POLYCHLORINATED BIPHENYL TOXICITY TO TWO PHYTOPLANKTERS AS A FUNCTION OF ENVIRONMENTAL CONDITIONS
A Dissertation presented by Robert Alan Michaels to The Graduate School in partial fulfillment of the requirements for the degree of . Doctor of Philosophy in Biology
State University of New York at
Stony Brook
August, 1979
Copyright by
Robert Alan Michaels
STATE UNIVERSITY OF NEW YORK
AT STONY BROOK
THE GRADUATE SCHOOL
Robert Alan Michaels
We, the dissertation committee for the above candidate for the
Ph.D. degree, hereby recommend CL. | of the d ee ‘ion.
Nae Brinkhuis
Hants 0’ —
Harold O'Connors
Robert Smolker
Cmte VI thew
George wir Nams Chmn.
Vi nleo?. Unita
Charles: F: Wurster
The dissertation is accepted by the Graduate School.
Original Signed By
JACOB BIGELEISEN, DEAN
Jacob Bigeleisen
(August, 1979)
Abstract of the Dissertation
POLYCHLORINATED BIPHENYL TOXICITY TO TWO PHYTOPLANKTERS AS A FUNCTION OF ENVIRONMENTAL CONDITIONS
_ by i Robert Alan Michaels Doctor’ of Philosophy ia Biology ‘
' State University of New York at Stony Brook
Batch unialgal cultures of one resistant and one sensitive marine phytoplankter were exposed to polychlorinated biphenyls (PCB's) under a variety of culture conditions, and the effects on growth and photosynthesis were determined to detect interactions between environmental conditions and toxicity. The hypothesis was tested that varying culture regimes would alter the xvelative growth and PCB-resistance of the organisms studied. These were the resistant, naked green flagellate, punaliella tertiolecta, and two isolates of the sensitive diatom, Thalassiosira pseudonana, one of estuarine and one of oceanic (Sargasso Sea) origin.
Susceptibility of the two 7. pseudonana isolates to PCB varied with nutrient concentration and salinity, parameters which differ between the habitats of origin of the isolates. Compared
with growth in a fully nutrient-enriched culture medium, low
iii
nutrient concentration inhibited growth of the estuarine isolate; sensitivity to PCB increased under this nutrient stress. In contrast, low nutrient concentration enhanced growth, but not PCB resistance,
of the oceanic isolate, reducing the differential in PCB resistance between these isolates.
Like low nutrient concentration, low salinity inhibited growth of the estuarine isolate of 7. pseudonana. Unlike the nutrient stress, low salinity increased resistance, enhancing growth of cells treated with PCB at low compared with normal salinity. Increased resistance with stress suggested a nonbiological explanation, e.g., that altered PCB distribution at low salinity reduced exposure of sensitive intracellular sites of action to the toxin. This hypothesis was refuted by adapting cells to low salinity, transferring them to normal salinity, and finding resistance to PCB equal to that exhibited at iow salinity. Genetic ox physiological adaptacion to low salinity, rather than altered PCB availability, produced the resistance.
Whereas 7. pseudonana gained resistance, D. tertiolecta lost resistance to PCB upon sudden exposure to low salinity. Unlike walled T. pseudonana, naked Dp. tertiolecta responded to sudden reductions in salinity by expanding in volume. Very low salinity caused lysis. PCB did not affect this process, since expansion in volume of PCB-treated cells was identical to controls. Induced sensitivity to PCB must have been caused physiologically by PCB gaining access to sensitive
sites of action, rather thaa structurally by weakening the cell membrane
iinity variation most important
To identify the factor in in modifying PCB susceptibility, altered ion concentrations and ratios were investigated. Variation in Na’, H , and in total ionic
oe . er 2+ variation in Kk and Ca
concentration modified PCB toxicit did not. Ing. pseudonana, when the nonelectrolyte glycerol was substituted for seawater salts to reduce total ion concentration while maintaining constant osmolarity, PCB-treated cells displayed veduced susceptibility as in the case of lowered salinity. Hence, the effect of ions exceeded any effect of osmolarity.
.. 14. 6. - + : : - Photosynthetic C-fixation per cell and cell density were
relationship between salinity and susceptibility of 7. pseudonana to PCB was identical whether the indicator of PCB effect was growch or photosynthesis per cell. At normal salinity photosynthesis per cell was inhibited up to 90% by 50 yg/1 of PCB, refucing claimed independence of PCB action and photosynthesis. PCB inhibition of photosynthesis per cell diminished. at low salinity, but not at high salinity, paralleling the effect of 208 oa growth.
The relationship between salinity and susceptibility of D. tertiolecta to PC% was more complicated when indicated by photosynthesis per cell than by growth. Although growth decreased at low salinity, photosynthesis pex cell increased. The increase was not
ensitization
w
as great as that of controls, however, paralleling the
of growth to PCB at low salinity. At normal or high salinity, photosynthesis per cell, like growth, was unaffected by PCB.
The resistance of p. tertiolecta to PCB did not vary with i. 7. pseudonana. Dp. tertiolecta
a
resistance was not conferred by release of a PCB detoxi
°
ier int
an
the medium, nor by possession of PCS-resistant ATPases. Thus, the \ .
differential in susceptibility to PCB, though somewhat variable,
Was pronounced under ali culture conditioas applied. In contrast,
the differential between isolates of 7. pseudonana depended upon
culture conditions, aad was not determined by habitat of origin.
va
To Jeannie
vii
3.
TABLE OF CONTENTS
ABSTRACT 2 6 ww ee ee ee et ee ee ee ee DEDICATION . 2 we ee ee ew ee ee ee eee ee TABLE OF CONTENTS . «we we ee ee ee we we ew ew LIST OF FIGURES . 2. 2 ee ee ee ee wee we LIST OF TABLES . 2. 2. ee ee we ee ee eee ee ACKNOWLEDGMENTS . . 1 ee 2 ee ee ee ew we ee
OBJECTIVES OF THE PRESENT INVESTIGATION ......-.
BACKGROUND . «we ee ee ee ew eee ee ee ee
POLYCHLORINATED BIPHENYLS .....+-+ +22 ee ee
CHLORINATED HYDROCARBON TOXICITY TO PHYTOPLANKTERS .
BIOLOGY OF DUNALIELLA TERTIOLECTA +
BIOLOGY OF THALASSIOSIRA PSEUDONANA
MATERIALS AND METHODS . 2... ee eee eee eee
ORGANISMS AND CULTURE CONDITIONS . . 1... es. ee
PCB STOCK SOLUTIONS AND INOCULATION .......
EXTRACELLULAR RELEASE PRODUCT EXPERIMENTS ....
Effects of medium filtrates on PCB toxicity to T. pseudonana . 2. 2 + «© © ee we we ee ee
Effects of medium hamogenates on PCB toxicity to fT. pseudonana . . 6 + © © we we ew ee ee
ATPase INHIBITOR EXPERIMENTS . . «eee ee eee
NUTRIENT LIMITATION EXPERIMENTS . 2. «1 2 ee eee
Single nutrient deficiencies .......e+.e..
viii
Page iii vii
viii xii xvi
xvii
17 26 31 36 36
43 44 44
TABLE OF CONTENTS (continued) Page Multiple nutrient deficiencies... 1... eee ee 45 Competition . 6. 6 ee ew ee ee ee ee ee ew 45 Senescent cells . 1. ee ee ee ee ew ee ew 46
Estuarine vs. Sargasso Sea isolates of T. pseudonana . s+ 1 ee ww ee we ee ew 49
SALINITY EXPERIMENTS . . - we ee ee ew ee ee ewe 50
PCB effect on p. tertiolecta size distribution at varying salinity . 1 2. ee eee ee ee ene 50
Effects of altered salinity on PCB toxicity to D. tertiolecta and T. pseudonana ......-. 51
PCB effect on photosynthetic M4e_fixation
at varied salinity . . 6. ee ee ew we ee wee 52
PCB toxicity to 7. pseudonana adapted to an altered salinity regimen... 2. 2. ee ee eee 54
ION EXPERIMENTS . «2 1 ee ee ee ee we we ewe 54
Osmotic vs. ionic effects on PCB toxicity .... 54
+ + . Effects of ca? and K concentrations on
PCB toxicity . 6. 6 6 ee ee ee ee ee eee 55
Effects of Na? concentration on PCB toxicity
Effects of Ho concentration on PCB toxicity ... 56
4. RESULTS 26 ww ee ee ee ee ee ee eee ee eee) «(8B EXTRACELLULAR RELEASE PRODUCTS . . . . ee ee ee ee 58
Effects of medium filtrates on PCB toxicity to T. pseudonana . . 2. «6 2 ee ew ee ee ee 58
Effects of medium homogenates on PCB toxicity to T. pseudonana «se ee ee ee ee we ee 61
TABLE OF CONTENTS (continued) Page ATPase INHIBITOR EXPERIMENTS . . «2 6 ere we we ee ee 61 Quabain effect on PCB toxicity .....-e.eeee 61
N, N'-dicyclohexylcarbodiimide (DCHCD) effect on PCB toxicity 2. 6 1 ee ee ee ee ee wee 68
Effects of ouabain and DCHCD combined on PCB toxicity 2. 6. 6 6 ee ee ee ee ee ee ee 68
NUTRIENT LIMITATION EXPERIMENTS . . . 2 2 2 we ee eee 69 Single nutrient deletions . . 1... 1 2 eee eee 69 Multiple nutrient deficiences . . . 2. 2. 2. ee eee 69 Competition... 2 6 ee ee ee ee ee eee 69 Senescent cells . 2. 2 ee ee eee ee eee 76
Estuarine vs. Sargasso Sea isolates of T. pseudonana . ss. 1 6 © © ew we we ee we ee ee 76
SALINITY EXPERIMENTS . «© ee ee ee we ee ee we 83
PCB effect on Dp. tertiolecta size distribution at varying salinity . 2. 6 1 ee ee eee eee 83
Effects of varying salinity on PCB toxicity to Dp. tertiolecta and T. pseudonana . . «+ s-e-e s 89
PCB effects on photosynthetic Mo. ixation at varied salinity... 6 ee ee eee eee ee we OT
T. pseudonana . . 1. 2 ee ee we ew we we ee es 97 D. tertiolecta . 1. 1. ee ee ee ee ee ee 103
PCB toxicity to 7. pseudonana adapted to an altered salinity regiment ....... +s... -. 110
ION EXPERIMENTS . 2. we ee ee ee we ew we ee ew ee 6110
TABLE OF CONTENTS (continued) Page
Osmotic vs. ionic effects on PCB toxicity to T. pseudonana . . 6s 6 6 ee ew ew we ew we we ew we 6110
Effects of ca** and xt concentrations on PCB
toxicity to T. pseudonana and Dp. tertiolecta... 113
Effects of Nat on PCB toxicity to T. pseudonana and p. tertiolecta .. 1... ee eee ew ee we 118
Effects of pH variation on PCB toxicity to [T. pseudonana and p. tertiolecta ......... 125
5. DISCUSSION... 1 ee ee ee ee ee ee ee ee ee ee 134 EXTRACELLULAR RELEASE PRODUCT EXPERIMENTS ...... 134
ATPase INHIBITION . 2. we ew ew ee ee ew ew ee ee 137 NUTRIENT LIMITATION . 1. we we ee ee we we we we ee 6A SALINITY EXPERIMENTS . 2 6 ee we ee ee we ew ew we ew ee) 6143
Growth . . Ce 3 Photosynthesis . 2. eee ee ee ee ee ew we ee 1A
ION EXPERIMENTS . 6 1 ee ee ew ee ew we ew ew we eh ee 6150
LITERATURE CITED . 1 + ee ee ee ee ee ee ee ee we 615K
Figure
6.
7.
10.
il.
LIST OF FIGURES
Possible response patters of PCB toxicity to environmental conditions for two hypothetical phytoplankton species, sj and s2, with tolerance limits ty and tg, respectively... +... ese e
Structures of selected compounds mentioned in this dissertation . 2 6 6 eee ee ee ee ee eee
Phase contrast light photomicrographs of Dunaliella tertiolecta (Chlorophyceae): resting cell from 1l-month-old culture, X 640 (top), and exponential phase cell fixed and stained in Lugol's iodine, oil immersion, X 1600 (bottom) ......-+-e eee
Phase contrast light photomicrographs of Thalassiosira pseudonana (Diatomatae): cells from senescent culture, X 640 (top), and exponential phase cells,
X 640 (bottom), stained with Lugol's iodine ....-
Filter sterilization of medium and transfer to dispensing carboy .... ee ee ee ee ee eee
One of two culture boxes, showing lighting arrangement of cultures . 2. 1 6 ee ee ee ee eee ee
Measurement of lag time, L (after Pirt, 87) .....
PCB (25 pg/1) effect on 7. pseudonana grown in EIO mixed with filtrates or sonicates of late-exponential cultures of 7. pseudonana or D. tertiolecta ...-.
Toxicity of 33 ypg/1 of PCB to 7. pseudonana with D. tertiolecta homogenate (solid symbols) or without D. tertiolecta homogenate (open symbols) .....
Effects of the ATPase inhibitors ouabain (107-3 M in culture medium) and DCHCD (10-3 and 1074 M in culture medium) with 1000 pg/1 of PCB (dashed lines) or without PCB (solid lines) on growth of Dp. tertiolecta
Effects of the ATPase inhibitors, ouabain qo73 M in culture medium) and DCHCD (10°* M in culture medium) with 50 pg/1 of PCB (dashed lines) or without PCB (solid lines) on growth of T. pseudonana.....-s
xii
Page
LIST OF FIGURES (continued) Figure Page
12. Effect of 640 pg/1 of PCB on growth of exponential D. tertiolecta cultures in single-nutrient-deleted media . 6 ew ew ee ee ee ee ee he eh 70
13. Effect of 1000 pg/1 of PCB on exponential (open circles) and senescent (solid circles) p. tertiolecta cells grown at three nutrient enrichment levels: £/2,
£/10, and £/20. 2 2. ee ee ee ee ee eee 72
14. Effect of nutrient concentration on T. pseudonana grown in medium with f/2 or £/40 enrichment .... 74
15. Growth of p. tertiolecta in dialysis bag cultures (circles; points are means of three replicates) vs. prescription bottles (square; unreplicated) .. 77
16. Effect of nutrient enrichment level on growth of T. pseudonana 3H and 13-1 .. 1. ww ee ee we 79
17. Effect of 50 pg/1 of PCB on T. pseudonana grown in £/2 or £/40 medium. 2... ew ew ee ee ee eee 81
18. Osmotic swelling of exponential p. tertiolecta cells placed in media of reduced salinity: control (bottom), and treated with 1000 yg/l of PCB (top) ....+.. 84
19. Osmotic swelling of senescent p. tertiolecta cells placed in media of reduced salinity: control (bottom), and treated with 1000 pg/l of PCB (top) ...... 87
20. Effect of salinity on Dp. tertiolecta resistance to 1000 pg/1 of PCB... ww ee ew ee we eee ee ee) 9D
21. 7. pseudonana 311 cell numbers vs. 15 yg/1 of PCB at 25 0/00 salinity (A) and 10 0/00 salinity (B) ... 93
22. 7. pseudonana 3H cell number vs. 50 pg/1 of PCB at four salinities: 5, 25, 40, 600/00 ........ 95
23. Growth (A) and 14¢_¢ixation per cell (B) of 7. pseudonana
3H at 25 0/00 salinity with and without 50 pg/1 of PCB «wwe eee eee eee ee ee ee ee ee) (9B
xiii
Figure
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
LIST OF FIGURES (continued)
Growth (A) and 140 fixation per cell (B) of T. pseudonana 3H at 25 0/00 salinity with and without 10. pg/l of PCB... . 2... eee eee
Comparison of the effects of 50 pg/l of PCB on growth (A) and 14¢-fixation per cell (B) of 7. pseudonana at three salinities: 6, 25, 37.5 0/00 .......
Effect of 50 pg/1 of PCB on 7. pseudonana after three days of growth at low or high salinity (6 or 37.5 0/00, respectively) ...... 2s ee eee
Effects of 1000 pg/1 of PCB on photosynthetic Mo_ fixation per cell of Dp. tertiolecta at three salinities: 4, 25, and 80 0/00.........
The effect of 7. pseudonana adaptation to low salinity on toxicity of 50 pg/1 of PCB at two salinities: 25 0/00 and 5 0/00 ........084868
Effects of 50 pg/l1 of PCB on growth of 7. pseudonana in three media: 25 0/00 EIO, 4 0/00 isosmotic glycerol-augmented medium, and 4 0/00 non-glycerolaugmented medium... 2. 6 6 ew ee ew we ew we ew
Effects of fivefold augmentation of ca2+ concentration (from 0.007 M ta 0.035 M) on toxicity of 50 ug/l of PCB to 7. pseudonana 3H and 100 pg/1 of PCB to D. tertiolecta grown at two salinities: 25 0/00 and 40/00. 6 wee ee ee ee ee
Effects of fivefold augmentation of Kt concentration (from 0.007 M to 0.035 M) on toxicity of 50 pg/1 of PCB to fT. pseudonana 3H and 1000 ug/1 of PCB to D. tertiolecta grown at two salinities: 25 0/00 and 4 0/00 . 2. 1 ee eee ee ee ee ee
Effects of NaCl substitution for Instant Ocean (EIO) salts on toxicity of 50 pg/1 of PCB to T. pseudonana 3H and 1000 ug/l of PCB to p. tertiolecta .....
Effects of varied NaCl concentration on toxicity of 10 pg/l of PCB to T. pseudonana 3H... 1 ee eee
xiv
Page
lil
Figure 34.
35.
36.
37.
LIST OF FIGURES (continued) Page pH tolerance of p. tertiolecta and T. pseudonana ... 126
Growth of 7. pseudonana 3i1 with and without 50 pg/1 of PCB at two pH's: 4.5 and 9.0 ........4.. 128
Growth of p. tertiolecta with and without 1000 pg/1 of PCB at two pH's: 3.8 and 9.8... ....2.644+2- «130
Ratios of cell densities over time of PCB-treated to
control cultures of 7. pseudonana 3H and D. tertiolecta at varying pH. .....-..+-+... 132
xV
LIST OF TABLES Table Page
1. The world's major trade names and producers of PCB (after Hutzinger et al., 55) ......2.2.266 13
2. Manufacturer's specifications of chlorine content of Aroclor preparations . . + 2 ee ee ee eee 15
3. Susceptibility of phytoplankters to various chlorinated hydrocarbons . 1. 6 6 6 ee ew ee ew we ee et 18
xvi
ACKNOWLEDGMENTS
Special thanks to the members of my doctoral committee, to George Rowland and Don Powers, to my family, and to my friends, who have been supportive in all ways during my graduate school
career, The research was performed at The Marine Sciences Research Center, State University of New York at Stony Brook, and was
supported in part by The Jessie Smith Noyes Foundation, The New
York State Sea Grant Institute, and The Rockefeller Foundation
(RF 76018).
xvii
Chapter 1 OBJECTIVES OF THE PRESENT INVESTIGATION
The literature on phytoplankton physiological response to exposure to chlorinated hydrocarbons under assorted growth conditions has revealed a wide range of response from stimulation to inhibition over a range of exposure from 10+ to 10° pe/1 (Table 3). Phytoplankton susceptibility appears not to vary systematically with taxonomic group or obvious morphological attributes of the test species. This investigation was undertaken to determine whether some environmental factors regulate response and whether these factors may explain the wide range of response exemplified by the phytoplankters chosen for study.
The types of factors contributing to the variability of response fall into three categories, of which the second and third are within the scope of this investigation: 1) molecular structure of the chlorinated hydrocarbon, 2) nature of the test organism, and 3) environmental conditions before and during exposure.
The delimitations of these categories for this investigation are specified below, and restrict it to a manageable complexity. Category 1, which would compare the toxicities of various chlorinated hydrocarbons, is ignored. Only the poly-
chlorinated biphenyl (PCB) Aroclor 1254 was tested, and only at
a few dosages. Category 2 is limited to one resistant and one sensitive species, Dunaliella tertiolecta and Thalassiosira pseudonana, respectively. These organisms were chosen because they are common species which are differentially susceptible to PCB, and because much is known about their biology and the history of the isolates in culture.
Most of the parameters investigated fall into category 3. They are ATPase inhibitors, extracellular release products, nutrient deletions, and the effects of varying osmotic pressure and the concentrations of specific ions on toxicity. The reasons for choosing these parameters are threefold: 1) they are readily manipulated and quantified, 2) experimental results suggested their importance (See Discussion.) during the course of the work, and 3) publications, to be cited, allude to their importance in related research.
Susceptibility of the test organisms to PCB under stress—- ful conditions of the test parameters was investigated to determine if simultaneously acting PCB and environmental stresses are tolerated less than each stress acting alone (36,97).
The pattern of phytoplankter susceptibility to PCB under varying levels of environmental stress may be portrayed graphically. Fig. 1 graphs susceptibility to PCB of two hypothetical phytoplankters as a function of environmental stress over the
tolerance range of the species. For simplicity the functions are
Fig. 1. Possible response patterns of PCB toxicity to environmental conditions for two hypothetical phytoplankton species, Sy and s9, with tolerance limits ty and tos
respectively
SUSCEPTIBILITY TO PCB————>
n bs)
1 H ' t : , 1 t Hott a —— . | 1 ' 1 Ss i i ! ' 1 I ! ! : ‘ { \ H t ' H ' ! ' \ 1 ' H H ! ' H ' 1 ' ' 1 ‘ t t 1 4 Ty To 7, To T Ty, a So } { ' Spot 1 { + 4 ' ' | ' i 2 TT,
ENVIRONMENTAL PARAMETER
nwt oe ee ee
depicted as being linear, but need not actually be linear. The degree to which environmental conditions regulate PCB toxicity to the hypothetical phytoplankters is represented by the slopes of the linear functions. The distance between them is the differential in toxicity.
Divergence or convergence of the lines illustrates how differential toxicity may be increased or decreased depending on the culture conditions during an experiment. If the lines were parallel for all phytoplankton species in a given experiment over the entire tolerance ranges for the environmental stress factor, then differential susceptibility would be independent of environmental stress. While it is extremely unlikely for the lines to be parallel, environmental stress would only be an important determinant of differential PCB toxicity if the rate of divergence or convergence of the lines were large relative to the distance between them. This situation is most likely to occur when the slopes of the lines are of opposite sign. In biological terms, this is when the susceptibility of the phytoplankters to the PCB increases for one species and decreases for the other in response to a variation in some environmental parameter.
A large amount of literature deals with PCB inhibition
of population growth or photosynthesis, and because these pro-
cesses are responsive to environmental conditions, they were
employed as the indicators of PCB toxicity under the test conditions mentioned in category 3. The relationship between photosynthetic and growth inhibitions was also investigated. Since Fisher (31) reported absence of photosynthetic inhibition per cell 48 hours after PCB inoculation and following growth rate recovery, the present investigation attempted to reconcile the seemingly conflicting reports by examining photosynthesis per cell prior to growth rate recovery. Further, the relationship of photosynthesis per cell and PCB was investigated under varying salinity to see whether the two indicators of inhibition
reponded similarly to an environmental stress.
Chapter 2 BACKGROUND POLYCHLORINATED BIPHENYLS
PCB's are chlorinated hydrocarbons structurally similar to DDT (Fig. 2). The international literature refers to PCB's under a variety of trade names. As of 1974 the major trade names and producers of PCB were those listed in Table 1 (55).
Most of the literature on PCB toxicity to algae refers to Monsanto's Aroclors. Their nomenclature consists of a 4digit number beginning with 12 to indicate chlorinated biphenyl or 54 to indicate chlorinated terphenyl, followed by two digits giving the weight percent of chlorine (Table 2).
Commercial Aroclors are composed of a variety of isomers of a number of individual PCB's. Individual PCB's are usually solids at room temperature, while commercial mixtures are mobile oils, viscous liquids (Aroclor 1254), or sticky resins due to mutual depression of melting points of their components.
PCB's are important industrially as plasticizers and heat-resistant electrical insulators. Annual world PCB consumption was about 250,000 metric tons (52). After 1970, Monsanto voluntarily restricted sales to uses’ in relatively closed systems
such as capacitors and transformers.
Fig. 2. Structures of selected compounds mentioned in this
dissertation
| oe pe-O>-a kero ci-c-Cl Ok . i) : cl Ci © PCB’ CHLOROBIPHENYLOL HPO __ ss cH) )-0H Cl Cl Cl Cl Cl C! Cl Oo Cl Se Cl cl CHLORINATED 4-CHLORO- BENZOIC AGID NAPHTHALENE BENZOIC
ACID
PCB's are important ecologically as recently recognized persistent mobile pollutants of aquatic ecosystems (40). The properties of PCB's which give rise to their importance in aquatic ecosystems are: 1) low solubility in water, 2) affinity for surfaces, 3) affinity for lipids, 4) resistance to degradation, and 5) ability to interact with biological systems to produce undesirable effects.
The solubility of a particular Aroclor formulation is a function of its component PCB's. In water, solubility of all PCB's is low, decreasing with increasing chlorination: Aroclor 1242 is soluble at 200 pg/1, 1248 at 100 yg/1, 1254 at 40 pg/l, and 1260 at 25 pg/1 (75). By comparison, DDT is soluble in water at about 1.2 pg/l (11).
PCB's are adsorbed to surfaces. In aquatic ecosystems, the bulk of the PCB is associated with particulate matter such as microorganisms and silt (27). For example, out of an initial
concentration of 10 pg/1 of Aroclor 1254 in river water, only
70% was recoverable from the water at time zero and 37% by week sixteen, the losses going to the glass walls of containers and to silt.
Because PCB's are lipophilic, uptake by bactera and protozoa occurs rapidly and results in many-fold higher doses in the biota than in the water (100). The ciliate Tetrahymena
pyriformis exposed to 10.0 ug/l in the growth medium contained
167.9 yg/l (dry weight) after seven days (24). The diatom Cylindrotheca closterium concentrated Aroclor 1242 from 0.1 pg/ml to 109.2 pg/ml, about three orders of magnitude (60). Mixed microbial populations from activated sewage sludge aerobically degraded commercial PCB preparations, with extent of degradation correlated with low chlorine content (76). Biphenyls are oxidized by pure cultures of Pseudomonas putida to benzoic acid, by Beijerinckia to a dihydrodihydroxybiphenyl, and by Achromobacter isolated from sewage effluent to benzoic acid and 4-chlorobenzoic acid (55, p. 147).
By and large,PCB's are very resistant to chemical and biological degradation, a property which, combined with lipophilicity and other solubility characteristics, permits biological concentration of PCB's in ascending trophic levels (55, p. 1). While PCB's in estuarine and near-shore areas exposed to fluctuating PCB inputs are biologically concentrated similarly to DDT, as described by Woodwell, Wurster, and Isaacson (114), another mechanism of biological concentration was proposed for the open ocean (45). North Atlantic plankton showed almost uniformly high PCB levels (100 to 900 ug/gm) in their fat, with residues in many predator-prey couples the reverse of what would be expected through biological concentration. The authors suggested that fat tissues have a definite capacity for residues regardless
of position in the food chain. A similar hypothesis of direct
absorption of PCB from the water in lentic environments was advanced by Hamelink (43).
PCB residues in fish have been high enough (i.e., 5 pg/gm, FDA "action level") to halt fishing in such waters as the Hudson River (53). In the Milwaukee River system, PCB's are ubiquitous from West Bend to Lake Michigan. The concentrations in fish from the river ranged from 2.3 to 405 pg/gm and in fish from Lake Michigan from 15 to 25 pg/gm. By contrast the river water ranged from 0.02 pg/1 in Milwaukee Harbor to 2.8 yg/l upriver near East Riverside Park (106). In the Great Miami River some fish exhibited PCB concentrations of over 150 pg/gm, the PCB apparently contributed by paper mills (62). ‘In Missouri, PCB residues in bluegills and channel catfish ranged from 2.9 to 703 ug/gm, with concentration factors of 26,300 to 52,000 times the concentration in water (102).
PCB's have acute toxic effects on humans, and are suspected of cumulative effects, including liver tumorigenesis (56) with chronic sub-acute exposure over a lifetime. Acute toxicity to humans may result in yellow atrophy of the liver (fatal), dermatitis, fatty degeneration of the liver (chronic exposure). The mechanisms of toxicity may vary with the degree of chlorination, and the products of metabolism (chlorobipenylols) are more toxic than the parent compounds (55, Ch. 1 and references therein).
PCB residues in human fatty tissue and milk (on a lipid
Table 1.
The world's major trade names and producers of PCB
(after Hutzinger et al., 55)
Trade Name of PCB
Aroclor Clophen Phenoclor, Pyralene Kanechlor Santotherm Fenclor
Producer
Monsanto Bayer Prodelec
Kanegafuchi Mitsubishi-Monsanto Caffaro
Sovol
Chemko
Country
U.S.A., Great Britain Germany France
Japan
Japan
Italy
U.S.S.R. Czechoslovakia
Table 2. Manufacturer's specifications of chlorine content of
Aroclor preparations
Aroclor
1221 32 42 48 54 60 62 68
Weight % Cl
20.5-21.5 31.5-32.5 42 48 54 60 61.5-62.5 68
Ave. No. of C1/molecule
1.15 2.04 3.10 3.90 4.96 6.30 6.80 8.70
Ave. Molecular Weight
192 221 261 288 327 372 389 453
basis) have repeatedly been reported in the microgram per gram range (55, p. 249). While PCB use remains legal, industrial atmospheric exposure limits have been set to below 0.5 and 1 mg/m for
PCB's containing 42% and 54% chlorine, respectively (55, p. 1). CHLORINATED HYDROCARBON TOXICITY TO PHYTOPLANKTERS
-At the base of aquatic food webs, phytoplankton display wide interspecific (71,73,80) and intraspecific (34) differences in susceptibility to PCB and other chlorinated hydrocarbons (Table 3). Differential toxicity to phytoplankton alters species composition in mixed cultures, both in the laboratory (33,39,81) and in nature (7,33). It is likely that interference with higher tropic levels occurs not only through direct toxicity by adsorption and absorption (43,45) and biological concentration (78,114), but by inducing changes in species composition during phytoplankton succession (67,73).
The morphological and physiological basis for differential chlorinated hydrocarbon toxicity to unicellular algae is not fully understood. Vadas (105) reported resistance of unicellular algae to DDT correlated with ability to osmoregulate over a wide range of salinities, and postulated a common mechanism. to account for this correlation. Batteron et al. (2) reported DDT-induced reversal of salinity (NaCl) tolerance in
a blue-green alga, which could be counteracted by ca2t, RNA but
Table 3. Susceptibility of phytoplankters to, various chlorinated
hydrocarbons
Logy9 Lowest Toxic Conc. (ng/1) 2 ‘TAXON SPECIES Logig Highest Non-Toxic Dose (ug/1) RUFERENCES Mo. ¢ x = 3 x FS & & Ao Ok % HE rs ¥ en ns 2 aR a OE LOS eS SE NE BPM LG CYANOPHYCEAE |Anacystis nidulans 2.9 2.0 | 3.0 1, 2 3.3 3.3 A. aeruginosa ae 3.3 cylindrospermum licheniforme 3.3 84 -0.7 |-0.7 Agmenellum guadruplicatum 1.3 3.0 1 5 EUGLENOPHYTA |Zuglena sp. Te 21 3.7 | 4,7 E. gracilis 2 2 3 4 23, 79, 88 CHLOROPHYCEAE |8.achiomonas submarina 2.7 105 2 Chlamydomonas reinhardtii 3.3 26, 78, 80 1.3 1 Dunaliella euchlora 14, 104 2 D. tertiolecta 3 3 3 3 10, 36, 71, 73, 89 ankistrodesmus sp. 3 17 0.7 A. falcatus 39 5 Chlorella sp. 2.8 1 7 3 13 C. pyrencidosa 17, 18, 46, 47 C. variegata 3.3 84 1 Pediastrum boryanum 79 5 Scenedesmis P+ vv 3.3 |S. obliquus 3.3 84 2.5 S. obtusiusculus ‘6 -1 | 3 2 S. quadricauda 3 +9, 71, 101 0.6 Selenastrum capricormutum 68 Desnococcus ap. 104 3 Platymonas sp. 14 2 P. subcordiformis 195 1 Pyramimonas sp. 115 1.3 1 CHRYSOPHYCEAE |monochrysis lutheri 104 adi coccolithus huxleyi 1.9 10, 31, 73 1 BACILLARIO- _ |Bellerochia sp. 34, “1 PHYCEAE Rhizosolenia setigera 36 aj. 1 1 Skelctonema costatum 10, 31, 73, 30, 115 rT Thalassiosira sp. 90 T. fluviatilis 1.9 10 1 lo ilo ° 7. pscudonana 1 1.9 10, 31, 33, 34, 36 2 ~ cylindrotheca closterium 1 € i . Fragillaria pinnata 34 3.3 3.3 . Gomphonema parvulum Ba 1 , Navicula pelliculosa 39, 74 3.3 [Ne seminulum 15 3.3 witzschia palea 3.3 84 1.6 1 Phacodactylum tricornatum 1.3 104 0.7 Synedra acus 39 0.7 =I PYRROPHYTA Exuviella paltica 0.3 | 0.3 89 1 amphidinium carteri 1.9 10 Peridinium trochoidium 115 RHODOPHYCEAE | Porphyridium sp. a 10, 105
not DNA levels were reduced by 100 pg/1 of Aroclor 1242 (60).
Chlorinated hydrocarbons inhibit growth and photosynthesis of many phytoplankters. A given level of resistance or susceptibility to a particular chlorinated hydrocarbon usually approximates sensitivity to other chlorinated hydrocarbons. Interspecific differences in sensitivity may span several orders of magnitude of chlorinated hydrocarbon concentration (Table 3). For example, the diatom T. pseudonana was sensitive to as little as 0.1 pg/1l of PCB in mixed algal culture (33) and, in monoculture, to as little as 25 yg/1 of PCB (80), 10 pg/1 of DDT, and 1 pg/1 of dieldrin and endrin (73), whereas the green flagellate p. tertiolecta was resistant to 1000 pg/1 of these compounds (80).
This generality was not applicable to the chlorinated naphthalenes, similar in structure to PCB's (Fig. 1), and varying from mono- to octachlorinated forms. Walsh, Ainsworth, and Faas (107) studied the toxicity of three commercial formulations of chlorinated naphthalenes to four marine phytoplankters including T. pseudonana and p. tertiolecta. All species were resistant to 1000 pg/1 of the most highly chlorinated formulation, and 7. pseudOnana was resistant to the least chlorinated formulation applied at 100 pg/1, a dose which inhibited growth of p. tertiolecta.
There was a negative correlation between degree of chlorination of chlorinated naphthalenes and toxicity. This
differs from the pattern of toxicity observed in the Aroclors
by Glooschenko and Glooschenko (39), in which the more highly chlorinated forms were most toxic. However, Hawes, Kricher, and Urey (46) reported that growth of Chlorella pyrenoidosa was more inhibited by Aroclors of lower chlorine content. No scheme can yet be postulated to explain this variation.
Chlorinated hydrocarbons induce a growth lag and sometimes reduce the maximum rate of exponential growth of sensitive phytoplankters. For example, cell division of the diatom Skeletonema costatum exposed to 80 pg/l (0.23 pM) of DDT was completely inhibited for nine days, after which cells divided at the same rate as controls (10). Retreatment of recovered cultures with DDT caused another nine day lag, indicating that there was a "true lag" (87) as opposed to an "apparent lag'' in which there is an unaffected, resistant subpopulation dividing at the same rate as controls throughout the experiment. Since the resistant subpopulation may initially be a small fraction of the total popula~
tion, cell division is undetected for what appears to be a lag
period during which the entire culture is not dividing. However, on retreatment with the test chemical, the resistant subpopulation, if there was one, will comprise the entire culture and the lag will not recur. Fisher (33) used this approach to demonstrate that 7. pseudonana exposed to 100 pg/1 of DDT did not acquire
resistance.
The blue-green algae Anacystis nidulans and Agmenellum
quadruplicatum exposed to several dose levels of dieldrin, endrin, aldrin, and five metabolites of these compounds, displayed reduction in the maximum rate of cell division (1). Cole and Plapp (18), using technical grade preparations, demonstrated that Aroclor 1254 was a stronger inhibitor of Chlorella pyrenoidosa than DDT or dieldrin. Aroclor 1254 reduced growth rate to 11% to 60% of controls, depending on dose. It is difficult to relate Cole and Plapp's constant 1000-pg/1 doses per 1 to 1000 pg/ml dry weight of algal biomass to the main body of literature in which biomass is expressed as cell numbers.
Aroclor 1254 reduces c. pyrenoidosa photosynthetic Me. fixation (18). When 1000 pg/1 of PCB was applied to 1000 pg of algae per ml of medium, multiple doses were inhibitory but a single dose was not. At 100 pg algae per ml, the multiple 1000-ng/1 dose had no effect but the single dose inhibited 46 eixation by 93% after seven days, a level of inhibition which was also observed at 10 and 1 yg algae per ml medium. No effort was made to measure the growth rate or viability of the cells at
the time the 4 eixation data were collected.
Hawes, Kricher and Urey (47) exposed Cc. pyrenoidosa to 100 ug/l or 1000 pg/l of Aroclor 1232. At 94 and 190 hrs., PCBtreated cultures displayed higher 14 gixation than controls ona per-cell basis. Cell density was reduced by PCB at 94 hrs. (47),
so that the enhancement of photosynthesis by PCB might in part
have been due to a reported inverse relationship between Chlorella population density and photosynthetic rate. Since recovery of cell density had taken place by 190 hrs., this inverse relationship could not have been present, and the PCB-treated
cultures were still fixing 14
C faster than controls on a per cell basis. Photosynthetic Mo uptake by a natural phytoplankton
community was inhibited by less than 10 pg/1 of Aroclors 1242 and 1254 in the Gulf of Mexico (77), and by Aroclor 1254 in the Long Island Sound (7). The Long Island Sound study revealed inhibition per cell and per unit chlorophyll a, as well as a downward shift in cell size distribution. Kricher and Bayer (61) reported inhibition of primary production by 1000 pg/1 of Aroclor 1232 in a natural phytoplankton population in a Massachusetts reservoir.
Although growth of the green flagellate p. tertiolecta
was unaffected by 1000 pg/1 of Aroclor 1254 @0), 100 yg/1 reduced
photosynthetic M4 c_uptake to 63% of controls (71). A 7000-pg/1
(20-pM) dose of DDT or DDE inhibited non-cyclic electron flow by 50% in isolated chloroplasts of p. tertiolecta (10). A 3.6-pg/1 dose of DDT inhibited photosynthetic 14 o-eixation by the green alga Selanastrum capricormutum, suppressing aspartic in favor of glycolic acid formation (68). This was interpreted as an
interruption of cyclic photophosphorylation.
In accordance with the observed susceptibility of its growth rate to inhibition by PCB (80), 10 pg/1 of Aroclor 1254 inhibited photosynthetic Mc¢_ fixation by Thalassiosira sp. isolated from Long Island Sound and grown in dialysis membrane bags suspended in a tidal channel (90). In re-examining the findings that DDT and PCB reduced photosynthesis of three phytoplankters, including T. pseudonana (34), Fisher (31) reported that while photosynthesis per culture was inhibited, the inhibition was produced by a reduction in the number of cells per treated culture. Interference by the chlorinated hydrocarbons with photosynthesis was not observed at the single time Fisher's samples were taken (48 hours after chlorinated hydrocarbon inoculation).
Fisher et al. (34) reported that estuarine isolates of three species of phytoplankton, including 7. pseudonana, were more resistant than Sargasso Sea isolates to PCB. He suggested that resistance to chlorinated hydrocarbons may be increased by adaptations to the unique selective pressures exerted on estuarine phytoplankton. He identified these pressures in estuarine habitats as exposure to metals and organic compounds, and unpredictable or large-scale fluctuations of temperature, salinity, and light. By contrast, the Sargasso Sea habitat
appeared to be stable and nutrient impoverished.
Fisher (32) theorized that this inter-habitat resistance
pattern was explainable by one never-tested aspect of the stability-time hypothesis of Slobodkin and Sanders. This hypothesis states that organisms of stable, regular environments are more vulnerable to novel or rare perturbations than are organisms adapted to physically rigorous and irregularly fluctuating habitats (95,99). Novel chemical stress represents a paradigm of these perturbations (32).
Fisher compared the toxicities of three chemicals which have never existed in the world oceans: adamantine, bromophenyloctane, and nitroethane to phytoplankters isolated from estuaries vs. the open ocean (Sargasso Sea). He claimed that the results confirm the hypothesis: estuarine isolates were more resistant than their Sargasso Sea counterparts. This suggested that algae of unpredictable habitats are "pre-adapted" to novel chemicals because adaptations to such habitats requires a "thick skin," i.e., a highly protective outer membrane (32).
Fisher demonstrated that growth of the green euryhaline flagellate, p. tertiolecta, reported to be resistant to 1000 pg/1 of PCB (80), was inhibited at low salinity (10 0/00) by as little as 10 pg/1 of PCB. The mechanism proposed for this induced susceptibility was osmotic swelling, allowing PCB molecules to come into closer contact with sensitive sites of action because of the
thinness of the distended cell membrane. In short, the hypothesis
implies that physical thinning of the cell membrane accounts for
the difference between sensitive and resistant cells. Inasmuch as Fisher et al. (33), citing Eppley et al. (29), reported that D. tertiolecta is an estuarine alga, Fisher's hypothesis states that unpredictable habitats like estuaries select for algae with "thick skins." This hypothesis will henceforth be referred
to as the "thick skin" hypothesis (See Discussion.).
BIOLOGY OF DUNALIELLA TERTIOLECTA
Division Chlorophyta Class Chlorophyceae Order Volvocales
Family Polyblepharidaceae
Dunaliella tertiolecta (Fig. 3) is one of twelve species of the genus, which includes Dp. salina, minuta, media, piercii, euchlora, bioculata, parva, primolecta, quartolecta, polymorpha, and minisculus (13). The genus possesses three of the four features diagnostic of the Division Chlorophyta (79): photosynthetic pigments include chlorophylls a and b; starch is the storage product; and there are two acronematic flagella. The fourth characteristic of the Chlorophyta is that cell walls contain cellulose; punaliella lacks a rigid cell wall (13,69), an observation verified by electron
micrography in p. salina (103) and p. tertiolecta (58,66).
Fig. 3.
Phase contrast light photomicrographs of punaliella tertiolecta (Chlorophyceae): resting cell from 11-monthold culture, X 640 (top), and exponential phase cell fixed and stained in Lugol's iodine, oil immersion,
X 1600 (bottom)
The genus is unicellular. Lacking gametangia surrounded by a sterile jacket of cells, it is placed in the class Chlorophyceae rather than the Charophyceae (22).
Dunaliella is in the order Volvocales, the most primitive of fourteen orders of the Chlorophyceae. This order includes unicellular, colonial, and multicellular forms, and is characterized by motile vegetative cells (79). Cells may reproduce vegetatively by longitudinal fission (13) or sexually via zoospores (65).
D. tertiolecta closely resembles Chlamydomonas, and was classified as "Chlamydomonas 111" by its collector in Oslo Fjord, Norway (13). Dp. tertiolecta is ubiquitous, also occurring in the open ocean (58), estuaries (41,71,73), rock pools (29), and tide pools (73). As expected of a ubiquitous marine alga, the species is euryhaline, i.e., tolerant of a broad salinity range (50,65). Other members of the genus range from halophilic (50), occurring in brine pools (37), to fresh water (85); many species tolerate wide changes in salinity.
Like other euryhaline flagellates lacking a cell wall,
D. tertiolecta osmoregulates by excretion of glycerol under reduced salinity to avert lysis by swelling (50). While there is some controversy over the need to osmoregulate at high salinity (96); several workers have demonstrated adjustment of glycerol
content as a function of external osmotic pressure (6,21,109)
and the degree of tolerance to high salinities is directly related to the maximum maintainable intracellular glycerol concentration (3,8,50).
Unlike the halophilic bacteria, which possess enzymes requiring high salt concentrations to function (63), enzymes of the halophilic algae are inhibited by salt concentrations at which the intact cell can survive (4,8,57). In protecting the intact cell, intracellular glycerol appears to act as a “compatible solute," that is, a substance which does not inhibit enzymes as do salts.
Since glycerol production is not light dependent, but glycerol is a photosynthetic product of punaliella, Ben-Amotz and Avron (6) postulated two mechanisms for glycerol production, one photosynthetic, the other degradative from starch reserves. The enzyme catalyzing the reaction Dihydroxyacetone + NADPH +
—s Glycerol in Dp. parva was shown to be an NADP dependent
dihydroxyacetone reductase with a pH optimum of 7.5 for glycerol production, and 9.0 for dihydroxyacetone production (5).
When plunged into hypertonic or hypotonic NaCl solutions, reversible volume change occurred in D. parva. Recovery of initial cell volume was unaffected by concentrations of the uncoupler FCCP high enough to completely block ATP synthesis. This observation disputes the hypothesis that volume recovery under hypertonic conditions is caused exclusively by intracellular glycerol synthesis,
assumed to be ATP dependent (38).
BIOLOGY OF THALASSIOSIRA PSEUDONANA
Division Bacillariophyta Class Diatomatae Order Centrales
Family Coscinodiscaceae
Thalassiosira pseudonana (Fig. 4), formerly cyclotella nana (54), is a small barrel-shaped centric diatom. It has been isolated from a variety of habitats. These include the Moriches and Great South Bays on the south shore of Long Island, New York (isolates 3H and 5A, respectively, 2.5 to 10 » in diameter),
Eel Pond, Woods Hole, Massachusetts (isolate e.p., 2.5 to 10 yp
in diamater), the edge of the continental shelf (isolate 7-15,
0.4 to 14 p in diameter), and the Sargasso Sea (isolate 13-1,
0.4 to 11 p in diamater) (42). It is a common river phytoplankter in the Weser in Germany, a river with attenuated flow due to
tidal pressure from the North Sea. Reproduction takes place in
the river before washout to the sea (92).
Phytoplankton blooms caused by waste from duck ponds were recorded in Moriches and Great South Bays since 1922 and were restricted to green algae until 1953. By 1957 centric diatoms dominated the bloom in a tributary of Moriches Bay. Previously only a minor component of the centric diatom blooms, T. pseudonana dominated the bloom in 1958, reaching 2 X 10°
cells per ml (42,93,94).
Fig. 4.
Phase contrast light photomicrographs of Thalassiosira pseudonana (Diatomatae): cells from senescent culture, X 640 (top), and exponential phase cells, X 640
(bottom), stained with Lugol's iodine
T. pseudonana is a photoautotroph. While facultative heterotrophy has been found among diatoms, it is almost exclusively restricted to pennates. Where heterotrophy has been demonstrated, specific uptake systems for the substrates have been found. As yet, no such uptake system has been found for glycerol or other alcohols in diatoms, although various workers have tested ethanol, mannitol, inositol, sorbitol and dulcitol on a variety of species (51, Ch. 6). Glycerol is a minor excretion product of T. pseudonana (48). No open-water centric diatom has been found capable of heterotrophy.
Guillard and Ryther (42) considered the isolates of T. pseudonana to be strains of a single species, although there was slight morphological and marked physiological differences between them. Strain 13-1 from the Sargasso Sea was most distinct morphologically in the structure of the girdle bands observed electron microscopically and physiologically in requiring relatively high temperature and salinity. Strain 13-1 was less euryhaline than the estuarine isolate 3H.
According to Eppley (28) isolate 3H is less efficient at silicic acid uptake than isolate 13-1, with half saturation constants of 0.98 vs. 0.19, producing growth rates of 3.6 vs. 2.1 div./day. The half saturation constants for nitrate
differed in the same direction, being 1.9 vs. 0.38.
All isolates require one vitamin, Bio (42). All occur singly or in pairs, rarely in chains of three or more. Joining is valve-to-valve, not by slime threads as in typical chain-forming diatoms.
T. pseudonana is diploid, reproducing sexually by auxospores. Asexual reproduction is by fission. Werner (110) reported that growth of T. pseudonana did not cease, as in some species, when it was grown in a silicon-limited medium. Instead
cells with thin walls were produced.
Chapter 3
MATERIALS AND METHODS
The medium is the message. Marshall McLuhan (understanding Media,72)
ORGANISMS AND CULTURE CONDITIONS
The species used were punaliella tertiolecta, a naked green flagellate, and Thalassiosira pseudonana (formerly cyclotella nana), Woods Hole clones 3H and 13-1. Stocks were batch cultured in an artificial seawater medium (enriched Instant Ocean, EIO). This was prepared by dissolving Instant Ocean (Aquarium Systems, Inc., Eastlake, Ohio) in double-distilled water to salinity 25 0/00, adding f/2 nutrient enrichment and adjusting pH to 7.80 (42). Media at other pH's were prepared by adding HCl or NaOH to EIO at pH 7.80.
Media were filter-sterilized through 0.22-p Millipore
“filters under 34 atm. of nitrogen gas pressure, and dispensed by gravity flow from spouted glass carboys (Fig. 5).
Stock and experimental cultures were maintained in an environmental chamber (Forma Scientific Co.) at 24°C under continuous fluorescent illumination at 2.7 X¥ 10 lux (Fig. 6), unless other conditions are stated.
For experiments, replicate 10-ml aliquots (usually three)
Fig. 5.
Filter sterilization of medium and transfer to dispensing carboy. T = cotton-filled thistle tube covered with aluminum foil, M = magnetic stirrer, C = clamp, F = in-line Millipore filtration apparatus containing two filters: 0.45- and 0.22-) pore sizes, S = rubber stopper
with aluminum foil on the underside, D = dispensing
tube, protected with removable sheath of aluminum foil. Connections are a combination of Tygon and glass tubing, firmly held by hose clamps. Carboys are four or eight liters. Pressurized carboy connected to nitrogen gas
tank is protected by electrical tape wrapping.
No REGULATED TO 34-ATM
Fig. 6.
One of two culture boxes, showing lighting arrangement
of cultures
of exponentially-growing cells were pipetted into sterile Teflon-lined screw cap test tubes from a common stock.
Initial cell densities varied from 5 X 10°
to 1X 10° per mi. Cell densities were determined by optical counts of cells fixed and stained in Lugol's iodine and Pasteur pipetted into
a Fuchs Rosenthal counting chamber. Test tubes were shaken and
agitated using a vortex mixer before sampling. PCB STOCK SOLUTIONS AND INOCULATION
Stock solutions of Aroclor 1254 (Fig. 2) were kept refrigerated in methanol solutions and diluted for use by a factor of 10, usually by inoculation by microsyringe of 10‘p1 into 100 ml of EIO. Sterile prescription bottles containing PCB-treated cultures were shaken vigorously before incubation. Controls received methanol and were also shaken.
Between treatments the microsyringe was thoroughly flushed with methanol. Test tube cultures derived from the prescription bottles were disposable following use. Nondisposable glassware was washed with soap, then rinsed at
least nine times: three times with warm tap water, three with
distilled water, and three with acetone.
EXTRACELLULAR RELEASE PRODUCT EXPERIMENTS
Effects of medium filtrates on
PCB toxicity to 7. pseudonana
To investigate the extent to which substances released by cells into the medium could modify PCB toxicity, exponentially growing stocks of p. tertiolecta and 7. pseudonana 3H were each divided into two subcultures. One of each pair of subcultures was centrifuged out of suspension and the superna tant medium filter-sterilized by gentle vacuum filtration through 0.45-p Millipore filters. The remaining subculture in each pair was sonicated to induce release of substances that might normally be retained within the cell, and then centrifuged and filtered as before. The filtrates were augmented with a volume of EIO equal to 10% of the filtrate volume to delay nutrient limitation. Lastly, the filtrates were again divided into two aliquots. One aliquot of each filtrate was heated in a water bath to 56°C for 30 minutes to inactivate proteins, and allowed to return to room temperature.
The heat-treated and non-heat-treated aliquots of each filtrate were then inoculated with 10° exponentially growing T. pseudonana 3H cells per ml, and shaken. These cultures were subdivided into two subcultures each, one to receive
25 ng/1 of PCB and the other methanol. Treated and control
subcultures were subdivided into three replicates, incubated, and sampled periodically to assess PCB toxicity in the differ-
ent media.
Effects of medium homogenates
on PCB toxicity to T. pseudonana
In a further attempt to discover a possible detoxifier produced by the resistant species, exponentially growing D. tertiolecta stocks were centrifuged, the supernatants decanted, and the pellets ground in a tissue grinder. Any substances not released previously by the filtration and sonication procedures might be released by this extreme disruption of the cells. Two 40-ml aliquots of p. tertiolecta stock at 5.45 X 10° cells per ml were pooled to produce the pellet. The debris was filtered under gentle vacuum through a 0.8-p: Millipore filter, and 30 ml of EIO were added to the filtrate. The pH was adjusted to 7.80 and the medium sterilized by vacuum filtration through a 0.45-p Millipore filter.
Thirty ml of exponentially growing 7. pseudonana stock cultures were augmented with 30 ml of EIO or with 30 ml of the homogenate medium. These cultures were each subdivided into two aliquots, one to receive 33 mg/l of PCB, the other
methanol. All four aliquots were dispensed in 10-ml amounts to
triplicate sterile test tubes, incubated, and sampled periodical-
ly to determine PCB toxicity.
ATPase INHIBITOR EXPERIMENTS
Inhibitors of cell membrane-bound ATPase systems were inoculated into exponentially growing cultures of p. tertiolecta and T. pseudonana. Ouabain was used as a selective inhibitor of the Nat,Kt,Mgt+ -ATPase system (Cirillo, personal communication). The effect of each inhibitor, and both acting together, on the growth of each species indicated the relative importance of the inhibited systems to each species.
By adding PCB in addition to selective ATPase inhibitors, it was possible to test whether algal susceptibility to PCB depended upon which ATPase system was inhibited, or whether
PCB action was independent of ATPases.
NUTRIENT LIMITATION EXPERIMENTS
Single nutrient deficiencies
D. tertiolecta exponential cells were centrifuged out of ELO, washed, and starved 23 hours by suspension in nonnutrient-enriched Instant Ocean buffered to pH 7.8. These starved cells were inoculated into 15 different nutrient media.
The control medium contained a full complement of all 14 enrich-
ment nutrients. Each of the remaining 14 media was deficient in one of the 14 enrichment nutrients. Each nutrient treatment was divided into PCB-treated (10 eng PCB per cell, 640 ps/1) and untreated groups, and each group was subdivided into two replicates. The effects of PCB on growth were determined for each nutrient treatment by sampling and counting
cells periodically, as described earlier.
Multiple nutrient deficiencies
Studies of growth and PCB susceptibility under nutrient limitation were performed as above, but instead of deleting single enrichment nutrients, the full complement of enrichment nutrients was varied simultaneously. Appropriate media were prepared by dilution with non-enriched Instant Ocean buffered to pH 7.80. The centrifugation and starvation steps were eliminated. Media were seeded with small volumes of cell
stocks which contained too little total nutrient material to
significantly alter enrichment levels. A higher PCB dose was employed for p. tertiolecta, and 1. pseudonana was also tested
in these studies.
Competition
The effect of competition for nutrients on PCB toxicity
to Dp. tertiolecta was investigated by enclosing triplicate control and PCB-treated cultures in dialysis bags suspended in buffered Instant Ocean. Each of the 6 dialysis bags contained 50 ml of culture. All were submerged in a common beaker containing 700 ml of buffered Instant Ocean, giving an equilibrium mixture of 30% nutrient enrichment. Competition was assumed to occur among cells within dialysis bags
and among dialysis bags within and between treatments. Senescent cells
Because senescent cells do not, by definition, immediately commence exponential division upon transfer to fresh medium, growth rate was an inappropriate measure of PCB effect on cells in this physiological state. PCB effect on senescent cells was indicated by alteration of the lag period preceding exponential growth upon transfer to fresh medium.
Prolongation of the lag phase was measured according to the method recommended by Pirt (87): From a point on the growth
curve at which p = 1 a line of slope Buax Was extended to-
Bmax ward the origin (Fig. 7). The value of the abscissa at the point of intersection with the initial cell density value of
the ordinate was the lag time.
Fig. 7.
Measurement of lag time, L (after Pirt, 87)
“A
LOG CELL DENSITY
CARRYING
CAPACITY
Estuarine vs. Sargasso Sea
isolates of 7. pseudonana
Two hundred ml of ELO with either f/2 or f/40 nutrient enrichment were placed into sterile 250-ml prescription bottles. The media differed only in the level of nutrient enrichment. Prescription bottles were inoculated with 104 exponentially growing T. pseudonana 3H or 13-1 cells per ml, then shaken. Each culture was subdivided into two 100-ml aliquots. Into one aliquot, 10 pl of PCB (Aroclor 1254) stock in methanol solution were inoculated by microsyringe to yield 50: pg/1 of PCB, The control received 10 pl of methanol. Lach of the eight resulting cultures was shaken, and from each, three 10-ml aliquots were withdrawn and pipetted into sterile disposable test tubes. The twenty-four resulting test tube cultures were incubated in an environmental chamber at 17°C under continuous fluorescent illumination at 2.7 X 10° lux. At intervals, about 1 ml of cell suspension was removed, stained with a drop of
Lugol's iodine, agitated with a vortex mixer, and counted
microscopically in a Fuchs Rosenthal counting chamber.
SALINITY EXPERIMENTS
PCB effect on D. tertiolecta size
distribution at varying salinity
Prior to becoming isosmotic with their medium, osmotically shocked punaliella cells undergo reversible volume shifts like osmometers (3). To investigate the effects of 1000 pg/1 of PCB on the size distributions of exponentially growing vs. senescent D. tertiolecta cultures exposed to salinities from 25 to 2.5 0/00, the following technique was used.
Measured aliquots of distilled water vacuum filtered through 0.22-p Millipore filters were added to cultures at 5minute intervals. Approximately 50-ml aliquots of PCB-treated or control cultures were withdrawn. These samples were counted and sized by an Electrozone Celloscope (Particle Data Corp., Elmhurst, I11.) bearing a 190-pm orifice and equipped with a PDP8/M computer functioning as a multichannel pulse analyzer (7).
The equipment was calibrated using standard 19.90-pm latex microspheres suspended in particle-free medium vacuum filtered through 0.45-p Millipore filters. To ensure that shifts in recorded cell size distributions over varying salinities were not artifacts of machine function at varying salinity, the latex microspheres were measured at each salinity
used in the experiments.
Effects of altered salinity on
PCB toxicity to p. tertiolecta
and T. pseudonana
Studies of growth and PCB susceptibility of p. tertiolecta and T. pseudonana were performed at varying salinity. Both hypersaline and hyposaline media were employed. Salinity was regulated without affecting nutrient enrichment or pH.
Hyposaline media were prepared by adjusting the proportion of EIO and double-distilled water enriched and buffered as ELO to the desired salinity below 25 0/00. Hypersaline media were prepared by dissolving the appropriate amount of Instant Ocean salts in double-distilled water to the highest desired salinity, adding f/2 nutrient enrichment, and adjusting pH to 7.80. Hypersaline media of less than the highest salinity were prepared by dilution with EIO or enriched double-distilled water, as described.
Small volumes of stock cell suspensions were inoculated
into the test media to yield the desired initial cell density. The initial inocula were too small to significantly alter the desired salinity treatment.
No attempt was made to acclimatize cells to extreme salinities by placing them first into media of intermediate
salinities. Inocula were subdivided into two aliquots, one to
receive PCB, the other methanol, as previously described.
PCB effect on photosynthetic
14¢c-fixation at varied salinity
T. pseudonana and Dp. tertiolecta were batch cultured as described. Replicate cultures in prescription bottles containing 150 ml of medium were inoculated with 10" exponentially growing cells per ml. The media were of low, normal or high salinity, as desired (See Results.).
Into two replicates, 15 ul of PCB stock in methanol were inoculated by microsyringe to yield 50 or 1000 pg/1 of PCB, for T. pseudonana or D. tertiolecta, respectively. Replicated controls received methanol. Cultures were shaken, then incubated in an environmental chamber at 24°C under continuous fluorescent illumination of 2.7 X 10° lux.
At intervals, about 2 ml of cell suspension was removed, stained with Lugol's iodine, and counted microscopically in a Fuchs Rosenthal counting chamber. At each counting time, triplicate 6-ml samples were withdrawn and pipetted into vials. One tenth ml of 14c-bicarbonate solution of specific activity 1 pCi/ml was added to each vial, comprising two illuminated samples and one dark sample. After incubation for two hours, samples were filtered under a gentle vacuum onto 0.45-p Millipore
filters, washed with medium, placed in scintillation vials with
10 ml of Aquasol (New England Nuclear Corp., Boston, Mass.),
and counted.
Resistance assay. Recovered cultures were re-exposed to PCB at the end of the experiment to determine whether any lag in growth induced by PCB was a "true lag" (87) or an "apparent lag" in which there is an unaffected, resistant subpopulation dividing at the same rate as controls throughout the experiment. If, as is likely, the resistant subpopulation is initially a small fractionof the total population, cell division would be undetected for what would appear to be a lag period in which the entire culture is not dividing. However on retreatment with the test chemical, the resistant subpopulation, if there was one,
would quickly dominate the culture, and the lag would not recur.
Viability determination. The minimum percent of viable,
i.e., dividing cells required to produce any increment in PCBtreated cultures, was calculated from the growth rate. The growth rate was taken to be the ph), to make the calculation of minimum percent viability conservative, that is, the lowest per-
cent permitted by the data.
Microscopy. The general appearance of the cells was noted
to determine whether there were vacant or chlorotic cells which
might indicate that some dead cells, i.e., debris was being
counted with the living populations whose Me eixation was
to be measured. An attempt was made to count only cells which looked and stained like controls to assure that viable cells
were being counted.
PCB toxicity to 7. pseudonana
adapted to an altered salinity
regimen
Subcultures of 7. pseudonana 3H were adapted to low salinity by repeated transfers through medium of salinity 2.5 0/00 for over two months. Following the adaptation period, the toxicity of 50 pg/1 of PCB was investigated as described previously. These toxicity investigations included normal and low-salinity adapted cells growing in media of low or
normal salinity. ION EXPERIMENTS
Osmotic vs. tonic effects on
PCB toxicity
Salinity variation encompasses changes in the osmotic pressure of the medium and in the concentration of electrolytes present in the Instant Ocean salts. To determine whether
variation in electrolyte availability results in variation in
the toxicity of 50 pg/1 of PCB to T. pseudonana 3H, electrolyte availability was varied independently of osmotic pressure.
T. pseudonana was grown as described in media of 4 0/00 or 25 0/00 salinity. A third treatment was included in which 0.62 M glycerol augmented the 4 0/00 medium such that the osmotic pressure of the glycerol-containing medium equalled that of the 25 0/00 salinity medium (0.74 M). Inasmuch as glycerol, in which PCB is insoluble (55), is a non-electrolyte, the electrolyte concentration of the glycerol-containing medium equalled that of the 4 0/00 salinity medium (0.12 M).
PCB was inoculated and cultures were incubated and periodically sampled as described. The toxicity of PCB in the three media was compared to see whether the glycerol-containing medium was more like the 4 0/00 salinity medium or the 25 0/00
medium.
Effects of Ca*t and xt
concentrations on PCB toxicity
The concentration of calcium and potassium in EIO is
2+ and ral to
0.007 M. To determine whether the ratios of Ca other ions in EIO can affect PCB toxicity, EIO was augmented fivefold to 0.035 M with the chloride of either ion. The
osmotic pressure and chloride content were altered very little,
since the ions each comprise only 0.9% of the total molarity of
EIO. PCB toxicity to p. tertiolecta and 1. pseudonana in the
iton-augmented media was determined as described.
+ Effects of Na concentration
on PCB toxicity
Sodium is a major ion of EIO, present at 0.313 M. Two methods were used to investigate its ability to affect PCB toxicity. Medium at 4 0/00 salinity was augmented as in the glycerol experiment. NaCl was added to achieve isosmosity with ELO. The toxicity of 50 or 1000 pg/1 of PCB to T. pseudonana or D. tertiolecta was assessed as previously described.
A more detailed investigation of Nae effects on PCB toxicity to T. pseudonana was also undertaken. A special medium was prepared containing 0.73 M NaCl enriched and buffered to pH 7.80 as EIO. This medium was combined in varying proportions
with EIO. Toxicity of 10 pg/1 of PCB in media of varying
percent NaCl-substitution was determined.
Effects of H’ concentration
on PCB toxicity
The effects of hydrogen ions on PCB toxicity to Dp. tertiolecta and T. pseudonana 3H were investigated. Two sets of seven sterile prescription bottles received 160 ml each of medium ad-
justed to pH 3.8, 4.8, 5.8, 6.8, 7.8, 8.8, or 9.8. These media
were inoculated with 10° exponentially growing p. tertiolecta or T. pseudonana cells per ml and shaken. Each culture was
divided into two 80-ml aliquots, one to receive PCB and one methanol. 7. pseudonana was treated with 25 pg/1 of PCB and
D. tertiolecta with 1000 pg/l. Triplicate 10-ml aliquots were withdrawn and placed in sterile disposable test tubes, incubated,
and sampled periodically. The remaining 50-ml cultures were
incubated and used to monitor pH at each sampling time.
Chapter 4
RESULTS
The data suggest that phytoplankton growing under
suboptimal conditions may be more vulnerable to
stress than when growing under optimal conditions. Fisher and Wurster, 1973 (36)
EXTRACELLULAR RELEASE PRODUCTS
Effects of medium filtrates on
PCB toxicity to T. pseudonana
The non-filtrate 7. pseudonana 3H controls exhibited no lag phase and a maximum growth rate (pax) of 2.6 divs./day (Fig. 8). A 25-ng/1 dose of PCB did not inhibit growth. All filtrates from p. tertiolecta or T. pseudonana cultures were inhibitory, whether they were sonicated or non-sonicated. The 25-pg/1 dose of PCB, which did not inhibit cultures lacking filtrate, further inhibited growth of all filtrate-treated cultures. Heating the filtrates to 56°C for 1/2 hour improved 7. pseudonana growth in both sonicated and non-sonicated filtrates of p. tertiolecta and T. pseudonana. This indicates that growth inhibition of the non-heated filtrates was not due to nutrient limitation, and that a growth-inhibitory product, inactivated by heat, was released into the filtrates. Since all filtrates increased
PCB toxicity, no detoxifier was released into the medium by
Fig. 8.
PCB (25 pg/1) effect on T. pseudonana grown in EIO
mixed with filtrates or sonicates of late-exponential cultures of 7. pseudonana or D. Tertiolecta. Numbers labeling curves refer to pre-heated (solid symbols, 56°C) or non-heated (open symbols, 24°C) medium. Controls were run only with non-heated medium. Dashed lines
are PCB-treated; solid lines are non-PCB-treated.
Points are means of three replicates.
rom THAL filtrates - THAL Sonicates
56 - - = 56/ / Eo / 24P © J “ a x“ 2 fea 3 as 2 2 <104 / 5 / 3 ‘e C7 oO N 3 10 ro st 1 2 rn 4 2 a x * 1 O° . 20 40 6 80 100 120 ° 20 40 60 80 100 120 Pad - j , Elapsed time (hours) 6 yo controls DUN filtrates OUN sonicates ~ (24°C )
Bn
Cell density (cells per mi)
2 1 2 v1 1 1 1 1 4 4 1 n 1 n 1 ra 1 4 60 60 100 9 20 40 60 80 100 120 140 0 20 40 60 80 109 120 Elapsed time (hours)
T. pseudonana or Dp. tertiolecta.
Effects of medium homogenates
on PCB toxicity to 7. pseudonana
D. tertiolecta homogenates produced by tissue grinding inhibited growth of 7. pseudonana 3H (Fig. 9). This was supported by a two-way analysis of variance of logarithmically transformed — cell count ratios (P * 0.005). The two-way analysis of variance revealed no significant interaction between PCB toxicity and
the presence of the homogenate. ATPase INHIBITOR EXPERIMENTS
Quabain effect on PCB toxicity
Statistically insignificant growth inhibition was produced by 1073-4 ouabain in T. pseudonana 3H and in Dp. tertiolecta (Figs. 10 and 11). Two-way analysis of variance of PCB or ouabain presence/absence was performed using logarithmically transformed cell counts. A dose of 1000 pg/1 of PCB stimulated growth of D. tertiolecta, whereas a dose of 25 pig/1 inhibited growth of T. pseudonana (P © 0.05). The interaction term for dependence of PCB toxicity on presence of ouabain was insignificant for
both species.
Fig. 9.
Toxicity of 33 pg/l of PCB to T. pseudonana with
D. tertiolecta homogenate (solid symbols) or without D. tertiolecta homogenate (open symbols). Dashed lines are PCB-treated; solid lines are controls.
Points are means of three replicates.
ce) %o
™ (lw dad $1192)
yisusp leap
60 €0 100 120
40 Elapsed
time (hours)
Fig. 10.
Effects of the ATPase inhibitors ouabain (1073-m in culture medium) and DCHCD aoe and 1074 in culture medium) with 1000-pg/1 of PCB (dashed lines) or without PCB (solid lines) on growth of p. tertio-
lecta. Points are means of three replicates.
Density (cells per mt)
Cell
Cell Density (cells per ml)
control
10° M
ouabain
ek a" 1S 30 45 60 75 90 o 1% 30 45 60 75 380 Elapsed Time (hours) . 104M = DCHCD 163M ocHCD ~ n ra n 2 . rn 4 Nowe sagen nzo ° 15 30.0 «(45 60 75 90 O 15 45 60 75 990 Elap sed Time (hours)
Fig. ll.
Effects of the ATPase inhibitors ouabain ao? M in culture medium) and DCHCD ao" M in culture medium) with 50 pg/1 of PCB (dashed lines) or without PCB (solid lines) on growth of T. pseudonana.
Points are means of three replicates.
10° M ouabain
n 1 1
—
164m DCHCD
°
15 Elapsed
30 45 60 time (hours)
N, N'-dicyclohexylcarbodiimide
(DCHCD) effect on PCB toxicity
D. tertiolecta growth was completely inhibited by 10. M DCHCD (e £ 0.001) but was completely unaffected by 1074 M DCHCD. ff. pseudonana growth was completely inhibited by 1074 M DCHCD (P * 0.001, Figs. 10 and 11).
PCB toxicity to T. pseudonana was significant (P <= 0.05) but independent of DCHCD presence. In contrast, PCB toxicity to D. tertiolecta depended upon whether DCHCD was present (interaction term significant at P * 0.05). There was a more rapid population decrease in the presence of 10 M DCHCD
combined with PCB than with DCHCD alone. There was stimulation
of p. tertiolecta growth without DCHCD present.
Effects of ouabain and DCHCD
combined on PCB toxicity
Combination of 107" M DCHCD and 10> M ouabain completely -3 suppressed T. pseudonana growth, as did 10 M DCHCD combined
with 10 M ouabain in D. tertiolecta.
NUTRIENT LIMITATION EXPERIMENTS
Single nutrient deletions
Fig. 12 depicts the growth and PCB susceptbility of D. tertiolecta cultures when one of the 14 enrichment nutrients was deleted. Cultures exhibited no lag, and grew exponentially at approximately the same u,,,+ Carrying capacity was reduced tenfold in the nitrogen-deleted cultures. None of the nutrient deletions significantly affected the resistance
of Dp. tertiolecta to the PCB.
Multiple nutrient deficiencies
Growth of p. tertiolecta exponential cells inoculated into nutrient limited £/10 and £/20 media was depressed relative to cells growing in EIO medium of f/2 enrichment (Fig. 13). Susceptibility to 1000 pg/1 of PCB was unaltered.
In contrast, toxicity to 7. pseudonana 3H depended upon nutrient level. Compared with growth in f/2 medium, growth
in £/40 medium was inhibited and toxicity of 50 pg/1 of PCB was
increased, as indicated by growth curves (Fig. 14).
Competition
D. tertiolecta dialysis bag cultures suspended in medium
of £/6 nutrient enrichment grew at about 1.5 divs./day, as if there
Fig. 12.
Effect of 640 pg/1 of PCB on growth of exponential D. tertiolecta cultures in single-nutrient-deleted media. Solid lines are non-PCB-treated, dashed lines
PCB-treated. Points are means of two replicates.
(CELLS PER ML)
CELL DENSITY
SILICON IRON .
NITROGEN "PHOSPHORUS
al gn n $$$
|. COPPER
0 200 300 40 0 200 300 400 0 200 300 400 © 200 300 400
‘ ELAPSED TIME (HOURS)
Fig. 13.
Effect of 1000 pg/1 of PCB on exponential (open circles) and senescent (solid circles) p. tertiolecta cells grown at three nutrient enrichment levels:
£/2, £/10, and £/20. Dashed lines are PCB-treated;
solid lines are controls. Points are means of
three replicates.
mi)
Cell density (celis per
On
On
~ - 1. Yo neenteanse tl
20 40 60 80
Elapsed time (hours)
Fig. 14. Effect of nutrient concentration on T. pseudonana grown in medium with £/2 or £/40 enrichment. Points
are means + range of three replicates.
CELL DENSITY (CELLS PER ML)
O- 100
ELAPSED TIME (HOURS)
were no nutrient control of growth. Doses of 1000 pg/1 of PCB did not inhibit growth of cells (Fig. 15). Since f£/10 enrichment was strongly growth limiting, it is assumed that
nutrient limitation produced some competition in this experiment.
Senescent cells
Senescent p. tertiolecta cells were unable to grow in the nutrient deficient media (Fig. 13). Consequently PCB effect on growth was measurable only in the senescent cultures grown in media with full nutrient enrichment. In this medium the lag period in PCB-treated cultures was five hours longer than in control cultures, an insignificant loss of resistance
to PCB due to senescence (Fig. 13).
Estuarine vs. Sargasso Sea
isolates of T. pseudonana
Sargasso Sea isolate 13-1 of T. pseudonana grew more rapidly in £/40 than in £/2 medium, whereas estuarine isolate 3H grew more rapidly in f/2 than in f£/40 medium (Fig. 16). The growth curves of cultures treated with 50 pg/1 of PCB are depicted with respective controls in Fig. 17. In £/2 medium PCB was less toxic to 3H than to 13-1, which was incapable of
growth. In £/40 medium PCB was toxic to both isolates, although
Fig. 15.
Growth of Dp. tertiolecta in dialysis bag cultures (circles; points are means of three replicates) vs. prescription bottles (squares; unreplicated). Cultures treated with 1000 pe/1 of PCB (dashed lines) in dialysis bags compete with controls (solid lines) for nutrients in a common f/6.6 EIO medium. PCB-treated and control prescription bottle cultures are not in
direct competition.
CELLS PER ML
DIALYSIS PRESCRIPTION BAGS. BOTTLES
50 lOO O 50
ELAPSED TIME (HOURS)
ere)
Fig. 16. Effect of nutrient enrichment level on growth of T. pseudonana 3H and 13-1. Points-are means of
t range of three replicates.
CELLS PER ML
On
° HOURS ELAPSED
Fig. 17. Effect of 50 pg/1 of PCB on 7. pseudonana grown in £/2 or £/40 medium. Points are means of three repli-
cates.
J, 7, 10 E10 10
3H
19 : - 10 1344 3H
fe 1321
CELLS PER ML
10 25 50 75 100 125 150 1750 25 50 75 100 125 150 175
ELAPSEO TIME (HOURS)
the difference in susceptibility was not as pronounced.
SALINITY EXPERIMENTS
PCB effect on D. tertiolecta
size distribution at varying
salinity
Fig. 18 (bottom) illustrates the size distribution of untreated exponential p. tertiolecta cells at varying salinity. The heights of the curves measure cell density. They diminish with salinity and in proportion to the amount of admixed cell-free diluent used to adjust salinity. At 25 0/00 salinity (highest peak) the mean equivalent spherical cell diameter was approximately 6.3 ». There was a stepwise displacement of peaks to the right with decreasing salinity, up to a mean equivalent spherical cell diameter of approximately 8.5 p at 2.5 0/00 salinity (lowest peak). This represents a 35% increase in diameter
and a 146% increase in cell volume.
The effect of 1000 pg/L of PCB on the initial cell size distribution and on the displacements of the distribution with dilution are depicted in Fig. 18 (top). The initial and final mean equivalent spherical cell diameters were not significantly different from the values for the untreated cells. PCB
did not alter the ability of the D. tertiolecta cells to resist
Fig. 18.
Osmotic swelling of exponential p. tertiolecta cells placed in media of reduced salinity: control
(bottom), and treated with 1000 pg/1 of PCB (top)
COUNTS
COUNTS
(pee T
+
SRB 4
B28 4
THB
PCB
BRA 4
See 4
+
CONTROL
oALINi i Tf
SALINITY
21.5
|
SIZE IN MICRONS
osmotic swelling over the entire range of salinities permitting growth.
Fig. 19 (bottom) illustrates the size distribution of untreated senescent Dp. tertiolecta cells at varying salinity.
At 25 0/00 salinity (highest peak) the mean equivalent spherical cell diameter was approximately 8.0 p, that is, 27% greater in diameter and 105% greater in volume than untreated exponential cells. Upon dilution, osmotic swelling and a gradual loss of definition of the peak occurred. The mean equivalent spherical cell diameter increased to approximately 9.5 p at a salinity of 7.5 0/00. Below this salinity peaks were not identifiable, suggesting lysis of the cells.
Senescent cells treated with 100 pg/1l of PCB (Fig. 19, top) displayed an initial mean equivalent spherical cell diameter of approximately 8.0 » and a final value of slightly less than 9.5 p at a salinity of 7.5 0/00. As with untreated cells, peaks were not identifiable at salinities below 7.5 0/00, and as
with exponential cells, PCB-treated cells did not display a
significant loss of ability to resist osmotic swelling compared
with their respective untreated controls.
Fig. 19.
Osmotic swelling of senescent D. tertiolecta cells placed in media of reduced salinity: control (bottom),
and treated with 1000 pg/1 of PCB (top)
COUNTS
COUNTS
Baa -- 7BR 4 aR + suit 4 Han + 328 4 288 +
IAB 4
CONTROL
SIZE IN MICRONS
“SALINITY
Effects of varying salinity on
PCB toxicity to p. tertiolecta
and T. pseudonana
At normal salinity (25 0/00), p. tertiolecta grew with a Bmax of 1.64 t 0.42 divs./day (mean over eight experiments t 95% confidence limits). Treatment with 1000 pg/L of PCB did not reduce Bnax (1-69 + 0.38 divs./day), but the lag phase required to reach Hmax @ppeared to be shortened by PCB. In one experiment, 2000 pe/1 of PCB did not inhibit p. tertiolecta.
D. tertiolecta proved to be a euryhaline alga. Under hypo-osmotic conditions (salinities 5, 2.5 0/00) p. tertiolecta became sensitive to 1000 pg/l of PCB (Fig. 20A). Growth of controls is shown in Fig. 20B. A two-way analysis of variance revealed significant (P € 0.005) interaction between PCB and salinity effect on cell density.
Under hyper-osmotic conditions (salinities 40, 60, 80,
100 0/00) p. tertiolecta exhibited decreased Bax and prolonged lag phase. It was unable to grow at 100 0/00 salinity. Toxicity of 1000 ng/1 of PCB was not apparent at these salinities (Fig. 20C & D), and PCB stimulated growth at 80 0/00 salinity (Fig. 20D).
In normal and hypo-saline media (25, 5, 2.5 0/00 salinity)
D. tertiolecta senescent cells displayed the usual prolonged
lag phase (Fig. 7). Whereas results in Fig. 20A indicate hypo-
Fig. 20. Effect of salinity on p. tertiolecta resistance to 1000 ug/l of PCB. Points are means of two or three replicates. The depicted interaction of PCB resis-
tance with salinity was verified statistically (P * 0.05)
by two-way analysis of variance.
9)
CELLS PER ML
ne
8 ppt D
Sac", 2 t
40 60 80 100 ELAPSED TIME (HOURS)
PCB-TREATED
CONTROL
osmotic stress produced susceptibility to 1000 pg/1 of PCB in
D. tertiolecta exponential cells, the susceptibility occurred at a salinity of 2.5 0/00 in this experiment, rather than at 5 0/00. Senescent cells did not display enhanced susceptibility to PCB at 5 0/00 salinity, and were unable to grow at 2.5 0/00.
At normal salinity (25 0/00), T. pseudonana grew faster than D. tertiolecta, with Prax of 2.69 + 0.48 ( n = 6 experiments). Treatment with PCB (15 pg/l) markedly inhibited growth (Figs. 21A and 22). Inhibition by 50 pg/1 of PCB (Fig. 22) was characterized by a prolonged lag phase and a more variable Puax (3.00 t 1.14).
In cultures which eventually grew exponentially, lag phase and the prolongation of lag phase were variable (coefficient of variation, CV = 79.3 and 93.3, respectively). PCB-treated cultures exhibited a lag phase of 24.5 hrs. longer than controls (11.8 hrs.). This 208% increase is equivalent to 2.75 division cycles at growth rate Unax*
Under hypo-osmotic conditions, T. pseudonana growth was
inhibited but cells appeared viable down to salinity 2.1 0/00.
Under hyper-osmotic conditions, 7. pseudonana could grow at salinity
40 0/00 but not at 60 0/00 or higher (Fig. 22). A two -way
analysis of variance revealed a statistically significant (P * 0.05) interaction between salinity and PCB inhibition of growth. In contrast to Dp. tertiolecta (Fig. 20), PCB toxicity to 7. pseudonana was
diminished at low salinity.
Fig. 21.
T. pseudonana 3H cell number vs. 15 ypg/1 of PCB at 25 0/00 salinity (A) and 10 0/00 salinity (B). Open circles, solid lines are controls; closed circles, broken lines are PCB-treated cultures. Points are
means t of three replicates.
CELLS PER ML
log 50. 100. O 50. 100 ‘150 ELAPSED TIME (HOURS)
Fig. 22.
T. pseudonana 3H cell number vs. 50 pg/1 of PCB at four salinities: 5, 25, 40, 60 0/00. Solid lines are controls; broken lines are PCB-treated. Points are means of three replicates. Curves are drawn from a larger data base, on which a two-way analysis of variance revealed a significant (P © 0.05) inter-
action between salinity and PCB inhibition of
growth.
CELLS PER ML
ELAPSED TIME (HOURS)
PCB effects on photosynthetic
140 fixation at_ varied salinity
T. pseudonana
Fig. 23 depicts the effects of 50 pg/1 of PCB on 7. pseudonana growth (A) and 140 eixation per cell (B) at normal (25 0/00) salinity. PCB induced a 44—hr. lag in exponential growth, and reduced the maximum exponential growth rate by 61%, from 3.3 to 1.3 divs./day.
One hour after PCB addition, M4 fixation per cell was reduced by 32%. By 6 and 12-hrs., Me eixation per cell was, respectively, 78% and 87% lower than controls.
By sampling frequently it was determined that there was no decline in cell density of PCB-treated cells during this initial 12-hr. period. The cells appeared viable, meaning that they looked and stained like the controls. Vacant and chlorotic cells appeared in PCB-treated cultures beginning at 24 hrs. At 120 ‘hrs. the frequency of vacant and chlorotic cells decreased, and by 148 hrs. the cells were indistinguishable in appearance from controls.
Resistance assay. Recovered cultures were retreated with PCB as before to determine whether the 44-hr. lag in growth was
a "true lag" or an apparent lag." The lag recurred, indicating that treated cultures had not acquired resistance to PCB. In-
creases in cell density in PCB-treated cultures over successive
Fig.
23.
Growth (A) and 74
C-fixation per cell (B) of
T. pseudonana 3H at 25 0/00 salinity with and without 50 pg/1 of PCB. Open circles, solid lines are controls; closed circles, broken lines are PCB-treated. Points are means + range of two
(14¢-£ixation) or three replicates
replicates (growth). Points in B on curve labeled PCB
ADJUSTED (open triangles) are estimates of maximum
140 eaxation per viable PCB-treated cell that would result under the assumption that all cells counted
at each sampling time were dead except those necessary
to generate the population sizes observed at the next
sampling time (See text.).
CELL DENSITY
M4g_eIXATION PER CELL (CPM X 107)
(CELLS PER ML)
CONTROL a
ee ee a
20 40 60 80 100 120 140
‘ ELAPSED TIME (HRS)
B
CONTROL
PROGRESS OF CL Tore GROWTH (CELLS PER ML)
samples were produced by a homogeneous population of inhibited cells dividing at up to 1.3 divs./day. Corroborating evidence comes from the absence of a decline in cell density at the beginning of the experiment. Cell density decline, i.e., mortality, would support the hypothesis of an "apparent lag"
with a resistant subpopulation.
Viability calculations. The lower density limit of viable
PCB-treated cells was estimated conservatively as the lowest density of dividing cells required to generate the population density counted at the next sampling time, assuming growth at Prax 1.3 div./day during the entire interval between successive samples. "PCB adjusted" (Fig. 23B) gives the maximum 14 C_eixation per PCB-treated cell that would result from this assumption of minimum viability. While the observed inhibitions at sampling times of 1 , 6 , and 12 hrs. were 32 , 78 , and 87%,
respectively, the inhibitions were 32 , 78 , and 77% under the
assumption of minimum viability (Fig. 23B).
The same experiment. was performed, this time using Fisher's PCB dose of 10 pg/1. The purpose was to see whether Fisher's contradictory result of no PCB inhibition of photosynthetic Me eixation was due to a low PCB dose or to recovery of cells prior to Fisher's 48~hr. sample. The results indicate little, if any, PCB inhibition of growth (Fig. 24A) or Me
fixation per cell (Fig. 24B), verifying Fisher's report. The
Fig. 24.
Growth (A) and Mou fixation per cell(B) of 7. pseudonana 3H at 25 0/00 salinity with and without 10 pg/1 of
PCB. Open circles, solid lines are controls; closed circles, broken lines are PCB-treated. Points are means t range of two replicates (14c-£4xation)
or three replicates (growth).
CELL DENSITY (CELLS PER ML)
on
an fo) T
> ° T
a ° T
C-FIXATION PER CELL (COUNTS PERMLX 104)
40 60 ELAPSED TIME (HRS)
B
CONTAOL
5 10 : PROGRESS OF CULTURE GROWTH (CELLS PER ML)
48-hr. sample showed higher PCB than control Mo eixation.
The effect of 50 pg/1 of PCB on 7. pseudonana Mo_fixation was measured at three salinities: 6, 25, 37.5 0/00. The purpose was to determine whether increased PCB resistance at low salinity correlated with relief from PCB inhibition of photosynthesis, and whether increased toxicity at high salinity correlated with increased inhibition of photosynthesis. Fig. 25A depicts the growth curves. These indicate that, at 6 0/00 salinity, PCBtreated cultures exhibited some growth, whereas the cell density of control cultures declined rapidly because of the low salinity. Fig. 26 illustrates that the difference between treated and control low-salinity cultures was visually obvious.
Fig. 25 gives Me eixation per cell. In accordance with the effects of PCB on growth, PCB inhibited 4o_ taxation per cell
at 25 0/00 and 37.5 0/00 salinity, but not at 6 0/00 salinity.
D. tertiolecta
4 1 C-fixation per cell of p. tertiolecta was measured at
4, 25, and 80 0/00 salinity. Photosynthetic Mo_fixation per cell increased sharply within 9 hrs. of transfer of D. tertiolecta to medium of 4 0/00 salinity, and gradually declined to normal within 49 hrs. of PCB inoculation (Fig. 27). PCB did not inhibit growth or photosynthetic 140 fixation at salinities of 25 0/00
and 80 0/00. However, at 4 0/00 salinity the peak following the
Fig. 25,
Comparison of the effects of 50 pg/1 of PCB on
growth (A) and 40 eixation per cell (B) of 7. pseudonana 3H at three salinities: 6 , 25 , 37.5 0/00. Open circles, solid lines are controls; closed circles, broken lines are PCB-treated. Points are means + range of
two replicates (4¢-£ixation) or three replicates
(growth).
3 Cl 7
Ss S.
CELL DENSITY (CELLS PER ML) 3,
o Shs
R CELL é T
E ) ow [s} T
(CPM X 10% °o
4C-FIXATION P s
A Sf-— L 1 60 “oO 20 40 4
“
L__ff 4 n 60 fo) 20 40
ELAPSED TIME (HOURS)
fF 60
Fig. 26. Effect of 50 pg/1 of PCB on 7. pseudonana after three days of growth at low or high salinity
(6 or 37.5 0/00, respectively)
Fig. 27.
Effects of 1000 pg/1 of PCB on photosynthetic
146 ereation per cell of p. tertiolecta at
three salinities: 4, 25, and 80 0/00. Open
>
circles, solid lines are controls; closed
circles, broken lines are PCB-treated.
Points are means + range of two replicates.
60T
(,Ol X Wdod) 1130 Yad NOILY XI5-O,,
ELAPSED TIME (HOURS)
abrupt rise in Mo-fixation per cell was lower in PCB-treated
than control cultures. A two-way analysis of variance of 14¢_ fixation per cell with and without 1000 pg/1 of PCB at 4 vs. 25 0/00 salinity, revealed a significant interaction (P € .05) between PCB toxicity
and salinity, as well as significantly higher M46 fixation per
cell at 4 0/00 than at 25 0/00 salinity (P = .01).
PCB toxicity to 7. pseudonana
adapted to an altered salinity
regimen
T. pseudonana adapted to low salinity ("hypo"), treated with 50 pg/1 of PCB, and returned to medium of normal (25 0/00) salinity, displayed greater resistance to PCB than non-adapted cultures. This dependence of toxicity on prior salinity regimen was determined by the significant interaction term (P < 0.05) of a two-way analysis of variance of growth vs. prior salinity regimen. Most of the differential in PCB toxicity to "hypo"
vs. normal Tf. pseudonana cells disappeared in culture at 5 0/00
salinity (Fig. 28).
ION EXPERIMENTS
Osmotic vs. ionic effects on
PCB toxicity to 7. pseudonana
PCB was toxic to T. pseudonana grown in glycerol-augmented
medium of 4 0/00 salinity (isosmotic with EIO at 25 0/00 salinity,
Fig. 28.
The effect of T. pseudonana adaptation to low salinity on toxicity of 50 pe/l of PCB at two salinities: 25 0/00 and 5 0/00. Open symbols, solid
lines are controls; closed symbols, broken lines are PCB-treated. Circles represent non-adapted; squares adapted to low salinity ("hypo"). Points are means
of three replicates.
Cell density (cells per ml)
25 ppt Salinity
5 ppt salinity
100 120 fe) Etapsed time (hours)
go
but containing the same electrolyte concentration as 4 0/00 medium lacking glycerol). PCB was less toxic in the glycerol-augmented medium than in EIO at 25 0/00 salinity, and about equally as
toxic as in non-glycerol-augmented 4 0/00 medium (Fig. 29).
This indicates that reduced concentrations of electrolytes, with no change in osmotic pressure, were sufficient to increase
T. pseudonana resistance to 50 pg/1 of PCB in low salinity medium. A two-way analysis of variance verified that the interaction of PCB toxicity with salinity was significant at
the 1% level.
Effects of ca’t and K* concentrations
on PCB toxicity to 7. pseudonana
and Dp. tertiolecta
The toxicity of 50 pg/1 of PCB to T. pseudonana at 25 0/00 salinity was unaffected by a fivefold increase in ca concentration. In contrast to other experiments, T. pseudonana was unable to grow at 4 0/00 salinity. ca2* alleviated the low-salinity inhibition, but did not reverse PCB toxicity (Fig. 30).
D. tertiolecta resistance to 1000 pg/1 of PCB was unaffected by a fivefold increase in ca2* concentration. This was true at both 25 0/00 and 40 0/00 salinities (Fig. 30).
Toxicity of 50 pg/l of PCB to 7. pseudonana was unaffected
by a fivefold increase in x’ concentration at either salinity.
Fig. 29.
Effects of 50 pig/1 of PCB on growth of T. pseudonana in three media: 25 0/00 EIQ, 4 0/00 isosmotic glycerolaugmented medium, and 4 0/00 non-glycerol-augmented medium. Open circles, solid lines are controls;
closed circles, broken lines are PCB-treated. Points
are means + range of three replicates.
(CELLS PER ML)
CELL DENSITY
ELAPSED TIME (HOURS) >
Fig. 30.
Effects of fivefold augmentation of ca2t concentration (from 0.007 M to 0.035 Mon toxicity of 50 pg/1 of PCB to T. pseudonana 3H and 1000 pg/1 of PCB to
D. tertiolecta grown at two salinities: 25 0/00 and
4 0/00. Open symbols, solid lines are controls; closed symbols, broken lines are PCB-treated.
Circles are EIO, hexagons EIO with ca2* augmentation.
Points are means of three replicates.
(CELLS PER ML)
CELL DENSITY
THAL
100 ELAPSED
° 50 TIME (HOURS)
CALCIUM= AUG}“MENTED
Unlike ca2*, xt augmentation decreased 7. pseudonana ability to
withstand the stressfully low salinity (Fig. 31). a As with ca” > Kt augmentation did not alter toxicity of
1000 pg/1 of PCB to p. tertiolecta at either salinity (Fig. 31).
Effects of Nat on PCB toxicity
to 7. pseudonana_and
D. tertiolecta
Medium at 4 0/00, augmented with NaCl to isosmosity with EIO at 25 0/00 salinity, permitted undiminished growth and resistance to 1000 pg/1 of PCB by p. tertiolecta. In contrast T. pseudonana was unable to grow in the NaCl augmented medium (Fig. 32).
The results of a more detailed study of NaCl effect on PCB toxicity to 7. pseudonana are depicted in Fig. 33. The abscissa represents stepwise increments in percent of enriched NaCl medium, that is, declining percent EIO. Fig. 33A gives the observed Uax and Fig. 33B gives the cell densities reached by 45 hrs. as a percent of controls. Both representations indicate decreasing growth at increased NaCl concentrations, as
well as decreased PCB toxicity.
Fig. 31.
Effects of fivefold augmentation of Kt concentration (from 0.007 M to 0.035 M) on toxicity of 50 pg/1 of PCB to T. pseudonana 3H and 1000 pg/1 of PCB to
D. tertiolecta grown at two salinities: 25 0/00 and
4 0/00. Open symbols, solid lines are controls; closed symbols, broken lines are PCB-treated. Circles are ELO, triangles ELO with ra augmentation. Points
are means of three replicates.
CELLS PER ML
THAL
CONTROL
K~AUGMENTED
Kore) ELAPSED
ISO O - 50 100 TIME (HOURS)
CONTROL,
Fig. 32.
Effects of NaCl substitution for Instant Ocean (EIO) salts on toxicity of 50 pg/1 of PCB to 7. pseudonana 3H and 1000 pg/1 of PCB to p. tertiolecta. Open circles, solid lines are controls; closed circles, broken lines are PCB-treated. Points are means of
three replicates.
CELL DENSITY (LOG CELLS PER ML)
10 P DUN + 1600 ptm PCB
DUN
5 10
THAL
Ss.) le THAL+ 50 ptm PCB
s0--7
° 20 40 60 80 100 120 ELAPSED TIME (HOURS)
12:
Fig. 33. Effects of varied NaCl concentration on toxicity of 10 pg/1 of PCB to T. pseudonana 3H. Open circles, solid lines are controls; closed circles, broken lines are PCB-treated. Points are means = range of three
replicates.
GROWTH RATE, U
CELL DENSITY-PERCENT EIO CONTROL
° a
CONTROL
. PERCENT NaCl- “SUBSTITUTED MEDIUM
Effects of pH variation on
PCB toxicity to 7. pseudonana
and p. tertiolecta
T. pseudonana displayed a narrower pH tolerance range than p. tertiolecta (Fig. 34). The upper and lower pH's at which phe 1/2) BS ase? obtained graphically by interpolation, were 4 and 9.5 for Dp. tertiolecta and 5 and 9 for T. pseudonana, respectively.
The effects of 50 pg/1 of PCB on growth of T. pseudonana at one high and one low pH are depicted in Fig. 35. At pH 9.0 PCB toxicity was acute. At pH 4.5, which itself inhibited growth, PCB toxicity was eliminated.
In contrast, D. tertiolecta became sensitive to 1000 pg/1 of PCB at pH 3.9. At pH 9.8, itself inhibitory, PCB stimulated growth (Fig. 36).
Fig. 37 gives the ratios of cell densities over time of treated to control cultures of each species at varying pH. The
graphs reveal opposite trends of pH effect on PCB toxicity to
the species.
Fig. 34.
pH tolerance of p. tertiolecta and T. pseudonana.
Solid lines are growth rates, pp, as a percent of maximum, Pmax? Over all pH values in this experiment. Dashed lines are absolute values of growth rates. The u's
were computed from cell densities after 64.5 hrs. of
growth of non-replicated cultures at each pH.
RCENT Uyjay
PE
—2
t +t
” XV
oO fe]
Mm LNa0USd
aT fo} N
Fig. 35.
Growth of T. pseudonana 3H with and without 50 pg/1 of PCB at two pH's: 4.5 and 9.0. Open symbols, solid lines are controls; closed symbols, broken lines are PCB-treated. Circles are pH 4.5, triangles pH 9.0. Points are means + of three
replicates.
CELLS PER ML
' 50 100 ELAPSED TIME (HOURS)
Fig. 36.
Growth of p. tertiolecta with and without 1000 pg/1 of PCB at two pH's: 3.8 and 9.8. Open symbols, solid lines are controls; closed symbols, broken lines are PCB-treated. Circles are pH 3.8, triangles pH 9.8.
Points are means ¢ of three replicates.
4.
a
wo
2 WYad s199
TIME (HOURS)
Fig. 37.
Ratios of cell densities oyer time of PCB-treated
to control cultures of 7. pseudonana 3H and
D. tertiolecta at varying pH. Ordinates are PCB-treated cell density divided by the sum of PCB-treated and control cell densities, so that equal growth of treated and control cultures is represented by the ratio
0.5. Shaded areas below 0.5 depict extent of PCB inhibition of growth. Points are means of three
replicates.
50 100 ELAPSED Time (HOURS)
D tertiolecta
FH 78
PHOS
PH 33,
50 100 ELAPSEO TIME (HOURS)
Chapter 5 DISCUSSION
It is sometimes held that no real progress has been made until a biological mechanism is placed on a firm molecular basis . . . Such a view denies the existence of different levels of organization at which one can meaningfully investigate biological processes . . . Perhaps we should be less apologetic and remember that the study of genetics was (and is) effective at levels other than the level of DNA, and that unless we have the right phenomenology we do not know what we are trying to explain or where to look for the explanation. . Lewis Wolpert ("Pattern Formation in Biological Development", 112)
EXTRACELLULAR RELEASE PRODUCT EXPERIMENTS
Acting jointly, inhibitors of growth or other processes are likely to interact, interfering with or enhancing inhibition. Competition among phytoplankters is mediated by resource utilization and by release of extracellular products which inhibit growth of conspecifics, growth of other species, or both (98,111). Phytoplankton succession is, therefore, sensitive to the presence or absence of release products in the medium (49). Since p. tertiolecta and T. pseudonana filtrates inhibited 7. pseudonana growth, substances present in filtrates could also modify the susceptibilities of the phytoplankters
to PCB, particularly if one species released a detoxifier
peneficial to the other.
Both p. tertiolecta and T. pseudonana are capable of metabolizing small amounts of intracellular DDT to DDE (10,91). Presumably this transformation is accomplished by enzymes, which have been associated with chlorinated hydrocarbon resistance. For example, resistance of houseflies to DDT is conferred by the heritable enzyme DDT-dehydrochlorinase (70). Similarly, dehydrochlorination was responsible for conversion of DDT to DDE in the diatom Cylindrotheca closterium (59). This particular degradation reaction was not equated with detoxification, since DDE proved to be toxic to several algae (89).
Since the growth inhibitors in the filtrates were partially inactivated by heating to 56°C (Fig. 8), they were probably enzymes. That the filtrates of both species also enhanced PCB toxicity to T. pseudonana implies the absence of a PCB detoxifier in D. tertiolecta, since a detoxifier would decrease toxicity.
The ability to degrade DDT to DDE enzymatically is not inconsistent with sensitivity to DDT or PCB. In general, only a small fraction of the toxin is degraded (10). The degradation product DDE may be more toxic than the parent compound (89,91). As yet, no case of algal degradation of PCB has been reported. Other phytoplankters reported capable
of the transformation of DDT to DDE include some sensitive
species: Skeletonema costatum (10,91), Thalassiosira fluviatilis (10), Chlorella sp. (17), Isechrysis galbana, Olisthodiscus luteus, Amphidinium carteri, and Tetraselmis chuii (91).
Since the filtrates of D. tertiolecta.enhanced PCB toxicity to 7. pseudonana, it is plausible that in mixed culture 7. pseudonana would be inhibited by a lower PCB dose than in pure culture. Mosser et al. (81) reported alteration of species composition of mixed cultures of these phytoplankters by lower doses of PCB (1 pg/1) and DDT (10 pg/1) than would inhibit either species in pure cultures. This was attributed to competition for a limiting nutrient in the mixed cultures, even though the nutrient enrichment level of their medium was initially hundreds of times what is found in natural marine ecosystems. It is possible that p. tertiolecta released products which enhanced the toxicity of PCB to 7. pseudonana, and that competition for a limiting nutrient played no part.
Expanding on thefindings of altered species composition,
Fisher et al. (33) reported that PCB reduced diversity of phytoplankton continuously cultured in natural Vineyard Sound seawater. It was implied here and stated elsewhere (9,12,25,83, 86,113) that reduced diversity is a general effect of chlorinated hydrocarbon or other ecosystem stresses. This
need not be the case if the stress eliminates a dominant
organism, allowing scarce organisms to proliferate. These scarce organisms, of which there could be many, may be more severely limited by the presence of a more effective competitor than by the introduced stress. The reports of PCB and DDT alteration of species composition represent a simple case in point (33,81). While its superior competitive ability would normally allow 7. pseudonana to eliminate Dp. tertiolecta
or make it scarce, in either case reducing diversity, PCB or DDT stress permitted coexistence at nearly equal abundance,
increasing diversity. ATPase INHIBITION
The evidence, though indirect, indicated that the
2+_atpases. Inhibition of these
dominant ATPases were the Mg with DCHCD stopped growth, indicating the absence of significant compensatory ATPase activity. Investigating salt tolerance of two blue-green algae, Batterton and co-workers likewise reported Me**-aTPase activity far exceeding Nat ,x'-ATPase activity (2).
The results illustrated in Figs. 10 and 11 do not refute the hypothesis that p. tertiolecta's great PCB resistance is attributable to a quantitative difference in the occurrence of the vg" -atPases, the major of the two ATPase systems.
There are two forms of this hypothesis. One is that
the size advantage of p. tertiolecta over fT. pseudonana accounts
to/
for the added ATPases. The other ascribes the difference to a higher concentration of the ATPases per unit cell size.
For the purpose of considering a toxin inhibiting a membrane-bound process common to both species, surface area rather than volume is the appropriate unit of size. This can be a critical distinction, because surface area varies as the square, but volume as the cube, of linear dimensions. The size advantage of p. tertiolecta would be much less if size is measured as. surface area, rather than volume.
The fact that. D. tertiolecta tolerated 1074 M DCHCD, which totally inhibited T. pseudonana indicates that D. tertiolecta contains more ATPase per cell, or that in this species DCHCD does not reach sensitive sites of action as readily. Since D. tertiolecta is resistant to at least 20 times the PCB dose to which 7. pseudonana is sensitive, it follows that a 2000% differential in size would be the minimum permitting size alone to provide the required differential in
ATPases. The estimate of Cox (19) for T. pseudonana surface
2 area is 185 p , and calculations from the upper limit in size of a D. tertiolecta cell assumed to be an ellipsoid with major
and minor axes 7 and 11 p (13) would give a surface area of
215 p 2 only 16% more than 7. pseudonana.* An equivalence of
*Several size estimates were actually considered in addition to the cited published estimates. 7. pseudonana was idealized as a cylinder of radius r, height h, with surface
one p. tertiolecta cell with 2.94 T. pseudonana cells was given by Fisher (30). Although this was probably a volume estimate, it falls short of providing the required 2000% differential in size which could refute the hypothesis that size alone, and not a difference in ATPase per unit cell size, accounts for the difference in ATPase per cell inferred from the data.
The primary toxic effect of PCB does not appear to be ATPase inhibition. ATPase inhibitors and PCB acting together did not interact, but produced additive inhibition. This non-interaction of PCB susceptibility with ATPase inhibitors, indicated for both species by two-way analyses of variance, is what would be expected for inhibitors of growth whose toxic activities are spatially or temporally separated inside the cell. It is likely, through redundancy, that inhibitors with toxic activities not separated spatially or temporally would interfere with one another, evincing less than additive inhibition. ATPases also appear to be unimportant in the mode of action of
chlorinated hydrocarbons on animals (R. D. O'Brien, personal
communication).
area 2mrh and volume rr2h. Dp. tertiolecta was idealized either
2 lat? as a sphere of radius r, surface area 4rr“, and volume —~_,
or as an ellipsoid with major and minor axes a and b, eccentricity
2 ab 4 ab2
€, surface area 2 b2 + eS sin” and volume 3 (108).
In contrast, Batterton et al. (2) reported DDT inhibition of ATPases in anacystis nidulans, and that this was reversed by cat, The absence of ca2t reversal of PCB toxicity to fT. pseudonana is another reason for believing that ATPase inhibition is not the primary toxic effect of PCB. That ca2* and PCB interacted with salinity but not with each other constitutes evidence of the independence of PCB and ATPase inhibitors in 7. pseudonana.
The contrast between DDT effect on a. nidulans and PCB effect on T. pseudonana and Dp. tertiolecta ATPases may be attributable to differences between the effects of DDT and PCB, or to differences between ATPases among species or between prokaryotic and eukaryotic algae. In support of the first alternative, Mosser et al. (82) reported that DDT counteracted the toxicity of PCB to T. pseudonana, indicating that the two compounds should not be treated interchangeably.
Three observations leave little room for a reliance of
Dp. tertiolecta ona qualitatively different sort of ATPase,
insensitive to PCB, conferring upon this species its remarkable tolerance to PCB and other chlorinated hydrocarbons: 1) the similarity of response of both species to the ATPase inhibitors,
suggesting possession of similar ATPases, 2) the absence of a
non-DCHCD-inhibitable ATPase in either species, precluding
supplemental, possibly PCB-resistant ATPases, and 3) the
apparent ineffectiveness of PCB as an ATPase inhibitor in T. pseudonana; indicating equal PCB-resistance of the ATPases
of both species.
NUTRIENT LIMITATION
The nutritional state of an organism can influence its ability to withstand stress. For example, the toxicity of PCB to 7. pseudonana increased under nitrate limitation (35). In comparing susceptibilities of phytoplankters to PCB, it is therefore necessary to take into consideration the suitability of the nutrient medium to each organism.
Important differences may exist in the suitability of a medium for different populations of a particular species. Geographically distinct isolates of phytoplankton, stable over years of laboratory culture, have been termed physiological "races." These races differ with respect to nutrient halfsaturation constants, growth rates, and tolerance ranges and optima for temperature and salinity (16). Each race appears to be best adapted to the conditions of its habitat of origin. 7. pseudonana exemplifies a species with several races, including the estuarine isolate, 3H, and the Sargasso Sea isolate, 13-1 (42).
Fig. 16 illustrates that 1/20 nutrient enrichment was
stressful to T. pseudonana 3H, whereas:full nutrient enrichment
was stressful to 13-1. Consequently, in medium of 1/20 enrichment, PCB toxicity to 3H increased, approaching toxicity to 13-1 (Fig. 17). This finding should introduce caution in the interpretation of reports that estuarine isolates of T. pseudonana and two other species of marine phytoplankters were more resistant to novel chemical stress than their racially distinct counterparts isolated from the Sargasso Sea (32,34). Insofar as toxicity was determined in highly enriched EIO, possibly more suitable to the estuarine than the Sargasso Sea isolates, the results may have been elicited by the test conditions rather than being independent of them.
Fig. 17 illustrates that nutrient overabundance as well as underabundance can constitute a stressful nutrient regimen and modify PCB toxicity. A corollary is that withdrawing nutrients can increase or decrease PCB toxicity depending upon the suitability of the initial conditions. Applying this to p. tertiolecta reveals that its resistance to PCB is independent of nutrient regimen. Resistance was retained despite single-nutrient deletions (Fig. 12), general nutrient reductions (Fig. 13), and competition for
reduced nutrients (Fig. 15).
SALINITY EXPERIMENTS
Growth
Estuarine isolates of T. pseudonana and two other species of marine phytoplankters were more resistant to PCB and other novel chemical stresses than their racially distinct counterparts from the Sargasso Sea. Part of this effect may be explained by the differential suitability of the test conditions (See Nutrient Experiments.). The salinity experiments illustrate another possibility. This is that adaptation by T. pseudonana 3H to a wide range of salinities encountered in an estuarine habitat has preadapted this isolate, and perhaps the estuarine isolates of other phytoplankters, to a wide range of stresses by novel chemicals.
The evidence for this is that reduced salinity caused 3H to resist a dosage of PCB which inhibited it in a medium of normal salinity (Fig. 22). Adaptation of 3H to low salinity, followed by transfer and exposure to PCB in medium of normal salinity, resulted in retention of the acquired resistance (Fig. 28). Therefore, altered availability of PCB in a low salinity medium could not have increased apparent resistance.
The adaptation to low salinity must have preadapted individuals to PCB.
A subtly different explanation was offered by Fisher (32).
This stated that relative toopen-ocean phytoplankton, estuarine algae are "intrinsically tolerant of physical/chemical stress" because, according to the stability/time hypothesis of Slobodkin and Sanders (99), they are "physically" rather than "biologically accommodated" (Introduction and 32). The subtle difference is that to become "physically accommodated" an organism must adapt to a physically rigorous habitat. Such
a habitat was defined as one in which extreme or unpredictably fluctuating physical conditions prevail. I shall argue that the Sargasso Sea is a physically rigorous habitat, to which its phytoplankters are adapted.
In being nutrient impoverished, the Sargasso Sea is physically rigorous. The stability/time hypothesis asserts that physically accommodated organisms, such as 3H, are prevented from exploiting relatively benign habitats containing biologically accommodated biota only by their inferior ability
to compete for resources. If competitors could somehow be
removed, physically accommodated individuals could spread to physically milder regions otherwise occupied by biologically accommodated individuals.
The fact that growth of unialgal cultures of 7. pseudonana 3H was depressed in 1/20 nutrient strength medium suggests that this alga could not grow in the Sargasso Sea. The Sargasso
Sea has nutrient levels much lower than 1/20 the strength of
ELO. For example, nitrate concentrations at the surface are usually 0.5 pM or below (16), compared with 840 pM in EIO, i.e., a differential of 1/1680 rather than 1/20.
Physical accommodation is therefore not a feature exclusive to the estuarine isolate. Neither is biological accommodation a feature exclusive to the Sargasso Sea isolate, 13-1. Although 13-1 did not grow as fast as 3H in EIO, it was capable of rapid growth, more rapid than, for example,
D. tertiolecta, a stress-resistant, physically accommodated but geographically ubiquitous phytoplankter. According to the stability/time hypothesis, biologically accomodated organisms, such as 13-1, should not be able to invade habitats occupied by physically accommodated organisms, such as 3H. In contrast, 13-1 grew in EIO. Whether it could grow in an estuary without competitors is undetermined.
In order for T. pseudonana 13-1 to cope with low levels of nitrate and other nutrients in the Sargasso Sea, it would probably have to enhance its active transport efficiency. Fisher suggested that this means exposing its membrane-bound functional units, e.g., active transport associated ATPases, to the external environment. This, he argued, would render Sargasso Sea clones more susceptible to chemical stresses. In comparison, estuarine phytoplankton possess a "thick skin", or an outer
membrane which protects functional units such as active transport
sites.
There is another way of looking at the problem facing a Sargasso Sea phytoplankter. To take up a nutritive molecule, the molecule would have to collide with a functional active transport unit. In a medium of low nutrient concentration, the way to increase the frequency of encounter would not be to externalize functional units, but to increase their number. This salution to the problem of low nutrient concentration would have the opposite effect to that predicted by the "thick skin" hypothesis. That is, more PCB molecules would be required for inactivation of a given fraction of the functional units. Hence, if the toxicity of the PCB is tested in a high nutrient level medium, as it was by Fisher et al. (34), the cell would possess functional units to spare, relative to individuals accustomed to high nutrient levels. On this basis, a
Sargasso Sea phytoplankter should be less sensitive to PCB than its estuarine counterpart.
A related problem arises from the physical accommodation
explanation of differential resistance in estuarine phytoplankters. It is that exposure to low salinity increased the sensitivity of p. tertiolecta to PCB, but decreased sensitivity of T. pseudonana 3H. Both of these are estuarine organisms.
Their opposite response to a specific selective agent of the
estuarine habitat, exposure to low salinity, illustrates the
importance of studying organisms in relation to their habitat.
The stability/time hypothesis makes predictions about the organisms from their habitat. While this seems plausible for some predictions, in thecase of the data presented here, the prediction is inaccurate.
Fisher's explanation of the induced sensitivity of D. tertiolecta was that osmotic distension and thinning of the cell membrane made p. tertiolecta more like a thin-skinned Sargasso Sea phytoplankter with exposed functional units. While osmotic shocks do not occur as suddenly in estuaries as in Fisher's experiments, thé induction of sensitivity falls short of illustrating how estuarine phytoplankters might be "intrinsically tolerant of physical/chemical stress"
(Also see Ion Experiments.).
Photosynthesis
In reassessing the findings that DDT and PCB reduced photosynthesis of three species of phytoplankters, Fisher (31)
reported that while photosynthesis per culture was inhibited,
the inhibition was produced by a reduction in the number of viable cells per treated culture. Interference by the chlorinated hydrocarbons with photosynthesis was not observed at the single time Fisher's samples were taken, 48 hrs. after chlorinated hycrocarbon inoculation. Since, in the present investigation, growth rate recovery of PCB-treated 7. pseudonana cells occurred
within 48 hrs., there was reason to re-examine whether PCB
would inhibit photosynthesis per cell prior to growth rate recovery, it.e., during the period of maximum PCB toxicity.
Failure to rule out several alternatives has prevented documentation of inhibition of photosynthesis by environmentally realistic amounts of PCB. These alternatives include: 1) transitory inhibition by PCB, followed by recovery,’ the inhibition being unobserved due to infrequent sampling; 2) lowering of photosynthesis in PCB-treated cultures due to reduced numbers of cells;
3) accidental inclusion of PCB-killed cells in counts of viable
cells, spuriously lowering the quotient: photosynthesis _ PCB-treated cell
by inflating the divisor; and 4) cell size reduction by PCB,
spuriously indicating inhibited photosynthesis. As indicated
in Results, three types of evidence justify interpreting the
data as PCB inhibition of photosynthesis per cell: 1) light
microscopy, 2) resistance assy, and 3) viability determinations. The alternatives to interpreting the data as reduced Mo
fixation per cell were refuted in turn. Frequent sampling
prior to growth rate recovery eliminated alternative 1). Cell
counts simultaneous with 40 §ixation measurements eliminated
alternative 2). Conservative estimates of minimum viability, hence
of maximum ~‘C-fixation per PCB-treated cell, eliminated
alternative 3). The light microscopic observations verified that control and PCB-treated cells were of about the same size, eliminating alternative 4). Differences in size sufficient to
account for inhibition of photosynthesis per cell of up to
87% would have been noticeable.
An independent estimate of viability of cells treated with 50 ug/l of PCB was reported by Mosser et al. (81). Agarplated cells showed only a slight increase in viability over three days, even though cell number in PCB-treated liquid cultures increased more rapidly. Using Mosser's data indicating about 80% viability, the inhibitions per cell at sampling times of 1,6, and 12 hrs. would be, respectively, 14 , 76 , and 77%, in reasonable agreement with the present investigation.
The PCB dose of 50 pg/1, compared with Fisher's dose of 10 ps/1, is within the range found in marine ecosystems (55). This 50-yg/1 dose must be considered low because the laboratory phytoplankton were not exposed to the substantial amounts of transferable, sediment-bound PCB available to cells in natural aquatic ecosystems (44). Because of its low solubility in water and high lipid affinity, only minor proportions of available PCB are found in analyses of water.
At varying salinity, PCB inhibition of photosynthesis
paralleled inhibition of growth in both species. The sharp increase in photosynthesis per cell displayed by Dp. tertiolecta, following exposure to low or high salinity, indicates that at normal salinity growth of Dp. tertiolecta is not rate-limited by the production of photosynthate. As a naked unicell reported
to excrete glycerol at low salinity, and to produce glycerol at
high salinity, steeply rising photosynthetic peaks in D. tertiolecta represent osmotic work. In other words, osmotic work is performed to become isotonic with the medium at the expense of stored, photosynthetically produced glycerol excreted or synthesized de novo (See Introduction.).
A capacity for excess photosynthesis, observed in D. tertiolecta, is not evident in T. pseudonana, and constitutes an important physiological distinction between the species which is related to their different structures. The absence of low-salinity induced growth inhibition of 7. pseudonana by PCB is due to this alga's mechanism of withstanding low salinity via its rigid cell wall. That the effect of PCB on T. pseudonana growth depends on salinity is attributable to some physiological process in this species other than activation of a physiological osmoregulatory mechanism at low salinity.
What this is is suggested by the ion experiments,
ION EXPERIMENTS
Salinity is a complex phenomenon, being an aggregate of a concentration of solutes per se and a specific composition of electrolytes of varying dissociation, size, net charge, and colligative properties, each with its own effect on cell physiology. In media of varying salinity, a phytoplankter is
separately affected, at times stressed, by the osmotic pressure
across the cell boundary and the specific ion composition. Either stress or both stresses could modify PCB susceptibility, possibly in different ways for different organisms.
There is disagreement about whether phytoplankton perform apparent osmoregulatory behavior to balance osmotic pressure across cell boundaries (e.g., 96) or for other purposes.
Such behaviors as polyol or proline regulation, which maintain isotonicity, are thought to serve a more important nonosmoregulatory function (96). These behaviors may be required
to maintain hydration of biopolymers under conditions of reduced water availability. Glycerol has been termed a "compatible solute" for D. tertiolecta, that is, a substance which prevents inactivation of enzymes by salts at high salinity (8).
Under reduced salinity achieving isotonicity would be more important for a naked cell like p. tertiolecta than for a walled cell like T. pseudonana. Exposed to a low enough salinity, D. tertiolecta but not T. pseudonana could swell and lyse in the absence of a physiological osmoregulatory behavior. Dp. tertiolecta's reported excretion of photosynthetically produced glycerol at low salinity represents such a behavior. The present investigation suggests that glycerol excretion was followed by a rapid increase in photo-
synthetic ~'C-fixation (Fig. 27), presumably because 7. pseudonana
lacks this physiological osmoregulatory behavior for dealing
with low-salinity stress.
The low-salinity-induced PCB inhibition of growth and photosynthesis in p. tertiolecta could reduce competitive ability upon exposure to widely fluctuating salinity in estuaries. At normal and high salinities, any PCB inhibition of photosynthesis in p. tertiolecta is not observed, apparently because it is compensated for by recruitment of photosynthetic apparatus not fully utilized at the submaximum photosynthetic rates occurring at these salinities. Therefore PCB inhibition of growth did not occur at normal and high salinities.
When 7. pseudonana was grown in medium of 2.5 0/00 salinity for several months, then transferred to medium of normal salinity and exposed to PCB, growth inhibition was reduced, as if the cells were exposed to the PCB at low salinity. This indicates that the resistance acquired to PCB by adaptation to low salinity was a feature of the cells, and not of the dynamics of PCB in a medium of low salinity. It also indicates that subcultures of a single population of phytoplankter can be induced to differ from other subcultures by exposure to a medium of altered salinity. Since estuaries differ in their salinity regimen from the Sargasso Sea, there appears to be no reason to invoke differences in habitat predictability to
explain intraspecific differences in PCB susceptibility between
T+. pseudonana populations derived from the two habitats.
The reduced PCB toxicity to T. pseudonana at low salinity was produced by altered concentrations of specific ions such as Nat, xt, Mg 2+, Ht, and C17, rather than by reduced osmotic pressure. This disputes the "thick skin" hypothesis, and agrees with with the finding of Batterton et al. (2) that induced susceptibility to DDT was due to ionic rather than
osmotic factors.
10.
LITERATURE CITED
Batterton, J. C., G. M. Boush, and F. Matsumura. Growth response of blue-green algae to aldrin, dieldrin, endrin and their metabolities. Bull. Environ. Contam. Toxicol. 6:589-94, 1971.
. » and + DDT: inhibition of sodium chloride tolerance by the blue-green alga Anacystis nidulans. Science 176:1141-3, 1972.
Ben-Amotz, A. Adaptation of the unicellular alga
Dunaliella parva to a saline environment. J. Phycol. 11: 50-54, 1975.
and M. Avron. Photosynthetic activities of .the
halophytic alga punaliella parva. Plant Physiol.
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Publication citation
Michaels, Robert Alan. Polychlorinated Biphenyl Toxicity to Two Phytoplankters as a Function of Environmental Conditions. Stony Brook, New York, USA; State University of New York at Stony Brook, Department of Ecology and Evolution, Marine Sciences Research Center (MSRC), Doctor of Philosophy (PhD) in Biology Degree, Prof. George C. Williams, Dissertation Committee Chairperson (Department of Ecology & Evolution); work performed in the laboratory of Dr. Charles F. Wurster, Marine Sciences Research Center, 182 pages including Front Matter, August 1979;