson, 1975) and a CalComp Plotter. Calculation of the algal growth kinetics was performed by using a Fortran rou- tine designed by the experimenter. All computations were performed by the Control Data Corporation Model 170 Cyber Series 750 computer maintained by the Michigan State Univers- ity Computer Laboratory. PRELIMINARY EXPERIMENTS The aim of this series of tests was to locate and attempt to minimize various sources of error in the experi— mental methods. The first two experiments dealt with sources of error encountered in the enumeration procedure of the AAP. The third experiment was a trial run of the two algal bio- assay methods chosen for use in this study. 30 EXPERIMENT 1 Cell Counting Procedure Error Purpose In this experiment, an attempt was made to locate and eliminate sources of error in the enumeration procedure of the AAP. Procedure Three 5 ml replicate samples were withdrawn from a stock culture of Chlorella vulgaris. Three 0.5 ml subsamples of each sample were filtered and fixed on glass microscope slides. Twenty microscope fields were counted for each sub- sample at 1,000X. A nested analysis of variance (Sokal and Rohlf, 1969) was used to test the significance of the vari- ance components in the model Yijk = u + Si + Fij + Cijk (19) where Yijk is the observed sample mean: u is the true popu— lation mean: Si is the effect due to sampling: Fij is the effect of subsamples within samples: and Cijk is the effect of cell counts within subsamples. Results The results of the cell counting procedure are tabulated in the Appendix (Table A1). The results of the analysis of '31 32 variance indicate that the variability due to sampling is not significant (Table 3). The low level of significance due to the variability involved in subsampling indicates variability due to the filtering procedure as well as sub- sampling. Since some clumping of the algal culture sampled was noted, and since pasteur pipettes were used in the sam- pling procedure, it was felt that the error due to subsampling makes up the majority of the variability in this term. Conclusions The majority of the variation in the enumeration pro- cedure was due to the cell counting. The sampling effect was not significant. Since some variation was due to sub- sampling, future samples were directly enumerated. Also, volumetric glassware was used. The clumping of the algal cells appeared to be a func- tion of culture age. This was determined by microscopic examination of cultures at different stages of growth. This effect was minimized by thoroughly mixing the culture prior to sampling and mixing the samples prior to filtration. As indicated in the analysis, the maximum effort should have been placed in counting rather than in sampling. Therefore, in the following experiments one sample was withdrawn from each culture flask at each sampling time and analyzed. 33 Table 3. Nested analysis of variance studying the vari- ability in the sampling, subsampling, and counting procedures used for the AAP. Source df SS MS F Total 179 3,115.4 Sample 2 38.9 19.5 0.577 ns Subsample 6 203.0 33.8 2.012 * Count 171 2,873.5 16.8 *** P = 0.99 ** P = 0.95 * P = 0.90 + P = 0.80 ns P = 0.80 (Not significant) EXPERIMENT 2 The Effects of Culture Concentration of the Cell Counting Procedure PUIEOSB In Experiment 1, all samples were of the same volume and were enumerated at the same magnification. This experiment was designed to test the effects of the use of various sam- ple volumes and microscope magnifications on the enumeration procedure. This gave an indication of the responsiveness of the modified McNabb (1960) method to differing biomass levels. Procedure A sample of 8-day-old Chlorella vulgaris culture was taken and various dilutions made (Table 4). These subsamples were filtered, fixed on slides, and enumerated. The results were analyzed by linear regression. Results One would have expected a linear relationship between the expected and observed cell numbers, if the volume of the sample filtered and/or the microscope magnification used made no difference in the enumeration procedure used here. The high degree of correlation found between these variables (r2 = 0.992, n = 6, log transformation) indicates that this 34 35 Table 4. Experimental setup and results from a test of the modified McNabb (1960) method of enumerating algal cells. Shown for cell count and cells/ml are the mean *1 standard deviation for ten replicates. Filtered Dilution volume Microscope Cell Cells/ factor (m1) magnification count ml 1 2.0 1,000 TNC -- 1 0.5 400 159 2.9x106 #53 *9.8x105 5 5.0 1,000 TNC -- 5 1.0 1.000 16 9.3x105 *3 t1.7x105 10 1.0 1,000 6 3.5x105 *2 *1.2x105 50 20.0 1,000 33 9.6x104 17 *2.0x104 100 20.0 1.000 9 2.6x104 *7 *2.0x104 500 20.0 400 14 6.4x103 *4 *1.8x103 1,000 20.0 400 6 2.8x103 *3 i1.4x103 TNC = Too numerous to count. 36 was the case for the range tested in this experiment .(Figure 1). It was noted that counting the Chlorella vulgaris cells at low magnification (400K) was difficult because of their small size. Increasing the sample size at low-culture densities eliminated the need for enumeration at this magnification. Conclusions From the results of Experiments 1 and 2, it was con- cluded that the modified McNabb (1960) method of enumerating algal cells was an acceptable alternative to indirect enume- ration by an electronic particle counter. The use of direct enumeration for the estimation of algal culture biomass is preferred by Miller et a1 (1978) over other methods such as absorbance and chlorophyll-a fluorescence. By varying the filtered volume and microsc0pe magnification, one can accu- rately estimate the algal culture density over a broad range (103 to 106 + cells/ml). 37 6" _ .__. T E ' i .9. - q 3 3 r- a O 351‘” .1 E - _ .0 'U I- .4 0 > L- O " - G D o — d §4— - " log ya -0.272 + 1.077 log at R I- -( 3 l l l l l l 1 1 l l 1 1 1 L l A l n 3 4 5 6 7 I09 Expected biomass (cells/ml) Figure 1.. The relationship between the expected and observed biomass estimates using the modified McNabb (1960) method of algal cell enumeration. EXPERIMENT 3 Trial Run of the AAP and pH Methods PUIEOSO This test was designed to familiarize the experimenter with the two algal bioassay methods chosen for use in this study. The purpose also was to isolate and correct proce- dural difficulties in these algal assay techniques. Procedure Ten culture vessels were each half filled with 500 ml of fresh nutrient medium (a = 1.77 meq CO32'--HC03' alk- alinity/l). Five of these vessels were randomly assigned to the pH method and five to the AAP. Measurements of light reaching the assay platform were taken and 25 of the 64 spaces on the assay platform were within the acceptable range of 400 j=40 footcandles of illumination (Figure 2). The test vessels were randomly assigned to 10 of these 25 spaces. Figure 3 shows the positions of the test vessels in relation to the photosynthetically active radiation (PAR) falling on the platform. Each flask was inoculated with 0.5 m1 of 6-day-old Chlorella vulgaris stock culture, giving a final concentra- tion of 5,300 cells/ml. The organic carbon content of the stock culture was 146 mg/l, as determined by carbon analysis. 38 Row 39 Figure 2. Column Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 3. The position of each test vessel is indicated by a circle. 4O Row Figure 3. Column Contour map of the photosynthetically active radiation (uE/m 2lsec) falling on the assay platform 1n Experiment 3. The position of each test vessel 18 indicated by a circle. 41 This equates to an experimental culture content of 0.0122 mM organic C/l. Daily samples of the AAP cultures were taken and the number of cells/m1 counted. Samples of the pH method cultures were taken on a daily basis and the pH determined. The pH of the AAP cultures was also monitored to ensure proper recarbonation of the medium by the pH control appa- ratus. The test was run at 27°C. The length of the test period was determined by examin- ing growth curves plotted as the test proceeded. The test continued until the stationary growth phase had been reached. Results The constant aeration of the medium kept the pH of the AAP cultures at a moderate level. The sharp rise in pH between days 4 and 5 (Figure 4) was the result of a break in the air delivery system. The return of the pH to a moderate level between days 5 and 6 illustrates the effectiveness of the pH control method used. The results of AAP culture pH control from Experiments 4 and 5 are also shown in Figure 4. In all three experiments, the pH control apparatus kept the pH of the AAP cultures within the guidelines of Miller et a1 (1978). The daily cell counts of the AAP cultures are tabulated in the Appendix (Table A2). These cell counts were used to produce the growth curves in Figure 5. The five AAP cultures remained in the lag phase of growth for approximately one day and reached their maximum standing crop biomass levels by day 5, at which time a lack of light due to shading effects 42 IO.5 —r I l 1 l 1 1 l I l T 10.0- '- 9’:- - I O o. 900'- / ‘ e o e — / . 8.5- 1 ._—.. Experiment 3 0—0 Experiment 4 H Experiment 5 8.0 J l l l l l I L_L J J O I 2 3 4 5 6 7 B 9 )0 H l2 Time (days) Figure 4. pH fluctuation of AAP cultures of Chlorella vulgaris from Experiments 3, 4 and 5. 43 7 1 1 1 r I I .J E \ - -"’- _ B 5 U n O 2 d z i I I l l J O 25 5O 75 IOO I25 I50 (75 Time (hr) Figure 5. AAP growth curves for Chlorella vulgaris cultures in Experiment 3. 44 within the cultures may have become the limiting growth factor. The statistical analysis of the data (Table 5) indicates that although the growth curves are fairly uniform, at several samplings there existed a significant difference between the biomass estimates of the cultures. It also was noted that the biomass order of the cultures changed on a daily basis (Table 5). These results indicate a nonuniformity of growth in the various cultures, independent of initial algal inoc- ulum. The variability of the growth rates of the five repli- cates is shown in Table 6. All five replicates reached their respective maximum specific growth rates (observed “max) in the interval between sampling times 21 and 44.5 hours. This observation is in accordance with the findings of Forsythe (1973) and Miller et al (1978) who stated that the observed “max is generally reached in the first few days of culture growth. The mean observed “max for the five replicates was 0.072 hour'l. The daily pH measurements of the pH method test cultures are tabulated in the Appendix (Table A3). The biomass curves for the pH method cultures (Figure 6) also are fairly uniform. As shown in Table A3, the pH measurements for all five repli- cates vary only by a few tenths of a pH unit throughout the experiment. Given the inherent variability of the pH meter (*0.1 pH unit), there was a high degree of replicability with the pH method in this experiment. The means and stan— dard deviations of the transformed data are given in Table 5. 45 Table 5. The statistical analysis of Experiment 3. The one- way analysis of variance F test and coefficient of variability are listed for each sampling period for the AAP and pH method, respectively. For the AAP, the replicates are listed in ascending order of biomass. Underlining indicates no significant dif- ference within the underlined group as judged by a Student-Newman-Keuls multiple comparison test (P a 0.95). For the pH method, the mean of 5 replicates *1 standard deviation is shown. Time AAP AAP pH method (hr) F value replicate (biomass) 21.0 7.08 *** 3 1 4 2 5 0.0241 *0.0090 44.5 1.51 ns 3 4 1 5 2 0.1274 t0.0325 68.5 21.17 *** 1 4 2 5 3 0.5447 *0.1373 93.5 0.72 ns 1 2 5 4 3 1.6127 *0.0744 116.5 3.39 ** 1 5 2 4 3 1.7636 i0.0439 140.5 2.58 ** 1 5 2 4 3 1.7636 1:0.0439 164.0 2.27 * 5 1 2 3 4 1.7636 t0.0439 *** P 3 0.99 ** P = 0.95 * P = 0.90 ns P = 0.80 (not significant) 46 Table 6. Specific growth rates (u) of Chlorella vulgaris cultures in Experiment 3, as calculated by the AAP. Maximum specific growth rates (“max) are underlined. Units of u are 1/hour. Replicates 1 Interval 1 2 3 4 5 1 0.0182 0.0191 .0.0047 0.0186 0.0235 2 0.0717 0.0719 0.0747 0.0714 0.0704 3 0.0503 0.0563 0.0635 0.0541 0.0570 4 0.0511 0.0452 0.0392 0.0495 0.0452 5 0.0093 0.0187 0.0195 0.0204 0.0092 6* 0.0065 -0.0029 -0.0023 -0.0024 0.0059 7 0.0078 0.0071 0.0046 0.0087 0.0036 *Individual replicates exhibited a decline in biomass. The group as a whole had an average u = 0.0006 hour-1. log Biomass (mmoles CH) 47 l 1 r l I I I q q q )- -( r 1 _ 3 l l l I l l 0 25 50 75 |00 l25 I50 I75 Time (hr) Figure 6. pH method growth curves for Chlorella vulgaris cultures in Experiment 3. 48 To estimate the photosynthetic C02 minima, the specific growth rates from the active phase of growth were plotted versus the corresponding average COZfo concentrations of the medium (Figure 7). The data were fit with a natural loga- rithmic regression curve and gave a COqu estimate of 9x10‘5 uM 002/1 (r2 = 0.970, n = 18). After correcting C02fo for C02fq, a double reciprocal plot of l/u vs l/COZfA was produced in order to estimate “max and Kco2f (Figure 8). A least-squares regression analysis of Figure 8 (r2 30.867, n =15) yielded KCOZf estimate of 0.0283 uM COZf/l and a u estimate of 0.0547 hour'1 for the combined max replicates. Conclusions The pH of the AAP cultures was maintained within the guidelines of Miller et a1 (1978) by constant aeration. This was the case for Experiments 4 and 5 as well as this experiment. The estimates of umax given by the two bioassay methods varied greatly. This may have been a function of the setups of the two methods and/or the variables used to estimate culture changes (cell counts versus carbon uptake). Because of these differences, the umax estimates from the two methods could not be compared statistically. The significant variations between replicates indicated a need for greater replication. However, because of the light-level restrictions and the time consumed in sampling 49 0 FTIIIIIII[WTIIlillllfirtlefirltwyu' h- c- P d I- -1 E . q \ :3-2 " r :1 )- T 0' _°_ " -1 (- J -3 .. Y - 0.0494 + 0.0053 In K )- q ' '1 -4lWa.thlln+lll-llllnlln -5 -4 -3 -2 -l O l 2 log C°2f0 (umoles / 'I) Figure 7. Specific growth rates of cultures of Chlorella vulgaris in Experiment 3 as a function of the correspoxfling free C02 concentrations of the medium. 50 5O 40)- l/u (hr) y ' I8.3 t 0.52)! IO - J o J J l l l J 0 5 l0 I5 20 25 30 35 I /C02fA (l/lUTlOIES) Figure 8. . Reciprocal plot of the specific growth rates of Chlorella vulgaris cultures in Experiment 3 versus the corresponding free C02 concentrations of the medium. 51 and enumeration, this was not possible in this study. There- fore, a more detailed analysis of variance, separating the error due to replication from that due to the treatment effects, was used in subsequent experiments. TOXICOLOGY EXPERIMENTS The following experiments were applications of the AAP and pH method algal bioassay techniques in assessing the effects of two toxicants on algal growth kinetics. The first two experiments dealt with the effects of cupric ion. The third experiment in this series was designed to assess the effects of p-cresol. The fourth experiment was a test of the degradation of p-cresol under algal bioassay conditions. 52 EXPERIMENT 4 The Effects of Cupric Ion on Chlorella vulgaris Culture Growth PUIEOSG This experiment was designed to test the effects of three concentrations of cupric ion on the growth kinetics of Chlorella vulgaris cultures. It was intended to show how a known algicide exhibits its inhibitory effect on algal growth in the two bioassay methods chosen for use in this study. Procedure Twenty-four test vessels were each half filled with 500 ml of fresh nutrient medium (a a 2.038 meq CO32_--HCO3‘ alkalinity/1). The average light intensity measurements (Figure 9) indicate that only 20 of the 64 spaces of the assay platform were in the acceptable range of 400 i40 footcandles of illumination during the test. However, at the beginning of the test, 24 of the 64 spaces were within this range, and the amount of bulb decay observed was not antici- pated prior to beginning the experiment. The 24 test vessels were randomly assigned to these spaces. The positions of the test vessels in relation to the photosynthetically active radiation falling on the platform are shown in Figure 10. 53 Row one 09: O OO O OO 4 000 -00 J,” 8 U N O N I Column Figure 9. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 4. The position of each test vessel is indicated by a circle. 55 8' ) 50”) “‘50 .. f 6- 0 0 0 0 Column Figure 10. Contour map of the pholsynthetically active radiation (uE/ m 2/sec) falling on the assay platform in Experiment 4. The position of each test vessel is indicated by a circle. 56 Twelve test vessels were randomly assigned to the AAP and twelve to the pH method. Within each method, three flasks were assigned to each of the three treatment levels of cupric ion. The remaining three vessels in each method were used as controls. One milliliter of cutrine (91.071 g Cu2+/l) was added to 999 ml of distilled deionized water to give a stock solu- tion concentration of 91.071 mg Cu2+/1. This stock solution was added directly to the test vessels within each method to give three replicates each of 0.05, 0.10, and 0.15 mg Cu2+/1. Each test vessel was inoculated with 0.5 ml of 7-day-old Chlorella vulggris stock culture to give a final concentra- tion of 3,400 cells/m1. Carbon analysis of the stock culture yielded a test culture concentration of 0.01133 mM organic carbon/1. Daily samples of the AAP cultures were taken and the number of cells/m1 computed. Samples of the pH method cultures were taken daily and the pH determined. The test was run at 27°C. The daily cell counts (cells/m1) from the AAP were tested for significant differences between treatments by a nested analysis of variance (Sokal and Rohlf, 1969) and Student-Newman-Keuls multiple comparison procedure (Steel and Torrie, 1960). ’The biomass estimates from the pH method (mM C fixed by the algae/l) were tested by an analysis of variance and Student-Newman-Keuls multiple comparison pro- cedure (Steel and Torrie, 1960). 57 Results The results of the daily cell counts of the AAP cultures are tabulated in the Appendix (Table A4). The resulting growth curves (Figure 11) indicate a graded response to the four levels of cupric ion tested, with the 0.15 mg Cu2+/1 treatment cultures exhibiting acute toxicity. All four treatments were judged significantly different from one another at various sampling times (Table 7). The three cupric ion treatments were judged significantly different from the control until day 6 (104.5 hr), when both the control and the 0.05 mg Cu2+/l treatment had almost reached their maximum standing crop biomass levels (Figure 11). The 0.10 mg Cu2+/l treatment remained significantly different from the control until day 10 (236 hr) when it too had almost reached the stationary phase of growth (Figure 11). The 0.15 mg Cu2+/1 treatment cultures remained in the lag phase of growth for several days and eventually entered the death phase (Figure 11). The specific growth rates calculated from these biomass estimates are shown in Table 8. The control, 0.05 and 0.10 mg Cu2+/l, treatments reached their maximum specific growth rates in interval 4 (between the 68 and 91.5 hr samplings). The specific growth rates indicate that the control cultures apparently reached the observed umax at an earlier time than did the 0.05 mg Cu2+/l treatment cultures, leading to the conclusion that the control may have had a higher umax than observed. The observed um of the 0.10 mg Cu2+/l treatment ax log Cele/ml 58 r 1 I I I I P 4‘. A T b ’./’ A q t 1 .. a l" ' " r- .1 l- .— _ e———e control . - . ' °——-o 0.05 mg Cu+2 ll - b J 41—4 0.10 mg Cu+2ll ‘ ‘_ - H 0.15 mg 00”" D -( )- . .1 l .. J l- .- 2 I I I I I I 0 50 100 150 200 250 300 350 Time (hr) Figure 11. AAP growth curves for Chlorella vulgaris cultures in Experiment 4. Each point represents the mean of three replicates. 59 Table 7. Statistical analysis of the AAP results from Experiment 4. F test values and associated sig- nificance levels from a nested analysis of variance are listed for each sampling time. The treatments are listed in ascending order of biomass (cells/ml) with those means judged not significantly different using a Student-Newman-Keuls multiple comparison procedure (P = 0.95) grouped by underlining. Time (hr) F value Treatment (mg Cu2+/l) 20.0 0.35 ns 0.15 0.05 0.00 0.10 44.0 82.16 *** 0.15 0.10 0.05 0.00 68.0 122.00 *** 0.15 0.10 0.05 0.00 91.5 129.20 *** 0.15 0.10 0.05 0.00 140.5 21.85 *** 0.15 0.10 0.05 0.00 # 164.0 6.01 ** 0.10 0.00 0.05 188.5 4.93 * 0.10 0.00 0.05 212.0 2.57 + 0.10 0.00 0.05 236.0 2.55 + 0.10 0.05 0.00 *** P = 0.99 ** P = 0.95 * P = 0.90 + P = 0.80 ns P = 0.80 (not significant) # The 0.15 mg Cu2+/l treatment was terminated after this sampling. 60 Table 8. Specific growth rates (u) of Chlorella vulgaris cultures in Experiment 4, as determined by the AAP. Maximum specific growth rates are underlined. Treatment (mg Cu2+/l) Interval Control 0.05 0.10 0.15* 1 -0.0015 -0.0030 0.0000 -0.0030 2 0.0481 0.0407 0.0035 -0.0071 0.0636 0.0585 0.0428 0.0057 4 0.0644 0.0648 0.0496 -0.0126 5 0.0238 0.0297 0.0352 -0.0248 6 0.0010 0.0044 0.0160 7 0.0081 0.0069 0.0218 8 -0.0007 -0.0011 0.0137 9 0.0074 0.0024 0.0117 10 0.0079 11 0.0009 *The u values obtained from the 0.15 mg Cu2+/l treatment cultures may be due to sampling error and are shown for discussion purposes only. 61 was much lower than the observed umax of either the control's or the 0.05 mg Cu2+/l treatment cultures. The differences between the u values of the control and 0.05 mg Cu2+/1 treatment cultures appears to have been mini- mal (Table 8). The u values obtained for the 0.15 mg Cu2+/l treatment cultures were probably due to sampling error. For the purpose of discussion, the 0.15 mg Cu2+/1 treatment was assumed to be acutely toxic. Because of these factors, models predicting u as a function of (Cu2+) were not max derived from the AAP data in this experiment. The rise in the u values of the 0.10 mg Cu2+/l treat- ment cultures late in the test (intervals 5 to 9) and the final maximum standing crOp biomass level reached by these cultures (Figure 11) seemed to indicate an ability of the algae to overcome the sublethal effects of cupric ion. This could have been the result of binding of cupric ion in older cells and a subsequent decrease in cupric ion concentration. Conversely, the cells could have excreted a binding agent into the medium. Other possibilities include microbial degradation of the chelating agent in cutrine and the subse- quent precipitation of cupric ion as an inorganic salt, or precipitation caused by physical/chemical changes of the medium. These hypotheses were not tested. Several observations were made upon microscopic exami- nation of culture samples. In the cultures receiving cupric ion treatment, the individual Chlorella vulgaris cells were 62 noticably chlorotic. This was especially prevalent in the 0.10 and 0.15 mg Cu2+/l treatments and particularly notice- able in the first few days of culture growth. The second observation was that the cells of the 0.15 mg Cu2+/l treat- ment were noticably smaller than the cells of the other three treatments. These observations are similar to those noted by Cedeno-Maldonado_et a1 (1972) and McBrien and Hassal (1965), and may indicate a decrease in the chlorophyll content of the cells. The daily pH measurements from the pH method cultures are tabulated in the Appendix (Table A5). The growth curves for the four cupric ion treatment algal cultures (Figure 12) show a graded response to the treatments. An analysis of variance and corresponding multiple range test revealed that the biomasg estimates of all three cupric ion treatments were significantly different from the control during most of the control cultures' active growth phase (Table 9). In the case of the 0.05 mg Cu2+/l treatment cultures this differ- ence was probably caused by an increase of the lag phases of growth. After these cultures entered the active growth phase, their growth curve parallels that of the controls. The 0.10 mg Cu2+/l treatment cultures' growth was more drawn out than either the control or 0.05 mg Cu2+/1 treatment cul- tures, lasting over 350 hours. Sampling of the 0.10 mg Cu2+/1 treatment cultures was stopped at 355 hours because there was insufficient culture volume remaining for repre- sentative sampling. The 0.15 mg Cu2+/1 treatment cultures log Biomass (mmoles CH) -2 63 e———e Control 4 0—0 0.05 mg GIFIL o——o 0.10m CHI 0-—O 0J5 mg of): ‘ I I I I I l I I l 0 40 80 l20 ISO '200 240 280 320 360 Time (hr) Figure 12. pH method growth curves for Chlorella vulgaris cultures in Experiment 4. Each point represents the mean of three replicates. 64 Table 9. Statistical analysis of the pH method results from Experiment 4. F test values and associated signi- ficance levels from an analysis of variance are listed for each sampling time. The treatments are listed in ascending order of biomass (mM C fixed by the algae/1), with those means judged not signifi- cantly different using a Student-Newman-Keuls mul- tiple comparison procedure (P = 0.95) grouped by underlining. Time (hr) F value Treatment (mg Cu2+/1) 20.0 1.36 ns 0.00 0.05 0.10 0.15 44.0 35.43 *** 0.15 0.10 0.05 0.00 68.0 169.07 *** 0.15 0.10 0.05 0.00 91.5 47.98 *** 0.15 0.10 0.05 0.00 117.0 53.50 *** 0.15 0.10 0.05 0.00 140.5 145.15 *** 0.15 0.10 0.05 0.00 # *** P = 0.99 ** P = 0.95 * P = 0.90 + P = 0.80 ns P = 0.80 (not significant) # The 0.15 and 0.10 mg Cu2+/1 treatment cultures remained significantly different from one another, as judged by t-tests (P = 0.99), for the remainder of the former's growth cycle. 65 never entered the active growth phase, indicating acute toxicity of cupric ion towards these cultures. The photosynthetic C02 minima was estimated from curves of u versus COZfO. The curves for the three treatments in which growth occurred (the control, 0.05, and 0.10 mg Cu2+/l) are shown separately in Figures l3, l4, and 15 for clarity. . The resulting COqu values from these plots are shown in Table 10. A linear regression analysis of the COqu concen- tration estimate versus the toxicant concentration indicated that the two variables were not linearly correlated (r2 = 0.047, n = 3). Double reciprocal plots of l/u vs 1/C02fA were used to estimate “max and KCOfA' Figures 16, 17, and 18 are the plots for the three treatment cultures which exhibited an active growth phase. The resulting KCOZfA and “max values from these Lineweaver-Burk plots are given in Table 10. The “max estimates from the pH method were lower than the observed “max values from the AAP, as was the case in the previous experiment. However, as can be seen in Figures 16, 17, and 18, the Michaelis-Menton growth kinetics line drawn through the data does not appear to fit the data as well as might be indicated by the derived regression coefficients (Table 10). To test the applicability of Michaelis-Menton kenetics to this experiment, two other transformations of the Monod model were used. The first is known as the Eadie- Hofstee plot (Lehniger, 1970) and takes the form u = “max — Kco2fA (“/COZfA) <20) 66 0' I ‘F l I I T 1 1 l I l [ l p d . 4 log IL (I /hr) “2’ __ y - 0.0441 + 0.0041 In x 4 -3— - -4 1 I 4 I 1 I L J 1 L l L 1 -5 -4 -3 -2 -| 0 l 2 log COZ‘I’O (umoles/l) Figure 13. Specific growth rates of the control Chlorella vulgaris cultures in Experiment 4- as a function of the C02f0 concentration of the medium. 67 0‘ T T I I I I 1 I I I ’1 j I .b d p .. E- u .- .4 -I— e—Ii . - “C - - .2 2-2 F “ :f " n g - d L .4 _3 _ y - 0.0471. 0.0049 In x b .l -4. l I I L 1 L l L 1 I n I n -5 -4 -3 -2 -I O I 2 log (50ng (umoles/I) Figure 14. Specific growth rates of the 0.05 mg Cu2+ll treatment C orella vulgaris cultures in Experiment 4 as a function of the C02f0 concentration of the medium. 68 o T I I T l l j l I I I I I - -+ - 4 I- -l b '1 -I- —I 0 r d log 11 (I /hr) “2’ 45* y . 0.0206 + 0.0017 In x _L I 1 I I 1 I _J L L -4 -5L -2 -l 0 log Cngo (umoles / I) Figure 15. Specific growth rates of the 0.10 mg Cu2+/I treatment Chlorella vulgaris cultures in Experiment 4 as a function of the C02f0 concentration of the medium. 69 Table 10. Kinetic constants for the Chlorella vulgaris cultures in Experiment 4, as determined by the pH method. 2 r2 for Treatment COzfq r for KCO2£A umax KCOZfA (mg Cu2+/l) (uM 002/1) c02fq (uM 002/1) (hr‘l) and umax Controla 2x10‘5 0.888 2.51x10-3 0.0429 0.858 Controlb -- -- 3.07x10'3 0.0458 0.618 Controlc -- -- 7.18x10'2 0.0514 0.996 0.05 72:10-5 0.991 2.52x10-3 0.0437 0.950 0.10a 51:10“6 0.721 5.77x10'4 0.0182 0.795 0.10b -- -- 6.29x10-4 0.0193 0.395 0.10C -- -- 3.25x10-1 0.0290 0.861 aKineticl constants derived from double reciprocal plots of /u vs 1/C02fA (Lineweaver-Burk equation). bKinetic constants derived from u/COZfA vs u plots (Eadie- Hofstee plot). cKinetic constants derived from COzfA/u vs C02£A plots. log 1111 (It) 70 2.0 r T T r I 1.8 '- y - 23.29 + 0.0821 1.8- 1.4" 1.2 - , . 1.0 1 I 1 I l J 1 I 1 -2 -1 O 1 2 3 log 1/CO2fA (llumoles) Figure 16. Reciprocal plot of the specific growth rates of the control Chlorella vugaris cultures in Experiment 4- as a function of the C02fA concentration of the medium. log tlu (hr) 2.0 1.8 1.8 1.4 1.2 1.0 71 y = 22.90 + 0.02): e’ O I: .0 C .J I L I 4 I 1 I 1 -1 0 1 2 3 log ”COZtA (l/umoles) Figure 17. Reciprocal lot of the specific growth rates of the 0.05 mg Cu 4'/1 treatment Chlorella vulggris cultures in Experiment 4 as a function of the C02f A concentration of the medium. log 1111 (hr) 72 22 - .1 2.0 l" — P cl 0 O 1.8 "' d ' e .. .0 1 g 0 1.6 - ° J n . .1 1.4 _ Y 1' 54.98 + 0.0311 ‘ 1 1.2 '- — p -l 1.0 I 1 I 1 J 1 I 1 ’1 0 1 2 3 4 log 1/C021.A (l/umoles) Figure 18. Reciprocal lot of the specific growth rates of the 0. 10p mg Cu +/l treatment Chlorella vulgaris cultures 1n Experiment 4 as a function of the CngA concentration of the medium. 73 The second rearrangement of the Monod model takes the form C02fA/u Kco = ZfA/umax + 1/umax (COZfA) (21) Plots of u/COzfA vs u and C02fA vs COzfA/u of the control and the 0.10 mg Cu2+/l treatments are shown in Figures 19, 20, 21, and 22. The resulting “max and KCOZfA values derived from these plots are given in Table 10. The results of the Eadie-Hofstee plots (Figures 19 and 20) indicate that both the “max and KCOZfA estimates for the control and 0.10 mg Cu2+/1 treatment cultures are of the same order of magnitude as predicted by the Lineweaver-Burk plots (Figures 16 and 18). However, the correlation coef- ficients for these plots are significantly lower than those for the Lineweaver-Burk plots. The Eadie-Hofstee plots magnified the departures from linearity which were not evident in the r2 values of the Lineweaver-Burk plots. The plots from the third transformation, COZfA vs COzfA/u, are shown in Figures 21 and 22. The kinetic constants derived from these figures were significantly higher than those derived by the previous transformations (Table 10) although the umax values were still lower than those obtained from the AAP method. Apparently, a higher order interaction exists in the treatment cultures, requiring a more complex model than the Monod model. Other interactions with nutrients, pH, light, algal by-products, or other factors may have been involved. The cause(s) of departure from Michaelis-Menton kinetics were 74 “LG . I r I l I I r r I “LZ - - e 0 0. “L4 '- - T. 5 r 4 1 O 3 “1.6 " '- y . 00458-000311 -13 _. .— -2 1 I 1 l 1 I 1 I 1 -3 -2 -l 0 l 2 log ulCOzfA (l/u moles hr) Figure 19. Eadie- Hofstee plot of the control Chlorella vulgaris cultures in Experiment 4. I09u(hr-l) 75 ' '-° l I l I I l— a -|.5 — . —-( e 0 e * g 0 e e '2.0 — -" _ y . l.926x 162-6286111021 _ -2.5 -- .— , l l l J ' 1 3'0-3 -2 -l 0 l 2 log u/COZfA (l/IUTIOIBS I'll”) Figure 20. Eadie-Hofstee plot of the 0.10 mg Cu2+ll treatment Chorella vulgaris cultures in Experiment 4. 76 Q 2 fl 2 — O E 3 3 5 N - 8 y-I.399+Ia472 x 0 .3 - . - -l 1;— _. -2 1 I 1 J 1 I l I t '3 -2 -| 0 I 2 log II/CO 2fA (llu moles) Figure 21. CngA/u vs. CngA transformation of the control Chlorella vulgaris cultures in Experiment 4. 77 2.5 1 I , 1 ' T 2.0 -— — I.5 — -1 < - _ L- a: ” LO _ — 2 O 5 ~ - 3 o é 0.5— ' _. 5 r. y- ll.2l8+ 34.47 x _ N O o o _ _ O .2 ._ _J O -O.5 — _ 4.0 — — — . '- O 4.5 L l 1 l L l 1 l l l -4 -3 -2 -| 0 log COZfA (I/umoles) Figure 22. CngA/u vs. CngA tranformation of the 0.10 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiment 4. 78 not investigated. Because of the deviation from the Monod model, models predicting u as a function of Cu2+ were not derived. Conclusions The results of the AAP and pH method indicated that cupric ion inhibits algal growth by prolonging the lag phase of growth. This finding, and the fact that the treatment cultures were chlorotic during this phase, supports the find- ings of Cedeno-Maldonado et a1 (1972). These researchers reported that cupric ion inhibits electron transport in chlorOplasts. The findings of Gross et a1 (1970), who reported that the chlorophyll content of cells exposed to cupric ion drops significantly, are supported by the results of this experiment. The smaller size of the Chlorella vulgaris cells exposed to the 0.15 mg Cu2+/1 treatment may have been due to an increase in cell permeability. Cupric ion reportedly has this effect on algal cells (McBrien and Hassal, 1965). ~ In this study, the 0.15 mg Cu2+/1 treatment was found to be acutely toxic to Chlorella vulgaris cultures. This is lower than the toxic level reported by McBrien and Hassal (1965) for Chlorella. The 0.10 mg Cu2+/1 treatment decreased the maximum specific growth rate and the saturation constant for COZfA of the algal cultures. The lag phase of growth of these cultures was prolonged; however, the cultures appeared to be able to recover their photosynthetic abilities, although at a diminished level. The 0.05 mg Cu2+/1 treatment 79 had only a slight effect on culture growth. Other concentra- tions of cupric ion between 0.05 and 0.15 mg/l needed to be assayed for their chronic effects on algal growth. Both the AAP and pH method showed similar relationships between the four treatments. At this juncture, neither method appeared to be superior in its ability to assess the toxic effects of cupric ion on algal growth.. The biggest advantage of the pH method over the AAP was the ease of sam- pling and measurement. Further study into the toxic effects of cupric ion was needed in order to test these two methods more completely. EXPERIMENT 5 Further Study into the Effects of Cupric Ion on Chlorella vulgaris Culture Growth PUIEOSB This test was a repeat of Experiment 4, testing several additional levels of cupric ion. The purpose was to assess the effectiveness of the two algal bioassay methods in detecting the chronic effects of cupric ion on Chlorella vulgaris culture growth. Procedure Measurements of light reaching the assay platform were taken before and after the experiment. The resulting con- tour map of light intensity is shown in Figure 23. Of the 64 spaces on the assay platform, 30 of these were within the acceptable range of 400'*40 footcandles of illumination, because of an adjustment made to the light support apparatus after the previous experiment. Twenty-four test vessels were each half filled with 500 ml of fresh nutrient medium (a = 1.811 meq CO32'--HC03' alka- linity/1) and were randomly assigned to 24 of the 30 avail- able spaces. The positions of these vessels in relation to the photosynthetically active radiation falling on the plat- form are shown in Figure 24. 80 81 V Row Column Figure 23. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 5. The position of each test vessel is indicated by a circle. 82 Column Figure 24. Contour map of the photosynthetically active radiation (uE/ m 2/sec) falling on the assay platform 1n Experiment 5. The position of each test vessel 13 indicated by a circle. 83 Four levels of cupric ion were tested. A cutrine stock solution (91.071 mg Cu2+/1) was added directly to the medium in the test vessels to give 6 replicates each of 0.075, 0.10, and 0.125 mg Cu2+/l. The remaining 6 test vessels were used as controls. Three replicates of each treatment were randomly assigned to the AAP and 3 to the pH method. Each flask was inoculated with 0.5 m1 of 5-day-old Chlorella vulgaris stock culture to give a final concentra- tion of 3,300 cells/m1. These test cultures had an initial organic carbon content of 0.0092 mM C/l, as determined by analysis with a carbon analyzer. Daily samples of the AAP cultures were taken and the number of cells/ml computed. Samples of the pH method cul- tures were taken daily and the pH determined. The size of the samples taken from the pH method cultures was decreased to 20 ml in an effort to increase the test length. The test was run at 27°C. The daily biomass estimates from the AAP cultures (cells/ml) were tested for significant differences between treatments by a nested analysis of variance (Sokal and Rohlf, 1969) and Student-Newman-Keuls multiple comparison procedure (Steel and Torrie, 1960). The biomass estimates from the pH method cultures (mM C fixed by the algae/l were tested by an analysis of variance and Student-Newman-Keuls multiple comparison procedure (Steel and Torrie, 1960). 84 Results The results of the daily cell counts of the AAP test cultures are tabulated in the Appendix (Table A6). The AAP growth curves for the four treatments show a graded response to cupric ion (Figure 25). The seeding of the test medium with a younger stock culture than previously used signifi- cantly decreased the lag phase of growth of the control cultures. The lag phase of the 0.10 mg Cu2+/l treatment cultures, the only cupric ion treatment from the previous experiment that was repeated here also was decreased. The results of the statistical analysis show a signifi- cant difference between the biomass estimates of the four treatments (Table 11). The treatment cultures remained sig- nificantly different throughout the active growth phase of the control cultures. The 0.075 mg Cu2+/l treatment cultures were judged not significantly different from the controls after both had reached the maximum standing crop biomass level. Statistical testing between the 0.10 and 0.125 mg Cu2+/l treatments showed that these cultures remained significantly different throughout the remainder of the test (Table 12). Although the control and 0.075 mg Cu2+/l treatment cul- tures were judged significantly different in terms of daily biomass estimates, the u values calculated for these cultures indicate that this difference may have been only slight (Table 13). The observed umax of the control cultures was only slightly higher than that of the 0.075 mg Cu2+/l log Cells / ml 85 T I l l l T l 6- .J 1- cl 5- - l- d l- . D d " . .——. control ‘ 4 " ' o——o 0.075 mg Cu‘Z/l "1 ' H 0J0 ' ‘ ’ - H O.l25 " " l l l l 0 IOO fizdoL Elf—"'0 ' Ti me (hr) Figure 25. AAP growth curves for Chlorella vul aris cultures in Experiment 5. Each point represents the mean of three replicates. 86 Table 11. Statistical analysis of the AAP results from the first eight sampling times of Experiment 5. F test values and associated significance levels from a nested analysis of variance are listed for each sampling time. The treatments are listed in ascending order of biomass (cells/ml) with those means judged not significantly dif- ferent, using a Student—Newman-Keuls multiple comparison procedure (P s 0.95), grouped by underlining. Time (hr) P value Treatment (mg Cu2+/l) 16.0 46.61 *** 0.125 0.10 0.075 0.00 39.5 68.02 *** 0.125 0.10 0.075 0.00 64.5 94.98 *** 0.125 0.10 0.075 0.00 91.0 58.80 *** 0.125 0.10 0.075 0.00 109.5 88.11 *** 0.125 0.10 0.075 0.00 134.5 135.13 *** 0.125 0.10 0.075 0.00 159.5 51.24 *** 0.125 0.10 0.075 0.00 182.5 133.42 *** 0.125 0.10 0.075 0.00 *** P = 0.99 ** P = 0.95 * P = 0.90 + P = 0.80 ns P = 0.80 (not significant) 87 Table 12. Statistical analysis of the AAP results from the last four sampling times of Experiment 5. Two tailed t-test values and associated significance levels are listed for each sampling time. The treatments are listed in ascending order of biomass (cells/m1). Time (hr) t value Treatment (mg Cu2+/1) 205.0 23.16 *** 0.125 0.10 229.0 13.25 *** 0.125 0.10 256.0 8.29 *** 0.125 0.10 277.0 4.21 *** 0.125 0.10 *** P = 0.99 ** P = 0.95 * P = 0.90 + P = 0.80 ns P = 0.80 (not significant) 88 Table 13. Specific growth rates (u) of Chlorella vulgaris cultures in Experiment 5, as determined by the AAP. Maximum specific growth rates are underlined. Treatment (mg Cu2+/1) Interval Control 0.075 0.10 0.125 1 '0.0773 0.0744 0.0268 0.0071 2 0.0641 0.0620 0.0560 0.0423 3 0.0623 0.0596 0.0536 0.0162 4 0.0333 0.0365 0.0536 0.0007 5 0.0129 0.0161 0.0407 0.0127 6 0.0104 0.0108 0.0044 0.0177 7 0.0020 0.0071 0.0144 0.0337 8 0.0025 0.0000 0.0069 0.0508 9 0.0035 0.0418 10 0.0045 0.0313 11 0.0032 0.0148 12 -0.0002 0.0054 13 0.0021 89 treatment cultures. Furthermore, the other u values for this treatment were only slightly lower than those recorded for the control cultures. This was the case up to the point where the control cultures' growth began to decrease. The observed umax for the 0.10 mg Cu2+/l treatment cul- tures occurred at a later time and was lower than the observed “max of the controls. The u values indicate that these cul- tures grew at a slower rate than did the control cultures. The 0.125 mg Cu2+/l treatment had an unusual effect on Chlorella vulgaris culture growth. The growth of these cultures appeared to have reached a minimum by interval 4 (between sampling times 64.5 and 91.0 hours), but then the cultures began growing again. The observed umax for these cultures appeared much later in the test and was the lowest umax recorded for any of the treatments. The reason for this two-step growth curve is unclear. Microscopic examination of the treatment cultures revealed that the Chlorella vulgaris cells in the cupric ion treatment cultures were lighter in color and smaller than their counterparts in the control cultures. This was partic- ularly evident in the 0.10 and 0.125 mg Cu2+/l treatment cultures. These conditions became less evident as the test proceeded. The daily pH measurements of the pH method cultures are recorded in the Appendix (Table A7). As was the case with the growth curves produced with the AAP biomass estimates, the pH method growth curves exhibit a graded response of the 90 algae to the cupric ion treatments (Figure 26). Both the lag and active phases of growth became more drawn out as the cupric ion level was increased. The 0.125 mg Cu2+/l treat- ment cultures did not exhibit the double growth curve anom- oly that was noted in the AAP results. As in the previous experiment, one treatment (0.125 mg Cu2+/l) had to be terminated prior to reaching the stationary phase of growth because of a lack of culture volume. These curves (Figure 26) and the AAP growth curves (Figure 25) indicate that at sublethal cupric ion concentrations, an increase of the cupric ion level does not affect the maximum standing crop biomass level reached by the Chlorella vulgaris cultures. The results of the statistical analysis of the four treatments indicated that the biomass estimates from the treatments were significantly different from one another throughout the active growth phase of the control cultures. This difference became more pronounced as the cupric ion concentration was raised (Table 14). Following the temina- tion of the control and 0.075 mg Cu2+/l treatments, the 0.10 and 0.125 mg Cu2+/l treatment cultures remained signifi- cantly different from one another for the remainder of the 0.10 mg Cu2+/l treatment cultures' growth cycle (Table 15). Even after 300 hours of testing, the 0.125 mg Cu2+/l treat- ment cultures continued to grow at a slow rate, showing the chronic effect of cupric ion. The photosynthetic C02 minima for the four treatments was estimated by curves of u versus Cngo. These curves are log Biomass (m moles CA) 91 ' r I I l I l I I 0 — ‘ _ ' — -1 ,— . _ . ._. Control . - o-—o 0.075 mg ou2*/1 ‘ _ . ' o——e 0.1 mg CUZ+/l ' _ o—o 0.0125 mg 003/1 -2 -4 1 J 1 l 1 l 4 l ICC 200 300 400 fime (hr) Figure 26. pH method growth curves for Chlorella vulgaris cultures in Experiment 5. Each point represents the mean of three replicates. 92 Table 14. Statistical analysis of the pH method results from the first six sampling times of Experiment 5. F test values and associated significance levels from an analysis of variance are listed for each sampling time. The treatments are listed in ascending order of biomass (mM C fixed by the algae/l) with those means judged not significantly different, using a Student-Newman-Keuls multiple comparison procedure (P = 0.95). grouped by underlining. Time (hr) F value Treatment (mg Cu2+/l) 16.0 31.61 *** 0.10 0.125 0.075 0.00 39.5 747.81 *** 0.125 0.10 0.075 0.00 64.5 527.06 *** 0.125 0.10 0.075 0.00 91.0 655.46 *** 0.125 0.10 0.075 0.00 109.5 704.85 *** 0.125 0.10 0.075 0.00 134.5 379.58 *** 0.125 0.10 0.075 0.00 *** P = 0.99 ** P = 0.95 * P = 0.90 + P = 0.80 ns P = 0.80 (not significant) 93 Table 15. Statistical analysis of the pH method results from the last seven sampling times of Experiment 5. Two tailed t-test values and associated sig- nificance levels are listed for each sampling time. The treatments are listed in ascending order of biomass (mM C fixed by the algae/l). Time (hr) t value Treatment (mg Cu2+/l) 159.5 5.04 ** 0.125 0.10 182.5 6.95 ** 0.125 0.10 205.0 9.07 ** 0.125 0.10 229.0 12.02 *** 0.125 0.10 256.0 25.32 *** 0.125 0.10 277.0 13.76 *** 0.125 0.10 302.5 8.90 ** 0.125 0.10 *** P = 0.99 ** P = 0.95 * P = 0.90 + P = 0.80 115 P = 0.80 (not significant) 94 shown separately in Figures 27, 28, 29, and 30 for clarity. The resulting COqu estimates from these plots are given in Table 16. The KCOZfA and “max values obtained from these plots are given in Table 16. As shown in Figures 31 through 34, the growth of the algal cultures (with the possible exception of the control treatment cultures) did not adhere to classical Michaelis- Menton kinetics, as was also the case in Experiment 4. Therefore, this model does not adequately explain the inter- actions in the treatment cultures, and extrapolation of the results in estimating “max or KCOZfA as a function of the Cu2+ concentration was avoided. Conclusions As in the previous experiment, the results indicated that cupric ion prolongs the lag phase of growth. The results of this experiment clearly indicated that cupric ion does cause a decrease in the maximum specific growth rate and subsequently retards the growth of the Chlorella vulgaris cultures. Cupric ion, in sublethal concentrations, does not appear to affect maximum standing crop biomass levels that the cultures reach. The results of both methods indicated a graded response of the algae to various cupric ion levels. However, this was more clearly shown by the pH method results than the AAP results. The kinetic constant “max had a greater range of values in the pH method than in the AAP and the change in "max due to cupric ion was more linear in the pH method. 95 W j I l I F I 1 I I I I I 1 Er . L . l- q -l - H L ’C - .C 2 -2 ~ g 1- U’ 2 D L 0 WI 0.0527 1» 0.0058 In x -3 — _ b -l -4 1 l _L A 1 l 1 l 1 _l 1 L 1 -5 -4 -3 -2 -l 0 l 2 mg C 021:0 (LUUOlES /I) Figure 27. Specific growth rates of the control cultures of Chlorella vugaris 1n Experiment 5 as a function of the C02f0 concentration of the medium. 96 /hr) 8 N .r log ull y - 0.0453 + 0.0054 In x F .- 1- -4 -4 L l 1 k 1 l 1 1 l 1 L 1 -5 -4 -3 -2 - 0 I 2 log 0021.0 (umoles/l) Figure 28. Specific growth rates of the 0.075 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiment 5 as a function of the C02fo concentration of the medium. 97 o I f l I l T I F T I r I 4 i. .. l- .1 ) r ’2 .- 5 :2" 1 1- g. O .. E . 3_ y - 0.0273 1010033 In x l- .. " 1 log COZfO (umoles/l) Figure 29. Specific growth rates of the 0.10 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiemnt 5 as a function of the C02f0 concentration of the medium. 98 OH 1 j 1 l l r 1 f j I I 1 1 b - " .1 -l L- .— L . 1‘ t .: >~2 - :t' )- O! 2 I- L _3 _ y . 0.0270 + 0.0037 In x _ " 4 L d -4 J l 1 I 1 l 1 1 l L l 1 -b -4 -3 -2 -| O l 2 log C 02fo (umoles/l) Figure 30. Specific growth rates of the 0.125 mg Cu2+ll treatment Chlorella vulgaris cultures in Experiment 5 as a function of the C02fo concentration of the medium. 99 Table 16. Kinetic constants for the Chlorella vulgaris cultures in Experiment 5, as determined by the pH method. 2 r2 for Treatment COzfq r for KCO2fA “max KCOZfA (mg Cu2+/l) (uM C02/1) c02fq (uM 002/1) (hr-1) and umax Control 1.21:10"4 0.983 1.76x10‘2 0.0575 0.929 0.075 2.2x10‘4 0.923 6.56x10'3 0.0411 0.841 0.10 2.5x10'4 0.733 2.14x10'3 0.0197 0.574 0.125 6.4x10-4 0.911 4.17x10-3 0.0180 0.790 log llll (hr) 100 2.0 F I 1 j j I T 1.8 1. a . J y 817.39 + 0.31): 1.6 - - b -l 1.4 - - 1- cl 1.2 L- . o a C 1.0 J J 1 l 4 l 1 L 1 ‘2 -1 0 1 2 3 log “COZfA (Ilumoles) Figure 31. Reciprocal plot of the specific growth rates of the control _ Chlorella vulgaris cultures in Experiment 5 as a function of the CngA concentration of the medium. - log llu (hr) 101 2.0 *r l 1 l T l 1 j F b ‘1 1.8 r- - ' y a 24.84 + 0.1611 ‘ 1.6 b —1 1.4 .. ' .. .0 r C 8 1.2 - . - - A 1.0 1 l l J l l 1 J l -2' -1 o 1 2 3 log ”COZfA (I/umoles) Figure 32. Reciprocal plot of the specific growth rates of the 0.075 mg Cu2+/l treatment Chlorella vulgaris cultures in Experiment 5 as a function of the CngA concentration of the medium. log llu (hr) 102 I I I I l I I T l 2.2 - 2.0 - 1.8 '- 1.6 - - 0' ' - b y-50.67+0.11x .. 0 1.4 - —1 0 I— . d O 1.2 - - 1.0 L l 1 l l l 1 i 4 -2 -1 0 1 2 3 log 1/CO 2M (llumoles) Figure 33. Reciprocal Elot of the specific growth rates of the 0.10 mg Cu +/l treatment Chlorella wilgaris cultures in Experiment 5 as a function of the COzfA concentration of the medium. log 1 [1.1 (hr) 103 2'4 1 I T I r I I I 22 1— r y = 55.61 + 0.23): 2.0 l" - 1.8 - .— 0 0 b . -I O 1.6 '- .— 0 . ‘ . .- 1.4 1 l 1 L 1 l 1 J a -2 -1 O 1 2 3 Figure 34. Reciprocal plot of the specific growth rates of the O. 125 mg Cu+2ll treatment Chlorella vulgaris scultures 1n Experiment 5 as a function of the C02fA concentration of the medium. 104 With the pH method it was not possible to produce a model which estimated u at various cupric ion and COZfA concentra- tions of the medium because of the lack of fit to Michaelis- Menton kinetics. However, the effort expended in sampling and enumeration in the AAP is much greater than that in the pH method. For these reasons, the pH method was judged to be superior to the AAP in this test. EXPERIMENT 6 The Effects of p-Cresol on Chlorella vulgaris Culture Growth PUI'EOSB This experiment was designed to assess the effects of three concentrations of p-cresol on the growth kinetics of Chlorella vulgaris cultures. The purpose was also to ascer— tain the mode of response of the algal cultures, to a com- pound of unknown toxicity, in the two bioassay methods used in this study. Procedure Light measurements of the assay platform were taken before and after the experiment. Twenty-six of the 64 avail- able spaces on the assay platform were found to be in the acceptable range of 400 *40 footcandles of illumination (Figure 35). Twenty-four test vessels were each half filled with 50 ml of fresh nutrient medium (a = 1.96 meq C032'-- HCO3' alkalinity/l) and these were assigned to spaces on the assay platform. The resulting positions, with respect to the photosynthetically active radiation falling on the assay plat- form, are shown in Figure 36. 105 106 Column Figure 35. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 6. The position of each test vessel is indicated by a circle. 107 Column Figure 36. Contour map of the photosynthetically active radiation , (uE/m / sec) falling on the assay platform 1n Experiment 6. The position of each test vessel 18 indicated by a circle. 108 Since the effects of p-cresol on algae had not been previously reported in the literature, the four p-cresol con- centrations tested were based on information from fish and invertebrate bioassays. The stock p-cresol solution (1,000 mg/l) was added directly to the medium in the test vessels to give six replicates each of 5, 10, and 15 mg p-cresol/l. The remaining six replicates were used as controls. Half of the replicates at each p-cresol level were assigned to the AAP and half to the pH method. Each flask was inoculated with 0.5 ml of 6-day-old Chlorella vulgaris stock culture to give a final concentra- tion of 3,300 cells/m1 (0.00975 mM organic C/l). Daily samples of the AAP cultures were taken and the number of cells/ml computed. These biomass estimates were tested for significant differences by a nested analysis of variance (Sokal and Rohlf, 1969) and a Student-Newman-Keuls multiple comparison procedure (Steel and Torrie, 1960). Samples of the pH method cultures were taken on a daily basis and the pH determined. The experiment was run at 27°C. The p-cresol concentration of the test cultures was monitored during the test by gas chromatography methods. A Varian Model 3700 gas chromatograph, fitted with a 6-foot- long (2mm ID) glass column containing 80/100 Chromosorb 101 (John's Manville Co.), was used for this purpose. One sam- ple of the 5 mg p-cresol/l treatment cultures and one of the 15 mg p-cresol/l treatment cultures from each of the two bioassay methods were taken at times 3, 42.5, and 114 109 hours. The samples were centrifuged for 5 minutes at 1,000 rpm to remove the algal cells. The samples were acidified with H3P04 to pH'2 and refrigerated until analysis. Prior testing showed that these steps minimized p-cresol loss from the samples. One subsample (1 ul) from each sample was anal- yzed in the aqueous phase by flame ionization detection. The column was kept at 220°C., the dector at 275°C., and the injection port at 270°C. during the analysis. The p-cresol peaks (time = 5.5 minutes from injection) were measured by planimetry. Results The daily cell counts from the AAP cultures are tabu- lated in the Appendix (Table A8). The AAP growth curves are shown in Figure 37. The growth of the control cultures exceeded that of the three p-cresol treatments' cultures throughout the test; however, the results of the statistical analysis do not show a graded response of the test cultures with increased p-cresol concentration. The results indicated no significant difference between the three p-cresol treat- ments' cultures throughout most of the test. Furthermore, the order (in terms of increasing biomass) of the three treatments varied from sampling time to sampling time (Table 17). A comparison of the observed umax values from the treat- ment cultures shows little effect due to p-cresol on the observed “max values attained by the treatment cultures. Although the values are dissimilar, the time at which each 110 7 T T 1 I I l- -( P G - a 6 - _ L- .1 )- d E . .. \ 1n = 5 '- m 0 d o» g -1 control - 5 mg p-cresol /l J 4 IO " - 15 " q )- .- 3 L 4 l L l 0 as so 75 ICC 125 Time (hr) Figure 37. AAP growth curves for Chlorella vulgaris cultures in Experiment 6. Each point represents the mean of three replicates. ' lll Table 17. Statistical analysis of the AAP results of Experiment 6. F test values and associated significance levels from a nested analysis of variance are listed for each sampling time. The treatments are listed in ascending order of biomass (cells/m1) with those means judged not significantly different, using a Student- Newman-Keuls multiple comparison procedure (P = 0.95), grouped by underlining. Time (hr) P value Treatment (mg p-cresol/l) 18.5 4.82 ** 10 15 5 0 42.5 27.73 *** 10 15 5 0 67.0 9.27 *** 15 10 5 0 91.5 2.66 + 15 5 15 0 114.0 5.46 ** 15 10 5 0 ** P = 0.95 * P = 0.90 + P = 0.80 ns P = 0.80 (not significant) 112 treatment's observed “max occurred varied. The trend appeared to be that as the p-cresol concentration was raised, the observed umax for each successive treatment occurred at a later time in the test. Since the observed u values were not statistically tested on a sequential basis, it is diffi- cult to say whether or not the values preceding the observed “max were significantly lower than the observed “max (Table 18). In addition, no morphological differences between test cultures were noted. From the result obtained by the AAP, the evidence of sublethal effects of p-cresol on Chlorella vulgaris cultures was inconclusive. The results of the daily pH measurements of the pH method cultures are recorded in the Appendix (Table A9). The daily rise in pH of the control cultures mimicked the results found in the preceding experiments. The pH of the p-cresol treatment cultures, however, decreased for the first few days of the test. The amount of pH decrease was propor- tional to the initial p-cresol content of the cultures. After the initial pH decrease, the pH of all the test cul- tures increased until all four treatments had virtually the same pH at 114 hours (Table A9). It was hypothesized that the initial pH decrease of the test cultures might be due to algal respiration caused by the p-cresol. However, the amount of carbon present in the algal inoculum, 0.00975 mM C/l, could not account for the drop in culture pH that was noted. Clearly, some other proton source must have contributed to this pH decrease. 113 Table 18. Specific trowth rates (u) of Chlorella vulgaris in Experiment 6, as determined by the AAP. Maximum specific growth rates are underlined. Treatment (mgp-cresol/l) Interval Control 10 15 1 0.0062 -0.0017 -0.0070 -0.0034 2 0.0652 0.0627 0.0607 0.0613 3 0.0650 0.0657 0.0668 0.0635 4 0.0599 0.0591 0.0571 0.0642 5 0.0196 0.0309 0.0340 0.0153 114 The gas chromatographic analyses of the initial samples (time a 3 hours) showed no decrease of the p-cresol concen- trations of the 5 and 15 mg/l p-cresol treatment cultures. By the time of the second sampling (time a 42.5 hours), the p-cresol concentrations of the 5 and 15 mg/l p-cresol treat- ment cultures were below the limit of detectability for the gas chromatography column used (determined to be approxi- mately 3 mg p-cresol/l). The p-cresol concentrations of the samples taken at 114 hours also were below the limit of detectability. Several of the test cultures, in particular those of the 15 mg p-cresol/l treatment had a whitish cast. Microscopic examination of these cultures revealed a high level of bacte- rial activity, as compared to the controls. Conclusions The effect of p-cresol on Chlorella vulgaris cultures was not determined due to problems encountered in the test. The results of the gas chromatographic analyses indicated that the p-cresol concentration of the medium was decreased by some mechanism. The results of the pH method test indi— cated that the p-cresol may have been degraded into constit- uent parts which caused a decrease in culture pH. The results of both bioassay methods indicated that these con- stituent parts apparently have no effect on Chlorella vulgaris culture growth. This conclusion supports the find- ings of Barthauer and Alred (1980). These researchers 115 stated that of all the known breakdown products of p-cresol, none was reported as being toxic. As previously noted, p-cresol may be degraded by a variety of bacteria. This appears to have been the case in this test. However, other breakdown mechanisms also may have contributed to the decrease of the p-cresol concentration of the medium. Photodegradation, volatilization, sorption onto algal cells, and biodegradation by algae could have contrib- uted to the p-cresol loss. Further investigation into these possible pathways of p-cresol degradation was called for. EXPERIMENT 7 Analysis of the Degradation of p-Cresol Under Algal Bioassay Conditions PUZ’EOSG This experiment was designed to ascertain the extent of p-cresol degradation under algal bioassay conditions due to four mechanisms. These were bacterial degradation, photoly- sis, sorption onto algal cells, and/or degradation due to the algae and volatilization from the medium. Procedure To test the extent of the effects of the four mechanisms listed above, the p-cresol concentration of eight treatments (with three replicates of each) was monitored by gas chro- matographic methods for several days. The flowchart given in Figure 38 shows the setup of the test and the eight treat- ments. For brevity, acronyms are used when the treatments are discussed in the text. For example, a vented test vessel containing sterile medium and incubated in the dark has the acronym SVD. All test vessels, closures, and other apparatus which came in direct contact with the medium were first washed, as previously described, and then autoclaved. The distilled water used in the medium preparation was membrane filtered 116 117 L D \ \ C’//////// V C V | //\ l //\ S S B A S S B A (SCL) (SVL) (BVL) (AVL) (8CD) (SVD) (BVD) AVD) < O U incubated under 400 140 footcandles of continuous illumination incubated in the dark closed culture vessel vented culture vessel sterile medium medium with bacteria added medium with Chlorella vulgaris culture added (nonaxenic) Figure 38. Flowchart of the experimental design of the p-cresol degradation test, Experiment 7. The acronyms for the eight treatments appear in parentheses. 118 (0.454 Millipore filter) into the autoclaved 18 1 preparation bottle. The nutrient solutions also were filtered in the same manner and the filters washed with several rinsings of distilled water to minimize nutrient loss. The test vessels used for the SCL and SCD treatments were 1 l erhlenmeyer flasks fitted with Number 9 solid rubber stoppers and capped with Parafilm. The test vessels for the remaining treatments were 1 1 erhlenmeyer flasks fitted with Number 9 one—hole rubber stOppers, in which the vent was placed. The vent was an inverted pasteur pipette containing a "glass wool“ plug and capped with a foam plug. This allowed for the free exchange of gases with the atmosphere in all six treatments, while minimizing the chance of bacterial contamination of the medium in the SVL and SVD treatments. Each test vessel was half filled with 500 m1 of sterile medium (a = 1.940 meq CO32'—-HCO3' alkalinity/l) by following accepted sterile procedure (Levy et al, 1973). All test ves- sels were inoculated with a sterile 1,000 mg/l p-cresol stock solution to yield a final concentration of 15 mg p-cresol/l. One hundred milliliters of nonaxenic Chlorella vulgaris stock culture were filtered through 1 u pore—size filter paper to remove the algal cells. The filtrate was examined microscopically and was found to contain only rod-shaped bacteria. The replicates of the BVL and BVD treatments were inoculated with 0.5 ml of this filtrate. The AVL and AVD treatment replicates were inoculated with 0.5 ml of nonaxenic S-day-old Chlorella vulgaris culture, to yield a 119 final concentration of 4,200 cells/ml. The remaining treat- ments (SCL, SCD, SVL, and SVD) were inoculated with 0.5 m1 of autoclaved distilled water. Light measurements of the assay platform were taken, and 27 of the 64 spaces were within the acceptable range of 400 *40 footcandles of illumination (Figure 39). The test ves- sels to be incubated in the light were then assigned to 12 of these 27 spaces. The positions of the test vessels in rela- tion to the photosynthetically active radiation falling on the assay platform are shown in Figure 40. The remaining 12 test vessels were placed in a dark chamber. The temperature of all cultures was maintained at 27°C. Samples of all treatment replicates were taken at times 0, 10, 22, 37.5, and 61 hours. The 10 ml samples were taken with volumetric pipettes by following sterile sampling proce- dure (Levy et al, 1973). The BLV, BVD, AVL, and AVD treat- ment replicate samples were centrifuged for 5 minutes at 1,000 rpm to remove bacteria and algae, respectively. All samples were fixed by the addition of H3PO4 to pH 2 and refrigerated until analysis. Prior to analysis, 1 ml of a 55 mg phenol/l standard solution was added to each sample to give an internal stan- dard concentration of 5 mg phenol/1. This step was intended to give greater precision in the estimation of the p-cresol concentration of the test cultures. A Varian Model 3700 gas chromatograph equipped with a 6-foot-long (2 mm ID) glass column containing 108 SP-1200/1% 120 1 1 1 1 i r 1 1 O O O O . .90 O a 3 d r2 “OH 320 280/ '* F“ f l l 1 1 1 I I I , l 2 3 4 5 6 7 8 13 Column Figure 39. Contour map of the light intensity (footcandles) falling on the assay platform in Experiment 7. The position of each test vessel is indicated by a circle. 121 Column Figure 40. Contour map of the photosynthetically active radiation (uE/mz/sec) falling on the assay platform in Experiment 7. The position of each test vessel is indicated by a circle. 122 H3PO4 on 80/100 Chromosorb W AW (Supelco, Inc.) was used for the analysis. One subsample (1 ul) from each sample was ana- lyzed in the aqueous phase by flame ionization detection. The column was kept at 160°C., the detector at 250°C., and the injection port at 190°C. during the analysis. The phenol and p-cresol peaks (time = 2.2 and 3.4 minutes from inje- ction, respectively) were measured by planimetry. Prior to the initiation of the experiment, several pos— sible sources of error were investigated. In the first test, the p-cresol concentration of centrifuged samples was com- pared to that of uncentrifuged samples. This was done to determine if any p-cresol was lost in centrifugation. The second test was designed to determine the conversion factor needed to convert units of phenol concentration to units of p-cresol concentration. This was determined by analyzing p—cresol standards of 0, 5, 10, and 15 mg p-cresol/l which contained a 5 mg phenol/1 internal standard. The conversion factor was determined by the equation p-cresol concentration area of phenol peak f = ( )( ) (26) area of p-cresol peak phenol concentration The average of the observed f values (f) was used as the conversion factor in the following equation. p-cresol peak area mg p-cresol/l = ( )(mg phenol/l)(f) (27) phenol peak area By measuring the phenol and p-cresol peak areas and using the known phenol concentration and f, the unknown p—cresol con- centration of the treatment replicates was determined. 123 Results The centrifuging process was found not to affect the p-cresol content of the samples. Therefore, only samples of treatments BVL, BVD, AVL, and AVD were centrifuged prior to analysis. The conversion factor, f, was determined to be 1.065 p-cresol units to l phenol unit. The results of the experiment are presented in Table 19. The treatments are presented in the order which would be expected (lowest to highest p-cresol concentration remaining) if all four factors which were tested were effectively decreasing the p-cresol concentration of the medium. This does not appear to be the case, however, as none of the three sampling times where p-cresol remained in the medium of each treatment (10, 22, and 37.5 hours) shows this trend. As in the previous experiment, most of the test cultures developed a whitish cast. Bacterial contamination was sus- pected not only in those treatments with known bacterial pop- ulations, but also in the treatments containing ”sterile” medium. Microscopic examination of samples revealed that rod-shaped bacteria were present in all test vessels. These bacteria had a mean length of 0.8 u, but most had a cellular width of less than 0.45 u, the pore size of the "bacterial" filter paper used for the sterilization process. This find- ing complicates the analysis somewhat; however, several conclusions can still be made. The results indicate little or no difference between the closed and vented flasks in terms of p-cresol loss. Also, as 124 Table 19. Results of the gas chromatographic analysis for p-cresol in Experiment 7. Each entry represents the mean p-cresol concentration (mg/1) of three replicates *1 standard deviation. Treatment Time (hr)* AVL AVD BVL BVD SVL SVD SCL SCD 10.0 11.9 13.1 11.7 11.9 13.1 12.8 12.8 12.9 $2.1 $2.2 $1.1 $1.3 $1.6 $2.5 $0.8 $0.9 22.0 11.1 11.9 11.0 11.1 11.6 11.8 11.2 11.2 $4.1 $2.9 $1.0 $0.8 $1.5 $1.8 $0.4 $0.6 37.5 9.1 6.2 7.3 5.0 7.7 7.5 9.8 8.8 $0.2 $0.9 $2.2 $1.0 $0.9 $1.9 $0.6 $1.1 6.10 nd nd nd nd nd nd 1.2 nd $1.0 * The initial p-cresol concentration at time = 15 mg/l for all replicates. nd = none detected 0 hours was 125 previously reported, centrifugation did not contribute to the loss of p-cresol from the medium. This leads to the conclusion that volatilization is not a significant pathway of p-cresol loss from the medium when compared to microbial action. The second hypothesis that photolysis could be causing the loss of p-cresol from the medium appears to be equally invalid. The data indicate that the test vessels incubated under continuous illumination did not have lower p-cresol concentrations than their counterparts in the dark. The third hypothesis was that the p-cresol sorbed onto the algae cells or was degraded by them. When compared to cultures containing only bacteria, the p-cresol concentration of the algal culture containing vessels was not lower. When compared to the biodegradation of the p-cresol by bacteria, the other three hypothesized pathways of p-cresol loss from the medium do not appear to be significant. It is apparent that the biodegradation of p-cresol by bacteria is the rate-determining factor in its loss from the medium in nonaxenic algal bioassays. Conclusions Due to the bacterial contamination of all of the treat- ment cultures, a statistical analysis of the experiment was not possible. However, the data indicate that bacterial degradation of p—cresol appears to be the most probable cause of the results found in the previous experiment. The observed results are in agreement with the findings of the 126 United States Environmental Protection Agency (1978). EPA's research indicated that under most conditions the bacterial degradation of p-cresol was the most significant factor in its loss from natural waters. SUMMARY The initial series of experiments were used to locate and eliminate various sources of error in the two algal bio- assay methods utilized in this study. The majority of error in the AAP was found to be in the cell-counting procedure. The corrections made in the procedure and analysis helped to minimize experimental error. The modification of the McNabb (1960) method of enumerating algal cells which was used in this study, was found to be accurate. The procedures chosen for the pH control of the AAP cultures, sampling for the pH method, and analysis for the pH method were found to be acceptable. Cupric ion was found to be acutely toxic to Chlorella vulgaris cultures at a concentration of 0.15 mg/l. This is lower than the level reported by McBrien and Hassal (1965) as being acutely toxic to Chlorella cells (0.27 ppm Cu2+). Cupric ion concentration of 0.05 mg/l had little effect on Chlorella vulgaris culture growth. Sublethal concentrations of cupric ion between 0.05 and 0.15 mg/l inhibited algal growth by: l. prolonging the lag phase of growth, 2. decreasing the maximum specific growth rate, and consequently 3. increasing the time needed to reach the maximum standing crop biomass level. 127 128 The extent of growth, the maximum standing crop biomass level, was not affected by sublethal concentrations of cupric ion. The results obtained and observations made are consis- tent with the previously reported findings that cupric ion inhibits algal photosynthesis (Cedeno-Maldonado et a1, 1972), decreases cellular chlorophyll content (Gross et a1, 1970), and increases cellular permeability (McBrien and Hassal, 1965). The cupric ion analyses indicate that of the two algal bioassay methods tested, the pH method was more sensitive in detecting the effects of cupric ion on algal growth kinetics. In addition, the sampling and testing procedures for the pH method were less time-consuming than the procedures used in the AAP. The test of the effects of p—cresol on algal growth kinetics was inconclusive. The p-cresol was rapidly degraded by bacteria present in the Chlorella vulgaris cultures. Further testing indicated that the bacterial degradation of p-cresol was the rate-determining factor in its loss from the algal nutrient medium, as compared to p-cresol loss due to photolysis, volatilization, and algal sorption and/or degra- dation of the p-cresol. The breakdown products of p-cresol had no effect on algal growth. The p-cresol toxicity test also indicated an inherent difficulty in the pH method. A compound tested by the use of this method which affects the pH renders this method useless. 129 The affect on culture pH violates the assumption that any rise or fall of culture pH is due only to algal activity. APPENDIX APPENDIX AAP and pH Method Data From the Experiments 130 131 Table A1. Cell counts as cells/ml from Experiment 1. Each entry represents the mean of 20 observations *1 standard deviation. Filter Sample number Cells/grid Cells/m1 1 1 14.4 $3.4 1,680,000 $397,000 2 15.3 $4.4 1,780,000 $513,000 3 17.1 $5.5 1,990,000 $642,000 2 1 14.9 $3.6 1,740,000 $420,000 2 17.5 $4.2 2,040,000 $490,000 3 17.8 $3.4 2,080,000 $397,000 3 1 15.8 $4.3 1,840,000 $502,000 2 16.1 $4.4 1,880,000 $513,000 3 17.2 $3.3 2,010,000 $385,000 132 Table A2. Cell counts as cells/ml from Experiment 3. Each entry represents the mean of 20 observations *1 standard deviation. Replicate Time (hr)* 1 21.0 7,800 7,960 5,850 7,870 8,770 $1,600 $1,300 $1,600 $1,170 $2,880 44.5 91,000 94,300 89,700 89,700 92,900 $16,000 $13,700 $18,800 $18,800 $18,100 68.5 368,000 487,000 6,64,000 421,000 495,000 $156,000 $108,000 $95,500 $80,500 $85,200 93.5 1&670,000 1£750,000 1&940,000 1&790,000 1&780,000 615,000 408,000 621,000 498,000 390,000 116.5 2,070,000 2,710,000 3,060,000 2,890,000 2,200,000 $596,000 $1,400,000 $1,280,000 $945,000 $802,000 140.0 2,410,000 2,530,000 2,900,000 2,730,000 2,530,000 $512,000 $615,000 $481,000 $481,000 $647,000 164.0 2,910,000 3,000,000 3,240,000 3,370,000 2,760,000 $635,000 $562,000 $1,030,000 $615,000 $671,000 * Each flask was inoculated with 5,300 cells/m1 at time = 0 hours. Table A3. 133 pH values for cultures of Chlorella vulgaris in Experiment 3. Replicate Time (hr)* 1 2 3 4 5 21.0 8.4 8.5 8.4 8.3 8.6 44.5 9.1 9.2 9.3 8.9 9.2 68.5 10.1 10.3 10.0 9.7 10.1 93.5 11.1 11.2 11.2 11.2 11.2 116.5 11.2 11.4 11.6 11.7 11.6 140.5 11.2 11.2 11.5 11.4 11.5 164.0 11.2 11.0 11.3 11.4 11.3 * pH = 8.3 at time 0 hours. 134 Table A4. Cell counts as cells/ml from Experiment 4. Each entry represents the mean of 60 observations i1 standard deviation. Treatment (mgCu2+/l) Time (hr)* Control 0.05 0.10 0.15 20.0 3,300 3,200 3,400 3,200 $1,200 $1,100 $850 $1,100 44.0 12,300 9,300 3,700 2,700 $3,700 $2,400 $1,600 $1,100 68.0 91,700 53,000 11,500 3,100 $25,400 $14,900 $9,140 $1,100 91.5 661,000 391,000 43,700 2,300 $260,000 $123,000 $35,800 $1,100 140.5 2,510,000 2,490,000 597,000 560 $914,000 $1,310,000 $795,000 $460 164.0 2,570,000 2&760,000 873.000 *** $819,000 857,000 $1,110,000 188.5 3,140,000 3,270,000 1,510,000 $880,000 $1,490,000 $1,230,000 212.0 3,090,000 3,190,000 2,090,000 $1,650,000 $1,040,000 $1,110,000 236.0 3,690,000 3,380,000 2,770,000 $1,110,000 $725,000 $831,000 259.0 ** ** 3,320,000 $998,000 331.0 3,530,000 $905,000 Each flask was inoculated with 3,400 cells/m1 at time = 0 hours. The previous day's result was the maximum standing crop biomass level for the control and 0.05 mg Cu2+/1 treatment . *** These cultures were terminated following this sampling. 135 Table A5. pH values for cultures of Chlorella vulgaris in Experiment 4. Treatment (mgCu2+/l) Time Repli- (hr)* cate Control 0.05 0.10 0.15 20.0 1 8.4 8.4 8.5 8.5 2 8.5 8.5 8.4 8.5 8.5 8.5 8.5 8.5 44.0 1 8 8 8.6 8.5 8.5 2 8.8 8.7 8.5 8.5 3 8.9 8.7 8.5 8.5 68.0 1 9.4 9.2 8.4 8.4 2 9.4 9.1 8.6 8.4 3 9.5 9.1 8.5 8.5 91.5 1 10.2 9.8 8.6 8.5 2 10.3 9.9 8.7 8.5 3 10.7 10.0 8.7 8.5 117.0 1 11.0 10.9 8.7 8.6 2 11.3 11.0 9.0 8.5 3 11.4 11.0 8.9 8.6 140.5 1 11.3 11.3 8.7 8.6 2 11.4 11.3 9.0 8.5 3 11.5 11.4 8.9 8.5 164.0 1 11.4 11.4 8.6 8.4 2 11.5 11.4 9.1 8.4 3 11.4 11.5 9.0 8.4 188.5 1 ** ** 8.7 8.4 9.2 8.4 9.2 8.5 212.0 1 8.7 8.5 2 9.4 8.4 3 9.5 8.4 * pH = 8.4 at time = 0 hours. ** The previous day's pH was taken as the maxima or minima and used in subsequent comparisons between treatments. Table A5 (cont'd) 136 Treatment (mg Cu2+/1) Time Repli- (hr)* cate Control 0.10 0.15 259.5 1 9.2 ** 2 9.8 3 10.2 306.5 1‘ 9.8 2 10.8 3 11.2 331.0 1 10.2 2 11.1 3 11.3 355.0 1 10.6 2 11.4 3 11.5 Table A6. Cell counts as cells/m1 from Experiment 5. 137 Each entry represents the mean of 60 observations 11 standard deviation. Treatment (mgCu2+/l) Time (hr)* Control 0.075 0.10 0.125 16.0 14,000 13,000 5,100 3,700 $6,000 $7,900 $1,700 $1,200 39.5 99,600 82,700 24,700 11,000 $35,000 $30,400 $15,100 $8,200 64.5 799,000 567,000 125,000 16,600 $244,000 $224,000 $43,300 $13,800 91.0 2,060,000 1,630,000 739,000 16,900 $524,000 $490,000 $302,000 $13,800 109.5 2,620,000 2,200,000 1,630,000 21,400 $581,000 $612,000 $281,000 $15,100 134.5 3,400,000 2,890,000 1,820,000 33,600 $783,000 $693,000 $588,000 $16,700 159.5 3,570,000 3,450,000 2,260,000 82,600 $766,000 $920,000 $844,000 $39,300 182.5 3,780,000 3,450,000 3,070,000 315,000 $930,000 $814,000 $686,000 $173,000 205.0 ** ** 3,320,000 875,000 $685,000 $450,000 229.0 3,700,000 1,930,000 $880,000 $551,000 256.0 4,030,000 2,890,000 $843,000 $654,000 277.0 4,010,000 3,240,000 $1,090,000 $907,000 302.5 3,420,000 $1,020,000 * Each flask was inoculated with 3, 300 cells/ml at time = 0 hours. 5* The previous day‘ 3 result was the maximum standing crop biomass level. 138 Table A7. pH values for cultures of Chlorella vulgaris in Experiment 5. Treatment (mg Cqu/l) Time Repli- (hr)* cate Control 0.075 0.10 0.125 16.0 1 8.6 8.6 8.4 8.5 2 8.6 8.6 8.4 8.4 3 8.6 8.5 8.3 8.4 39.5 1 9.4 9.2 8.7 8.5 2 9.4 9.2 8.7 8.6 3 9.4 9.2 8.6 8.5 64.5 1 10.4 9.8 9.0 8.6 2 10.5 9.9 9.0 8.6 3 10.5 '9.8 9.1 8.6 91.0 1 11.2 10.7 9.4 8.7 2 11.3 10.8 9.4 8.7 3 11.3 10.7 9.6 8.7 109.5 1 11.3 11.0 9.6 8.7 2 11.3 11.1 9.5 8.7 3 11.3 11.1 9.8 ‘8.8 134.5 1 11.3 11.2 9.8 8.7 2 11.3 11.3 9.8 8.7 3 11.4 11.3 10.2 8.9 159.5 1 ** 11.4 10.2 8.7 2 11.4 10.1 8.7 ' 3 11.4 10.7 9.0 182.5 1 11.4 10.7 8.7 2 11.4 10.5 8.7 3 11.4 11.0 9.1 205.0 1 ** 10.9 8.7 2 10.9 8.7 3 11.2 9.2 229.0 1 11.1 8.8 2 11.1 8.8 3 11.3 9.2 * pH 2 8. 4 at time = 0 hours. ** The previous day' 3 pH was taken as the maxima and was used in subsequent comparisons between treatments. 139 Table A7 (Cont'd) Treatment (mg Cu2+/l) Time Repli- (hr)* Cate Control 0.075 0.10 0.125 256.0 1 ** 11.2 9.1 2 11.2 9.1 3 11.3 9.6 277.0 1 11.2 9.4 2 11.3 9.5 3 11.3 9.8 302.5 1 11.3 9.9 2 11.3 10.0 3 11.3 10.4 326.5 1 ** 10.3 10.4 3 10.8 350.5 1 10.6 2 10.8 3 10.9 374.5 1 11.0 2 11.0 3 11.1 398.5 1 11.0 2 11.1 3 11.2 140 Table A8. Cell counts as cells/m1 from Experiment 6. Each entry represents the mean of 60 observations *1 standard deviation. Treatment (mg p-cresol/l) Time (hr)* Control 5 10 15 18.5 3,700 3,200 2,900 3,100 $1,000 $900 $1,100 $1,100 42.5 30,400 22,600 18,500 20,300 $9,400 $11,600 $9,800 $9,500 67.0 268,000 209,000 185,000 162,000 $61,500 $71,900 $72,900 $51,900 91.5 1,640,000 1,230,000 991,000 1,250,000 $427,000 $356,000 $525,000 $414,000 114.0 2,470,000 2,540,000 2,220,000 1,770,000 $638,000 $663,000 $508,000 $563,000 * Each flask was inoculated with 3,300 cells/m1 at time = 0 hours. 141 Table A9. pH values for cultures of Chlorella vulgaris in Experiment 6. 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