United States Environmental Research
Environmental Protection Laboratory
Agency South Ferry Road
Narragansett, Rl 028B2
Research and Development EPA-600/9-79 -035
Advances in Marine
Environmental Research
Proceedings
of a Symposium
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EPA-600/9-79-035
September 1979
pv hsz.
Advances in Marine
Environmental Research
Proceedings of a
Symposium
Francine Sakin Jacoff,
Editor
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Narragansett, Rhode Island 02882
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DISCLAIMER
This report has been reviewed by the Narragansett Environental
Research Laboratory, U.S. Environmental Protection Agency,
and approved for publication. Mention of trade names or
commercial products does not constitute endorsement or
recommendation for use.
ii
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DEDICATION
ADVANCES IN MARINE ENVIRONMENTAL RESEARCH
A decade ago, there were only a handful of scientists throughout the world
engaged in the field of science called ecology..With rising social consciousness
and an escalating series of local, national and global environmental problems,
there was an outcry for the application of scientific analysis to these problems.
The result of this was an evolution of a new field, called pollution research,
which had as its cornerstone the science of ecology.
This volume includes papers that will discuss many of the specific aspects of
marine ecology and marine pollution research. You will find authors who are
studying the transformation and movements of pollutants in chemical systems,
as well as those who are attempting to miniaturize and model ecosystems with
the microcosms. The papers contained herein are a benchmark of marine
pollution research.
We are dedicating the volume to one of the founders of modern ecology and
marine pollution research, Eugene P. Odum. Dr. Odum has dedicated his life to
understanding the holistic processes of ecosystems and man's interaction with
these complex biological, physical and chemical systems. His pioneering work
in wetlands and radioecology led to his synthesized works in
FUNDAMENTALS OF ECOLOGY. The more that we attempt to understand
and unravel the complexities of modern marine ecosystems, the more we
recognize that the basic principles espouse by Eugene Odom are true. Not
only are we realizing that we cannot uncouple the various components of
ecosystems, but that man himself is coupled into these complex systems.
Do not read the volume with the expectation of understanding all the
answers to major marine pollution problems today—but read it as a
state-of-the-art document outlining our advances in a rapidly changing and
evolving science. Throughout all the papers, attempt to follow Odum's
guidance, and to understand how the discussion of various parts of the problem
can be combined into holistic concepts that have eluded us in the past. Our
field of marine ecology has not evolved to that of a predictive science; we lack
basic hypotheses and understandings to make it so.
I hope that the reader will view these papers through Odum's
"macroscope," and in this way gain insights into the holistic view of our
oceans and coastal waters that will allow man to live in closer harmony with
the sea.
Eric D. Schneider, Director
Environmental Research Laboratory
Narragansett, R.I.
iii
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KEYNOTE ADDRESS
DEDICATION OF NEW WING OF
NARRAGANSETT EPA LABORATORY
JUNE 1977
Delivered by
Eugene P. Odum, Director
Institute of Ecology, University of Georgia
The theme of my address at today's dedication is that the time has come to
adopt a holistic approach to researching and managing our environmental
problems. This is not to say that we abandon the traditional reductionist way
of science which involves dividing up a complex problem into small com-
ponents that are then assigned to specialists for detailed study. Rather, we
perhaps need to follow the general procedure we use in microscopy, namely,
shifting back and forth between powers so as to examine the subject at
different levels of organization. To put it another way, we need to develop the
"macroscope" as a tool as well as the microscope. Most of all, we need to
promote integrated team research as well as reward the individual effort that is
the traditional, and too often the only, criterion for promotion in universities
and research institutions.
Reductionisxri in science has led to important discoveries in physics,
chemistry, molecular biology and genetics, but this approach comes up short in
ecology where the exciting problems, and also those of most concern to
society, lie at the ecosystem level rather than at the molecular level. The
Environmental Protection Agency was organized by society to fight cancer at
the ecosystem level, not at the cell or organism level. Theories, and tools, must
be organized accordingly, since procedures appropriate for one level of concern
may not be appropriate at all for another level of study.
Holism as a basic operational principle or paradigm rests on the theory of
hierarchal systems, a theory not yet fully understood nor accepted by many
scientists. Since there is both continuity and discontinuity in the evolution of
the universe, development may be viewed as continuous because it is
never-ending, but also discontinuous because it passes through a series of
different levels of organization with vertical as well as horizontal integration.
The keystone in the theory of hierarchal organization is the concept of
emergent properties. As components, or subsets, are combined to produce
larger functional wholes, new properties emerge that were not present or not
evident at the next level below. In speaking of these matters in general lectures,
I often use water as an example. Water has many unique properties not shared
iv
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by the components, hydrogen and oxygen. To cite a few, it is a liquid,
chemically inert, and has its maximum density at 4°C; in contrast to the two
gaseous components having none of these characteristics. It is obvious that the
holistic approach of studying water as water (as a whole molecular complex)
would reveal these important integrative, or "emergent", properties more easily
and quickly than the reductionist approach keying on the study of the
component parts. Thus, it would be very difficult, if not impossible, to deduce
the maximum-density-at-4°C property of water from knowledge of the
properties of hydrogen and oxygen as they occur in their separate states.
Thus, the forest is indeed more than a collection of trees, to quote an old
adage. As a specific example of emergent properties at the ecosystem level, I
might cite the work my brother and I did on a coral reef on a Pacific Atoll, as
was alluded to by Frank Lowman in his introduction. We measured the
metabolism of the intact reef by monitoring oxygen changes in the water
flowing over the reef. We also did a detailed trophic analysis as a means of
charting major energy flows, and were able to construct an energy budget for
the whole system. It became evident from these analyses that coral animals and
associated algae were much more closely linked metabolically than had
previously been supposed, and that the inflow of nutrients and animal food
from surrounding ocean water was inadequate to support the reef community
if corals and other biota were functioning in ordinary food chains. We
theorized that the observed very high rate of productivity for the reef as a
whole was an emergent property resulting from symbiotic linkages that
maintain efficient energy use and nutrient recycling between autotrophic and
heterotrophic components. I believe we can'say that subsequent work on
Pacific reefs has verified this hypothesis.
As an interesting aside, we suggest that these coral reef discoveries have at
least philosophical significance for urban-industrial man. The Pacific coral reef
as an oasis in a desert ocean can stand as an object lesson for man who must
now realize that mutalism between autotrophic and heterotrophic components,
and between producers and consumers in the societal realm, coupled with
efficient recycling of materials and use of energy, are the keys to maintaining
prosperity in a world of limited resources. Only by moving up in our thinking,
in our research, and in our management to the ecosystem level in the hierarchal
system can we accomplish this vital mission. During the industrial revolution
mankind essentially "uncoupled" himself from nature. Because the individual
in industrial societies no longer is directly dependent on the natural
environment for his day-to-day needs, he forgets how dependent we really are
on natural processes that produce food, recycle water, purify air, and so on.
Our food, for example, comes in on a long and complex chain of production,
processing, and transportation so we are not really aware of where it came
from or how much energy was expended, or how much pollution created, and
v
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so on. It is definitely time to recouple the house of man and the house of
nature and assess and manage them as one integrated ecosystem.
In recent months the writing of environmental impact statements, as
required by NEPA, has been criticized in the pages of Science and other
professional magazines as being superficial and exercises of bureaucratic
futility. As I see it, current impact assessment is not so much bad or inadequate
science as it is wrong-level applied science, a viewpoint that has not been
emphasized in recent discussions of the subject. In other words, if NEPA is to
survive the economic and political pressure of the future, assessment must
evolve as rapidly as possible from the present largely descriptive component
approach to a more holistic approach which combines the use of broad
ecosystem-level indices of structure and function with specific local or
population factors (i.e., "red flags") that are of special public concern (such as
fish or game, or an endangered species). Also, economic and ecologic
considerations must be integrated, not undertaken as separate studies without
common denominators. This can be done, and if I had time I could describe
two cases where we were successful in such a merger. (Write me and I'll send
reprints.)
Finally, the impact-assessor and the decision-maker should be part of the
same team, or at least sit around the same table to review all the alternatives. In
other words, a good assessment cannot be made piecemeal any more than one
can understand water or a coral reef by component study alone.
So much for general theory; now for some suggestions for EPA and
Directors of EPA laboratories. In pursuing its mission to reduce and control
pollution, EPA has so far concentrated efforts in two areas: (1) monitoring
technology, designed to determine the what, where, and how much of
undesirable inputs into our environment, and (2) control technology and
regulations designed to roll back the tide of effluents which threaten our health
and the quality of our life. These efforts, of course, are appropriate and need
to be continued without let-up, but they are essentially negative in approach
since they indicate to industry and to people in general what they must not do,
but not what they can do. I believe the time has come to add two positive
dimensions to the menu; namely, (1) waste-management systems that couple
in-house waste treatment with the assimilatory capacity of surrounding natural
ecosystems that serve as the ultimate tertiary treatment plants, and (2) a
merging of ecologic and economic assessments, along lines mentioned in my
earlier review of theory so as to demonstrate what we all believe to be true;
namely, that the economic return of clean environments is greater than the
short-term gains that may result from ignoring or postponing pollution
abatement.
vi
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As an example of the first of these suggested new research areas, I can cite a
project that we at the University of Georgia have undertaken under contract
with a large industrial company. In this case the company proposes to build a
chemical plant on a site that is adjacent to an extensive area of natural
wetlands, both swamp forests and marshes, which we believe have considerable
capacity to assimilate and recycle nutrients and bio-degradable wastes. By both
inventory and experimental procedures we are in the process of determining
just what this "tertiary" treatment capacity is with the understanding that the
company will then design their in-house treatment facilities so as to remove the
toxic substances and release into the wetland environment only that which can
be assimilated. Such a procedure I like to call "reciprocal design" in that both
the industrial engineer and the ecologist have the same objective; namely,
essentially zero pollution after effluents have passed through both the
man-made and the natural filters. In this case, the company owns the wetlands
which, when used in the manner described, become a highly valued part of a
total waste management system. I believe there would be much to be gained if
EPA laboratories entered into "reciprocal design" contracts with industry, and
thus become partners, rather than adversaries in the pursuit of common goals.
Merging econornics and ecology may prove difficult, but it does make
common sense since the two words have a common Greek root, "oikos"
meaning "household"; ecology literally is "the study of the household," and
economics "the management of the household." The trouble is that "nature's
house" is entirely external to "man's house" in current economic procedures,
so that the very valuable and necessary work of nature, such as the tertiary
treatment of wastes just discussed, is not included in economic cost-accounting
or in the workings of the market system. In discussing theory, I made a point
of the need to recouple the "houses" of man and nature, so we can follow up
by suggesting that the best practical way to do this is to find ways to
internalize into the economic system what are now considered to be the "free
goods and services" of nature.
I will close by mentioning several special marine research challenges, since
this laboratory focuses on coastal and marine environments. Microbial
components and transformations in marine and estuarine environments are the
least known, yet the most important aspects when it comes to systems
metabolism and the impact of man-made perturbations. Microbial activities in
the anaerobic layers of sediments and how these activities are coupled with
those in the aerobic layers and water columns provide especially difficult, but
challenging, problems. The role of the sea in global cycles of carbon, nitrogen
and sulfur need further study. For example, the sea has not proved to be as
efficient a "sink" for CO2 released into the atmosphere by fuel-burning and
deforestation, as had once been predicted. Finally, the impact of estuaries and
vii
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coastal wetlands on continental shelf waters needs close reexamination on local
and regional scales. We now have a pretty good understanding of upwelling
processes, but not of outwelling processes. On the basis of our early work at
Sapelo, we thought that the salt marsh estuaries exported large quantities of
detritus, but now we are not so sure if it's POC, DOC, or living biomass that
outwells, if indeed there is a net export at all. There is likely wide regional
variation in import and export flows of carbon and nutrients along our
coastline, and these need to be quantified if we are to anticipate the fate of
pollutants which in the future are going to be introduced offshore (offshore
drilling, etc.) as well as inshore.
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TABLE OF CONTENTS
TITLE PAGE
Preparation and Characterization of a Marine Reference Material 1
for Trace Element Determinations
The Release of Heavy Metals from Reducing Marine Sediments 9
The Use of Introduced Species (Mytilus edulis) as a Biological 26
Indicator of Trace Metal Contamination in an Estuary
Trace Metal Speciation and Toxicity in Phytoplankton Cultures 38
A Simple Elution Technique for the Analysis of Copper in 62
Neanthes arenaceodentata
Geochemistry of Fossil Fuel Hydrocarbons in Marine Sediments: 68
Selected Aspects
Identification of Environmental Genetic Toxicants with Cultured 79
Mammalian Cells
Development of a Bioassay for Oils Using Brown Algae 101
Effects of No. 2 Heating Oil on Filtration Rate of Blue Mussels, 112
Mytilus edulis Linne
Lobster Behavior and Chemoreception: Sublethal Effects of 122
Number 2 Fuel Oil
Influence of No. 2 Fuel Oil on Survival and Reproduction of 135
Four Marine Invertebrates
Extraction of Environmental Information Stored in Molluscan 157
Shells: Application to Ecological Problems
Laboratory Culture of Marine Fish Larvae and Their Role in 176
Marine Environmental Research
Laboratory Culture of the Grass Shrimp, Palaemonetes vulgaris 206
Evaluation of Various Diets on the Lipid and Protein Composition 214
of Early Life Stages of the Atlantic Silverside
ix
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TABLE OF CONTENTS (Continued)
TITLE
The Combined Effect of Temperature and Delayed Initial Feeding
on the Survival and Growth of Larval Striped Bass Morone
saxatilis (Walbaum)
The Evolution of the Bugsystem: Recent Progress in the Analysis
of Bio-Behavioral Data
The Effects of Temperature, Light, and Exposure to Sublethal
Levels of Copper on the Swimming Behavior of Barnacle Nauplii
Use of a Laboratory Predator-Prey Test as an Indicator of
Sublethal Pollutant Stress
Burrowing Activities and Sediment Impact of Nephtys incisa
Second Generation Pesticides and Crab Development
Some Suggestions for the Collection and Analysis of
Marine Environmental Data
Kaneohe Bay: Nutrient Mass Balance, Sewage Diversion, and
Ecosystem Responses
Replicability of MERL Mocrocosms: Initial Observations
Turbulent Mixing in Marine Microcosms — Some Relative Measures
and Ecological Consequences
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PREPARATION AND CHARACTERIZATION OF
A MARINE REFERENCE MATERIAL FOR
TRACE ELEMENT DETERMINATIONS
Peter F. Rogerson and Walter B. Galloway
Environmental Research Laboratory
U.S. Environmental Protection Agency
South Ferry Road
Narragansett, R.I. 02882
ABSTRACT
A reference material for marine molluscan trace element determinations has
been developed. It consists of 637 clams, Arctica islandica, that have been
homogenized together and subsequently divided into 476 samples. A
representative subsample of these has been analyzed for trace element
concentrations. Of the 14 elements measured, 10 had relative standard
deviations from the mean of 7% or less.
INTRODUCTION
The study of pollution in marine systems often involves the measurement of
trace element concentrations in organisms over extended periods of time (1, 2).
Development of valid time trends from such data requires a strict quality
control program at every stage of data collection, from field sampling through
final statistics, to ensure that data from any single point in time is comparable
to that collected at all other times. This paper describes some of the efforts
undertaken to provide control over the laboratory analysis of marine organisms
for trace element concentrations. Specifically, we describe the preparation and
characterization of an in-house reference material which can be used as a
benchmark sample for quality control, a known sample for methods
development, or an intercalibration sample.
EXPERIMENTAL
Marine molluscan samples are prepared for flame atomic absorption
spectroscopy as follows (3):
1. Thaw sample.
2. Using stainless steel instrument, shuck into a dry, labeled, tared
beaker. Determine wet weight.
1
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3. Cover beaker with watch glass and place in drying oven. Dry to
constant weight at 95°C (about 48 hours).
4. Cool and determine dry weight.
5. Add concentrated nitric acid in sufficient quantity to cover
organisms, adding acid in 20 ml increments. Cover beaker with
watch glass. (Use same amount of acid for all samples within a
group.)
6. Let sample cold digest until tissue is well broken down; i.e., 4-24
hours.
7. Heat gently, to about 40°C, being careful to avoid frothing over.
Continue until frothing stops.
8. Heat to 85°C while covered, and bring sample to near dryness. {Be
careful not to take to complete dryness at any time.)
9. Remove from hot plate and add 20 ml of concentrated nitric acid.
Repeat step 8.
10. Repeat step 9 until digestion is complete, which is indicated by pale
yellow color, clarification of the liquid, and no trace of lipids.
(Treat all samples within a group in identical fashion.)
11. Take to near dryness (about 5 ml remaining), cool, and add 20 ml
of 5% nitric acid, getting all soluble residue into solution.
12. Filter sample into 50 ml volumetric flask through Whatman #42
Filter Paper which has been prerinsed with 5% nitric acid.
13. Rinse beaker 2-3 times with 5% nitric acid and pour through filter.
14. Rinse funnel down and bring up to volume with 5% nitric acid.
15. Mix well, transfer to acid stripped 60 ml polyethylene bottle, and
hold for A.A. analysis.
NOTE: All glassware is detergent washed, soaked in 10% nitric acid, and
copiously rinsed with deionized water.
Each group of 15 samples is accompanied by a complete reagent methods
blank. Analysis is performed on a Perkin-Elmer model 603 Atomic Absorption
2
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spectrometer according to the manufacturer's instructions. Operation is
facilitated by the use of an autosampler (P.E. Auto 200) and an ASR-33
teletypewriter. To ensure against instrument drift, a calibration standard is
included with each 15 samples. To check for unknown matrix effects, a known
spike is added to an aliquot of one of the samples and an equal quantity of 5%
nitric acid. From this, a spike recovery is calculated for each group of 15. For
each group, a sample of the reference material described in this paper is
included.
Preparation of Reference Material
Clams (Arctica islandica) were collected by commercial dredge from Block
Island Sound, and frozen prior to use. At the time of preparation, they were
cleaned, thawed and shucked as if for analysis, except that extracellular fluid
was drained and discarded. A total of 637 clam meats was pureed and
homogenized in a stainless steel, 40 quart mixer, of the sort found in many
commercial kitchens (Hobart VCM-40). Samples of approximately 60 g wet
weight were removed, placed in 120 ml acid-stripped polyethylene bottles,
sequentially numbered, and frozen for future analysis. This procedure yielded
476 samples.
Characterization of Reference Material
From the 476 samples, a total of 65 were selected for investigation of the
homogeneity of the material. These consisted of every tenth sample, and two
blocks of 10 consecutive samples from each end of the sequence. These
samples were analyzed for 14 trace elements by the above procedure, and the
results examined for homogeneity.
RESULTS AND DISCUSSION
The concentrations of 14 metals in the reference material on a wet weight
basis are listed in Table 1-1. Only data on a wet weight basis will be discussed
because of some anomalous wet to dry weight ratios indicating some samples
were not uniformly dried. On the basis of several criteria, the 14 metals may be
divided into two groups, A and B. Group A consists of the 10 metals Cd, Cr,
Cu, Fe, Mg, Mn, Ni, Pb, V, and Zn, for which the relative standard deviations
are less than 7%, as seen in Table 1-2. Three of these metals are graphically
represented in Figure 1-1. Note the similarity of the graphs. This similarity may
be quantified for these metals by determining the 45 pair-wise correlation
coefficients. Almost all of the coefficients indicate positive correlation at the
95% confidence level, with many of them being much more highly significant.
3
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Table 1-1, Trace Element Concentrations in Reference Material, ug/g (Wet)
GROUP A
GROUPB
Sample
Number
Cd
Cr*
Cu
Fe
Mg
Mn '
Ni
Pb
V
Zn
At
Ca
Co
Ti
10
0.18
1.02
2.87
92.3
816
3.25
1.57
1.27
0.9
17.5
19.3
422
0.22
1.0
20
0.18
0.94
2.82
90.8
1040
3.19
1.45
1.29
0.9
17.7
16.7
541
0.19
0.9
30
0.18
1.00
2.90
86.4
857
3.25
1.48
1.30
0.9
J 7.6
16.4
499
0.27
0.9
40
0.17
0.98
2.85
92.3
776
3,19
1.49
1.31
0.9
17.5
18.5
419
0.16
1.0
50
0,18
0.96
2.85
91.1
760
3.19
1.55
1.32
0.9
17.7
18.2
387
0.21
1.0
60
0.18
0.98
2.85
90.3
830
3.28
1.47
1.28
0.9
17.8
15,9
437
0.27
0.9
61
0.17
1.00
2.84
88.3
855
3.14
1.51
1.26
0.9
17.3
17.9
453
0.23
0.7
62
0,18
1.01
2.83
88.2
870
3.18
1.51
1.26
0.9
17.6
19.0
462
0.12
1.1
63
0.17
1.00
2.84
87.0
757
3.07
1.51
1.25
0.9
17.0
16.9
389
0.15
0.9
64
0.18
0.98
2.84
87.6
888
3.16
1.54
1.32
0.9
17.6
16.7
459
0.15
0.6
65
0.18
1.06
2.87
95.4
776
3.25
1.53
1.33
0.9
16.8
27.5
396
0.25
0.9
66
0.17
0.99
2.81
91.3
790
3.15
1.54
1.30
0.9
17.4
18.3
411
0.23
1.0
67
0.17
0.94
2.75
88.1
801
3.02
1.43
1.25
0.8
17.2
16.6
461
0.19
0.6
68
0.18
1.02
2.82
91.5
840
3.21
1.48
1.30
0.9
17.9
18.9
563
0.21
0.9
69
0.18
1.05
2.82
85.9
767
3.06
1.44
1.26
0.9
17.4
15.6
489
0.11
0.8
70
0.17
1.12
2.82
95.6
792
3.10
1.54
1.25
0.9
17.3
17.2
479
0.19
1.1
80
0.18
1.02
2.83
95.4
742
3.22
1.49
1.26
0.9
17.8
20.0
369
0.24
1.1
90
0.17
1.05
2.87
92.6
775
3.20
1.47
1.28
0.9
17.9
20.1
368
0.23
1.2
100
0.17
1.02
2.79
92.2
731
3.17
1.46
1.26
0.8
17.5
18.6
403
0.23
1.0
110
0.18
1.03
2.79
90.4
765
3.10
1.51
1.24
0.9
17.5
20.0
369
0.20
0.6
120
0.17
1.00
2.75
85.5
765
3.05
1.50
1.24
0.8
17.6
19.1
400
0.21
0.7
130
LOST
140
0.16
1.04
2.86
90.0
813
3.14
1.49
1.24
0.8
17.4
18.8
432
0.23
1.0
150
0.17
1.02
2.68
88.7
865
3.14
1.42
1.13
0.8
17.3
19.8
589
0.22
0.8
160
0.18
1.05
2.82
91.1
803
3.26
1.52
1.27
0.9
17.4
21.7
409
0.18
0.6
170
0.18
1.01
2.84
94.6
839
3.t8
1.52
1.24
0.9
17.3
20.2
414
0.18
1.3
180
0.16
1.00
2.74
94.5
849
3.09
1.38
1.19
0.9
17.6
21.3
481
0.21
0.3
190
0.17
1.08
2.84
92.0
781
3.27
1.50
1.26
0.9
17.3
22.1
430
0.25
<0.3
200
0.17
1.03
2.86
90.0
808
3.11
1.49
1.25
0.9
17.6
20.2
439
0.19
1.1
210
0.18
1.08
2.84
96.4
894
3.16
1.49
1.30
1.0
17.5
21.3
464
0.17
1.4
220
0.18
1.05
2.85
87.5
824
3.12
1.47
1.29
0.9
17.2
20.4
412
0.20
0.9
230
0.18
1.02
2.76
93.2
680
3.01
1.51
1.20
0.9
17.6
20.1
337
0.11
0.4
240
0.18
1,01
2.81
87.4
781
3.18
1.46
1.30
0.8
17.2
18.0
389
0.15
1.0
250
LOST
¦"Corrected for blank
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Table 1-1. (Continued)
GROUP A
GROUP B
Sample
Number
Cd
CR*
Cu
Fe
Mg
Mrt
Ni
Pb
V
Zn
Al
Ca
Co
Ti
260
0.17
1.03
2.84
85.3
767
3.17
1.47
1.29
0.9
17.6
19.5
444
0.20
1,1
270
0.17
1.06
2.83
86.0
730
3.07
1.56
1.28
0.9
17.3
18.6
375
0.21
0.9
280
0.16
0.99
2,82
92.2
790
3.18
1.49
1.31
0.9
17.6
17.5
424
0.18
1.1
290
0.16
1.03
2.81
89.3
782
3.10
1.22
1.27
0.9
17.5
18.4
446
0.18
1.1
300
0.17
1.00
2.82
89.0
777
3.08
1.38
1.31
0.9
17.2
16.9
417
0.22
0.9
310
0.17
1.04
2.83
91.9
748
3.22
1.49
1.28
0.9
16.9
18,0
401
0.19
1.1
320
0.17
1.00
2.78
88.0
758
3.12
1.50
1.27
0.9
16.8
15.9
381
0.19
0.8
330
LOST
340
0.17
1.02
2.78
89.4
793
3.03
1.45
1.29
0.8
17.0
14.9
454
0,24
0.6
350
0.20
1.08
3.13
99.2
898
3.43
1.67
1.42
0.9
19.0
19.1
452
0.21
1.0
360
0.16
0.94
2.56
75.4
649
2.80
1.40
1.18
0.8
15.3
14.8
327
0.19
0.8
370
0.18
1.04
2.83
79.4
782
3.22
1.47
1.21
0.8
16.7
12.7
456
0.15
0.6
380
0.19
1.05
2.87
83.8
870
3.10
1.45
1.24
0.8
17.1
15.4
463
0.15
1.0
390
0.18
0.94
2.85
87.0
806
3.13
1.54
1.28
0.9
17.4
18.1
462
0.13
0.6
400
0.19
0.96
2.82
87.0
747
3.13
1.50
1.24
0.9
17.1
14.6
496
0.22
0.5
401
0.18
1.00
2.84
84.9
775
3.10
1.52
1.27
0.9
16.9
14.6
369
0.19
0.6
402
0.18
1.04
2.86
87.4
821
3.15
1.49
1.33
0.9
17.2
16.5
420
0.18
1.0
403
0.18
1.12
2.84
78.5
823
3.29
1.45
1.30
0.8
17.0
14.7
734
0.23
0.5
404
0.18
1.00
280
80.2
787
3.07
1.44
1.29
0.9
16.9
12,8
406
0.13
0.6
405
0.17
0.88
2,80
88.6
775
3.10
1.48
1.30
0.8
17.3
16.1
427
0.14
0.5
406
0.17
0.90
2.82
90.7
759
3.21
1.47
1.30
0.8
16.7
15.8
394
0.18
0.7
407
0.17
1.00
2.80
87.2
803
3.15
1.45
1.26
0.9
17.6
17.4
421
0.20
0.8
408
0.18
1.04
2.90
90.0
774
3.13
1.44
1.30
0.9
17.6
19.1
437
0.20
0.9
409
0.17
1.01
2.82
85.6
810
3.11
1.41
1.32
0.9
16.9
15.4
470
0.19
0,5
410
0.18
1.03
2.80
84.6
885
3.14
1.48
1.29
0.9
17.9
18.8
453
0.16
0.8
420
0.17
1.03
2.82
88.4
776
3.15
1.45
1.29
0.9
17.4
17.0
422
0.18
1.0
430
0.17
1.02
2.85
91.1
854
3.20
1.40
1.27
0.9
17.3
17.0
452
0.21
1.0
440
0.15
0.93
2.59
77.4
781
2.87
1.36
1.17
0.8
15.7
14.6
456
0.21
0.7
450
0.17
1.00
2.75
87.3
869
3.10
1.43
1.26
0.9
17.2
15.7
468
0.20
0.9
460
0.19
1.07
2.76
89.1
823
3.45
1.56
1.33
1.0
17.9
18.2
492
0.23
0.8
470
0.14
0.99
2.82
83.7
782
3.23
1.45
1.32
0.9
16.9
15.9
439
0.21
<0.4
*Corrected for blank
-------
3,10-
(+10%)
2.82-
(Mean)
2.54-
(-10%)
Cu pg/g, Wet
n »n i I n
11111111 h 111 h i n li n 111111111111
11111
3.47-1
(+10%)
3.15
(Mean)
2.84-
(-10%)
19.0
(+10%)
17.3
(Mean)
15.6
(-10%)
60 70
Mn jjg/g, Wet
200
300
400
410 470
410 470
200 300
400
410 470
REFERENCE SAMPLE NUMBER
Figure 1-1. Trace element distributions
in reference material.
6
-------
Table 1-2. Relative Standard Deviations
Group A
Metal
Mean, ug/g Wet
St. Dev.
Rel. Std. Dev.
Cd
0.17
0.01
6
Cr
1.01
0.05
5
Cu
2.82
0.07
3
Fe
88.7
4.5
5
Mg
802
58
7
Mn
3.15
0.10
3
Ni
1.48
0.06
4
Pb
1.27
0.04
3
V
0.9
(0.05)
6
Zn
17.3
0.5
3
Group B
Al
17.8
2.5
14
Ca
439
61
14
Co
0.19
.04
21
Ti
0.8
0.3
38
In contrast are the group B metals which consist of Al, Ca, Co, and Ti. As
shown in Table 1-2, these metals are characterized by a much higher relative
standard deviation, from 14% to 38%. When plotted in a fashion similar to
Figure 1-1, they show a much higher degree of scatter. Accordingly, the
correlation coefficients show no real correlations at the 95% confidence level.
Although this material has been prepared as a reference material, it is in no
way equivalent to a standard reference material such as those developed by the
National Bureau of Standards (NBS) (4). The NBS Standard Reference
Materials are certified as to their trace element content on the basis of analysis
by at least three independent techniques. Our material is an internal reference
material, not a certified standard.
This material is now in routine use within our laboratory. One sample,
randomly chosen from the sequence, is included with each group of 15
unknown samples prepared and analyzed. Thus, there is continuous verification
of analytical results irrespective of operator, instrument settings, or standards.
In addition, this material is ideal for methods development and intercalibration
because of its extremely low variability. Individual organisms from a wild
7
-------
population can have a range of metal concentrations up to an order of
magnitude, thus obscuring differences between techniques that may be small
but real. Comparison of clam homogenate values, however, will render obvious
differences of less than 25% which would be arduous or impossible to discern
using natural populations.
CONCLUSIONS
We have shown that a satisfactorily homogeneous reference material can be
prepared with a minimum of specialized equipment. The material has a low
relative standard deviation for the 10 metals Cd, Cr, Cu, Fe, Mg, Mn, Ni, Pb, V,
and Zn. For reasons that are not always clear, the other metals analyzed Al, Ca,
Co, and Ti have higher relative standard deviations. The material is useful as a
benchmark reference material so that analyses at different times can be shown
to be intercomparable. Thus, legal defensibility of time series studies of
pollution sites can be greatly enhanced. The material is also ideal for methods
development and intercalibrations because of its very low relative standard
deviation. Therefore, with cooperation from a cafeteria kitchen, it becomes a
relatively simple task to prepare such a reference material when a certified
standard from a recognized supplier such as NBS is not available.
ACKNOWLEDGEMENTS
We gratefully acknowledge the statistical consulting of Drs. J. Callahan and
J. Heltshe of the University of Rhode Island, Mrs. M. Boyce of the Roger Williams
Dining Hall, University of Rhode Island, and the assistance of Dr. R. Payne
and Messrs. B. Reynolds, D. D'Alessio, F. Storti, E. Truesdell, and C. Young of
this lab.
REFERENCES
1. Eisler, R. 1973. Annotated Bibliography on Biological Effects of Metals in
Aquatic Environments, 287 pp. Washington, D.C.: U.S. Government Printing
Office (U.S. Environmental Protection Agency, Rep. No. R3-73-007).
2. Eisler, R. and M. Wapner. 1975. Second Annotated Bibliography on
Biological Effects of Metals in Aquatic Environments, 400 pp. Springfield,
Virginia: National Technical Information Service. (U.S. Environmental
Protection Agency Rep. No. 600/3-75-008).
3. Pesch, G., B. Reynolds, and P. Rogerson. 1977. Trace Metals in Scallops
from Within and Around Two Ocean Disposal Sites. Mar. Pollut. Bull. 8 p.
224.
4. Anonymous. 1975. Catalog of NBS Standard Reference Materials. NBS
Special Publication 260. p. 32.
8
-------
THE RELEASE OF HEAVY METALS
FROM REDUCING MARINE SEDIMENTS
Michael L. Bender
Richard J. McCaffrey
J. Douglas Cullen
Graduate School of Oceanography
University of Rhode Island
Kingston, Rl 02881
ABSTRACT
We address the hypothesis that metals forming soluble sulfides are released
from nearshore anoxic sediments, while those forming insoluble sulfides are
retained. As a test, we have studied pore water chemistry, benthic fluxes, and
water column distributions of heavy metals in Narragansett Bay, Rhode Island.
The results show that metal forming soluble sulfides (Mn and Fe) have high
pore water concentrations and are released to the Bay waters, while metals
forming insoluble sulfides have low pore water concentrations and negligible
benthic fluxes.
INTRODUCTION
Several authors have suggested that heavy metal concentrations in reducing
marine waters are strongly influenced by sulfide solubility: metals forming
relatively soluble sulfides (such as Fe and Mn) will have relatively high
concentrations, and those forming relatively insoluble sulfides (such as Cd and
Cu) will have very low concentrations (5, 13, 4). A corollary of this suggestion
is that benthic fluxes (fluxes of dissolved chemicals from sediments to the
overlying water) out of anoxic sediments should be high for those metals
forming soluble sulfides, and negligible or even negative for those metals
forming insoluble sulfides. These hypotheses, if correct, have important
implications. They imply that estuarine and reducing nearshore and continental
shelf sediments are not sites where insoluble sulfide-forming metals are readily
remobilized: metals reaching these sites by inorganic scavenging or biological
removal are, most likely, permanently sequestered as long as sulfide is being
produced in the sediment porewaters. Secondly, the hypotheses indicate that,
to avoid release of metals forming insoluble sulfides, sewage sludge and dredge
spoils polluted with heavy metals should be dumped under sites of high organic
productivity, where continued deposition of organic matter maintains the
conditions which prevent their release to the overlying water. Of course, other
considerations (such as the presence of organic toxins) may show another
course of action to be more prudent.
9
-------
We have carried out a series of experiments aimed in large part at testing the
two hypotheses outlined above. We have proceeded by 1) determining the
concentrations of elements forming relatively soluble sulfides (Mn and Fe) and
elements forming relatively insoluble sulfides (Ni, Cu and Cd), along with
nutrients, pH, total CO2 (TCCy, SO^-, and S~ in anoxic Narragansett Bay
sediments; 2) carrying out tracer experiments to determine the rate at which
organisms enhance the benthic flux of chemicals by pumping water across the
sediment-water interface, and using these results, along with diffusive flux
estimates, to predict total fluxes across the sediment-water interface;
3) measuring benthic fluxes directly via bell jar experiments, and using the
results both to check the prediction of the pore water data and flux model, and
to make direct determinations of metal benthic fluxes; and 4) working out the
mass balance of heavy metals in Narragansett Bay at one point in time, using
the Bay itself as a gigantic bell jar and thereby checking on the generality of
our benthic flux measurements.
In this paper we summarize the results of this work.
PORE WATER WORK
Samples were collected in plexiglas box cores or PVC pipe. One centimeter
sediment slices were placed in polyethylene centrifuge bottles in a helium
atmosphere and centrifuged at in situ temperatures to separate pore waters.
Supernatant waters were filtered through acid-washed Nuclepore filters. The
concentration of TCO2 was determined by gas chromatography. Sulfate was
measured by BaSo^ coprecipitation using ^3ga as a tracer. Sulfide and
ammonia were determined colorimetrically by the methods of Cline (3) and
Solorzano (10), respectively. Phosphate and silicate were determined by a
modified autoanalyzer procedure (11, 12). Dissolved manganese and iron were
determined by flameless atomic absorption spectrometry using either a
Perkin-Elmer 503 or 360 AAS with model 2100 graphite furnace deuterium arc
background corrector. Standards were prepared in Sargasso Sea water. Further
details of sample collection and the above analytical methods are given by
McCaffrey (8).
Cd and Ni were determined by flameless atomic absorption after sample
concentration using a modification of the Co-APDC coprecipitation technique
of Boyle and Edmond (2). A 0.2 ml aliquot of a 4mM C0CI2 solution is added
to the pore water sample at pH 2, followed by addition of 0.4 ml of a 2% W/V
solution of APDC. The sample is shaken vigorously and allowed to sit for up to
30 minutes. It is then filtered through a 2.5 cm diameter, 0.4 p micron
Nuclepore filter held by an acid-washed MiHipore filter apparatus. The filter is
washed with several ml of deionized water, and placed in a polypropylene vial
containing 1.0 ml of redistilled 3N HNO3. The precipitate is dissolved by
10
-------
ultrasonication for 1 hour. The filter is then removed and the solution analyzed
by flameless atomic absorption.
The yields, as determined by addition for Ni and Cu and tracer experiments
for Cd, are 0.76+.06%, 0.75±.10, 0.70±.08 for Ni, Cu and Cd, respectively. The
precision is about ±10% at concentrations well above the detection limits of
0.1 ppb for Cd and 1.0 ppb for Cu and Ni.
Equipment and reagents are all carefully cleaned prior to Ni, Cu and Cd
analysis. Polypropylene vials are soaked in 6M HC1 for 18 hours, rinsed with
deionized water, and ultrasonicated twice for three hours each time in 6M HC1.
They are rinsed at least three times and dried. C0CI2 6H2O is purified using
Dowex-l-X8 anion exchange resin. It is put on in 9M HC1 and eluted in 4M
HC1. The APDC solution is purified by filtration through a 0.4 /zm micron
Nuclepore filter followed by five extractions with 20 ml MIBK (methyl
isobutyl ketone). Filters are acid washed, as all glassware used in the filtration
is continuously soaked in acid. Blanks are below the detection limits.
Typical summer pore water profiles are shown in Figures 2-1 and 2-2 for a
long core and four short cores from the Jamestown North study site in
Narragansett Bay (located about 0.5 km north of Jamestown Island in 5-10 m
of water). These profiles are discussed in detail by McCaffrey et al (8).
Concentrations of all constituents are far higher in the top centimeter than in
bottom water (TCO2 increases from 2.0 to 2.8 mM, NH3 from ~5 to 100 juM,
PO4 = from 1-25 nM, etc.). From 1-20 cm the profiles are flat (TCO2, NH3,
H4Si04) or show decreasing concentrations with depth (PO4 =, Mn++). The
lack of a systematic increase is ascribed to transport of metabolites out of the
sediments by the pumping activity of organisms, rather than by ionic or
molecular diffusion. The sharp concentration decreases observed for Mn++ and
PO4 = are ascribed to inorganic reaction in the sediment column. Below
approximately 25 cm, SO4 = concentrations decrease and concentrations of
other metabolites increase. Organisms are assumed to be absent, or at least
ineffective water transporters, below this depth. Metabolite concentrations
change sympathetically approximately as predicted by organic matter
decomposition: TCO2 increases twice as fast as SO4 = decreases NH3 increases
about 1/7 as rapidly as TCO2, and PO4 = increases about 1/150 as rapidly as
tco2.
An important point about the flat portion of the profiles is that the TCO2
value is considerably higher than can be accounted for by O2 reduction, and
implies anoxic diagenesis. The bottom water TCO2 and O2 concentrations are
2.0 and 0.15 mM, respectively. When all O2 is consumed, the TCO2
concentration will rise to 2.15 mM. NOj reduction could conceivably increase
the value to 2.2 mM. SC>4~ reduction must be postulated as the agent causing
the further increase to 2.8 mM.
11
-------
from a Jamestown North long core (JN-8) (from McCaffrey et al, 1977).
-------
0
2
4
6
8
10
12
14
16
18
20
2C02(mM) NH3(^.M) PO^tyiM) Si02 (/xM) Mn(ppb)
Note: Heavy lines show inferred concentration gradients at the sediment-water
interface (from McCaffrey etal, 1977).
-------
Nutrient and metal data for Jamestown North core 11 (a short core
collected on 7/13/76) and 12 (a long core collected on the same date) are given
in Tables 2-1 and 2-2. Metabolite concentrations vary in the manner discussed
earlier.
The metal concentrations are striking. Mn and Fe values are similar to values
reported elsewhere for anoxic sediments (11). Cu and Cd concentrations, on
the other hand, are very low, being comparable to or less than Bay bottom
values of about 2 ppb (Cu) and 0.2 ppb (Cd) at the Jamestown North location.
There is some scatter in the Cu and Cd data. This is in part due to
contamination. For example, the 58-60 cm sample in Jn-12 has Mn, Fe and Cd
values which are all higher than values in surrounding samples, apparently due
to contamination. On the other hand, there may be real variations, such as a Cu
maximum at 3-6 cm in JN11. Nevertheless, the main conclusion is clear: of the
four metals we analyzed, those forming relatively soluble sulfide (Mn and Fe)
gave pore water concentration far above ambient bottom waters, whereas those
forming highly insoluble sulfides (Cu and Cd) gave concentrations comparable
to or less than bottom water values. We would not expect such sediments to
release Cu and Cd to the bottom waters at significant rates.
MODELLING SEDIMENT-WATER EXCHANGE
To understand how fluxes of constituents between pore and overlying
waters at Jamestown North depend on pore water and bottom water
distributions, McCaffrey et al (8) constructed a simple model in which we
consider both exchange mechanisms — simple diffusion (transport along
gradients due to the thermal motion of ions and molecules) and advection
(transport in water which is moving as a result of the irrigation, feeding and
burrowing activities of the benthic fauna).
Conceptually, the diffusive flux is easy to calculate - it is simply the
product of a diffusion coefficient and a concentration gradient. Diffusion
coefficients in Narragansett Bay sediments at 25°C were taken as half the value
at 25°C in deionized water. Concentration gradients are not well known: the
gradients are very steep in the top 1 centimeter and zero below, and the pore
water concentrations do not allow us to accurately estimate the gradients at
the interface. We have assumed that the concentration near the interface is as
shown in Figure 2-2. Diffusive fluxes were calculated from the product of the
gradient and the diffusion coefficient.
Aller (1) and others have stressed the importance of organisms in
sediment-water exchange. In calculating advective fluxes, it is necessary to
know the rate at which organisms move water. The activity of organisms may
either be modelled as a random, or "biodiffusion" process, or as an ordered, or
"biopumping" process. Since organisms pump water into the sediment to
14
-------
Table 2-1. Nutrient and Metal Concentrations in Pore Waters from a Short Core
from the Jamestown North Study Site (JN-11)
Depth
tco2
so4"
s~
nh3
II
O
Q.
Mn
Fe
Cd
Cu
Ni
(cm)
(mM)
(mM)
(mM!
(mM)
(mM)
(ppb)
(ppb)
(ppb)
(ppb)
(ppb)
0
1
2.83
26.2
<0.003
75
13.0
2500
2000
<10
2.6
5.8
1/2-1
2.73
25.7
82
31
1580
5700
<.10
<1
3.4
1-2
2.89
<0.003
101
50
890
5700
<.10
1.6
3.1
2-3
2.98
25.8
104
61
700
5500
<10
1.8
1.6
3-4
2.75
25.2
<0.003
90
35
290
3200
.19
7.0
2.2
4-5
2.56
26.0
<0.003
88
35
230
3100
<10
3.2
1.8
5-6
2.58
24.7
<0.003
82
27
240
2400
<10
2.6
1.8
6-7
2.71
25.8
<0.003
71
22
240
1800
<10
2.2
2.0
7-8
2.94
25.1
<0.003
72
22
230
1440
<10
1.8
3.0
8-9
2.52
26.0
<0.003
68
18.8
195
800
<10
<1
1.6
9-10
2.47
25.4
<0.003
62
13.0
220
250
<10
<1
1.9
10-11
2.40
26.2
<0.0)3
58
15.0
170
<10
<1
2.3
11-12
2.75
26.3
<0.003
57
12.1
160
85
.16
1.4
4.1
12-13
2.35
26.5
<0.003
51
11.1
155
42
.16
<1
2.6
13-14
2.50
26.2
<0.003
45
10.1
148
44
<10
8.4
13.4
14-15
2.50
24.3
0.003
41
8.7
122
101
.10
<1
2.6
-------
Table 2-2. Nutrient and Metal Concentrations in Pore Waters from a Long Core
from the Jamestown North Study Site (JN-12)
Depth
tco2
so4-
S=
nh3
p
-------
supply oxygen and pump water out to expel waste products, we regarded the
biopumping model as more appropriate. In the model of McCaffrey at al (8), it
was assumed that organisms pumped water across the sediment-water interface
at a certain "biopumping rate" (with units of volume of water per unit surface
area per unit time). The biopumping rate was determined experimentally by
bringing box cores of Jamestown North sediment into the laboratory, spiking
the supernatant solution with ^^Na, and measuring the decrease in the
supernatant ^2|sja concentration with time. The experiments gave a
biopumping rate of 0.7±0.3 cm^ crrf^ day"*. The biopumping flux is then
taken as the product of the biopumping rate and the difference between pore
water and bottom water concentrations.
Model diffusive and advective fluxes for summer Jamestown North
sediments are given in Table 2-3. Surprisingly, both fluxes are of the same
magnitude.
RESULTS OF BENTHIC FLUX MEASUREMENTS
In order to test our predictions that nutrient and manganese fluxes have the
values calculated from the model outlined in the previous section, and to make
direct measuremertts of copper and nickel fluxes, we have measured benthic
fluxes in the field using the "bell jar" instruments developed and extensively
deployed by Hale (7) and Nixon et al (9). In these experiments, PVC pipe
halves with closed ends PVC flanges around the base are placed on the
sediment. At the start of the experiment, a sample is withdrawn from the
Table 2-3.
Advective flux
Measured
Diffusive
calculated from
fluxes:
Flux
biopumping model*
x ± 1o(n)
H4Si04
0.3
0.3
1.2±0.2(7)
nh3
0.19
0.07
0.27±0.08(15)
po4=
0.018
0.02
0.07±0.02(9)
sco2
0.9
0.6
2(1)
Mn++
0.02
0.01
0.049±0.018(14)
* Assuming a biopumping rate of 0.7 cm cm" day"
17
-------
chamber from a valve at one end; a second sample is withdrawn at the end of
the experiment. An ambient bottom water sample is also taken, and a dark
bottle is filled with water at the start of the experiment and sampled at the
end. The samples are analyzed for metals and nutrients after filtration. Benthic
fluxes are calculated from the change in the concentration of a constituent in a
chamber, the mean height of the chamber (chamber volume/enclosed sediment
surface area), and the length of the experiment. Dark bottle "fluxes" are
calculated in the same manner as benthic fluxes, taking the dark bottle
concentration at the end of the experiment as the final concentration, and the
ambient bottom water concentration as the initial concentration. The dark
bottle results are important for metals; because if they are equal to zero within
analytical uncertainties, they indicate that metal analyses are not seriously
affected by sample contamination during collection.
Metal concentrations were measured with the techniques used for pore
waters. The ambient water column metal concentrations at Jamestown North
are about 10 ppb (0.2 juM) for Mn and Fe, 3 ppb (0.06 juM) for Ni, 2 ppb (0.04
MM) for Cu, and 0.1 ppb (.001 mM) for Cd. Precisions in individual metal
analyses are ±10%; hence uncertainties in fluxes are 40 pM m"2 day"' for Mn
and Fe, 8 mM m"2 day"' for Cu and Ni, and 2 /iM m"2 day"' for Cd. Histograms
of dark bottle results at Jamestown (Figure 2-3) are roughly as predicted from
these errors.
Results of experimentally determined fluxes of Mn++ and nutrients at
Jamestown North are given in Table 2-3, along with model fluxes for these
chemicals presented earlier. The model fluxes agree quite well with the
measured values, indicating that the system is well characterized.
A histogram of Jamestown North metal fluxes is shown in Figure 2-4 , and
averages are tabulated in Table 2-4. Manganese fluxes are similar to the model
values. Pore water iron concentrations are similar to those of manganese, and
comparable fluxes are predicted. Observed iron fluxes are about an order of
magnitude lower than manganese (and predicted) fluxes; this is ascribed to
rapid oxidation of iron in the supernate following diffusion out of sediments.
Nickel, copper, and cadmium fluxes are predicted to be negligible (see
discussion of pore water values, above) and in fact measured fluxes are
generally equal to zero within the analytical uncertainty.
From the concentration of constituents in the Bay, the average height of the
water column (taken as 10 m) and the fluxes, we can calculate doubling times
of metals in the Bay with respect to benthic fluxes. These values are given in
Table 2-4. Upper limits on Cu, Ni and Cd doubling times were calculated taking
the flux as less than or equal to the sum of the mean flux and one standard
deviation. Doubling times are to be compared with residence times of water in
Narragansett Bay of about one month. The results show that, in the
18
-------
DARK BOTTLE RESULTS AT
JAMESTOWN (^M m"2 day-1)
Fe :
l*j I i i i i I i i i i«T»i i i i I i»i i i I
<-500 -500 -250 0 250 500
Mn
I I ' I ' I I I I '*1*1 I M I I I I I I lii
•1000 -500 0 500 1000 >1000
Ni
• •
I i i i i I i i»i*i T»i«i i i 1 i i i i I u
-50 - 25 0 25 50 >50
(_
)
Cu
•
•
i 11 i i i i
1
"l LI.I.I Mill
¦50 -25
25 50
Cd
•
• •
i i i i i«i i
1
•i*i i.i 1 l l l l 1
-2.0 -1.0
1.0 2.0
Figure 2-3. Histograms of dark bottle results at Jamestown North,
calculated as fluxes (see text). Units are m'2 day*^.
19
-------
TRACE METAL FLUXES AT
JAMESTOWN (yu.M m"2doy"')
N
Fe
ISJ
*1 I I I I I l*l»l l*J
<-500 -500 -250 0 250 500 >500
+ FLUX —
Mn
•
• •
• •
"a AAA
• WWW
• •••
i i i i i i i i i r
¦ 1*1 l.l.l.l I.I.I 1
1000 -500 0 500 1000 >1000
Ni :
•
•
•
• •
•
•
1 1 1 1*1 1*1
• 1 1 1*1 1 1 1 1 1 1
¦50 -25 0 25 50
(I )
C u
•
• •
\ i i i i \ i i.i.i*
t •
•i*i i ) i i i i i i
-50 -25
Cd
i i»i i i«i»i«i*i
o
(2)
25 50
i i I i i i i
-2.0 -1.0 0
1.0 2.0
Figure 2-4. Histograms of heavy metal fluxes at Jamestown North
(units are juM m"2 day'^).
20
-------
Table 2-4. Benthic Fluxes Measures at the Jamestown
North Study Site and Estimate Doubling Times
for Cu, Ni, Mn and Fe in Narragansett Bay
Mean flux and Concentration of Time for benthic flux
standard deviation dissolved metal in to double water column
(/igcm"2 day"^(1) Narragansett Bay (ppb) concentration (days)
Cd
Cu
Ni
Mn
Fe
-0.0029+0.0043
-0.00910.044
-0.035±0.064
2.1±0.8(2'
0.17+0.23
10
0.10
2.0
3.0
>71
>57
>100
5
NOTES:
(1) Based on twelve determinations.
(2) Calculated excluding one anomalously high value believed to reflect
contamination.
summertime, release of manganese from sediments is a major source of
manganese in Narragansett Bay, but release of nickel, copper and cadmium are
probably not significant.
TRACE METAL BUDGETS IN NARRAGANSETT BAY:
Mn AND Cu AS EXAMPLES
The preceeding discussion suggests that benthic fluxes will be a source of
Mn, but not Cu, to the waters of Narragansett Bay. The distribution of these
metals in the Bay provides a check on these conclusions, as will be seen from
the following discussion.
Graham et al (6) did a mass balance for dissolved and particulate manganese
in Narragansett Bay in the Summer of 1973. Their results for the distribution
of dissolved manganese in the main part of the Bay are shown in Figure 2-5. It
is readily seen that dissolved manganese is not a single-valued function of
salinity: at a given salinity, surface waters have far lower manganese
concentrations than deep waters. The manganese concentration of surface
samples is far less than that expected from a simple conservative mixing model
in which the deep water concentration is approximately equal to the
conservative concentration. Graham et al (6) interpreted these results as
indicating that manganese is scavenged (presumably by oxidation and
precipitation) throughout the waters of the estuary, and bottom waters are
enriched relative to surface waters by the benthic flux of manganese.
21
-------
30
-Q
Q.
Q.
26
- 24
UJ
to
<
o
20
o
UJ
>
12
O
cn
co 8
oOn
NISKIN BOTTLE, SURFACE O
(1 m depth )
NISKIN BOTTLE, BOTTOM ~
° fP Dg
ia
"24 26 28 30 32
SALINITY, %o
34
Figure 2-5. Dissolved Mn vs. salinity in Narragansett Bay,
in the Summer of 1973.
Dissolved copper and manganese (along with O2, nutrients and other metals)
were measured in the waters of Narragansett Bay in the Spring of 1977 by the
Narragansett Bay Study Group (in preparation). At this time of year,
manganese was found to be nearly conservative, showing no surface water
depletion and only a small deep water enrichment. The contrast between
manganese behavior in the spring and summer probably reflects slower
oxidative precipitation, slower benthic fluxes, and more rapid flushing of the
Bay under springtime conditions of lower temperatures and higher runoff.
22
-------
The dissolved copper distribution in Narragansett Bay in the springtime of
1977 is shown in Figure 2-6 (results of the Narragansett Bay Study Group).
Concentrations were determined using the methods outlined earlier for pore
waters. Several copper values fall far above a conservative mixing line, and are
believed to reflect contamination. Most values, however, appear to define a
simple conservative mixing line. These results show no evidence for an input of
copper into the Bay by diffusion out of sediments. The Bay water results will
be discussed in more detail elsewhere.
J8
NISKIN BOTTLE, SURFACE O
(1 m depth)
NISKIN BOTTLE, BOTTOM ~
PLASTIC BUCKET A
~ A
O
A
£D
u
~ s
~
~
24 26 28 30 32
SALINITY, %o
A
-A
34
Figure 2-6. Dissolved Cu vs. salinity in Narragansett Bay,
in the Spring of 1977 (Narragansett Bay Study Group, in prep.).
23
-------
SUMMARY
The results summarized here are consistent with the hypotheses of earlier
workers that metals forming highly insoluble sulfides will be sequestered in
anoxic marine sediments.
Our conclusions reflect results of a limited study on a small number of
metals during one or two seasons in a single estuary. The conclusions are thus
preliminary, and cannot be extrapolated to other seasons, metals or estuaries.
Organic complexing, in particular, may render certain heavy metals far more
soluble than would be expected from sulfide solubilities calculated, considering
inorganic ion pairing only.
ACKNOWLEDGMENTS
We are grateful to Scott Nixon, Candace Oviatt and colleagues, for their
generous cooperation in collecting samples, and for the loan of sampling
equipment. We also wish to express our appreciation to Nile Luedtke, who
participated extensively in the pore water and benthic flux determinations, and
scuba divers Paul Benoit, George Morrison, Allen Myers and Bob Pavia for their
skill and care in obtaining in situ benthic flux samples. This work was
supported by a grant from the Environmental Research Laboratory of the
Environmental Protection Agency.
REFERENCES
1.Aller, Robert C. 1977. The Influence of Macrobenthos on Chemical
Diagensis of Marine Sediments. Ph.D. Thesis, Yale University, 600 pp.
2. Boyle, Edward G. and John M. Edmond. 1975. Determination of Trace
Metals in Aqueous Solution by APDC Chelate Co precipitation. In:
Advances in Chemistry Series, No. 147, Analytical Methods in
Oceanography (Thomas R.P. Gibb, Jr., ed.) American Chemical Society,
pp. 44-55.
3. Cline, J.D. 1969. Spectrophotometric Determination of Hydrogen Sulfide
in Natural Waters. Limnology and Oceanography 74:454-458.
4. Elderfield, H. and A. Hepworth. 1975. Diagenesis, Metals and Pollution in
Estuaries. Marine Pollution Bulletin 6:85-87.
5. Goldberg, E.D., W.S. Broecker, M.G. Gross and K.K. Turekian. 1971.
Marine Chemistry. In: Radioactivity in the Marine Environment, National
Academy of Sciences, pp. 137-146.
24
-------
6. Graham, W.F., M.L. Bender and G.P. Klinkhammer. 1976. Manganese in
Narragansett Bay. Limnology and Oceanography 21:665-673.
7. Hale, Staphen. 1974. The Role of Benthic Communities in the Nutrient
Cycles of Narragansett Bay. M.S. Thesis, University of Rhode Island, 129
pp.
8. McCaffrey, Richard J., Allen C. Myers, Earl Davey, George Morrison,
Michael Bender, Nile Luedtke, Douglas Cullen, Philip Froelich and Gary
Klinkhammer 1977. Benthic Fluxes of Nutrients and Manganese in
Narragansett Bay, Rhode Island. Submitted to Limnology and
Oceanography.
9. Nixon, Scott W., C.A. Oviatt and S.S. Hale 1976. Nitrogen Regeneration
and the Metabolism of Coastal Marine Bottom Communities. In: The Role
of Terrestrial and Aquatic Organisms in Decomposition Processes (J.M.
Anderson and A. Macfayden, eds.) Blackwell Scientific Publication,
Oxford, pp. 269-283.
10. Solorzano, L. 1969. Determination of Ammonia in Natural Waters by the
Phenol-Hypochlorite Method. Limnology and Oceanography 74:799-801.
11. Technicon 1973. Technicon Industrial Method 155-71/W. Orthophosphate
in Water and Seawater. Technicon Industrial Systems, Tarrytown, N.Y.
10591.
12. Technicon 1973. Technicon Industrial Method 186-72/W. Silicate in Water
and Seawater. Technicon Industrial Systems, Tarrytown, N.Y. 10591.
13. Thompson, John, Karl K. Turekian and Richard J. McCaffrey 1975. The
Accumulation in and Release from the Sediments of Long Island Sound.
In: Estuarine Research, Vol. I, Chemistry, Biology and the Estuarine
System, Academic Press, New York, pp. 28-44.
25
-------
THE USE OF INTRODUCED SPECIES
(MYTILUS EDULIS)
AS A BIOLOGICAL INDICATOR OF TRACE
METAL CONTAMINATION
IN AN ESTUARY
D.K. Phelps and W.B. Galloway
EPA, Environmental Research Laboratory
Narragansett, R.I. 02882
The use of introduced as well as indigenous marine species as biological
monitors or indicators of water quality is being evaluated at the Environmental
Research Laboratory, Narragansett (ERLN). This paper presents data that
demonstrate the edible blue mussel,Mytilus edulis, to be an effective indicator
of metal pollution when introduced along a gradient of anthropogenic stress.
M. edulis were collected from a commercially-fished mussel bed in Narragansett
Bay, Rhode Island, and held in a laboratory seawater system for six days.
Sub-groups were deployed in polluted and clean sections of that estuary,
respectively, for a period of four weeks.
Atomic absorption analyses revealed that M. edulis from the polluted section
had significantly higher levels of lead, nickel, and copper when compared to
M. edulis from the clean part of the estuary and those retained in the seawater
system at the laboratory as controls. No differences were apparent between the
three groups in the case of cadmium, chromium, vanadium, and zinc; however,
comparisons between introduced M. edulis and indigenous Mercenaria
mercenaria, demonstrated M. edulis to be an effective surrogate biological
monitor for M. mercenaria in the case of lead, nickel, and copper.
INTRODUCTION
The area of study is Narragansett Bay, Rhode Island, U.S.A. (Figure 3-1).
The Bay has been described as "abnormally stressed" by man's activities in its
upper reaches, and as being divisible into a polluted upper Bay, a transitional
zone, and a lower Bay having water of high quality (1). Bottom water salinities
range 28-31°/oo in the upper reaches, and 30-32°/oo in the lower Bay.
Temperature seasonally escalates from -l^C to 26°C in various sections of
the estuary.
26
-------
Figure 3-1. Area of study.
27
-------
Major environmental differences within the system are attributable to a
history of pollution effects in the upper reaches of the Bay (Figure 3-1). Major
sources of pollution are domestic waste treatment plants, which include
industrial effluents from such activities as metal plating and jewelry
manufacturing, and urban runoff.
Fine sediments in the upper area (Stations 1 and 2) are anaerobic,
characteristically having the redox boundary at the sediment-water interface, as
well as having a strong odor of F^S. In the lower Bay (Stations 3 and 4), fine
sediments have a well-defined aerobic layer with a redox boundary defined
between 5 and 10 cm below the sediment-water interface (2). Metals in upper
Bay sediments include typically elevated levels of zinc (337 ppm), lead (167
ppm), copper (493 ppm), and chromium (208 ppm) compared to lower Bay
levels of zinc (119 ppm), lead (40 ppm), copper (48 ppm) and chromium (53
ppm) (2). In addition, higher concentrations of hydrocarbons have been
reported in upper Bay sediments compared to levels found in the lower Bay
(3). Over the past few years, a transect of stations, indicated in Figure 3-1 as 1,
2, 3, and 4, has been used to study the effects of pollution from north to south
in the Bay.
M. mercenaria is a molluscan species indigenous to all areas of the Bay.
Phelps and Myers (4) compared levels of aluminum (Al), cadmium (Cd),
cobalt (Co), copper (Cu), lead (Pb), manganese (Mn), nickel (Ni), silver (Ag),
titanium (Ti), vanadium (V), and zinc (Zn) between M. mercenaria collected from
the "polluted" upper Bay and the "clean" lower Bay. Of particular interest is
the fact that after a thirty-day period of depuration, M. mercenaria from the
upper polluted part of the Bay retained significantly higher levels of Cd, Cu,
Ni, Pb, and Ti compared to M. mercenaria collected from the lower "clean" part
of the Bay (Figure 3-2).
This paper reports on the use of M. edulis as an introduced biological
monitor for trace metals. The specific goals of the study were:
1. To observe whether or not introduced M. edulis bioaccumulation would
reflect the spatial differences in trace metal levels previously observed
in sediments and indigenous M. mercenaria.
2. If such differences were reflected by M. edulis, over what time frame
were differences observable?
3. If quantitative differences were reflected, how do metals accumulated
by introduced M. edulis, compare qualitatively to metals accumulated by
indigenous M. mercenaria?
28
-------
1000 —
mil DIRTY MERCENARIA
CHI CLEAN MERCENARIA
A UNDEPURATED
B DEPURATED
Cu Ni Pb
MERCENARIA
Figure 3-2. Metal levels in indigenous Mercenaria mercenaria from
clean and polluted parts of Narragansett Bay, Rl.
Note: Range shown is one standard deviation on either side of the mean.
The mussels used in this study were collected at one time immediately south of
Popasquash Point, Narraganasett Bay, R.I. (Figure 3-1), and held in a
laboratory flow-through system for six days prior to the first deployment.
29
-------
Field Methods
Since metals were chosen as the initial pollutants of interest, a completely
metal-free apparatus was desired for deploying mussels in the field. In view of
potential use by other investigators, a simple, relatively inexpensive apparatus
which was easy to deploy and service was a secondary goal of the design
process. The present apparatus is shown in Figure 3-3.
A plastic float is attached to a concrete weight by approximately two meters
of polypropylene line. The mussel holding baskets, 15 x 15 x 15 cm
polypropylene test tube baskets with snap-on lids, are suspended from the line
approximately 1 meter above the sediment surface.
A M. edulis monitoring station is deployed in a two-step operation. The
apparatus, minus the mussel baskets, is lowered to the bottom in 5 to 8 m of
water, after which typically eight mussel baskets are taken down and
suspended in groups of four from the line by scuba divers. The
deployment operation requires 10 to 15 minutes on-station and subsequent
sampling only about five minutes. Each basket of 20 mussels serves as a
subsample, thereby allowing removal of the desired sample without disturbing
the remaining mussels. Samples for metals analyses are immediately transferred
to "ziploc" polypropylene bags and frozen until analyzed.
plastic
float
polypropylene
line
mussel
baskets
anchor
Approx. I m
Figure 3-3. Schematic of subsurface mussel station.
30
-------
The first collection was made from each station after an exposure period of
three weeks. A second collection was made one week later.
Analytical Methods
The mussel tissue is analyzed for metal content by flame Atomic Absorption
Spectrometry after wet digestion in concentrated nitric acid. Each sample is
oven dried to constant weight, then digested in concentrated nitric acid in a
simple reflux system. The digestate is filtered on transfer to 50 ml volumetric
flasks, brought up to volume, and analyzed on a Perkin-Elmer Model 603
Atomic Absorption Spectrophotometer using Deuterium arc background
correction where necessary. The raw data are reduced to ug of metal/gram of
tissue, on both a wet and dry weight basis, by computer.
RESULTS
Results of analyses for Cd, Pb, Ni, Cu, chromium (Cr), V, and Zn on M. edulis
from Stations 2 and 3, as well as from controls, are presented in
Figures 3-4 and 3-5 and Table 3-1.
Except in the case of Pb and V, where the numbers of samples having
detectable limits were below the minimum number required for the statistic,
the standard deviation is comparable between a sample of 41 or 10 (Table 3-1).
This fact establishes that 10 M. edulis are a reasonable sample size with the
noted exceptions of Pb and V. After four weeks, Cd levels in M. edulis from
mid-Bay, Station 3, were slightly lower than either laboratory-held M. edulis or
those collected from the polluted area at Station 2. However, the overlap in Cd
values between the three sites renders differences insignificant (Figure 3-4).
Lead, in those four animals from the polluted area having
detectable levels, was higher than those levels detected in the laboratory-held
animals, and the single animal having detectable levels from Station 3.
However, due to the low number of sampling points, statistical significance
cannot be established for these data (Figure 3-4).
Nickel levels in M. edulis from Station 2 are significantly higher than those
measured in laboratory-held animals. M. edulis from Station 3, while not being
significantly different from Station 2, or laboratory-held animals, clearly fall in
a range midway between those polluted and clean areas. A gradient of Ni, with
highest levels in the polluted area of the Bay, diminishing in the mid-Bay,
having lowest levels in the lower Bay, is demonstrated by these data. It is of
interest to note-that only 19 of the 41 laboratory-held animals had detectable
levels of Ni.
31
-------
100
10.0
"O
0)
E
CT>
CD
E
en
A.
1.0
0.1
Ni
-- U
Cu
Pb
L -
u
Cd
Cr
--F
L' Laboratory held animals at time = 0
U: Animals held in unstressed area (Sta. 3, Fig. 3-1)
for 4 week exposure
S: Animals held in stressed area (Sta. 2, Fig. 3-1)
for 4 week exposure
Zn
L U S _
I000
I00
I0.0
I.O
Note: Bar lines, when present, indicate one standard deviation on either side
of the mean; otherwise total range is indicated.
Figure 3-4. Metal levels in Mytilus edulis introduced into
stressed and unstressed environments, Narragansett Bay, Rl,
September-October, 1976.
32
-------
100
iO.O
<1)
5E
-------
Table 3-1. Metal Levels in Mytilus edulis
Cd
Pb
Ni
Cu
Cr
V
Zn
Lab-held
2.89
9.7
5.7
8.5
2.35
10.7
170
41 specimens
.78
1.9
1.6
2.2
1.52
4.3
52
(time = 0)
(40)
(10)
(19)
(41)
(41)
(22)
(41)
Stressed
3.04
12.6
15
19
3.76
10.5
199
10 specimens
.65
10.8-
3.69
2.8
.69
4.7-
83
15.5
11.4
(time = 4 weeks)
(10)
( 4)
(10)
(10)
(10)
( 5)
(10)
Unstressed
2.42
7
9.1
12
2.53
8.4
149
10 specimens
.47
7
2.7
1.4
.50
6.4-
22
11.5
(time = 4 weeks)
(10)
( 1)
(10)
(10)
(10)
( 5)
(10)
Metal levels in Mytilus edulis expressed above in ug/g dry weight as:
Mean
Standard deviation or range
(no. of organisms above detection limit)
Copper levels in M. edulis are significantly higher in that group of animals
from polluted Station 2 than either Station 3 or laboratory-held animals.
However, the latter two animal groups have statistically similar levels. Copper
data show a sharp drop from the polluted area to the levels in both lower Bay
groups (Figure 3-4).
Chromium levels in M. edulis from the three groups are not significantly
different (Figure 3-4).
Vanadium levels were above detection in only about one-half of the
individuals collected from each station. While the data are too sparse for
statistical analysis, no difference between the levels at the three stations is
apparent (Figure 3-4).
Variability in levels of Zn is so great within each station that meaningful
comparison between stations is not possible.
34
-------
After a three-week period, M. edulis from polluted Station 2 had accumulated
significantly higher levels of Ni and Cu than had M. edulis from the clean Bay
stations (Figure 3-5). Similarly, Station 2 values after three weeks were
significantly higher than values from control animals sacrificed at time zero.
However, levels remained the same in all animal groups after one additional
week of exposure.
DISCUSSION
Currently, M. edulis is being used as an indigenous biological monitor
for a variety of materials including petroleum hydrocarbons, chlorinated
hydrocarbons, and transuranics, in addition to trace metals in coastal waters of
the United States (5). Similar activity is underway in the United Kingdom and
Germany as well. Because of its ubiquitous distribution, M. edulis is being
considered as an international monitoring organism.
To date, the use of M. edulis appears to be limited to that of an
indigenous monitor. We are not aware of an approach similar to that reported
here where M. edulis is used as an introduced biological monitor.
The results indicate that M. edulis as an introduced species does reflect
elevated levels of metals previously observed in the polluted section of
Narragansett Bay, in the sediments and the indigenous mollusc, Mercenaria
mercenaria. Nickel and copper were concentrated to significantly higher levels
in M. edulis introduced into the polluted section of the Bay compared to those
introduced in the clean lower Bay, and those held in our laboratory
flow-through system. Lead levels were observed to be higher in animals from
collecting Station 2, than in either collecting Station 3 or the laboratory;
however, statistical significance was not established due to the small data set.
Cadmium, chromium, vanadium, and zinc were not reflected at higher levels in
M. edulis from the polluted area.
With one exception, that being in the case of Cd, these results reflect those
reported by Phelps and Myers (4) for M. mercenaria collected in the same parts of
Narragansett Bay. In that study, M. mercenaria from the polluted area were shown
to concentrate to-higher levels, but not depurate, Cd, Pb, Ni, and Cu compared
to lower Bay animals. No differences were observed in levels of V and Zn
between the two groups of M. mercenaria before or after depuration. Thus,
Mytilus edulis, when used as an introduced monitor, is demonstrated to reflect
metal levels observed in the major resident or indigenous species in three out of
the four metals of note.
Higher metal accumulations were established after three weeks of
monitoring by M. edulis introduced in the polluted section of the Bay for Ni and
35
-------
Cu. Higher ievels of Pb appeared after four weeks of exposure. The relatively
quick response time established in the case of Ni and Cu represents an obvious
advantage to the use of M. edulis as a biological monitor. For these metals,
M. edulis quickly reflects a situation which effects the long-lived indigenous
species, M. mercenaria.
These results encourage further study. Our three specific goals listed in the
Introduction have been answered in the affirmative:
1. Introduced M. edulis from polluted areas reflect elevated levels of metals,
as did sediments and M. mercenaria reported in previous work.
2. M. edulis displays a relatively short response time in accumulating
elevated metal levels — three weeks in the case of Ni and Cu, and four
weeks in the case of Pb.
3. A/, edulis, the introduced biological monitor, took up three of the four
metals previously demonstrated to have been accumulated and retained
by the resident species, M. mercenaria.
The results reported here are based on data collected when the annual
temperature cycle was declining toward winter levels. This fact may account
for the leveling-off of Ni and Cu observed between weeks three and four at
Station 2.
Further studies along the transect in Narragansett Bay, and in other
comparable areas, including a complete annual temperature cycle, are being
carried out to supplement the knowledge gained in this study on the use of
M. edulis as an introduced biological indicator of man's impact on the
environment.
REFERENCES
1. Phelps, D.K., G. Telek, and R. L. Lapan, Jr. 1975. Assessment of Heavy
Metal Distribution Within the Food Web. In Marine Pollution and Waste
Disposal. Pearson and Frangiapane, editors. Pergamon Press, pp. 341-348.
2. Myers, A.C„ and D.K. Phelps. 1978. Criteria of Benthic Health: a Tran-
sect Study of Narragansett Bay, Rhode Island. Final Report. Contract
No. P.O. 53203, URI Division of Marine Resources, Graduate School of
Oceanography, Kingston, R.I.
3. Farrington, J.W. and J.G, Quinn. 1973. Petroleum Hydrocarbons in
Narragansett Bay. I. Survey of Hydrocarbons in Sediments and Clams. Est.
and Coastal Mar. Sci. 1:71 79-
36
-------
4. Pheips, D.K. and A. Myers. 1977. Ecological Considerations in Site
Assessment for Dredging and Spoiling Activities in Ecological Research
Series, EPA-600/3-77-083, 266-286.
5. Goldberg, E. 1978. Annual Report, Mussel Watch Program. EPA Contract
No. R-80421501.
37
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TRACE METAL SPECIATION AND TOXICITY
IN PHYTOPLANKTON CULTURES
F.M.M. Morel, N.M.L. Morel, D.M. Anderson,
D.M. McKnight and J.G. Rueter, Jr.
Division of Water Resources
and Environmental Engineering
Civil Engineering Department
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
ABSTRACT
The toxicity of trace metals to phytoplankton has been demonstrated to
depend on metal ion activities. The various chemical processes that control
metal speciation, and thus activities in aquatic systems, are inorganic
complexation, chelation, precipitation and adsorption. For example, the
activity of metals such as mercury, cadmium or lead are controlled in saline
waters of low organic content by the formation of chloride and bromide
inorganic complexes. For mercury, this is also the case in typical
phytoplankton culturing media. Artificial chelating agents permit convenient
manipulation of metal ion activities in algal toxicity experiments. However,
kinetic phenomena can result in transient peaks in metal ion activities and lead
to large overestimations of toxicity. The release of metal complexing agents by
algae is not expected, in general, to affect markedly the chemistry of metals in
highly chelated artificial media except in cases of high specific affinity. The
greatest complication in interpretation of phytoplankton toxicity
experiments arises from the presence of solids in the culture medium. These
can precipitate during the preparation of the medium, or as a result of the
pH increase due to photosynthetic carbon uptake. The kinetics of
precipitation of these solids, their aging and the adsorption of trace metals
on their surface, lead to variations in metal activities that are difficult to
quantify, and do not permit proper assessment of the toxic effects.
Understanding the global aquatic chemistry of trace metals in algal culture
media, is a sine qua non prerequisite to proper design and interpretation of
toxicity experiments.
INTRODUCTION
Using copper as the principal example, this study aims at establishing a
chemical framework for the study of laboratory and natural processes involving
trace metal toxicity to phytoplankton. If it is a reasonable assumption that the
38
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mechanisms of metal toxicity are similar in many phyla, this framework should
be useful for toxicity studies with many aquatic organisms, and can serve as the
general basis for design and interpretation of such studies.
Although the importance of both organic complexing agents and trace
metals in phytoplankton cultures has been recognized for some time, the
critical role played by the speciation of trace metals in controlling their
toxicity and availability to algae has just begun to be understood. It has now
been established that it is the activity of the free ions, rather than the total
metal concentrations, which determine the toxicity of metals to
phytoplankton (46, 2). The study of the chemical processes which govern the
activity of a given trace metal in culture media becomes then a prime area of
concern to phycologists interested in metal toxicity experiments. An
enumeration of these processes includes inorganic complexation, chelation,
precipitation and adsorption. In addition, indirect chemical effects involving
several interacting chemical species in the medium can influence trace metal
activities in unobvious ways. In this study, these basic principles of Aquatic
Chemistry (43) that apply to metals in phytoplankton cultures will be
discussed systematically.
There are but a few free metal activities that can be experimentally mea-
sured in the range and under the conditions of interest. There are also few
metallic complexes which can be analytically determined in chemical
systems as complex as culturing media. Therefore, theoretical equilibrium
calculations will be used throughout this paper to assess metal speciation
and activities. The assumption of equilibrium is a reasonable one when the
proper precautions are taken during medium preparation. Thermodynamic
calculations provide, then, a convenient means of illustrating the critical
chemical principles, even if they have inherent uncertainties. Complications
introduced in the chemistry of the system by kinetic phenomena, or by the
influence of the algae, will be discussed for each of the examined processes.
Copper has been the metal of choice in studies of metal toxicity of
phytoplankton because it has been postulated that cupric ion toxicity might
play a role in the ecology of phytoplankton in some natural waters (6, 37, 7,
10). In keeping with this situation, this paper will focus, albeit not exclusively,
on copper which provides a rather good example for metal speciation and
toxicity in phytoplankton cultures.
EQUILIBRIUM SPECIATION OF METALS IN CULTURING
MEDIA
Before studying in detail the role of chemical processes in controlling metal
speciation, it seems useful to examine what metal species are expected to be
important in typical culturing media.
39
-------
In artificial media where the analytical concentrations of the components
are precisely known, the exact composition of the system, including all soluble
and insoluble species of the various metals and their activities, can be
computed if a state of equilibrium or partial equilibrium is established (43). It
should be underlined that such calculations of chemical speciation depend on
correct identification of the principal species, and knowledge of the
corresponding equilibrium constants. The calculations presented here were
performed with the computer programs, REDEQL (25, 21) and MINEQL (49)
which contain a list of possible species, and a selection of the necessary
thermodynamic constants from a variety of sources (36, 38, 33).
Results of chemical equilibrium computations of three media recipes, the
freshwater medium WC (11) and the seawater media F/2 (12) and Aquil (26),
are shown in Table 4-1. Possible adsorption processes are not considered in
these calculations. Note that some heavy metals (Pb, Cd, Hg, Ni, Co, Cr) which
are not part of the recipes, have been added in trace amounts (lO'^M) to
illustrate how they would be speciated if they were present as contaminants in
the media. Such low metal concentrations affect the rest of the chemical
system negligibly. Heavy metal speciation in all media is completely
dominated by the chelation with ethylenediaminetatraacetate (EDTA) which is
included in the recipes for the very purpose of chelating metals. An important
exception is mercury, which, according to the computations, is present entirely
as chloride complexes in F/2 and Aquil and half as hydroxide species in WC.
For all metals, the free ion activities are several orders of magnitude smaller
than their total concentrations. In all media, iron and manganese oxides and
calcium phosphate (hydroxylapatite) are computed to precipitate at
equilibrium. Calcium carbonate (calcite) is also shown to be saturated in the
seawater media. Actual precipitation of these various solids is dependent on
kinetic processes as will be discussed later.
The trace metal chemistry of such culturing media can be grossly affected by
the presence of algal cells due to metal uptake. For example, typical values for
the uptake of copper by phytoplankton are in the range 10"^ to 10"^
moles/cell (44, 35, 16). With the algal densities and the copper concentrations
commonly used, a sizeable part of the total concentration of copper in the
medium can thus be taken up by the cells. This underlines the necessity of
"buffering" metal ion activities in toxicity experiments in order to render these
activities relatively insensitive to total metal concentrations. The use of various
chelating agents for this purpose will be discussed later.
INORGANIC COMPLEXATION
It has been observed in several instances that the toxicity of metals such as
lead, cadmium, mercury or silver to a variety of organisms, from bacteria to
40
-------
Table 4-1. Equilibrium Trace Metal Speciation in Typical Algal Growth Media
WC(EPTA»1.2 10~5M) F/2(EDTA-1.2 I0~3M) Aqull (EDTA - 5 x 10~6M)
Analytical Computed Major Analytical Compared Major Analytical Computed Major
Concentration Activity of Species concentration Activity of Species Concentration Activity of Species
M free ion. M M free ion, M M free ion. M
C-log) (-log) (-log)
Iron
l,2xllf 5
21.5
FeEDTA
22%
1.2xl0~5
21.5
Fe(0H)3(S)
99%
4.5x10 7
21.5
FeEDTA
6%
Fe(OH)
(S) 76%
Fe(OH>3
(S) 94%
Manganese
8.9x10™7
14.7
Mn02(S)
100%
a.9xio"?
14.7
Mn02(S)
100%
2.3xlG~8
14.7
Mn02(S)
100%
Copper
4.0xl0'8
14.8
CuEDTA
100%
4.0xl0-8
13.3
CuEDTA
99%
l.OxlO-9
14.4
CuEDTA
100%
CdEDTA
98%
Cadmium
l.OxlO"9
14.8
CdEDTA
100%
l.OxlO-9
13.3
CdEDTA
99%
l.OxlO-9
12.8
CdCl+
11
-8
-8
_Q
CdCl j
1%
Zinc
7.9x10
12.2
ZnEDTA
100%
7.9x10
10.7
ZnEDTA
100%
4.0x10
11.5
ZnEDTA
100%
Nickel
l.Gxltf9
16.8
NiEDTA
100%
l.OxlO"9
15.3
NiEDTA
100%
1.0xl0~°
14.8
NiEDTA
100%
Mercury
1.Qxltf 9
20.4
HgEDTA
49%
l.OxlO-9
23.6
HgCl 2"
87%
1.0x10 9
23.6
HgCl42~
87%
Hg(OH)2
51%
HgCl3"
13%
HgClj^
13%
Lead
1.0xl0~9
16.3
PbEDTA
100%
l.OxlO-9
14.8
FbEDTA
100%
l.OxlO"9
14.3
PbEDTA
99%
Cobalt
5.Ox10"8
13.9
CoEDTA
100%
S.OxlO™8
12. 3
CoEDTA
100%
l.OxlO-9
11.9
CoEDTA
99%
Chromium
l.OxlO*9
23.1
CrEDTA
loo?*
l.OxlO-9
21.5
CrEDTA
100%
l.OxlO-9
21.1
CrEDTA
100%
-------
fish, decreases with increasing salinity of the water (45). One possible
explanation for such a phenomenon is the decrease in metal ion activity
resulting from the formation of ion pairs with the major anions of seawater.
Figure 4-1 shows, for example, how the speciation of mercury and the activity
of the mercuric ion vary in function of salinity in the medium F/2. As salinity
increases the bromide complexes of mercury replace the EDTA chelate as the
major species, followed by the chloride complexes as the salinity approaches
that of seawater. In natural systems, in the absence of strong chelating agents,
the same phenomenon would extend to other metals such as lead and
cadmium. Table 4-2 illustrates this point by giving the major species of the
various metals in Aquil where EDTA has been reduced to 10'^M. Besides the
chloride complexes, a number of carbonate (Cu, Pb), sulfate (Zn, Mn, Co) and
hydroxide (Zn, Pb, Co, Cr) complexes become significant. Because the kinetics
of formation of the various inorganic complexes of metals are typically fast
(43), equilibrium is a good assumption in this instance, and the thermodynamic
calculations should give accurate values of metal activities.
The role of carbonate complexation in decreasing the toxicity of metals in
unchelated media has been verified for copper on Daphnia magna (3), for
function of salinity at pH = 8.1.
Note: Top: mercuric complexes as a percent of the total mercury (10"®M);
Bottom: the activity of the mercuric ion. All other trace metals remain bound
to EDTA throughout the salinity range. SW represents seawater, salinity 33%o.
42
-------
Table 4-2. Speciation of Trace Metals in Aquil with
EDTA Concentration Reduced to 10"%l.
Major Species
Percent of Total Metal
Iron
Fe(OH) 3(S)
100%
Manganese
Mn02(S)
100%
Copper
Cu2+
1%
CuEDTA
87%
CuCOo
12%
Cadmium
Cd2+
3%
CdCI3+
39%
CdCI2
42%
CdEDTA
6%
Zinc
Zn2+
60%
ZnSC>4
8%
ZnCI+
3%
ZnCI3
1%
ZnEDTA
27%
ZnOH+
1%
Nickel
NiEDTA
99%
Mercury
HgCI42-
87%
HgCI3-
13%
Lead
PbC03
21%
PbCI3
50%
PbCI2
5%
PbCI+
3%
PbEDTA
20%
PbOH+
1%
Cobalt
Co2+
51%
CoSO^
11%
CoCI+
21%
CoEDTA
15%
CoOH+
2%
Chromium
CrEDTA
57%
Cr(OH)4-
40%
Cr(OH)2+
3%
copper on some fishes (31), and for cadmium on a grass shrimp (45). Although
there has been no experiment reported to date that provides direct evidence for
the importance of inorganic complexation in controlling metal toxicity to
phytoplankton, this result can be inferred from data with these other
organisms, and from the general demonstration that metal ion activities are the
important parameters of toxicity to algae.
43
-------
Through uptake of carbon dioxide for photosynthesis, algae can modify the
inorganic species of metals by decreasing the total concentration of carbonate
in the system and increasing the pH. As pH increases, the hydroxyl ion activity
increases and so does the importance of metal hydroxide complexes. The effect
on the carbonate ion activity and on the metal carbonate complexes is less
straightforward, and depends on the original pH of the medium. In seawater
media (pH 8), and in freshwater media around neutral pH, the carbonate
complexes will increase with CO2 uptake due to the predominance of the
resulting pH increase over the total carbonate decrease. Such variations in
metal chemistry can be alleviated by bubbling air in the cultures, thus insuring
a steady concentration of carbonate in the medium.
CHELATION
The history of the development of artificial culturing media for algae is in
part that of the replacement of "growth factors" and "soil extracts" by
chelating agents (17). The exact role of these chelating agents in promoting
algal growth has been a subject of some controversy (6, 14). It is now well
established that they do control the toxicity of various heavy metals — copper
in particular (46). Whether they also increase the availability of some metallic
nutrients — chiefly iron — is yet unproven. Figure 4-2 shows the percentage of
chelated metal and the metal activities in Aquil (with contaminant metals) as a
function of the concentration of EDTA, by far the most widely utilized
chelating agent in algal media. Note that the order in which the metals are
chelated by EDTA is not simply related to either the metal ion activities or
their affinities for EDTA (Fe>Cr>Cu>Ni >Pb>Zn, Cd, Co).
Other chelating agents which are commonly used include nitrilotriacetate
(NTA), citrate and various amino acids. "Tris" (tris(hydroxymethyl)amino
methane) commonly used as a pH buffer for biological experiments has
received much use in recent studies of copper toxicity to phytoplankton (44).
Used in conjunction with EDTA which chelates the other metals at a very low
concentration, Tris permits a convenient manipulation of the cupric ion
activity. Figure 4-3 illustrates this point by comparing how the cupric ion
activity varies with total copper in Aquil (EDTA = 10'^-^M) and in a modified
Aquil recipe containing Tris (EDTA = lO'^M and Tris = 10'^M). Around
[Cu2+] = 10'^M, where many toxicity studies are run, the cupric ion activity
in the Tris medium is less sensitive to variations in total copper concentration
than in the EDTA medium. However, with the proper precautions, both media
yielded the same results in a study of copper toxicity to Gonyaulax tamarensis
(2).
44
-------
B
-log (EDTA)t (M)
Figure 4-2. The effect of EDTA on the speciation of metals
in Aquil.
Note: Total metal concentrations are given in Table 4-1, with Pb, Cd, Hg, Ni,
Co, and Cr added as contaminants (10"^M). A) The percent of each metal that
is chelated (MeEDTA) versus total EDTA concentration, (M); B) Metal ion
activity (M) versus total EDTA concentration (M).
45
-------
-log (Cu)y
Figure 4-3. Computed activity of the cupric ion (M) versus
total copper concentration for two Aquil recipes.
Note: Chelated with: A) 10'6-3M EDTA plus 10"3M Tris and B) 10"5-3 EDTA.
Although the forward kinetic constants of chelate formation are invariably
very large, resulting in quasi instantaneous kinetics in simple systems, the
situation can be very different in systems as complex as culturing media. For
example, when copper was spiked in Aquil cultures of G. tamarensis, a
dramatic short term toxic response was observed much above that expected for
the calculated equilibrium activity of the cupric ion (2). This phenomenon
which was not observed when Tris replaced EDTA as the major copper
chelating agent, was attributed to the slow kinetics of the metal exchange
reaction:
Cu2+ + CaY - CuY + Ca2+
This appears as a reasonable explanation, since the calcium chelate is the major
form of EDTA in Aquil and the dissociation is slow. No such phenomenon can
occur with Tris, whose major species in culturing media are the various
protonated forms of the ligand. This can be checked directly by monitoring the
cupric ion activity with a mixed sulfide electrode (34, 13) in chemical systems
similar to the culturing media. Figure 4-4 presents the results of such an
experiment, and leaves no doubt as to the slow kinetics of copper reaction with
46
-------
Figure 4-4, Effects of dissociation of Ca-EDTA on short term
cupric ion activity after addition of 10~45M Cu(N03>2.
Note: Background electrolyte 0.5 m KN03, pH 8.25 <2 x 10"3M NaHC03
bubbled with air), EDTA, temperature 23°C. A Radiometer
selectrode (F 3000), an Orion d/j reference electrode, and an Orion pH
electrode were used. In both experiments, the cupric ion activity (M) was
calculated using the Nernst equation and data from 10'^, 10"® and 10"®M
Cu(N03>2 solutions at pH 4:00 in 10"^N KNO3 background electrolyte. A)
Calcium (10"2M) in equilibrium with EDTA prior to copper addition; B) No
calcium present.
47
-------
EDTA in the presence of an excess of calcium: an initial peak in cupric ion
activity is measured by the electrode, and it takes about four hours to
approach the equilibrium value. Such phenomena have to be taken into
account when studying the toxicity of metals to any aquatic organism, as
transient effects can lead to large overestimations of toxicity.
The release of chelating metabolites has been widely assumed as a
conditioning mechanism for culture media (39). As is the case for natural
waters, most of the chemically quantitative work on this topic has focused on
the synthesis and exudation of iron chelating agents, particularly hydroxamates
(19, 30). What seems often overlooked is that hydroxamic acids do not chelate
exclusively iron, and that their binding of other metals can result in sizable
decrease of these metals' activities (1).
By direct potentiometric techniques, extracellular metabolites of algae have
been characterized in terms of copper complexing capacity and affinity (48).
According to this work, the ligand produced by the algae under the conditions
of the experiments is characterized by a constant of approximately unity for
the reaction:
Cu2+ + HY~ = H+ + CuY
If one assumes the ligand to be copper specific, the effect of its release in Aquil
and Aquil with Tris is shown in Figure 4-5. Note that a significant decrease in
the cupric ion activity does not begin until the total ligand concentration
reaches 10'^M, an upper limit for the measured ligand releases. Although
[Cu2+] start decreasing at a slightly lower ligand concentration when the
copper concentration is elevated, the release of such relatively weak
complexing ligand has little overall effect on the cupric ion activity in a well
chelated medium. Ligands, with higher affinity for copper, appear to be
released by some blue green algae (22).
In principle, phytoplankton could modify the trace metal chemistry of the
medium by assimilating artificial chelating agents. However, this potential
problem is avoided by using EDTA or NTA which have been shown not to be
assimilated by algae (23). Although photodegradation of EDTA and NTA has
been reported (40), the light intensities normally used for culturing
phytoplankton are insufficient to promote it in the laboratory.
PRECIPITATION
According to the computations of Table 4-1, the precipitation of several
solids is calculated to be thermodynamically favorable in typical culturing
media. Visible precipitates are indeed a common observation of users of algal
48
-------
10
11
-
i
A
i
^3 12
o
-
-
cn
O
1 13
B
14
-
C
-
15
-
i
i
8 6 4
-log (Y)t
Figure 4-5. The effect of different molar concentrations of
copper specific metabolite (Y) on the activity of the cupric ion
for three variations of Aquil medium.
Note: A) 10'5-3M EDTA plus 10"6M Cu; B) 10"6-3M EDTA plus 10_3M Tris;
C) 10"5-3M EDTA.
media. This is especially true following autoclaving, which brings about a large
pH increase by eliminating carbon dioxide from the system. This problem has
been studied by researchers involved in the design of culturing media (32, 8,
12, 18, 26). The increase in temperature and pH during autoclaving decreases
the solubility of calcium carbonate, and results in the precipitation of a
magnesium rich solid (this suggests the solid to be magnesium calcite, although
aragonite has been identified in such precipitates). Hydrous oxides of iron and
manganese can also precipitate under such conditions, depending on the
chelating agent concentration and the pH reached during autoclaving. When
such precipitates occur, phosphate becomes largely associated with the solid
phase, presumably in some calcium precipitates (apatite or CaHPO^.), or as an
adsorbate on the various solids. Depending on the initial concentration of
silicic acid and on the nature of the container, which can increase the silicate
concentration of the solution by dissolution, some amorphous or crystalline
form of SiC>2 can form in the medium.
49
-------
I
IRON HYDROXIDE
CALCIUM CARBONATE
I
MANGANESE OXIDE
I
CALCIUM PHOSPHATE
II. ¦
LEAD OXIDE
COBALT HYDROXIDE
MAGNESIUM HYDROXIDE
STRONTIUM SULFATE
6 7 8 9 10 pH
Figure 4.6. Saturation pH for various metal solids in Aquil.
These various solid formation processes are dependent on kinetic factors
which are controlled by the particular temperature and pH regime of the
medium. These, in turn, depend on the conditions and duration of autoclaving,
as well as on the size of the containers and the mixing conditions. Precipitates
are rarely seen with filter sterilization. Figure 4-6 shows the onset of saturation
for various solids as pH is increased in the medium Aquil, normally designed to
avoid precipitates. None of the four solids that are saturated at pH = 8 in Aquil
are actually seen to precipitate, even after autoclaving if the volumes are kept
smaller than 100 ml. If larger volumes are autoclaved, immediate bubbling with
carbon dioxide prevents precipitation. Avoidance of calcium carbonate
precipitate is very important for maintaining iron and manganese in solution, as
the presence of" CaCO^s) will catalyze the formation of hydrous oxides of
manganese and iron (43). For other trace metals the formation of these
precipitates creates difficulties mostly through adsorption processes (see next
section).
For toxicity studies, trace metals are sometimes introduced in algal cultures
in excess of the chelating agent concentration. Precipitates are then often
expected to form mostly oxides, hydroxides and carbonates, depending on the
metal. For example, a hydroxide (Cu(OH)2), an oxide (CuO, tenorite) and a
carbonate (Cu2C03(0H)2, malachite) become quickly saturated in Aquil when
copper exceeds the EDTA concentration. Although the hydroxide is not the
50
-------
thermodynamically stable form, it probably is the one which forms initially in
the medium for kinetic reasons. Regardless of the precise nature of the solid,
good agreement has been obtained between calculated and measured copper
concentration in the solid phase in Aquil medium with a high EDTA
concentration, 2 hours after addition of excess copper (29). It is worth noting
that the precipitate was very finely dispersed, and that centrifugation was
necessary to separate it from the aqueous phase. Ignorance of the formation of
a precipitate can obscure completely the meaning of otherwise well controlled
experiments. In terms of metal ion activity, the situation is complicated by the
change in the nature of the precipitate which might evolve from an active form
to a more stable one. In copper saturated media, the cupric ion activity has
been measured potentiometrically to decrease markedly over 24 hours, the rate
of decrease becoming very small thereafter (22). Such conditions can create
large uncertainties in toxicity experiments.
ADSORPTION
The common notion that chelating agents make iron available to algae,
seems to be supported by experiments where addition of iron or EDTA salts
provide similar growth and carbon uptake enhancement in a variety of algal
cultures (3). However, aluminium salts have also been observed to enhance
carbon uptake (24). Following Stumm and Barber (41), it is now a prevalent
interpretation of such experiments to attribute part, or all of the beneficial
effect of the metal additions to a scavenging of other toxic metals by
adsorption on precipitating iron or aluminium hydrous oxides. Figure 4-7
illustrates the beneficial effect of iron additions to a Pyramimonas culture, and
demonstrates how iron and copper behave antagonistically under controlled
conditions (28). The growth rate of Pyramimonas is reduced at a total copper
concentration of 1.2 10'^M, and completely stopped at 4.4 10'^M when the
iron concentration is low (1.2 10'^M). Increasing the iron concentration by a
factor of 10 completely blocks the toxic effect of the same copper
concentrations. The question to be resolved is how much of this
"detoxification" of copper by iron is due to adsorption processes, effectively
removing the copper from solution and decreasing the cupric ion activity, and
how much is due to a genuine physiological antagonistic effect at the cellular
level. In a recent study of the adsorption of copper on hydrous iron oxide in
seawater (48), it has been observed that under conditions similar to the
experiments of Figure 4-7, iron adsorbs copper up to a Fe/Cu molar ratio of
1/3. Adsorption can then certainly account for all of the antagonistic effects in
the Pyramimonas experiment. What becomes more difficult to explain is the
lack of antagonistic effect at the low iron concentration (1.2 xlO'^M) since
even then the highest copper concentration (4.4 x 10'^M) should be entirely
adsorbed. Note, however, that this is a domain of concentrations where copper
starts saturating the colloidal iron surface, and there must be a titration effect
51
-------
Control
4* ICf8M
~
o
A
1.2 x IO"7M
•
~
4.4 x I0~7M
¦
~
Expt. #1 Expt.#2 Expt.#3
-Experiments #1,2,3 done with inocula from
different cultures
-No EDTA
Figure 4-7. Antagonistic effects of iron and copper on
the growth rate of Pyramimonas 1 in artificial seawater medium
with the usual supplements of F/2 medium except for EDTA
which is not added.
Note: Experiments 1, 2, and 3 represent inocula from different cultures.
"Fresh" iron is FeCl3 solution prepared the day of the experiment and
sterilized by filtration.
52
-------
(like that of copper on EDTA in Figure 4-3) where the cupric ion activity
increases rapidly with increasing copper concentration. Exact quantitification
of this phenomenon awaits a better mathematical description of adsorption
processes on hydrous iron oxides in seawater. Despite great recent advances in
the modeling of adsorption in aqueous systems (50, 42, 15), it is still the least
quantifiable chemical process in thermodynamic calculations. The presence of
precipitates in a culture medium modifies its global trace metal chemistry to an
unpredictable degree. This creates the most common difficulty in interpreting
experiments on toxicity of metals to a variety of organisms. Note that
adsorption on the walls of a glass culture vessel is equally hard to predict.
Choice of container material which minimizes adsorption of solutes is critical
to the design of trace metal toxicity experiments.
Adsorption on the surface of algal cells can also be important for the trace
metal chemistry of the medium in dense cultures. There is, however, no
practical way to distinguish it from intracellular uptake. The effects of cellular
uptake processes including adsorption on the cell surface, have been discussed
earlier.
INDIRECT CHEMICAL EFFECTS
The general principles of coordination, precipitation, and adsorption which
have been discussed heretofore, are readily understood and their importance in
toxicity studies is usually recognized. What is less often perceived is the global
interdependency of the chemistry of culture media, the indirect interactions
(43, 27). For example, upon variations in the total copper concentrations, it is
natural to relate the observed effects to changes in the cupric ion activity.
However, as illustrated in Figure 4-8, activities of the zinc and ferric ions are
also increased when the total copper is augmented in Aqtiil. Conceivably, any
or all of these increased activities could be responsible for the observed effects.
It is then a difficult choice to either maintain all metal activities constant by
adhoc modification of all analytical concentrations—a method which
multiplies the workfor medium preparation and can create other interpretive
ambiguities—or to perform the multitude of necessary controls on an al-
ready arduous series of experiments. Table 4-3 shows how the total metal
concentrations have to be varied concomitantly with that of copper, to vary
exclusively the cupric ion activity in Aquil with two EDTA concentrations
(35).
The indirect interactions illustrated in Figure 4-8 are almost exclusively
mediated by EDTA, which chelates all the interdependent metals. In principle,
a convenient way to avoid the complications created by these interactions is to
reduce them to a minimum. This can be achieved by uncoupling the system
using more specific complexing agents. Figure 4-8 shows how the metal
53
-------
A
-log (Cu)T
Figure 4-8. Variations in metal activities (M) of manganese,
zinc and iron with total copper concentration.
Note: A) Aquil medium, IQ'^M EDTA; B) Aquil with EDTA,
10"^M Tris,
54
-------
Table 4-3. Calculation of Total Metal Concentrations Needed
to Change the Cupric Ion Activity in Aquil While Maintaining
the Other Metal Activities Constant
(-log (concentration) or [activity], M)
(EDTA)t
[Cu2+1
(Copper)^.
(lron)T
(Mang)-j-
(Zinc)-j-
(Cobalt) j
8.5
3.30
7.00
7.63
8.7
8.6
3.3
10.9
3.70
4.72
6.40
6.49
6.7
11.3
4.0
4.6
6.30
6.40
6.6
9.8
4.35
6.45
7.20
8.30
8.49
4.3
10.9
4.70
5.72
7.20
7.49
7.7
11.3
5.00
5.58
7.15
7.4
7.6
activities vary with total copper in modified Aquil medium containing "Tris", a
ligand known to chelate mostly copper (44). Upon variations in copper
concentration, the other metals are seen to have a much more constant activity
in Aquil with Tris than in Aquil with only EDTA.
One of the principal ways by which indirect chemical effects can be initiated
is through pH variations. For example, pH has an indirect effect on metal
complexation due to the acid-base properties of the coordinating ligands.
Figure 4-9 illustrates this effect for Mn, Cu and Zn in Aquil, with EDTA and
Aquil with Tris. In this case, Tris mediates a much greater indirect effect than
EDTA: zinc and especially cupric ion activities are markedly depressed by
increasing pH in the Tris medium, while the activities of all three ions remain
essentially constant in the EDTA medium. Increases in pH, which can be
brought about by photosynthetic carbon uptake if the aeration of the culture
is insufficient, can also result in precipitation as illustrated in Figure 4-6.
Adsorption on the fresh precipitate will follow, resulting in an unquantified
decrease in the soluble concentration of trace metals. It is apparent that pH is a
major factor in determining directly and indirectly the activity and toxicity of
trace metals, and should be monitored regularly in metal toxicity experiments.
CONCLUSION
The chemistry of metals in the external milieu of algal cells is only one of
the determinants of their toxicity. The literature on bacteria and higher cells
abounds with examples of how the sensitivity of a particular strain or clone to
a particular toxicant, depends markedly on the physiological status of the cells
(9, 4). Although it is often recognized that the same situation should apply to
phytoplankton, this concept has received scant attention in recent algal
literature. It stands to reason that the previous history of an algal cell, its
55
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Figure 4-9. Variations in metal activities with pH.
Note: A) Aquil medium, 10"®-%! EDTA; B) Aquil with 10"®-%! EDTA,
1(T3M Tris.
56
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nutritional status, and the particular phase of the cell cycle during which the
experiment is conducted — to name but a few obvious determinants of
physiological status — must affect its sensitivity to trace metals. Batch culture
experiments which, so far, have been used principally for metal toxicity
studies, have inherent restrictions to resolve the importance of these
physiological factors. Toxicity studies in continuous phytoplankton cultures
promise to be enlightening in this respect; they also promise to accentuate the
difficulties in controlling precisely the chemistry of the system.
It is hoped that the conceptual framework presented here will help in
designing and interpreting experiments where physiological responses to trace
metal toxicity are clearly assessed, distinctly from purely chemical effects in
the growth medium. It is also hoped that this study will help to increase
phytoplankton physiologists' awareness of the important chemical processes
which can affect their studies. It is, for example, surprising that so little
attention has been paid to the possible importance of phosphate speciation in
nutrient uptake experiments. Understanding the ecology of phytoplankton
requires detailed resolution of the cells' physiological responses to the total
aquatic chemistry of their environment.
ACKNOWLEDGMENTS
We thank S.W. Chisholm for her critical review of the manuscript and R.C.
Selman for her excellent job in typing the manuscript. This work was funded
by National Science Foundation grant no. DES75-15023, Environmental
Protection Agency grant no. R-803738 and the office of Sea Grant in the
National Oceanic and Atmospheric Administration grant no. 04-6-1584407.
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Natural Waters, P. C. Singer (ed.), Ann Arbor Science, Mich.
7. Davey, E.W., M.J. Morgan, and S.J. Erickson. 1973. A Biological
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8. Droop, M.R. 1961. Some Chemical Considerations in the Design of
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10. Gachter, R., K. Lum-Shue-Chan, and Y.K. Chau. 1973. Complexing
Capacity of the Nutrient Medium and its Relation to Inhibition of Algal
Photosynthesis by Copper, Schweizerische Zeitschrift fur Hydrologie.
35:252-260.
11.Guillard, R.R.L., and J. Lorenzen. 1972. Yellow-Green Algae with
Chlorophyllide-C, J. Phycol. 8:10-14.
12. Guillard, R.R.L., and J.H. Ryther. 1962. Studies on Marine Planktonic
Diatoms I. Cyclotella nana Hustedt and Detonula Confervacea (Cleve)
Gran, Can. J. Microbiol. 8:229-239.
13. Hansen, E.H., C.G. Lamn, and J. Rizicka 1972. Anal. Chim. Acta. 59:403.
14. Jackson, G.A., and J.J. Morgan 1977. Trace Metal-Chelator Interactions
and Phytoplankton Growth in Seawater Media, Limnol. Oceanogr. (in
press).
15. James, R.O., and T.W. Healey. 1972. Adsorption of Hydrolyzable Metal at
the Oxide-Water Interface, J. Colloid and Interface Sci. 40:42.
16. Jensen, A., B. Rystad, and S. Melsom. 1976. Heavy Metal Tolerance of
Marine Phytoplankton II. Copper Tolerance of Three Species in Dialysis
and Batch Cultures, J. Exp. Mar. Biol. Ecol. 22:249-256.
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17. Johnston, R. 1964. Seawater, the Natural Medium of Phytoplankton. II.
Trace Metals and Chelation, and General Discussion, J. Mar. Biol. Assoc.
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18. Jones, G.E. 1967. Precipitate from Autoclaved Seawater, Limnol.
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19. Lange, Willy. 1974. Chelating Agents and Blue-Green Algae, Can.J.
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20. Lockhart, H.B., Jr., and R.V. Blakeley. 1975. Aerobic
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21. McDuff, R.E., and R.M. Morel. 1972. T.R. #EA-73-02, Keck Laboratories,
California Institute of Technology, Pasadena, Calif.
22. McKnight, D.M. Unpublished results.
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25. Morel, F.M., and Morgan, J.J. 1972. A Numerical Method for
Computing Equilibrium in Aqueous Chemical Systems, Envir. Sci. and
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of the Algal Growth Media "Aquil" and "Fraquil", Technical Note No. 16,
Water Quality Lab., Ralph M. Parsons Laboratory for Water Resources and
Hydrodynamics, Dept. of Civil Engineering, Mass. Instit. of Technol.,
Cambridge, Mass.
27. Morel, F.M., R.E. McDuff, and J.J. Morgan. 1973. In: Trace Metals and
Metal-Organic Interactions in Natural Waters, Interactions and Chemostatis
in Aquatic Chemical Systems, P.C. Singer (ed.), Ann Arbor Sci.
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of Pyramimonas 1 by Manipulation of the Trace Metal Chemistry of the
Medium, Technical Note No. 17, Water Quality Lab., Ralph M. Parsons
Laboratory for Water Resources and Hydrodynamics, Dept. of Civil
Engineering, Mass. Instit. of Technol., Cambridge, Mass.
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29. Morel, N.M.L., J.G. Rueter, and F.M. Morel. 1977. Copper Toxicity to
Skeletonema Costatum, J. Phycol. (in press).
30. Murphy, T.P., D.R.S. Lean, and C. Nalewajko. 1976. Blue-Green Algae:
Their Excretion of Iron Selective Chelators Enables Them to Dominate
Other Algae, Science 192:900-902.
31. Pagenkopf, G.K., R.C. Russo, and R.V. Thurston. 1974. Effect of
Complexation on Toxicity of Copper to Fishes, J. Fish. Res. Bd. Can.
31:462-465.
32. Provasoli, L., J.J .A. McLaughlin, and M.R. Droop. 1957. The Development
of Artificial Media for Marine Algae, Arch. Mikrobiol. 25:392-428.
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Standards (USA) Spec. Pub. 314.
35. Rueter, J.G. 1977. The Response of Skeletonema costatum to Copper
Relating to the Question of Medium Conditioning, Master's Thesis, Mass.
Instit. of Technol., Cambridge, Mass.
36. Sillen, L.G., and A.E. Martell. 1964, 1971. Stability Constants, Special
Publication, No. 17 and No. 25, The Chem. Soc. of London.
37. Smayda, T.J. 1974. Bioassay of the Growth Potential of the Surface Water
of Lower Narragansett Bay over an Annual Cycle Using the Diatom
Thalassiosira pseudonana (Oceanic Clone, 13-1), Limnol. Oceanogr.
19:889-901.
38. Smith, R.M., and A.E. Martell. 1976. Critical Stability Constants, Vol. 4,
Inorganic Complexes, Plenum, N.Y.
39. Steemann Nielsen, E., And S. Wium-Anderson 1971. The Influence of Cu
on Photosynthesis and Growth in Diatoms, Physiol. Plant. 24:480-484.
40. Stolzberg, R.J., and D.N. Hume 1975. Rapid Formation of Iminodiacetate
from Photochemical Degradation of Fe(III) Nitrilotriacetate Solutions,
Environ. Sci. and Technol. 9:654.
41.Stumm, W. 1969. Personal Communication to Barber, Referenced in
(1973) Barber, Trace Metals and Metal-Organic Interactions in Natural
Waters, P.C. Singer (ed.), Ann Arbor Science, Ann Arbor, Mich.
60
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42. Stumm, W., Herbert Hohl, and Felix Dalang 1976. Interaction of Metal
Ions with Hydrous Oxide Surfaces, Croatica Chemica Acta. 48:491-501.
43. Stumm, W., and J.J. Morgan 1970. Aquatic Chemistry, Wiley-Interscience,
New York.
44. Sunda, W.G. 1975. The Relationship Between Cupric Ion Activity and the
Toxicity of Copper to Phytoplankton, Doctoral Thesis, Woods Hole
Oceanographic Instit., Woods Hole, Mass.
45. Sunda, W.G., D.W. Engel, and R.M. Thuotte 1978. Cadmium Toxicity to
the Grass Shrimp, Palaemonetes pugio, as a Function of Free Cadmium Ion
Concentration (to be published in Envir. Sci. and Technol.).
46. Sunda, W.G., and R.R.L. Guillard 1976. Relationship Between Cupric Ion
Activity and the Toxicity of Copper to Phytoplankton, J. Mar. Res.
34:511-529.
47. Swallow, K.C. 1977. Adsorption of Trace Metals by Hydrous Ferric Oxide
in Seawater, Ph.D. Thesis, Dept. of Chemistry, Mass. Instit. of Technol.,
Cambridge, Mass.
48. Swallow, K.C., J.C. Westall, D.M. McKnight, N.M.L. Morel, and F.M. Morel
1977. Potentiometric Determination of Copper Complexation by
Phytoplankton Exudates, Limnol. Oceanogr. (in press).
49. Westall, J.C., J.L. Zachary, and F.M. Morel 1976. MINEQL, a Computer
Program for the Calculation of Chemical Equilibrium Composition of
Aqueous Systems, Technical Note No. 18, Water Quality Lab., Ralph M.
Parsons Laboratory for Water Resources and Environmental Engineering,
Dept. of Civil Engineering, Mass. Instit. of Technol., Cambridge, Mass.
50. Yates, D.E., S. Levine, and T.W. Healey 1974. Site Binding Model of the
Electrical Double Layer at the Oxide/Water Interface, Far Soc. I., J. Chem.
Soc. 70:1802.
61
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A SIMPLE ELUTION TECHNIQUE FOR
THE ANALYSIS OF COPPER IN
NEANTHES ARENACEODENTATA
Gerald L. Hoffman and Raymond M. Zanni
U.S. Environmental Protection Agency
Environmental Research Laboratory
South Ferry Road
Narragansett, R.I. 02882
ABSTRACT
It is common practice to dissolve the tissue of marine organisms completely
with acid prior to metal analysis with atomic absorption. However, it may not
be necessary to completely destroy the organic matrix with acids prior to metal
analysis. It has been determined that a simple 5 percent HNO^ elution of a
freeze-dried Neanthes arenaceodentata is sufficient to extract Cu quantitatively
from this marine polychaete. This type of elution, rather than complete
dissolution, has several advantages when analyzing small (1 mg to 10 mg)
organisms. The two major advantages are (1) blank values are lower, and
(2) the technique is less tedious and time consuming.
INTRODUCTION
High temperature ashing and/or various acids (HNO3, H2SO4, HCIO^, etc.)
are generally used to break down, oxidize, and solubilize marine organisms
prior to metal analysis by atomic absorption. If metal levels are high, and the
organisms weigh several grams, the techniques of dry ashing and wet ashing are
usually successful. However, solubilizing individual organisms that weigh 1 mg
to 10 mg with standard techniques without contaminating the final solutions
for the element of interest is difficult.
Matsunaga (1) has shown that it was possible to extract Hg completely from
various types of fish muscle with IN HC1 containing cupric chloride. In his
study, no attempt was made to solubilize the fish muscle tissue. Therefore, we
reasoned that a simple elution with 5 percent HNO3 might be sufficient to
extract metals from small marine organisms. The feasibility of extracting Cu by
this elution technique was tested on the polychaete, Neanthes arenaceodentata.
62
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EXPERIMENTAL METHODS
Apparatus
All atomic absorption analyses were made using a Perkin-Elmer atomic
absorption unit (Model 360) coupled to a Perkin-Elmer heated graphite
atomizer (Model No. HGA-2100). The weight measurements of the worms
were made with a Perkin-Elmer microbalance (Auto balance Model No.
AD-2Z). Freeze-drying of the worms was accomplished using a Virtis (Model
No. 10-145MR-BA) lyophilizer. Low temperature ashing of the samples was
done with a L.F.E. (Model LTA-505) low temperature asher.
Reagents and Materials
Ultrex HNO-j was used throughout the analytical elution and dissolution
procedures. Copper standards were made up from a stock solution of ALPHA
atomic absorption standard copper. All 2/5 dram polyethylene snap-cap vials
were acid washed in concentrated HNO^ for two days, soaked in demineralized
water for two days, and finally rinsed five times with copious quantities of
demineralized water. The vials were allowed to air dry in a class-100 clean
bench.
Procedure
The 61 polychaete specimens used in this study were raised in the
laboratory. Complete details of the methods to raise the worms are given by
Pesch and Morgan (2). Live polychaete samples were removed from the
seawater tanks with the aid of a nylon brush and rinsed in control seawater for
approximately one minute, and then placed in precleaned polyethylene vials
(1.2 ml capacity) fitted with snap-caps. The samples were frozen and then
freeze-dried for 24 hours. The freeze-dried worms were then weighed. The
average weight was 8.7 ± 4.7 mg. One ml of 5 percent HNO3 was added to the
worms in their respective vials. The sample vials were capped and allowed to
stand at room temperature for two days. The worms were then transferred
from the first extraction vial (A) to precleaned tared vials (B) with the aid of a
teflon fiber. The tared vials (B) containing the wet acid leached worms were
again weighed. One ml of 5 percent HNO^ was added to the B vials. The (B)
vials were capped and allowed to stand at room temperature for two days. The
worms were again transferred to pretared vials (C) and weighed wet. The
worms were then freeze-dried and weighed again. During these transfer steps
care was taken so that the worms did not disintegrate. The insoluble worm
carcasses were then destroyed by low temperature ashing. The freeze-dried
worms were inserted into teflon beakers (10 ml capacity) and ashed for 24
hours at the following conditions: O2 flow 50 cc/min; and RF power of 50
63
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watts. After ashing, the inorganic residue was transferred back into the (C)
vials. The transfer was facilitated by adding 0.1 ml of ultra-pure concentrated
HNO3 to the teflon beaker and slowly picking up the inorganic residue into the
drop of HNO3 as it was rolled around the inside of the beaker. The HNO3 does
not wet the inside of the beaker and can be quantitatively transferred into the
polyvial. The (C) vials were capped and allowed to stand at room temperature
for several days to insure dissolution of the particulate residue. One ml of
demineralized water was added to the (C) vials after the dissolution period. The
final acid concentration in the (C) vials was approximately 1.6 N in HNO3. All
three vial solutions (A, B and C) were then analyzed for their Cu content.
RESULTS AND DISCUSSION
Three of the (A) vial solutions were monitored for increases in Cu content
during the first 15 hours of the extraction process. This data is plotted in
Figure 5-1. It can be seen from Figure 5-1 that the extraction appears to be
fairly rapid, and approaches a constant value at 15 hours. This particular data
was the major reason for selecting a two-day elution time for the rest of the
worms processed.
TIME
Figure 5-1. Extraction of Cu from Neanthes arenaceodentata
with 5 percent HNO3 as function of time.
64
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The weight measurements made during the various processing steps allowed
the calculation of the amount of 5 percent UNO3 transferred with each worm
from vial (A) to vial (B), and from vial (B) to vial (C). Therefore, the amount
of solubilized Cu transferred during the transfer steps could be calculated. The
amount of 5 percent HNO3 transferred from the (A) vial to the (B) vials ranged
between 7 and 20 percent of the 5 percent HNO^ present in the (A) vials.
Figure 5-2 shows the calculated mass of Cu that should be present in the (B)
vials versus the measured mass of Cu present in the (B) vials. The solid line
represents a perfect correlation, and is not the calculated regression line. This
plot shows that there is very little copper that cannot be accounted for in the
second elution that has not been transferred from the first solution.
Figure 5-3 shows a plot of the original freeze-dried weights of the worms,
versus the freeze-dried weights of the worms after two elutions in 5 percent
HNOj. It is interesting to note that the worms lost 50.5 ±7.2 percent of their
weight with the two elutions. The Na concentrations were measured in all
samples, and indicated that only about half of the freeze-dried weight loss
could be attributed to the loss of solubilized NaCl. Most of the unexplained
weight loss probably comes from solubilized organic matter. On a qualitative
basis, this was confirmed by the color of the (A) and (B) solutions which were
pale yellow in color, and contained very surface active compounds. Even
though the worms may have been slowly dissolving in the 5 percent HNO3,
only a few of the worms had broken down into two or more pieces, and
generally from a physical appearance looked unchanged.
O.IO
— 0.08
3
tf)
cn Oj06
<
S
a 0.04
3
<
LU
2 0.02
0
0 0.02 0.04 0.06 0.08 0.10
CALCULATED MASS (//g)
Figure 5-2. Calculated mass of Cu in the (B) vials versus the
measured mass of Cu in the (B) vials.
65
-------
14
o>
E
I
o
~ 10
Q
UJ
-------
Using this elution technique it is possible to extract and analyze large
numbers of worms for copper very simply, since only the first extract need be
analyzed. The methods and conditions used in this study should not be
considered to be the ultimate in elution of metals from marine organisms. It
may be possible to use other dilute acids (i.e. HF, HC1, H2SO4, etc.) that may
be more effective for the elution of other metals from different species. If
elution rather than total destruction of the animal matrix is desirable, then a
thorough study should be made of the effectiveness of the procedure chosen.
Dilute acid elution has several advantages over complete destruction of the
sample matrix. The first and most important is the potential for providing
lower blanks. The second is the simplicity involved, which allows processing
100 small organisms in approximately 8 contact hours. The worms need not be
removed from their respective extraction vials prior to analysis, since they sink
to the bottom of the vial and do not interfere with HGA atomic absorption
analysis. The samples do not need to be analyzed within any constrained time
frame. Some samples have been analyzed repeatedly over a period of several
months, and have shown no tendency for a concentration change with respect
to Cu. The worms also do not decompose over this period of time, as they
appear to be permanently preserved. At this time, we have used this elution
technique to analyze over 1000 small marine organisms for Cu.
ACKNOWLEDGMENTS
We thank Ms. Carol E. Pesch and Mr. Douglas Morgan for providing the test
animals for this study, and Dr. P. Rogerson for consultation during the course
of this work.
REFERENCES
1. Matsunaga, K., T. Ishida, and T. Oda 1976. Extraction of Mercury from Fish
for Atomic Absorption Spectrometric Determination. Anal. Chem. 48:1421.
2. Pesch, C.E., and D. Morgan. 1977. Influence of Sediment in Copper Toxicity
Tests with the Polychaete Neanthes arenaceodentata. Submitted for
publication to Water Research.
67
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GEOCHEMISTRY OF FOSSIL FUEL
HYDROCARBONS IN MARINE SEDIMENTS:
SELECTED ASPECTS
John W. Farrington
Associate Scientist
Department of Chemistry
Woods Hole Oceanographic Institution
Woods Hole, Massachusetts 02543
ABSTRACT
Three investigations are described which illustrate recent advances in
analytical chemical and geochemical research on fossil fuel hydrocarbons in the
marine environment.
First: The application of quantitative gas chromatography-mass
fragmentography to measure selected aromatic hydrocarbons in marsh and
coastal sediments. Instrument precisions of 2 to 3% for 50 x 10"^ g of
naphthalene and ) -methylnaphthalene are achieved. The detection limit for
naphthalene (signal/noise ratio of 2:1) is estimated to be 5 x 10"** g/g dry
weight of sediment with 25-50 g dry weight silt-clay coastal sediments. Using
this method No. 2 fuel oil aromatic hydrocarbons incorporated into marsh
sediments were precisely measured in samples taken within one week of a spill,
and eight months after a spill.
Second: Several sections from a core in Buzzards Bay, Massachusetts have
been analyzed for alkanes, cycloalkanes. and aromatic hydrocarbons. This is an
initial attempt at investigating an historical record of anthropogenic fossil fuel
inputs to coastal sediments. The results indicate an increase of an order of
magnitude in concentrations of fossil fuel hydrocarbons from circa 1810 to
1840 to the present. The aromatic hydrocarbon distributions indicate urban air
hydrocarbons as the major source.
Third: The input of fossil fuel hydrocarbons from sewage sludge and dredge
spoils in the New York Bight is discussed. An estimated 3.6 x 10 tons of fossil
fuel hydrocarbons are discharged each year by dumping in this area.
INTRODUCTION
Research concerned with chemical pollutants in the environment can be
most easily divided into two broad areas of investigation: biological effects and
68
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biogeochemistry. The latter is the category encompassing the three fossil fuel
pollution investigations summarized in this paper. Each investigation is, or will
be, the subject of one or more papers, and the reader is referred to these papers
for details and further discussion. Biogeochemical research delves into the
sources, distributions, pathways of transfer, reactions, intermittent and
ultimate fates of pollutants in the environment.
The incorporation of fossil fuel hydrocarbons into surface sediments as a
result of oil spills or chronic effluent releases (2, 6, 15), and the resulting
long-term slow (years) chemical and biochemical removal processes, was a
major finding of oil pollution research between 1969 and 1974. An important
concern evolving from these findings was the question of the distribution and
long-term fate of fossil fuel hydrocarbons in surface sediments. The fossil fuel
components causing the greatest concern were the aromatic hydrocarbons,
although recent research has documented that nitrogen containing compounds
such as p-toluidines and degradation products such as phenalene-l-one, are also
very toxic to certain marine species (18, 19). Thus, there was a need for
investigations of aromatic hydrocarbons in surface sediments. This led to a
search for a means to accurately measure individual aromatic hydrocarbons at
the 1 to 100 x 10"^ g/g dry weight concentration level in sediments.
Quantitative gas chromatography-mass spectrometry or mass fragmentography
has evolved as one of the more discriminating and sensitive methods to apply
to this problem (10, 11,13).
Our quantitative GC-mass fragmentographic method is described in detail in
another paper (8). We have determined the precision of the method as 2 to 3%
based on repeated injections of standard aromatic hydrocarbons for 50 x 10"^
g and about 12% for 1 x 10"^ g. This compares favorably with quantitative gas
chromatographic determinations. However, the GC-MF technique has the very
powerful added advantage of allowing mass spectrum to be scanned to insure
more complete identification of the compounds measured. A comparison of
GC-MF determination of the weight percent of selected aromatic hydrocarbons
in the API reference No. 2 fuel oil with earlier GC measurements (17) is
presented in Table 6-1. We think that the agreement is quite good. We have
applied this technique to measuring selected aromatic hydrocarbons in marsh
sediments exposed to a No. 2 fuel oil spill.
RESULTS AND DISCUSSION
Bouchard No. 65 Oil Spill—October, 1974
On October 12, 1975 the Bouchard Barge No. 65 spilled No. 2 fuel oil into
Buzzards Bay, Massachusetts. A small amount of this oil entered Windsor Cove
69
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Table 6-1. Weight Percent of Selected Aromatic
Hydrocarbons in Fuel Oils.
Naphthalene
Cj-Naphthalenes
C^-Phenanthrenes
C2"Phenanthrenes
API No. 2 Fuel Oil
{Present Study)
-
2.0 ± 0.3
0.24 ± .02
0.23 ±.03
API No. 2 Fuel Oil
(Warner, Ref. 17)
0.40
2.7
0.27
0.19
Bouchard Barge No. 65
No. 2 Fuel Oil Spilled
October, 1974.
Buzzards Bay, Mass.
0.17 ±.01
0.95 ± .05
0.37 ± .02
0.33 ± .04
(Figure 6-1) and a sheen of oil with accompanying fuel oil odor was present in
some marsh and intertidal areas of the cove. We selected two sites for a small
study of the long-term fate of this fuel oil in sediments; a marsh area and an
intertidal area. The locations of these sites were carefully recorded, and cores
have been taken every fall in October, and every spring in May or June since
October, 1974. We did not intend, nor do we pretend, to offer an in-depth
study of the geographical extent of the spill or long-term fate at several stations
as was conducted for the West Falmouth oil spill (2, 3, 5). Funding, manpower,
laboratory space and other commitments to oil pollution research prevented
such a study. Also, it was our understanding that Commonwealth of
Massachusetts laboratories were conducting a survey of the geographical
extent, and long-term fate of the oil.
Our intent was to compare the long-term fate of the oil at the two locations
described, with earlier studies of the West Falmouth oil spill. In essence, there
was a near duplicate experiment in progress. The West Falmouth oil spill
involved No. 2 fuel oil spilled in late September, 1969 a few miles away from
where the Bouchard Barge No. 65 spilled No. 2 fuel oil in October, 1974
(Figure 6-1). Was the West Falmouth oil spill really unique with respect to
longevity of the spilled oil in marsh and intertidal sediments as some have
suggested? This was the primary focus of our investigation. The complete set of
data of our study will be presented elsewhere. We have applied the GC-MF
technique to a set of marsh cores from October, 1974 and May, 1975. This
data is presented in Table 6-2. Note that the concentrations of aromatic
hydrocarbons in the 14-18 or 15-20 cm core section are the concentrations
present in marsh sediments prior to the spill. The concentrations of aromatic
hydrocarbons in the surface sediments, 0-6 cm and 0-5 cm, clearly show at
least two orders of magnitude elevation in concentration as a result of the fuel
oil spill; and elevated concentrations are still present in May, 1975, although
they have decreased by a factor of about 5 to 6. The longevity of the aromatic
hydrocarbons in the marsh sediment is still under investigation.
70
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Figure 6-1. Buzzards Bay, Massachusetts.
71
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Table 6-2, Concentrations of Naphthalenes/Phenanthrenes in
Windsor Cove Sediments (jug/gram dry wt. sediment).
Component
Oct.
Depth
MARSH
Cone.
May
Depth
Cone.
Naphthalene
0-6 cm
9.2
0-5 cm
0.63
14-18 cm
0.024
15-20 cm
0.011
^-Naphthalenes
0-6 cm
370
0-5 cm
41
14-18 cm
1.1
15-20 cm
0.33
C2"Naphthalenes
0-6 cm
1380
0-5 cm
260
14-18 cm
5.1
15-20 cm
1.5
Cg-Naphthalenes
0-6 cm
3040
0-5 cm
590
14-18 cm
12
15-20 cm
3.4
C^-Phenanthrenes
0-6 cm
500
0-5 cm
97
14-18 cm
2.2
15-20 cm
0.94
C2"Phenanthrenes
0-6 cm
480
0-5 cm
96
14-18 cm
2.2
15-20 cm
1.1
The data in Table 6-2 demonstrate the usefulness of the technique of
quantitative GC-MF analysis in biogeochemieal studies of oil spills. The data
also demonstrates that fuel oil aromatic hydrocarbons have survived for at least
seven months in the marsh sediments in concentrations well above background.
Thus, for this time period, the West Falmouth spill was not unique. Further
investigation will determine if the parallel between the fate of the petroleum
compounds from the two oil spills will continue.
Historical Record of Fossil Fuel Hydrocarbons
in Buzzards Bay, Massachusetts
Our gas chromatographic measurements of hydrocarbons in surface
sediments at several locations in Buzzards Bay, Massachusetts indicated the
presence of an unresolved complex mixture of hydrocarbons with a wide
molecular weight range, indicating that these hydrocarbons might be from
chronic oil pollution (8). However, there were several other sources such as
natural diagenetic processes and weathering of ancient sediments to be
considered (8). In order to assist in evaluating the source of these
72
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hydrocarbons, we obtained sediment cores at several locations in coastal areas
of the western North Atlantic (8). At Station P Pb-210, geochronology
measurements were obtained (7). These measurements and the measurements
of Pu-239/240 and Cs-137 at this same location by others (4) allowed us to
estimate sedimentation rates. We then measured hydrocarbon concentrations in
several core sections at Station P. We also applied quantitative GC-MF analyses
to measure phenanthrene and Cj and C2 phenanthrenes. The results of these
measurements, as reported in (8), are given in Table 6-3. It is clear that circa
1900 concentrations of hydrocarbons constituting the unresolved complex
mixture increased, as did the concentrations of phenanthrenes. The ratios of
the Cj and C2 phenanthrenes are not those found in spilled oil. Instead, the
ratios indicate that these aromatic hydrocarbons are from pyrolytic sources
(13, 20). Our hypothesis is that these hydrocarbons are primarily from direct
and remobilized urban air hydrocarbons (8). We have determined that there is a
trend of decreasing concentrations of UCM hydrocarbons with increasing depth
in a core at another station in Buzzards Bay (8), and a station in the Gulf of
Maine (Figure 6-2).
Furthermore, similar results have been reported for Lake Washington,
Seattle, Washington sediments (16). A much more detailed analysis of the
aromatic hydrocarbons in three sections of another core from Station P,
Table 6-3. Hydrocarbons and Chlorinated Hydrocarbons
in Station P Core Sections
Section
Average Time
of Deposition
UCMa
(A*g/g)
Phenanthrenes
(ng/g)
C0 C1
c2
0-1 cm
74
NAb
44
38
1-2 cm
105
34
27
28
8-12 cm
1940
44
NA
31
29
20-24 cm
1900
12
15
11
10
54-58 cm
1790
5.2
3.7
2.9
2.8
58-62 cm
1780
6.2
NA
NA
NA
aMixture of alkanes and cycloalkanes — indicates petroleum hydrocarbons.
L
NA — not analyzed.
73
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Figure 6-2. Gulf of Maine.
74
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Buzzards Bay, have recently been completed (12). These analyses, also by
GC-MF, greatly extend the earlier analyses for the Buzzards Bay station, and
establish that polynuclear aromatic hydrocarbons (PAI1) from a combustion
source increase by at least an order of magnitude in sediment deposited after
about 1850. Another recent paper (14), has reported a detailed study of PAH
in a core from Lake Constance in the Federal Republic of Germany. The
surface sediments of this core contained increases of PAH concentration of 50
to 100 times that of sediments deposited circa 1800. The PAH composition
again indicated a pyrolytic source.
The implications of these findings are that roastal and lacustrine
environments, especially the benthic ecosystems, have been exposed to
increased PAH concentrations over the past several decades. Whether or not
this chronic long-term increase in PAH concentration reflects a substantial
environmental risk is not known, and a detailed discussion is beyond the scope
of this paper. It is important, though, to consider that many of the PAHs are
known to have adverse effects on marine organisms (13). The benthic
ecosystems may have been "stressed" by PAH pollution for some time. This is
an important point to keep in mind when considering control stations for
studying oil spills in coastal areas. For example, the control stations for the
studies of the effects of the West Falmouth oil spill on subtidal benthos were
not very far from the two stations, P and D, we have sampled in Buzzards Bay.
Does this mean that these stations are truly "normal" with respect to the
effects of aromatic hydrocarbons, or have they also been subtly, chronically
affected by the increasing amounts of PAH deposited from direct and
remobilized urban air PAH?
New York Bight Surface Sediments
We have estimated the rate of fossil fuel hydrocarbons discharged by
dumping in the New York Bight is about 3.6 x 10^ tons/year (9), or about 2%
of the estimated global discharge of 180 x 10^ tons per year of petroleum
hydrocarbons from routine operations and spills associated with outer
continental shelf drilling and production (3). The composition of PAH in the
New York Bight dump site surface sediments indicates that these hydrocarbons
are primarily of pyrolytic origin (13). The fossil fuel hydrocarbons most likely
are from urban air fallout, and are swept into storm sewers and municipal
sewers by rain water, and are either discharged to New York harbor or become
associated with the sewage sludge in the treatment plants. Dredge spoils from
the harbor and sewage sludge are then dumped in the New York Bight resulting
in delivery of PAH and other pollutants to the continental shelf area. Since
there are other dump sites off the East coast of the U.S., the input of
petroleum hydrocarbons from this source must be larger than in the New York
Bight alone. Thus, significant and measurable quantities of contaminant
75
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hydrocarbons are already being deposited in continental shelf areas off the
eastern United States before Outer Continental Shelf oil and gas drilling and
production have begun. This must be taken into account when assessing
potential environmental impacts of OCS operations, now and in the future.
SUMMARY AND GENERAL DISCUSSION
Aromatic hydrocarbons are incorporated into surface sediments as a result
of oil spills, and the chronic dribbling of urban air hydrocarbons into the
marine environment. These compounds are known to have adverse effects on
marine organisms under certain conditions. The challenge posed is to conduct
experiments which will investigate how bottom current resuspension,
bioturbation by animals, and long-term microbial and chemical processes act
individually and collectively on the aromatic hydrocarbons in surface
sediments. Are these compounds in the sediments incorporated into benthic
organisms? At what rate and under what conditions? We need to relate
chemical analyses by some means to biological availability.
Some recent investigations conducted on a short-term two-week exposure of
sipunculid worms suggest that naphthalenes can be ingested from naphthalene
contaminated sediments (1). Two weeks of "depuration" in a clean
environment removed all measurable quantities of naphthalenes from the
worms (1). The exposure time was very short. What happens when exposure of
the benthic organism is continuous for years, as is probably the case for low
concentrations of polynuclear aromatic hydrocarbons in Buzzards Bay, and
higher concentrations near the New York Bight area?
ACKNOWLEDGMENTS
I wish to thank R.A. Hites, R.E. LaFlamme, B.W. Tripp, N.M. Frew and J.M.
Teal for enjoyable collaboration and discussion of much of the research
discussed and referenced in this paper. This paper presents a synopsis of several
papers and acknowledgments to agencies providing financial support are found
in the referenced papers.
The compilation of data and writing of this paper were supported by U.S.
Environmental Protection Agency Grant R803902. This paper is Contribution
Number 4041 of the Woods Hole Oceanographic Institution.
REFERENCES
1. Anderson, J.W., L.J. Moore, J.W. Blaylock, D.L. Woodruff and S.L.
Kiesser. 1977. Bioavailability of Sediment-Sorbed Naphthalenes to the
Sipunculid Worm, Phascolosoma agassizzii. Chapter 29 in Fate and Effects
76
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of Petroleum Hydrocarbons in Marine Organisms and Ecosystems, Wolfe,
D.A. (ed.), Pergamon Press, New York.
2. Blumer, M. and J. Sass. 1972. Oil Pollution: Persistence and Degradation of
Spilled Fuel Oil. Science 176:1120.
3. Blumer, M. and J. Sass. 1972. The West Falmouth Oil Spill, Data Available
1977. II. Chemistry Technical Report No. 72-19, Woods Hole
Oceanographic Institution, unpublished manuscript.
4. Bowen, V.T. 1975. Transuranic Elements in Marine Environments. In: U.S.
Energy Research and Development Administration Health and Safety
Laboratory Report HASL-291 April 1,1975, pp. 157-179.
5. Burns, K.A. and J.M. Teal. 1971. Hydrocarbon Incorporation into the Salt
Marsh Ecosystem from the West Falmouth Oil Spill. Technical Report No.
71-69, Woods Hole Oceanographic Institution, unpublished manuscript.
6. Farrington, J.W. and J.G. Quinn. 1973. Petroleum Hydrocarbons in
Narragansett Bay. I. Survey of hydrocarbons in Sediments and Clams
(Mercenaria meacenaria). Estuarine and Coastal Marine Science 1:71.
7. Farrington, J.W., S.M. Henrichs and R. Anderson. 1977. Fatty Acids and
Pb-210 Geochronology of a Sediment Core from Buzzards Bay,
Massachusetts. Geochemica et Cosmochimica Acta 41:289.
8. Farrington, J.W., N.M. Frew, P.M. Gschwend and B.W. Tripp. 1977.
Hydrocarbons in Cores of Northwestern Atlantic Coastal and Continental
Margin Sediments. Estuarine and Coastal Marine Science.
9. Farrington, J.W. and B.W. Tripp. 1977. Hydrocarbons in Surface
Sediments of the Western North Atlantic. Geochimica et Cosmochimica
Acta 41:1627-1641.
10. Frew, N.M. and J.W. Farrington. 1977. Mass Fragmentographic
Determination of Aromatic Hydrocarbons in Marsh and Coastal
Sediments. Unpublished report.
11. Hase, A., P.H. Lin and R.A. Hites. 1976. Analysis of Complex Polynuclear
Aromatic Mixtures by Computerized GC/MS. In: Carcinogenesis, Jones,
P.W. and Freudenthal, R.I. (eds.), Raven Press, N.Y., Vol. I.
12. Hites, R.A., R. LaFlamme and J.W. Farrington. 1977. Polycyclic
Aromatic Hydrocarbons in Recent Sediments: The Historical Record.
Science 198:829-931.
77
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13. LaFlamme, R.E. and R.A. Hites. 1977. The Global Distribution of
Polycyclic Aromatic Hydrocarbons in Recent Sediments. Geochimica et
Cosmochimica Acta (in press).
14. Miller, G., G. Grimmer and H. Bohnke. 1977. Sedimentary Record of
Heavy Metals and Polycyclic Aromatic Hydrocarbons in Lake Constance.
Die Naturwissenshaften (in press).
15. National Academy of Sciences. 1975. Petroleum in the Marine
Environment. Washington, D.C.
16. Wakeham, S.G. and R. Carpenter. 1976. Aliphatic Hydrocarbons in
Sediments of Lake Washington. Limnology and Oceanography 21: 711.
17. Warner, J.S. Battelle Memorial Institute. Unpublished data.
18. Winters, K., R. O'Donnel, J.C. Batterton and C. Van Baalen. 1976.
Water-soluble Components of Four Fuel Oils: Chemical Characterization
and Effects on Growth of Microalgae. Marine Biology 36:269.
19. Winters, K., J.C. Batterton and C. Van Baalen. 1977. Phenalene-l-one:
Occurrence in a Fuel Oil and Toxicity to Microalgae. Marine Biology (in
press).
20. Youngblood, W.W. and M. Blumer. 1975. Polycyclic Aromatic
Hydrocarbons in the Environment: Homologous Series in Soils and Recent
Marine Sediments. Geochimica et Cosmochimica Acta 39:1303.
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IDENTIFICATION OF ENVIRONMENTAL
GENETIC TOXICANTS
WITH CULTURED MAMMALIAN CELLS
Alexander R. Malcolm, Robert R. Young,
and Carolyn J. Wilcox
U.S. Environmental Protection Agency
Environmental Research Laboratory
Narragansett, R.I. 02892
ABSTRACT
Experiments designed to detect small-scale mutations leading to auxotrophy
were carried out in vitro with the Chinese hamster ovarian (CHO) cell system
(5-bromodeoxyuridine/visible light selection) initially described by Puck and
Kao (43). The system was standardized with ethylmethanesulfonate (EMS), a
known mutagen previously demonstrated to be active in CHO cells (27), and
5-bromodeoxyuridine (BrdU), another known mutagen (7) utilized in the
selection procedure, but not previously evaluated for mutagenic activity in the
CHO Cell/BrdU-VL assay. Both EMS and BrdU routinely yielded glycine,
hypoxanthine or triple-requiring (glycine/hypoxanthine/thymidine)
auxotrophs and showed dose response. For a series of inorganic compounds
known to be or suspected of being genetic toxicants, statistically significant
numbers of auxotrophs were obtained only with the chloride salts of cadmium
and manganese. Neither cadmium nor maganese were consistently mutagenic,
cadmium showing activity in about 20 percent of experiments, manganese in
50 percent of experiments. It was not possible to demonstrate dose response
with these compounds. A water extract of JP-5 jet fuel was also found to be
mutagenic in a single test. Variant cell types other than auxotrophs were
isolated from cell populations treated with three different carcinogenic agents
(EMS, CrOg, PbAc2-3H20) but not from control experiments. These cells,
exhibiting either a rounded cell morphology or potential contact inhibition,
may reflect mutation in additional loci of possible value as genetic markers.
Other data are presented to illustrate special problems associated with the
application of in vitro cell systems.
INTRODUCTION
Serious concern for the possible effects of genetic toxicants in the
environment developed approximately a decade ago with the discovery of
79
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chemical mutagens capable of inducing high frequencies of mutation at high
levels of survival (17). Concern also stemmed from the realization that man was
greatly expanding the number of compounds theoretically capable of
increasing mutation frequencies beyond present 'spontaneous' levels. As
recently stated at an open meeting sponsored by the United States Department
of Health, Education and Welfare on the value of selected test systems to
detect and assess the mutagenic activity of chemicals (21), a human disease
burden exists which is of genetic origin. Increases in mutation frequency can be
expected to enlarge this burden, and many classes of chemicals already in the
environment are known to include genetic toxicants. Although uncertainty
remains regarding the precise impact such compounds might have upon human
health, there is justification for apprehension (15).
Genetic toxicology, a new branch of toxicology concerned with the
identification and evaluation of DNA-damaging agents (carcinogens, mutagens
and some teratogens), may be broadly divided into (a) screening tests for
identification of potential toxicants, (b) procedures for estimating risk, and (c)
techniques for population monitoring. Screening involves primarily the use of
rapid, inexpensive assays which detect agents capable of damaging or altering
DNA. Because DNA is chemically and structurally similar in most organisms,
and is considered the probable target of genetic toxicants, any organism or
appropriate part thereof may be theoretically employed as a screening tool.
Accordingly, viruses (18), a variety of microbial systems (3,36), cultured
animal cells (14, 39, 26), Drosophila (2, 48), and various subcellular assays
designed to measure effects directly on DNA (47, 49) are widely used for
screening purposes. Several short-term tests utilizing intact mammals are also
available for screening (31).
This paper is concerned with the application of an in vitro mammalian cell
assay utilizing nutritional markers as an indicator system for genetic toxicants
detected as mutagens. Major objectives are to (a) outline techniques for
measuring the acute toxicity of chemicals to cultured cells, (b) qualitatively
describe the CHO Cell/BrdU-VL system as an assay for mutation, (c) present
data relative to the mutagenic potential of a series of compounds known to
accumulate in the tissues of edible marine organisms (41, 45, 52) or which have
been associated with the occurance of neoplasias in such organisms (8, 51), and
(d) illustrate some additional end points, as well as some potential problems,
pertinent to the application of in vitro cell assays. A detailed description of
equipment, reagents, special techniques and experimental procedures relevant
to the CHO Cell/BrdU-VL system will not be given here. A general protocol for
this assay has been published by Kao and Puck (28). Our modifications to their
procedure will be described elsewhere (33).
80
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METHODS
Acute toxicity
Before compounds can be evaluated for mutagenic activity, their acute,
physiological toxicity must be determined. This is accomplished by measuring
the ability of single cells to produce macroscopic colonies arising in
experimental dishes following exposure to specific concentrations of the test
agent for specified periods of time. Relative plating efficiency (RPE), defined
as the ratio of macroscopic colonies arising in experimental dishes, to those
appearing in controls, may be plotted against dose to yield survival curves of
the type shown in Figure 7-1. Concentrations of compound to be tested for
genetic activity are selected from such curves. The exponential portion of each
curve is described by equation [1] where (S/SQ) is the surviving cell fraction or
percent RPE, (n) is the hit or target number (30) and (D/DQ) is relative dose
(44). The target number is defined operationally by the intersection of the
exponential portion of the curve with the ordinate axis when the former is
extrapolated back. Relative dose is defined as the ratio of the experimental
molar concentration of toxicant (D) to that increase in molar concentration
(Dq) required to reduce the cell population by the fraction (1/e). The value of
Dq is, for each compound, obtained from a plot of molar concentration versus
surviving cell fraction. Because chemicals differ in their molar toxicity by
orders of magnitude,1 relative dose provides a convenient way to depict survival
data for many compounds simultaneously. It is noted that the random hit
model expressed by equation [1] was derived for radiation effects (30), and
requires interpretive modifications when describing cell inactivation by
chemicals (32). The use of plating efficiency to assess acute chemical toxicity
has been described elsewhere (34).
= [i]
The CHO Cell/BrdU-VL System
Figure 7-2 represents a simplified and generalized protocol for inducing,
isolating and characterizing mutant cells. Initially, cells are inoculated into
dishes or flasks and allowed to attach to the plastic substratum. Following
attachment, cells are exposed to the test agent at one or more concentrations.
The cells are then washed free of the test compound and fresh medium added.
During the expression period, cells are grown under nonselective conditions,
permitting induced genetic damage to become fixed into DNA, and ultimately
to become expressed at the cellular level. The length of the expression period is
a function of the system employed, the genetic markers involved, and the
conditions of the experiment. Selection represents the application of a set of
conditions permitting mutant cells to survive while eliminating wild-type cells.
Selective conditions employed are specific for the type of mutant sought. Once
potential mutants have been isolated, they may be subjected to genetic analysis
for confirmatory purposes and for further characterization.
81
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RELATIVE DOSE
Figure 7-1. Typical 16-hour survival curves for five test compounds.
Note: The curves are constructed from relative plating efficiency data by plotting the surviving cell fraction (S/S0) against relative
dose (D/D0). Concentrations or doses of toxicant employed in mutagenesis experiments are selected from such curves.
-------
GENERALIZED AND SIMPLIFIED PROTOCOL FOR POINT MUTATION IN CELLULAR SYSTEMS
CELL INOCULATION
AND
EXPOSURE TO
TEST AGENT
MUTANT
EXPRESSION
MUTANT CELL
SELECTION
MUTANT CELL
ANALYSIS
ATTACHMENT
Figure 7-2. A generalized and simplified protocol for the
induction, isolation and characterization of mutations
in cellular systems.
Figure 7-3 represents a protocol containing the same basic features as that in
Figure 7-2, but is specific for the CHO Cell/BrdU-VL system. In this technique,
CHO-K1 cells are cultured in two types of media. One medium (F12D)
contains the minimal nutritional requirements for the growth of single cells
into macroscopic colonies with high efficiency. A second, enriched medium
(F12) is constructed from the minimal by addition of nine nutrilites (alanine,
glycine, aspartic acid, glutamic acid, lipoic acid, vitamin Bj2> inositol,
A B
chemical
mutant cell
selection
5-bromodeoxyuridine
colony of
mutant =
cells
exposure
to
white
light
mutant cell growth
< ©
E D
Figure 7-3. Schematic representation of the Chinese hamster
ovarian cell system (BrdU-visible light selection procedure) for
the detection of small-scale mutations.
Note: (A) Population of wild-type cells. (B) Nutritional mutant (auxotroph)
following induction and expression. (C) Wild-type cells with BrdU-containing
DNA. (D) Surviving mutant cell following selective elimination of wild-types
via the combined action of BrdU and white light. (E) Colony of mutant cells
which grew in enriched medium. (After Kao and Puck (28)).
83
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thymidine and hypoxanthine) not required exogenously by the cells for
optimal growth. Mutants are detected by screening populations of cells
exposed to test compounds for nutritionally deficient forms (auxotrophs)
requiring one or more of the nine nutrilites omitted from F12D medium.
Operationally, 10 cells are exposed in four parallel cultures to single or
multiple doses of the test agent, following inoculation and cell attachment
(point A, Figure 7-3). Because even induced mutation is a rare event, there will
generally be, following expression, a small number of mutant cells growning
among millions of nonmutants in enriched medium. To identify the mutants, it
is necessary to introduce a procedure which will eliminate the prototrophic
(wild-type) cells, while allowing auxotrophs to survive. This is accomplished by
taking advantage of the fact that mutants auxotrophic for one or more of the
nine nutrilites omitted from F12D medium will be unable to grow in this
medium, whereas wild-types will. Thus, at point B, Figure 7-3, F12 medium is
replaced with F12D medium. This initiates the selective process by effectively
terminating protein and nucleic acid synthesis. The thymidine analog, BrdU, is
then added to the F12D medium from which it is incorporated into the DNA
of wild-type cells (point C, Figure 7-3). Subsequent illumination of the cell
population with white light is lethal to those cells having incorporated
sufficient BrdU. Mutant cells do not incorporate BrdU, and survive the
selective process (point D, Figure 7-3). Wild-type cells survive selection to an
extent approaching 0.02 percent. In the presence of F12 medium, mutant cells,
along with some wild-types, grow into macroscopic colonies (point E, Figure
7-3). These are picked and tested for mutant identification. It is important to
note that the use of a known mutagenic agent (BrdU) in the selective process is
of no consequence, as the only cells incorporating BrdU are wild-types destined
for death. Mutants to be isolated are existent in the population at the end of
the expression period prior to the application of selective conditions.
The procedure illustrated in Figure 7-3 and described above was applied in
collecting the mutagenesis data presented below. Figure 7-4 shows the lighting
apparatus used to illuminate cell populations following exposure to BrdU.
Figure 7-5 shows cell survival in four randomly selected dishes several days
after illumination. Cells not killed in selection have grown into macroscopic
colonies. Because mutant and wild-type cells differ only in their requirements
for exogenous nutrilites, they may be distinguished only by analysis of their
growth properties in enriched and deficient media or by biochemical analysis.
Statistical Analysis of Data
When testing compounds for mutagenic activity, we usually want to know if
the number of mutants per unit number of viable cells screened is sufficiently
larger in experimental, versus control situations, to support a conclusion of
84
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Figure 7-4. Illumination apparatus for the inactivation of cells
with BrdU-containing DNA.
Note: Plastic culture dishes containing 2 x
white fluorescent light for 60 minutes.
10 cells each are subjected to
Figure 7-5. Macroscopic colonies in four randomly selected dishes
approximately seven days after illumination with white light.
Note: These clones, originating from cells surviving selection, are tested to
determine if any are auxotrophic mutants.
85
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induced mutation by the test compound. In the CHO Cell/BrdU-VL assay, each
cell screened will either be auxotrophic for one or more of the nine nutrilites
omitted for F12D (operational definition of mutant), or it will not be
(operational definition of wild-type). The actual criteria employed to classify
mammalian cell variants as true mutants are somewhat complex, and have been
reviewed recently (16, 40, 46). For the purpose of this paper, the operational
definitions given above shall be used.
In the standard procedure, a sample of 10^ cells from each test or control
population is distributed among fifty 60 mm dishes, and subjected to the
selective process. Surviving clones are then sampled and tested for the presence
of auxotrophs. The total number of auxotrophs expected per 10^ viable cells is
then estimated from the data by equation [2], where (y) is the estimated
number of auxotrophs, (x) is the number of auxotrophs observed, (n) is the
number of replica experiments, (A) is the total number of cells surviving
selection, (B) is the total number of colonies picked and tested, (C) is the
initial number of 60 mm dishes, (D) is the final number of dishes (some may be
lost to contamination during the course of the experiment), and (E) is the
absolute plating efficiency (defined as the ratio of macroscopic colonies
produced to cells inoculated) as measured in low density control dishes.
Because mutants are randomly distributed among wild-types in mixed
populations, the probability that any given survivor will be a mutant should be
constant over all survivors. Moreover, as only a small number of mutants is
generally found in any given population, the distribution of mutants in such
populations should be Poisson. Accordingly, mutagenesis data from sets of
replica experiments were tested for goodness of fit to a Poisson model and
found to be consistent with this type of distribution (33).
For two independent Poisson variables (X, Y), a new statistic (V) has been
proposed by Best (9) for testing the difference between two Poisson
expectations (e.g., the estimated mean number of mutants in experimental (X)
versus control (Y) populations). This statistic, given by equation [3], is similar
in performance to the more familiar square root of the Poisson Index of
Dispersion (20), except in the tails of the distribution where (V) is superior.
Although (V) is a function of the Poisson variables (X, Y), (V) itself shows an
approximately normal distribution. This statistic may be pa-rticularly applicable
to mutagenesis data where the difference in variance observed between
experimental and control populations is large. This is the situation at the
present time with the CHO Cell/BrdU-VL system where mutants are rarely
observed in control populations. All mutagenesis data considered below were
scaled via equation [2] and compared to an historical control (Y) in
accordance with equation [jjand appropriate confidence limits. The model given by
86
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equation [3] is at present a proposed one and may not be the final model of
choice.
(y)
(V)
(x/n) [(A)/(B)3 [(C)/(D)(E)] [2]
(2X + 3/4)1/2 - (2Y + 3/4)l/2 [3]
RESULTS
Control Investigations
With the exception of the spontaneous proline auxotroph isolated as the K1
subclone of the CHO cell (44), other spontaneous auxotrophs with
requirements for one or more of the nutrilites omitted from F12D medium had
not been previously reported for the CHO Cell/BrdU-VL system. Such
auxotrophs could easily be suppressed in stock cell populations by maintaining
cells in minimal rather than enriched medium. This was not done in order to
determine if spontaneously arising auxotrophs could indeed be identified in
control or stock populations. Table 7-1 summarizes data from 16 different
control experiments carried out over a period of several months. Two glycine
mutants were identified among 989 clones picked and tested. The observed
frequency of spontaneous auxotrophy is thus two mutants per 1.2 x 10^ viable
cells. These data, in combination with data for the other parameters of
equation [2], were utilized to obtain an estimate of 0.331 mutants per 10^
viable control cells. This value was substituted for (Y) in equation [3].
Table 7-1. Summary Mutagenesis Data From Several
Control Experiments
(n)
Viable
[(A)/(B)]
[(C)/(D)(E)]
Auxotrophys isolated (y)
cells
Gly Hyp (x)
16
1.2 x 107
1888/989
(800)/(770) (.750)
2 0 2 0.331
The scaling of (x) by equation [2] does not consider the fact that mutant
cells may be lost to the effects of starvation during selection. In fact,
reconstruction experiments employing known numbers of mutants have
demonstrated that this type of loss does occur for the three types of
auxotrophs observed (27, 33). Consequently, equation [2] underestimates
actual mutant frequencies.
87
-------
Induced Mutation with Standard Mutagens
The standard mutagens, EMS and BrdU, were evaluated for mutagenic
activity at several doses. Data pertinent to the mutagenicity of these
compounds are presented in terms of the parameters of equation [2] in Table
7-2. The estimated number of mutants per 10^ viable cells (7) is plotted in
Figure 7-6 as a function of relative dose. By expressing the mutagenesis data in
terms of mutant cell frequencies per 10^ viable cells, meaningful comparisons
between different compounds or different doses of the same compound could
be made. Applying the data to equation [3], EMS was mutagenic at all doses
tested (a< 0.05). BrdU was mutagenic at five of seven doses tested, and
produced a complex dose-response pattern similar to those observed with
hycanthone methanesulfonate, and other compounds in different assays (12,
13).
Induced Mutation with Other Compounds
Forward mutation experiments employing doses of toxicant generally
yielding 20 percent survival or greater were carried out in replica with several
inorganic compounds, and an aqueous extract of JP-5 jet fuel. The data are
presented in Table 7-3 in terms of the parameters of equation [2]. These
compounds or mixtures were selected for evaluation as mutagens because they
were either known to have, or were suspected of having, carcinogenic
properties. Their mutagenic response in the CHO Cell/BrdU-VL system can be
divided into three classes: (1) Experiments with the oxides of arsenic and
selenium, lead acetate, and the chloride salts of cobalt and nickel, failed to
produce any auxotrophs; (2) tests with beryllium and chromium usually
produced auxotrophs, but never in sufficient numbers to support a conclusion
of induced mutation; (3) Experiments with cadmium chloride, manganese
chloride, and an aqueous extract of JP-5 jet fuel, also produce auxotrophs,
sometimes in sufficient numbers to suggest induced mutation by these
compounds. As indicated in Table 7-3, it was possible to obtain relatively large
numbers of auxotrophs with CdC^- Usually, however, observed mutant
frequencies were low. Cadmium chloride was found to be significantly
mutagenic in about 20 percent of experiments, as was the extract of JP-5 jet
fuel. Manganous chloride was observed to be mutagenic in approximately 50
percent of experiments.
Isolation of Nonauxotrophic Variants
Two classes of variants, other than auxotrophs, were isolated from cell
populations treated with known genetic toxicants. One class consists of cells
exhibiting a rounded morphology, and represents cells unable to stretch out on
88
-------
Table 7-2. Data From Forward Mutation Experiments With EMS and BrdU
Experiment
Molarity
(n)
Viable
[(A)/(B)]
[{C)/( D HE)]
Auxotrophs isolated
(y)
(D/D0)
Cells
Gly
Hyp
(x)
1 (EMS)
1.50 x 10"3
0 17
4
2.5 x 10®
339/138
(200)/(183) (0.625)
3
8
11
11.8**
2 (EMS)
1.75 x 10"3
4
2.4 x 106
605/173
(200)/( 180) (0.603)
8
3
11
17.6**
2.54
3 (EMS)
3.00 x 10"3
4
2.8 x 10®
436/163
(200)/(200) (0.703)
44
2
46
43.8**
4.35
1 (BrdU)
7.00 x 10"5
4
1.6 x 106
949/361
{200) /(180) (0.400)
2
1
3
5.5*
1.42
2 (BrdU)
1.00 x 10"4
1
1.2 x 105
169/081
(050)/(050)(0.120)
4
1
5
86.9**
2.45
3 (BrdU)
1.30 x 10"4
1
1.6 x 105
161/046
(050)/(034){0.160)
0
0
0
0.0
3.62
4 (BrdU)
2.40 x 10'4
1
21 x 105
248/127
(050)/(050)(0.210)
0
0
0
0.0
4.90
5 (BrdU)
3.50 x 10"4
1
3.2 x 105
326/145
(050)/(048) (0.320)
2
1
3
22 1 * *
7.14
6 (BrdU)
4.60 x 10"4
1
2.8 x 105
214/128
(050(/(050) (0.280)
1
4
5
30.1**
9.38
7(BrdU)
5.70 x 10"4
1
7.2 x 105
160/109
(050)/( 049) (0.720)
6
1
7
14.5**
11.63
* Denotes significance at the 95% confidence level
; ** denotes significance at the 99% confidence level.
-------
RELATIVE DOSE
Figure 7-6. Mutation frequency induced by EMS and BrdU
as a function of dose.
the plastic substratum and assume the standard epithelial morphology (Figure
7-7). Although this type of cell produces colonies of sufficient size to permit
cloning, the cells are continuously in a rounded state, as if entering mitosis.
When grown in medium containing 10 percent fetal calf serum, some of the
clones assume a more normal morphology, and may represent mutants with
increased serum requirements (27). To date, this type of variant has been
observed only in populations treated with EMS, BrdU, and compounds of
chromium, cadmium and lead.
A second type of variant, appearing to possess the property of contact
inhibition (1, 19), was isolated from cell populations treated with the known
carcinogens, EMS, CrOg and PbAc2'3H20. These were detected as wild-type
cells surviving selection, and which possessed a pronounced fibroblastic
morphology (Figure 7-7). When cells were grown into confluent monolayers,
unlike the transformed CHO-K1 cell, they ceased to divide and assumed a state
of contact inhibition, or a state resembling that of contact inhibition. Dense
monolayers remained for as long as two weeks without medium changes, and
without significant deterioration. Confluent sheets of cells could be easily
trypsinized and dispersed into uniform, single-cell populations. Upon replating
in fresh growth medium, cells grew with a generation time of approximately 14
hours, ceasing to divide when the monolayer again became confluent.
90
-------
Table 7-3. Data From Forward Mutation Experiments with Selected Additional Compounds
Chemical
Molarity
(n)
Viable
[(A)/(B)]
[(C) /(D) (E) ]
Auxotrophs isolated
(y)
(D/D0)
Cells
Gly
Hyp
(x)
As2°3
3.0 x 10"5
4
3.1 x 106
541/250
(200) /{192) (0.770)
0
0
0
0.0
3.85
BeC12
5.0 x 10"4
4
3.2 x 106
680/230
(200) /(200) (0.865)
2
0
2
1.7
2.50
CdC12
5.0 x 10"7
4
2.5 x 106
1347/162
(200)/( 173)(0.615)
36
0
36
140.7**
4.73
Cr03
7.0 x 10"6
4
2.2 x 106
253/188
(200) /(166) (0.549)
2
0
2
1.5
3.68
CrC13'6H20
3.0 x 10"3
4
3.1 x 106
623/304
(200) /(163) (0.780)
1
0
1
0.8
3.15
CoC12"6H20
1.5 x 10"4
4
2.1 x 106
168/090
(200)/( 1891(0.525)
0
0
0
0.0
1.98
NiC126H20
2.0 x 10~4
4
3.2 x 106
458/238
(200)/(200X0.800)
0
0
0
0.0
2.56
MnC124H20
3.5 x 10"4
4
25 x 106
769/357
(201)/(191 X0.618
4
0
4
3.7*
3.37
PbAc23H20
5.0 x 10"4
4
3.2 x 106
592/359
(201 )/(201 M0.800)
0
0
0
0.0
Se02
1.5 x TO"5
4
2.3 x 106
276/156
(2001/(1801(0.575)
0
0
0
0.0
0.26
JP-5
Aq. extract
4
2.9 x 106
892/391
(200)/(187)(0.738)
7
0
7
5.8*
1:15 dilut.
* Denotes significance at the 95% confidence level; ** denotes significance at the 99% confidence level.
-------
m w
« s
%
afc,
' =| * *
* %** "* %
* « *'
"00 m
,mrn r m m§0M^ m t m i *
*'? • *. • ,% *
A*' i * | #' A »/ # *%f* # * 4
- A#«/t«%• //~ «
y* -j"£\- -
%: •
• •» t* «.'* «," *v«w '•'
7-7a.
• » - »hVv #*
* * # ~ **&*¦%..%%* *
:«* *:.
p
;||P
* J»
v-r
** *> $
L* **-o
#1^
•V V'*<¦.*'.
v.-'." ;•?.•¦•' ',
* , t l'' * •* *
** A •, . •
7-7b.
Figure 7-7. Epithelial and Fibroblastic Morphologies Exhibited by
the CH0-K1 Cell.
Note: (A) shows the standard epithelial morphology usually adopted by the
cell. (B) illustrates the highly fibroblastic form assumed by a potentially
contact-inhibited cell isolated from a population treated with the known
carcinogenic agent chromium trioxide.
92
-------
Preliminary experiments suggest that these cells show contact inhibition of
replication in colonies containing at least 100 cells, as well as in dense
monolayers (35). All such clones tested to date appear stable in their ability to
express the apparent contact-inhibited state.
Mutation Frequency and Expression Time
Mutation is a complex biological process involving much more than
interaction of mutagen with DNA, or more generally, with the DNA-replicating
system. The mutagen-DNA interaction usually results not in mutation, but in
the creation of premutational lesions which become expressed as mutations
only after a series of additional events has occured (6). For example,
auxotrophic mutants usually become expressed as soon as the cells become
depleted of normal gene product. The length of time required for this and
other preliminary events to occur may vary with the nature of the gene
product, the specific mutagen utilized, and the doses employed (5, 7). To
determine the influence of expression time on observed mutation frequency
for the mutant phenotypes reported above, experiments employing variable
expression time were carried out with EMS and BrdU. Doses used were those
giving maximal observed mutation frequency when the expression time was
five days (3 x 10"^'molar for EMS, 1 x 10"^ for BrdU). Expression time was
varied between two and eleven days. The data are given in Table 74, again in
terms of the parameters of equation [2]. Observed mutation frequency is
plotted in Figure 7-8 as a function of expression time.
Optimal expression time is defined as the interval between mutagen
quenching, and the application of selective conditions yielding maximal
mutation frequency when dose is held constant. This is observed to be five
days for BrdU and eight days for EMS. Moreover, for a two-day expression
period, statistically significant numbers of mutants could always be identified
in populations exposed to EMS, whereas none are found in populations treated
with BrdU. Once optimal expression time is exceeded, mutant cell frequency
decreased rapidly, suggesting that these types of mutants are at a replicative
disadvantage relative to wild-type cells under nonselective conditions. The fact
that optimal expression time was different for the two mutagens when tested
at doses showing similar toxicity, suggests that expression time may be mutagen
dependent. This lends support to the contention that assays utilizing variable
expression time shall be required when screening compounds for mutagenic
activity (4). This may be particularly important for weak mutagens.
DISCUSSION
The genetic toxicology of inorganic compounds has largely been ignored,
despite the fact that many are ubiquitous, highly toxic, and implicated as
93
-------
Table 7-4. Auxotroph Frequency as a Function of Expression Time for Single Doses of EMS and BrdU
Chemical
Expression
In)
Viable
HA)/IB)]
UO/IDME)]
Auxotrophs isolated
tv>
(days)
cells
Gly"
Hyp
GHT"
(x)
EMS
2
1
1.2 x 10®
109/050
(051i/(051)(.120)
3
0
1
4
072.7**
5
1
3.4 x 105
368/143
(051 )/(049)(.338)
12
6
1
19
149.9**
8
1
5.9 x 105
408/147
(051 )/(051) (.593)
41
5
0
46
213.5**
11
1
6.1 x 105
531/105
(051)/(051)(.610)
11
1
0
12
99.5**
BrdU
2
1
1.3 x 10®
086/040
(051)/(032)(.125)
0
0
0
0
0.0
5
1
5.0 x 104
180/113
(0511/(045) (.005)
3
0
0
3
108.1"
8
1
6.3 x 105
569/107
(0511/(043) (.633)
3
1
0
4
39.1**
11
1
5.8 x 105
414/075
(051 )/(049)(.578)
1
1
0
2
6.8**
Doses: EMS; 3.0 x 10'3 molar (D/D0 = 4.35}. BrdU; 1.0 x 10"4 molar (D/D0 = 2.04).
GHT" designates an auxotroph with simultaneous requirements for glycine, hypoxanthine and thymidine.
** Denotes significance at the 99% confidence level.
-------
DAYS
Figure 7-8. Mutation frequency induced by EMS and BrdU
as a function of expression time.
human carcinogens. Neglect of the inorganics in this regard seems due, in part,
to the fact that such compounds comprise a relatively small percentage of
known or potential genetic toxicants, are difficult to handle in many
experimental situations, appear to mediate genetic effects by obscure
mechanisms, and have not been active in many major assays. Some metals have
shown activity in DNA repair tests employing microbial systems (37), and in
reverse mutation assays with E. coli (50). Recently, selenate and some
compounds of chromium have been shown to be active in selected strains of
Salmonella typhimurium (38). Perhaps the best assay developed to date for
predicting the potential genetic toxicity of metals is the in vitro
DNA-synthesizing system described by Sirover and Loeb, and which detects
copying errors in replicating DNA (47). The fact that the carcinogenic metals
were not generally active in the CHO Cell/BrdU-VL system may reflect a
sensitivity problem related to the loss of mutants during selection to the effects
of starvation. Because mutation is periodically observed with certain of these
compounds, factors other than assay sensitivity may be involved. These factors
could be uniquely important to the expression of mutagenic activity by metals
and may not be presently under control or consideration. Extensive testing
with cadmium chloride and beryllium chloride indicates that the problem does
not lie with expression time.
95
-------
The isolation of potentially contact-inhibited cells from populations treated
with known carcinogenic agents is interesting for several reasons. Among the
more intriging ideas is the possibility that such cells represent back
transformation to the noncancerous state. The CHO cell possesses many of the
properties of transformed cells, including the loss of contact inhibition (42,
44). In accordance with the somatic cell mutation hypothesis for cancer (10,
22), agents inducing cell transformation through mutation should, in at least
some cases, be capable of inducing back transformation in individual cells via
true reverse mutation or via forward mutation at suppressor loci. Indeed,
spontaneous revertants of cells transformed to the malignant state in vitro by
viruses and chemicals have already been described (23, 24). Reversible
conversion of transformed cells to the contact-inhibited state has been
observed during exposure of such cells to dybutryl cyclic AMP (25) or
concanavalin A (11). It remains to be shown if the same agents capable of
inducing transformation form normal to malignant state can also reverse it,
such that the revertants are stable in the absence of inducing agent. Although
virtually all cell-transforming agents tested to date are also mutagens, it is not
yet possible to say if the phenomenon of cell transformation involves mutation
(29). Investigation of the properties of these cells is continuing.
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100
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DEVELOPMENT OF A BIOASSAY FOR
USING BROWN ALGAE
OILS
M. Dennis Hanisak
Richard L. Steele
U.S. Environmental Research Laboratory
Narragansett, Rhode Island 02882
ABSTRACT
Bioassay procedures were developed to observe the effects of No. 2 fuel oil,
two jet fuels, and a crude oil on the growth and early development of Fucus
zygotes and Laminaria gametophytes. These algae are as sensitive, or more so,
than fish and invertebrates previously tested in oil bioassays. Fucus sperm and
Laminaria spores are extremely sensitive to oil, with dramatic effects at the
levels of 2 ppb. These results indicated that these species are potentially good
bioassay organisms, and also that chronic, low-level pollution could
significantly alter the community structure in marine ecosystems.
INTRODUCTION
Algae are the primary producers in the marine ecosystem. Yet, despite their
importance, little is known on how they are affected by specific pollutants in
their environment. A bioassay is one method of studying pollution effects on
organisms. This paper reports on bioassay procedures developed with two
brown algal genera, Fucus and Laminaria.
In order to be a useful bioassay organism, an alga should be readily available
in nature, easily maintained in the laboratory, hardy enough to grow in culture,
yet sensitive enough to respond to low levels of pollution encountered in
nature, and preferably, of ecological or economic importance. Both Fucus and
Laminaria have most, if not all, of these qualities; yet, because of differences in
their habitats, they might react differently to an oil spill. Fucus, being an
intertidal species, would be subjected to repeated immersion in the water mass
and be coated with oil when the tide is out. Laminaria is a subtidal species and
would normally be subjected only to concentrations of oil present in the water
column.
101
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Although Fucus and Laminaria are both brown algae, their life cycles are
quite different. The life cycle of Fucus (Figure 8-1) is very much like that of an
animal. Mature, diploid thalli produce haploid eggs and sperm which fuse to
form zygotes that ultimately develop into new diploid thalli. The life cycle of
Laminaria (Figure 8-2), in which an alternation of generations occurs, is much
more complicated than that of Fucus. The macroscopic, blade-like, diploid
sporophyte alternates with microscopic, filamentous, haploid gameto-
phytes. The variability and complexity of algal life cycles provide several
opportunities to study the effect of a specific pollutant on growth and
development. In the present study, the effects of different petroleum
products on the growth of Fucus zygotes and Laminaria gametophytes
were observed.
MATERIALS AND METHODS
Both Fucus and Laminaria plants were collected from various locations in
and around Narragansett Bay (i.e., Camp Vamum, a National Guard
installation, the dock of the Environmental Research Laboratory, Narragansett,
R.I., and Monohan's Cove, Narragansett, R.I.). In developing techniques,
several species were used, including Fucus vesiculosus, F. edentatus, F.
distichus, Laminaria saccharine, and L. digitalis.
The Fucus species represent both monoecious and dioecious types. In
deciding which species of Fucus and Laminaria to use, little difference was
noted in preliminary response among various species. Data presented herein
represent the responses of Fucus edentatus and Laminaria saccharina, but are
representative of other species in both genera.
For Fucus, methods of procurement of eggs and sperm were evaluated (4,
5), and a technique was devised that is applicable to all species tested. The
method is essentially a combination of other methods reported in the
literature, and consists of the following: receptacles (fertile plant tips) that
appeared most erumpent and mature, even to the point of being partially
eroded, were collected from mature plants. These receptacles were observed
to produce the highest numbers of viable eggs and sperm. Receptacles were
rinsed in sterile charcoal filtered* seawater at 30 ppt. salinity, and were
placed in a moist chamber overnight. The moist chamber consisted of large
150 x 25 mm plastic petri dishes (Falcon Plastics) containing filter paper of
the same diameter moistened with sterile seawater.
* Cartridge filtration through Commercial Filter Corporation honeycomb
wound filters, 15 p porosity, and .22 j* porosity pleated Gelman filters.
102
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CONCEPTACLE SPERMATANGIA
Figure 8-1. The Life Cycle of Fucus.
SGAMETOPHYTE
Figure 8-2. The Life Cycle of Laminaria.
103
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These plates were placed in a 12°C culture chamber overnight. Receptacles
were then placed in sterile charcoal filtered seawater the next morning. Eggs
and sperm were immediately released, and fertilization was observed within 15
to 20 minutes. Zygotes were immediately pipetted into culture dishes (60 x 15
mm plastic petri dishes, with 2 mm square grids — Falcon Plastics) while
keeping the eggs suspended in seawater by stirring. Twenty four hours after dispens-
ing into culture dishes, the toxicant was introduced by allowing the zygotes to
settle on the bottom of the dish, the seawater removed, and replaced with
seawater containing the toxicant at the test levels. In a few cases, the tips were
pre-treated with the toxicant, and in these cases, the above sequence was
suitably modified.
The growth medium for the Fucus assays was, in all cases, sterile charcoal
filtered seawater. The parameter measured for these experiments was the
increase in length after 12 days of growth.
Methods of procuring Laminaria spores were similar to those of Fucus
gametes. Sporogenous plants were collected and then washed in deionized
water to remove surface contaminants. Small pieces (2-3 cm square) of
sporogenous tissue were placed into moist chambers overnight. These pieces
were placed into sterile seawater the following morning, and spores were
released in abundance. Spores were dispensed into culture dishes at
concentrations sparse enough to allow counting and to prevent overcrowding,
but dense enough (ca. 100-260 eggs) for good statistical data. In all cases, the
culture medium was Provasoli's Enriched Seawater (6). The parameter
measured was the increase in diameter of the gametophyte after 21 days of
growth.
All assays were conducted at 400 ft-c of continuous cool white fluorescent
light. Except for the first series of experiments to determine the optimal
temperature salinity combinations for the assays, the temperature and salinity
were 18°C and 30 o/oo for Fucus and either 12 or 18°C and 30 o/oo for
Laminaria. For the various tests, the petroleum product (either No. 2 fuel oil,
JP-4, JP-5, and Willamar crude) at concentrations ranging from 0-2000 ppm
was equilibrated with seawater, proper dilutions made, and added to the
cultures. The oil-seawater mixtures were analyzed by infrared
spectrophotometry (Perkin-Elmer Model 621) to determine the amount of
dissolved product causing toxicity.
Observations and measurements of Fucus zygotes and Laminaria
gametophytes were made with a Unitron inverted microscope (Model BMIC).
Approximately 10-20 individuals were measured per dish. Four replicates were
performed for each treatment.
104
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RESULTS
Fucus zygotes were much more tolerant of salinity-temperature extremes
than were Laminaria gametophytes (Table 8-1). This is probably a reflection of
their habitats; Fucus, being intertidal and growing in a more variable
environment than the subtidal Laminaria, is adapted to a wider range of
environmental conditions. Optimal growth of Fucus was at 18°C and 30-42
ppt while that of Laminaria was at 12-18°Cand 24-36 ppt. There appeared
to be some seasonal variability in Laminaria\ optimal temperature for growth.
Fertile Laminaria collected during the colder winter months produced spores
that germinated and grew slightly better at 12°C than at 18°C; the converse
was true for Laminaria collected during the warmer spring months. In the
following experiments to determine the toxicity of different oils, the standard
conditions were 18°C and 30 ppt for Fucus and 12°C and 30 ppt for
Laminaria.
Of the four types of oil tested, No. 2 fuel oil was the most toxic to Fucus
zygotes, and the jet fuels, JP-4 and JP-5, were least toxic (Table 8-2). There
appeared to be a slight stimulation at lower levels (200 ppb or less), except for
Willamar crude. This may be due to a surfactant effect on the part of the oil.
Above those levels, these oils became increasingly deleterious to growth.
The toxicity of the four oils to Laminaria gametophytes was similar to
that of Fucus zygotes, although at lower levels, Laminaria response was not
comparable to Fucus (Table 8-3). Number two fuel oil was still the most
toxic, although not as relatively toxic as it was to Fucus. The jet fuels, JP-4
and JP-5, were somewhat more toxic to Laminaria than they were to Fucus.
The lower growth rate of Laminaria gametophytes compared to Fucus
zygotes is probably a reflection of their different growth habits. Laminaria
gametophytes are much smaller, and grow in a more radial fashion than do
Fucus zygotes.
Preliminary experiments on application of oil during gamete release in
Fucus, and spore release in Laminaria, indicate that these brown algae may be
extremely sensitive to oils (Table 8-4).
Concentrations greater than 20 ppb of No. 2 fuel oil were completely toxic
to Laminaria spores. Even at 2 ppb, significant inhibition of the resulting
gametophytes occurred. Fucus was even more sensitive. At 2 ppb, fertilization
of eggs was blocked, apparently due to a toxicity effect on the sperm.
DISCUSSION
It is somewhat difficult to compare the results of these bioassays with
brown algae, with those developed with other organisms by different
105
-------
Table 8-1. Effects of Various Temperature and Salinity Combinations
on the Growth of Fucus edentatus Zygotes and Laminaria saccharina
Gametophytes.
Length* of Fucus after 12 Days
Diameter* of Laminaria after 21 Days
Mm
Temperature, C
Temperature, C
6
12
18
24
30
6
12
18
24
30
6
102 ±0
190 ± 23
0 + 0
0±0
0±0
0±0
0±0
0± 0
0±0
0±0
12
153 ± 5
273 ±15
341 ± 13
140 ±0
0±0
0±0
0+0
93 ±7
0±0
0±0
18
165 + 3
419± 13
586 ±4
331 ±28
0±0
0±0
102 ±7
133 ±8
0 ±0
0±0
24
151 ± 1
383 ±9
645 ±36
400 + 13
0±0
0±0
168 ±9
196 ± 8
0±0
0±0
30
153 ± 20
380 + 3
704 + 13
406 ±9
0 + 0
0±0
265 ±2
262
0±0
0±0
36
147 ±17
395 ± 14
701 ±18
400 ± 15
0±0
0±0
238 ±8
184
0±0
0±0
42
122 + 20
285 + 14
712 ±20
365 ±17
0±0
0±0
88+2
78
0±0
0±0
48
102 + 13
260 + 12
601 ±68
342 ± 19
0±0
0±0
0±0
0±0
0±0
o
+l
o
54
0 + 0
254 ±8
458 ±16
295 ± 14
0±0
0±0
0±0
0±0
0±0
0 ±0
60
0±0
0±0
0±0
0±0
0±0
0±0
0 + 0
0±0
0±0
0 ±0
•Mean ± SE
-------
Table 8-2. Effects of No. 2 Fuel Oil JP-4, JP-5 and Willamar Crude Oil
on Growth of Fucus edentatus Zygotes.
Numbers Shown are in Lengthf of Juvenile Plants
After 12 Days.
Added
Total
Extractable
Hydrocarbons
No. 2 Oil
JP-4
JP-5
Willamar Crude
0.0
*
694 ± 19
811 ± 48
811 ± 48
776 ± 13
2ppb
*
703 ±6
758 ± 28
807 ±31
764 ± 20
20ppb
*
748 ± 32
884 + 37
913 ±43
748 ±7
200ppb
*
697 ±9
818 ±39
923 ± 37
750 ±4
2ppm
(0-30ppb)
671 ± 21
854 ± 23
881 ± 24
735 ± 19
20ppm
(1-3ppm)
0 ±0
781 ±17
753 ±4
656 ± 14
200ppm
(18-28ppm)
Q±Q
655 ± 28
445 ±22
399 ±9
2ppt
(45-50ppm)
0±0
0±0
0±0
0 + 0
* Dissolved hydrocarbons undetectable by spectrophotometry due to extremely minute amounts. Values are range of
measurement and include all the petroleum products.
fMean ± SE
-------
Table 8-3. Effects of No. 2 Fuel Oil, JP-4, JP-5 and Willamar Crude Oil
On Growth of Laminaria saccharina Gametophytes.
Numbers Shown are mm in Diameterj
Gametophytes After 21 Days.
Added
Total ixtractable
Hydrocarbons
No. 2 Oil
JP-4
JP-5
Willamar Crude
0.0
*
186.4+2.3
184.01 3.5
188.01 2.5
198.0 ± 7.0
2ppb
*
158.0+2.5
161.01 2.5
152.014.8
188.514.7
20ppb
*
150.0+5.4
148.211.3
151.21 5.7
194.21 5.2
200ppb
*
136.1 ±3.1
140.5+ 1.2
132.2 + 3.4
175.51 5.6
2ppm
(0.30ppb)
120.210.9
117.312.1
129.21 5.3
157.015.9
20ppm
(1-3ppmj
105.91 2.8
110.0 + 2.3
102.8 + 3.1
144.8 ± 3.2
200ppm
(18-28ppm)
**010
**010
010
96.812.2
2ppt
(45-50ppm)
0±0
010
010
0±0
* Dissolved hydrocarbons undetectable by spectrophotometry due to extremely minute amounts. Values are range of
measurement and include all the petroleum products.
** Germinated but dead after 3 days.
tMean ± SE
-------
Table 8-4. Growth of Fucus Zygotes and Laminaria Gametophytes
After Treatment in Oil/Water Mixture During Gamete and Spore Release.
Treatment was with No. 2 Fuel Oil.
Added
Totat Extractable
Hydrocarbons
Length (^m ± SE)
of Fucus zygotes
after 13 days growth
Diameter (nm ± SE)
of Laminaria
gametophytes after
21 days.
0
*
699 ± 21
164.4 + 9.2
2ppb
*
0±0
60.8 ± 1.2
20ppb
*
0± 0
**0 ±0
200ppb
*
0±0
0±0
2ppm
(0-30ppb)
0 ±0
0±0
20ppm
(1-3ppm)
0±0
0±0
200ppm
(18-28ppm)
0±0
0 ±0
* Dissolved hydrocarbons undetectable by spectrophotometry due to extremely minute amounts
Values are range of measurement and include all the petroleum products.
** Spores germinated but were dead after day nine.
-------
investigators using different techniques and response parameters. Based on the
bioassays with Fucus and Lamimria, it appears that they were not as sensitive
to oil as some microalgae that have been studied (7), but they were similar or
slightly more sensitive than bioassays developed with fish and invertebrates (2,
3). Although the toxicity values obtained in these studies were not directly
applicable to Fucus and Laminaria (Pulich used doubling times and Eisler used
LC50 values) similar values can be derived.
The extreme sensitivity of the reproductive stages, i.e., eggs and sperm in
Fucus, and spores in Laminaria, indicate that either of these organisms might
become a useful bioassay tool.
In one experiment, Fucus receptacles were allowed to stand in various
concentrations of oil 5 hours before being placed in moist chambers overnight.
After completing the experiment in the usual manner, and allowing gamete
release to occur in sterile seawater, the deleterious effects on the sperm were
not observed. However, growth of the juvenile plants was reduced, being
similar to that in Table 8-2, even though the zygotes were not in oil solutions
during or after fertilizations. Further experiments with two-week old juvenile
Fucus plants indicated that as the plant gets older, sensitivity to oils decreases.
Thus, the most critical stage of the life cycle, and the one most sensitive to oil,
is the reproductive phase.
Continued development and refinement of these bioassay procedures is
needed, as well as a survey of other seaweeds, to see how representative these
results are for seaweeds in general. The development of a bioassay in a
flow-through system would be more representative of natural conditions.
The deleterious effects of low-level oil pollution on the reproductive cycle
of these algae can easily be visualized, especially in areas of chronic pollution,
such as those found near harbors, marinas, and similar installations. By
preventing the completion of the life cycle, the community structure of algae,
as well as that of higher trophic levels, will be altered. Greater effort should be
made to examine chronic, long-term effects of oil pollution on the marine
ecosystem.
REFERENCES
1. Dawson, E.Y. 1956. How to Know the Seaweeds. Wm. C. Brown,
Publishers, Dubuque, Iowa.
2. Eisler, R. 1975. Acute Toxicities of Crude Oils and Oil-Dispersant Mixtures
to Red Sea Fishes and Invertebrates. Israel Journ. of Zool. 24:16.
110
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3. Eisler, R., and G.W. Kissel. 1975. Toxicities of Crude Oils and
Oil-Dispersant Mixtures to Juvenile Rabbit Fish, Signanas rivulatus. Trans.
Am Fish. Soc. 104:571.
4. McLachlan, J. 1974. Effects of Temperature and Light on Growth and
Development of Embryos of Fucus edentatm and F. distichus ssp.
distichus. Can. Jo urn. Bot. 52:943.
5. Pollock, E.G. 1970. Fertilization in Fucus. Planta 92:95.
6. Provasoli, L. 1968. Media and Prospects for the Cultivation of Marine
Algae, p. 63 In A. Watanabe and A. Hatteri, Ed. Cultures and Collections of
Algae. Proc. U.S.-Japan Conf. Hakone, Sept. 1966, Jap. Soc. Plant Physiol.
7. Pulich, W.M., K. Winters, and C. Van Baalen. 1974. The Effects of No. 2
Fuel Oil and Two Crude Oils on Growth and Photosynthesis of Microalgac.
Marine Biology 26:87.
Ill
-------
EFFECTS OF NO. 2 HEATING OIL
ON FILTRATION RATE OF BLUE MUSSELS,
MYTILUS EDULIS LINNE
J. G. Gonzalez, D. Everich, J. Hyland, and B. D. Melzian
U.S. Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
ABSTRACT
Reductions in gill filtration rates were observed for adult blue mussels,
Mytilus edulis, that were exposed in a continuous flow-through dosing system,
to three concentrations of the water-accommodated fraction of No. 2 fuel oil.
The oil concentrations were measured routinely by infrared spectrometry, and
averaged 0.019 ppm, 0.06 ppm, and 0.64 ppm throughout the exposure period.
Filtering rates for healthy, unexposed mussels ranged from 7.2 to 30.9 ml/min,
depending on ambient water conditions. In comparison to controls, filtering
rates decreased as the oil concentration increased, with significant reductions
occurring at all dose levels within 48 hours of exposure. Continued oil
exposure up to two weeks produced progressively higher reductions in filtering
rate. When returned to uncontaminated water for two weeks, the mussels
resumed their normal feeding rates, revealing that the effect was reversible.
Mussels collected from a small oil spill site exhibited similar responses.
INTRODUCTION
Bivalve mollusks are of considerable value to ecologists studying the effects
of pollution, because many of the species are sedentary filter feeders, and are
likely to accumulate contaminants from their surroundings. Mytilus edulis, the
blue mussel, has become one of the most widely studied members of the group
since it has a worldwide distribution; it is easy to maintain in the laboratory;
and it is exploited commercially, particularly in European countries. Also,
Mytilus, because of its intertidal existence, is particularly vulnerable to oil
exposure.
Several investigators have demonstrated a reduced feeding rate in mollusks
exposed to environmental stress. Galtsoff et al (3) reported fifty percent
reduction in gill ventilation rate of oysters exposed to an extract of crude oil.
112
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They attributed this reduction to an anaesthetic effect upon the gill cilia.
Preliminary investigations at the Environmental research Laboratory,
Narragansett, revealed that feeding rates of clams, Mercenaria mercenaria, and
scallops, Argopecten irradians, were notably diminished after exposure to No.
2 fuel oil (7).
A reduced feeding rate has also been noted for My tilus edulis. For instance,
Abel (1) observed reduced filtration rates in mussels exposed to various
pollutants including copper, zinc, mercury, cyanide, thiocyanate, and sulfide.
Gonzalez and Yevich (5) reported that Mytilus show a significant decrease in
filtering rate when exposed to high temperatures in the laboratory. Gilfillan
(4), investigating the response of Mytilus to seawater extracts of crude oil,
reported a decrease in both food consumption and assimilation, and an increase
in respiration. The combined effects resulted in a reduction in the net carbon
flux at oil concentration as low as 1 ppm.
The Oil Pollution Research Branch at the Narragansett Environmental
Research Laboratory has been assigned the task of evaluating the effects of
very low levels of oil on ecologically and commercially important marine
species. Such levels may not immediately lead to death of the organisms, but
may ultimately jeapordize their long-term success at survival. Since Mytilus
edulis is an important species in the marine community, and since a change in
filtration rate appears to be a well-defined response to environmental
disruption, we conducted an investigation to elucidate the behavioral effects of
very low levels of oil, and to evaluate the recovery potential of the stressed
animals.
METHODS
Adult Mytilus edulis were collected from the southeastern shore of
Conanicut Island, Rhode Island, in April, 1976. In the laboratory, the animals
were measured and separated into four groups of 50 individuals each. Mean
shell length of the mussels was 4.63 cm. Each group of mussels was maintained
for a two-week acclimation period in a plastic coated wire cage that was
suspended in a one meter diameter fiberglass tank. The tanks were supplied
with continuously renewed unfiltered seawater, which allowed the mussels to
feed on natural plankton from the incoming water.
After the acclimation period, the filtration rate was measured for each of the
four groups of mussels. Next, these animals were placed for two weeks in a
flow-through oil exposure system designed by Hyland etal. (6). One group was
placed in each of three nominal oil concentrations—0.01 ppm, 0.1 ppm, and 1
ppm— and one group was held under control conditions. Filtration rates were
measured at various intervals during the two-week oil exposure period. All
113
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animals were then transferred to control conditions, and their filtration rates
measured periodically to determine recovery.
Gill filtration rate was measured indirectly by recording the rate at which
the animals removed food particles from the surrounding water. Each cage of
50 mussels was transferred from its exposure or recovery tank to individual
glass aquaria containing equal and known quantities of Isochrysis galbana. The
algal suspension was maintained by aeration. Subsequently, three replicate 25
ml water samples per aquarium were withdrawn at intervals during a three-hour
feeding period. The number of algal cells in each sample was counted on a
Coulter electronic cell counter, and the average percent reduction through time
recorded for each aquarium.
Filtration rates were expressed both graphically and numerically. Feeding
curves were generated by plotting percent food particles removed versus time
to allow visual comparison between the feeding rates of the control and oiled
mussels for each of the various exposure or recovery periods. Results were
analyzed statistically by performing linear regressions on the natural log
transformed data, and comparing the regression lines (9). Actual filtration
rate—the rate at which a solution is pumped through the gills of the animal in a
given time period—was determined numerically with the aid of the following
formula (8):
Filtration rate (ml/min/mussel) = vol- solution (ml) x In CQ
(no. animals) x A T min)
where CQ and Ct represent food concentrations at the beginning and end of a
particular feeding interval (AT). The solution is based on the assumption that if
filtration rate remains constant over the feeding interval, then the rate at which
particles are removed from suspension will decline exponentially, as described
by the curve e"x. For a given group of mussels, filtration rate was finally
expressed as the average of those values calculated separately for each interval
during which the mussels were actively removing food particles. Averaging is
necessary to correct for the fact that the calculation of filtration rate can vary
slightly depending on the magnitude of the time interval selected, a result of
the fact that particles are not always removed at an exact exponential rate.
The flow-through oil exposure system is designed to dose marine animals
with the water-accommodated fraction of No. 2 fuel oil at three nominal
concentrations—0.01 ppm, 0.1 ppm, and 1 ppm. The W.A.F. contains finely
dispersed oil as well as the water-soluble components, but does not include the
whole oil slick. The system simulates an area of chronic petroleum
hydrocarbon pollution, such as one that might exist near a sewage outfall, an
oil refinery, or an area consisting of sediments that have been heavily
contaminated with oil.
114
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Temperature, salinity, dissolved oxygen, and pH were routinely measured in
the dosing tanks, and averaged 16°C, 31 ppt, 8.09 ppm, and 8.03 respectively.
Hydrocarbon concentration was also determined routinely by infrared
spectrometry, following the techniques described in Hyland, et al. (6). The
actual oil concentrations measured according to this method, are somewhat
different from the nominal ones mentioned previously. Accordingly, during the
exposure period the 0.01 ppm tank averaged 0.019 ppm above the natural
background hydrocarbon concentration; the 0.1 ppm tank averaged 0.06 ppm;
and the 1 ppm tank averaged 0.64 ppm.
RESULTS AND DISCUSSION
Figure 9-1 illustrates the feeding activity of Mytilus edulis prior to oil
exposure. There appears to be little difference in the shapes of the four feeding
curves; and, in fact, statistical analysis revealed no significant differences (P<
0.05). Typically, 80 percent of the food particles were removed by the mussels
in 15 minutes, and 95 percent in 30-minutes, at which point maximum filtering
activity was reached. Over this 30 minute interval, the average filtration rate
for the four groups was calculated as approximately 18.1 ml/min, which is
representative of values reported elsewhere in the literature (2). The values
ranged from 15.6 for the control; to 18.7 for the 1 ppm group, and 19.1 for
both the 0.01 and 0.1 ppm groups (Table 9-1).
Figure 9-1. Pre-exposure: Comparison of Filtering Activity of
Mytilus edulis Prior to Exposure to W.A.F. No. 2 Fuel Oil.
115
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Table 9-1. Filtration Rates for Control and Oil-Exposed Mussels.
NOTE: Mean and standard error (in parentheses) are both given.
0.01
0.1
1.0
Control
ppm
ppm
ppm
Pre-exposu re
15.6
19.1
19.1
18.7
(0.4)
(0.4)
(1.9)
(1.5)
48-hr. Exposure
19.3
10.5
5.2
2.2
(3.3)
(0.9)
(0.5)
(0.2)
2-wk. Exposure
17.8
5.3
1.8
0.3
(1.5)
(0.3)
(0.5)
(0.04)
24-hr. Recovery
15.8
9.9
3.9
0.8
(0.2)
(1.2)
(0.2)
(0.1)
2-wk. Recovery
30.9
30.9
17.2
(6.5)
(6.5)
(1.6)
After 48 hours of exposure (Figure 9-2), the filtration curves for the three
experimental groups began to diverge, while the control curve retained
pre-exposure characteristics. Mussel feeding activity in all three oil
concentrations was significantly reduced from that of the control, with the
highest concentration producing the most severe reduction. Filtration rates for
the 0.01 ppm, 0.1 ppm, and 1 ppm polluted mussels, decreased to 10.5, 5.2
and 2.2 ml/min, respectively, while the control group filtered at an average rate
of 19.3 ml/min. Figure 9-3 illustrates that continued oil exposure produces
progressively lower filtration rates. Mussels exposed for two weeks to 0.01 ppm
required two hours to filter what the controls filtered in 30 minutes. Similarly,
after three hours, animals exposed to 1 ppm had only consumed approximately
35 percent of the algae; while the controls far surpassed this in less than 10
minutes. After two weeks of exposure, filtration rates for the three exposed
groups decreased to 5.3, 1.8, and 0.3 ml/min, while the control group filtered
at an average rate of 17.8 ml/min.
Following the two-week exposure period, the animals were returned to clean
water. Some evidence of recovery was noted after 24 hours in clean water
(Figure 9-4); however, the filtration curves for all three exposure groups were
still significantly different from the control. Filtration rates increased to 9.9
ml/min for the 0.01 ppm group, 3.9 ml/min for the 0.1 ppm group, and 0.76
ml/min for the 1.0 ppm group.
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Figure 9-2. 48-h9ur Exposure: Comparison of Filtering Activity
of Mytilus edulis Exposed to W.A.F. No. 2 Fuel Oil.
Figure 9-3. Two-week Exposure: Comparison of Filtering
Activity of Mytilus edulis Exposed to W.A.F. No. 2 Fuel Oil.
117
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Figure 9-4. 24-Hour Recovery: Comparison of Filtering
Activity of Mytilus edulis After 24 Hours of Recovery from
Two Weeks Exposure to W.A.F. No. 2 Fuel Oil.
Gradual improvement of all groups was observed as the animals remained in
clean water. After two weeks, recovery was almost complete (Figure 9-5). The
control and 0.1 ppm groups both filtered at an accelerated rate of 30.9 ml/min,
and the 1.0 ppm group filtered at a rate of 17.2 ml/min, characteristic of
pre-exposure rates. The higher filtration rates observed may reflect increasing
ambient water temperatures at the time of testing. Temperatures increased
from 11°C at the time of pre-exposure testing, to 19°C during this latter
testing period. Due to a laboratory failure resulting in reduced water flow, and
subsequent anaerobic conditions in the recovery tank which held the 0.01 ppm
exposure group, it was necessary to discard these mussels without
demonstrating their complete recovery. However, since mussels at higher oil
concentrations did recover, it seems reasonable to assume recovery for the 0.01
ppm exposure group as well. There remained a significant difference between
the 1 ppm exposure group and the controls after two weeks in clean water;
however, after one month of recovery, they actually fed slightly better than
the controls.
Based on the current investigation, it appears that the adverse effect of oil
on filtration rate of mussels is reversible, provided the stressed animals are
returned to unpolluted conditions. However, the data also strongly suggest that
recovery does not occur under conditions of continued exposure. Further
investigation is currently in progress to determine the implications of reduced
118
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i ft
A
CONTROL o
O.I ppm 4
1.0 ppm ¦
J I
0
30
60
90
120
150
180
TIME (minutes)
Figure 9-5. Two-week Recovery: Comparison of Filtering
Activity of Mytilus edulis after Two Weeks of Recovery from
Two Weeks Exposure to W.A.F. No. 2 Fuel Oil.
feeding over a long period of time as a result of continued oil exposure. A
question confronted, for example, is whether mussels exposed for several
months to chronic inputs of oil reveal reduced growth.
The laboratory experiments reported herein were designed to investigate
responses to chronic oil pollution, and not the acute phenomena which occur
immediately after an oil spill. However, in November, 1976, a small spill of No.
6 fuel oil occurred at Quonset Point, Rhode Island. The resulting slick drifted
across the western passage of Narragansett Bay, and impacted approximately
one mile of shoreline on Conanicut Island. This incident provided an
opportunity to investigate the effects of spilled oil on filtering activity in
mussels, and thus supports the laboratory results with field data. Accordingly,
48 hours after the spill, Mytilus were collected from the polluted site and from
an unimpacted area nearby. Filtration rates were measured in the laboratory,
and feeding curves were generated for both groups (Figure 9-6). Compared to
controls, a small but statistically significant reduction in feeding activity was
observed in oiled mussels. For example, over a period of 45 minutes, the
controls had removed approximately 96 percent of the food particles, while
the polluted mussels removed only 84 percent. Filtration rates were calculated
as 7.2 ml/min (S.E. = 1.0) for the controls, and 4.9 ml/min (S.E. = 0.5) for the
oiled group. The relatively low value obtained for the control group is most
likely a reflection of low winter ambient water temperatures (5-6°C). One
119
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0 30 60 90 120 150 180
TIME (minutes)
Figure 9-6. Oil Spill: Comparison of Filtering Activity
of Mytilus edulis Collected From a Clean Area and from an
Area Impacted by an Accidental Spill of No. 6 Fuel Oil.
week after the spill, another collection was made. Test results indicated that
feeding activity of the oiled mussels had improved to the point that no
differences could be found between control and oiled groups.
In conclusion, the investigation demonstrates that (1) under laboratory
conditions an adverse reduction in filtration rate occurs in Mytilus edulis at
very low levels of continuous oil exposure; (2) the effect is reversible, since
recovery will gradually occur if the stressed animals are returned to unpolluted
conditions; and (3) a similar effect occurs in response to spilled oil in the
natural environment.
ACKNOWLEDGEMENTS
Grateful appreciation is extended to Ms. Terry Richie and Dr. James Heltshe
for their assistance with the statistical analyses.
REFERENCES
l.Abel, P.D. 1976. Effects of Some Pollutants on the Filtration Rate of
Mytilus. Mar. Pollut. Bull. 7(12): 228-231.
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2. Foster-Smith, R.L. 1975. The Effect of Concentration of Suspension on
Filtration Rates and Pseudofecal Production for Mytilus edulis L.,
Cerastoderma edule (L.) and Venerupis pullastra (Montagu). J. Exp. Mar.
Biol. Ecol. 17: 1-22.
3. Galtsoff, P.S., H.F. Prytherch, R.O. Smith and V. Koehring. 1935. Effects of
Crude Oil Pollution on Oysters. In: Louisiana Water, Bull. U.S. Bur. Fish.
48(18): 144-210.
4. Gilfillan, E.S. 1975. Decrease of Net Carbon Flux in Two Species of Mussels
Caused by Extracts of Crude Oil. Mar. Biol. 29(1): 53-57.
5. Gonzalez, J.G. and P. Yevich. 1976. Responses of an Estuarine Population
of the Blue Mussel Mytilus edulis to Heated Water from a Steam Generating
Plant. Mar. Biol. 34: 177-189.
6. Hyland, J.L., P.F. Rogerson, and G.R. Gardner. 1977. A Continuous Flow
Bioassay System for the Exposure of Marine Organisms to Oil, pp. 547-550.
In. Proc. 1977 Oil Spill Conference (Prevention, Behavior, Control,
Cleanup), March 8-10, 1977, New Orleans, LA. U.S. Coast Guard,
Environmental Prbtection Agency, and American Petroleum Institute.
7. Hyland, J.L., P.P. Yevich, and P.F. Rogerson. 1976. Unpublished Data. U.S.
Environmental Protection Agency, Narragansett, Rhode Island.
8. Quayle, D.B. 1948. Biology of Venerupis pullastra (Montagrie), Ph.D.
Thesis, University of Glasgow.
9. Snedecor and Cochran. 1967. Statistical Methods (6th ed.). Ohio State Univ.
Press. Ames, Iowa. pp. 432-436.
121
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LOBSTER BEHAVIOR AND CHEMORECEPTION:
SUBLETHAL EFFECTS OF
NO. 2 FUEL OIL
Jelle Atema, Elisa B. Karnofsky
and Susan Oleszko-Szuts
Boston University Marine Program
Marine Biological Laboratory
Woods Hole, Massachusetts 02543
ABSTRACT
Lobsters (Homarus americanus) were exposed in a flow-through oil dosing
system to the water-accommodated fraction of #2 fuel oil. Behavioral
observations of feeding efficiency and general behavior, showed that 5-day
exposure to 0.08 and 0.15 ppm caused significant delays in feeding, without
causing severe neuromuscular defects. Exposure to 1.5 ppm caused gross
neuromuscular defects within 24 hours. Recovery was proportional to the
gravity of observed defects. Neurophysiological experiments on antennular
chemoreceptors of behaviorally observed animals showed that oil is perceived
as a chemical stimulus, and can change normal responses to food juices.
Oil-exposed animals show abnormal, bursting spike patterns, both
spontaneously and in response to food juice. It remains to be proven that low
level exposure effects are due to oil interference with chemoreception. This
is a report on preliminary data.
INTRODUCTION
Each year more evidence appears which demonstrates the importance of
chemical signals in the lives of marine animals. The following are just a few
examples of the broad categories of behavior where chemical signals are of vital
importance: feeding behavior, both the predator's detection of live prey and
the scavenger's localization of dead bait; the prey's alarm and escape behavior;
mating behavior and mate selection; parental brood recognition; and the
selection of suitable geographic locations, as in larval settling and homestream
return of migratory species. Interference with chemical signals or with the
receptors that evolved to receive them could therefore jeopardize animal
survival without causing immediately obvious deleterious effects on the
individual. Man's chemical discharges into the environment, such as large
amounts of petroleum hydrocarbons in coastal areas, may cause such
interference.
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Speculations about petroleum hydrocarbon interference with the processes
of chemoreception have appeared with a certain regularity in the literature,
starting with Blumer (4). The reasons for this speculation are obvious:
petroleum hydrocarbons are a mixture of organic chemical compounds, some
of which are related to compounds such as pheromones and alarm substances,
which are utilized by animals for their orientation and communication. These
communication signals may have chemical features, such as carbon skeleton,
functional groups, volatility, and solubility, in common with compounds in
petroleum (7). In an oil-polluted environment, different petroleum compounds
will be in solution or in emulsion in the water column, while the heavier
fraction can become part of the benthic mud and affect the benthic ecosystem
for many years, as shown by Blumer and Sanders, among others (5,9). One
can thus envisage the scenario when these chemical look-alikes mimic or mask
the reception of biologically important signals. Mimicked signals may result in
"false alarm", i.e., animals may look for imaginary food or mates, or avoid
predator danger where there is none. If their chemical signals are masked,
animals cannot respond to them and may miss opportunities to feed, or mate,
or escape. A third possibility less frequently mentioned is that animals may
become subject to two competing signals (3), for instance, an attractant signal
from food (or mate) and a repellent signal from oil. In such cases
chemoreception would be perfectly normal, but the animal may not be able to
decide whether to feed or hide. Such delays may be more critical than apparent
at first glance: even a slight delay in responding to food can put an individual
at a significant disadvantage when competing with an unimpaired conspecific,
or in escape from predators.
Thus far, some cases of mimicked food attraction and delayed food
responses have been observed, as well as increases and decreases in alarm and
attraction behavior (1, 8, 10, 11). However, specific effects of oil on
chemoreception itself have never been documented. Studies showing oil
interference with chemoreception will provide us with a general understanding
of the effects of oil pollution, since the processes of chemoreception —
although essentially unknown — are probably similar in all animals. This would
be especially true if similar effects for petroleum fractions were found in
different animals. Interference with chemoreception or chemically mediated
behavior also may be one of the most sensitive measures of low level oil
pollution, since the much more obvious neuromuscular abnormalities appear at
higher, although still sublethal, levels of oil exposure.
For this study we chose the bait localization behavior of the lobster,
Homarus americanus. The lobster uses two chemoreceptor organs. Aesthetasc
hairs on the antennules represent their sense of smell, and function probably to
detect distant chemical signals in low concentration. Hairs on the walking legs
and maxillipeds are the equivalent of taste, and are essential in picking up food
and bringing it into the mouth while testing its palatability for ingestion. In
123
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this study, using the water-accommodated fraction (WAF) of #2 fuel oil, we
are mainly concerned with distance chemoreception of the antennules.
The first purpose of these experiments is to determine the range of #2 fuel
oil exposures that affect the feeding behavior of lobsters without causing
neuromuscular disturbance. Since chemoreception provides an important input
into their feeding behavior, we then apply neurophysiological techniques to
measure the effects of oil exposure on chemoreceptors in animals, where
sublethal behavioral abnormalities have been shown. This is the second goal of
these experiments. The results reported here are preliminary; the methods
are established.
MATERIALS AND METHODS
Flow-Through Oil Dosing System
In order to measure actual exposure levels, a continuous flow-through oil
dosing system is necessary. The flow-through system (Figure 10-1) consisted of
two head tanks, one control and one experimental. The experimental head
tank, 8' x 11" x 8", was fitted with three baffles to aid in the layering of the
oil after mixing. Its inflow was 4,000 ml/min. Oil was introduced via a syringe
pump at a fixed rate into the center of the fast jet of seawater, causing rapid
emulsification. The overflow of the head tank was skimmed off into a
collecting tank where the oil layer was siphoned off occasionally. From the
head tank, the oil-water mixture entered six 100-liter tanks individually.
The overflow from the individual tanks entered a holding tank where
lobsters were stored for neurophysiological preparations on oil-exposed
animals. The overflow from the collecting box and the holding tank entered an
acrylic-fiber filter box, where oil was removed before the water entered the
drain (Figure 10-1). The control head tank, 4' x 11" x 8", supplied four
individual 100-liter tanks. Its inflow was 2,600 ml/min. Individual tanks, both
experimental and control, had inflows ranging from 400-460 ml/min. Water
quality — salinity, temperature, ammonia, pH and C>2 content — and flow rate
to individual tanks were measured every other day.
Behavior
Two male and two female lobsters served as controls, three males and three
females as experimentals. From our holding facility, we chose lobsters which
had molted within two to eight weeks of the start of the experiment, to avoid
effects of pre-molt behavior during observation. The animals were measured for
close size match, and put in individual tanks containing a glazed clay shelter
and a pebble substrate. They were fed twice daily until all animals were feeding
normally. Then a base line for feeding behavior was determined over a five-day
period. During the whole experiment lobsters were observed daily in the early
morning (7-9 am) and late afternoon (4-6 pm). One-minute behavior recordings
were followed by the addition of food, which was lowered on a string from the
right or left front corners, alternately. Apart from general behavior (about 25
124
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INFLOW
DRAIN
Figure 10-1. Diagram of Flow-through Oil Dosing System.
NOTE: See text for details.
different postures and movements) the times for alert (first observable response
to food), wait (period between alert and leaving shelter to search), and search
(time after first leaving shelter until food touched with maxillipeds) were
recorded. On the morning of the 6th day, #2 fuel oil was introduced at a
predetermined flow rate. Lobster behavior was recorded 6 hours later and
subsequently twice daily, as above. After five days, oil introduction was
stopped. All recordings were continued as before for another five days to
determine behavioral and chemical recovery rates.
Oil Chemistry
Concentrations of total hydrocarbons in the water column were determined
by infrared spectroscopy before, during, and after introduction of oil. On the
day before oil was added, a 2-1 water sample from every tank was extracted
with 50 ml CCI4. A second extraction was performed 12 hours and a third 24
hours after onset of oil mixing. Extractions were continued daily for the
remaining four days of oil exposure, and on the first and second day
post-exposure to determine how quickly oil left our system. A small number of
CTbCli extractions were performed for gas chromatographic analysis. The oil
used in. the study was an Exxon #2, provided by the EPA Environmental
Research Laboratory, Narragansett, Rhode Island.
125
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Chamber ("Olfactometer").
NOTE: Stimulus is injected with a syringe (A) into a seawater flow (B) over
the dissected lobster antennule. The antennule is perfused through a micro-
pipette with oxygenated lobster saline (C) which exits the antennule into a
bath of saline (D). To make recordings, one small bundle of nerve fibers is
lifted from the saline bath into the air with a platinum hook electrode (E).
Seawater and saline baths are separated by a Sylgard cork, through which the
antennule passes.
Neurophysiology
Electrophysiological data were obtained from chemoreceptors of oil-exposed
and normal lobsters, some of which had been observed behaviorally. This
permits a comparison between the neural chemosensory input the animal
received, and the resultant behavior after processing through higher nerve
centers. Such a comparison is a necessary step in determining whether oil
interferes with behavior through chemoreception.
To measure neurophysiological activity, the lateral flagellum of the
antennule of a lobster was removed and placed in fresh seawater. The cut
proximal end was inserted through a Sylgard cork. Three to four cuticular rings
were removed. The distal tip was cut and the antennule placed in a lobster
saline bath in the stimulation-recording chamber (Figure 10-2). A micropipette
was inserted snugly into the distal tip, and perfusion with oxygenated lobster
126
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saline started within 10 minutes after the removal of the antennule from the
animal. Test chemical stimuli were injected into the continuously flowing
saline bathing the antennule. Recordings were made by picking up a small
nerve bundle with a platinum electrode. The signal was amplified via a
Tektronix Type 122 Preamplifier, and displayed on conventional recording
equipment for later analysis.
It is commonly accepted that neurophysiologically determined thresholds of
sensory receptors lie an order of magnitude above the behaviorally determined
thresholds. Thus, to document the effects of #2 fuel oil on lobster antennular
chemoreception, we used the following test series: (1) mussel juice; (2) #2 fuel
oil, 10 ppm: (3) mussel juice plus oil; (4) artificial seawater; and (5) mussel
juice. Stock solutions were made at one time and refrigerated. Artificial
seawater was made according to the MBL formula: 420 mM NaCl, 9 mM KC1,
9 mM CaCl 2'2H20, 23 mM MgCl2'6H20, 26 mM MgS04'7H20, 2 mM
NaHCOj (pH 7.3). This was used to eliminate introduction of day-to-day
variations in natural seawater. Mussel juice was made by homogenizing 10 g
wet weight of Mytilus edulis tissue in 100 ml artificial seawater. The suspension
was centrifuged at 27,000xg for 20 minutes and the pellet discarded. The
supernate was frozen in small aliquots until needed. The WAF of 10 ppm #2
fuel oil was made at the start of each preparation, due to the lability of the
oil-water suspension.
This protocol allowed us to compare the response to Stimulus (1) with the
response to Stimulus (5) for nerve fiber damage or fatigue, or lasting effects of
the prior oil test stimulus. Stimulus (2) and Stimulus (3) were used to
determine a) whether lobster antennules can detect oil as a chemical stimulus,
and b) if the presence of oil changes the response to mussel juice. Stimulus (4)
was used to determine the sensitivity of the preparation to a chemically neutral
stimulus; this allowed us to measure mechanoreceptor activity which can be
subtracted to discover purely chemosensory responses in the other tests.
RESULTS
Chemistry
Water quality measurements showed that for all experiments, salinity and
pH remained constant, ammonia remained undetectably low, and 02 remained
at saturation. Temperatures fluctuated with ambient water temperature, and
are listed below with each experiment.
Gas chromatography showed that the water-accommodated fraction
recovered from the lobster tanks closely resembled whole #2 fuel oil. Infrared
spectroscopy of CCI^-extractable lipids showed moderate (±20%) daily
fluctuations within and between individual tanks. The dosing system was
capable of maintaining relatively similar exposure levels.
127
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Behavior
In the first experiment, the recovered oil level in the exposure tanks was
about 0.08 ppm total hydrocarbon. The temperature gradually rose from 22°
to 24.5° C over the 15-day experimental period. Behavioral changes were
observed in the morning alert times, which, when comparing oil-exposed days
with pre-exposure days, slowed in experimental animals (p < 0.05). Total food
localization time was also slower (p < 0.025), perhaps as a result of slower
alerting. Control animal behavior did not change (Table 10-1). In this first
experiment, defensive postures and sometimes erratic and frantic behavior was
observed in most of the exposed lobsters, and not in control animals. Defensive
postures are characterized by wide open seizer claws, held close to the body,
while the animal sits retreated far into its shelter. Erratic and frantic
movements are sudden, unprovoked seizer snapping, jerky body movements
and twitches.
In the second experiment, the recovered oil level was about 0.15 ppm;
temperature was a constant 10° C. Neither oil-exposed nor control animals
showed significant changes in feeding behavior in the morning obervation,
when comparing pre-oil with oil exposure period. In the afternoon observation,
oil-exposed animals did not change their search speed but their alert was
delayed (p < 0.05). Control animals had a faster search time in the afternoon
(p < 0.005). Both control (p < 0.01) and experimental animals (p < 0.05)
showed shorter wait times (Table 10-1).
In the third experiment the temperature rose from 11° to 13.5° C, and the
recovered oil level was 1.5 ppm. At 30 hours the lobsters showed gross
neuromuscular defects, and oil inflow was stopped. In this experiment,
behavior in post-exposure recovery period was compared with pre-exposure
behavior. Experimental lobsters were slower in all phases of feeding behavior
during the five-day recovery period than in the five-day pre-oil period
(p < 0.001). Even five days after exposure to 1.5 ppm #2 fuel oil for 30 hours,
half the lobsters did not feed within the 10-minute limit (Figure 10-3). Control
lobsters showed no significant differences (Table 10-1). The animals during this
last experiment showed three levels of effects, some animals being affected
much more than others. A description of the three levels follows (see also
Figure 10-3).
• Most extreme (two lobsters) —After 30 hours of oil exposure, these
lobsters were found outside their burrows lying on their backs, pleopods
twitching or still, tail half curled, walking legs twitching, antennae and
antennules limp, gill bailers moving slightly. Occasionally the back and tail
were arched and then curled. At times, attempts were made to right itself.
Body jerked after food entered into tank. No recovery occurred in five
days.
128
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Table 10-1. Significant Changes in Feeding Behavior of Lobsters
Before and During Exposure to #2 Fuel Oil (WAF)*.
Level of
Exposure
0.08 ppm
0.15 ppm
1.5 ppm
Observation
Time
AM
PM
AM
PM
AM
PM
Cont Exp Cont
Exp Cont
Exp
Cont Exp Cont
Exp Cont
Exp
Alert
s(0.05) =
=
=
s(0.0S) =
s(0.001) =
=
Wait
=
=
-
f(0.01) f<0.05) =
s(O.OOI) =
stO.001!
Search
=
=
=
f (0.005) =
s(0.025) =
s(0.001)
Total
s(0.025) =
f(0.05) =
—
=
s(0.01) =
s(0.001)
* In 1.5 ppm exposure significant differences are listed for the
pre-exposure period compared with the post-exposure period; the other
two experiments list differences between pre-exposure and exposure
periods,
NOTE: Measured times were equal {=), slower (s), or faster (f) than in
the pre-exposure control period. Significance levels are given in
parentheses.
-------
Q_
Q_
2.00 -
o
Q I .00
cr
UJ
>
o
o
UJ
or
.05
EFFECT OF NO. 2 FUEL OIL ON
LOBSTER FEEDING BEHAVIOR
110
z —I
o m
en
0 12 24 36 48 60 72 84 96 108 120 132 144 (HRS.)
PRE OIL
POST
Figure 10-3. Effect of 1.5 ppm No. 2 Fuel Oil (WAF)
on Lobster Feeding Behavior.
NOTE: Large bars indicate number of lobsters not feeding within 10 minutes
of food introduction. Broken line represents estimated oil levels in lobster
tanks based on actual recovery measurements (indicated by dots and stand-
ard deviation bars). Hatched horizontal bar shows period of oil introduction
into the system. "Pre" is the 5 day pre-exposure period: all lobsters feed and
recovered oil remains below 0.05 ppm. "Oil" is the exposure period: highest
mean exposure level 1.7 ppm, oil stopped after 30 hrs. "Post" is the recovery
period: oil quickly leaves the system, feeding behavior remains seriously af-
fected for the entire 5-day period.
• Moderate (three lobsters) — These animals were generally out of their
burrows in two alternating stances. In one, the tail and head were down,
the antennae were folded back, the antennules beat slowly, and the
animal lay down low on the walking legs. This appeared to be a resting
position. Then the tail and head arched up, the claws were opened and
held close to the body, the antennae and antennules were held straight up
and together, animal stood high on walking legs, and made frequent tail
flips. Poor coordination was exhibited. Frequent aggressive lunges were
made with open, raised seizer claw, or jabs with both claws at no obvious
target. Lobsters were unaware of the presence of food, at times walking
right over it without responding. If they did pick up the food, they
continued to wander aimlessly around the tank with the food clutched
tightly in the maxillipeds. Recovery in five days was almost complete in
two animals; one animal did not recover.
• Light (one lobster) — This animal remained in its burrow but was high on
its walking legs, "spider" position and shaking. There were sporadic alerts
not necessarily related to food. It exhibited no search. After two days
recovery, there were slow hesitant approaches to the food with tail flips at
the slightest irregularity. At the end of five days there was complete
recovery.
130
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Neurophysiology
The preliminary results from neurophysiological experiments on the
lobster's olfactory chemoreceptors are presented with a few examples in Figure
10-4. Details are provided in the figure legend. These examples show that (1)
the water-accommodated fraction of #2 fuel oil itself can be perceived as a
stimulus by the primary receptor cells, (2) that the presence of oil in a mussel
juice food stimulus can change the response pattern of the small nerve bundle,
and (3) that exposure to oil causes abnormal bursting patterns (See Figure
10-4).
Generally, differences in chemoreceptor responses between mussel and
mussel-plus-oil are more distinct in oil-exposed lobsters than in controls. One
other striking feature is the tendency of oil-exposed individuals to exhibit
irregular bursts, or frequent small clusters, of spikes, both spontaneously and in
response to stimuli (Figure 10-4), This may be a general injury response (6)
here caused by oil exposure. However, it has also appeared in nerves from
animals which were exposed to very low levels of oil (0.3 ppm) in response to
mussel-plus-oil stimuli, but not to mussel alone.
DISCUSSION
Our experiments have shown thus far that the original hypothesis that oil
pollution may interfere in a number of different ways with chemoreception,
and hence marine animal behavior, is not unreasonable. Behavioral experiments
on the efficiency of the lobster's chemically mediated feeding behavior have
shown that exposure to #2 fuel oil (WAF) causes sipifieant delays after five
days at exposure levels as low as 0.08 and 0.15 ppm. Increased dosage caused
increasingly severe effects. Also behavioral recovery was a function of exposure
level. At higher exposures (1.5 ppm) serious neuromuscular abnormalities
appeared within 30 hours. Lobsters showed great individual differences in
behavioral effects and recovery. The range of exposure levels where behavior
was affected, but no serious neuromuscular defects appeared, proved to be
surprisingly narrow. However, further experiments are required for complete
documentation of this point.
Parallel neurophysiological experiments on the effects of such exposures on
chemoreceptor performance showed that the receptors perceive oil as a
chemical stimulus, that the presence of oil could modify normal responses, and
that oil-exposed lobsters ofen showed abnormal receptor activity, both
spontaneously and in response to food stimuli.
Based on these results, it appears that #2 fuel oil (WAF) interferes with
lobster behavior in a number of ways. At low exposure levels (0.1 ppm range),
131
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J OIL
I"" rrWtNi"!' Ir"l"f I 1
| MUSSEL
miiinmi ii inn ii' iiii»i
J MUSSEL + OIL
NOTES;
a. Control lobster, response to oil stimulus,
b. Control lobster, response to mussel juice stimulus, i i
c. Control lobster, response to mussel juice-plus-oil stimulus. 1 sec
d. Oil-exposed lobster, response to mussel juice stimulus. Note the differences
between b and c which were recorded from the same nerve bundle a few min-
utes apart. Apparently the addition of oil changes the response to mussel
juice. Note also the differences between b and d. The irregular bursting pat-
terns seen in d are observed in oil-exposed lobsters, not in control lobsters.
While there still is a response to mussel juice, the response pattern appears
erratic. It is not known if this erratic pattern results in a different interpreta-
tion by the lobster.
-------
the observed effects on behavior may be due to oil-induced changes in
chemoreception. At higher exposure levels (1 ppm range), the behavioral
effects appear neuromuscular, with a loss of coordination and equilibrium.
These preliminary results are being corroborated at different exposure levels.
In additional experiments, we will attempt to provide a correlation between
behavioral and neurophysiological results of oil exposure to determine if the
observed behavioral effects of oil are caused by a malfunctioning
chemoreceptor system.
ACKNOWLEDGEMENT
We would like to thank L. Ashkenas, B. Bryant and T. Dourdeville for
expert technical assistance. We are indebted to B. Melzian and D. Stenzler
for help in designing and construction of the flow-through oil dosing
system. Dr. B. Ache and B. Johnson provided us with the design and
training for the neurophysiological recordings. We would also like to thank
Dr. D.C. Miller for valuable discussions during the course of these
experiments. Financial support was provided by grants from the U.S.
Environmental Protection Agency (R-803833) and the U.S. Energy
Research and Development Administration (E(l 1-1)2546).
REFERENCES
1. Atema, J. 1976. Sublethal Effects of Petroleum Fractions on the Behavior
of the Lobster, Homarus americanus, and the Mud Snail, Nassarius
obsoletus. In: Estuarine Processes; Uses, Stresses and Adaptation to the
Estuary, Wiley, M. (ed.), Academic Press, New York, Vol. 1. pp. 302-312.
2. Atema, J. 1977. The Effects of Oil on Lobsters. Ocean us 20: 67.
3. Atema, J. and L. Stein. 1974. Effects of Crude Oil on the Feeding Behavior
of the Lobster, Homarus americanus. Envir. Poll. 6: 77.
4. Blumer, M. 1970. Oil Contamination and the Living Resources of the Sea.
FAO Technical Conference on Marine Pollution and Its Effects on Living
Resources and Fishing, Rome, December 9-18.
5. Blumer, M. and J. Sass. 1972. Oil Pollution: Persistence and Degradation of
Spilled Fuel Oil. Science 176: 1120.
6. Dethier, V.G. 1971. A Surfeit of Stimuli: A Paucity of Receptors. Amer.
Sci. 59: 706.
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7. Jaenicke, L., D.G. Miiller and R.E. Moore. 1974. Multifidene and
Aucantene, C-l 1 Hydrocarbons in the Male-Attracting Essential Oil from
the Gynogametes of Cutleria multifield (Phaeophyta). J. Amer. Chem. Soc.
96: 3324.
8. Mitchell, R., S. Fogel and I. Chen. 1972. Bacterial Che mo reception: An
Important Ecological Phenomenon Inhibited by Hydrocarbons. Water Res.
6: 1137.
9. Sanders, H.L., J.F. Grassle, G.R. Hampson, L. Morse, S. Garner-Price,
and C.C. Jones. 1980. Anatomy of an Oil Spill: Long Term Effects from
the Grounding of the Barge Florida off West Falmouth, Massachusetts.
J. Mar. Res. 38:265-380.
10. Takahashi, F.T. and J.S. Kittredge. 1973. Sublethal Effects of the Water
Soluble Component of Oil: Chemical Communication in the Marine
Environment. In: The Microbial Degradation of Oil Pollutants, Ahearn,
D.G. and Meyers, S.P. (eds.). Publ. No. LSU-SG-73-01. Center Wetland
Resources, Louisiana State Univ., Baton Rouge, La. 259 pp.
11. Walsh, F. and R. Mitchell. 1973. Inhibition of Bacterial Chemoreception by
Hydrocarbons. In: The Microbial Degradation of Oil Pollutants, Ahearn,
D.G. and Meyers, S.P. (eds.). Publ. No. LSU-SG-73-01. Center Wetland
Resources, Louisiana State Univ., Baton Rouge, La. 275 pp.
134
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INFLUENCE OF NO. 2 FUEL OIL
ON SURVIVAL
AND REPRODUCTION
OF FOUR MARINE INVERTEBRATES
J. A. Pechenik, D. M. Johns, and D. C. Miller
Environmental Research Laboratory
U.S. Environmental Protection Agency
Narragansett, Rhode Island 02882
ABSTRACT
Responses to the water accommodated fraction of No. 2 fuel oil were
determined in three marine gastropods (Nassarius obsoletus, Crepidula
fornicata and Urosalpinx cinerea), and one Crustacean (Cancer irroratus).
Experiments were conducted in either flowing or static systems at the
following nominal oil concentrations: 0.0 ppm (control), 0.01 ppm, 0.1 ppm,
1.0 ppm. Mortality of adults and larvae was consistently pronounced only at a
nominal concentration of 1.0 ppm. Toxicity to adult N. obsoletus at this
concentration was greater during the winter than during the summer. Presence
of sediment accelerated mortality during the summer, but had no effect on
winter mortality. Exposure of adult N. obsoletus and U. cinerea to oil
concentrations as low as 0.01 ppm and 0.1 ppm, respectively, interferred with
normal patterns of egg capsule deposition. Exposure to oil did not alter the
number of eggs/capsule in TV. obsoletus or U. cinerea, and embryos produced
by oil-exposed snails were viable. Fecundity of N. obsoletus may be reduced at
a nominal concentration of 0.10 ppm. Growth rates of larval N. obsoletus and
C. fornicata were reduced at nominal levels of 0.01 ppm and greater. Larvae of
C. irroratus reared at a nominal concentration of 0.1 ppm weighed less at all
zoeal stages relative to controls, even though carapace length of each larval
stage, and time required to reach the megalops stage of development, were not
altered.
INTRODUCTION
Lethal effects of petroleum hydrocarbons have been documented for a
variety of marine organisms (21), including zooplankton (20) and both adult
and developmental stages of benthic invertebrates (2, 9, 11, 19). Sublethal
concentrations of hydrocarbons are also known to interfere with aspects of
135
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invertebrate reproduction, such as sperm motility and fertilization success (22,
25), and embryonic cleavage rates (1, 22). Reduced egg production has been
reported for oil-exposed Mytilus edulis (7) and Eurytemora affinis (5), and
development of some larval Crustacea (16, 32, 33) and bivalves (9, 24, 25) is
delayed after exposure to sublethal oil concentrations. Oil-induced changes in
larval behavior of Homarus americanus (32) and Cancer irroratus (6) have also
been demonstrated. Recently, Anderson et al (3) have reported effects of low
hydrocarbon levels on hatchability of fish embryos and on heart beat rate of
larval fish.
The general objective of this investigation was to elucidate some sublethal
effects of No. 2 fuel oil (introduced as the water accommodated fraction,
WAF) on aspects of the reproductive and developmental biology of several
common coastal invertebrates. Specific topics studied include egg capsule
deposition, fecundity, hatchability and larval growth rates. The lethal dose of
the oil was also determined for adult and larval Nassarius obsoletus, and larvae
of Crepidula fornicata and Cancer irroratus, in order to establish sublethal
exposure levels.
A review by Moore and Dwyer (21) suggest that larval organisms are more
sensitive to hydrocarbon toxicity than are adults, yet tolerance data on adults
and larvae of the same species are infrequently reported. Culliney et al (12)
suggest that the high surface/volume ratio in larvae and their "obligatory
exposure to whatever may be in the water" would make larvae particularly
susceptible to toxic substances, such as oil, at very low concentrations. Our
study includes work on both adults and larvae of N. obsoletus to further
examine this hypothesis.
MATERIALS AND METHODS
Experiments were conducted using the gastropods Nassarius obsoletus,
Crepidula fornicata and Urosalpinx cinerea, and the crustacean Cancer
irroratus. Adults were exposed to oil using the flow-through oil-dosing system
described by Hyland et al (15). Briefly, unfiltered seawater and No. 2 fuel oil
enter a mixing chamber. The WAF produced is then metered into exposure
tanks where it is diluted to the desired concentration by controlled flow of
untreated seawater. Total hydrocarbon concentrations in control (for
background) and experimental tanks are monitored three times/week by
infrared spectrophotometry, and flow rates are adjusted to maintain desired
WAF exposure levels. Nominal total hydrocarbon concentrations (WAF) were:
0.0 ppm (control), 0.01 ppm, 0.1 ppm, and 1.0 ppm. Because measured
hydrocarbon concentrations varied with time in the flow-through system, the
nominal concentrations (cited as "X" ppm in the text) indicate only the order
136
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of magnitude dose level employed. Mean total hydrocarbon concentrations (±
s.d.) measured during each experiment are given with the results; between
treatment mean hydrocarbon values were significantly different for all
experiments (P < 0.05).
All three gastropod species studied produce egg capsules. While C. fornicata
broods its capsules, U. cinerea and N. obsoletus attach capsules to solid
substrates and then abandon them, making these capsules easy to collect and
count. Descriptions of larval development have been published for N. obsoletus
(29), C. fornicata (34) and C. irroratus (28).
Adult Mud Snail Survivorship and Egg Capsule Deposition
Experiments with adult mud snails were conducted in the flow-through
dosing system described above. Adults of N. obsoletus were collected from
Bissell Cove, Rhode Island, and groups of 35-100 individuals placed in circular
plastic containers (26 cm diameter, 6 cm high) and completely submerged in
the dosing tanks. The top and sides were perforated to permit water
circulation. Surface area of the top and side of each container was
approximately equal. Snails were fed shredded Mercenaria mercenaria tissue
weekly, and the number and position of deposited egg capsules were recorded
before capsules were removed each week. All container surfaces were wiped
clean after each examination. Dead snails were counted and removed
periodically. The mean number of eggs per capsule was determined for N.
obsoletus in all treatments. Since N. obosoletus is primarily a deposit feeder
(30), and sediments are known to accumulate petroleum hydrocarbons from
seawater (15, 17), one experiment was run with mud added to evaluate its
influence on toxicity.
Reproduction of Urosalpinx cinerea
Specimens of U. cinera were collected at Jamestown, R.I., in May, 1976,
and groups of ten individuals were placed in perforated plastic freezer
containers. Three boxes were submerged in the flow-through system at each of
the following nominal oil levels: control (0.0 ppm), 0.01 ppm, 0.1 ppm.
Freshly collected barnacles were provided weekly as food. Once each week,
deposited egg capsules were counted and then removed. In July, a sample of
egg capsules was taken from each treatment level to determine the mean
number of eggs encapsulated. The number of females present in each container
was determined in the middle of the experiment using the live-sexing technique
of Hargis (13).
137
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Larval Survival and Growth
Survival of larvae exposed to No. 2 fuel oil (WAF) was assessed primarily
under static conditions. Glass scintillation vials were completely filled with
water siphoned from the flow-through dosing tanks. Fifteen to 20 two to three day
old N. obsoletus or C. fornicata larvae were pipetted into each vial, and
Isochrysis galbana was provided as food at an initial density of about 1x10^
cells/ml. Vials were then tightly capped to minimize volatilization of
hydrocarbons. Each experiment was conducted in triplicate. Larvae were
counted daily and survivors were transferred to fresh medium. The experiments
were conducted at room temperature (21-23°C), well within the range for
good larval growth (10, 31).
One preliminary flow-through experiment was conducted with about 100
two-day old C. fornicata larvae (approximately 420 (im in shell length), using a
system similar to that described by Calabrese and Rhodes (10). Water from the
oil-dosing tanks was siphoned at approximately 50 ml/minute into
flow-through chambers containing larvae, and 200 ml I. galbana suspension
added at the beginning and end of each day as a feeding supplement.
Completely filled, capped quart glass jars were used to determine the effects
of the oil on growth and survival of larval C. irroratus. Seventy-five Stage I zoea
were added to each jar. Larvae were fed Anemia salina nauplii provided in excess
numbers. Larval mortality was determined daily, and survivors were transferred
to fresh medium. These experiments were conducted at 15°C, the optimal
temperature for development of C. irroratus (28), and under a 12L:12D
photoperiod. In a separate series of experiments, at least five individuals of
each larval stage were harvested from control and "0.1" ppm levels to monitor
growth. Larval carapace lengths were measured using an ocular micrometer.
Dry weights were then determined using a Perkin-Elmer electrobalance after
the larvae were rinsed with distilled water and dried at 80°C for 24 hours in
pre-weighed foil pans.
RESULTS
Adult and Larval Survival
Exposure to a nominal concentration of 1.0 ppm was found to be lethal for
all adults and larvae tested. Concentrations of "0.1" ppm and "0.01" ppm
were sublethal to all test organisms for the particular exposure periods of our
experiments.
Mortality of N. obsoletus adults did not exceed five percent in any
experiment at control, "0.01" ppm or "0.1" ppm exposure levels. Substantial
138
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mortality occurred only at "1.0" ppm. Pronounced seasonal variation in
toxicity was evident (Figure 11-1). In one winter exposure, mortality at "1.0"
ppm reached 50 percent within approximately 30 days, with the remaining
snails dying during the subsequent 30 days. Similar results had been obtained
in a preliminary experiment initiated the preceding February. A very different
mortality profile was seen at "1.0" ppm in two summer exposures. In 1976,
approximately 40 percent of the snails were still living at the end of three
months (Figure 11-1). In 1977, presence or absence of sediment in the holding
containers was added as another variable. While summer toxicity of the WAF
was still relatively low, mortalities were substantially increased by the presence
of mud (Figure 11-2). In contrast, the mortality pattern was unaffected by
sediment in the winter.
DAYS OF EXPOSURE
Figure 11-1. Survival of adult N. obsoletus exposed to
"1.0" ppm No. 2 fuel oil (WAF).
NOTE: Control mortalities were less than five percent. Winter experiment was
run 10/22/76 - 12/15/76 (100 snails/treatment). Summer experiment was run
7/6/76 - 10/12/76 (20 snails/treatment). Mean oil hydrocarbon concentrations
± s.d. (N) measured were: 1.25 ppm ±0.34 (25), winter; 0.94 ppm ±0.44
(31) summer.
139
-------
DAY
Figure 11-2. Survival of adult N. obsoletus exposed to
"1.0" ppm in the presence or absence of sediment.
NOTE: Winter exposures were initiated 10/22/76 (50 snails/treatment). Sum-
mer exposures were initiated 7/12/77 (25 snails/treatment). Control mortal-
ities less than 5 percent. Mean oil hydrocarbon concentrations ± s.d. (N) were:
1,25 ppm ±0.34 (25), winter; 0,70 ppm ±0.27 (23), summer.
Larval mortality of N, obsoletus was high in four to eight day experiments
at "1.0" ppm, and relatively low at lesser concentrations (Table 11-1).
Substantial batch variability in larval tolerance was observed (Figure 11-3).
Whereas 50 percent mortality was recorded at "1.0" ppm after three-days in
experiment "A", the 50 percent level was not exceeded until day eight in a
second experiment using a different hatch of larvae. Indeed, no mortality
occurred until day four in experiment "B".
All C. irroratus larvae exposed to "1.0" ppm died within four days. At
"0.1" ppm and "0.01" ppm, about 33 percent of the larvae were still living
after three weeks (Figure 11-4). Survival to the mega lops stage, attained after
25-28 days, was: control, 58%; "0.01" ppm, 34%; "0.1" ppm, 30%; "1.0"
ppm, 0%.
140
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Table 11-1. Larval Mortality of N. obsoletus after Exposure to No. 2 Fuel Oil (WAF).
Control
Nominal Oil Concentrations
0.01 ppm 0.1 ppm
1.0 ppm
Experiment A (4 days)
Oil concentration (ppm)
0.0
0.0085 ± 0.0055(2)
0.093 ±0.017(3)
0.98 + 0.28(3)
Mean mortality (%)
11.6
0.0
5.9
81.6
Range of mortality (%)
0.0-20.0
_
0,0-8.3
53.6-100.0
Experiment B (8 days)
Oil concentration (ppm)
0.0
0.012 ± 0.0089(4)
0.11 ±0.024(4)
0.83 ±0.19(4)
Mean mortality (%)
5.9
26.1
13.7
65.5
Range of mortality (%)
0.0-10.5
12.5-40.0
6.7-23.5
55.0-83.3
NOTE: Experiment "A" employed 15 larvae/vial arid ran 4 days. Experiment "B" employed 20 larvae/vial and ran 8 days. The
range of mortality observed in the 3 replicates run at each concentration is indicated. Oil concentrations are reported as mean
ppm ± s.d. (N).
-------
DAYS OF EXPOSURE
Figure 11-3. Batch variability in N. obsoletus larval
survival upon exposure to "1.0" ppm fuel oil (WAF).
NOTE: Experiment "A" employed 15 larvae/vial and ran 4 days. Experiment
"B" employed 20 larvae/vial and ran 8 days. The range of mortality observed
in the 3 replicates run at each concentration is indicated.
Static exposures of larval C. fornicata ran only three days. No mortality was
observed at any oil level tested. However, no larvae were observed swimming at
"1.0" ppm by day two. Activity was normal at lower oil concentrations.
Moreover, the guts of larvae at "1.0" ppm were empty of food by the second
day, whereas veligers at lower oil concentrations continued to feed during the
three-day experiment. In the flow-through exposure, veligers held at "1.0"
ppm also stopped swimming by the second day of the experiment. These larvae
were alive but emaciated by day four, and dead by day six. Crepidula fornicata
larva had good survival at lower oil levels, although growth rates were affected
as discussed below.
Gastropod Reproduction
Adults of N. obsoletus collected in February, 1976, and October, 1976,
deposited their first egg capsules in laboratory control containers on 4/30/76
and 5/6/77, respectively, when the water temperature warmed to
approximately 10°C. Sastry (27) also obtained egg capsules at 10°C from AC
obsoletus collected in January at Beaufort, North Carolina.
142
-------
100
80
_l
<
> 60
>
tr
3
20
0
0 4 8 12 16 20
DAY
Figure 11-4. Survival of C. irroratus during development
at different oil concentrations.
NOTE: Mean oil concentrations ± s.d. (N) were "0.01" ppm: 0.011 ppm
±0.009 (14); "0.1" ppm: 0.094 ppm ±0.027 (14); "1.0" ppm: 0.913 ppm ±
0.210 (14).
Onset of egg capsule deposition by snails exposed to "0.01" ppm and "0.1"
ppm was delayed by about two weeks relative to controls in 1976, and delayed
up to one week in 1977. Encapsulated embryos produced by these oil-exposed
snails and transferred to control conditions developed to hatching without
noticeable abnormality. In a mid-summer experiment, control individuals
produced many capsules within three days of collection from the field, but
those held at "1.0" ppm never deposited any capsules.
Egg capsule production is used here as an index of fecundity for both N.
obsoletus and U. cinerea, since exposure to oil did not alter the average number
of eggs per capsule (Table 11-2).
Egg capsule production by N. obsoletus held at "0.1" ppm may be reduced
relative to control, and "0.01" ppm snails (Table 11-3, line "e"), although
these data are insufficient for statistical analysis. Results are inconclusive due
to the death of an unknown number of females during the test breeding period,
precluding accurate calculation of individual fecundity.
143
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Table 11-2. Effect of Exposure to No. 2 Fuel Oil (WAF) on the Distribution
of Eggs Among Egg Capsules of N. obsoletus and U. cinerea.
Nominal Oil Concentration
Species Control 0.01 ppm 0.01 ppm
N. obsoletus
Mean oil concentration
No. capsules examined
Mean eggs/capsule ± s.d.
F =0.013 (P > 0.25)
U. cinerea
Mean oil concentration
No. capsules examined
Mean eggs/capsule ± s.d.
F = 0.46 (P> 0.25)
NOTE: F-values calculated by one-way analysis of variance. Oil concentration is given as mean ppm ± s.d.
(N).
0.0 0.021 ±0.012(15) 0.086 ± 0.048(60)
8 8 8
62.8+ 14.8 61.5 ± 11.1 64.8±11.6
0.0 0.016 ± 0.011(18) 0.067 ± 0.033(22)
20 20 20
8.5 ±1.5 8.6 ±1.8 8.1 ±2.5
-------
Table 11-3. Effect of Exposure to No. 2 Fuel Oil on
Fecundity of N. Obsoletus.
Nominal Oil Concentration
Control
0.01 ppm
0.10 ppm
A
B
A
B
A
B
a. Total capsules
11,892
6,771
6,442
10,983
7,509
7,419
b. No. live snails 6/3
33
34
34
28
33
37
c. No. live snails 8/17
31
28
33
28
30
35
d. No females 8/17
14
11
11
14
14
20
e. Maximum capsules/female*
849.4
615.5
585.6
784.5
536.4
370.9
f. Minimum capsules/female**
743.2
398.3
536.8
784.5
441.7
337.2
* Assuming all dead individuals were female (a/d)
** Assuming no dead individuals were female
NOTE; Each container initially held 35 snails. Two groups of snails were exposed at each oil level. Dates of exposure were
2/13/76— 8/9/76. Measured oil concentrations are given in Figure 11-5.
-------
Oil exposure modified the normal pattern of egg capsule deposition by adult
N. obsoletus (Figures 11-5 and 11-6). Control snails tended to climb up the
sides of the containers and deposit egg capsules mostly on the underside of the
lid (Table 11-4). In the intertidal zone where spawning occurs, this behavior
would contribute to placement of egg capsules into high-humidity
environments where exposure of developing embryos to desiccation stress
would be minimized (23). In contrast, M obsoletus exposed to the "0.01" ppm
and "0.1" ppm oil deposited capsules primarily on the container sides (Table
11-4). This effect was consistently most pronounced in May and June, at the
beginning of the reproductive season (Figures 11-5 and 11-6). After 15-20
days, lid deposition began to increase for oil-exposed snails in both runs.
However, lid deposition by control snails remained greater than that by
UJ
a
Figure 11-5. Influence of No. 2 fuel oil on egg capsule
deposition behavior of N. obsoletus, 1976.
NOTE: Exposures were initiated 2/13/76. Each point represents data pooled
from 2-4 replicate containers holding fifty snails each. The total number of
capsules deposited were: 19,492 (control): 9,894 ("0.01" ppm); 11,287 ("0.1"
ppm). Arrows indicate the transfer of two control containers to "0.01" ppm.
Mean total petroleum hydrocarbon concentrations ± s.d. (N) at each nominal
concentration were "0.01" ppm: 0.020 ± 0.008 (9); "0.1" ppm: 0.082 ±0.044
(20).
146
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3QIS NO S3~inSdVO ON / Qll NO S3inSdV3 ON
Figure 11-6. Influence of No. 2 fuel oil on egg capsule
deposition behavior of N. obsoletus, 1977.
NOTE: Exposures initiated 10/22/76. Open circles represent data from con-
trol snails, closed circles represent data from snails held at "0.1" ppm. Mean
oil hydrocarbon concentration at "0.1" ppm ± s.d. (N) was 0.077 ppm ±0.026
(34). Arrows indicate reversal of treatments.
147
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Table 11-4. Placement of Egg Capsules by ISI. obsoletus.
Nominal Oil Level
Capsules on Lid
Capsules on Sides
Lid/Side Ratio
Oil Concentration
1976
Control
19,092
3,570
5.3
0.0
0.01 ppm
7,215
2,456
2.9
0.0201 ± 0.008(9!
0.1 ppm
10,112
4,816
2.1
0.082 + 0.044(20)
1977
Control
6,393
852
7.5
0.0
0.1 ppm
3,445
6,967
0.5
0.081 ±0.025(17)
NOTE: Summary of results obtained in 1976 {4/30 — 8/9) and 1977 (5/18 — 6/29). Oil concentrations are given as mean ppm
± s.d. (N).
-------
oil-exposed snails, with one exception (i.e., 6/10/76). After transfer of two
control containers to "0.1" ppm, lid deposition decreased and remained
suppressed for some 30 days (Figure 11 -5). In a similar experiment conducted
the next spring, control containers were transferred to "0.1" ppm and
containers held at "0.1" ppm were transferred to control conditions. Shifts in
deposition patterns were again observed (Figure 11-6), although not until after
four weeks for the former control group of snails.
With oyster drills (U. cinerea), there was no demonstrable effect of oil on
fecundity when tested by one-way analysis of variance (P > 0.25), nor
did exposure to "0.01" ppm and "0.1" ppm have any statistically significant
effect on egg capsule placement. Egg capsule deposition behavior of oyster
drills was affected by oil, however. No drills held at "0.1" ppm deposited
capsules on the undersides of the container lids, in contrast to 13% to 14% lid
deposition in 0.01 ppm and control treatments, respectively (Table 11-5).
Larval Growth
The influence of No. 2 fuel oil (WAF) on larval growth of three invertebrate
species is summarized in Table 11-6. Growth of N. obsoletus larvae (jum shell
length) was dramatically impaired at "0.01" ppm and "1.0" ppm, but was only
slightly reduced at "0.1" ppm. This curious response pattern was observed in
two experiments involving different hatches of larvae. In contrast, the effect of
oil on larval growth of C. fomicata correlated positively with increasing oil
concentration in the static experiments. Reduced growth was also evident at
"0.1" ppm in the single flow-through experiment conducted. Growth of C.
irroratus larvae, measured as change in mean dry weight/individual, declined
relative to controls as development proceeded at the "0.1" ppm concentration.
The dry weight of oil-exposed larvae was only 70.1 percent of control weight
for the Stage IV zoea, and only 63 percent of control weight for the Stage V
zoea. However, these same C. irroratus larvae exhibited no differences in
molting frequency ,or in carapace length with respect to control individuals.
Cancer irroratus larvae in all sublethal oil treatments (i.e., all below "1.0" ppm)
reached the megalops stage in 25-28 days.
DISCUSSION
The concentration of hydrocarbons lethal to larvae is generally believed to
be about one-tenth of the concentration lethal to adults (14, 21). In our study,
a nominal concentration of 1.0 ppm was lethal to adults and larvae alike,
although 50% mortality of larval N. obsoletus occurred much sooner for larvae
than for adults. Exposure to "0.01" ppm and "0.1" ppm produced mainly
sublethal effects in both larvae and adults of this species.
149
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Table 11-5. Effect of Exposure to No. 2 Fuel Oil (WAF) on the Egg Capsules
Deposition Patterns of U. cinerea.
Substrate
Control
Nominal Oil Concentration
0.01 ppm 0.1 ppm
F
Underside of lid
12.9%
14.0%
0.0%
2.0
Bottom Substrates (rock and shells)
58.8%
53.2%
40.0%
0.3
Sides
28.3%
32.8%
60.0%
1.1
Total No. Capsules Deposited
583
808
477
NOTE: Table entries are percent of total egg capsules deposited on each surface. Data are means of three replicates at each
concentration. F-values {d.f. = 2, 6} calculated by one-way analysis of variance on percentages of total egg capsules deposited.
Oil concentrations are given in Table 11-2.
-------
Table 11-6. Effect of Exposure to No. 2 Fuel Oil (WAF) on Larval Growth Rates,
Calculated as (Control Growth - Experimental Growth)/Control Growth x 100%.
Species
Days
Exposed
0.01 ppm
Percent Reduction in Growth
Nominal Oil Concentration
0.1 ppm 1.0 ppm
No. Larvae/
Treatment
N. obsoletus
4
44.7%
7.9%
94.7%
45
N. obsoletus
8
52.4%
6.1%
97.6%
60
C. fomicata
3
11.7%
69.1%
100%
60
C. fomicata
3
—
70.6%
100%
40
C. fomicata (flow-through)
14
44.8%
—
50
C. irroratus
22
37.0%
—
75
NOTE: Growth of veliger larvae was measured in terms of shell length (/Jim) while growth of crab zoea was measured in terms of
dry weight {/ug). All experiments were conducted under static conditions, except as indicated. Measured hydrocarbon
concentrations are given in Table 11-1 and Figure 11-4 legends. Each entry is based on the pooled data from 2-3 replicates at
each concentration.
-------
Experiments on pollutant toxicity are usually conducted under constant
conditions of light, temperature, and salinity. The results of such experiments
may not be applicable to the field, where environmental conditions vary. A
case in point is the observed seasonal variation in oil toxicity to adult N.
obsoletus, with toxicity being accentuated in winter. Egg capsule deposition
patterns of oil-exposed N. obsoletus also showed temporal variability. Patterns
were least like those for control snails when water temperature was low at the
beginning of the breeding season, but changed substantially as water
temperatures rose. Similarly, Krebs and Burns (17) have observed that fiddler
crabs (Uca pugnax) exposed to No. 2 fuel oil in the field show abnormal
behavior only at temperatures near the lower limit of their normal range of
activity.
There are several possible explanations of the greater toxicity of this oil at
low temperatures. Low temperature may directly increase the relative
concentration of the more toxic oil fractions present in the water, either
through altered solubility, volatization, or shifts in bacterial activity. This
hypothesis is currently being explored. It is also possible that seasonal changes
in toxicity result from changes in the physiological state of the animals and/or
from additive effects of low temperature and oil stress.
There is currently little information on how petroleum hydrocarbons enter
aquatic animals, but recent evidence indicates that uptake of oil through
ingestion of contaminated food may be at least as important as diffusional
uptake (8, 11, 18). This is consistent with our observations of higher mortality
of adult N. obsoletus in the presence of sediment. This occurred only during
the summer, when N. obsoletus is actively deposit-feeding (30). The sediment
effect did not occur during the winter, when the snails are inactive.
We observed several sublethal effects on invertebrate reproduction,
including possible reduction in the fecundity of N. obsoletus. Although there
was no alteration in the number of eggs per capsule, the number of capsules
produced appeared to decline. One possible variable influencing egg capsule
production may be date of initiation of oil exposure relative to the onset of
oogenesis. In this study, U. cinerea were exposed to hydrocarbons after
gametogenesis was completed, and egg capsule depostion was already
underway, which might explain the absence of a fecundity response for this
species. More data are needed to resolve this issue.
The observed alteration of egg capsule deposition behavior with respect to
substrate orientation has significant ecological consequences for N. obsoletus.
The egg capsules and embryos of N. obsoletus are not well adapted for
deposition in the exposed intertidal zone; successful pre-hatching development
of this species is apparently dependent instead upon the proper placement of
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the capsules on substrates (23). Interference, with normal patterns of egg
capsule deposition behavior could substantially increase pre-hatching mortality
from desiccation stress (23).
The reduction in larval growth rate observed at sublethal oil concentrations
could result from increased energy expenditure, decreased ingestion rate,
decreased assimilation efficiency, or a combination of these factors. Present
evidence suggests that the reduced growth observed was due at least in part to
reduced food intake. Larvae of C. fornicata and N. obsoletus held at "1.0"
ppm ceased feeding at least one to two days before they died. The larval guts
of C. fornicata were empty of food by the second day of each experiment,
even though the velar lobes remained extended and ciliary activity was
observed. Tissues in these individuals became dramatically shrunken within
several days after initiation of exposure to oil. Veligers held at oil
concentrations of "0.10" ppm showed no such morphological abnormality, but
preliminary experiments (Pechenik, unpublished) reveal decreased ingestion
rates at this concentration, relative to ingestion rates of control larvae.
It is not yet possible to precisely predict the threshold oil concentrations at
which lethal or sublethal effects occur. The potential for seasonal changes in oil
toxicity has already been discussed. Moreover, most laboratory experiments
conducted to date, including many in the present study, have used static
exposures in which the dosing medium is replenished at one to two day
intervals. Due to loss of volatile fractions from these aqueous mixtures, the
initial hydrocarbon concentrations cited represent only maximum
concentrations which the animals experienced during a test (6, 32). Atkinson
et al (4) reported that 90 percent of the benzene initially present in a test
solution is lost from undisturbed cotton-plugged flasks within a 24 hour
period. Our containers were kept tightly sealed in the static experiments,
minimizing such loss. Some loss of hydrocarbons through volatilization could
have occurred during transfer of the medium from the flow-through tanks to
the experimental containers, however. Finally, oil concentrations are generally
reported as total hydrocarbon content, as measured by infrared
spectrophotometry. Yet, toxicity to animals is probably due to only a small
fraction of the hydrocarbon compounds present in the water accommodated
fraction used (17), a fraction which can vary qualitatively and quantitatively
over the period of an investigation. The concentration of specific oil fractions
present during an experiment is generally unknown. Better control and analysis
of oil exposures conditions are needed if we wish to accurately determine
threshold concentrations of oil which are toxic to marine organisms.
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ACKNOWLEDGEMENTS
We wish to thank A. Ackenhusen, L. Halderman, E. Kenyon and N. Miller
for their assistance in various aspects of this study. L. Halderman and S.
Sosnowski provided helpful criticisms during manuscript preparation. The
flow-through dosing system was maintained by R. Pruell and B. Melzian.
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3. Anderson, J.W., D.B. Dixit, G.S. Ward, and R.S. Foster. 1977. In:
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6. Bigford, T.E. 1977. Effects of Oil on Responses to Light, Pressure and
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8. Boehm, P.D., and J.G. Quinn. 1976. The effect of Dissolved Organic Matter
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9. Byrne, C.J., and J.A. Calder. 1977. Effect of the Water-Soluble Fractions
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10. Calabrese, A., and E.W. Rhodes. 1974. Culture of Mulinia lateralis and
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Thalassia Jugoslavica. 10: 89.
11. Corner, E.D.S., and R.P. Harris. 1976. In: Effects of Pollutants on Aquatic
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12. Culliney, J.L., P.J. Boyle, and R.D. Turner. 1974. In: Culture of Marine
Invertebrate Animals, Smith, W.L. and Chanley, M.H. (eds.), Plenum Publ.
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13. Hargis, W.J., Jr. 1957. A Rapid Live-Sexing Technique for Urosalpinx
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14. Hyland, J., and E.D. Schneider. 1976. Petroleum Hydrocarbons and their
Effect on Marine Organisms, Populations, Communities, and Ecosystems.
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15. Hyland, J.L., P.F. Rogerson, and G.R. Gardner. 1977. In: Proceedings of
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16. Katz, L.M. 1973. The Effects of Water Soluble Fraction of Crude Oil on
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17. Krebs, C.T., and K.A. Burns. 1977. Long-term Effects of an Oil Spill on
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18. Lee, R.F., C. Ryan, and M.L. Neuhauser. 1976. Fate of Petroleum
Hydrocarbons Taken Up from Food and Water by the Blue Cr^b
Callinectes sapidus. Mar. Biol. 37: 363.
19. Lee, R.F., M. Takahashi, J.R. Beers, W.H. Thomas, D.L.R. Seibert, P.
Koeller, and D.R. Green. 1977. In: Physiological Response of Marine Biota
to Pollutants, Vernberg, F.J., Calabrese, A., Thurberg, F.P., and W.B.
Vernberg (eds.), Academic Press, New York, pp. 323-342.
20. Lee, W.Y., and J.A.C. Nicol. 1977. The Effects of the Water Soluble
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21. Moore, S.F., and R.L. Dwyer. 1974. Effects of Oil on Marine Organisms: A
Critical Assessment of Published Data. Water Res. 8: 819.
22. Nicol, J.A.C., W.H. Donahue, R.T. Wang, and K. Winters. 1977. Chemical
Composition and Effects of Water Extracts of Petroleum on Eggs of the
Sand Dollar Melitta quinquiesperforata. Mar. Biol. 40: 309.
23. Pechenik, J.A., 1978. Adaptations to Intertidal Development: Studies on
Nassarius obsoletus. Biol. Bull. 154:282.
24. Renzoni, A. 1973. Influence of Crude Oil, Derivatives and Dispersants on
Larvae. Mar. Poll. Bull. 4: 9.
25. Renzoni, A. 1975. Toxicity of Three Oils to Bivalve Gametes and Larvae.
Mar. Poll. Bull. 6: 125.
26. Rossi, S.S., and J.W. Anderson. 1976. Toxicity of Water-Soluble Fractions
of No. 2 Fuel Oil and South Louisiana Crude Oil to Selected Stages in the
Life History of the Polychaete, Neanthes arenaceodentata. Bull. Environ.
Contam. Toxicol. 16:18.
27. Sastry, A.N. 1971. Effect of Temperature on Egg Capsule Deposition in the
Mud Snail Nassarius obsoletus (Say). Veliger 13: 339.
28. Sastry, A.N. 1977. The Larval Development of the Rock Crab, Cancer
irroratus Say, 1817, Under Laboratory Conditions. (Decapoda, Brachyura).
Crustaceana 32: 155.
29. Scheltema, R.S. 1962. Pelagic Larvae of New England Intertidal
Gastropods. I. Nassarius obsoletus Say and Nassarius vibex Say. Trans.
Amer. Microsc. Soc. 81: 1.
30. — 1964. Feeding Habits and Growth in the Mud-Snail Nassarius obsoletus.
Ches. Sci. 5: 161.
31. — 1967. The Relationship of Temperature to the Larval Development of
Nassarius obsoletus (Gastropoda). Biol. Bull. 132: 253.
32. Wells, P.G. 1972. Influence of Venezuelan Crude Oil on Lobster Larvae.
Mar. Poll. Bull. 3: 105.
33. Wells, P.G., and J.B. Sprague. 1976. Effects of Crude Oil on American
Lobster (Homarus americanus) Larvae in the Laboratory. J. Fish. Res. Bd.
Canada. 33: 1604.
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Meeresunters. 5: 169.
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EXTRACTION OF ENVIRONMENTAL
INFORMATION STORED
IN MOLLUSCAN SHELLS:
APPLICATION TO ECOLOGICAL PROBLEMS
Donald C. Rhoads and Richard A. Lutz
Department of Geology and Geophysics
Yale University
New Haven, Connecticut 06520
ABSTRACT
Ecological stress, when broadly defined, is responsible for most, if not all,
growth patterns within the molluscan shell. As the type of pattern deposited is
largely a function of the specific biological or environmental stress involved,
considerable ecological information is stored within the exoskeleton. The
resulting record is in the form of either (1) microstructural growth increment
sequences or (2) changes in the shell structural type (e.g., nacreous, prismatic,
crossed-lamellar, etc.) or relative proportions of structures within the shell.
Microstructural growth increments, heretofore interpreted as resulting from
varible despositional rates of calcium carbonate and organic matrix, are viewed
as refections of periodic shell dissolution-deposition cycles.
Changes in the type of crystalline structure deposited under various
environmental conditions within the inner shell layer of several species of
bivalves have been defined. During periods of extreme ecological stress, such as
prolonged exposure to sub-freezing temperatures, extensive dissolution and
"reworking" of this inner layer occurs in a number of species.
Extraction of environmental information recorded within the shell is
facilitated through examination of polished thin sections, acetate peels,
fractured shells, polished and etched shell sections, and growth surfaces using
polarizing, optical, and scanning (and, occasionally, transmission) electron
microscopy. Application of these techniques to long-term monitoring of
ecologically stressed environments is discussed.
INTRODUCTION
Ecology has been defined as the study of relationships between organisms
and their environment (28). In functioning ecosystems it is possible to make
direct observations of these relationships in real time. Organism-environment
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relationships may not always be directly measured, however, requiring an
indirect or deductive approach; for instance, we may wish to assess the effect
of a storm, pollution event, change in salinity, temperature, etc. on a species
population after the event has taken place. In the absence of data about
pre-disturbance rates of growth, death, and reproduction, we are totally
dependent on indirect techniques. This kind of after-the-fact problem is
common in paleoecology and promises to be an increasingly important
approach in pollution biology^.
Subject matter of the present article has been extracted and condensed from
initial drafts of a manual which is currently being prepared for the
Environmental Protection Agency . The purpose of this manual is to bring
together and organize paleoecological literature so that it may be of use to the
pollution biologist confronted with after-the-fact monitoring problems.
The Skeletal Record
Skeletonized organisms provide an opportunity for deducing ecologic
relationships in the past. The skeleton often contains a record of dynamic life
and death processes, and provides both ontogenetic and demographic^
information. Ontogenetic data are related to the life history of an individual.
Growth rates may be resolved to a high level of resolution from mineralized
tissue showing growth banding correlated with lunar and/or solar cycles, or
seasonal changes in water temperature, salinity, day length, primary
productivity, etc. Biological events, such as season of reproduction and death,
may also be recorded. Demographic data are related to population structure
and its maintenance; growth, mortality, recruitment, and migration. The unit
of study is a single species population. In the present article, we will limit our
discussion to extraction of ontogenetic data.
Paleoecologic research over the past decade has developed many techniques for
reconstructing paleoenviroments (30-32). Much of this literature is unknown to
neontologists.
2
Preparation of the manual is supported by Environmental Protection Agency grant
R804-909-010.
3
The term monitoring is used here to describe reconstruction of an organism s history
of growth, reproduction, and mortality as preserved in its skeletal parts. Inferences
about environmental causes for the observed record are, by definition, indirect and
deductive.
4
Demography, taken literally, means writing about the people (Gr. demos, the people +
to write). The term was originally used to describe statistical studies of human
populations; births, deaths, marriages, etc. We use demography in a broader sense; the
statistical description of populations of any taxonomic group.
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Although all organisms with either an exoskeleton or endoskeleton can
potentially provide ontogenetic data, bivalve molluscs are the most universally
used group for obtaining these types of information for three reasons:
(1) Most members of the Class Bivalvia are preservable, and are common
faunal elements in both recent and fossil assemblages. Many species are
present in areas impacted by pollution and are represented in both
early and late stages of ecological successions following seafloor
disturbance.
(2) Preparation of the shell for obtaining ontogenetic information is easily
done. This involves sectioning or fracturing the shell along a plane
passing from the oldest part of the shell, the umbo, to the growing edge
along the maximum axis of growth (30, 32). Coiled or otherwise
torqued shells (e.g. gastropods) make this technique impossible with
present methods.
(3) Most research relating shell parameters to environmental conditions is
based on bivalves.
Data from Living and Dead Molluscs
The relationship of a species to its environment has been conceptualized in
the niche model (28). The species of interest is able to grow and reproduce as
long as the organisms' functional range (biospace) is not exceeded by the
ambient environment. Not all parts of the realized biospace promote equal
growth or fecundity. Different combinations of niche parameters will be
manifested in changed rates of growth, survivorship, or reproductive success.
All of these manifestations are capable of being preserved within the shell. We
therefore have a record of an organism's responses to changing niche conditions
preserved in shells of individuals, composing either the living or death
assemblage.
Suboptimal niche conditions can be thought of as ecological stress.
Ecological stress is responsible for most, if not all, growth patterns within
individual shells. In this regard, an ecological stress, such as a pollution event,
can be assigned dimensions in both space and time. These dimensions are
important when considering species appropriate for establishing after-the-fact
relationships. Ideally, the spatial distribution of a species should overlap and
extend beyond the affected area. Populations falling outside of the polluted
area can be used as control or reference populations. If the pollution event is
lethal to part or all members of the population occupying the affected seafloor,
after-the-fact study will include a comparison of both living and death
assemblages. If the effect is sublethal, living assemblages alone will be used.
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The duration of the pollution event should be considered relative to the
mean life span of individuals (turnover rate). Again, the ideal situation is one
where the species overlapping the affected area is one with a low turnover rate,
and a life span that is long relative to the duration of the pollution event. If the
ecological stress is sublethal, a record of growth before, during, and after the
stress event, may be recorded within the living population, and can be
compared with that of the reference population outside the affected area. If
the stress results in high mortality, the death assemblage may be all that
remains to document the event.
MOLLUSCAN GROWTH PATTERNS
Environmental information is stored within the molluscan shell in the form
of either (1) microstructural growth increment sequences or (2) changes in the
shell structural type (e.g. nacreous, prismatic, crossed-lamellar, etc.) or relative
proportions of structures within the shell. These two distinct types of records
and their usefulness in ecological studies are discussed below. Much of this
discussion is taken directly from a recent article by Lutz and Rhoads (26).
Microstructural Growth Patterns
During the past decade, numerous workers (2-4, 13, 14, 30-32) have
described microstructural increments within the molluscan shell. As a result of
marked periodicity associated with many of these structures, they have proved
useful in geophysical studies for defining changes in the earth's rotational rate
(3, 29-31), in ecological and paleoecological studies for assessing the effects of
various biological and environmental stresses (9, 14, 18, 30, 32), and in
archaeological studies for reconstructing migration patterns of prehistoric
hunter-gatherers (6, 7, 19). When shells are viewed in cross-section (procedural
details outlined in Methods section below), these microstructural patterns are
seen as alternating bands of shell material ranging in thickness from 10° to 10^
Many, if not all, microstructural periodicity structures within the molluscan
shell are a reflection of variations in the relative proportions of organic material
(conchiolin) and calcium carbonate (aragonite or calcite). Alternation of
calcium carbonate-rich layers and organic-rich regions or lines has been well
documented for numerous recent and fossil species through detailed studies of
shell thin sections, acetate peels, and polished and etched surfaces, employing
polarizing, optical, and scanning electron microscopy (see Methods section).
"Daily" growth increments have been reported by several workers (2, 13, 14,
18, 30-32). These "daily" lineations were originally interpreted as reflections
of solar time (13, 14, 17, 30, 31). Recent studies, however, have revealed a
complex relationship between incremental growth, and lunar and solar cycles.
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Although a one-to-one correspondence has not been established, the deposition
of increments in bivalves is highly correlated with shell valve movements (27,
30, 35, 36). As the valves of many species are generally closed during low tide,
and open during high tide, a high positive correlation also exists between the
number of increments and the number of tides to which an organism has been
subjected. While valve-movement rhythmicity is generally most pronounced in
intertidal individuals, subtidal specimens of at least one species (Mercenaria
mercenaria) exhibit biological rhythms in relative harmony with the tidal cycle.
There is general agreement among growth line workers that when the valves are
open and the organism is actively pumping, a layer is deposited which is rich in
calcium carbonate relative to adjacent shell material. The origin of alternating
layers or lines relatively rich in organic content has recently been theorized by
Lutz and Rhoads (26). The following few paragraphs summarize this theory
which is based on recent studies of molluscan anaerobiosis and mechanisms of
shell formation.
During aerobic metabolism, molluscs deposit calcium carbonate, in the form
of either aragonite or calcite, together with organic material, resulting in shell
construction. Such metabolism is usually highly correlated with periods of
active pumping- during high tide in well-oxygenated waters. As the
concentration of dissolved oxygen falls, such as in the microenvironment
created by the organism during periods of shell closure, anaerobic respiratory
pathways are employed and levels of succinic acid (or other acidic
end-products) within the extrapallial fluid rise. The acid produced is gradually
neutralized by shell calcium carbonate, leading to increased levels of Ca++ and
succinate (or other end-products) within the extrapallial and mantle fluids (8).
As a result of this decalcification, the ratio of relatively acid-insoluble organic
material to calcium carbonate increases at the mantle-shell interface. One need
not invoke the complication of increased concentration of organic material in a
given volume, although a collapse of unsupported matrix structures or
movement of the mantle as a compensatory response to the increased
mantle-shell distance could result in increased concentrations of freed organic
material in specific regions of the extrapallial fluid. With the return of
oxygenated conditions and resumption of aerobic metabolism, and assuming
shell deposition during this post-anaerobic period proceeds via a process similar
to that occurring immediately prior to anaerobiosis, deposition of calcium
carbonate and organic material within an area already containing organic
material should result in an increase in the organic/ CaCOg ratio within the
specific shell region. The end-product of this process, from a strictly structural
viewpoint, is one growth increment.
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Methods
Microstructural increments can be studied in thin sections of shell material,
in acetate peel replicas of acid-etched shell sections, or under the scanning
electron microscope (fractured or polished and etched shell sections).
Acetate peels are the easiest and most rapid method of preparation for
examination of most molluscan shells. The basic method of preparation, as
outlined by Rhoads and Pannella (32), is as follows:
Shells are embedded in a block of epoxy resin (e.g., Epon 815 resin with
DTA hardener, 10:1 ratio, under vacuum; Miller-Stephenson Chemical
Company, Danbury, Connecticut) to avoid shell fracture during sectioning. The
plane of the cross-section passes from the umbo to the shell edge along the axis
of maximum growth (30, 32). This cut is oriented so that growth increments
intersect the plane of the section at right angles. The cut shell surface is
polished sequentially with 350, 600, and, finally, 2600 or 3000 grade
carborundum grits. The polished surface is then etched with 0.1 N HC1 for
periods varying from a few seconds to a few minutes. Optimal etching time is
related to shell structure, mineralogy, organic content, and state of
preservation. It is recommended that a series of test etching times be carried
out to determine optimum etching periods for a particular set of specimens.
Etched shell surfaces are flooded with acetone, and a piece of sheet acetate
is applied to the etched shell surface and weighted to avoid bubble formation.
After the acetone (solvent) has evaporated (approximately 30 minutes), the
acetate is removed from the shell and examined under the microscope (or used
as a negative by placing directly in a photographic enlarger and printing). This
technique yields excellent results for most species.
Thin-sections are necessary for the examination of growth increments which
are not structurally discontinuous, but instead recognizable only by dark and
light color bands (32). For example, the growth increment boundaries in the
deep-water species, Nucula cancellata and Calyptogena ponderosa, are
indistinct and recognizable only by color variations of the bands, each band
consisting of one dark and one light layer. The initial procedure for making
thin-sections is the same as that for preparation of acetate peels, however, after
the cut shell surface has been polished, it is glued to a glass slide using epoxy
resin. The majority of the embedded shell and remaining embedding material is
cut away using a diamond rock cutting saw, and the new exposed surface is
polished sequentially until a 0.03 mm thick section of material is left on the
slide. A cover slip is glued onto the newly polished surface using epoxy resin,
and the material examined using optical or polarizing microscopy. Thin
sectioning of shells is difficult, because shells tend to fracture when sectioned
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and the micro-growth lines are obscured. In addition, it is difficult to avoid the
formation of bubbles beneath the coverslip which obscure features. Equipment
for preparing thin sections is available in rock preparation laboratories found in
most geology departments. Alternatively, there are commercial firms (e.g.,
Rudolf von Huene, Pasadena, California) that will prepare thin sectioned
material.
Ecological Applications
Several workers (9, 14, 18, 30-32) have suggested that information about
physiological and environmental conditions may be recorded and stored in
molluscan shells. Various studies in which workers have used microstructural
increments within shells to extract such information are discussed below under
appropriate sub-headings.
Seasonal Cycles
Seasonally caused annual growth rates and patterns are observable in all
bivalves collected in climatic zones, ranging from cold-temperate to
sub-tropical. In many species, as winter approaches, there is a gradual slowing
down of the deposition rate, and the microstructural increments become
gradually thinner. This slowing down of growth in the autumn culminates in a
marked depositional break at the time of the first freeze. These depositional
breaks are characterized by indentations of the outer shell layer, a dark band of
organic-rich shell material extending downward from the base of the
indentation, small daily growth increments on either side of the break, and a
change in the shell structure near the break (9). These winter breaks may not
be as marked in specimens living subtidally (32), although Mercemria
mercenaria from water depths of eight meters clearly show a distinct winter
break (9).
Through careful examination of microstructural patterns, Farrow (13)
found that part of a population of the shallow sub tidal cockle, Cerastoderma
edule, from the Thames estuary in England, stopped growing during winter due
to sub-zero temperatures. Tevesz (34) observed that Gemma gemma grew very
little in the winter. Growth increments were very closely spaced and the inner
shell layer had a brownish hue. During the summer, G. gemma grew rapidly;
microstructural increments were widely spaced, and the inner shell layer was
clear and translucent in appearance.
Through an examination of numerous acetate peels, Evans and LeMessurier
(12) were able to demonstrate striking winter growth rate differences between
two sympatric species of bivalves. They found winter growth of the
rock-boring clam, Penitella penita, to be approximately 75 percent of the
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summer growth rate, while growth of the cockle, Clinocardium nuttalli, which
inhabited a neighboring mud flat, was slowed by as much as a factor of 19.
In addition to rhythms based on periodic environmental fluctuations,
biological rhythms, such as breeding periods, are also reflected in growth
increment clustering. "Breaks" due to spawning events are less severe than
winter breaks. They are preceded by little or no slowed growth, and recovery is
more rapid than after winter breaks. In most bivalves, spawning occurs during
the summer, sometimes more than once a year. In Mercenaria mercenaria,
reproductive breaks do not occur until the second year of growth (32).
Semiperiodic and Random Events
Depositional breaks in bivalves also result from semiperiodic or random
events such as storms, unseasonable temperatures, attacks by predators, and
environmental pollution disturbances. The irregular nature of such events
makes them easily separable from periodic or cyclical shell-secretion rhythms
(18, 30, 32).
Storm breaks, a common feature, may have different characteristics
depending on the severity of the storm and depth at which the bivalves live
below the water surface. In any case, these breaks appear suddenly and are
followed by a rapid return to increments of pre-storm width (9). Shuster (33)
noted that during storms, silt became trapped between the mantle and the shell
in Mya arenaria and was subsequently incorporated into the shell. Trapped silt
within shell indentations formed by storm breaks has also been observed in
Mercenaria mercenaria (9, 18).
The Season, Age, and Frequency of Reproduction and Death
Growth patterns can supply detailed information on the age of individuals
at time of death and their season of death. The age at death will be:
2
where Ad is age at death and N$ and Nw are, respectively, the number of
summer and winter bands in the shell (32). The season of death is determined
by relating the position of the last increment at the margin of the shell to the
seasonal growth pattern. For example, a margin preceded by a complete
summer depositional record represents death in early fall. A margin which
follows a long period of winter growth represents late winter or early spring
death. Often, the shell margin is preceded by a few days of growth slowdown,
and comparison of several individuals may be necessary in order to determine if
the slowing down in growth represents a moribund condition prior to death, or
is related to seasonal changes (32). By counting the number of increments in a
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dead shell, it is also possible to relate the season of death to absolute age at
death.
The age at sexual maturity and season of reproduction can be determined
by relating the position of spawning breaks to absolute age and seasonal
pattern of growth. An illustration of the usefulness of growth patterns in
determining age and season of reproduction is given by Rhoads and Pannella
(32). They examined a population of Gemma gemma from an intertidal muddy
sand flat on Long Island Sound. Summer growth patterns in G. gemma
consisted of thick increments (7-25 11) and were readily distinguished from
winter ones which were thin (1-3 ju). A period of decreased growth was seen in
shell sections and was interpreted by them as reflecting reproductive events
which occurred at the beginning of summer deposition. These thin increments,
if related to spawning, should be associated with a spawning break in the shell
margin. Rhoads and Pannella (32) determined that the periods of highest stress
and mortality were different for juvenile and mature bivalves. Specimens 3.2
mm (generally less than 6 months old) died with greatest frequency from
summer to mid-autumn. Older individuals died primarily in late fall or early
winter.
Ontogenetic Records of Environmental Change.
In addition to episodic and periodic events, variations in environmental
parameters including food supply, the type of substratum, salinity, oxygen
content, turbidity, agitation, temperature, and population density can
influence growth of bivalves. Hallam (15) reviews these various environmental
parameters as causes of stunting and dwarfing in living and fossil marine
benthic invertebrates. Several studies conducted within the past few years have
used microscopic growth increments within shells to define the effects of
various environmental perturbations on bivalve growth. Rhoads and Pannella
(32), for example, through careful examination of both acetate peels and thin
sections, have demonstrated that examination of both acetate peels and thin
sections, have demonstrated that Mercenaria mercenaria grows faster in sandy
sediments than in mud when other variables are eliminated. Farrow (13) used
microstructural growth increments within the shell of Cerastoderma edule to
illustrate that dense populations of the cockles had a much shorter growing
season than sparse populations. An inverse relationship between individual size
and population density of cockles was also noted. In a subsequent study,
Farrow (14) used growth increments within the outer shell layer of this species
to demonstrate that individuals living high in the intertidal zone were stunted.
The higher shore cockles were situated near the high water mark, and,
consequently, were aerially exposed for several days during neap tides.
Following neap tide deceleration, there was a resumption of vigorous growth.
Many of the high intertidal cockles were some two-thirds the size of individuals
lower in the intertidal zone, where growth was more continuous.
165
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In a recent study, Kennish and Olsson (18) studied the effects of thermal
discharges on the microstructural growth of Mercenaria mercenaria in Barnegat
Bay, New Jersey. They found that clams from within a mile and a half of the
mouth of Oyster Creek, which carries the heated effluent from the Oyster
Creek Nuclear Power Plant, had a much higher number of breaks in their shells,
thinner shells, and slower summer growth than did clams farther from the river.
Counting the growth increments back from the shell margin, they determined
that many of the breaks occurred concurrently with rapidly decreasing water
temperatures, resulting frojn abrupt shut-downs of the power plant, or rapidly
increasing temperatures associated with abrupt renewal of plant operations.
The growth rate of M. mercenaria generally increases with increasing
temperatures and peaks between 20-24°C; Haskin (communicated to Kennish
and Olsson) found decreased growth above 26°C. The thermal effluent raised
the water temperature in areas around the mouth of Oyster Creek 3-5°C above
ambient. Kennish and Olsson (18) also suggested that the thermal effluent may
be adversely affecting physiological functions other than growth. At the station
nearest the effluent, no spawning breaks were observed within the shells, while
they were seen in specimens from all the control sites.
Shell Structural Changes
In addition to changes in patterns of microstructural growth increment
sequences, changes in the type of crystalline structure deposited under various
environmental conditions have been observed within the shells (particularly
within the inner shell layers) of numerous species of bivalves. Dodd (10)
described environmentally-controlled variation in the relative proportions of
nacreous and calcitic prismatic structures within the innermost shell layers of
Mytilus californianus. Lutz (23) found annual variation in the thickness of
nacreous laminae within the inner shell layer of Mytilus edulis, and suggested
that such variation might be growth rate and/or temperature dependent, with
relatively fine laminae being formed with increased growth rate and rising
temperatures in the late spring. Bryan (5) examined the effects of oil spill
remover (detergents) on the shell of the intertidal gastropod, Nucella lapillus,
following the Torrey Canyon spill in March of 1967. The addition of toxic
detergent BP 1002 applied to the Kuwait crude oil spill was effective in
temporarily sealing the shell edge by continuing the inner nacreous layer to the
outer surface. Subsequent shell growth on thin nacre produced a growth mark
and lines of weakness in the shell. Kennish and Olsson (18) observed
transgressing regions of crossed-lamellar structure within the outer shell layer
of Mercenaria mercenaria associated with shell deposition occurring during
periods of extreme ecological stress (winter freezes, high summer temperatures,
and thermal shocks from abrupt changes in operations of a nuclear power
plant). Farrow (14) noted similar transgressing regions of crossed-lamellar
structure within the outer layer of Cerastoderma edule associated with winter
166
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growth, particularly in specimens sampled from high elevations (highly stressed
environments) within the intertidal zone.
Lutz and Rhoads (26) have recently presented evidence that structural
changes within the shells of certain bivalve species may reflect periodic
dissolution and "reworking" of primary depositional structures during periods
of extreme environment stress. Here, alternating periods of aerobic and
anaerobic metabolism provide the driving forces for shell deposition and
dissolution, respectively. Parallel annually-formed sub-layers of nacre and
simple aragonitic prisms (24, 25) within the inner shell layer of the Atlantic
ribbed mussel, Geukensia demissa (Figure 12-1), for example, were interpreted
as reflective of seasonal metabolic changes. In populations from Gulf of Maine
waters, nacre deposition was restricted to the relatively warm months of the
year (24, 25). During both the fall and spring, nacreous tablets on the inner
shell layer growth surface became smaller and less regular, showing visible signs
of erosion in the form of marked pitting and "hollow crystals", as well as
increased proportions of fine-grained structures. Differential dissolution of
calcium carbonate and organic material was also often observed at the inner
layer growth surface during these seasons (Figure 12-2). During the colder
months of the year (January — March, with water temperatures below 3°C),
shell erosion became visible to the naked eye, the entire inner shell surface
often presenting a chalky white appearance. Ultrastructurally, this surface
appeared uniformly fine-grained or "homogeneous" (Figure 12-3). Similar
visible erosion has been reported in Mercenaria mercenaria after long periods of
valve closure (11). The ability of G. demissa to endure anaerobiosis for
extended periods has been well documented (20, 22), as has the relative
increased efficiency in this species of some of the citric acid cycle enzymes in
an anaerobic direction (16). The observed shell erosion may well be a reflection
of buffering of acid end-products from anaerobic metabolism during the colder
months, when oxygen transport into the cells should theoretically be reduced
relative to that occurring at higher temperatures (21). Wibur (37) has suggested
that during periods of "adverse environmental conditions", shell decalcification
may predominate over growth. The often-seen gradation in fractured, as well as
polished and etched, vertical shell sections of G. demissa nacreous laminae into
finely grained structures (suggestive of massive erosion), instead of regular
prisms, (Figure 12-4) tends to support this view.
SUMMARY
(1) Environmental and biological events are recorded in the molluscan shell
in the form of small-scale growth increments and/or changes in shell
structure.
167
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Figure 12-1. Parallel annually-formed sub-layers of nacre and
simple aragonitic prisms within the inner shell layer of the
Atlantic ribbed mussel, Geukensia demissa.
NOTE: (A) Scanning electron micrograph of vertical fracture surface x240.
(B) Acetate peel of polished and etched longitudinal shell section x4Q0.
168
-------
Figure 12-2. Scanning electron micrographs of the inner
shell layer growth surface of Geukensia demissa
showing natural shell dissolution.
NOTE: The differential solubility of calcium carbonate and organic matrices is
apparent. Stereo pairs were taken with a 6° angular displacement between ex-
posures. (A) x5000. (B) x20000.
169
-------
shell layer of Geukensia demissa showing fine-grained
structures reflective of extensive shell dissolution during the colder
months of the year (February sample).
NOTE: Stereo pair was taken with a 6° angular displacement between expo-
sures. x3000.
170
-------
NOTE: (A) Scanning electron micrograph of vertical fracture surface showing
gradation of nacreous tablets into fine-grained structures at top and gradation
of prisms into nacre at bottom x2000. The most recently deposited crystals
are at the bottom of the micrograph. (B) Acetate peel showing similar grada-
tions of nacre into fine-grained structures and prisms into nacre x125. Again,
the most recently deposited crystals are at the bottom of the micrograph.
171
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(2) Alternating aerobic-anaerobic metabolic cycles are proposed as the
physiologic mechanism for forming shell periodicity structures. Aerobic
respiration is associated with shell calcification. Shell closure,
accompanied by anaerobic metabolism, results in shell decalcification;
acidic end-products are neutralized by dissolution of shell calcium
carbonate.
(3) Shell growth patterns can be easily studied by preparing shell thin
sections or acetate peel replicas of acid-etched shell sections. Scanning
electron microscopy of fracture or polished and etched shell sections
can also be employed.
(4) Patterns of growth increment sequences and shell structural changes are
related to seasonal climatic cycles and, on shorter time scales, to lunar
and solar periodicities. Semiperiodic or random events, such as storms,
sedimentation events, or biological events (e.g., reproduction) are
superimposed as "noise" on the geophysical cycles. Causal effects for
this "noise" can be deduced by detailed studies of the growth record.
(5) Shell growth patterns have proven useful in paleoecologic
reconstructions. Detailed analysis of these patterns also promises to be
an efficient manner in which to conduct after-the-fact or retrospective
monitoring studies of pollution events.
ACKNOWLEDGEMENTS
Parts of this manuscript are a summary from a document prepared by Dr.
Josephine Yingst; a contributor to the E.P.A. shell-growth manual. We thank
A.S. Pooley, E. Tveter Gallagher, and A. Krishnagopalan for technical
assistance with the scanning electron microscopy; W.C. Phelps for preparation
of specimens; and W.K. Sacco for his assistance in photograph reproductions.
This research was supported in part by Environmental Protection Agency grant
R804-909-010 and NOAA grants 04-6-158-44056, SGI-77-17, and
04-7-158-44034. Contribution number 108 from the Ira C. Darling Center,
University of Maine, Walpole, Maine, 04573.
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LABORATORY CULTURE OF
MARINE FISH LARVAE
AND THEIR ROLE IN
MARINE ENVIRONMENTAL RESEARCH
E.D. Houde and A. K. Taniguchi
Rosenstiel School of Marine
and Atmospheric Science
University of Miami
Miami, Florida
ABSTRACT
The capability to predictably culture marine fish larvae beyond embryonic
and yolk-sac stages has been developed during the past 15 years. This has led to
advances in our understanding of how environmental variables affect survival
and eventual recruitment of fishes. Most marine fish larvae are planktonic
carnivores and consume living prey less than 150 /um in breadth when they first
feed. The most important advance in culture technology was the determination
of kinds and concentrations of prey that enable larvae to survive and grow at
predictable rates, permitting ecological, physiological, and behavioral research
to be undertaken. Prey concentrations necessary for growth and survival of
some typical marine teleost larvae, usually range from 10^ to 10"^ per liter.
Best survival rates, fastest growth, and lowest variability, have been obtained at
the 103 per liter concentration. Growth efficiencies and food consumption by
marine fish larvae are comparable to other predatory zooplankton. Some
knowledge about effects of predation on marine fish larvae survival has been
obtained, but further study is necessary, especially to determine how
environmental factors modify predator effects. Some areas of environmental
research, using cultured marine fish larvae, are reviewed. These include the
roles of physical and chemical variables, other than man-induced environmental
perturbations, and some effects of environmental change caused by man's
encroachment upon and alteration of marine habitats. Other important
advances include development of field bioassay methods to determine if
plankton standing stock can support fish larvae; development of biochemical
and histological techniques to evaluate larval condition; and the recent
discovery that larvae can be accurately aged using daily otolith increments.
Some ideas for productive future research are proposed.
176
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INTRODUCTION
Fishes are large and conspicuous members of marine communities. They
have important commercial and recreational value, and their abundance can
fluctuate widely in response to environmental variability or heavy exploitation.
Fluctuations in abundance of fishes usually are caused by large annual
differences in recruitment, which are related to mortality experienced by a
cohort during the larval stage (28, 29). It has been difficult to evaluate
potential factors that could affect recruitment in studies carried out on natural
populations at sea because of the problem in estimating egg or larval
abundances over extensive ocean areas, and because of an unpredictable
environment whose effects cannot be controlled. During the past 15 years a
capability has been developed by several laboratories to routinely culture
marine fish larvae beyond embryonic and yolk-sac stages to the juvenile stage.
Experiments on these laboratory cultured species has resulted in significant
advances in our understanding of how environmental factors affect survival and
growth of larvae.
Several papers recently have reviewed aspects of marine fish larvae culture
(42, 50, 64, 65, 67). Although May's (67) evaluation of the critical period
hypothesis, and Iwai's (50) review of feeding by fish larvae, included
discussions of both laboratory and field-oriented studies, they did not
specifically make conclusions about larval requirements based on experimental
research. Blaxter's (14) general review of egg and larval development of fishes
did summarize results of laboratory studies. We review some important results
of recent experimental research on marine fish larvae and make conclusions
about effects of environmental factors based on laboratory studies. Emphasis is
on studies of species that have typical, pelagic larvae and includes the period
from initiation of feeding until transformation to juvenile. Research on embryo
and yolk-sac stages, aquaculture-oriented studies, and work on non-pelagic or
non-typical larvae are not emphasized, although important contributions have
been made in recent years.
Two major areas of research are reviewed and discussed. These are 1) the
role of the food supply, the ability to feed, and the effects of predators; and 2)
the role of physical and chemical variables, other than those due to man's
impact on the environment. In addition, new techniques that hold promise for
advancing environmental research on fish larvae are outlined and discussed.
FOOD REQUIREMENTS
The most important advance in larval culture technology during the past 15
years has been the determination of kinds and concentrations of living prey
that give predictable survival and growth rates. The ability to undertake
177
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meaningful ecological, physiological and behavioral research developed once
larvae could be routinely cultured. A myriad of foods has been used to rear
marine fish larvae (64), but five foods have been more successful in recent
years for meeting larval nutritional requirements. These are the rotifer
Brachionus plicatilis, the nauplius of brine shrimp Artemia salina, copepods
from wild plankton collections, the harpacticoid copepods Tisbe and Tigriopus
spp., and the naked dinoflagellate Gymnodinium splendens.
Prey Concentrations
Marine fish larvae are visual feeders, with limited ability to search a volume
of water for suitable food items during a unit of time. Suitable items usually
are living organisms of a size that can be ingested, are nutritionally adequate,
and are present at concentrations which allow a larva to encounter enough
items during a day to meet its metabolic demands and to provide some excess
for growth. Typical marine fish larvae are 2-3.5 mm long when they begin to
search actively for food. Acceptable prey usually are 20-150 jum in breadth (7,
31, 56, 92). Some large and rather atypical larvae, like Atlantic herring, Clupea
harengus, or plaice Pleuronectes platessa, can begin feeding on items in excess
of 300 p.m in breadth (10, 80, 82). Perhaps not surprisingly, required
concentrations of prey for newly-feeding larvae have been shown to vary
greatly in laboratory studies, the variation in large part reflecting size
differences in the prey that has been offered.
Prey concentrations that have been used successfully to rear larvae have
ranged from 1 x 10* to 2 x 10^ per liter, although required concentrations for
significant survival probably lie in the range 10* to 10^ per liter. The highest
reported concentrations (1-2 x 10^ per liter) were of the large dinoflagellate
Gymnodinium splendens, which can be used to culture northern anchovy
larvae during the first week of life (47, 57, 95). Lowest concentrations (4-42
per liter) were of brine shrimp Artemia salina nauplii used to culture Atlantic
herring larvae (82, 83). Neither G. splendens nor A. salina is usually available to
marine fish larvae in nature, although Kiefer and Lasker (53) recently have
shown that G. splendens may be present at 1-4 x 10 per liter in the
chlorophyll maximum layer of the Southern California Bight. Northern
anchovy larvae can concentrate in the chlorophyll maximum layer and can feed
on G. splendens when its concentration exceeds 2x10^ per liter (56). The
most common prey reported from stomach analyses of marine fish larvae in
nature are nauplii and other stages of copepods. Using copepod nauplii as food
Houde (45) reported 10 percent survival at metamorphosis when per liter
nauplii concentrations were 34 for sea bream Archosargus rhomboidalis, 107
for bay anchovy Anchoa mitchilli, and 130 for lined sole, Achirus lineatus.
Other studies with wild plankton (predominantly copepod nauplii) as prey
have reported higher concentrations required for significant survival than those
178
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reported by Houde (45). O'Connell and Raymond (73) estimated that more
than 1000 nauplii per liter were required by northern anchovy larvae. Haddock
Melanogrammus aeglefinus, larvae required 500-3000 per liter (58) and winter
flounder, Pseudopleuronectes americanus, required 300-3000 per liter (60). It
is possible that some reported prey concentrations required by larvae could be
too high. Saksena and Houde (84) needed 1500-2000 nauplii per liter to
successfully rear about 10 percent of bay anchovy larvae, but more recent
experiments, with refined culture methods (45), have demonstrated that only
100 nauplii per liter are necessary to attain that survival rate. In some research,
such as toxicological studies to determine effects of pollutants on larval
survival, potential survival rates higher than 10 percent are required. For those
studies, copepod nauplii concentrations of 1000 per liter or higher should be
routinely employed (44, 45) (Table 13-1).
For six cases where copepod nauplii were fed to similar-sized larvae, the
relationship between percent survival and nauplii concentration can be
compared (Table 13-1). Haddock larvae had the highest required prey
concentration, more than 2000 nauplii per liter being required for 10 percent
survival. Winter flounder and northern anchovy larvae had an expected survival
of 10 percent when nauplii were available at approximately 1600 and 1000 per
liter, respectively. But, bay anchovy and lined sole required only about 100
nauplii per liter and sea bream needed less than 50 per liter to attain 10 percent
survival. All of these species consume prey of similar types and sizes; the
differences in requirements among species have not been explained.
Temperature may play a role because the three species with lowest required
prey concentrations were reared at 26-28°C, while the three with higher
requirements were reared at 7-17°C. If searching ability and capture efficiency
are enhanced at higher temperatures, required prey concentrations may
decrease accordingly.
Rotifers, Brachionus plicatilis, are often used at high densities by
aquaculturists to successfully rear fish larvae, but the minimum concentration
required by larvae usually has not been determined. Hunter (46) estimated that
105 rotifers per liter were required by newly-feeding northern anchovy larvae
to meet metabolic demands, a number that must be exceeded for larvae to
grow. Lined sole larvae required from 60-120 rotifers per liter for 10 percent
survival to metamorphosis (Houde, unpublished data).
Concentrations of microzooplankton in marine waters are not frequently
reported, but when suitable collection techniques have been used observed,
concentrations often are in the ranges of required prey densities determined in
the laboratory (Table 13-2). Concentrations of suitable prey are exceptionally
low in oceanic waters compared to coastal waters, and larval survival may
depend upon the occurrence of relatively dense prey patches in oceanic areas.
179
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Table 13-1. Copepod Nauplii Concentrations Used as Prey
to Rear Six Species of Marine Fish Larvae and
Corresponding Percent Survivals.
Reference
Species
Stage to which
larvae
were reared
Temperature
(°C)
Nauplii
Concentra-
tion
(no./1}
Percent
Survival
O'Connell and
Northern anchovy
12 days after
17
10
0.0
Raymond (1970}
(Engraulis mordax)
hatching
100
0.5
1000
12.0
4000
56.0
8000
25.0
14000
30.0
Laurence (1974}
Haddock
metamorphosis
7
10
0.0
(Melanogrammus aegfefinus)
100
0.0
500
1.1
1000
7.9
3000
13.9
Laurence (1977)
Winter flounder
metamorphosis
8
10
0.0
(Pseudop/euronectes americanus)
100
0.0
500
2.6
1000
3.8
3000
34.2
Houde (in press)
Bay anchovy
metamorphosing
26
50
11.6
(Anchoa mitchil/i)
100
4.7
1000
48.2
5000
63.9
Houde (in press)
Sea bream
metamorphosis
26
10
3.9
{Archosargus rhomboidalis)
25
7.3
50
12.7
100
37.7
500
72.4
Houde (in press)
Lined sole
nearly
28
50
1.4
(Achirus lineatus)
metamorphosed
100
13.3
1000
54.3
180
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Table 13-2. Reported Concentrations of Some Microzooplankton Suitable as Prey for
Marine Fish Larvae from Coastal and Estuarine Areas.
Reference
Place
Organisms
Concentration
Burdick (1969, cited
Kaneohe Bay,
copepod nauplii
50-100/1 common
in May, 1974)
Hawaii
200/1 sometimes present
Duka (1969)
Sea of Azov
Acartia clausi nauplii
62-65/1
Other copepod nauplii and
copepodids
>30/1
TOTAL
>90/1
Mikhman (1969)
Gulf of Taganrog,
early stages of copepoda
39-546/1
Sea of Azov
Hargrave and Geen (1970)
two eastern
copepod nauplii and
Canada estuaries
copepodids
>60/1
Reeve and Cosper (1973)
Card Sound,
copepod stages 20-200 m
range 23-209/1
South Florida
in breadth
mean for 28 collections 72/1
Tintinnids
range 40-369/1
Heinle and Flemer (1975)
Patuxent River
Eurytemora affinis nauplii
> 100/1 frequently
estuary
and copepodids
> 2000/1 occasionally
Houde (unpublished data)
Biscayne Bay,
copepod nauplii and copepodids
usually 50-100/1
South Florida
< 100 Mm in breadth
Tintinnids
frequently > 100/1
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Even in rich coastal waters, daily variability in microzoo plankton
concentrations occurs over order of magnitude ranges. Laboratory studies have
shown that larvae deprived of food pass a "point of no return," after which
they cannot initiate feeding (21, 67). This point can occur at only 0.5-2.5 days
after yolk absorption for species at 20-32° (43, 57). Thus, unstable conditions
that lead to temporary low prey concentrations probably are an important
cause of mortality, even in areas where mean prey levels are high enough to
sustain larvae.
Growth of larvae in relation to prey concentration can be determined in the
laboratory. There are, of course, factors other than density of prey which
influence larval growth. The size of prey, their caloric value, their percentage
protein, and their digestibility are important. The effect of temperature makes
it difficult to compare growth among species of larvae, even when similar foods
have been used. Despite limitations in the comparative approach, larval growth
responses to changes in food concentration can be demonstrated in the
laboratory, and results extended to explain how densities of prey influence
growth of wild populations.
A relationship between size at 16 days after hatching and copepod nauplii
concentration was demonstrated for larvae of bay anchovy, lined sole, and sea
bream (44, 45). Lengths and mean dry weights of survivors increased rapidly
when prey level was raised from approximately 50 to 500 nauplii per liter.
Lengths and weights tended toward asymptotes at food levels higher than 1000
per liter, although significant, additional growth could be obtained at higher
prey levels. Laurence's data (58) on haddock larvae at six weeks of age show a
similar relationship for prey concentrations in the range 500-3000 copepod
nauplii per liter. Weights of winter flounder at 7 weeks of age in relation to
copepod nauplii concentration also approached an asymptote at 1000 per liter
prey level (60). O'Connell and Raymond (73) also found this type of
relationship between length of northern anchovy larvae at 12 days and
copepod nauplii concentration, except that prey ranged from 1000-14,000
nauplii per liter and the asymptotic size was not attained until prey level was
approximately 8000 nauplii per liter.
Specific growth rates of marine fish larvae relative to prey concentration
have been obtained in only a few instances. Specific growth rates (in dry
weight) of haddock larvae were 7 percent, 8 percent, and 9 percent per day at
copepod nauplii concentrations of 500,1000 and 3000 per liter (58). The rates
for winter flounder larvae, at the same nauplii concentrations were similar, 6
percent, 8 percent and 9 percent (60). Temperature for haddock experiments
was 7°C and for winter flounder it was 8°C. Specific growth rates of sea bream
and bay anchovy larvae at 26°, and lined sole larvae at 28°C can be estimated
from Houde's data (45). The rates were 16,20, and 28 percent per day for sea
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bream at 50, 100 and 500 per liter copepod nauplii concentrations; they were
16, 17 and 25 percent per day for bay anchovy at 50, 100 and 1000 per liter
nauplii concentrations; but, lower rates of 7, 9 and 17 percent were obtained
for lined soles at 50, 100 and 1000 per liter nauplii concentrations. Depending
on prey concentration, the length of the larval stage can be highly variable. In
the case of sea bream, specific growth rates at 100 and 50 per liter nauplii
concentrations indicate that duration of the larval stage at those prey levels
could be 1.4 to 1.7 times as long as at the 500 per liter level. Even if starvation
was not a direct cause of mortality at low prey levels, the indirect effects of
increased time of exposure to predators and possible enviornmental stresses
during the larval stage, must have important consequences on the numbers that
eventually metamorphose.
The density of prey, expressed as numbers per liter, provides a useful
measure of availability of prey for capture by larvae, but does not necessarily
provide a measure of energy available for growth and metabolism. Energy
available is a function of prey density, prey size, and the ability of larvae to
ingest particular prey, which is related to mouth size in many instances (10,
90). The kinds of prey also could influence the availability of energy, either
through differential ability of prey to escape capture by larvae, or through
differences in caloric content of prey. Few studies concerned with marine fish
larvae have taken a bioenergetic approach to examine nutritional requirements.
Such studies can provide the means to estimate amounts of ingested energy
used for growth and metabolism. Estimates of required food intake, specific
ration, growth efficiency and the critical minimum prey level all can be
determined on a caloric basis using this method. When used in conjunction
with studies on feeding by larvae in relative to prey concentration, valuable
insight into nutritional requirements and feeding dynamics can be obtained.
Recent work by Laurence (60) on winter flounder larvae is the best example of
the use of a bioenergetic model for marine fish larvae.
The winter flounder larvae model (60) predicted critical food
concentrations in the range 2.1-5.7 cal per liter, corresponding to 300-800
copepod nauplii per liter. Highest prey concentrations were required by
newly-feeding larvae, suggesting that food was most critical at that time.
Smallest larvae required most of the daylight period to obtain a minimum
ration. Relatively high metabolic energy demands were made by the smallest
larvae, reflecting their low efficiency in capturing food. Metabolic demands
were lowest at high prey concentrations because larvae expended less energy in
searching when food was readily available. Thus, for winter flounder larvae it
appears that food consumption needs to be higher at low prey concentrations
than at high prey concentrations. Estimated minimum consumption ranged
from 18-230 nauplii per day over a range of larval dry weights from 10-1000
Atg. Specific rations (pg consumed per jug larva x 100) decreased from nearly
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300 percent for newly-feeding larvae to 27-31 percent for the smallest larval
stages (10-75 ng), and continued to increase slowly for older larvae, ranging
from about 18-33 percent for metamorphosed individuals. Laurence (60)
predicted a continuous decrease in growth efficiency as prey concentrations
were decreased, but so few values of growth efficiency are available for fish
larvae that it is not possible to say whether this relationship will hold for other
species.
Some valuable insight into feeding by marine fish larvae recently has been
gained by combining results of bioenergetic studies on larvae with studies on
feeding behavior and feeding ability of larvae. Blaxter and Staines (23)
estimated swimming ability and feeding efficiency of herring, plaice, pilchard
Sardina pilchardus, and sole So lea solea larvae. From their estimates they
calculated the volume of water that could be effectively searched by larvae
when they initiated feeding, and at sizes up to metamorphosis. Because
swimming distances and volumes searched per unit time increased rapidly as
larvae grew, larvae presumably needed higher prey concentrations during the
youngest feeding stages. A similar approach was used by Rosenthal and Hem pel
(82), who in addition estimated the digestion time for herring larvae. They
were then able to calculate the daily ration and required densities of prey
(Artemia nauplii) for herring larvae at the end of the yolk-sac stage (10-11 mm)
and at 13-14 mm length. Estimated ration was 40 Artemia nauplii per day at
10-11 mm and 50 per day at 13-14 mm. Required Artemia concentrations for
larvae to obtain the rations at each of those length-classes were 4 to 42, and 2
to 25 per liter, respectively. Hunter (46) further extended the method by
incorporating metabolic demands of larvae, and caloric values of prey (the
rotifer Brachionm plieatilis and the dinoflagellate Gymnodinium splendens)
into the prediction of food requirements. He concluded that first feeding
northern anchovy larvae required 105 rotifers per liter or their caloric
equivalents (e.g. 1785 Gymnodinium per liter) to just meet metabolic
demands. Larvae at 10 days (5,9 mm) required only 34 rotifers per liter. In all
of the examples, the relatively poor swimming ability and the low prey capture
efficiency of first feeding larvae were demonstrated. This implies, as did
Laurence's study (60), that food concentration is most critical at the first
feeding stage and, when low, could be a significant cause of larval mortality in
the sea.
It is possible to make many conclusions about larval food requirements
based on dry weights of larvae, dry weights of prey, prey selection by larvae,
digestion time, and estimates of the caloric values of the prey (cal/g ash free).
Using these methods, Stepien (92) showed how feeding rates, specific rations
and growth efficiency of sea bream larvae varied in relation to larval age, and to
temperature for a single prey concentration. At 1000 copepod nauplii per liter,
feeding rates for first feeding larvae (2-3 days after hatching) varied from 7.2
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nauplii/hr/larva at 23°C to 17.6 nauplii/hr/larva at 29°C. These rates increased
exponentially as larvae grew, so that larvae were consuming 53.8
nauplii/hr/larva at 23°C and 142.7 nauplii/hr/larva at 29°C at 16 days of age.
Rations, in terms of numbers of nauplii and dry weight consumption, were
then calculated. Specific ration also was calculated, and it tended to decrease as
larvae grew, particularly at the highest temperature (29°C), where it was 220.8
percent at two days after hatching but decreased to 79.7 percent at 8 days.
This result is similar to that of Laurence (60) for winter flounder (8°C), where
specific ration decreased from over 300 percent for the smallest larvae to about
30 percent for metamorphosed individuals. Mean gross growth efficiency of sea
bream (92) varied from 23.9 to 30.6 percent, the highest value being obtained
to the lowest temperature (23°C); there was no evidence that growth
efficiency changed with age. Mean growth efficiencies were similar to those for
winter flounder (60), except that first feeding winter flounder had low growth
efficiencies, which increased rapidly during the first few days of active feeding.
The relatively high growth efficiency of sea bream, when it begins to feed,
suggests that food is less critical for it than for winter flounder at that stage, a
suggestion supported by the relatively low required prey concentration for
survival of sea bream larvae (45).
Starvation Criteria
Because starvation is suspected as a major cause of larval mortality,
biochemical, histological, and behavioral criteria have been developed for some
species to show changes that occur when the food supply is inadequate. These
techniques eventually may be used to characterize starving larvae collected at
sea. Biochemical methods also have been used to show how laboratory-reared
larvae differ from wild larvae. When supported by morphometric data,
biochemical criteria hold promise to evaluate how types and amounts of food
affect larval condition.
The biochemical composition of laboratory-reared, larval Atlantic herring
and plaice was studied by Ehrlich (32, 33). He found that water, triglyceride,
carbohydrate, nitrogen, carbon and ash content varied as a percentage of body
weight as larvae grew, In starving larvae both relative (percentage) and absolute
changes in amounts of those substances were measured, the relative changes
often being a better measure of starvation than the absolute changes.
Percentage of water increased about four percent in starved larvae of both
herring and plaice, while percentages of triglyceride, carbohydrate, and carbon
decreased. Percent nitrogen decreased in starved plaice larvae but did not
decrease in herring; absolute amounts of nitrogen decreased in both species.
Ash percentage of both species increased rapidly during starvation. Ehrlich (32,
33) concluded that the "point of no return" was not defined by an abrupt
change in the chemical composition at some point during starvation, but rather
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that a continual change in chemistry occurred until the larvae became
moribund.
In a similar study Anraku and Azeta (6) compared cultured and wild larvae
and juveniles of the sea bream Chrysophrys major. They did not examine larvae
less than 10 mm length, but for large specimens the cultured individuals tended
to have a lower percentage of water, higher percentages of carbon and
hydrogen, and a percentage nitrogen that did not differ from wild specimens
until 20 mm length, when percentage nitrogen decreased in cultured
individuals. Differences in food of cultured and wild specimens were the
probable cause of differences in body chemistry. Starved individuals of sea
bream showed effects similar to those for herring and plaice — i.e. increased
percentage water and decreased percentages of carbon, nitrogen and hydrogen.
Histological changes in laboratory-reared larvae are indicative of starvation.
Recent studies indicate that these criteria could be used to recognize starving
or poorly nourished larvae in the sea. Umeda and Ochiai (96) examined fed and
starved yellowtail Seriola quinqueradiata larvae, Ehrlich et al (34) examined
herring and plaice larvae, and O'Connell (72) examined northern anchovy
larvae. In all of these studies there were some similar findings. Intestinal
epithelial cells atrophied in starving larvae and the intestine degenerated. The
liver also degenerated in yellowtail, plaice and northern anchovy. O'Connell
(72) and Umeda and Ochiai (96) examined the pancreas and found that its
condition was markedly deteriorated in starved anchovy and yellowtail larvae.
O'Connell (72) examined several other histological characters and found that
starved anchovy larvae also had separations of muscular fibers and little
intermuscular tissue, as well as notochord shrinkage. Using a discriminant
function analysis he was able to discriminate 90 percent of starving larvae from
fed larvae when four or more good histological characters were used. Ehrlich et
al (34) found that there were good morphological characters associated with
the histological changes, especially in herring larvae where severe head
shrinkage and gut shrinkage caused a decrease in the "pectoral angle", and an
increase in the eye height to head height ratio. Histological criteria as indicators
of impending starvation seem excellent. They are relatively time consuming
compared to morphometric analyses, but perhaps are more effective to
distinguish starvation effects.
The concentration of prey affects larval behavior. Wyatt (100)
demonstrated that duration of plaice larvae activity (searching behavior) was
inversely related to prey concentration, and that starving larvae increased their
time spent searching for food. This behavior presumably is adaptive and
increases the probability of encountering prey when it is scarce. Using vertical
migration as an index of activity, Blaxter and Ehrlich (20) found that fewer
herring and plaice larvae vertically migrated after periods of starvation, and
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that starved larvae tended to be neutrally buoyant because of a relative increase
in body water. They speculated that under starvation conditions larvae would
be relatively inactive, suspended in midwater, and thus more susceptible to
plankton net sampling than well nourished larvae. Blaxter and Ehrlich's (20)
results differ somewhat from Wyatt's (100), partly because of the different
criteria used to define activity.
Behavior of northern anchovy larvae in dense patches of prey,
Gymnodinium splendens and Brachionus plicatilis, was investigated by Hunter
and Thomas (49). In dense patches larvae swam slower and covered smaller
areas. Reversals in swimming direction occurred more frequently in patches of
food than in non-patch situations. The evidence strongly suggested that
northern anchovy larvae were able to maintain themselves in suitable patches
of prey, and that such an adaptation would allow larvae to take advantage of
prey patchiness in the sea.
PREDATION
Predation almost certainly is the greatest direct cause of mortality to marine
fish larvae, but there have been few attempts to evaluate its impact in
laboratory studies. The food supply of larval fishes and other environmental
factors can modify the predation mortality experienced by a cohort. As
Cushing (29) noted, larvae that have an adequate food supply grow fast and
swim well. Thus, they presumably avoid predation by growing quickly through
the larval phase, when they are most vulnerable to a variety of planktonic
predators. Similarly, pollutants or toxicants that retard larval growth or modify
behavior could lead indirectly to increased predation mortality.
Four recent laboratory investigations have examined the potential impact of
predators on larvae. Three species of pontellid copepods could more than meet
their metabolic requirements by preying upon yolk-sac larvae of northern
anchovy (62). Predation in 3500 ml beakers increased as anchovy larvae
concentration was raised. Two of the copepods, Labidocera jollae and L.
trispinosa, were only efficient as predators on yolk-sac larvae, but a third
species, Pontellopsis occidentalis, also was able to prey on more developed,
faster swimming larvae. The presence of alternate prey (Artemia salina nauplii)
reduced larval mortality caused by Labidocera spp. in direct proportion to the
numbers of Artemia that were present. In similar experiments, but using the
euphausiid Euphausia pacifica, as an anchovy larva predator, Theilacker and
Lasker (94) demonstrated that larval, juvenile and adult stages of the
euphausiid could meet their daily carbon requirements by preying upon
northern anchovy yolk-sac larvae. There was no strong evidence that anchovy
concentration, when above 10 per 3500 ml, or the presence of alternate prey
{Artemia nauplii) significantly influenced the predation rate on anchovies, at
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least by juvenile euphausiids. Euphausiids, like pontellid copepods, were most
successful at capturing yolk-sac larvae. Based on laboratory results and known
abundances of pontellid copepods, euphausiids, and yolk-sac anchovy larvae in
surface waters off the coast of California, it is possible that copepods and
euphausiids can have a significant influence on anchovy larvae survival (62,94).
Yolk-sac larvae of the Pacific herring Clupea harengus pallasi were offered as
prey to the amphipod Hyperoche medusarum in experiments conducted in 500
ml beakers (99). The numbers of larvae preyed upon increased as both predator
and larval densities increased. But, the number of prey attacked per hour per
predator decreased as predator abundance increased. The number of herring
larvae attacked per hour increased, but the rate of increased slowed as the
concentration of herring larvae was raised. When "flatfish" larvae were
provided as alternate prey, the amphipods showed a preference for herring.
Amphipods such as Hyperoche medusarum may be an important source of
mortality to Pacific herring larvae in the sea, especially when the
newly-hatched larvae are concentrated near the spawning areas.
Kuhlmann (54) investigated the chaetognaths Sagitta setosa and S. elegans
and their possible role as predators on several species of fish larvae. Despite the
often observed phenomenon in plankton samples of larval fish in chaetognath
guts, S. setosa and S. elegans did not prefer the fish larvae in laboratory
experiments when cope pod prey was present in sufficient quantity. Kuhlmann
(54) did not believe that the chaetognaths were important predators on larval
fishes. He did find that both S. setosa and S. elegans consistently ate fish larvae
after starvation periods of 24 to 48 hours if copepods were not offered as
alternate prey.
ROLE OF NATURAL PHYSICAL AND CHEMICAL VARIABLES
A review of literature dealing with effects of natural environmental factors
on development of marine fishes reveals a wealth of information on egg and
yolk-sac stages (e.g., 14, 36, 38). However, these factors have not been
intensively studied for larval stages from the time of first feeding to transition
to the juvenile.
Light
Blaxter (16, 18) discussed the preferences of fish for light of specific
intensities. This preferendum may vary from day to night and may not be
available at the preferred intensity in some shallow water situations where little
vertical migration is possible. The evidence reviewed by Blaxter (16)
demonstrates that the light preferendum is variable among species and also
among individuals.
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Marine fish larvae typically are visual feeders and require a minimal light
intensity above 10"^-10 lux to feed optimally (14, 16). Light levels reported
by aquaculturists for successful culture of marine fish larvae have ranged from
250-10,000 lux (9, 42). A 500-3000 lux range has been used most often. A
minimum light intensity is necessary for initial detection of prey, visual
recognition and prey selection. At light intensities close to the threshold level
there is a gradual reduction in larval activity and in feeding performance (16).
Blaxter (19) recently has summarized information on the anatomy of eyes in
fish larvae and also has discussed the development of vision. A pure cone
retina, which requires relatively high light intensities to be effective, appears to
be typical of larval stages of fish and such retinas have been identified in the
Atlantic herring (22), the plaice (12) and other species (19, 23). In general, for
the few species of larvae that have been studied, the light intensity range in
which feeding activity decreases is approximately 10^-10"^ lux (11, 12, 13).
This is near the dusk-dawn light intensity range of 10^-10"^ lux (16).
Laurence (60) estimated the number of hours required daily by winter
flounder larvae at 8°C to consume a ration that exceeds the maintenance
ration. For a prey concentration of 3.4 cal/1 (= 500 copepod nauplii/1), the
minimum suitable for survival and growth, first feeding winter flounder larvae
would require about 20 hours per day to consume the maintenance ration. The
diurnal light period, when light intensities are above 10^ lux, is most important
at low prey concentrations. For prey concentrations exceeding 6.8 cal/1 (=
1000 nauplii/1) winter flounder larvae could meet their daily food
requirements in less than 10 hours and at prey levels above 13.6 cal/1 (= 2000
nauplii/1) established feeders could meet requirements in only 5 hours. It is
apparent that the seasonal variation in day length and light intensity are
important elements in any model predicting survival of fish larvae, particularly
in high latitudes where seasonal variation is greatest.
Possible harmful effects of natural sunlight on larvae are poorly known.
Ultraviolet light near the sea surface may be deleterious to pelagic eggs (63).
Some information on effects of ultraviolet light on pelagic embryos may apply
to larval stages. Pommeranz (75) tested effects of ultraviolet light on plaice
embryos, using an artificial UV intensity of 0.05 ly/min (1 ly = l cal/cm ^) at
the water surface of 350 ml incubators with a 200 ml/min water exchange.
Although results were not conclusive, lower percentages of embryos survived in
12 hours and 24 hour-exposed incubators than in control incubators which
were in the dark. Pommeranz (75) also exposed plaice embryos to natural
daylight, natural daylight with UV wavelengths filtered out, and natural
daylight with long wave infrared above 1400 nm filtered out. Average light
intensities in two experiments were 257 ly/day and 462 ly/day. Only the high
intensity experiment caused high mortality of embryos (35 percent).
Ultraviolet light was considered to be the lethal agent because corresponding
mortalities were not observed in incubators where ultraviolet was filtered out.
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Temperature
Brett (25) refers to temperature as a polymorphic environmental factor that
may be a lethal agent, a controlling factor regulating metabolism and
development, a limiting factor restricting activity and distribution, a masking
factor interacting with other environmental factors, or a directing agent such as
a thermal gradient. It appears that the major temperature effect on marine fish
larvae is that of a controlling factor regulating metabolic and developmental
rates. In turn, those rates can affect survival of larvae through their influence
on establishment of exogenous feeding and regulation of food requirements
(e.g. 43, 57, 59, 60). For clupeiform, perciform, and pleuronectiform larvae, a
6-10°C range has been reported in which culture attempts are most successful
(8, 10, 43, 57, 59, 61), although some survival can be obtained over wider
temperature ranges.
There are few temperature effect-metabolic rate studies on marine fish
larvae. Laurence (59) examined growth and metabolism of feeding winter
flounder larvae at 2°, 5° and 8°C. Larvae reared at 5° and 8°C were tested,
until metamorphosis and the specific growth rate at 8°C (10.1 percent/day)
was significantly higher than that at 5°C (5.8 percent/day). The growth rate at
2°C (2.6 percent/day) was less than at 5°C but not significantly less.
Metamorphosis took 49 days and 80 days at 8° and 5°C, respectively. At 2°C
larvae did not survive more than six weeks after yolk absorption. Power
functions describing oxygen consumption of winter flounder in relation to
body weight had exponential coefficients lower than the expected theoretical
value of 0.80 (0.49 for 8°C, 0.56 for 5°C, 0.54 for 2°C). When separate power
functions were fitted for larvae and for metamorphosed juveniles, the
exponential coefficients for larvae closely agreed with the theoretical 0.80
value for all three temperatures, but the coefficients for metamorphosed
juveniles were lower.
Hoss et al (41) examined the effect of a rapid 12°C rise in temperature
(thermal shock) on growth of pinflsh Lagodon rhomboides and spot
Leiostomus xanthurus, and oxygen consumption of pinfish, to determine if
growth and metabolism could be used to detect sublethal effects of power
plant thermal pollution. The fish that they used were transformed juveniles, in
most respects (5.15-7.89 mg dry weight for pinfish, 11.23-23.70 mg for spot).
No significant difference in growth was observed for thermally shocked and
control groups. Oxygen consumption rates of experimental and control pinfish
indicated that a 12°C shock produced a slight increase in consumption rate
which returned to normal levels within a few hours. Their determinations (41)
of critical thermal maxima and survival after acute thermal shocks may not
represent responses that might be obtained for smaller larvae.
Time-temperature exposure histories are critical for determining thermal
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effects of entrainment (87, 89), but there are no such studies that include
marine fish larvae from first feeding to metamorphosis stages.
During the past 15 years temperature responses often have been investigated
in conjunction with effects of other environmental factors, usually variations in
salinity for embryos and yolk-sac larvae (e.g. 66, 86). Multi-dimensional
analysis has led to use of response surface models which permit evaluation of
interacting effects such as between temperature, salinity, oxygen, and dose
time (1). However, most of this research has dealt with the egg, embryo, and
pre-feeding larval stages (e.g. 2, 3, 4, 5, 68).
Salinity
The developing eggs and yolk-sac larvae of many marine teleosts are known
to tolerate wider ranges of salinity than they are likely to encounter under
natural conditions (e.g. 2, 5, 36, 38, 68, 77, 86), but there are few studies
dealing with salinity tolerances of typical pelagic marine fish larvae during the
actively feeding stages.
In unaltered environments, the effect of changes in salinity on larval survival
may be minimal, since pelagic larvae usually will be retained within a water
mass that does not undergo extreme salinity changes. In the lower latitudes,
where time for larval development to metamorphosis is short, the probability
of an extreme salinity change that might cause mortality seems even less
probable than in higher latitudes. Holliday (36), in reviewing data on salinity
tolerances of Atlantic herring and plaice, observed that newly hatched larvae
had a wider tolerance range for salinity than did metamorphosed juveniles.
Tolerance to high salinities decreased from about 60°/oo at hatching to about
40°/oo after metamorphosis, while low salinity tolerance changed little during
development, ranging from about 2-8°/oo for both species. Kurata (55)
obtained similar results for Pacific herring, C. harengus pallasi larvae which
could tolerate a salinity range of approximately 2-60°/oo at 10 days after
hatching, but only 2-42°/oo at 20 days.
There are several investigations on salinity tolerances of non-typical or
non-pelagic marine fish larvae, from which conclusions about tolerances of
marine fish larvae in general perhaps can be inferred. For mummichogs
Fundulus heteroclitus the range of salinity tolerance was very wide,
0.39-100.00°/oo (51). California killifish larvae F. parvipinnis also had a wide
salinity tolerance, but the tolerance for low salinities decreased with age (76).
Two atherinids, the California grunion Leuresthes tenuis and the Gulf grunion
L. sardina, were tested for salinity tolerances during the larval stage (78, 79).
Gulf grunion had a wider salinity tolerance range than did California grunion,
but in both species the tolerance range decreased with age. A reasonable
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conclusion, based on limited data, is that newly hatched larvae of marine fishes
are unlikely to suffer mortality as a direct effect of salintiy, but that older
larvae are more vulnerable and could be killed by physiological stresses caused
by salinity extremes.
Oxygen uptake of anesthetized Atlantic herring eggs and newly hatched
larvae did not differ significantly at test salinities of 5, 15, 35 and 50°/oo (38).
For larvae there was variable oxygen uptake, the rates sometimes being 10X
the pre-transfer oxygen consumption rate. For example, for a transfer from 35
to 5°/oo at 8°C, larval oxygen consumption went from about 0.07 ill
C^/larva/hour to as high as 0.7 jul C^/larva/hour within one hour after
transfer. Oxygen uptake then fluctuated before returning to normal about five
hours after transfer to the test salinity. Such fluctuations occurred for six-eight
hours following transfer and were believed caused by osmoregulatory
imbalance prior to acclimation to the treatment salinity.
Dissolved Oxygen
Vernberg (97) remarked that effects of low oxygen levels on animals are not
easily determined under natural conditions because anoxic situations are
always accompanied by other factors such as increased carbon dioxide and
hydrogen sulfide concentrations. The effects of temperature and salinity on the
solubility of oxygen also complicate the analysis of direct oxygen effect.
Dissolved oxygen requirements of developing eggs and larvae of Salmonidae
and other freshwater or estuarine species have been investigated many times
(e.g. 35, 91, 98). There are few studies on marine fish larvae to determine their
tolerances to low oxygen tensions (30, 85). De Silva and Tytler (30) found that
the incipient lethal oxygen level (LD^q) for Atlantic herring and plaice varied
with development from the yolk-sac stage to metamorphosis. At 10°C the
LD^q for yolk-sac larvae was 1.93 ml/1 for herring and 2.73 ml/1 for plaice.
After larvae had been feeding for two weeks the LD^q was 3.08 and 2.66 ml/1
respectively. At 56-63 days after hatching for herring and 42^-9 days after
hatching for plaice, gills developed and the LD^q levels fell to 2.91 and 2.52
ml/1, respectively. At metamorphosis, 70-80 days after hatching for herring
and 77-84 days after hatching for plaice, the LD^q was 2.17 and 1.69 ml/1,
respectively. De Silva and Tytler (30) also measured routine metabolism of
herring larvae from 7-62 days after hatching and plaice larvae from 5-75 days
after hatching at 10°C. For the relationship between oxygen consumption and
weight, they obtained exponential coefficients of 0.82 for herring and 0.65 for
plaice. These values are higher than the values 0.49-0.56 obtained by Laurence
(59) for winter flounder from hatching through metamorphosis, although
Laurence obtained a coefficient of 0.80 when he excluded metamorphosed
individuals from his analysis.
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In surface waters of the euphotic zone dissolved oxygen usually ranges from
4-8 ml/1 with supersaturation (> 6-9 ml/1) possible in highly productive
shallow coastal waters. Kalle (52) reported that in coastal areas with high
primary production, oxygen super-saturations up to 120 percent are not
unusual during periods of intensive solar illumination. In shallow waters,
temporary super-saturations may approach 500 percent (52). Mortality or
stress of fish larvae due to low oxygen tensions probably occurs only under
unusual conditions in the sea.
Miscellaneous Environmental Factors
Environmental factors such as turbidity, mechanical stresses, and shear
forces likely to be found in nature have not been studied experimentally with
regard to effects on marine fish larvae. A few investigations of effects of these
factors on embryonic stages indicate that embryos are resistant to high
sediment suspensions and mechanical forces which are present in the
environment (e.g. 71, 75, 88).
Schubel et al (88) observed that striped bass Morone saxatilis eggs could
tolerate silt loads'up to 500 mg/1. They noted that turbidity in areas being
dredged could be as high as 1000 mg/1, which would cause significant embryo
mortality, but that such high concentrations rarely occurred. Hoss et al (40)
tested larvae of seven estuarine species with three concentrations of sediment
extracts (the supernatent from 500 g of Charleston Harbor sediment shaken in
one liter of filtered seawater). Under their laboratory conditions, survival of
larval pinfish and menhaden Brevoortia tyrannus was 25-0 percent at the 75
and 100 percent test concentrations. The supernatent water and sediments
were not analyzed for toxic substances by the authors, but they cited
references to relatively high levels of lead, copper, zinc and chromium in
Charleston Harbor sediments.
Pommeranz (75) investigated mechanical properties of plaice eggs by
deforming them with a lever. The force to burst the chorion varied with time
from fertilization and ranged from about 1.5 g during the 30 minutes after
fertilization to a mean of about 700 g and 600 g for gastrula and embryo
stages, respectively. For comparison purposes, Pommeranz (75) cited one
rough estimate of the pressures developed by a spilling breaker in the open sea
as approximately 0.1 kg/cm^.
Morgan et al (71) subjected striped bass and white perch Morone americana
embryos and yolk-sac larvae to experimental shear forces of 0-86 dynes/cm^
over exposure times of 1-20 minutes. The estimated median lethal shear (LS^q)
that could kill 50 percent of the embryos and larvae ranged from 120
dynes/cm^ for a 20 minute exposure, to 785 dynes/cm^ for a one minute
193
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exposure. Estimated LS^g values for striped bass yold-sac larvae were 785
dynes/cm^ and 300 dynes/cm^ for 1 and 4 minute exposures, respectively.
White perch yolk-sac larvae were more vulnerable, their LS^q values were 415
dynes/cm^ and 125 dynes/cm^ for 1 and 4 minute exposures respectively.
Calculated average shear force in the Chesapeake and Delaware Canal, where
striped bass eggs occur, was only 13.8 dynes/cm^, far below the estimated
LSjq values. The authors (17) also related these LS^q values to expected shear
forces of 72-230 dynes/cm^ that might be present in the water box of a power
plant cooling system. The 230 dynes/cm^ shear approaches the 4 minute LS^g
value for striped bass yolk-sac larvae and exceeds it for white perch.
Chipman (27) reviewed literature on effects of naturally occurring ionizing
radiation on marine animals. He found no convincing evidence to demonstrate
that marine animals showed any response, functional or structural, to ionizing
radiation levels present in the environment. In marine animals observable
effects are primarily at the cellular level, and the radiation tolerance is a
function of the dose-rate, time patterns of exposure and metabolic rate;
consequently, effects would be most evident during embryonic development
(27).
FUTURE RESEARCH
Both laboratory and transitional laboratory-field studies will extend our
knowledge of environmental effects on larval stages of marine fish. A recent
colloquium on larval mortality and the recruitment problem has defined some
areas in need of research (48). Emphasis of that colloquium was to advocate
research related to starvation and predation, the two factors that probably have
the greatest effect on recruitment of year classes. Environmental stresses from
man's activities are additional threats, particularly to estuarine species or those
found over the continental shelf. Pollution effects on embryos can cause gross
functional and structural abnormalities that may produce yolk-sac larvae
incapable of surviving to the exogenous feeding stage (81). Larvae can be
equally vulnerable to deleterious effects of pollutants, and their responses to
this stress may be reflected in impaired predator avoidance behavior and food
capture efficiency. More subtle effects could involve functional disruptions of
metabolism, temperature and salinity tolerance, and enzyme-substrate
interactions. Both direct and indirect effects of environmental modification on
recruitment need to be determined.
The ability to culture larvae widens the possibilities for laboratory research
which will help interpret results of field studies. The larval stage is a dynamic
one, characterized by fast growth, sometimes spectacular developmental
changes, and frequent shifts in behavior. Typical toxicity bioassays, where
times to 50 percent mortality are estimated, may not be the best approach to
194
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determine how environmental factors affect survival of a larval cohort.
Environmental factors act in concert, and it is the sum of experiences over the
entire embryo and larval stages that determines whether a good or poor year
class results. A bioassay for 96 hours, testing one or two factors, usually can
provide only a rough evaluation of the potential effect of the factor(s) on
recruitment. More meaningful conclusions can be drawn from investigations
that encompass the entire larval period. Many studies of that land have been
carried out on larvae of freshwater fishes (69), but the difficulties in rearing
larvae of marine species have limited most bioassay research to embryo and
yolk-sac larva stages.
Experiments in large volumes of seawater, either in plastic bag enclosures,
such as those used in recent Controlled Ecosystems Pollution Experiments
(CEPEX) (70) or in large tanks (74) hold great promise because whole
communities can be entrapped in such volumes. Effects of predation and
competition can be evaluated. Direct and indirect effects of added pollutants
on each trophic level can be observed. Recruitment success or failure by fishes
in such systems can be interpreted in the context of observed changes that
took place in the plankton community during the course of larval
development.
Other approaches include transitional studies that combine laboratory and
field experiments. The "field bioassay" developed by Lasker (56) uses
laboratory-reared larvae in shipboard experiments, in which larvae are reared in
natural seawater sources to evaluate the potential of particular water masses to
support larval survival and growth. The recent discovery, based on laboratory
studies, that daily growth rings are present on otoliths of larvae, will allow
better estimates of larval growth and mortality rates in the sea (26, 93), and
also will allow comparison of growth in the laboratory with growth under
natural conditions.
Except for swimming-feeding behavior of a few species and behavioral
responses to varying light levels (12, 15, 17) little is known about normal
behavior patterns of larvae or changes in behavior induced by environmental
effects. Behavioral studies not only can increase our understanding of how
pollutants affect larval behavior, but they also can provide important insight
into how predation and competition operate during the larval stage.
There are many techniques presently available that allow environmental
factors and their effects on marine fish larvae to be evaluated. In the next 10
years, culture of marine fishes will be routine procedure at many laboratories;
and as more data accumulate, some of the seemingly contradictory results
obtained to date, especially with regard to critical food concentrations, will be
resolved. Additional species of marine fishes need to be tested for larval
195
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tolerances to environmental factors. Present day literature is dominated by
research on herring, plaice, and northern anchovy; the first two species are
rather atypical pelagic, marine fish larvae because of their unusually large size
and advanced development at hatching. Refinement of culture methods,
improved techniques for handling and testing delicate larvae, and examination
of multiple factor effects and interactions throughout the period of larval
development will help us to better understand how the environment acts on a
cohort of larvae. This knowledge can be incorporated into predictions of
recruitment success based on probable influences of environmental factors
operating during the larval stage.
ACKNOWLEDGEMENTS
Support from Environmental Protection Agency Grant R804519 made the
preparation of this paper possible.
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90. Shirota, A. 1970. Studies on the Mouth Size of Fish Larvae. Bull. Jap.
Soc. Sci. Fish. 36: 353-368.
91.Siefert, R.E., W.A. Spoor, and R.F. Syrett. 1973. Effects of Reduced
Oxygen Concentration on Northern Pike (Esox lucius) Embryos and
Larvae. J. Fish. Res. Board Can. 30: 849-852.
92. Stepien, W.P., Jr. 1976. Feeding of Laboratory-Reared Larvae of the Sea
Bream Archosargus rhomboidalis (Sparidae). Mar. Biol. 38: 1-16.
93. Struhsaker, P. and J.H. Uchiyama. 1976. Age and Growth of the Nehu,
Stolephorus purpureus (Pisces: Engraulidae), from the Hawaiian Islands as
Indicated by Daily Growth Increments of Sagittae. Fish. Bull., U.S. 74:
9-17.
94. Theilacker, G.H. and R. Lasker. 1974. Laboratory Studies of Predation by
Euphausiid shrimps on Fish Larvae, pp. 287-299. In: J.H.S. Blaxter (ed.)
The Early Life History of Fish. Springer-Verlag, New York, Heidelberg,
Berlin.
95. Theilacker, G.H. and M.F. McMaster. 1971. Mass Culture of the Rotifer
Brachionus plicatilis and its Evaluation as a Food for Larval Anchovies.
Mar. Biol. 10: 183-188.
204
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96. Umeda, S. and A. Ochiai. 1975. On the Histological Structure and
Function of Digestive Organs of the Fed and Starved Larvae of the
Yellowtail. Seriola quinqueradiata. Jap. J. Ichthyol. 21: 213-219.
97. Vernberg, F.J. 1972. Dissolved Gases, 9.3 Animals, pp. 1491-1526.In: O.
Kinne (ed.) Marine Ecology, Vol. 1, Pt. 3. Wiley Interscience, New York.
98. Voyer, R.A. and R.J. Hennekey. 1972. Effects of Dissolved Oxygen on
Two Life Stages of the Mummichog. Prog. Fish-Cult. 34: 222-225.
99. Westernhagen, H. and H. Rosenthal. 1976. Predator-Prey Relationship
between Pacific Herring, Clupea harengus pallasi, Larvae and a Predatory
Hyperiid Amphipod, Hyperoche medusarum. Fish. Bull., U.S. 74:
669-674.
100. Wyatt, T. 1972. Some Effects of Food Density on the Growth and
Behaviour of Plaice Larvae. Mar. Biol. 14: 210-216.
205
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LABORATORY CULTURE OF THE
GRASS SHRIMP
Palaemonetes vulgaris
by
Thomas E. Bigford
Environmental Research Laboratory
United States Environmental Protection Agency
Narragansett, Rhode Island 02882
ABSTRACT
Experiments have been undertaken to test the feasibility of hatching,
rearing, and breeding an in-laboratory population of the grass shrimp,
Palaemonetes vulgaris. Primary objectives include continual availability of all
life stages (for use in experiments or as food organisms) and comparisons of
lab-reared and field-collected animals.
Systems have been designed for culturing the grass shrimp throughout its
life cycle. Larval survival percentages reached 70 percent in the beaker and
"hatching jar" culture systems. Up to 75 percent of these metamorphosing
larvae survived to adult stages. Both Artemia salina and the flake food Tetra
Marin were proven to be successful diets for/1, vulgaris.
Results indicate that P. vulgaris can be maintained and propagated in the
laboratory. Larvae hatched in the lab have been induced to produce normal
larvae within as little as 90 days. This generation time is apparently shorter
than the time in field populations.
INTRODUCTION
Most marine biology research efforts require a consistent supply of
experimental animals. Field-collected organisms often confer variability due to
individual differences in life history, nutrition, etc. Many of these problems can
be controlled by culturing the animals under rigorous, well-documented
laboratory conditions.
The purpose of this study was to develop and standardize laboratory
holding and culture techniques for the grass shrimp, Palaemonetes vulgaris
(Say). Establishment of suitable methods would permit testing of the
206
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feasibility of hatching, rearing and breeding a laboratory population of the
shrimp for use as experimental animals. Of primary consideration in this study
was the development of flow-through culture systems for the various life
stages. Static designs represent poor simulations of field conditions and may
impose unnecessary stresses on the animals (8). A secondary concern was to
determine suitable diets for the juvenile and adult grass shrimp. Broad (1)
found brine shrimp, Artemia salina, nauplii to be a very satisfactory larval
food. Success for the project must be measured in terms of growth, survival,
and population reproduction.
The advantages of using lab-reared organisms are countered by several
anomalous characteristics of organisms maintained in the lab. Morphological
changes, as compared to field animals, have been noted by Paul Yevich
(Environmental Research Lab, Narragansett, R.I.; personal communication) in
many marine animals. However, the increased control of age, nutrition, and
prior exposure to environmental variables would appear to outweigh slight
changes in morphology.
The grass shrimp, Palaemonetes vulgaris (Say), was selected for these studies
for several reasons: the shrimp is a common estaurine species available to
researchers along the Atlantic and Gulf of Mexico coasts (9); the animal is
relatively easy to rear in the laboratory; and the life cycle can be greatly
compressed in the lab (4).
EXPERIMENTAL
Several ovigerous grass shrimp were collected by dip net on 14 July 1976 in
the Pettaquamscut River estuary adjacent to Narragansett Bay, Rhode Island.
Mid-summer salinities at the collection site range from 25-30o/oo depending
on the tidal cycle; water temperature was 21.5°C.
Egg-bearing females were isolated in six £ (1.6 U.S. gal.) tubs^ at 21.0-
23.5°C and oceanic salinities (29-34.5o/oo). Photoperiod was maintained at
ambient levels of L14:D10. Water was changed daily and aerated gently.
Shrimp were offered food during this holding period but they rarely fed.
Larvae hatched after 1 to 17 days of holding and were immediately pipetted
into the flow-through system shown in Figure 14-1. About 200 larvae were
held in this two £ system for 21 days, by which time all shrimp had reached the
late larval stages. Developing larvae were fed excess quantities of newly hatched
and one-day old brine shrimp nauplii, Artemia salina (San Francisco Bay
Brand).
^ Rubbermaid Commercial Products, Inc., Winchester, VA.
207
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h2o
NOTE: Designed by Dr. W. B. Vernberg under E.P.A. Grant R 802071 and
modified for use in the culture of larval and juvenile Palaemonetes vulgaris.
All surviving juvenile P. vulgaris (about 140) were transferred on day 22
(post-hatch) to the 20 £ "hatching jar"^ commonly used in hatching and
rearing larval fish (Figure 14-2). This 30 cm diameter, clear acrylic tank, was
modified from the manufacturer's design by placing a 400 nm nylon mesh
across the outflow ramp, thereby eliminating the need for a mesh over the top
of the entire system. This smaller outflow area effectively decreased the
chances of impinging larvae on the mesh. Gentle aeration and a water flow of
50 ml/min created a satisfactory circulation pattern. This hatching jar system
^Midland Plastics Co., Brookfield, WI.
208
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AIR SEAWMER
INFLOW INFLOW
Figure 14-2. The 12 8 Hatching Jar System Used to Culture
Juvenile and Adult Grass Shrimp, Palaemonetes vulgaris.
NOTE: 20 and 48)2 sizes are identical in design.
was used for 14 weeks, during which time a combination of thawed and live
juvenile brine shrimp were fed in excess, with unconsumed food removed by
siphon once each week.
All grass shrimp were transferred from the 20 2 jar at age 17 weeks. Of these
shrimp, 40 were used in a diet study, and the remaining 66 were placed in a 48
£ hatching jar, a scaled-up version of the 20 £ size (Figure 14-2). In the diet
study, 40 shrimp were divided evenly between two 4fi systems resembling
that shown in Figure 14-1. One group of 20 shrimp was reared on lab-reared
Artemia adults while the other group was fed 243-400 jum pieces of the
commercial flake fish food, Tetra Marin. Animals were fed daily in excess.
Growth in carapace length, survival, and the incidence of ovigerous females
were considered in determining the suitability of each diet.
209
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RESULTS AND DISCUSSION
Attempts to culture and maintain a laboratory population of P. vulgaris
have been successful thus far. Ovigerous females were obtained from adults
hatched in the laboratory and cultured for 90 days. These egg-bearing females
have yielded morphologically normal larvae, thereby indicating that the eggs
resulting from lab-reared females are viable. After 16 months of culture, a total
of 21 ovigerous females have been collected from the system. Six of these
shrimp are females that also bore eggs in the first laboratory spawning season
(November, 1976 to January, 1977 or 9 to 10 months ago). Problems in
controlling photoperiod and water temperature have limited successful hatches
to only two females. Little (4) has discussed how manipulation of these two
environmental factors can be used to induce winter breeding in grass shrimp.
The diet study (Table 14-1) has indicated on a gross scale that a flake food
can be used as a diet. Ovigerous females were collected from the four £
flow-through systems used for both the brine shrimp and Tetra Marin diets.
Therefore, growth, survival and reproduction are acheivable with the live and
dried foods.
All of the systems and techniques mentioned herein have yielded
satisfactory results. However, some minor problems remain. One such problem
is cannibalism, especially in the 20 and 48 2 hatching jars used as holding tanks
for juvenile and adult grass shrimp. Obvious solutions include increasing the
food available, either as more food per day, or as multiple daily feedings, or
decreasing the density of shrimp. A certain degree of cannibalism is to be
expected in mass cultures during periods of molting.
A second problem, also in the hatching jar systems, relates to the physical
design of the container. The concave bottom of the jar, coupled with a circular
flow, causes a centrifuging of the shrimp into the center near the bottom. A
flatter bottom with a larger bottom surface area to volume ratio could be a
solution. The 40 £ kriesel systems (3) used in lobster culture efforts have the
desired flatter bottoms, and also jetted water inflow along the sides that create
a more uniform distribution of the shrimp. Preliminary studies indicate
that the kriesel design will be very successful for juveniles and adults.
Compared to growth in the four £ beaker systems (See Table 14-1), the kriesel
has yielded significantly higher growth rates. Mean carapace length in the
kriesel after nine weeks was 6.8 ± 0.69 mm (range 5.9 to 7.9 mm), a size not
attained until an age of about 20 weeks in the beaker.
One last problem is animals flipping out of the systems, especially the 48 C
jar. This appears to happen in conjunction with a molt, and the subsequent
cannibalism pressure from other shrimp in the system. A simple solution to the
210
-------
Table 14-1. Summary of growth, measured via carapace length,
and survival of juvenile and adult Palaemonetes vulgaris
cultured in excess concentrations of live, adult Artemia and
ground Tetra Marin.
Artemia salina
Tetra Marin
Date of
Age
Mean
No. of
Mean
No. of
Sampling
(Weeks)
Length (mm)
S.D.
Range
Survivors
Length (mm)
S.D.
Range
Survivors
11-6-76
14
6.4
± 0.65
5.1-7.4
20
6.5
+ 0.66
5.4-7.7
20
12-14-76
20
-
-
-
19
-
-
-
18
2-7-77
28
7.5
± 1.05
6.2-10.4
18
8.0
±0.96
5.4-9.9
18
5-23-77
45
-
-
-
17
-
-
-
15
NOTE: Initial counts of shrimp were 20 per four £ beaker. Carapace lengths were measured from tip of rostrum to posterior edge
of carapace.
-------
escape problem is a cover over the system. However, such a design may change
the cause of mortality from escape and desiccation to cannibalism.
Each of these systems emphasizes low maintenance and unlimited scale-up
potential. Care was taken during the design phase to avoid sharp corners, excess
mesh area, or eddying currents. Hartman (2) has shown that brachyuran larvae
become impinged in corners that break spines or setae and impede molting. He
also mentioned the importance of tapered walls so that larvae and food do not
become caught in eddying currents.
Even with these problems, survival of all life stages has been high. Larvae
cultured in the beaker systems have shown approximately 70 percent survival
when fed Artemia nauplii in excess. In this experiment, survival of juveniles
and adults reared in the 20 £ hatching jar was 75 percent (since
metamorphosis) over a 14 week period. The diet studies have shown similar
survivals in smaller cultures.
Several other comments are worthy of mention. The ages at transfer from
one system to another represent the schedule used in this study and most likely
could be altered without problem. Also, one key factor to consider in the two
£ and four £ flow-through systems and the hatching jars, is mesh size. A mesh
should be chosen that will permit debris to pass through, yet retain both larvae
and food organisms. For these reasons a 243 or 400 ;um mesh was used.
Use of A. salina nauplii as a diet for larval grass shrimp has been
substantiated by several investigations (1, 6). The study by Broad (1)
confirmed that diets including brine shrimp were more successful in terms of
survival and development than diets lacking this animal tissue. Provenzano and
Goy (6) established the possibility of using Artemia from several locations,
including San Francisco, Canada and, to a lesser degree, Utah.
Establishment of a laboratory population of grass shrimp should lead to
increased use of the animal in bioassays. Nimmo et al (5), based on cadmium
bioassays, concluded that adult P. vulgaris were "acutely and chronically"
more sensitive than the pink shrimp, Penaeus duorarum. Studies by Shealy and
Sandifer (7) have shown the susceptibility of P. vulgaris larvae to mercury.
Conversely, Vernberg et al (8) reported that P. pugio is quite resistant to
cadmium bioassays. Apparently animal age and species are important; P.
vulgaris may prove to be a better pollution indicator than P. pugio.
REFERENCES
1. Broad, A.C., 1957. The Relationship Between Diet and Larval Development
of Palaemonetes. Biol. Bull. 112:162-170.
212
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2. Hartman, M.C., 1977. A Mass Rearing System for the Culture of Brachyuran
Crab Larvae. In: Proceedings of Eighth Annual Meeting of World
Mariculture Society, Jan. 7-14,1977. San Jose, Costa Rica.
3. Hughes, J.T., R.A. Shleser and G. Tchobanoglous., 1974. A Rearing Tank
for Lobster Larvae and Other Aquatic Species. Prog. Fish. Cult. 36:129-132.
4. Little, G., 1968. Induced Winter Breeding and Larval Development in the
Shrimp, Palaemonetes pugio Holthius (Caridea, Palaemonidae). Crustaceana
2 (suppl.): 19-26.
5. Nimmo, D.W.R., D.V. Lightner and L.H. Bahner, 1977. Effects of Cadmium
on the Shrimps, Penaeus duorarum, Palaemonetes pugio, and Palaemonetes
vulgaris. In (F.J. Vernbergefa/, Ed.) Physiological Responses of Marine
Biota to Pollutants. Academic Press, New York. pg. 131-183.
6. Provenzano, A.J., Jr. and J.W. Goy, 1976. Evaluation of a Sulphate Lake
Strain of Artemia as a Food for Larvae of the Grass Shrimp, Palaemonetes
pugio. Aquaculture 9:343-350.
7. Shealy, M.H., Jr. and P.A. Sandifer, 1975. Effects of Mercury on Survival
and Development of the Larval Grass Shrimp Palaemonetes vulgaris. Mar.
Biol. 33:7-16.
8. Vernberg, W.B., P.J. DeCoursey, M. Kelley and D.M. Johns, 1977. Effects of
Sublethal Concentrations of Cadmium on Adult Palaemonetes pugio under
Static and Flow-Through Conditions. Bull. Environ. Contam. Toxical.
17:16-24.
9. Williams, A.B., 1965. Marine Decapod Crustaceans of the Carolinas. Fish.
Bull. 65:1-298.
213
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EVALUATION OF VARIOUS DIETS ON
THE LIPID AND PROTEIN COMPOSITION
OF EARLY LIFE STAGES OF THE
ATLANTIC SILVERSIDE
Kenneth L. Simpson, Leslie M. Richardson and
Paul S. Schauer
Department of Food Science and Technology,
Nutrition and Dietetics
University of Rhode Island
Kingston, R.I. 02881
ABSTRACT
A study was performed to evaluate the effect of various natural and
artificial diets on the lipid and protein composition of.laboratory cultured
Atlantic silversides, Menidia menidia. Results were compared to analyses of
wild silversides, which constituted the biochemical control.
The best growth and survival of juvenile silversides was obtained on a live
3-day-old brine shrimp nauplii diet. Substantially lower growth and survival
were obtained on a freeze-dried brine shrimp diet and the artificial diets.
Amino acids were incorporated into the tissue of batch cultured silversides
fed a live 3-day-old brine shrimp diet by the fifth day of culture. Thereafter,
the profiles changed very little, except for the levels of histidine and arginine in
the 58-day-old silversides. The amino acids of the cultured fish fed natural or
artificial diets were quite similar. Bioavailability studies are necessary to
ascertain the degree of incorporation and assimilation of dietary amino acids.
The whole body fatty acid composition of cultured fish reflected the
composition of their diets. Fish fed a live brine shrimp nauplii diet had higher
total lipid levels and lower polyunsaturated fatty acid levels than wild
silversides. Cultured fish may store large amounts of lipids in order to facilitate
the bioaccumulation of long chain polyunsaturated fatty acids. The
incorporation of cod liver oil into a diet previously containing a soybean oil
increased the levels of the polyunsaturated fatty acids in the fish. The resulting
fatty acid tissue levels resembled the long chain fatty acids of the wild fish
lipids more closely than the profiles of fish fed brine shrimp nauplii.
214
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INTRODUCTION
The Atlantic silverside, Menidia menidia, is a marine fish used in bioassay
studies due to its relative sensitivity to environmental contaminants (17).
Bioassays using silversides have been relatively short-term studies, principally
because of the dependence upon wild fish populations. Before long-term
studies are possible, the dietary aspects of laboratory culture technology must
be developed. The diet can affect the organism's ability to respond in a
reproducible fashion. Additionally, the diet is an important feature in the
ability of cultured fish to reach maturity, and spawn viable eggs necessary for
multi-generation bioassay evaluations.
Live brine shrimp, Artemia salina, have been used world-wide in the
laboratory culture of larval marine fish (4). Silversides used in toxicological
bioassays by the Environmental Protection Agency's Environmental Research
Laboratories have commonly been fed brine shrimp as their primary diet.
However, the difficulty of culturing large volumes of biochemically similar
brine shrimp (8), coupled with increased costs and decreased availability (32)
of cysts has mandated the need for an artificial diet. Providing an adequate,
practical, and economical diet is a major factor limiting culture of most marine
fish reared on either a laboratory or commercial scale. Based on these facts,
the University of Rhode Island, Food Science & Technology, Nutrition and
Dietetics Department collaborated with the Environmental Research
Laboratory to evaluate a number of artificial diets that could replace brine
shrimp.
In our study, we were attempting to produce a cultured fish that could
respond in bioassays in a similar manner to wild fish, and provide comparable
growth and survival as brine shrimp fed juvenile fish. This paper discusses the
effects of various diets on the protein and lipid composition of laboratory
reared silversides.
EXPERIMENTAL
General
This study consisted of three parts: 1) a two month batch culture of
silversides fed 3-day-old brine shrimp, 2) a preliminary evaluation of an
artificial Atlantic salmon, Salmo salar, diet comprised of a soybean oil base
and 3) an expanded study using brine shrimp and a number of artificial diets.
Culture
The collection of the gravid female silversides, the stripping and fertilization
of the eggs, the hatching and feeding procedures, and the culture systems used
215
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in this study have been previously documented (7). Gravid fish were collected
from Bissel Cove, Narragansett Bay (R.I.) and transported in aerated containers
to holding tanks located at the Environmental Protection Agency Laboratory,
Narragansett, R.I. Eggs were stripped from females onto nylon monofilament
screens with a mesh size of 400ju, and fertilized by bathing them in the milt of
two to three males (5). They were then suspended in egg hatching jars, 15 cm
in diameter, modified from the original design of Buss (12) by the addition of a
bottom center drain.
After hatching, the fish were transferred to a 720 liter holding tank and fed
live 3-day-old brine shrimp. Fish were periodically removed during the two
month batch culture study for biochemical analyses. Fish used in both the
preliminary and expanded diet evaluations were cultured for approximately
two weeks. The jars used for hatching of the eggs, were also used as the culture
vessels in the artificial diet studies. For the eight-diet expanded study each jar
was stocked with 50, 23-day-old fish (individual mean weights, 8.90 mg)
obtained from the batch culture population; Two replicates were run for each
diet fed group.
Diets and Feeding Procedures
The wild plankton (Diet 1) were collected from a number of locations in the
west passage of Narragansett Bay, R.I. and from local estuarine areas with a
243/u mesh conical plankton net (Table 15-1). The plankton population was
comprised of a mixture of copepods, primarily Acartia tonsa, and some
invertebrate larvae (22). The plankton samples were transported to the
laboratory in insulated containers and held at 20°C.
The live brine shrimp nauplii (Diet 2) (San Francisco Bay Brand, USA) were
incubated for 24 hours in two liter separatory funnels containing filtered
seawater (29.0 to 31.0 o/oo salinity, 20 to 22°C) and harvested after 72 hours.
A starved group served as a control (Diet 3). The freeze-dried brine shrimp
(Diet 4) was obtained by freezing the live brine shrimp to -38°C and then
drying at 4/i/Hg pressure for 24 hours.
Diets 5 through 9 were the artificial formulations. Tetra Marin (Diet 5) is a
commercial flake diet used in aquarium fish applications and consists of
unknown proportions of meals from fish, crab, mussel, lobster, beef heart, and
brine shrimp. In addition, it is made up of such components as halibut liver,
Calanus finmarchicus, kelp, oatflour, wheat germ, Agar-Agar, seaweed, and
bone charcoal. The other four artificial diets were modified formulations
originally prepared to suit the requirements of Atlantic salmon. The diets were
prepared by the Tunison Laboratory of Fish Nutrition, U.S.F.W.S., Cortland,
New York. The composition of these diets is given in Tables 15-2 and 15-3.
216
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Table 15-1. Description of the Experimental Diets
1
Experimental Diets Source and Description
1.
Wild plankton
Collected in West Passage, Narragansett
Bay by conical net with 243/x mesh opening
and retained on a 116ju mesh sieve.
2.
Brine shrimp
nauplii, live
San Francisco Bay brine shrimp, Artemia
salina, eggs hatched and harvested after
72 hours in filtered and autoclaved seawater
of 20-22°C and 20-30 o/oo salinity.
3.
Starved
Unfed
4.
Brine shrimp
nauplii, freeze-dried
Nauplii as obtained in diet #2, then
freeze-dried 24 hours to constant weight.
5.
Tetra Marin
Lot #125244. Tetra Marin Staple Food,
Tetra Werke Dr. rer. nat. Baensch, Melle,
West Germany
6.
Artificial, CM-1
Cortland #1 diet with cod liver oil.
(See Table 15-2)
7.
Artificial, C-1
Cortland #1 diet with soybean oil.
(See Table 15-2)
8.
Artificial, CMP-1
Semi-purified diet with cod liver oil.
(See Table 15-3)
9.
Artificial, CMP-2
Semi-purified diet with cod liver oil and
an amino acid supplement. (See Table 15-3)
1
All diets were ground to a coarse powder of 400m size or less.
Fish were fed four times daily at a level of five percent body wet weight. A
compensation factor was provided to accommodate the flushing action of the
system (7).
Protein Analysis
All fish were starved for 24 hours prior to sacrificing to reduce the stomach
contents. The diet and fish samples were acid hydrolyzed according to the
217
-------
Table 15-2. Composition of the Artificial Diets,
CM-1 and C-1
Components
Percent Composition
CM-1
C-1
Herring meal
40.00
40.00
Soybean oil
10.00
10.00
Corn gluten meal, 60%
10.00
10.00
Wheat middlings, standard
9.00
9.00
Brewers dried yeast
5.00
5.00
Dried condensed fish solubles
5.00
5.00
Dried Whey
5.00
5.00
Meat and Bone Meal
5.00
5.00
Soybean oil
10.00
Cod liver oil
1
Mineral mixture
10.00
0.40
0.40
Vitamin mixture^
0.70
0.70
^ Mixture provided the following compounds in g/kg diet: MgSO^, 2.0; ZnSO^HjO, 0.3;
FeS04-7H20, 0.3; CuSC>4, 0.3; KlOg, 0.0091 and MnS04-H20, 1.0.
2
Mixture provided 10,000 IU Vitamin A as retinyl palmitate; 4,000 IU Vitamin D as
Cholecalciferol; 75 IU Vitamin E as dl-a-tocopheryl acetate; and the following amounts
(milligrams) of other vitamins per kilogram of diet: menadione dimethylpyriminidol
bisulfate, 10.0; thiamine HCL, 4.0; riboflavin, 30.0; calcium pantothenate, 150.0;
niacinamide, 300.0; pyridoxine-HCL, 20.0; d-biotin, 6.0; folacin, 15.0; Vitamin
0.002; L-ascorbic acid, 1000; inositol, 500.0; butylated hydroxytoluene (100%), 100.0;
and choline chloride (70%), 1330.0.
methods of Spackman et al (31) and Moore and Stein (27) with modifications
by Niederwieser and Pataki (29), Blackburn (10) and Hirs (21). Amino acid
analyses were performed on a Technicon Auto Analyzer (NC-2P) with a 25 cm
column. An electronic integrator (Columbia Scientific Supergrator 2) was used
to compute the absolute amounts of each amino acid.
Protein content was assayed by microkjeldahl according to Hiller et al (20),
and the moisture content was determined using procedures described by
Chibnall etal (13).
218
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Table 15-3. Composition of the Artificial Diets,
CMP-1 and CMP-2
Composition
Percent Composition
CMP-1
CMP-2
Casein
40.00
40.00
Gelatin
10.00
10.00
Cod liver oil
10.00
10.00
Sucrose
10.00
10.00
Dextrin, white Technical
Cellulose^
10.00
10.00
4.26
7.16
Choline chloride (70%)
0.30
0.30
L-glutamic-HCI
1.20
L-ascorbic acid
0.30
0.30
L-glutamic acid
6.30
NaCI
0.50
0.50
Mineral mixture
3
Vitamin mixture
6.40
6.40
0.50
0.50
DL-Methionine
0.20
0.20
L-tryptophan
0.04
0.04
Amino Acid mixture^
-
4.60
^ Solka floe. Brown Company, Berlin, N.H.
2 Minerals in g/kg diet: CaHP04H20, 18.04; CaC03, 19.04; KH2P04, 14.03; NaHC03,
8.82; MnS04 H20, 0.35; FeS04H20. 0.50; MgS04,3.02; KI03, 0.01; CuS04-H20,
0.03; ZnC03, 0.15; CsC12-6H20, 0.002; NaMo04-H20, 0.008; and NajSeOg, 0.002.
3
The vitamin mixture included 10,000 IU Vitamin A as retinyl palmitate; 4,000 IU
Vitamin D as Cholecalciferol; 75 IU Vitamin E as dl-a-tocopheryl acetate; and the
following amounts of vitamins in mg/kg diet: thiamin'HCL, 40.0; menadione
dimethylpyrimidinol bisulfite (Vitamin K), 2.0; riboflavin, 30.0; D-calcium
pantothenate, 150.0; niacin, 300.0; pyridoxine'HCL, 20.0; d-biotin, 0.5; folic acid,
15.0; Vitamin B^2> 0.3; Ethoxyquin (100%), 200.0; and myo-inositol, 500.0.
4
Amino acids in g/kg diet: L-threonine, 7.0; L-valine, 5.0; L-cystine, 3.0; L-lsoleucine,
10.0; L-leucine, 8.0; lysine'HCL, 2.0; and L-arginine'HCL, 11.0.
Lipid Extraction and Analysis
Samples were collected, weighed, measured, lyophilized, and stored at
-20°C under nitrogen. Several small fish or approximately one gram of each
diet for each sample were rehydrated with 5 ml distilled water. Samples were
extracted twice in a Sorvall Omni-mixer (60 ml capacity), according to the
Bligh and Dyer (11) technique as modifed by Kates (23). Lipids were
219
-------
determined gravimetrically. The lipid material was saponified with 10 ml 0.5 N
potassium hydroxide-methanol.
Fatty acids were methylated with 14 percent boron trifluoride-methanol
(28). Fatty acid methyl esters (FAME) were injected into a single column
Varian Aerograph 1200 gas-liquid chromatography unit operated isothermally
at 180°C and equipped with a flame ionization detector. FAME were separated
on a 15 percent diethylene glycol succinate (DEGS) column, on 100-120 mesh
Chromosorb W-HP, 2.1 m long x 3.2 mm O.D., supplied with 75 ml/min flow
of nitrogen as the carrier gas and a three percent ethylene glycol succinate
polyester-Z (EGSP-Z) column (same dimensions as DEGS) on 100-120 mesh
Gas Chromosorb Q support with 40 ml/min nitrogen. Identification and
quantification of the FAME were made with an electronic integrator (Hewlett
Packard 3380A) supplied with the relative retention times of authentic
standards and literature values for published oils (2). Cod liver oil was used as a
secondary standard (3) and heptadecanoic acid (17:0) was used as an internal
standard (16). Unresolved chromatogram peaks were detected by comparing
the profiles of the two individual column separations.
RESULTS AND DISCUSSION
Artemia Diet—Batch Culture
The total protein levels and amino acid profiles are given in Table 15-4 for
the wild silversides, their eggs, and laboratory cultured fish of various ages. The
cultured fish had been fed the live 3-day-old brine shrimp diet. The amino acid
spectrum of the silversides was very similar to the spectrum of migrating
Atlantic salmon (15).
The brine shrimp analysis was similar to the results of Gallagher and Brown
(18) who also analyzed San Francisco Bay brine shrimp. These authors stated
that methionine in the brine shrimp may be limiting compared to standard egg
albumin levels. However, our results showed that the methionine levels in brine
shrimp were very similar to the level found in the silverside eggs. The major
differences between the 3-day-old brine shrimp and the silverside eggs were the
lower levels of threonine, serine, proline, valine, and leucine, and the higher
levels of arginine in the brine shrimp.
The silverside eggs contained higher levels of threonine, serine, proline,
alanine, leucine, and tyrosine, and lower levels of glycine and methionine than
were found in the wild fish. The amino acid profile of the 5-day-old silversides
changed substantially from the profile of the eggs. Most of the changes resulted
in a general decrease in amino acids from the egg to the larval stage.
220
-------
Table 15-4. Amino Acid Profiles of Silversides,
Expressed as Gram Amino Acid Per 100 Gram Protein
Amino Acid
Wild
Silversides
Eggs
Unfert.
5-day
Cultured Silversides
10-day 25-day
58- day
Brine
Shrimp
Diet
Aspartic Acid
9.3
8.7
10.2
9.9
9.9
10,2
8.9
Threonine
4.3
6.4
4.6
5.1
4.7
4.4
3.1
Serine
4.3
8.2
4.9
4.9
5.2
4.6
4.4
Glutamic Acid
14.1
13.7
15.2
14.4
15.2
13.8
11.6
Proline
3.9
7.5
4.2
4.3
4.5
4.8
3.1
Glycine
6.0
3.7
5.8
6.0
6.4
6.6
4.3
Alanine
6.0
7.7
4.7
4.5
4.9
6.2
6.0
Valine
5.3
6.8
5.5
5.6
5.8
5.6
5.1
Methionine
3.6
Z1
2.5
2.8
2.2
3.3
2.3
Isoleucine
4.6
5.9
4.8
5.1
4.4
4.0
4.9
Leucine
7.7
10.6
7.7
7.8
7.2
7.5
8.9
Tyrosine
3.5
5J
3.9
4.0
3.9
3.2
4.2
Phenylalanine
4.2
4.7
4.7
4.6
4.6
4.3
4.5
Htstidine
3.6
2.9
2.9
2.9
2.7
5.1
1.2
Lysine
&9
&3
8.3
8.7
8.3
8.9
9.2
Arginine
6.3
7.1
7.3
7.1
7.3
6.5
10.5
% Protein*
15
NA2
13
12
NA
14
NA
% Moisture
NA
84
79
81
81
79
NA
^ wet weight basis
2
NA " not available
Tryptophan was not determined by the procedure used in this study.
Table 15-5. Fatty Acid Composition of Unfertilized Eggs
and 2- and 15-Day-Old Silversides Fed 3-Day-Old Brine Shrimp Nauplii
Wild
2-Day-Old
15-Day-Old
Unfertilized
Silversides
Sac fry (6.0 mm)
Silversides
Fatty Acids
Eggs
(20.5 mm)
Silversides
{11.0 mm)
14:0
2.501
1.34
1.14
0.69
14:1
0.27
0.14
0.06
0.30
15:0
0.77
0.55
0.36
0.33
15:1
0.15
0.14
0.02
0.19
16:0
18.67
22.55
22.67
16.29
16:1
7.40
5.83
4.48
10.06
17:1
1.28
0.14
0.13
1.49
18:0
5.99
9.44
9.74
7.34
18:1u>9
14.19
10.40
12.83
25.33
18:2oj6
1.58
1.23
0.68
2.24
18:3oj6
0.55
0.38
0.32
0.52
18:3co3
1.47
0.88
0.44
2.49
18:4to3
0.88
0.75
0.47
0.34
20:1w9
1.36
0.71
0.20
0.49
20:4cj6-
2.71
3.75
2.52
5.13
20:5gj3
8.09
7.37
4.90
7.84
22: 5oj3
3.72
J.34
2.33
3.26
22:6cj3
27.15
35.53
36.05
14.96
% oil2
13.9
8.5
NA
NA
oj3/w6 Ratio
8.5
8.6
12.6
3.7
' weight percent
o
based on dry weight
221
-------
From the fifth to the 58th day of culture, the amino acid profiles did not
change markedly. The only changes which occurred were a decrease in glutamic
acid and an increase in alanine and histidine. Compared to the 3-day-old brine
shrimp diet, the 58-day-old fish differed only in the levels of histidine and
arginine. Therefore, it seems that the dietary amino acids were absorbed and
deposited as early as the fifth day of life.
Table 15-5 shows the fatty acids of unfertilized silverside eggs and 2 and
15-day-old fry fed on 3-day-old brine shrimp nauplii. The unfertilized eggs had
a whole body lipid level of 13.9 percent and the fatty acids 20:5co3 and
22: 6oj3 comprised more than 35 percent of the total fatty acids. The co3 acids
exceeded the u>6 component by greater than eight times. It would appear that
the high energy level coupled with the large co3 polyunsaturated fatty acid
(PUFA) component are indicative of their metabolic and physiological
importance in the early life stages of silversides.
In the 2-day-old yolk sac fry the acids 16:0, 18:0 and 22:6w3 were
preferentially retained from the energy rich egg, while 16:1, 18:1 co9, 20:5to3
and those acids which comprise individual contributions of less than 4 percent
each showed reduced levels. The gj3/w6 ratio of the 2-day-old yolk sack fry
increased to 12.6, from the egg level of 8.5. A similar pattern of fatty acid
retention and utilization was found by Hayes (19) and his associates in the
total lipids of developing steelhead trout, Salmo gairdneri.
The brine shrimp diet was composed largely of 16:0, 16:1, 18:lw9, and
20:5co3 but contained no 22:6co3 (Table 15-6). The analyses of silversides fed
this diet (Tables 15-5 and 15-6) showed that the fatty acids 16:1 and 18:lo;9
increased from the 2-day-old yolk sac fry levels, while 16:0 and 22:6co3
decreased. It is evident that the fish change their concentration of fatty acids
to reflect the general composition of their diets. Other researchers have made
the same correlation between the diet and tissue fatty acids of cultured fish (1,
9, 14, 24, 25).
In silversides cultured for 137 days (30) the level of 20:5w3 and 22:6w3
represented as little as three percent of the total fatty acid composition. The
level of these two fatty acids in the wild fish represent an amount about ten
times this level. Additionally, the wild fish had an oil content of only about
eight percent, whereas 137-day-old cultured fish had a lipid level of 21.4
percent (30). Thus, brine shrimp fed fish did not closely resemble the lipid
content of their natural counterparts. Since the oj3 acids have been shown to
play a chief role in the metabolism of fish, it would seem that the amount of
lipid storage may be related to a certain minimal amount of cj3 PUFA, namely
22:6co3. A mechanism may exist which enhances the absorption and
deposition of lipids to ensure a minimal 22:6co3 tissue level. Therefore, the
222
-------
Table 15-6. The Major Fatty Acids of the 3-Day-Old
Brine Shrimp Diet and 25 and 58-Day-Old Silversides
3-Day-Old
25-Day-Old
58-Day-Old
Brine
Juvenile
Juvenile
Shrimp
Silversides
Silversides
FAME
(Diet #2)
(13.05 mm long)
(22.24 mm long)
16:0
11.45
16.77
21.05
16:1
16.49
8.35
12.76
18:0
4.10
8.72
9.19
18:1cj9
34.34
27.44
36.87
18:2cj6
4.78
2.36
3.19
1 8:3cj3
4.67
2.85
2.26
20:1to9
0.55
0.56
0.70
20:4co6
3.13
5.99
3.79
20:5co3
13.31
7.89
3.87
22:5cj3
—
2.23
1.73
22:6co3
-
13.67
1.97
% oil
10.00
12.90
12.40
co3/co6 ratio
2.27
3.19
1.41
3-day-old brine shrimp diet may lead to critical nutritional problems if used in
a long term study.
When compared to the wild fish (Table 15-5), the cultured fish have a far
lower oj3 fatty acid level and much higher level of the co6 acids. The co3/w6
ratio of the wild fish lipid was 8.0, more than two times the cultured fish
levels. The wild fish fatty acid profile was similar to the egg and 2-day-old yolk
sac fry values, as would be expected. In the wild fish the fatty acids 20:5co3
and 22:6o>3 represented about 40 percent of the total fatty acid composition.
Preferably, the cultured fish should resemble the wild juvenile fish in our
experiments.
Artificial Diets
Since the amino acid profiles of the Atlantic salmon and wild silversides
were comparable, a commercial salmon diet was tried in the preliminary
evaluation of the artificial diets. Compared to the brine shrimp fed fish, growth
and survival in the test diet fed group was very poor. Fish on the salmon type
diet exhibited some scoliosis. Two factors which could have contributed to this
223
-------
problem were the leaching of dietary components from the artificial diet when
it became water soaked, or perhaps an inadequate lipid composition (soybean
oil). In reference to the latter point, the fatty acid composition of the artificial
diet is shown in Table 15-7 along with the spectrum for soybean oil and that of
silversides cultured on the salmon type diet. The soybean oil diet and the oil
resemble each other to some degree, since 16:0, 18:1cj9, and 18:2o>6are the
major fatty acids of both analyses. Likewise, silversides fed this artificial diet
closely resemble the lipid make-up of the diet they were fed. However, it is
evident that fish fed the artificial diet were not similar in fatty acid
composition to the wild fish, and thus this diet had not accomplished the
major goal of providing a laboratory cultured fish of similar biochemical
composition to the wild fish (Table 15-5).
Based on the biochemical analyses of the diets and cultured fish, and the
results of poor growth and survival, the salmon diet was modified by the
addition of a marine oil (cod liver oil). The soybean oil based diet was again fed
to verify past results. In addition to these two diets, the live brine shrimp diet
was used along with: a freeze-dried form of brine shrimp, a wild plankton diet,
Tetra Marin—a commercial aquarium food, and two semi-purified diets with
various amino acid compositions (Table 15-3). The cultured fish were again
analyzed for protein and lipid composition.
The growth and survival results of fish fed these experimental diets are
presented in Table 15-8. The live brine shrimp diet gave the best survival (97
Table 15-7. The Major Fatty Acids of Soybean Oil,
an Artificial Diet (#7) with a Soybean Oil Base,
and Silversides Cultured on the Diet
Artificial Diet
Soybean
Silversides Fed
With Soybean Oil
Oil
the Artificial Diet
FAME
(13.97 mm long)
16:0
13.05
12.52
17.43
16:1
2.43
-
3.74
18:0
2.45
4.69
7.41
18:1w9
36.12
19.25
36.37
18:2cj6
29.81
54.90
21.22
18:3co3
1.88
7.83
0.51
20:1 co9
2.94
-
2.95
20: 5oj3
1.62
22:6gj3
2.08
4.76
a)3/w6
0.19
0.14
0.25
224
-------
Table 15-8. Percent Survival and Weight Gain for Silversides
Cultured on Various Experimental Diets
(Modified from Beck and Poston (7))^'^
Experimental Diet
Survival
%
Weight Gain
mg %
1. Wild plankton
54.0
4.3
48.3
2. Brine shrimp-live
97.0
36.6
411.2
3. Starved
0.0
-
-
4. Brine shrimp-dried
66.0
4.5
50.6
5. Tetra Marin
95.0
4.4
49.4
6. Artificial, HPM-1
51.0
0.6
6.7
7. Artificial, CHP-1
28.0
1.2
13.5
a Artificial, MP-1
65.0
-0.7
-7.9
9. Artificial, MP-2
61.0
-0.5
-5.6
Diet #1 data only one replicate, diets 2, 4-9 are averages of two replicates.
2
Fish were 23 days old at onset of the study and were cultured for 23 days.
percent) and the best weight gain (411 percent). Normal growth of the wild
fish has been estimated at 12 mm per month during the growth period (6).
None of the other diets gave an appreciable weight gain; in fact some groups
actually lost weight. With the exception of Tetra Marin, all the artificial diets
produced a relatively poor survival rate.
Table 15-9 shows the fatty acid composition of the natural and artificial
diets. The effect of the dietary fatty acids on the cultured fish lipids is
presented in Table 15-10. The diet profiles and the respective cultured fish
profiles were quite similar. The cultured fish fed the cod liver oil based diets
(Diets 6, 8, 9) and those fed Diet 5 contained a much higher level of 22:6co3
than the brine shrimp fed fish (Table 15-6 and 15-10) or the soybean oil fed
fish (Table 15-7 and 15-10). These fish more closely resembled their wild fish
counterparts. A lipid modification of the salmon diet had therefore effected a
biochemical change in the fish to a more "wild like" laboratory fish. The
replacement of the soybean oil in Diet 7 by cod liver oil (Diet 6) doubled the
survival level, however growth was only one-half as great. It is very difficult to
draw a direct correlation between the dietary lipid composition of the
various diets and survival, since the experimental design of this study was quite
unlike the classical nutrition experiments.
The comparison of the amino acid profiles of fish fed the natural and
artificial diets indicated little variation between the treatment groups (Table
225
-------
Table 15-9. Percentage Composition of Fatty Acids from Total
Lipids of the Various Experimental Diets
Fatty Acid
#1
#2
Experimental diets
m #5
#6
#7
#8
#9
14:0
7.42
1.16
1.33
1,39
4.50
1.90
4,39
4,72
14:1
1.64
1.10
1.14
0,14
0.40
0.16
0.51
0.46
15:0
0.87
0.53
0.57
0.18
0.31
0.15
0,33
0.30
15:1
0.15
0.38
0.36
0.06
0.09
0.02
0.13
0.12
16:0
26.30
11.45
12.27
16.83
13.17
13.00
10.84
11.59
16:1
10.50
16.49
17.05
4.74
9.03
2.45
11.21
11.48
16:4
0.45
-
-
0.06
_
0.08
0.40
0.19
17:1
0.24
2.72
2.55
-
0.18
0.06
0.27
0.20
18:0
5.24
4.10
2.16
3.64
0.66
2.45
2.01
2.02
18: 1cj9
9.48
34.46
36.43
22.40
24.36
35.56
25.05
26.73
18:2cj6
1.93
4.78
2.98
31.38
7.64
29.52
3.66
2.83
18:3u>6
1.13
0.75
0.81
-
-
-
-
_
18:3oj3
270
4.67
4.49
5.31
1.05
1.90
1.04
0.81
18:4w3
3.46
0.73
0.75
0.25
1.99
0.70
3.89
2.12
20:0
0.13
-
0.03
0.18
-
-
-
_
20:1«9
0.36
0.55
0.62
3.43
9.40
3.49
9.77
10.28
20:2«6
0.40
-
0.04
0.08
0.19
-
0.27
0,29
20:4w6
1.22
3.13
2.72
0.36
0.33
0,11
0.26
0.30
20:5w3
12,58
13.31
12.20
3.46
8.36
1.48
9.94
9.45
20:0
-
-
-
-
0.36
-
0.39
0.43
22:1
-
-
_
1.99
6.95
4.41
5.03
5.01
22:5o>6
-
-
-
-
0.04
-
—
-
22:5cj3
0.03
-
-
0.21
0.62
0.06
0.81
0.81
22:6^3
13.44
-
-
3.72
9.69
1.99
10.60
10.09
co3/gj6 ratio
6.88
2.16
2,66
0.41
2,65
0,21
6.27
6.81
-------
Table 15-10. Percent Composition of the Fatty Acids from Total Lipids of
Silversides Cultured for 23 Days on the Various Experimental Diets.
23-Day
Experimental Diets
Wild
Fatty Acid
Old
1
2
4
5
6
7
8
9
Fish
14:0
0.53
1.26
1.39
0.90
1.08
2.27
1.46
2.53
3.51
1.34
14:1
0.29
-
0.32
0.44
-
-
0.06
0.17
-
0.14
15:0
0.32
0.56
0.51
0.66
0.52
0.45
0.35
0.45
0.83
0.88
15:1
0.09
0.18
0.14
0.31
0.33
0.15
0.18
0.27
0.48
0.14
16:0
16.77
21.43
15.66
16.91
19.28
17.94
17.95
19.56
20.68
22.55
16:1
a35
4.13
13.09
8.51
3.14
5.23
2.92
6.36
4.64
5.83
17:1
0.90
-
0.63
1.33
1.88
0.98
0.11
0.99
-
0.14
18:0
8.72
13.31
6.32
6.09
13.58
7.40
7.99
9.16
9.53
9.44
18:1 oj9
27.44
12.52
33.83
27.22
15.41
24.27
34.20
24.72
24.60
10.40
18:2oj6
2.36
1.30
3.45
2.85
13.54
3.06
18.39
2.78
1.82
1.23
18:3^6
0.42
1.95
1.14
1.45
1.71
1.31
2.45
0.58
0.58
0.38
18:3a>3
2.85
0.56
3.05
1.92
1.48
0.48
0.52
0.18
0.80
0.88
18:4co3
0.26
0.30
0.44
0.76
0.46
0.42
0.21
0.36
0.36
0.75
20:0
0.20
0.38
0.03
0.21
1.34
-
-
0.18
0.15
-
20:1to9
0.33
1.14
0.79
1.35
1.74
7.81
2.78
6.10
5.58
0.71
20:2w6
-
0.46
-
0.06
0.29
0.68
0.08
0.05
0.15
0.09
20:4w6
5.99
3.32
3.93
5.37
3.49
2.66
1.48
2.97
2.54
3.75
20:5u>3
7.89
5.14
4.60
6.89
1.76
3.00
0.44
2.42
2.22
7.37
22:1
-
0.14
-
0.37
0.51
3.91
1.27
2.28
2.44
0.20
22:4cj6
-
0.27
-
-
0.29
-
-
-
0.03
0.24
22:5cj6
-
-
0.03
-
-
-
-
-
-
—
22:5oj3
2.23
2.60
4.70
2.63
1.30
0.55
-
0.65
1.47
1.35
22:6cj3
13.67
29.03
5.83
13.57
16.62
17.47
6.95
17.24
17.99
35.53
% oil 12.88 11.27 19.90 10.79 11.72 12.82 NA 15.45 12.44 NA
co3/w6 ratio 3.07 5.15 2.18 2.65 1.12 2.84 0.36 3.27 4.46 8.06
-------
15-11). Although histidine and methionine were present in greater amounts in
Diets 6 and 9 than in the other treatment groups, no relationship is obvious
between these amino acid levels and growth and survival. The higher amounts
of leucine in the brine shrimp fed fish could indicate a role of this amino acid
in their greater growth and survival. However, this was not evident in the
freeze-dried brine shrimp fed group.
No substantial differences were found in growth and survival of fish fed the
nonsupplemented amino acid diet (Diet 8) versus the supplemented diet (Diet
9). Therefore, it is difficult to say the quantity of dietary amino acids influence
the metabolism and utilization of the various diets. Bioavailability studies will
be necessary to ascertain the degree of incorporation of the supplemented
amino acids.
CONCLUSIONS
1. The best growth and survival of juvenile silversides was obtained on a live
3-day-old brine shrimp nauplii diet. Substantially lower growth and
survival was obtained on the artificial diets.
2. Freeze-dried brine shrimp provided less growth than a live brine shrimp
diet. Live brine shrimp must contain some component which is removed
or altered upon freeze drying.
3. It is difficult to say whether protein (amino acids) in the diets was a
factor in the differences in growth and survival of cultured fish fed the
artificial diets and the brine shrimp fed fish. The brine shrimp fed fish
did, however, contain higher levels of leucine than all other cultured
groups. Bioavailability studies will be necessary to ascertain the degree of
assimilation and incorporation of dietary amino acids.
4. Whole body lipid fatty acid composition of cultured fish changed to
reflect the composition of their diets. Fish fed the brine shrimp diet had
higher fat levels and lower polyunsaturated fatty acid levels than wild
fish. Cultured fish appear to be storing large amounts of lipids in order to
obtain a threshold level of the polyunsaturated fatty acids.
5. Fish fed a cod liver oil based diet more closely resembled their wild
counterparts. However, growth and survival were poor compared to
3-day-old brine shrimp fed fish.
ACKNOWLEDGEMENTS
The experimental part of the project required a close working arrangement
between scientists of the marine fish culture team at the Environmental
228
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Table 15-11. Amino Acid Profiles of Silversides Fed the Various Artificial
and Natural Diets Expressed as Gram Amino Acid Per 100 Gram Protein
Experimental Diets
Amino Acid
1
2
4
5
6
8
9
Aspartic Acid
10.1
9.9
10.5
10.2
10.2
9.7
10.0
Threonine
4.5
4.0
4.9
*4.4
4.4
5.0
4.3
Serine
4.5
4.2
5.0
4.7
4.7
5.0
5.4
Glutamic Acid
15.4
16.3
14.8
15.4
14.3
15.2
14.1
Proline
3.9
3.7
4,0
4.6
4.6
4.5
4.7
Glycine
6.7
6.8
7.0
6.6
7.2
6.0
6.4
Alanine
6.4
6.7
6.4
7.4
5.2
5.6
6.2
Valine
5.2
4.5
5.0
5.1
5.4
5.6
5.7
Methionine
1.5
2.4
1.5
2.5
1.9
2.3
3.2
Isoleucine
5.1
4.6
4.8
4.8
4.5
4.2
4.5
Leucine
7.9
11.4
7.8
7.4
7.7
8.1
7.8
Tyrosine
23
3.2
2.4
3.3
3.7
3.1
3.5
Phenylalanine
4.5
4.2
4.6
4.4
4.8
4.8
4.9
Histidine
3.3
2.7
3.5
3.4
4.7
3.8
3.3
Lysine
8.5
9.2
9.1
9.0
8.6
7.9
8.1
Arginine
7.3
6.2
7,4
7.3
6.9
7.0
7.1
Ammonia
19.6
16.2
17.8
16.1
17.4
18.4
16.9
i
% Protein
19
17
17
16
13
15
14
% Moisture
76
75
NA2
77
NA
77
NA
^ Net weight basis
o
NA = not available
-------
Protection Agency, under the direction of M r. Allan Beck, and foodscientists
at the University of Rhode Island. This cooperative effort has promoted an
investigation that neither group could have accomplished easily on an
independent basis.
This work was supported by the Environmental Protection Agency under
Grant #R-803818 and the Rhode Island Agricultural Experiment Station. This
manuscript is Rhode Island Agricultural Experiment Station Contribution
Number 1766.
Thanks is given to Allan Beck, Grace MacPhee, Bruce Lancaster and
Drs. Robert Barkman and Hugh Poston for their contributions.
Additionally, we thank the personnel at the Tunison Laboratory of Fish
Nutrition for formulating and pelleting the artificial diets, and providing
advice and guidance to the total project. Thanks is also given to Cindy
Seidel for typing of the manuscript.
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29. Niederwieser, A. and G. Pataki, 1971. New Techniques in Amino Acid,
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31. Spackman, D.H., W.W. Stein and S. Moore, 1958. Automatic Recording
Apparatus for use in the Chromatography of Amino Acids. Anal. Chem.
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Bossuyt, E. Bruggeman, G. Personne and D. Versichele, 1977. Artemia
salina as life Food in Aquaculture. In: Fundamental and Applied Research
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THE COMBINED EFFECT OF TEMPERATURE
AND DELAYED INITIAL FEEDING ON THE
SURVIVAL AND GROWTH OF
LARVAL STRIPED BASS
Morone saxati/is (WALBAUM)
Bruce A. Rogers
Graduate School of Oceanography
University of Rhode Island
Kingston, R.I. 02881
Deborah T. Westin
Graduate School of Oceanography
University of Rhode Island
Kingston, R.I. 02881
ABSTRACT
Rearing temperature and the time of first feeding interact to determine the
degree of survival and rate of growth in larval striped bass. Between 15 and
27°C, temperature affects the rate of growth and development in fed groups,
and the time to death by starvation in unfed lots. Delayed first feeding retards
structural development. The 'point-of-no-return' in striped bass is very near the
stage of complete mortality due to starvation. Unfed groups survived up to 22
days after hatching at 24°C and 32 days at 15°C. Larvae fed late into
starvation survived and continued to grow at a rate somewhat higher than that
observed in earlier fed groups at all temperatures. Larvae which has survived
delayed development were indistinguishable on the basis of external
morphology from much younger individuals reared under more favorable
conditions. The effects of nutritional and thermally induced developmental
retardation are discussed in terms of how they may affect larval growth and
mortality rate estimates used in assessing the effects of estuarine power plants.
INTRODUCTION
Many estuarine and marine fish species, including the striped bass, Morone
saxatilis (Walbaum), produce large numbers of relatively small pelagic eggs at
spawning. These smaller eggs contain fewer yolk reserves. After a relatively
short incubation period, they hatch into prolarvae that are, in general, at a
more rudimentary stage of structural development than those of species
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producing fewer but larger eggs (3, 17). Development continues while the
young larvae drift in the water column, absorbing their yolk and developing
the mouth parts and swimming ability to capture food and avoid predators.
Although the methods used to determine the extent of first year mortality in
natural populations are at best imprecise (25), it is clear that among high
fecundity species, losses early in life are extremely high, with the highest
mortality rates among the early larval stages.
As early as the end of the last century, Fabre-Domergue and Bietrix (9)
encountered heavy mortality among laboratory reared marine fish larvae which
had exhausted their yolk reserves. Hjort (12) concluded, based on his studies of
year to year fluctuations in Norwegian cod and herring abundance, that
year-class strength was probably determined early in the larval development of
these species. The term "critical phase" or "critical period" has been used, in a
general sense, to refer to that span of time in the early development of the
individuals comprising a particular year-class during which the ultimate number
of recruits is determined (11). In a narrower usage "critical period" may be
used to refer to that point in development of the larval fish at which all sources
of endogenous (yolk) nutrition have been consumed, and active feeding must
commence if death by starvation is to be avoided. Hjort (12) proposed death
following yolk exhaustion as only one of several possible mechanisms by which
events early in development might affect the subsequent size of a given
year-class. In 1956, Marr (23) reviewed the available evidence in support of the
existence of a "critical period". He concluded that there was little evidence to
suggest that mass starvation occurred in the sea among larvae that had recently
absorbed their yolk, or that survival curves for natural populations revealed any
noticeable inflection at the point of yolk absorption. 18 years later, May (25)
noted that little new data has been gathered since Marr's review that could
contribute meaningfully toward the resolution of the problem of whether or
not a "critical period" at yolk absorption exists as a widespread phenomenon
among fish species. He suggested that while among high fecundity species,
year-class strength is certainly determined during early development as Hjort
maintained, the physiological mechanisms that have evolved to meet
environmental challenges that confront the developing larva must be addressed
on a species by species basis.
The prolarva, from the time it is hatched until it captures its first meal, is
reliant on its yolk reserves to provide the structural materials for continued
ontogenetic development, as well as to provide energy to fuel its maintenance,
activity, and growth needs. Unless sufficient satisfactory food is taken after the
exhaustion of yolk reserves, structural tissue already laid down is metabolized
to support the continued costs of swimming in search of prey, until the larvae
is so debilitated by the effects of starvation that it is unable to capture and
utilize suitable prey when it does become available. Blaxter and Hempel- (4)
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termed th'is ecological death-point the "point-of-no-return" (PNR). Starved
larvae may live after the PNR has been reached but with no likelihood of
ultimate survival. The time span between the development of the ability to
feed and PNR determines how important the period of transition to exogenous
feeding will be to the survival of larvae of a particular species.
The rate of growth and development of larval fish is very much temperature
dependent. Among the studies in which the relationship between rearing
temperature and larval growth rate have been demonstrated are those of
Kramer and Zweifel (16), Houde (14), and Shelbourne et al (33). In all these
studies, larval growth rate increased with increasing temperature, except where
survival limits were approached.
Weight specific metabolic rate (oxygen consumption) also increased with
increasing temperature among fish larvae of the same weight in studies such as
those of Holiday et al (13) and Laurence (20).
The striped bass, Morone saxatilis (Walbaum), is a commercially important
anadromous teleost native to the Atlantic coast of North America. The natural
range of the striped bass extends along the Atlantic coast of North America
from the St. Lawrence River to Louisiana, with its center of abundance
between Cape Cod and Cape Hatteras (26). There have been many introduced
populations ranging from the extremely successful Pacific coast estuarine
population, introduced in the 1880's, to the many landlocked populations
which have been established in natural and man-made freshwater
impoundments in the southeastern states.
Sexually mature striped bass enter and ascend rivers to the spawning
grounds within the period between March and July. Peak spawning generally
occurs at a water temperature on the spawning grounds of 15 to 18°C.
Spawning sites are typically well into the freshwater portion of the estuary,
although often within the range of tidal influence (35). Each ripe female may
produce from one to three million eggs. Newly shed eggs are 1.28 to 1.38 mm
in diameter. Upon water hardening they swell to a diameter of approximately
3.0 mm. Newly hatched larvae average 3-4 mm in length, and have a large yolk
sac and oil globule (22). For several days after hatching, young prolarvae spend
much of their time in a vertical, head-up position drifting in the current. Larvae
develop functional mouth parts and are capable of feeding within 2-10 days
after hatching at normally encountered river temperatures. Yolk is generally
completely absorbed by the time the larva reaches 6 mm in length.
Metamorphosis into essentially adult form occurs by the time the larvae are
approximately 17 mm in length, generally 2 to 3 weeks after hatching. Feeding
larvae are capable of consuming relatively large organisms as their first food.
Planktonic crustacea and their developmental stages predominate in their diets
through most of their first year (27, 10).
236
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The striped bass is a well-studied species and there is a voluminous literature
relating to its biology (30). Relatively little attention, however, has been paid
to the ecology of early life stages, the period during the life of the animal
which is most important in the determination of year-class strength. Mansueti
(22) presented descriptions of the eggs and larvae from collections from the
Roanoke and Patuxent Rivers, and provided observations on the feeding and
early growth of larvae in captivity. Doroshev (8) reviewed aspects of egg and
larval development and added anecdotal observations on metabolic rate and
graying rates of larvae. Bayless (2) provided a manual of culture methods as
practiced in South Carolina hatcheries, but did not give many details of larval
requirements beyond the yolk sac stage. Short term lethal temperature levels
for eggs and larvae were presented by Albrecht (1), Davies (7), Shannon and
Smith (32), Shannon (31), and Morgan and Rasin (28). Observations of prey
selectivity among late larvae were reported by Meshaw (27) and Gomez (10).
Daniel (6) presented data on the effect of food density on larval survival.
In many spawning rivers on the Atlantic coast, major conflicts have arisen
over the effect of power plant operations on striped bass recruitment, as a
result of the entrainment of eggs and larvae in cooling water intakes, and later
the impingement of juveniles on intake screens. Entrainment losses are highest
among striped bass under approximately 3 cm in length. The duration of the
period of major entrainment losses is a direct function of the time required for
young bass to develop from semiplanktonic eggs to early juveniles large enough
to escape intake currents. An assessment of plant impact must take into
account the duration of entrainable life stages. To date, only crude estimates
have been used in plant impact models involving striped bass (e.g., 21, 36).
The purpose of the present study was to determine in what way
temperature and an initial delay in the onset of active feeding work together to
affect the rate of survival and growth of striped bass larvae. Temperature is a
controlling factor which may be expected to have a profound effect on the
metabolic demands of the developing larva. The availability of food determines
the extent to which these demands can be met. Temperature and delayed first
feeding may be expected to interact in a manner which would largely
determine the life span and early growth trajectory of the developing larva. By
observing how water temperature and feeding level affect growth, better
predictions of stage duration, hence vulnerability to power plant entrainment,
may be made.
MATERIALS AND METHODS
Source of Study Material
Eggs from Maryland used in the 1976 experimental series were netted from
the Nanticoke River during the spawning season, using a 1 x 2 meter, 947
237
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micron mesh neuston net. Eggs were also obtained from a striped bass hatchery
run by the state of South Carolina at Moncks Corner, South Carolina. Eggs
were air-shipped to the University of Rhode Island, where all experiments
reported here were performed.
Experimental Procedures
For the duration of the relatively short incubation period, eggs were
maintained in static 208 liter polyethylene drums filled with dechlorinated tap
water. Best hatching success was observed when bacteria were controlled using
an antibiotic. The antibiotic dosage used was 50,000 I.U./liter penicillin G plus
50 mg/liter streptomycin sulfate. A strong air stream maintained the eggs in
suspension and maintained an adequate dissolved oxygen level. Dead eggs
floated to the surface and were removed as they were discovered. One-half of
the volume of the tanks was replaced daily. Water temperature was maintained
at laboratory room temperature, 14-16°C, during incubation.
The experimental containers used in growth experiments consisted of four
liter glass beakers. Prolarvae were stocked into these containers usually within
24 hours of the time they were hatched. Larvae stocked at yolk absorption
were held in their incubation containers until visible vestiges of yolk had
disappeared. At stocking, all were of the same chronological age and had been
exposed to the same conditions prior to the beginning of the experiment. No
antibiotic was used in larval growth or survival experiments. The water used in
all experiments was raised to 5°/oo salinity by mixing dechlorinated tap water
with seawater (32°/oo, which had been passed through a cartridge filter rated
to retain particles larger than 5 microns). Water in each container was changed
every two days.
Constant temperatures of 15, 18, 21, 24, and in some cases 27°C, were
maintained in the test containers by keeping them immersed in temperature
controlled water baths. Bath temperatures were controlled using Haake (model
E-52) 1000 watt heater-thermoregulators operating against a cooling coil in
each bath. Temperature excursions of no more than 0.25°C were normally
encountered. The temperatures used span the range that might be encountered
by developing larvae in nature. Bath temperatures were monitored on a
strip-chart recorder and measured manually at least twice a day during the
course of experiments.
In initial experiments, dissolved oxygen, pH, ammonia, and salinity
measurements were made regularly. Dissolved oxygen was determined using the
Y.S.I. D.O. probe, supplemented periodically with determinations using the
azide-modification of the Winkler titration. The pH was measured using an
Orion pH electrode. Ammonia was determined using a micro-modification of
238
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the indophenol technique of Solorzano (34). Salinity measurements were made
using an American Optical salinity refractometer. With frequent water changes,
most of the water quality parameters changed insignificantly during the course
of each experiment. In later experiments where the number of individual
treatments became unmanageable, regular monitoring of all variables except
temperature was discontinued, and a stringently maintained schedule of water
changes observed.
Feeding larvae were supplied with newly hatched Artemia nauplii at least
twice a day in quantities sufficient to permit a portion to remain until the next
feeding. Artemia nauplii proved to be a satisfactory diet for striped bass
through the early juvenile stage.
Larval growth was measured in terms of dry weight. Prior to weighing,
larvae were lifted on a No. 6 sable brush, dipped in distilled water to remove
any adherent salt or debris, blotted on filter paper and placed on a tared
weighing pan. Pans were cut out of aluminum foil with a paper punch and
ashed 4 hours at 500°C before use. Dry weight determinations were made after
the specimen on its tared pan had been dried to a constant weight in a heated
vacuum desiccator at 80°C over silica-gel. On larvae up to metamorphosis,
weights were determined using a Cahn "Gram" electrobalance. Weights were
read to the nearest microgram.
Design of Experiments
Temperature-delayed feeding-survival experiments were performed during
the springs of 1976 and 1977. A total of three experiments were performed,
each identical in its design. In each, five containers were placed in each of four
constant temperature water baths. Each container was stocked with 100
prolarvae at the temperature at which they had been held prior to the
beginning of the experiment. Stocked containers were assigned to particular
temperature treatments by lot. The acclimation period from holding
temperature to the experimental temperature treatment was at most one hour.
In each temperature treatment, the larvae in one container were offered a
diet of newly hatched live Artemia nauplii at the beginning of the experiment.
Food was withheld from another container throughout the observation period.
The time of first feeding for larvae in each of the remaining three containers of
starved larvae in each temperature treatment was determined on the basis of
observations of the apparent state of health of individuals in each population.
Each container was checked for mortality several times each day throughout
each experiment, and all dead larvae removed.
239
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Similar stocking and treatment procedures were used in
temperature-delayed feeding-growth experiments. Food was withheld from one
group at each temperature, and one group at each temperature was given food
at the beginning of the experimental period. Initial dry weight measurements
were made on a sample of 20 larvae at the beginning of the experiment. At the
time each group was given its first food, a sample of 10 larvae from the unfed
lot was weighed. At the end of the observation period all of the larvae in each
treatment were measured and weighed. In cases where an intermediate growth
observation was made between the time of first feeding, and before the end of
the experiment, a sample of 10 larvae was used to establish growth of the
population to this point.
RESULTS
Figure 16-1 shows the effect of delayed initial feeding on groups of larvae
maintained at four temperatures. The survival time of the unfed control
decreased with increasing temperature. The time to 50 percent mortality for
unfed groups was 19, 21, 25 and 27 days after hatching among groups
maintained at 24, 21, 18 and 15°C, respectively. Survival among early fed
groups was generally highest in each temperature treatment. Among groups in
which food was provided for the first time after up to 50 percent of the
population had died, a portion of those remaining alive survived through the
end of the observation period. A "point-of-no-return" beyond which survival
could not occur even when food was provided, was nowhere in evidence in
these experiments. In each temperature treatment the longer food was
withheld, the greater the total mortality each group suffered. In cases where
some additional mortality was observed after food was presented, there was
generally evidence that the dead larvae had captured at least some food before
expiring.
Figure 16-2 shows the result of an experiment in which initial feeding was
progressively delayed in a series of experimental groups of larvae held at five
test temperatures. Changes in dry weight were used here to measure the rate of
growth or shrinkage in fed and unfed groups at each temperature. Among
starved lots, longevity increased and the rate of weight loss decreased at lower
temperatures. Growth in dry weight increased rapidly once food was provided.
In general, the effect of delayed initial feeding was to defer the attainment of a
temperature-specific rate of growth of which larvae fed to satiation were
capable.
Larvae receiving their first food at day six after hatching at 15°C, had
scarcely recovered their initial weight at hatching by the end of the 25-day
observation period. Other groups which received their first food at day six after
240
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Figure 16-1. The Effect of Delayed Feeding on the Survival of
Striped Bass Stocked at Yolk Absorption at 24, 21,18 and 15°C.
NOTE: Initial population 100 larvae. Numbered arrows indicate the order and
time of first feeding. Group 5 was unfed.
241
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Figure 16-2. The Effect of Temperature and Delayed Feeding on
the Growth in Dry Weight of Striped Bass Larvae Stocked at
Hatching at 27, 24, 21,18 and 15°C.
NOTE: Each sample contains 10 individuals. Numbered arrows indicate time
of first feeding for each population. Symbols identify groups which received
their first food at the same time. The location of symbols denotes sample
means. Vertical bars indicate range of weights in each sample.
242
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hatching, attained a mean dry weight of 0.5 mg at 25, 19,18 and 12 days after
hatching in temperature treatments of 18, 21, 24 and 27°C, respectively.
There was a tendency for later fed groups at each temperature to grow at a
greater rate than those receiving their first food earlier in the experiment. Table
16-1 shows the instantaneous growth coefficients calculated for delayed
feeding groups at each temperature, using the same data that were presented
graphically in Figure 16-2. In each case, the growth rates of the earliest fed
groups were lower than those of groups which had been starved before
receiving their first food. This growth compensation seldom permitted later fed
groups to overtake those fed earlier^ but did serve to partially offset the growth
setback that resulted from later initial feeding.
An additional effect of delayed initial feeding was a retardation of structural
development which was observed at all temperatures. Figure 16-3 shows an
example of the degree of developmeantal retardation which may occur among
larvae of the same chronological age as a result of a delay in the timing of
initial feeding. Among developing larvae, each developmental event appeared to
coincide with the attainment of a particular larval length or dry weight. As a
result, factors like temperature and nutritional state have a marked effect on
the degree of structural development larvae of a particular age may achieve.
DISCUSSION
Among the fish species that have been investigated in the past, there appear
to be several alternative patterns of survival following the delayed initial
feeding of larvae which have consumed most or all of their yolk reserves. The
northern anchovy, Engraulis mordax (Girard) (19) and the herring, Clupea
harengus (4), both may survive food deprivation to a point beyond which
continued survival is possible but ultimate recovery is not. The grunion,
Leuresthes tenuis (Ayres) (24), on the other hand, can recover from food
deprivation nearly up to the point of death through starvation. Observations
reported here using striped bass and those of May (24) using grunion, are very
similar. For neither species are the concepts of a "point-of-no-return" or of a
"critical period" at yolk absorption appropriate. In the striped bass, as in the
grunion, protracted food deprivation results in a suspension of further
structural development, and a gradual reduction in dry weight during starvation
as the costs of continued maintenance are met at the expense of body tissues.
The experiments of May (24) was performed at one temperature. In this
study a range of temperatures was used. Temperature has been repeatedly
shown to have a controlling influence on the rate of growth of fish larvae
maintained on unlimited rations (e.g., 14, 16, 20). Similarly, temperature
affects the rate of weight loss during starvation (18). Data presented in Figure
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Table 16-1. Instantaneous Growth Coefficients for Delayed
Feeding Groups at Five Constant Temperatures.
Initial
Final
Days Since Hatching
Instantaneous
Temperature
Dry Wt.
Dry Wt.
Growth
<°C)
(mg)*
(mg)
Day of First Feeding
Day Measured
Coefficient**
27
0.211
0.863
3
19
8.803
0.157
2.542
6
19
21.419
24
0.155
0.413
6
19
7.538
0.100
0.289
11
19
13.266
21
0.170
0.593
6
21
8.330
0.140
0.376
10
21
8.981
0.120
0.180
14
21
5.793
0.102
0.111
17
21
2.114
18
0.190
0.451
6
24
4.802
0.135
0.257
14
24
6.438
0.116
0.205
17
24
8.135
15
0.198
0.231
6
25
0.811
0.145
0.174
14
25
1.657
0.125
0.195
17
25
5.558
Determined by interpolation when the actual weight was not available (See Figure 16-2).
Instantaneous growth coefficient (29) = loge wt2 — loge wt-|
t2-ti
where wt^ and wt2 are dry weight at times t.| and t2> respectively.
-------
( Buu ) i H 9 I 3 M A H Q
Figure 16-3. The Effect of Temperature arid Delayed Feeding on
the Growth in Dry Weight of Striped Bass Larvae Stocked at
Hatching at 21 °C.
NOTE: Each sample contains 10 individuals. Numbered arrows indicate time
of first feeding for each population. Symbols identify groups which received
their first food at the same time. The location of symbols denotes sample
means. Vertical bars indicate range of weights in each sample. Development
at twenty-one days after hatching is shown for larvae receiving their first food
on days six and fourteen.
245
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16-2 of this study indicate that at lower temperatures the differences in size
between early-fed and starved larvae was not great even three weeks after
hatching. At higher temperatures both the rate of weight loss in starved
populations, and the rate of weight gain in early fed populations increased
markedly, with the major effect being seen on the rate of individuals in groups
which were fed an unrestricted ration shortly after yolk absorption.
There are vast differences between the conditions that exist in the
laboratory and those in the natural habitat of the striped bass. These
differences limit, to some extent, use of laboratory observations as an aid in
interpreting conditions in the field. In these studies the only measurable
mortality was that most closely associated with the availability of food. Losses
due to predation, probably the most important sources of mortality in nature
(5), were not involved at all here. It has frequently been suggested that the
most likely victims of predation in nature might be individual larvae that have
been weakened by the effects of starvation (5,11,15).
Experimental groups in this study which received food were fed to excess.
Therefore, the difference in growth attainment between starved and fed groups
was probably at a maximum. Under conditions of restricted prey density, even
larvae fed early in development might not have enjoyed as great a growth rate.
At the same time, satisfactory food is probably never totally absent in nature
as it was among the starved groups in this study.
Artemia nauplii are a frequently used laboratory diet for the larvae of fish
species that appear to require live food. Although Artemia nauplii appear to
support a satisfactory rate of growth in laboratory populations, there is little
nutritional information available to serve as a basis for comparison between
Artemia and the variety of micro-crustacea that comprise the natural food of
striped bass larvae (27).
In nature, striped bass larvae are present on their estuarine nursery grounds
during the spring under conditions of rapidly rising water temperatures. An
average temperature rise of 1°C per week is typical in the Hudson River
estuary during the period of larval striped bass abundance (36). Constant
temperatures were used in these laboratory studies.
With these reservations in mind, some statements may still be made about
the probable early growth pattern of striped bass larvae under natural
conditions. Data presented here indicate that the size and developmental stage
of early striped bass larvae of a given chronological age are intimately related to
their thermal and nutritional history. In well studied estuaries, the probable
temperature history of a group of larvae spawned at a particular time and
location may be estimated with some accuracy. However, a basis for
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determining the nutritional history of a given group of larvae is not readily
obtainable. Even where data are available on the spacial distribution and
density of potential food organisms, the frequency with which larvae actually
encounter suitable prey can never be known with any degree of accuracy (11).
In assessing the effects of power generating plants on striped bass
populations, it is necessary to estimate the rates of natural and plant induced
mortality among the pelagic larvae. Life-stage duration estimates, coupled with
estimates of stage-specific vulnerability to plant entrainment, may be used to
determine the extent of losses that may be attributed to the operation of a
particular plant.
Larval mortality rates in nature are frequently estimated on the basis of the
relative frequency of occurrence of larvae of various presumed age-classes in
ichthyoplankton collections made throughout the period of larval abundance
in the water column. The results of this study suggest that the occurrence of
large numbers of early post yolk sac larvae in such collections may be a
reflection of a period of suspended or slowed growth among larvae which are
being subjected to heavy competition for the available food. Without some
knowledge of hatching time, temperature regime, and feeding history, there
appears to be no way that such larvae may be aged accurately on the basis of
size and/or structural development alone.
The use of fixed stage duration estimates in predictive models, especially for
that stage immediately following yolk absorption, could lead to serious errors
in the resulting estimates of stage-to-stage mortality rates.
ACKNOWLEDGEMENTS
This study was performed while under contract to the U.S. Environmental
Protection Agency (Contract #68-03-0316). The authors wish to thank Janice
Steele for typing the manuscript, and Margaret Leonard for drafting the
figures.
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7. Da vies, W.D. 1973. The Effects of Total Dissolved Solids, Temperature,
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8. Doroshev, S.I. 1970. Biological Features of the Eggs, Larvae and Young of
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10. Gomez, R. 1970. Food Habits of Young-of-the-Year Striped Bass, Roccus
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13. Holliday, F.G.T., J.H.S. Blaxter and R. Lasker. 1964. Oxygen Uptake of
the Developing Egg and Larvae of the Herring (Clupea harengus). J. Mar.
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14. Houde, E.D. 1974. Effects of Temperature and Delayed Feeding on
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15. Hunter, J.R. (ed.). 1976. Report of a Colloquium on Larval Fish Mortality
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Tech. Rept. NMFS Circ.-395:l-5.
16. Kramer, D. and J.R. Zweifel. 1970. Growth of Anchovy Larvae (Engraulis
mordax Girard) in the Laboratory as Influenced by Temperature. Calif.
Mar. Res. Comm., Cal. COFI Rept. 14:84-87.
17. Lagler, K.F., J.E. Bardach and R.R. Miller. 1962. Ichthyology. John Wiley
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18. Lasker, R. 1964. An Experimental Study of the Effect of Temperature on
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Embryos and Larvae. Copeia 1964(2):399-405.
19. Lasker, R., H.M. Feder, G.H. Theilacker and R.C. May. 1970. Feeding,
Growth, and Survival of Engraulis mordax Larvae Reared in the
Laboratory. Mar. Biol. 5:345-353.
20. Laurence, G.C. 1975. Laboratory Growth and Metabolism of the Winter
Flounder Pseudopleuronectes americanus from Hatching Through
Metamorphosis at Three Temperatures. Mar. Biol. 32:223-229.
21. Lawler, J.P., R.A. Norris, G. Goldwyn, K.A. Abood and T.L. Engler. 1974.
Hudson River Striped Bass Life Cycle Model. In: Entrainment and Intake
Screening. Proc. 2nd Workshop on Entrainment and Intake Screening (L.D.
Jensen, ed.). The Johns Hopkins University, Baltimore, Maryland, pp.
83-94.
22. Mansueti, R. 1958. Eggs, Larvae and Young of the Striped Bass, Roccus
saxatilis. Ches. Biol. Lab. Contr. No. 112:1-35.
23. Marr, J.C. 1956. The "Critical Period" in the Early Life History of Marine
Fishes. J. Conseil Inter. Explor. Mer 21:160-170.
24. May, R.C. 1971. Effects of Delayed Initial Feeding on Larvae of the
Grunion, Leuresthes tenuis (Ayres). Fish Bull. 69:411-425.
25. May, R.C. 1974. Larval Mortality in Marine Fishes and the Critical Period
Concept. In: The Early Life History of Fish (J.H.S. Blaxter, ed.).
Springer-Verlag, New York, pp. 3-19.
26. Merriman, D. 1941. Studies on the Striped Bass (.Roccus saxatilis) of the
Atlantic Coast. U.S. Fish & Wildl. Serv., Fish. Bull. 50(35):l-77.
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27. Meshaw, J.C., Jr. 1969. A Study of Feeding Selectivity of Striped Bass Fry
and Fingerlings in Relation to Zooplankton Availability. M.S. Thesis, North
Carolina State Univ., Raleigh, 58 pp.
28. Morgan, R.P. II and V.J. Rasin, Jr. 1973. Effects of Salinity and
Temperature on the Development of Eggs and Larvae of Striped Bass and
White Perch. Appendix X to Hydrographic and Ecological Effects of
Enlargement of the Chesapeake and Delaware Canal. Contract No.
DACW-61-71-C-0062, Army Corps of Engineers, Philadelphia District. NRI
Ref. No. 73-109, 37 pp.
29. Ricker, W.E. (ed.). 1971. Methods for Assessment of Fish Production in
Fresh Waters. IBP Handbook No. 3. Blackwell Scientific Publications,
Oxford, 384 pp.
30. Rogers, B.A. and D.T. Westin. 1975. A bibliography of the Biology of the
Striped Bass, Morone saxatilis (Walbaum). Univ. of R.I., Mar. Tech. Rept.
No. 37:1-134.
31. Shannon, E.H. 1970. Effect of Temperature Changes upon Developing
Striped Bass Eggs and Fry. Proc. of the 23rd Ann. Conf. Southeastern
Assoc. Game and Fish Comm., pp. 265-274.
32. Shannon, E.H. and W.B. Smith. 1967. Preliminary Observations of the
Effect of Temperature on Striped Bass Eggs and Sac Fry. Proc. 21st Ann.
Conf. Southeastern Assoc. Game and Fish Comm., pp. 257-260.
33. Shelbourne, J.E., J.R. Brett and S. Shirahata. 1973. Effect of Temperature
and Feeding Regime on the Specific Growth Rate of Sockeye Salmon Fry
(Oncorhynchus nerka), with a consideration of size effect. J. Fish. Res. Bd.
Canada 30:1191-1194.
34. Solorzano, L. 1969. Determination of Ammonia in Natural Waters by the
Phenolhypochlorite Method. Limn, and Oceanogr. 14:799-801.
35. Talbot, G.B. 1966. Estuarine Requirements and Limiting Factors for
Striped Bass. In: A Symposium on Estuarine Fisheries, Amer. Fish. Soc.,
Spec. Publ. 3:3749.
36. United States Nuclear Regulatory Commission. 1975. Final Environmental
Statement Related to Operation of Indian Point Nuclear Generating Plant,
Unit No. 3. In the Matter of Consolidated Edison Co. of New York, Inc.
Docket No. 50-286. Volume I and II.
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THE EVOLUTION OF THE BUGSYSTEM:
RECENT PROGRESS IN THE ANALYSIS OF
BIO-BEHAVIORAL DATA
Robert S. Wilson
Department of Biology
Yale University
New Haven, Conn. 06520
John O. B. Greaves
Electrical Engineering Department
Southeastern Massachusetts University
North Dartmouth, Mass. 02747
ABSTRACT
Experimental investigation of the movements of organisms often entails the
acquisition and processing of large samples of spatio-temporal data. An
interactive, interpretive, on-line computer-television system (viz., the
Bugsystem) was developed in order to expedite such analyses. Aspects of the
structure of this prototype system are outlined. Its effectiveness is evaluated
with regard to the problems confronting the bio-behavioral researcher.
A second generation system has been developed under a research grant from
the Environmental Protection Agency. Utilizing new hardware and software, it
in many ways constitutes a generalization of its prototype. We describe
features of the refined system which provide for the following: a large degree
of machine-independence, significant expansion of the size of data records,
inclusion of experimental parameters and variables within the data structure,
investigation of rotational and flectional movement, statistical analysis, and
tracking of organisms in three dimensional space. Current utilization of the
Bugsystem for research in behavioral physiology and the potential for future
development are discussed.
INTRODUCTION
The fundamental focus of behavioral research is the description and
explication of what individual organisms do. Because those biological activities
most often classified as "behavior" consist largely (although not exclusively) of
the movements of organisms, quantitative investigation of behavior is often
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dependent upon the collection of temporal sequences of spatial information.
Cinematography is the classical method used to gather such information.
Behavioral variables such as the position of an organism, its orientation, and
angles of flection of its appendages are extracted from the motion pictures by
means of frame-by-frame analysis. An important advantage of this technique is
its flexibility; it may, in principle, be employed in the investigation of any
overt behavior which can be photographed. However, two factors preclude the
widespread use of this method: (1) manual quantization via frame-by-frame
analysis is a lengthy and tedious process; and (2) once the data is obtained, a
substantial amount of subsequent data processing may be required in its
analysis.
The Bugsystem (2, 4, 5) was designed to enable the acquisition and
processing of video data by a minicomputer. Behavioral data are initially
recorded using standard closed-circuit television equipment. These data are
analyzed by replaying a video tape into a specially desiped video-to-digital
processor (christened the "Bugwatcher"). This device acts as an edge detector,
greatly reducing the information flow to the computer and thereby allowing
the real-time collection of spatial coordinates delineating the outlines of
moving organisms. Frame-by-frame analysis of digitized video data is achieved
through the use of specially designed programs tailored to the task of
quantizing behavioral variables of interest to the researcher.
The original version of the Bugsystem was developed by Greaves and
implemented on an IBM 1800 computer at the University of California, Santa
Barbara. This prototype system, previously described by Greaves (5), has been
utilized by Hand and Schmidt (6) and by Wilson (8) to investigate the
photokineses and phototaxes of marine dinoflagellates. However, several
features of this system severely limited the domain of its application.
Supported by a research grant from the Environmental Protection Agency, we
have developed a second generation Bugsystem. Our explicit goal in the design
of this system was to provide a flexible tool for the quantitative investigation
of behavior, a system capable of realizing much of the potential of
frame-by-frame analytic techniques.
The purpose of this paper is to describe this second generation Bugsystem,
emphasizing the way in which certain hardware and software refinements have
expanded the scope of questions which may be conveniently answered by
means of "bugwatching." We discuss the way in which the user interacts with
the system via a specially formulated "Behavioral Research Language" and the
way in which this language has been implemented upon physical machines. We
also describe the input of data to the system, the processing and display of
behavioral data, and a variety of experimental strategies accommodated by the
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system. Finally, we outline work now in progress to further generalize the
Bugsystem to provide for the analysis of movement in three dimensions.
DEFINING A BEHAVIORAL RESEARCH LANGUAGE
The Behavioral Research Language, or BRL, is a high level operator based
language that is tailored to the unique problems associated with the input,
scaling, analysis and display of video images of moving objects. BRL is an
interpretive language which runs as an application program on a good sized
minicomputer and relies heavily upon user interaction with a storage graphics
terminal to input, plot, edit and transform the data through image processing
functions. Image processing generally culminates in the computation of paths
or trajectories of the objects moving before the video camera. Sets of
trajectories may be merged together and the data may be transformed to yield
time series of behavioral variables (e.g., linear velocity, angular velocity,
direction of travel, etc). These results may then be analyzed statistically, and
the resultant data sets either listed numerically or plotted on the graphics
terminal. Thus, the Bugsystem consists of two basic subsystems: (1) an unique
image processing system for the frame-by-frame analysis of video data; and (2)
a signal processing system for the statistical analysis of equispaced time series.
The key element to understanding and using BRL lies in grasping the
operator-operand-resultant nature of specifying functions or commands to the
system. The general command syntax is as follows:
*Operator/sw/sw Operand-name/sw/sw Resultant-name/sw/sw nl, . .. n5,
Where is the prompting character, "Operator" is one of the available
functions (of which there are currently 88, with the list still growing),
"Operand-name" is the name of the input (or operand) data set, and the
numeric constants "nl" through "n5" are optional numeric constants which
govern details of the function of certain operators. The "/sw" denote optional
"switches" (the word is taken from minicomputer jargon) which are used as
operator modifiers or to supply special information to the operator being used.
All data sets are disk resident and are specified by a four-letter name that can
be used to denote an experimental condition, a two-letter extention that
specifies the type of data represented (e.g., "VI"-video, "PA"-path,
"LV"-linear velocity, "CA"-catergorized, etc.), and a six letter front name that
can be used to identify the species studies andjor the date of the experiment.
The front name must be specified only when starting the system and remains
unchanged unless it is explicitly modified by the LOAD operator. Operator
names will henceforth be in bold upper case letters in the text.
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For example, to plot all of the data in the video file called BUGS.VI, the
command would appear as "*PLOT BUGS.VI". To plot only the first twenty
frames of this file, one would enter "*PLOT BUGS,VI 1, 20". To calculate the
linear velocities for the paths in the file BUGS.PA, the command line is
"*LVEL BUGS.PA BUGS.LV". Note that in this language the loops required
to access all data elements within a data set are not explicitly stated. The
operator automatically processes all of the elements of the operand data file
unless directed to a particular subset (e.g., "*PLOT 1, 20", as illustrated
above).
Three other aspects of BRL are worth including here. The first of these
concerns the way in which data are represented within the Bugsystem: a data
set consists of one or more vectors of variable length. While performing image
processing operations, each vector represents one video frame; one element of
such a vector represents a single point in two dimensional space. As the analysis
of the data proceeds through successive application of operators to operand
data sets, a single vector may represent an organism's path (i.e., a time series of
cartesian coordinates in two space as in the file "BUGS.PA" illustrated above)
or a real function defined over the length of such a path (e.g., the estimate of
instantaneous linear velocity as previously illustrated by the file "BUGS.LV").
Finally, in the statistical analysis of such data a vector may correspond to a set
of statistical parameters, a collection of "bins" or categories established for
histograming, an estimate of an autocorrelation function, etc.
The second aspect of the language to be considered here are the
self-documenting aspects of BRL. No one can be expected to memorize all of
the 88+ operator names, what they do in detail and the various switches and
numeric constants which they expect. To help in this regard, the NAMES
operator lists on the terminal the names of all the keyboard operators.
Moreover, entering "*Operator/HELP" for any of the available operators will
cause the system to print a full page of information describing what the
operator does, the types of operands for which the operation is defined, what
constants and switches are expected and an example of the operator's use.
The third aspect of BRL to be considered involves the construction of user
programs. BRL was designed primarily to be an interactive language: the user
normally enters commands at the terminal one at a time and thereby directs
the analysis to its desired end. It is also possible to create a disk file of
commands consisting of operators and operand specifications and to direct the
system to execute this stored sequence of operations (a computer program
written in BRL) via the USER operator.
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THE IMPLEMENTATION OF BRL
The implementation of BRL by means of the second generation Bugsystem
differs significantly (both in hardware and in software) from that of the
prototype system. This section outlines the main features of the new system,
giving reasons for their importance.
A New Bugwatcher
The Bugwatcher hardware was redesigned to utilize medium scale
integration (MSI) circuitry. Algorithmic state machine (ASM) charts were
employed to formulate and to document the new design. Functionally, the
new Bugwatcher is similar to its earlier counterpart: it extracts digital X-Y
coordinate pairs representing points belonging to image outlines from the video
raster scan and stuffs these coordinates into the computer's direct memory
access (DMA) channel. The computer program to input these data buffers them
and writes them to disk, thereby allowing the collection of extremely long data
records. Within such a record each frame of video data is represented as a
vector. Each video vector sent by the Bugwatcher to the computer contains not
only a variable length list of coordinate data, but also includes a leading header
of fixed length. The elements of this header are referred to as "vector
attributes" and are employed to encode information associated with each
vector. Video data possess four 16-bit words of attribute information supplied
by the Bugwatcher hardware: (1) a unique word consisting entirely of zeroes
used by the software to delimit frame boundaries; (2) a descriptor word which
contains an encoding of the frame rate at which the video data were digitized
and the on-off status of tone stimulus markers; (3) a total frame counter that
can be used to determine relative or absolute time intervals between data
segments recorded at varying time intervals; and (4) auxiliary digital input
which allows an encoding of an experimental variable (e.g., the direction of the
source of stimulation) to be automatically associated with each video frame.
Representation of stimulus conditions is discussed in more detail below (see
"Coupling to Research Environments"). This division of vectors into attributes
and data applies to all vectors manipulated by Bugsystem software. However
the meaning of each attribute depends on the type of data; e.g., one of the
attributes of a path (represented as a single vector) is the starting frame
number.
The Implementation Language—FORTRAN IV
The prototype BRL system was implemented entirely in assembler language
on an IBM 1800 computer and ran as a stand-alone system with no operating
system support. This made it non-portable and difficult to maintain and
expand. The new BRL system was to overcome these major shortcomings;
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thus, we chose FORTRAN IV as the language with which to implement the
Bugsystem. FORTRAN IV has become a standard language among
minicomputers and has simplified the tasks of maintaining Bugsysem software
and training new programmers to implement new BRL operators. The
importance of these aspects cannot be overestimated since, all in all, ten
different programmers have added software to the system during its three years
of development, each requiring instruction on the software conventions and
use of system utility subrouting packages. But at least they knew the
FORTRAN language.
The software was first operational on a PDP 11/45 computer at
Southeastern Massachusetts University under the DOS-9 operating system. It
was then implemented on a Data General ECLIPSE S/200 under the RDOS
operating system. Some assembler language subroutines had to be recoded,
including the software drivers that handle the direct memory channel to the
Bugwatcher. But the transition to the new computer went fairly smoothly. The
PDP 11 system was maintained for development purposes after the ECLIPSE
was sent to its Narragansett home at the EPA lab. Due to malfunctioning
DOS-9 software, the PDP 11 operating system was changed to RT 11—a change
that required as much or more development effort than changing computers!
Virtual File Structure
Certainly one of the major disadvantages to using minicomputers for large
application software projects is the address space limitation imposed by its
small word size. This system was no exception. To overcome this limitation so
as to allow both programs and data to fit within allocated memory, both
program structures and data structures were designed so that only pieces of
either resided in memory at any given time. Manufacturers of minicomputers
recognize this problem and provide software support for manipulating
overlayed programs; i.e., programs consisting of parts which are swapped in and
out of main memory. However, software support was not available for similarly
overlaying data sets. The earlier prototype system did not allow data sets to be
any larger than the memory buffer on the IBM 1800—a simple solution, but
not acceptable in the newer system. Certain Bugsystem applications require the
acquisition and analysis of behavioral records consisting of many frames of
video data or the trajectories of hundreds of organisms. The system required
the potential to manipulate data structures ten to hundreds of times the size of
main memory available for data.
One of the early software design goals was to simplify the process of adding
new BRL operators. Each operator was to be implemented in FORTRAN as a
program overlay. An operator would be required to access as many as three
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simultaneous data sets (e.g., the arithmetic operators PLUS, SUBTRACT,
MULTIPLY and DIVIDE each require two operands and generate one
resultant) within a labeled common buffer of 8192 integer values (or 4096
single precision values). Within the code that implements a new operator, files
are accessed with a complete set of virtual file handlers coded in FORTRAN.
These routines provide services for opening an existing file, creating a new file,
reading a vector into the buffer from disk, writing a vector from the buffer to
the disk and closing a file. To minimize disk access time and thereby insure
optimum response time to the user, techniques employed in managing other
virtual memory systems were adapted to the Bugsystem. All data sets, stored
on disk as contiguous files, are accessed directly using multiple block transfers.
If, for example, a command is issued from an applications program to read a
given vector within a file, then the software first determines if the vector is
resident within the buffer. If it is resident, then the routine immediately
returns to the calling program providing the length of the vetor and a pointer
into the buffer to the first element of the vector. If it is not resident, its logical
address within the file is computed and it—and the vectors which succeed
it—are read into the labeled common area. A similar algorithm is employed
when writing a vector into a file; i.e., a disk write is not required unless the
output buffer area is full.
Funneling all file input/output through a common set of routines has
significant advantages. The development of new BRL operators is simplified
insofar as the underlying applications programs do not each separately (and
redundantly) require the algorithms needed to manipulate large files. Another
advantage lies in the increased portability of the software. Versions of
FORTRAN supported by different machines and operating systems vary most
markedly in their non-standardized methods of accessing files. Machine and
operating system dependencies are thus isolated in a manageable number of
software modules, yielding more portable and more easily maintainable
software.
COUPLING TO RESEARCH ENVIRONMENTS
Images
Primary input to the Bugsystem consists of digitized video data. If the video
images are sufficiently "clean" (i.e., possess high contrast and lack structural
complexity), input of data to the computer is accomplished automatically in
real time. Video tapes are replayed into the Bugwatcher which compares the
incoming video signal to a "video threshold" set by the user. Those points
within the image where the video signal crosses the video threshold are
displayed on a video monitor. The user adjusts the video threshold to make
these points coincide with the outlines of moving organisms and selects a frame
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rate (ranging from sixty frames/sec to one frame/min) appropriate for the
relative speed of the organisms. When the BUGWATCHER INPUT operator is
executed, the computer accepts digitized image information from the
Bugwatcher at the selected frame rate. Each threshold point is represented in
Cartesian coordinates with 8-bit resolution for each of two orthogonal
components. A video frame is represented within the resultant data structure as
a data vector with a variable number of such points as its data elements. An
entire record (or "video file") consists of a temporally ordered sequence of
such vectors.
The Bugsystem was originally developed for the investigation of the
behavior of motile microorganisms (2). In this application, the organisms are
viewed under dark-field illumination swimming within a well slide upon the
stage of a compound microscope. However, automatic digitization of data is
possible for any study of moving objects for which "clean" video records are
available. One of us (Wilson) is currently using the second generation system to
study the effects of plane polarized light upon the behavior of aquatic
arthropods. The animals move freely within a cylindrical aquarium (diameter «
20 cm) under bright-field illumination and are viewed using a macro lens
attached to the television camera. Use of video tape as a storage medium allows
experiments to be conducted in a laboratory remote from the site at which the
data are analyzed.
Occasionally video recordings are not "clean" enough to allow fully
automated digitization (e.g., data collected in the field) or the digitized images
are too crude to provide information about details of an organism's anatomical
structure (e.g., the orientation of its eyes). A technique has been developed to
expedite manual analysis of such data. Using the PICK operators, the video
tape is examined frame-by-frame. The user selects points upon the screen of a
video monitor using a video cursor controlled by a JOYSTICK. A synthetic
video signal representing a tiny bright dot is sent to the Bugwatcher which, in
turn, sends digitized video information to the computer. Because averaging
algorithms are employed, this method affords higher spatial resolution than
does fully automated input: each coordinate in the resultant data structure
may have 8-9 significant bits in comparison to the 7-8 bits of the normally
digitized input data.
Input of video data to the computer effects a mapping of the image as seen
upon a television screen onto a two dimensional representational space.
Distances within this space ("Bugspace") are measured in arbitrary internal
units (or "Bugwatcher units") and, therefore, must be scaled. Using the LIVE
INPUT operator, the Bugwatcher processed image of a ruler (or any object of
known length)—recorded under the same conditions used in gathering
behavioral data—is displayed on the screen of a CRT terminal and two points
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are selected using the terminal cursors. Thus, the ratio between the distances
separating the points in Bugspace and in physical space yields a factor or
proportionality between Bugwatcher units and conventional units of spatial
measurement (e.g., nm, mm, cm, etc.).
Experimental Parameters and Experimental Variables
Biological interpretation of behavioral data requires that the behavior of
organisms be related to experimental conditions prevailing at the time of
observation. Consequently, we have developed methods to associate
experimental parameters and experimental variables with sets of data. An
experimental parameter is a quantity characterizing a condition which is
constant throughout any given record but may vary from record to record
(e.g., temperature, concentration of a pollutant, etc.). Using the PARAMETER
operator, such numerical constants may be inserted into the set of attributes
belonging to each data vector. Parameters may be deleted, modified or listed.
They may be used to organize graphical displays (e.g., average linear velocity as
a function of temperature) or to modify the execution of certain operations.
An experimental variable is a quantity whose value changes during a single
record. Times at which simple step changes in stimulus conditions (e.g.,
switching a light on and off) occur may be indicated by the presence or
absence of tones stored on the audio track of a video tape. The Bugwatcher
possesses four tone generators to produce such temporal markers during the
course of an experiment; it also possesses external connections which allow the
simultaneous gating of laboratory apparatus. When the video tape is replayed
into the Bugwatcher these tones are detected. As discussed above, the second
attribute in each data vector sent to the computer contains four bits dedicated
to representing the presence or absence of the four tones.
We are presently developing a technique which provides for the
representation of stimuli which vary continuously over time. The stimulus level
will be encoded by means of frequency modulation (fm) on the audio track of
the video tape. When the tape is replayed into the Bugwatcher the fm signal
will be digitized and represented with 10-bit precision by the fourth attribute
of each data vector sent to the computer. The APPEND operator will be
employed to extract this information from each data vector, scale it and store
as an equispaced time series. Again these data may be used to organize
graphical displays or they may enter into computations involving time series of
behavioral variables. One of us (Wilson) is preparing to employ this method to
investigate behavior evoked by rotation of the plane of polarized light. The
organisms swim beneath a polaroid filter whose angle is controlled by an analog
servomechanism. The filter may be rotated so as to describe quick jumps,
ramps, harmonics, etc. A signal directly proportional to the angle of the filter
will be encoded and analyzed.
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PROCESSING AND DISPLAY
Image Processing
As discussed above, input of video data to the computer entails substantial
preprocessing of pictorial information. A data vector within a resultant video
file is a list of contemporaneous points; an organism's outline is represented
within this data structure as a localized set of points. The user can display such
data graphically (using the PLOT operator) or alpha-numerically (using the
LIST or EXAMINE operators). Video files may be edited both in time and in
space. The EDIT operator allows one to save (or delete) temporally contiguous
sets of data vectors. Thus, the user could EXAMINE the data to ascertain the
frame at which the status of a tone had changed (indicating a change in
stimulus conditions, e.g., switching on a blue light) and then EDIT the data to
insure that this change occurs on frame number 100. The MASK operator
allows one to save (or delete) points within rectangular or circular regions of
the image plane. Thus, the user could MASK out all points within a video file
which correspond to a particle of detritus within the experimental preparation.
Finally, the user may APPEND additional information to a video file (text
describing the conditions of the experiment, numerical constants, time series of
tone states or time series of experimental variables).
Analysis of video data by means of the Bugsystem proceeds by abstracting
one (or more) points from each point set delineating an organism's outline. In
an investigation of translational movement this task is easily defined: unlike
either rotational or flectional movement, quantitative description of the
translational component of an individual organism's behavior does not require
detailed knowledge of the organism's external anatomy. The body of the
organism is represented by a single point, viz., its "center of mass".
Translational movement is defined as displacement of this point from.one
position in space to another.
Mapping outlines into centrally located points is usually achieved by means
of the CENTROID operator whose command syntax is exemplified by the
entry
*CENT BUGS.VI BUGS.CE N1, N2, N3.
Each vector in the resultant file "BUGS.CE" corresponds to a vector in the
operand file "BUGS.VI". Each element of a resultant data vector is a
"centroid": a point in Bugspace whose X and Y coordinates are, respectively,
the average X and Y coordinates of an "acceptable set" of points in the
corresponding operand data vector. The numerical parameters "nl", "n2" and
"n3" are required to characterize an "acceptable set" of operand data points.
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An initial member (having non-zero coordinates) of such a set is chosen from
the operand data vector, the coordinates of this point are set equal to zero in
the operand data and a mask (width = 2'nl, and height = 2"n2) is centered upon
this point. The set is augmented if an operand point (having non-zero
coordinates) falls within the mask. The mask is then centered upon the new
point and the search continues. The search terminates when the set is deemed
"acceptable" if it possesses at least n3 members. Ideally, this process yields one
centroid corresponding to each outline—unless the outlines of two organisms
are merged. The user can PLOT the centroids over the original data to confirm
this correspondence and recompute the centroids using new parameters if the
correspondence is not adequate. The structure of "centroid data" is quite
similar to that of video data except that each coordinate of a centroid is
represented with 15-bit (rather than 8-bit) precision in attempt to exploit the
greater accuracy resulting from the averaging procedure. Centroid data is
displayed and edited in the same fashion as video data.
The PICK operator provides an alternative method to abstract points of
interest from video data. The use of this operator for direct analysis of a
videotape, thereby, bypassing the acquisition and processing of video files, is
discussed above (see "COUPLING TO RESEARCH ENVIRONMENTS"). The
user may also PICK points associated with each outline by means of the
terminal cursors. The PICK operator is indispensable for investigations of
rotational and flectional movement. For example, the user might elect to study
the orientations of the longitudinal body axes of a group of organisms (i.e.,
simple rotation in a single plane). The longitudinal axis may be defined as a
vector extending from the tail to the head of an organism. Thus, the user
selects an ordered pair of points corresponding to each outline by entering
*PICK/VI BUGS.VI BUGS.TH
and using the cursors to specify first the "tail" and then the "head" associated
with each outline. The resultant file "BUGS.TH" possesses the structure of
centroid data with each "tail point" immediately followed by the correlated
"head point". These points are then segregated using the PLUCK operator. For
example, the two commands
*PLUC BUGS.TH BUGS.T 1, 2
*PLUC BUGS.TH BUTS.H 2, 2
respectively produce the files "BUGS.T"—containing all first elements of the
ordered pairs (viz., the "tail points)—and "BUGS.H"—containing all second
elements of the ordered pairs (viz., the "head points). This type of analysis
may be extended to encompass larger collections of points (or n-tuples),
thereby providing for the study of flectional movement (e.g., the angles of
propulsive appendages with respect to the longitudinal axis).
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Regardless of the type of movement under investigation, the next stage of
image processing entails the computation of paths or trajectories through
Bugspace. A path or trajectory is a time-ordered set of points represented as a
single data vector and characterized as follows: All points in a path are selected
from an operand file consisting of centroids or having the structure of centroid
data. The manner in which points are represented in a path is identical to the
way they are represented in a frame of centroid data. Each path starts within a
specific frame and its starting frame number is represented as the fourth
attribute of the vector. Over the temporal interval during which a path is
defined one (and only one) point is selected from each corresponding vector
(or frame) of the operand file. Adjacent pairs of points within a path are
selected on the basis of their spatial contiguity within adjacent frames.
The search for paths is performed by the PATH operator whose command
syntax is exemplified by the entry
PATH BUGS.CE BUGS.PA nl, n2, n3, n4, n5.
The numerical parameters "nl" through "n5" control various details of the
search, nl specifies the width (in Bugwatcher units) of a square mask used as a
criterion of spatial contiguity. n2 is the maximum number of times to expand
the mask if no contiguous point is located within the frame being searched. n3
is the n4 minimum number of points to be accepted as a valid path. n4 is the
minimum average displacement in Bugspace between consecutive frames for a
set of points to be accepted as a valid path; this parameter may be used to
"weed out" stationary artifacts (e.g., a particle of detritus). n5 is the maximum
number of frames to "look ahead"; i.e., if no contiguous point is found in the
current frame, then the next frame may be searched. If a point within the new
frame qualifies, the missing point is computed by linear interpolation.
Ideally, every path would correspond to the movement of a single point
(associated with the outline of one individual organism) through Bugspace and
each such movement would be represented by one path. In practice, the PATH
operator may commit two types of errors. The operator may overlook certain
segments of continuous movement. Such omissions may yield an abnormally
short path, or they may result in a one-to-many (even a one-to-none)
correspondence between real word trajectories and paths. Alternatively, the
operator may confound certain segments of the continuous movements of two
(or more) organisms. If there were but one organism in the field of view at any
time, then errors of omission could be abolished by using a large mask (why
not let it include all of Bugspace?) and allowing the program to look ahead
several frames. But with several organisms represented within each frame there
is clearly a tradeoff in choosing parameters so as to reduce the incidence of the
two types of errors. The user may PLOT the paths over the centroids (or over
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the original video data) to check their fit and then recompute the paths using
new parameters. Often several iterations of this process are required to obtain
an optimum combination of parameters. Since the parameters depend largely
upon the magnification, the density of organisms and the way in which they
move, the same "optimum set" of parameters (retained in memory for the
user's convenience) are generally used to compute paths for all replicates of an
experiment.
Proper selection of pathfinding parameters can significantly reduce the
number of erroneous paths but cannot be relied upon to eliminate all errors.
Consequently, we have developed programs which allow the user to interatively
detect and correct mistakes within path files. Path editing programs (including
CHOZ, EDIT, MERG and JOIN) allow the user to preform the following basic
operations: (1) delete a path; (2) truncate a path; (3) cut a path into two
smaller paths; and (4) join two paths (assuming they do not overlap in time).
Once a valid collection of paths has been obtained, the user may proceed
directly to the extraction of time series of behavioral variables (as discussed
below) from the path files. Before doing so, however, there are several
additional procedures which the user may choose to apply to the path data.
Since the use of these procedures (and the order in which they are applied) is
dependent upon the overall design of the experiment, we will first illustrate
them by means of a specific example.
Wilson video taped the behavior of Daphnia pulex (a small freshwater
crustacean, commonly known as a "water flea") in Talbot Waterman's
laboratory at Yale University. Twelve animals were observed from below swimming
against a brightly and uniformly illuminated background. A variable
polarizer/depolarizer was interposed between the chamber containing the
animals and the light source. The tape consisted of 23 separate video records.
Before each recording the polarizing filter was rotated to a randomly chosen
angle and, also in random sequence, the device was adjusted so as to polarize or
depolarize the illumination. Each record began with the image of a strip of
plastic attached to the filter in order to indicate the angle of the filter and the
magnification of the image. The plastic strip was then removed and the animals
were observed swimming under constant conditions. In all, 13 records were
obtained under polarized light and 10 records were obtained under unpolarized
light. Each recording lasted two minutes.
The video tape was analyzed with the aid of the ECLIPSE at Narragansett,
R.I. For each record the LIVE INPUT operator was used to determine the
angle of the filter with respect to the Bugsystem reference frame (which is
fixed with respect to the raster scan of the video signal). As discussed above,
the appropriate spatial scale factor (approximately 0.40 mm/Bugwatcher unit)
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was also computed. The BUGWATCHER INPUT operator was then used to
generate one video file (480 frames of data at ten frames/sec. or 48 sec. of
data) for each record on the tape. Wilson then MASKed the data, saving only
those points within a centered circular region of Bugspace. The centroids and
then the paths were separately computed for each masked video file using the
same set of centroid and pathfinding parameters. Very few pathfinding errors
resulted from these computations, but these were corrected by editing the
separate path files. Wilson then employed the PARAMETER operator to
associate the angle of the filter with respect to the Bugsystem reference frame
(an experimental parameter) with each path. This angle was the same for every
path within a given path file. By repeated use of the MERGE operator, Wilson
condensed the data to produce two exceedingly large files: one containing all
paths observed under polarized light (154 paths, 13210 data points) and
another containing all paths observed under unpolarized light (166 paths,
13691 data points). These paths were still represented in Cartesian coordinates
relative to the Bugsystem reference frame. However, orientation with respect
to the plane of polarization should only be manifest with respect to the
reference frame of the filter. Using the ROTATE operator, the two frames of
reference were made to coincide: every path within each merged file was
rotated in Bugspace (about an axis passing through the center of this space)
through an angle obtained by negating the appropriate experimental parameter
for each path. All resultant paths for Daphnia swimming under polarized light
are PLOTed in Figure 17-1. Many of these paths may be seen to be aligned
approximately orthogonal to the axis of the filter (i.e., perpendicular to the
E-vector of the polarized light).
The preceading analysis illustrates the use of three operators (vis., the
PARAMETER, MERGE and ROTATE operators) to organize path data prior
to the computation of behavioral variables. The ROTATE operator was
implemented to expedite studies of animal orientation. Using this operator, all
spatial data in a file may be rotated through a constant angle, each path may be
rotated through a constant angle associated with that path or each point may
be rotated through an angle associated with a corresponding moment in time.
The last option enables the investigator to study orientation with respect to a
moving stimulus (e.g., be MERGED) whenever the respective files may be
taken to be replicates of the same experiment. Not only does this simplify the
bookkeeping tasks associated with subsequent analysis, but, in addition, allows
a set of similar data to be treated as a single sample by statistical operators. The
fundamental advantage of an operator-based interactive system for the analysis
of behavioral data is its flexibility: the operators which the user chooses to
apply to the data—and the order in which they are applied—can be selected to
correspond to the design of the original experiment.
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Figure 17-1. Merged Path File of Daphnia Pulex Swimming with
Respect to the Reference Frame of the Linear Polarizer.
NOTE: The E-vector is horizontal to the field of view. The experiments and
analyses which gave rise to these data are discussed in the text.
Generating and Transforming Time Series Data
Time series of X and Y coordinate values are generated from path data by
my means of the SPLIT operator whose command syntax is illustrated by the
entry
*SPLI BUGS.PA BUGS.X BUGS.Y.
Each data vector in the resultant files "BUGS.X" and "BUGS.Y" is,
respectively, a time series of X and Y coordinate values. The resultant data sets
are in one-to-one correspondence and each element is represented as a single
precision floating point number. Like the paths from which they are derived,
resultant time series may start and end at arbitrary points in time. Therefore,
the number of series defined at any given moment is also arbitrary. Other
computer systems have been developed for the analysis of equispaced time
series (7). After image processing has been completed, the operators available
on the Bugsystem are unique only insofar as they possess the sophistication,
required to manage large collections of arbitrarily derived series (e.g., Figure
17-2).
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Figure 17-2. Linear Velocity as a Function of Time for 128 Paths
of the Fairy Shrimp Eubranchipus Vernalis in Polarized Light.
In principle, all behavioral variables which may be investigated using the
Bugsystem can be generated by simple arithmetic transformation of series of X
and Y coordinates. Greaves (4) has discussed the computation of certain
behavioral variables (viz., linear velocity, net to gross displacement ratio,
direction of travel and angular velocity) using arithematic operators
implemented within the prototype system. Such operators transform every
element of every data vector within a file, treating each vector as a separate
unit of data. The present Bugsystem is also provided with a wide selection of
simple arithematic operators. However, we have condensed the computation of
certain frequently calculated behavioral variables into single operators which
require path data as input. As an example, let us consider the RATE OF
CHANGE OF DIRECTION operator whose command syntax is illustrated by
the entry
*RCDI BUGS.PA GUBS.RD nl.
Every element in the resultant file "BUGS.RD" is the unsigned rate at which
the corresponding path changed its direction of travel at a given moment. RCD
is the absolute value of angular velocity expressed in degrees per second (The
parameter "nl" is the frame rate, required to convert from degrees per frame.)
The importance of this variable in ascertaining mechanisms responsible for
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certain animal aggregations has been widely discussed (3). RCD may also be
computed by a sequence of simple arithematic operations as follows:
(a) * SPLIT BUGS.PA BUGS.X BUGS.Y
(b) *CDIF BUGS.X BUGS.DX
(c) *CDIF BUGS.Y BUGS.DY
(d) UCTAN BUGS.DX BUGS.DY BUGS.DI
(e) *CDIF BUGS.DI BUGS.AC
(f) *MULT/CO BUGS.AC BUGS.AV nl
(g) *ABSV BUGS.AV BUGS.RD
Where "CDIF" is the CENTRAL DIFFERENCE operator (a discrete
approximation to the differential operator), "CTAN" is the CONTINUOUS
ARCTANGENT operator, "MULT/CO" denotes multiplication by a constant
and "ABSV" is the ABSOLUTE VALUE operator. The resultant "BUGS.DI"
of step (d) is the direction of travel measured in degrees with respect to the
Bugsystem reference frame; it could have been generated from the original path
data using the DIRECTION OF TRAVEL operator. The resultant "BUGS.AV"
of step (0 contains angular velocities (measured in degrees per second); this file
could have been produced using the ANGULAR VELOCITY operator. Other
operators have been developed to evaluate LINEAR VELOCITY and NET TO
GROSS DISPLACEMENT RATIO functions defined upon path data.
Simple Statistical Processing
For the purpose of statistical analysis two different types of data
structure—representing two levels in a structural hierarchy—may be
distinguished as "samples": vectors and files. Many statistical operators
recognize this distinction. For example the STATISTICAL PARAMETER
operator estimates parameters such as the mean, variance, standard deviation,
skewness, kurtosis, etc. The command
~STAT/VE BUGS.LV BUGS.ST
produces the resultant file "BUGS.ST', containing one data vector (i.e., a list
of statistical parameters) for each data vector in the operand, whereas the
command
*STAT/FI BUGS.LV BUGS.ST
produces only one resultant data vector characterizing the entire file. In either
case, the user can LIST the resultant parameters. Similarly, the SLOT operator
provides for estimation of density and distribution functions via histograms
both for individual vectors and entire files. These data may be displayed
graphically (Figure 17-3) or LISTed on the terminal or the line printer.
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Figure 17-3. Linear Velocity Histograms for E. Vernalis in
Polarized Light (the Data Displayed in Figure 17-2)
and in Unpolarized Light
NOTE: Dashed lines denote the estimated mean of each distribution.
We chose to store statistical parameters within data vectors, rather than
merely computing and displaying them, in order to allow them to enter into
subsequent calculations. For example, assume the user had computed statistical
parameters for each data vector in a file of instantaneous linear velocities (e.g.,
the "STAT/VE" example given above); each estimate of the mean is thereby an
average for each path. The user may then investigate the distribution of these
path averages. The estimated means are first isolated using the STRIP operator:
~STRI/MN BUGS.ST BUGS.MN.
The resultant file "BUGS.MN" contains a single data vector; each element of
this vector is a mean ("/MN") stripped from one operand data vector. The user
may wish to compute statistical parameters for the new set of data, or explore
its frequency distribution via histograms (Figure 17-4). The user may also
MERGE such files so that each data vector corresponds to a single
experimental condition. The vectors may then be compared with one another
(e.g., one-way analysis of variance, chi-square tests, etc.).
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Figure 17-4. Average Linear Velocity Histograms for E. Vernalis in
Polarized Light and in Unpolarized Light.
NOTE: Dashed lines denote the estimated mean of each distribution.
We have made special provision within the Bugsystem for the manipulation
and statistical analysis of angular data. For example, the command
*STAT/AN/FI BUGS.DI BUGS.ST
produces a single data vector whose elements are statistical parameters
appropriate for circular distributions. These include the length and direction of
the mean vector (and related measures) as discussed by Batschelet (1).
Moreover, because the Bugsystem is well suited for investigations of animal
orientation (and because this is a major interest of one of its codevelopers) we
have implemented an extensive polar graphics package within the Bugsystem.
Figure 17-5 illustrates the use of polar wedge histograms to represent angular
density functions.
More Advanced Statistical Operations
These fall into two major categories: operators directed toward the analysis
of time dependence of behavioral variables and operators used to explore
mutual relationships between variables (e.g., Figure 17-6). In the first category,
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Figure 17-5. Polar Wedge Histograms of Instantaneous Direction of
Travel with Respect to the Filter Reference Frame for D. Pulex.
NOTE: (A) in polarized light (evaluated for the path plotted in Figure 17-1)
and (B) in unpolarized light. The percent of sample within each angular
category is indicated on the radius. The dashed circles denote expectation for a
circular uniform distribution.
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Figure 17-6. Dependence of RCD (Rate of Change of Direction)
Upon Direction of Travel with Respect to the Axis of the
Polarizer for D. Pulex.
NOTE: (A) in polarized light and (B) in unpolarized light. Data was processed
by partitioning RCD into disjoint subsets on the basis of the correlated
direction of travel. The estimated mean (± the standard error of the mean) for
each subset is indicated in the figures.
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the Bugsystem includes several ensemble operators which compute a statistic
(e.g., an estimate of the mean or mean vector or even a histogram) for every
frame defined within the operand data. The Bugsystem also includes serial
correlation operators (viz., AUTOCORRELATION and CROSS
CORRELATION). Programs providing for analysis in the frequency domain,
sinusoidal, regression and polynomial regression are currently under
development.
REFERENCES
1. Batschelet, E. 1965. Statistical Methods for the Analysis of Problems in
Animal Orientation and Certain Biological Rhythms. AIBS Monograph.
Washington, D.C. pp. 1-57.
2. Davenport, D., G.J. Culler, J.O.B. Greaves, R.B. Forward and W.G. Hand.
1970. The Investigation of the Behavior of Microorganisms by
Computerized Television. IEEE Trans. BME 17: 230-237.
3. Fraenkel, G.S. and D.L. Gunn. 1961. The Orientation of Animals. Dover.
New York, pp. 1-367.
4. Greaves, J.O.B. 1971. An On-Line Television Computer System for the
Study of the Behavior of Microorganisms. Ph.D. Dissertation. Dept. Elec.
Eng., Univ. of California, Santa Barbara.
5. Greaves, J.O.B. 1975. The Bugsystem: The Software Structure for the
Reduction of Quantized Video Data of Moving Organisms. IEEE Proc. 63:
1415-1425.
6. Hand, W.G. and J. A. Schmidt. 1975. Photo tactic Orientation by the Marine
Dinoflagellate Gyrodinium dorsum Kofoid. II. Flagellar Activity and
Overall Response Mechanism, J. Protozoel. 22: 494-498.
7. Martin, W. and K. Brinkman. 1976. A Computer Program System for the
Analysis of Equispaced Time Series. J. Interdiscipl. Cycle Res. 7: 251-258.
8. Wilson, R.S. 1976. Light Elicited Behavior of the Marine Dinoflagellate
Ceratium dens. Ph. D. Dissertation. Dept. Biol. Sci., Univ. of California,
Santa Barbara.
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THE EFFECTS OF TEMPERATURE, LIGHT
AND EXPOSURE TO SUBLETHAL LEVELS
OF COPPER ON THE SWIMMING BEHAVIOR
OF BARNACLE NAUPLII
William Lang
Sarah Lawrence
Don C. Miller
U.S. Environmental Research Laboratory
Narragansett, Rhode Island 02882
ABSTRACT
The "Bugsystem" is a computer-television system developed to
accurately track and analyze swimming patterns of aquatic organisms.
Video images of test animals are converted to time sequence X-Y
coordinates to allow rapid computer analysis of linear or angular velocity,
rate of change of direction, direction of travel and other parameters. Initial
experiments using barnacle nauplii (Balanus amphitrite, B. improvisus, B.
venustus, Chthamalus fragilis) indicate larval swimming speeds are affected
by temperature and light regime. Response to temperature appears to be a
function of species tested and, perhaps, geographic location of the adult
population. Changes in linear velocity induced by acute light intensity
variation are of short duration. Mean linear velocities of nauplii are altered
by 24 hour exposure to copper as low as 20 ppb. Linear velocities of exposed
populations increase relative to controls at low copper levels, and then
decrease as lethal levels are approached. Copper will also alter the
swimming pattern of exposed larvae.
INTRODUCTION
In view of concern that bioassays directed solely toward determining lethal
concentrations of pollutants may not accurately reflect levels doing harm to
the environment, attention has been directed toward sublethal effects of
pollutants— "effects which do not immediately, or directly, lead to death, but
which nevertheless cause disturbances which may be of ecological significance
(1)." Existing studies using pathological, physiological, and behavioral
parameters indicate approximate thresholds for sublethal responses are often
10-20 percent of LC50 levels or less (7, 22). It is generally recognized that
behavioral responses of marine animals are often highly sensitive to stress (18)
and that juvenile or larval stages of many marine organisms represent that part
of the life-cycle most susceptible to stress (4, 8, 16). Logically, larval
273
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behavioral responses to pollutants would represent a potentially significant
field of study; however, devising a means to easily record and rapidly quantify
swimming and other responses of small larvae has limited efforts in this
direction (8,12, 17, 24).
Development of the Bugsystem at this laboratory has provided the
technology to rapidly analyze the swimming patterns of large sample numbers
of organisms of a wide size range (Wilson & Greaves, this volume, report 17).
With this potential we are presently investigating the use of behavioral
bioassays for marine larvae. The following results represent initial studies using
larvae of common barnacle species.
EXPERIMENTAL
The spontaneous locomotory activity for second stage nauplii of four
barnacle species (Balanus amphitrite amphitrite, B. improvisus, B. venustus,
Chthamalus fragilis) was investigated. Of primary concern in this initial study
was the mean linear velocity (MLV) of sample groups and changes in MLV
induced by water temperature, light regime, and 24 hour exposure to sublethal
copper levels.
Source of Larvae
Second stage Balanus nauplii used in all experiments were released from
adult barnacles maintained at 20 ± 2°C, constant illumination in 30-32°/oo, 1
Id filtered seawater from Narragansett Bay. Balanus amphitrite and B.
improvisus were initially collected near Georgetown, South Carolina, and
maintained under the above laboratory conditions 2-12 weeks prior to larval
release. B. venustus adults were collected in Narragansett Bay; adults released
larvae within two weeks after capture. Chthamalus fragilis nauplii, also from
Narragansett Bay, were obtained from ripe egg masses incubated for 24 hours
at 20°C. In some copper experiments, stage II nauplii were reared to later
stages on a mixed algal diet of'Tetraselmis suecia and Thalassiosira pseudonana.
Methods of maintaining laboratory populations of barnacles and rearing of
nauplii are further described by Lang (13,14).
Video Recording
To obtain video tapes of swimming patterns for Bugwatcher analyses, ca.30
nauplii were placed in 25 ml beakers with a water depth of about 10 mm. The
beaker with nauplii was put within a cylindrical flat black metal lenshood (light
shield) attached to a 72 mm diameter #25 deep red glass filter (Figure 18-1).
This complex was centered upon a Wild M-5 dark field illumination stage fitted
with an "800 nm" interference filter and 22 mm diameter diaphram (Figure
274
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Figure 18-1. Diagram of Video Recording Equipment.
NOTE: A) TV camera, B) Photo tube, C) Wild M-5 microscope, D) wide-angle
attachment, E) metal light shield with deep red glass bottom, F) container with
test organisms, G) 800 nm interference filter, H) metal diaphram, I) clear glass,
J) dark field stage with halogen light. Components E-H are shown separated
from each other for graphic clarity.
18-1). Video images were obtained using a Cohu 4400 television camera
attached to the M-5 microscope body. The field of view recorded was 10 x 10
mm. Optimum image contrast was obtained using a halogen light source with
dark field optics at 8.5 volts (Figure 18-1).
Spectroradiometer (ISCO-SR) readings indicate light transmitted through
the stage filters was as low as 680 nm. Peak transmission occurred between
810-840 nm. With the exception of light experiments, nauplii were moved
directly from constant light temperature boxes to the microscope stage. Room
lights were extinguished and, following a two-minute acclimation period, larvae
were taped for 3-5 minute intervals using filtered substage illumination.
Swimming parameters reported were determined by analysis of 30-60 second
portions of these tapes; results are pooled from replicate samples.
275
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Response to Light
Nauplii used in all experiments were light adapted as fluorescent bulbs in
the temperature boxes were on continuously. When transferred to the darkfield
stage, larvae tended to disperse to the beaker walls with ceiling lights on. With
these lights extinguished, nauplii tended to swim away from the beaker walls.
Direction of travel upon entering or leaving the camera field exhibited no
particular orientation. Ongoing studies indicate dark adapted B. amphitrite
nauplii will exhibit a weak photonegative response to substage light over a
five-minute period (Forward & Lang, personal observation).
Balanus spp. stage II nauplii exhibit similar response to sudden changes in
light intensity. When overhead white room lights are turned on, Balanus nauplii
will approximately double linear velocities, then within 4-6 seconds return to
initial swimming speeds (Figure 18-2). Turning overhead lights off has
essentially the opposite effect; nauplii will cease locomotion for about five
seconds and then return to initial swimming speeds (Figure 18-3).
SECONDS
Figure 18-2. Example of running average linear velocity
(mm/sec) for sample of ten stage II Balanus venustus
nauplii exposed to sudden light increase.
NOTE: Dashed line indicates time at which overhead white light stimulus was
applied. Filtered (820 nm peak transmission) substage light was present
throughout experiment for recording purposes.
276
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5
4
3 -¦
l/\ I) P^Va
MM/SEC
2 -¦
0
0
20
25
SECONDS
Figure 18-3. Example of reaction of a single stage 11 Balanus
venustus nauplius to sudden light decrease.
NOTE: Dashed line indicates time at which overhead white light was extin-
guished. Filtered (830 nm peak transmission) substage light was present
throughout experiment for recording purposes.
Chthamalus fragilis exposed to similar light changes exhibited little response
in terms of MLV. The distinctive response seen with Balanus nauplii was clearly
absent.
Response to Temperature
Newly hatched nauplii from the same brood (incubated at 20°C) were
subdivided and placed into various temperature boxes for 24 hours to test the
effects of temperature on swimming velocity (Table 18-1). The metal
lightshield, glass filters, and beaker with larvae were equilibrated to the test
temperature, then transferred immediately to the microscope stage for brief
taping. Readings with a temperature probe indicated a maximum 2°C shift
toward ambient occurred during taping.
Even with the potential of a 2°C deviation in test temperatures, certain
geographical distinctions are suggested between swimming velocity and
temperature (Table 18-1). Data from Balanus amphitrite and B. improvisus
nauplii from South Carolina adults suggest a direct relationship of increased
swimming velocity with increased temperature. In contrast, B. improvisus
277
-------
Table 18-1. Mean Linear Velocities (MLV) of Stage II
Barnacle Nauplii Exposed to Different Temperatures
for 24 Hours.
Species
Temp.
(°C)
N
MDP
(Sec)
MLV + sd
(mm/sec)
T test
BA
28
12
8.5
0.81 ± 0.23
NS
BA
20
24
9.0
0.71 ±0.34
BA
15
22
7.1
0.58+0.20
P=.20
BIS
25
26
7.4
1.18 + 0.56
NS
BIS
20
26
6.0
1.09 ±0.43
BIS
15
25
ao
0.72 ± 0.43
P=.05
BIN
26
13
7.3
1.56 ±0.62
P=.10
BIN
22
17
9.4
2.43 ± 0.64
BIN
10
14
ao
1.25 ±0.64
P=.05
CF
25
26
7.2
1.46 ±0.48
P=.05
CF
20
40
6.4
1.92 ±0.67
CF
15
40
6.0
1.89 ±0.62
NS
nauplii from Rhode Island adults exhibited a decreased velocity above 22°C. A
comparable reduction in velocity with Rhode Island nauplii also occurred with
Chthamalus fragilis above 20°C. Yet overall, naupliar swimming speeds appear
to be greater in the Rhode Island animals when measured within this
temperature range.
Brood Variability
Linear swimming velocity in nauplii obtained from different broods of
South Carolina adults was assessed under similar temperature, salinity, and
light regimes to evaluate brood variability. Linear velocities were found to be
similar among six broods (Table 18-2). Examples of MLV distributions within
test groups of B. amphitrite, C. fragilis, and B. improvisus are shown in Figure
18-4. Control groups usually have linear velocity distributions which
approximate normal or are skewed to the left.
278
-------
Table 1&-Z Mean linear velocites (MLV) of various hatches of
stage 11 Balanus amphitrite nauplii at 20°C.
Hatch
N
MDP
MLV±sd
No.
(sec)
(mm/sec)
3
40
7.0
0.75+0.34
8
57
5.3
0.72 ± 0.20
9
14
6.1
0.65 ± 0.43
14
24
9.0
0.71 ± 0.23
24
23
7.4
0.72 ±0.27
25
20
7.0
0.85 ± 0.33
NOTE: N = number of paths analyzed, MDP = mean duration of paths
analyzed. H
o
30
20-
0
30
JJ
D
MM/SEC
Figure 18-4. Examples of mean linear velocity (mm/sec) dis-
tribution within five test groups of stage II barnacle nauplii:
NOTE: A) Balanus improvisus from Rhode Island at 20°C, B) Chthamalus
fragifis at 20°C, C) Balanus amphitrite control, D) B. amphitrite with 24 hr.
exposure to 20 ppb Cu, E) B. amphitrite with 24 hr. exposure to 350 ppb Cu.
279
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Response to Copper
Having characterized linear swimming velocities for B. amphitrite nauplii
under defined conditions, the effects of sublethal levels of copper on swimming
speeds were investigated. A primary stock solution of 10,000 ppm Cu++ in
dilute nitric acid was adjusted to 1-4 ppm secondary stock solutions using
deionized water. Final test solutions were obtained by serial dilutions with 1
filtered natural seawater at 32-34°/oo. Total copper was determined by heated
graphite absorption on a HGA-2100 coupled to a Perkin-Elmer 360 atomic
absorption spectrophotometer.
Newly hatched B. amphitrite nauplii were exposed to various copper levels
for 24 hours at 20°C. Replicate samples of nauplii for each exposure level were
then video taped in a darkened room using the described darkfield
illumination. Total mortality at 24 hours post-exposure was determined.
Larvae were not fed during the experiment. Analyses indicated a 3-15 percent
decrease in total copper occurred during a 24-hour static exposure. In the first
test, nauplii were exposed to levels ranging from control (3 ppb) to
approximately 50 ppb Cu (Table 18-3). No increased mortality was observed;
however, MLV of nauplii exposed to 10 through 47 ppb Cu nearly doubled
relative to control values. Close agreement was observed in replicate samples.
In a second test, three higher copper levels were added: 120,185, 350 ppb.
After the 24 hour Cu exposure period, nauplii were transferred to clean filtered
seawater with the mixed algal diet. Significant mortality at 24 hour
post-expossure occurred only at 350 ppb Cu. However, a delay in molting was
Table 18-3. Mean Linear Velocities (MLV) of
Balanus Amphitrite Stage II Nauplii Exposed to Sublethal
Copper Levels for 24 Hours at 20°C
Copper
No.
Mean Path
MLV±sd
Mortality 24 Hr.
(ppb)
Animals
Duration (sec)
(mm/sec)
Post Exposure
3
16
6.4
0.71 ± 0.27
3%
3
14
6.2
0.65 ± 0.44
5%
10
12
5.0
1.24±0.48
4%
12
10
7.3
1.18 ±0.51
2%
14
21
7.6
1.09 ±0.54
2%
14
6
7.3
1.06 ±0.76
4%
30
10
7.8
1.07 ±0.54
2%
47
15
6.4
1.25 ±0.47
5%
47
11
7.0
1.16 ±0.48
5%
280
-------
seen at 48 hours post-exposure; only 35 percent of nauplii exposed to 185 ppb
Cu had molted to Stage III, as opposed to nearly 70 percent at lower copper
levels. (Figure 18-5.) Analysis of variance for naupliar MLV indicated a
significant (p=0.01) difference between control (2 ppb), intermediate (18-186
ppb), and highest (350 ppb) copper levels. Swimming speeds significantly
increased at sublethal copper levels but rapidly declined at or near the lethal
level (Figure 18-6.) The shift in distribution of linear velocities for test groups
at these three exposure levels is clearly illustrated by frequency histograms
(Figure 18-4).
In the third experiment, B. improvisus nauplii (from South Carolina) were
exposed to Cu levels ranging from control (3 ppb) to 190 ppb for 24 hours at
25°C. Following video taping at 24 hours, nauplii were transferred to filtered
seawater with algal diet and reared at 25°C. At 24 hours, post-exposure
mortality at 190 ppb was 100 percent; at 150 ppb, 20 percent. Rearing to
cyprid stage indicated no significant mortality differences between controls
and nauplii exposed up to 98 ppb Cu. However, development time appeared
delayed by Cu exposure as low as 50 ppb (Table 18-4).
Cu ppb
Figure 18-5. Mortality 24 Hours Post Exposure and Percent
Larvae Molting to Stage III, 48 Hours Post Exposure, for
Balanus amphitrite Stage II Nauplii Exposed to Various Copper
Concentrations for 24 Hours at 20°C.
281
-------
1.5]
00
k>
1.0
o
UJ
<0
x
5
5
as
NOTE: Squares indicate Ba/anus im-
provisus nauplii at 26°C; stars indicate
Balanus amphitrite nauplii at 20°C,
Standard deviation bars and number of
paths are indicated.
5 10 50 100 500
Cu ppb
Figure 18-6. Mean linear velocity (mm/sec) of stage II barnacle
nauplii following 24 hour exposure to different copper
concentrations.
-------
Table 18-4. Percent mortality and development time to cyprid
of Balanus improvisus larvae following 24 hour exposure of
stage II nauplii to different copper concentrations at 26°C.
Cu
N
Mortality
Days to Cyprid
(ppb)
(%)
Range
Mean
3
75
19
7-15
10.2
25
81
25
7-15
10.7
47
59
19
8-16
11.7
77
64
19
9-16
12.6
98
77
25
9-14
11.6
150
76
61
8-15
10.3
190
70
100
-
NOTE: During the post-exposure period, larvae were reared on a mixed algal
diet of Tetraselmis suecia and Thalassiosira pseudonana at 26 ± 1 °C.
Swimming speeds of the above nauplii were significantly (p=0.01) increased
at 25 and 50 ppb Cu relative to controls, nearly indentical at 98 ppb Cu, and
significantly decreased at 150 ppb Cu (Figure 18-6). Although MLV of control
and 98 ppb Cu populations were equal, the rate of change of direction (RCDI
— absolute value of angular velocities in degrees/second) was substantially
different (Table 18-5). These data show that the nauplii not only changed
swimming speeds but also tended to change swimming patterns with increasing
concentrations of Cu. Examples of path outlines and their computer assigned
RCDI values are shown in Figure 18-7.
Table 18-5. Rate of Change of Direction (RCDI) and
Mean Linear Velocities of Balanus Improvisus Stage II
Nauplii after 24 Hour Exposure to Indicated
Copper Levels at 26°C
Cu
N
Linear Velocity
RCDI
(ppb)
(mm/sec)
(deg./sec)
3
40
0.82
130
24
40
1.43
140
57
40
1.23
151
98
40
0.83
166
151
29
0.49
188
283
-------
Figure 18-7. Examples of computer tracked paths for stage II
Balanus improvisus nauplii and assigned rate of change of
direction values (degrees/sec.): A) 114, B) 53, C) 152, D) 373.
NOTE: Paths A, B are typical of control conditions; path C occurs more fre-
quently with copper present; path D was observed only above 50 ppb copper.
DISCUSSION
Initial results have demonstrated possible use of invertebrate larval
swimming behavior as a sublethal response index. It has also been shown that
for this index to be reliable, the effects of basic experimental variables such as
temperature and light regime on the swimming response of test organisms
should be understood.
Although previous studies on cirriped and brachyuran larvae (2, 11, 25)
indicate no phototactic response is evident above 650 nm, cirriped nauplii
appeared to exhibit a weak response to the present substage light. Spectral
sensitivity of the species tested appears to extend further into the red than
previously reported.
Balanus venustus and B. amphitrite nauplii exhibited two responses to
sudden changes in light. The cessation of swimming by nauplii following a
sudden light decrease is similar to the "sinking response" described for
284
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brachyuran larvae (10). The increase in swimming velocity of larvae following
light increase cannot be fully described without consideration of possible
directional response. Of practical relevance to our video taping and analysis
procedures is that both responses are of brief duration, limited to the first
three to five seconds of an acute illumination change, and can be detected from
calculations of the MLV. The significance of these responses are not yet
understood. Similar behavioral characteristics were not observed in stage II
nauplii of Chthamalus and are also reported lacking in Balanus balanoides (6).
Temperature is known to directly affect swimming rate of invertebrate
larvae (15, 23). All temperatures tested here on second stage nauplii were
within ranges allowing complete development of the barnacles (14). It is clear
for all species tested that small temperature shifts can alter swimming speeds.
The basic influence of temperature observed on larval swimming rates is
probably primarily a function of species and thermal history, yet initial results
with these barnacle nauplii suggest other factors may prove significant. For
example, Balanus improvisus collected from Rhode Island and South Carolina
and maintained at identical laboratory conditions for over one month, released
larvae having apparently different swimming rates relative to temperature.
Maximum MLV occurred at 25°C for South Carolina larvae and at 22°C for
Rhode Island larvae. Similar differences in response were observed in B.
amphitrite from South Carolina and C. fragilis from Rhode Island. Replicate
tests with different hatches are needed to confirm whether geographical
variations persist.
To determine whether swimming patterns of barnacle nauplii are altered by
toxic substances, stage II nauplii were exposed to different copper concen-
trations. Our exposure time to copper was limited to 24 hours. No algal
food was added during this period to preclude complexing of the metal by the
algae. Deprivation of food for 24 hours is not deleterious to the larvae. A 24
hour LC50 of between 200-350 ppb Cu for B. amphitrite nauplii at 20°C and
between 150-200 ppb Cu for B. improvisus nauplii at 26°C was observed.
Similar toxic concentrations have been reported for Balanus crenatus nauplii
(19) and Balanus eburneus nauplii (5). Cyprid larvae or adults were more
resistant to copper in both these studies.
Differences in LC50's observed for B. amphitrite and B. improvisus nauplii
may be related to temperature. Higher temperatures can increase copper
toxicity (3) or at least give this appearance in short-term experiments (21).
Weiss (26), however, found B. amphitrite to be more tolerant of Cu than B.
improvisus at settlement. In either case, toxic effects of Cu are often
cumulative (3); both LC50 levels and sublethal effects probably occur at lower
concentrations with increased exposure times.
285
-------
For 24 hour exposures, concentrations of Cu below 100 ppb were clearly
sublethal to the nauplii tested. Delay in development of B. improvisus nauplii
occurred at 50 ppb Cu and changes in swimming behavior were evident at
15-25 ppb Cu. At the lowest Cu test levels, responses were restricted to
increased MLV, but at higher sublethal concentrations MLV was depressed and
swimming patterns became atypical. A stimulatory effect of very low levels of
copper on swimming activity has also been reported for brook trout (9.5 ppb
Cu) (9) and with sea urchin sperm (<20 ppb Cu) (28).
Forward and Costlow (12) also observed increased swimming activity of
crab larvae exposed to 0.1 ppm of an insect juvenile hormone mimic, although
larval development was not perceptively affected until 1.0 ppm was reached.
On the other hand, sublethal concentrations of mercury and oil are reported to
depress activity of marine crustaceans at nearly all levels tested (8, 18).
Stebbing (23) suggests that apparent stimulatory effects of heavy metal ions on
growth in marine hydriods and other groups are often only temporary and may
represent a normal response to stressors.
Observations on swimming of B. improvisus nauplii indicate that not only
the linear velocity, but also the pattern is altered by Cu. Nauplii swimming in
convoluted paths (Figure 18-7) tends to increase in number in the presence of
copper above control levels. As copper concentrations exceed 50 ppb, paths
with a distinct "wobble" became evident (Figure 18-7). This latter pattern is
possibly a consequence of impaired or abnormal beating of appendages. This
would lead to reduced feeding abilities, as feeding in cirriped larvae is a direct
function of appendage movement. The increased development time to cyprid
observed at higher sublethal copper concentrations may be the result of
difficulties in feeding.
The present study has consistently observed altered swimming behavior of
cirriped larvae at Cu concentrations far below 24 hour toxic levels. Basic
changes in swimming speed per se may prove useful indicators of pollution
stress, but also of great interest are additional effects on larval motile responses
to environmental stimuli or cues (light, chemical, gravity, etc.). The latter may
prove more meaningful in predicting safe levels of pollutants. If short-term
behavioral reaction can be satisfactorly correlated with long-term detrimental
effects, the potential exists for rapid screening of toxic levels using this motile
behavioral qualification technique. Further studies relating observed behavioral
responses to other physiological parameters, and ultimately larval success, are
planned.
286
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ACKNOWLEDGEMENTS
We wish to acknowledge the assistance of Gerald Hoffman and Raymond
Zanni for providing copper analysis; and Richard Steele and Leslie Mills for
culturing algae used to rear larvae.
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Mortility of Sea Urchin Spermatozoa. Biol. Bull. 147: 236-246.
289
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USE OF A LABORATORY PREDATOR-PREY TEST
AS AN INDICATOR OF SUBLETHAL
POLLUTANT STRESS
Christopher Deacutis
U.S. Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
ABSTRACT
A method is presented to quantify the effects of sublethal stress on newly
hatched and older ichthyoplankton using predation vulnerability as a
measurable parameter. A laboratory predator-prey system was developed and
tested using sublethal thermal shock (10°C above ambient water temperature)
as the stressing factor. Fundulus majalis was chosen as the predator and larvae
of Menidia menidia and Paralichthys dentatus as prey organisms. Predation
interactions were quantified by recording all attacks, escapes, and captures,
allowing comparison of escape probabilities (no. escapes/ attack) for control
and shocked prey groups.
Predator escape ability of four and six week old larvae M. menidia was
significantly impaired following a 15 minute, +10°C thermal shock in summer
(thermal test exposure = 30.0°C). Newly hatched and two week old shocked
M. menidia were not significantly different from controls. Tests with P.
dentatus showed an increase in total number of escapes following 10°C
thermal shock in late fall tests (thermal test exposure = 25.2°C).
The potential for laboratory predator-prey tests as behavioral bioassays to
assess sublethal pollutant stress is evaluated, with consideration given to the
several techniques developed to date.
INTRODUCTION
The present study was undertaken to develop a laboratory predator-prey
test system to evaluate relative ecological fitness of larval fish following a
sublethal pollutant stress. Thermal shock was employed in this case. Behavioral
bioassays are considered to be more sensitive indicators of low-level stress in
comparison with mortality bioassays (22). Hence, behavioral tests should serve
to identify the less conspicuous, but nonetheless important limiting effect that
real-world sublethal stress can have on organisms. In the case of laboratory
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predator-prey tests, changes in prey escape success serve to indicate changes in
ecological fitness, which can affect natural mortality rates in localized
populations.
MATERIALS AND METHODS
For this study, larval prey species were restricted to those available from
laboratory culture. Wild ichthyoplankton were not considered because of the
potential for damage due to capture methods, and the difficulty in acquiring
adequate numbers of a single species of the same age. The cultured larval prey
species used were Menidia menidia and Paralichthys dentatus. Six hatched lots
500/lot) of M. menidia were reared to six weeks of age and tested during
this period. Studies with P. dentatus were limited to newly hatched larve only,
as this species experiences high mortality at time of first feeding. All larvae
were reared at the prevailing Narragansett Bay water temperature (M. menidia,
summer—20.5 ±0.7°C',P. dentatus, late fall—15.1 ±0.8°C).
An attempt was made to correlate prey and predator species. Fundulus
majalis, a carnivorous near-shore predator, was chosen as a spatially coexisting
predator of estuarine larval fish (6). Larvae of P. dentatus are not highly
correlated with near-shore predators since they are usually found offshore at
hatching. However, this species was utilized to provide a larval fish with
different swimming abilities. Paralichthys dentatus relies on high fecundity for
successful development and eventual recruitment. Larvae of this reproductive
strategy are usually weak-swimming relative to larvae of a species such as M.
menidia, which has a lower fecundity, but relies on advanced morphological
development and strong swimming capabilities at hatching.
Biological variables controlled for this study include: reproductive condition
of predator (a L:D 10:14 photoperiod was used to minimize reproductive
development interference); nutritive condition (all predators were fed a mixed
daily diet of Tetramarin flake food and adult frozen Artemia salina until 48
hours prior to a test); predator size in relation to prey size (preliminary tests
indicated selection of a predator size of 6-8 cm total length (TL)); feeding
periodicity (all tests were performed at the same time of day); and hunger state
(all predators were starved 48 hours prior to a test).
Forty-eight hours prior to a test, each predator was placed into an
experimental predation tank, which consisted of a polypropylene tub 30 cm
diameter x 12.5 cm deep, with a clear Plexiglas bottom (Figure 19-1). Test
tanks received a continuous flow of filtered seawater ('v 400 ml/min.) pumped
directly from Narragansett Bay. All predation tests occurred between 1300 and
1500 hours at ambient bay water temperatures.
291
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II
+
B
Figure 19-1. Predation tank and inflow funnel (A) and (B)
Observation Bench, front and side view (normally covered
with black plastic).
Thermal Shock Procedures
Larval Menidia menidia were transferred in 100 ml polypropylene beakers
with a horizontal slit 1 cm x 2 cm wide cut 1 cm above the bottom, and
covered with 240 u nylon screening. Larvae were placed into the beakers in
groups of 10, four hours prior to a test and maintained at ambient water
temperature (20.5 ± 0.7°C). For the shock tests, a seawater bath was preheated
292
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to 30.0 ± 0.6°C (A T = 9.7 ± 0.7°C) and beakers were placed into the heated
water at six minute intervals. Containers were aerated throughout the shock
procedure. As a 15 minute exposure period was completed, a beaker was
immersed in seawater 1°C above ambient for a five minute cooling period. The
larvae were introduced into predation tanks via a funnel filled with incoming
seawater (Figure 19-1). Larvae were added at six minute intervals, and most
larvae were eaten by the predator within the first three minutes following
introduction. All control larvae were treated in the same manner as shocked
larvae, but with transfer containers held at ambient water temperature rather
than a higher temperature.
Larvae of Paralichthys dentatus are prone to damage in screened beakers
because of weak swimming ability and great sensitivity to handling (Grace
MacPhee, personal communication). Therefore, intact 100 ml polypropylene
beakers were used as transfer vessels. Ambient bay water temperature during
these tests was 15.1 ± 0.8°C. Groups of 10 larvae were shocked by gently
pouring the contents of each 100 ml beaker into a glass culture bowl (12.5 cm
dia.) containing 100 ml of seawater preheated to 25.2 ± 0.8°C (AT = 10.1 ±
0.6°C). After the 15 minute exposure period, larvae were siphoned into the
predation tank using silicon tubing (9.5 mm dia.). Introductions of larvae to
the thermal treatment were again staggered at six minute intervals, as with M.
menidia. Control larvae were treated in the same manner as shocked larvae, but
with transfer to 100 ml of seawater at ambient water temperature.
Quantifying Predator-Prey Interactions
During the predation tests, all attacks, captures, and escapes were observed
from below and recorded using an Esterline-Angus event recorder. The best
visual field for recording observations was achieved by placing two opposing
light sources (two fluorescent bulbs) above, yet just outside of the visual range
of an observer directly below the tanks, and placing a flat black background
over the tanks (Figure 19-1). This system permitted accurate recording of
predator-prey interactions involving organisms as small as four mm.
Significance of changes in escape probabilities, expressed as no. escapes/attack,
were tested using the Wilcoxon distribution-free rank sum test (13).
RESULTS
Results of predation tests for Menidia menidia are grouped by prey age
categories (Table 19-1). The two oldest larval groups of M. menidia (four week
and six week old) experienced a significant decrease (P < .01) in the number of
attacks, escapes and escapes/attack for shock tests relative to control groups.
The two youngest age groups of this species (newly hatched and two week old)
did not show significant treatment differences in any of the parameters
measured.
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Larval Paralichthys dentatus of only one size class (4 mm TL) were tested.
The results stand in contrast to the findings with older Menidia larvae. A
significant increase in escape ability (P < .01) occurred in P. dentatus larvae
following thermal shock (Table 19-1).
DISCUSSION
Relationship of Results to Upper Thermal Limits
Results of tests with four and six week old M. menidia indicate a possible
adverse effect following an acute thermal increase to 30.0°C from an
acclimation temperature of 20.5°C. The magnitude of this thermal elevation is
close to the one hour TLM value of 31.4°C given by Hoff and Westman (14)
for juvenile M. menidia acclimated to 20°C. The present study points to the
increased sensitivity of behavioral stress indices to monitor effects of
short-term or low level pollutant stress. Indeed, these findings strongly contrast
the view of Austin et al (1). Based on mortality studies of a 13 minute shock of
14°C above a 20°C acclimation temperature, he concluded that this treatment
would not have any important effects on survival of larvae of this species.
The absence of significant differences in escapes in newly hatched and two
week old stressed M. menidia vs. the controls may be real, or could be due to
the low number of tests run and the high variability observed within the shock
groups. More data are necessary before final conclusions can be made on the
sensitivity of these younger larvae to thermal shock.
The increase in escape probability following thermal shock with larvae of P.
dentatus may be due to an increase in altertness or in frequency of locomotory
movements. Because the ambient water temperature was lower in tests with P.
dentatus (15.1°C), the thermal shock did not approach reported lethal levels
(32.0°C CTM at 15°C acclimation, Hoss et al (25)). Increased escape ability
has been reported by Coutant (8) in juvenile Salmo gairdneri when thermal
shock temperatures are well below lethal levels.
Potential Mechanisms of Thermal Shock Effects on Predator
Avoidance
Although the phenomenon of differential predation in thermally shocked
fish is now well documented (8, 25, 27), causal mechanisms for changes in
vulnerability following thermal shock are not known. It has been demonstrated
that the central nervous system is highly sensitive to temperature fluctuations
(4, 23). The thermal sensory receptors are believed to consist of cutaneous free
nerve endings (3), yet behavioral response to thermal shock is not necessarily
limited to free nerve endings. Blood chemistry, membrane permeability, and
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Table 19-1. Results of Predation Tests: Pooled Data for
Each Age Category (Mean and 1 S.D.)
Mean
Mean
Tot. # Escapes
Total #
Group
# Attacks
# Escapes
Tot. # Attacks
Introduced
S
C
S
C
S
C
S C
M. menidia
1-3 day; 4mm
64.8
63.0
17.3
17.0
0.268
0.270
150 150
N = 3 S; 3 C
(11.0)
( 7.6)
(12.0)
( 4.6)
(0.139)
(0.043)
2 week; 9.8 mm
53.0
61.4
26.0
33.0
0.491
0.537
120 150
N = 4 S; 5 C
(19.5)
( 9.7)
(16.5)
( 8.6)
(0.131)
(0.056)
4 week; 12.2 mm
60.4**
74.3
32.4**
45.6
0.536**
0.613
600 420
N = 20 S; 14 C
(13.1)
(13.1)
(12.1)
(12.9)
(0.089)
(0.067)
6 week; 23.5 mm
25.8**
35.5
15.3**
25.4
0.592**
0.715
160 160
N = 8 S; 8 C
( 7.1)
( 8.9)
( 6.3)
( 9.3)
(0.097)
(0.089)
P. dentatus
1-3 day; 4 mm
66.0*
56.8
17.8**
8.4
0.270**
0.148
240 249
N - 5 S; 5 C
( 8.6)
( 2.9)
( 6.3)
( 2.5)
(0.061)
(0.038)
NOTE: S = Shocked Group (15 min. duration, M. menidia
AT = 9.7°C, P dentatus
AT =10.1°C),C= Control Group. *P<.05. **P<.01.
-------
other physiological effects have all been demonstrated to occur following
thermal shock, and may cause behavioral changes (23). Laudien (18) and
Murray (21) both note that the lateral line system in fish is highly sensitive to
rapid temperature changes. It is possible that disruption of normal lateral line
function may potentially decrease response to a predator's attack. Blaxter (7)
considers the free neuromast system in larvae to play an important role in
avoiding predation.
There is evidence that the lateral line may indeed be disrupted by a thermal
shock. Dijgraaf (10) demonstrated that the spontaneous discharge frequency
varies with temperature in the lateral line of Xenopus (Amphibia). Murray
(20), also working with Xenopus, noted that a sudden temperature increase of
10°C would decrease or even completely inhibit the spontaneous discharge
frequency, followed by compensation back to normal levels. Sudden cooling
would cause a sudden increase in frequency. If free neuromast and developed
lateral line receptors of fish larvae react similarly to those of Xenopus
following thermal shock, there are two periods when the normal receptor
frequency would be altered and signal information from the system possibly
masked or inhibited. The first would occur upon contact with water of
increased temperature. Thus, upon interception with a thermal discharge, and
if the temperature differential is high enough, complete inhibition may occur,
cutting off all signals from the lateral line momentarily. Inhibition of
spontaneous discharge probably does not pertain to the present study, since
predators are absent during the initial 15-minute thermal shock period.
However, this initial neural inhibition could render larvae which pass through a
thermal discharge plume more vulnerable to predation. Next, following such a
thermal shock, the larvae experience rapid cooling, which could result in a
sudden increase in lateral line discharge frequency and possible distortion or
masking of near-field environmental stimuli. This latter effect may be involved
in the present study since cooling of larvae occurs just prior to the predation
interaction.
Evaluation of Laboratory Predator-Prey Tests as Sub-lethal
Indicators
Laboratory predator-prey tests, such as the one described here, can be
valuable as a means of observing subtle, but ecologically significant effects of
low pollutant levels. In developing such tests, it is important to evaluate the
strengths and limitations inherent in laboratory techniques utilized by other
investigators (2, 8, 11, 12, 16, 25, 27). One must consider which primary
predation factors are being measured by each method. Bams (2) states that a
differential predation situation is determined by three primary factors:
discovery rate of the prey by the predator; attack rate on the prey; and capture
rate of the prey.
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Discovery rate is assumed to be approximately equal in all methods cited
here since exposure of all prey groups to the predators is complete and equal.
This parameter is best measured by recording the reactive distance to the prey
item (Beukema, 5), a difficult task which is not addressed in any of the above
techniques.
Attack rate can influence differential predation rate in that certain
characteristics of the prey may be perceived by the predator and produce
active selection. This behavior could occur in predation tests where
simultaneous introduction of treated and control prey groups occur, as in the
methods employed by Bams (2), Coutant (8), and Kania and O'Hara (16).
However, as Bams noted, this parameter cannot be quantified by these
methods because group identity of individual prey is not discernible during
attacks.
Differences in capture rate between prey groups are a result of differences in
prey ability to evade an attacking predator. The techniques used by Bams (2),
Coutant (8), and Kania and O'Hara (16) cannot discern between differences in
capture rate and differences in attack rate since the overall result of predation
is measured, and not individual attacks and captures. It is in this regard that the
method devised by Yocum and Edsall (27) is superior, because it specifically
measures the actual instantaneous predation rate as affected by changes in the
rate of capture. Although Sylvester (25) also measured rate of capture (in
terms of mean survival time), Yocum and Edsall found excessive variance
between predator groups in time-to-capture when using his method. Of the
various techniques, those of Yocum and Edsall (27), Bams (2) and Coutant (8)
are considered best suited for laboratory predator-prey studies. The technique
used by Kania and O'Hara (16), is similar to that of Coutant and Bams, but
with the addition of an escape area. This modification limits its application to
those prey species with a specialized behavioral characteristic of shallow water
refuge. Also, there is a probable complication of learning, as prey become
familiar with the predator's area, and the "safe", shallow, screened area used in
the tests. Finally, the 60 hour test duration is fairly lengthy.
In this particular study with larval fish prey, Bams' (and Coutant's) method
of simultaneous presentation of prey from different treatment groups could
not be utilized, as a tag to distinguish prey treatment groups is necessary.
Common methods for identification, such as fin clipping and cauterization
branding, were not feasible with larval prey. A visual dye is unacceptable
because of potential alteration to predator-prey relationships due to
conspicuousness of prey and color preferences in the predator. A number of
fluorescent dyes were tested, but successful dyes were found to alter normal
behavior in fish larvae (Pseudopleuronectes americanus). Efforts to label fish
larvae with a radioisotope were also unsuccessful. Due to these difficulties
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Yocum and Edsall's technique of recording individual attacks, captures, and
escapes was adopted.
Both methods have intrinsic advantages and disadvantages. Bams' method
allows groups of predators to select between treated and untreated prey
simultaneously. However, the test statistic used by Bams is a biased estimate of
instantaneous mortality rate (2, 8). The method used by Yocum and Edsall
records the actual instantaneous mortality rate in terms of attacks, escapes, and
captures. These parameters allow a more accurate representation of changes in
escape capabilities. In this latter method, prey treatment groups are separated,
and the predator does not simultaneously compare prey groups. In a thermal
plume area, where predators have been observed to attack thermally-shocked
prey (8), it is not likely that shocked and unshocked prey would be in close
proximity to one another. However, in studying the effects of other pollutants,
simultaneous comparison of prey behavior may be an important factor in
differential predation, and should be considered.
A number of biological variables which should be considered in designing a
laboratory predator-prey test system, including coexistence of predator and
prey in nature (spatial and seasonal); plausible prey-predator relationship; size
relation prey-predator; reproductive condition of predator; nutritive condition
of predator and prey; feeding periodicity of predator; and hunger state of
predator. Control of many of these variables has already been described in the
Methods section. Perhaps one of the most difficult to control is satiation
(hunger state) of the predator. Satiation state may affect prey risk (5). If a
predator is less motivated to eat, attack efficiency may not be as high, thus
artificially increasing escape rate of prey as measured by Yocum and Edsall's
method. Prey size will affect time to satiation in a predator, and must,
therefore, also be controlled. Optimal prey size can be estimated by calculating
a prey thickness to predator mouth size ratio with 0.5 as optimal (17, 26).
However, even with an optimal prey size and a set deprivation schedule,
individual variability is often substantial. Procedures to categorize motivation
state of the predator are recommended for laboratory predator-prey tests in
order to eliminate this variability. In the present study, the total mean number
of larvae captured per test was calculated for all tests within each prey age
group. A minimum percentage of this mean was chosen as an indicator of
adequate feeding motivation. A 75 percent limit described a minimum level of
22 larvae captured in tests with four week old M. menidia. Because six week
old larvae were larger, the capture minimum was narrowed to 80% of the mean
total larvae captured, giving a lower limit of eight larvae. All tests in which this
minimum capture level was not reached were excluded from statistical
treatment of data. In tests with larvae younger than four weeks, predators did
not reach satiation before completion of the test, and establishment of a
minimum capture level was unnecessary.
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Laboratory predator-prey testing techniques should prove to be a useful
tool in future pollution research. As noted, the various techniques available
offer different approaches to the question of changes in prey vulnerability. The
relative merits of each must be weighed with due consideration to the normal
ecology of the predator and prey utilized, and the biological variables which
must be controlled.
ACKNOWLEDGEMENTS
I would like to thank both Dr. Don C. Miller who suggested this project, and
the staff of the EPA, Environmental Research Laboratory for their cooperation
and helpful suggestions. I give special thanks to Miss Elaina Kenyon for her
invaluable assistance. This work was completed in partial fulfillment of the
requirements for Master of Science, Univ. Rhode Island.
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1. Austin, H.M., A.D. Sosnow, and C.R. Hickey, Jr., 1975. The Effects of
Temperature on the Development and Survival of the Eggs and Larvae of
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2. Bams, R.A., 1967. Differences in Performances of Naturally and
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3. Bardach, J.E., 1956. The Sensitivity of the Goldfish (C. auratusL.) to Point
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4. Battle, H.I., 1926. Effects of Extreme Temperature on Muscle and Nerve
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6. Bigelow, H.G. and W.C. Shroeder. 1953. Fishes of the Gulf of Maine. U.S.
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8. Coutant, C.C.. 1973. Effect of Thermal Shock on Vulnerability of Juvenile
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10. Dijgraaf, S., 1962. The Functioning and Significance of the Lateral-line
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pugio by exposure to Sublethal Doses of Parathion. Trans. Am. Fish. Soc.
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15. Hoss, D.E., W.F. Hettler, Jr., and L. Coston, 1974. Effects of Thermal
Shock on Larval Estuarine Fish-Ecological Implications with Respect to
Entrainment in Power Plant Cooling Systems. In: J.H.S. Blaxter (ed.), The
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Importance in Food Selection by the 15-Spined Stickleback, S. spinachia.
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20. Murray, R.W., 1956. The Thermal Sensitivity of Lateralis Organs of
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BURROWING ACTIVITIES AND
SEDIMENT IMPACT OF NEPHTYS INCISA
Wayne R. Davis
Don C. Miller
Environmental Research Laboratory
U.S. Environmental Protection Agency
Narragansett, R.I. 02882
ABSTRACT
It is suggested here that benthic deposit feeders are an important faunal
group contributing to the flux of materials, including pollutants, between the
benthos and overlying water. The present study has documented the burrowing
and feeding activities of one dominant deposit feeder, the polychaete worm.
Nephtys incisa, at a series of test temperatures spanning the annual thermal
range (0-24°C) of Narragansett Bay, R.I. New burrow development and feeding
are coupled events as the worm penetrates and ingests sediment. Each new
burrow is usually continuous with recently abandoned burrows, which results
in extensive perforation of the benthic sediment. Then as Nephtys ventilates its
burrow for respiratory purposes, sediment oxygenation along the entire
subsurface burrow network also occurs. Rate of new burrow building ranges
from one/20 days at 0°C to one/day at 24°C.
It is hypothesized that Nephtys burrowing, feeding and irrigation activity
contributes significantly to substrate conditioning for development of the
aerobic benthic compartment. Doubtless, pollutant diagenesis is also directly
influenced by this creation of an oxidative environment, resulting in significant
pollutant fluxes to and from the benthos.
INTRODUCTION
The polychaete worm, Nephtys incisa, is common in silty-clay sediments of
the northern Atlantic estuarine and coastal waters. Its dominance in fine
sediment is a unique departure from other Nephtys species, all reported to be
active carnivores inhabiting poor to well-sorted sands (Clark, 1962; Clark etal,
1962). To better understand the anomalous, silty-clay habitat preference ofvV.
incisa, information regarding its in-sediment activities was pursued, primarily
through the use of laboratory microcosms.
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Soft-bodied organisms that burrow into the sediment generally do so for
predator avoidance, at the minimum. Those which burrow continuously
through the sediment, such as the Nephteidae, Nuclionid bivalves and
Haustorid amphipods, do so- to obtain food either as predators or
deposit-feeders. This vagile or wandering mode of life requires adaptations, not
only for burrowing, but also for obtaining sufficient food and dissolved oxygen
in this environment. The specific questions that have been investigated concern
adaptations for burrowing, feeding and irrigation in N. incisa. The present
paper will address certain questions of burrowing activities, while the two
subsequent papers (Davis, 1979 b,c) will deal with feeding and irrigation
activities in N. incisa. These activities are interrelated in that continuous
sediment burrowing is generally a feeding adaptation and necessitates further
adaptation to obtain well-aerated seawater while moving through the sediment.
N. incisa occurs in estuarine, shallow coastal waters and across the Atlantic
continental shelf from Chesapeake Bay northward to Nova Scotia, Greenland
and Iceland, and along the European coast from the North Sea, the Baltic Sea,
and south into the Mediterranean (Pettibone, 1963; Thorson, 1946; Bellan,
1969). Reported population densities include 600/m^ in Long Island Sound
(Sanders, 1956), 300-600/m^ in Narragansett Bay (Davis, Phelps and Morrison,
unpublished), and up to 1500/m^ in Buzzards Bay (Sanders, 1960). Population
age structure has been examined temporally by Sanders (personal
communication) who has observed three and sometimes four year classes, with
each new year class appearing as 2 mm worms during early spring. Density of
N. incisa can be correlated with sediment clay-silt content (Sanders, 1956),
pollution gradients (Farrington, Quinn and Davis, 1973), and possibly
meiofauna density (Tenore et al, 1977).
The types of burrows found among infaunal polychaetes range from totally
permanent to highly temporary. In the case of completely vagile worms such as
the Nephteidae, Nereidae and Glyceridae, this in-sediment wandering may lead
to burrow galleries and multiple openings to the surface. This mode of
burrowing is generally adapted to exploit debris or prey on the sediment
surface while minimizing exposure to predators. Two or more openings to the
water also permit efficient one-way irrigation to obtain dissolved oxygen.
Glycera alba produces such a gallery, using various burrow openings as prey
vibration conduits and will even intercept moving prey at other gallery
openings (Ockelman and Vahl, 1970). Nereis diversicolor has a similar gallery
to better exploit debris on the sediment surface. An interesting adaptation for
secondary filter feeding in this species was described by Harley (1950). Under
certain conditions the respiratory irrigation stream is directed into a mucous
funnel which is then eaten. Other vagile polychaetes burrow to exploit
subsurface organic-containing sediments, for example the Capitellidae,
Maldanidae and Paraonidae. The capitellid Heteromastus filiformis develops
303
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semipermanent vertical burrows to reach deeper sediment in which it
continually burrows and deposit-feeds. Oxygen exchange occurs near the
sediment-water interface using modified posterior segments when the worm
surfaces to defecate (Linke, 1939). Paraonis spp. continually burrow to form a
deposit-feeding ring within a single stratum of high organic content (Gripp,
1927, cited in Schafer, 1962). When the concentric burrowing reaches 8-10 cm
in diameter, the worm burrows to a new stratum to begin another feeding ring.
Partially or non-vagile families such as the Arenicolidae and Chaetopteridae
possess U-shaped burrows and irrigate for the dual purposes of feeding and
respiratory exchange. Families with the least sediment contact, termed
tubicolous polychaetes, include the Sabellidae, Onuphidae and Serpulidae.
These worms develop permanent tubes lined with mucopolysaccharides, shell
debris, sand grains or calcite. They generally feed and ventilate above the
sediment-water interface. There are many exceptions; for instance, the
tubicolous Pectinariidae drag their sand grain tube horizontally through the
sediment using it as an irrigation tube to the surface as they deposit-feed
below.
The present investigation provides information on the microhabitat of N.
incisa by describing the nature of its burrow and examining some of the
variables influencing burrowing. Employing laboratory in situ microcosms,
coupled with direct observation in the field for verification, this investigation
has addressed such topics as the form and make-up of the N. incisa burrow,
how it is constructed, what is its horizontal and vertical extent, and how the
rate of burrowing is influenced seasonally as the worm is exposed to
temperatures which can range from 0-24°C.
MATERIALS AND METHODS
Nephtys incisa used in this study were collected from a station north of
Conanicut Island, Narragansett Bay, Rhode Island (Figure 20-1). This benthic
station is characteristic of a large portion of the Narragansett Bay, where a
clayey-silt sediment covers 60-75 percent of the Bay bottom (McMasters,
1960). Previous studies (Davis, et al., unpublished) found N. incisa density to
drop off rapidly in the sandy sediment toward the Bay mouth and decrease
gradually toward the northern head of the estuary, the latter possibly due to a
pollution gradient (Farrington, et al., 1973).
N. incisa were collected by gently sieving Smith-Mclntire grab samples and
by SCUBA diver-collected box cores, which were then transported intact to the
laboratory and held in flowing seawater. When temperature change was
required, it was shifted at a rate of 2°C per week, which is comparable to the
rate of temperature change in the field (Figure 20-2).
304
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M ASSACHUSETTS
305
-------
1970 1971 1972 1973
Figure 20-2. Narrangansett Bay bottom water salinity, dissolved
oxygen and temperature (monthly measurements at sampling
site, % mile north and southwest of site).
NOTE: Each circle represents a single measurement made on a single day (3
replicate measurements).
Burrowing activities were described and quantified by observing single
worms in sediment-filled thin aquaria (2 cm thick, 14 cm wide and 15 cm
deep). These aquaria were maintained in flow-through systems at appropriate
temperatures. The thin aquaria allowed free three-dimensional movement
(worms can easily turn in 1 cm thick aquaria), yet also permitted direct
observation of some activities with a stereomicroscope when the worms
burrowed along the glass. The glass walls are considered to represent an
obstacle to the worm not unlike buried rocks or bivalve shells, which are
306
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commonly observed in the field. Experimental worm density was one per
experimental aquarium or one worm/28 cm^. The aquaria were kept in the
dark except for a two-minute interval once weekly when both sides of the
squaria were photographed.
Patterns and rates of burrowing and sediment aeration were described from
sequential photographs of the worm burrows constructed against the glass wall
of the sediment-filled thin aquarium. Sediment aeration by local burrowing and
irrigation was indicated by a change in sediment color from dark to light and
provided a record of the worm's present or past location. This method relies on
oxygen-sediment-col or relationships proposed by Fenchel and Riedl (1970),
Hayes (1964), Teal and Kanwisher (1961), Rhoads, Aller and Goldhaber
(1977), and Aller and Yingst (1978). The absence of oxygen generally leads to
a dominance of reduction reactions (eH<0) including formation of iron
sulfides which blacken the sediment. At the point of change from dark to light
color in the sediment, values for both eH (volts) and dissolved oxygen (mg/1)
begin to increase from 0.0. Presence of oxygen is key to substrate oxidation
reactions (eH<0). By quantifying the development of this color discontinuity
against the thin aquarium glass wall where worms are burrowing, it is possible
to document three parameters of burrowing activity: 1) the spatial-temporal
extent of burrowing, 2) the effective new surface area of the sediment-water
interface, and 3) the extent of sediment aeration. This record, visible against
the aquarium wall, can be photographed at appropriate time intervals and
activity quantified by counting burrows, measuring the surface area of burrow
linings and by planimetry, measuring the volume of aerated sediment.
Horizontal burrowing patterns were also described by recording temporal and
spatial appearance of new burrow openings at the sediment surface in large
sediment-filled dishes (single .2-3 g worms in 3 x 6 x 4" deep sediment trays).
RESULTS
Description of the Burrow
Nephtys incisa actively penetrates fine sediments and establishes an
open-ended burrow with no visible modification of the burrow wall except
packing. It is not known if mucous, exuded onto parapodial setae during
feeding (Davis, 1979b), is present in the burrow wall. The burrow is often
W-shaped, but many variations exist. Back and forth motion of the worm with
packing of the burrow wall by setal bundles creates a section which is closely
fitted to the front and mid section of the worm. This precise fit permits
flow-through irrigation by the parapodial cilia (Davis, 1979c). The occupied
burrow is often continuous with a recently abandoned burrow posteriorly,
which then continues to receive oxygenated water before it returns to the
surface. Abandoned burrow segments gradually fill with suspended particulates
307
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and/or from the collapse of old burrow walls. There is also avalanching of
surface floe into efferent and afferent burrow openings. The volume of
suspended sediment transported in this manner into the deeper benthos then is
proportional to total burrow volume.
Method of Burrow Building
New burrow building is accomplished as N. incisa first penetrates the wall of
its existing burrow with an undulating proboscis, displacing small amounts of
sediment. Worm position for this initial step is maintained through hydrostatic
enlargement of the posterior segments. The worm next penetrates the sediment
by lengthening the anterior segments and finally, as the head penetration
reaches its forward limit, the pharynx everts, creating a bulbous cavity in the
sediment. This type of sediment penetration has been described as "bolting"
by Schafer (1962). He states that this is a common form of sediment
penetration and is accomplished by contracting all body musculature. The
resulting pressure forces coelomic fluid into the anterior region, forcing those
segments without longitudinal muscles to expand and finally everting the
pharynx at peak pressure. When the pharynx is re-inverted, N. incisa swallows a
slurry of sediment which was created as the compacted sediment was
penetrated. The whole sequence is repeated until the worm occupies the new
burrow fully and has established a new opening to the surface. The entire
process can usually be accomplished in less than an hour.
Spatial Extent of Burrowing
A series of observations were made to determine if burrowing followed
some functional pattern vs random burrowing and also to determine the
horizontal and vertical scope of burrowing. A typical sequence of new burrow
formation is illustrated in Figure 20-3. This figure is a two-dimensional
reconstruction of a three-dimensional activity which is typically only partially
visible against the glass wall. It represents an example of burrowing but does
not indicate a predictable pattern of burrowing. Figure 20-3 shows actual
tracings from weekly photographs of a different burrow building sequence (a
single worm over a six-week period at 18°C). Observations were also made with
worms in large dishes of sediment so that horizontal movement could also be
assessed without wall interference. Each new afferent burrow opening was
mapped, with new openings connected as a series of vectors (Figure 20-4). At
the time of the last recording, the worm is probably lying in a burrow between
opening 10 and 11, with the head located toward opening 11. The magnitude
of each horizontal vector was found often correlating with the size of the
worm (Figure 20-5), with burrow length approximately 2-3 times the length of
N. incisa. Yet exceptions do occur, as shown in Figure 20-4 by the short
distance to afferent openings 2 and 4. By observing 30 six-week sequences of
308
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NOTE: A. Diagram of the reconstructed sequence of new burrow building
(burrows nos. 2 and 3) by a single worm, with collapse of original burrow (No.
1). "A", anterior, "P", posterior; direction of movement. B. Diagram of the
vertical burrow network developed by a single worm and its persistence over
time (18°C).
309
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Figure 20-4. Horizontal burrowing pattern of a single Nephtys
incisa as indicated by the sequence of new afferent burrow
openings appearing on surface of large sediment-filled box
over a period of 2 weeks (18°C).
NOTE: Afferent opening no. 11 indicates most recent burrow opening.
I
S
5
18
16
14
12
IO
8
6
4
2
.02 .03 .04 .06 .08 IO
.20 .30 .40
WORM WEIGHT (g)
Figure 20-5. Relationships of size of N. incisa to burrow length
using a flexible cm rule.
NOTE: Each vertical bar represents the range of 4-7 observations of a single
worm of the indicated weight. N = 7 worms.
310
-------
vertical and horizontal burrowing, it was concluded that no pattern of burrow
gallery formation existed but that burrowing was a meandering extension of
past burrows.
Depth of sediment penetration likewise correlates with worm size (Figure
20-6). The lower vertical limit of burrows for first-year worms (up to 0.1 g wet
weight) is 2-3 cm beneath the sediment-water interface. Second-year worms
(up to 0.4 g) limit burrowing to 7-8 cm. Three-year old worms (up to 1 g) may
burrow as deep as 14-15 cm. N. incisa of all sizes may be found near the
0,
WORM WEIGHT (g WET WT)
.2 .4 .6 .8 1.0
1 1 1 1 1— I I I 1
10
Figure 20-6. Nephtys incisa Mean Depth of Burrowing in
Relation to Worm Size.
311
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sediment-water interface, indicating that N. incisa may perforate sediment at
all levels down to its size-limited depth.
Temporal Aspects of Burrowing
Rate of new burrow formation is temperature-dependent. Figure 20-7
illustrates both the numbers of new burrows formed per week and its
reciprocal, duration of burrow occupancy, at five temperatures spanning the
annual thermal range. At 0°C, burrow turnover averages one new burrow built
every two weeks. At 6°, an average of 1.5 burrows are built per week; 3-3.5 at
12°; 3.5-4 at 18° and 6.5-7 burrows were observed per week at 24°C.
BURROW FORMATION RATE (PER WEEK)
— row -c.ui0i~JCO(£
-------
Burrow-Sediment Relations
The sediment color along present and past burrows was also useful in
semi-quantifying the role of N. incisa in 1) aeration of sediment and 2) in
increasing the effective surface area of the sediment-water interface into the
benthos. There is always some oxygen diffusion across any sediment-water
interface, assuming the overlying water is oxygenated. Wherever burrows
penetrate the sediment and are irrigated with oxygenated water, a halo of light
brown or yellow oxygenated sediment soon develops around the burrow. The
transition of yellow-brown to black, 2-5 cm deep, approximates the limit of
oxygen penetration. Thus an increase in "aerobic" sediment is described by the
expression:
^halo ~ ^burrow-halo system " ^burrow
This volume was estimated here by measuring the light brown oxidized zone
visible against the thin, sediment-filled glass aquaria using planimetry. This
subsurface oxygenation persists for some time after a worm abandons the
burrow, since efferent oxygenated irrigation water typically continues to
course through old burrows. The rate of increase in sediment aeration
following introduction of a single worm in a thin aquarium at 18°C is
summarized in Figure 20-8. The 2-5 mm thick "aerobic" halo is continuous
with and as thick as the aerobic zone at the sediment-water interface (see
Figure 20-3). The dotted line in Figure 20-8 represents the depth of aerobic
sediment in an aquarium without any worm present, that is, the aerobic zone
at the sediment surface. Any increase above this level represents that resulting
as a consequence of burrow irrigation activities. By comparing the aeration
rates depicted in Figure 20-8, it is apparent that the slope of the curve increases
with water temperature. This rise in sediment aeration eventually levels off in
time as an equilibrium develops between oxygenation of new burrow sediment
and chemical reduction of oxygenated sediment along old abandoned burrows.
Figure 20-8 summarizes the relationship between temperature and rate of
Nephtys sediment aeration observed in laboratory in situ thin aquaria. It is
apparent that the extent of sediment oxygenation is positively correlated with
temperature, i.e. more oxygen is delivered to deeper layers during warmer
seasons. Hence, even though oxygen demand by the benthos is at its maximum
during warm periods, the actual sediment aeration through Nephtys burrowing
and irrigation can be even greater.
DISCUSSION
N. incisa burrows through sediment using adaptations previously described
for other Nephtys and Nereis species (Schafer, 1962) and for Arenicola marina
(Wells, 1952). This specialized locomotion called "bolting" refers to the head
313
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NOTE; The quantity of aerated sediment visible against the thin aquarium
wall is expressed as mean depth of aerated sediment, although its distribution
is related to burrow location as indicated in Figure 20-3.
NOTE: Dotted line represents mean depth of aerobic sediment in sediment-
filled, thin-walled aquaria in absence of benthic organisms. Mean aerobic sed-
iment depth is measured through planimetry of vertical burrowing pattern (e.g.
Figure 20-3).
314
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being forced into consolidated sediment. This is accomplished by contracting
all body muscles, creating coelomic fluid pressure to expand anterior segments.
The hard, bullet-shaped muscular proboscis is forced into the sediment and the
pharynx finally everted when coelomic pressure reaches its peak. At this point
N. incisa inverts its pharynx, literally sweeping an emulsion of sediment into
the midgut, carried by fleshy, finger-like papillae at the tip of the everted
pharynx. Immediately the posterior region of the worm crawls into the new
cavity through peristaltic contractions. The bolting action is then repeated
until a new burrow to the surface is complete. Other means of polychaete
locomotion are also used by N. incisa for sediment penetration. Body
undulation common to nereids is typically used by N. incisa to enter sediment
from the water column. This more rapid sediment penetration is used only
when N. incisa lacks a sufficient anchor on the sediment. Peristaltic locomotion
in the Capitellidae is limited to movement within a burrow cavity. Both
undulation and peristalsis involve a wave of segmental muscle contraction along
the body from head to tail if locomotion is directed forward (Schafer, 1962).
Burrow maintenance by N. incisa appears limited to packing loose sediment
against the burrow wall as observed in Nereis spp. and is termed "wallpapering"
(Schafer, 1962). Burrow wall integrity may also be maintained by mucous
since it is readily observed covering the setae. In addition, Schafer suggested
that the iron oxides in the surrounding sediment (oxide halo) is itself a local
"cementing" of sediment.
N. incisa develops temporary burrows, but unlike the continuous burrowers
(e.g. Paraords, Heteromastis or Pectinaria), N. incisa rapidly completes a new
open burrow and then remains in it from one day during the summer to three
weeks in the winter. Such a burrowing sequence suggests that N. incisa is
abandoning discreet burrows rather than continuously meandering. The period
of temporary burrow residence, occupied with feeding and irrigation activities,
will be addressed in the following two papers (Davis, 1979 b,c).
The magnitude and orientation of new burrow construction may offer
insight to in-sediment adaptations such as feeding or predator avoidance. In N.
incisa the scalar values of burrowing depth and breadth appear to be strictly
worm-size related, with larger worms burrowing increasingly deeper and
covering greater horizontal distances. The vector measurement of sequential
burrow direction appears to be random. This in contrast to other vagile
polychaete burrowers. Glycera and Nereis, for instance, develop burrow
galleries that maximize the worm's ability to exploit large areas of sediment
surface for prey and food debris respectively. The deposit-feeders Paraonis and
Heteromastis burrow in patterns termed "guided meandering". This systematic
exploitation presumably minimizes repeated ingestion of sediment recently
eaten. N. incisa shows no indication of such adaptations.
315
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A general expression describing this depth-related burrowing intensity may
be stated:
i= 1
(1)IZ=S DYi-BTY1+Y2"--Yn
i = n
where Iz is the depth-related burrowing intensity, Dyj is the mean length of
burrowing by the year class (from Figure 20-6), Bj is the
temperature-dependent burrowing rate (Figure 20-7) and Yj is the size of the
2nd year class.
The influence of N. incisa on increasing the sediment-water interface surface
area is best described by computing the surface area of the burrow wall since
the burrow is continuously irrigated and may be stated:
i= 1
(2) S.A.l = 2 Lyi CYj + Cy2 ""' CYn
i = n
where S.A.^ is the burrow lumen wall surface area of a population of N. incisa,
LYj is the mean burrow length of the Yi year class and Cyn is the mean
circumference of the burrow of the nth year class.
Burrow irrigation by N. incisa results in the oxidation of surrounding
sediment. The degree of oxidation has only been expressed in a qualitative
sense here. However, since the thickness of the sediment "halo" is virtually
identical to the oxidized zone at the sediment-water interface, the influence of
N. incisa on oxygenating subsurface sediment can be quantititively expressed
by calculating the volume of light brown aerobic sediment surrounding the
burrow for each year (size) class and extrapolating this figure over the density
of that size class in a square meter of sediment:
i^N.incm = * Vhalo' [Yl+Y2 Yn]
i = n -*
where 0 is the quantity of oxygen-containing sediment, is the volume of
the oxygenated halo (Figure 20-8) and Yj is the density of the 1st year class in
worms/m^.
The silt-clay habitat of N. incisa is unique within the genus Nephtys,
virtually singular in its sand-dwelling, predaceous life mode. This departure in
316
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sediment preference may be related to its equally unique deposit-feeding habit
since silt-clay sediments are typically rich in organic matter. Motility of TV.
incisa in fine sediment can best be described as an extended period of open
burrow habitation followed by its extension of the burrow developing another
temporary burrow. This habit is different from infauna that constantly burrow
as predators, or continual deposit-feeders, or those which reburrow only
because of complete burrow destruction. Whatever the purpose(s) for high
burrowing rates by N. incisa, reported densities of 600 — 1200 per may
significantly perforate the top 10 - 15 cm of sediment during warmer months
(such as suggested by model #1 above). This perforation probably results in a
large increase in the surface area of the sediment-water interface (model #2).
Since both present and some past burrows are irrigated, this expansion of
surface area may result in a general biological model for sediment-seawater
exchange, assuming a gradient as in dissolved oxygen (model #3).
ACKNOWLEDGMENT
We gratefully acknowledge the stimulating discussions and criticisms of Drs.
Allen C. Myers, K. John Scott, and Robert B. Whitlatch. This work is a portion of
the first author's dissertation and much is owed to his major professor, Dr. Winona
B. Vernberg.
REFERENCES
1.Aller, Robert C. and Josephine Y. Yingst. 1978. Biogeochemistry of
Tube-Dwellings: A study of the Sedentary Polychaete Amphitrite ornata. J.
Mar. Res. 36(2):201-265.
2. Bellan, Gerard. 1969. Contribution a l'Etude des Annelides Polychetes de
la Region de Rovinj (Yougoslavia). Jugoslavenska Akademija Znanosti I
Umjetnosti. pp. 25-55.
3. Clark, R.B. 1962. Observations on the Food of Nephtys. Limnol.
Oceanogr. 7(3):380-385.
4. Clark, R.B., J.R. Alder and A.D. Mclntyre. 1962. The Distribution of
Nephtys on the Scottish Coast. J. Anim. Ecol. 31:359-372.
5. Davis, Wayne R. 1979b. Feeding Activities in Nephtys incisa. Part II of
Ph.D. Thesis, University of South Carolina.
6. Davis, Wayne R. 1979c. Irrigation Activities in Nephtys incisa. Part III of
Ph.D. Thesis, University of South Carolina.
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7. Farrington, John W., James G. Quinn and Wayne R. Davis. 1973. Fatty
acid composition of Nephtys incisa and Yoldia limatula. J. Fish. Res. Bd.
Canada. 30:181-185.
8. Fenchel, T.M. and R.J. Riedl. 1970. The Sulfide System: A New Biotic
Community Underneath the Oxidized Layer of Marine Sand Bottoms.
Marine Biology. 7(3):255-268.
9. Harley, Margaret B. 1950. Occurrence of Filter-Feeding Mechanism in the
Polychaete Nereis diversicolor. Nat. 165:734-735.
10. Hayes, F.R. 1964. The Mud-Water Interface. Oceanogr. Mar. Biol. Ann.
Rev. 2:121-145.
11. Linke, O. 1939. Die Biota des Jadebusenwattes. Helgol. wiss. Meeresunters.
1:201-348.
12. McMasters, Robert L. 1960. Sediments of Narragansett Bay System and
Rhode Island. J. of Sedimentary Petrology. 30(2):249-274.
13. Ockelman, Kurt and Ola Vahl. 1970. On the Biology of the Polychaete
Glycera alba, Especially Its Burrowing and Feeding. Ophelia. 8:275-294.
14. Pettibone, Marian H. 1963. Marine Polychaete Worms of the New England'
area. Part 1. Museum of Natural History, Smithsonian Institution.
15. Rhoads, Donald C., Robert C. Aller and Martin B. Goldhaber. 1977. The
Influence of Colonizing Benthos on Physical Properties and Chemical
Diagenesis of the Estuarine Seafloor. In: Ecology of Marine Benthos. Ed:
Bruce C. Coull. Univ. So. Carolina Press, Columbia, So. Carolina, pp.
113-138.
16. Sanders, Howard L. 1956. Oceanography of Long Island Sound,
1952-1954: X. The Biology of Marine Bottom Communities. Bull. Bingham
Oceanogr. Coll. 15:345-414.
17. Sanders, Howard L. 1960. Benthic Studies in Buzzards Bay. III. The
structure of the soft-bottom community. Limnol. and Oceanogr.
5(2): 138-153.
18. Schafer, Wilhelm. 1962. Ecology and Palaeoecology of Marine
Environments. Oliver and Boyd. Edinburgh, Scotland. 568 p.
19. Teal, John M. and John Kanwisher. 1961. Gas Exchange in a Georgia Salt
Marsh. Limn, and Oceanogr., 6:388-399.
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20. Tenore, Kenneth R., John H. Tietjen, and John J. Lee. 1977. Effect of
Meiofauna on Incorporation of Aged Ellgrass, Zostera marina, Detritus by
the Polychaete Nephthys incisa. J. Fish. Res. Bd. Canada. 34:563-567.
21. Thorson, Gunnar. 1946. Reproduction and Larval development of Danish
Marine Bottom Invertebrates, with Special Reference to the Planktonic
Larvae of the Sound (Oresund). Meddelelser fra Kommissionen for
Danmarks fiskeriog Havundersogelser. 4(1):523 p.
22. Wells, G.P. 1952. The Proboscis Apparatus of Arenicola. J. Mar. Biol. Ass.
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SECOND GENERATION PESTICIDES
AND CRAB DEVELOPMENT
John D. Costlow and C. G, Bookhout
Duke University Marine Laboratory
Beaufort, North Carolina 28516
ABSTRACT
A number of compounds have been introduced recently as potential
substitutes for the traditional "hard" pesticides in the control of insect
populations. Some of these compounds, juvenile hormone mimics or analogs,
are intended to simulate the activity of naturally occurring juvenile hormones
and prevent metamorphosis or. in the case of insect growth regulators, control
differentiation or specific physiological processes at specific stages of
development. Because of the phylogenetic relationship between insects and
crustaceans, one might legitimately expect that those compounds which alter
or interfere with the developmental pattern of insects could also have similar
effects on the developmental stages of marine crustaceans.
In salinities of 20 and 35 ppt. 100 percent mortality of megalopa of C.
sapidus occurred when exposed to 10 ppin MONO-585 while 1 ppm reduced
survival from 100 percent to 40 percent. 100 percent mortality in the zoeal
stages of R, Imrrisii was observed with a dilution of 1.0 ppm in reduced
salinities but at 20 and 35 ppt. survival was unaffected. The concentration of
10 ppm MONO-585 was lethal in all experimental salinities. Exposure of C.
sapidus megalopa to 0.1 ppm Methoprene resulted in reduced survival only
when lower temperatures (20-25°C) were used. Juvenile crab stages I through
IV were unaffected by the concentrations of Methoprene used.
The findings of these experiments and their possible significance to normal
development of larvae of these two species within the natural environment are
considered.
INTRODUCTION
A number of compounds have been introduced recently as potential
substitutes for the traditional, "hard" pesticides (DDT, Malathion, Dieldrin,
etc.) in the control of insect populations. Some of these compounds, juvenile
320
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hormone mimics or analogs, are intended to simulate the activity of naturally
occurring juvenile hormones and prevent metamorphosis or, in the case of a
second group, insect growth regulators, to control differentiation or specific
physiological processes during development.
Several authors have reported the effects of juvenile hormone mimics on the
development of insects (15, 17, 19 and others). Only a few studies, however,
are reported on the effects of these compounds on other invertebrates (9,10,
13). Of these Gomez et al (9) and Ramenofsky et al (13) first described the
effect of two juvenile hormone mimics on development and metamorphosis of
the cirripede, Balanus galeatus, and Tighe-Ford (1977) subsequently reported
juvenile hormone analog effects on another species of barnacle, Elminius
modestus. Studies on representative species of other marine Crustacea are
limited, but do include the effect of two juvenile hormone mimics on larval
development of the mud-crab, Rhithropanopeus harrisii (1, 2, 4). A study by
Forward and Costlow (8) describes the manner in which one of these
compounds may affect the behavior of crab larvae. Payen and Costlow (11)
studied the effects of juvenile hormone mimics on gametogenesis of adult
Rhithropanopeus harrisii.
Because of the phylogenetic relationships between insects and crustaceans,
one might legitimately expect that those compounds which would alter or
interfere with the developmental pattern of insects could also have similar
effects during the development of marine decapods.
The present study was undertaken to further explore the effects of two
compounds, methoprene (Zoecon Corporation) and MONO-585 (Monsanto
Corporation) on the development of larvae of estuarine crabs. Specifically,
experiments were designed to determine if these compounds would affect
survival of the larvae, alter the number of larval stages, change the time
required for development of all stages and metamorphosis, or affect the
frequency of molting within the early juvenile crab stages after metamorphosis.
A second portion of the experiment was designed to determine if effects of
these compounds would be altered by changes in such environmental factors as
salinity and temperature.
The two species which were selected for study were the small mud-crab,
Rhithropanopeus harrisii (Gould), and the megalopa of the commercial blue
crab, CaBinectes sapidus Rathbun.
MATERIALS AND METHODS
Following the general rearing procedures described by Costlow and
Bookhout (5) and Costlow, Bookhout and Monroe (7) ovigerous females of C.
321
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sapidus and R. harrisii were brought in from the waters of the Newport Estuary
in the vicinity of Beaufort, North Carolina, and maintained in salinities and
temperatures most closely approximating those of the experimental conditions
until hatching of the larvae occurred. With the experiments on
Rhithropanopeus harrisii, the larvae were set up in separate experimental series
consisting of 50 larvae per species, and maintained in temperature controlled
cabinets with a photoperiod of 12 hours light and 12 hours dark, until the
fourth juvenile crab stage was reached.
In experiments with Callinectes sapidus, which involved only the megalopa
stage, larvae were maintained through the seven zoeal stages of 30 ppt, 25°C
until the final zoeal molt. At that time, 20 megalopa were transferred to each
of the experimental salinity and temperature conditions, and maintained in a
photoperiod consisting of 12 hours light and 12 hours dark until the fourth
juvenile crab stage was reached. Within each of the experimental series a
control series was maintained, and at least one acetone-control series was
maintained, since both methoprene and MONO-585, only slightly soluble in
water, were prepared from the pure compound as an acetone stock solution of
1 ppt.
D
The two compounds used in this experiment were methoprene (Altosid :
ZR-515; isopropyl 1 l-methoxy-3, 7, 1 l-trimethyldodeca-2, 4-dienoate)
manufactured by Zoecon Corporation, Palo Alto, California, and MONO-585
(2, 6-di-t-butyl-4- (aadimethylbenzyl) phenol) manufactured by Monsanto
Chemical Company, St. Louis, Missouri.
In the experiments on Rhithropanopeus harrisii larvae involving MONO-585,
dilutions of 10, 1 and 0.1 ppm were used in combination with 25°C, known
from previous work to be the optimum temperature for development (7), and
salinities of 5, 20 and 35 ppt.
In experiments with Callinectes sapidus megalopa, a variety of salinities,
constant temperatures, and cyclic temperatures were combined with the
dilutions of MONO-585 (10, 1, 0.1 ppm) or methoprene (0.1 and 0.01 ppm).
These included, depending on the particular series, salinities ranging from 5 to
35 ppt and temperatures, constant or cyclic, ranging from 20°C to 35°C. The
specific conditions for individual experimental series will be considered in
connection with the results.
Larvae in all series were checked each day for survival and stage of
development, the numbers being recorded for each experimental series.
Individual megalopa were segregated in plastic compartmented boxes to avoid
cannabalism, and also to facilitate recording the time of metamorphosis to the
first and subsequent crab stage for each individual.
322
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RESULTS
Effect of MONO-585 on Development
Survival of the zoea of R. harrisii was unaffected by the presence of 1.0 and
0.1 ppm MONO-585 when the larvae were maintained in salinities of 20 and 35
ppt (Figure 21-1). In the reduced salinity of 5 ppt, however, total mortality
within the zoeal stages was observed with a dilution of 1.0 ppm, while survival
at 0.1 ppm was higher than that observed for either the seawater control or the
acetone control (Figure 21-1). A concentration of 10 ppm MONO-585 was
lethal in all three experimental salinities and none of the zoeae developed
beyond the first stage.
Megalopa of R. harrisii were affected by the presence of 1.0 ppm
MONO-585 when combined with a high salinity of 35 ppt but survival of this
last larval stage was only slightly reduced at 20 ppt (Figure 21-1). There were
no reductions in survival of megalopa in 0.1 ppm, regardless of the salinity.
In those experimental salinities in which some development occurred, the
time required for development from hatching to the megalopa, megalopa to the
crab, and hatching to the time of final metamorphosis to the crab, was
unchanged by the presence of either 1.0 or 0.1 ppm MONO-585 (Figure 21-2).
The development pattern followed the sequence of four zoeae and one
megalopa normally observed for R. harrisii and no additional or supernumerary
larvae were noted.
As indicated in Figure 21-3, total mortality of megalopa of C sapidus was
observed in all series maintained at 5 ppt, including the control. In salinities of
20 ppt and 35 ppt, 10 ppm MONO-585 resulted in total mortality. One ppm
reduced survival from 100 percent observed in the controls to 40 percent,
regardless of salinity, and 0.1 ppm reduced survival to approximately 90
percent. Time for metamorphosis of the megalopa, from the final zoeal molt to
the appearance of the first juvenile crab, varied from a mean of approximately
8 days to 11 days, but the presence of MONO-585 did not appear to be related
to this variability (Figure 21-4).
When cultured in 5°C, 24 hour cyclic temperature (20-25°; 25-30°; and
30-35°: Costlow and Bookhout, 1971) there was no significant change in
survival of control series or those series of megalopa maintained at 0.1 ppm
MONO-585 (Figure 21-5). There was, however, some reduction in survival of
megalopa maintained in 1.0 ppm MONO-585 coupled with all three cyclic
temperatures. The greatest reduction in survival occurred when the compound
was combined with a salinity of 35.0 ppt, but this effect was reduced when the
cyclic temperature was increased to the maximum level of 30-35°C.
323
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RHITHROPANOPEUS HARRISII
M-0585 25° C
PERCENT SURVIVAL
00
<100
o
o 80
V-
X 60
o
< 40
20
0
r—
0 0
1—1
r—
|—
0
1—
1—1
—
0
r-
r—
100
m
CE SO
o
o 60
1-
I*
< 20
O
Ui
2 0
0 0
0
0
100
a. 8°
O
< 60
e>
Ui
2 40
0
H 20
I-
1
r—
i—|
0 0
0
0
C AC 10 1 0.1 ppm C AC 10 1 0.1 ppm C ACI0 1 OJppm
5% 20* 35%
Figure 21-1. Survival of Larvae of Rhithropanopeus Harrisii
Maintained at 25°C, 5 ppt, 20 ppt and 35 ppt when Exposed to
Three Dilutions of MONO-585.
NOTE: C-control; AC-acetone control: 10, 1, and 0.1 ppm-dilutions of
MONO-585.
324
-------
RH1THR0PAN0PEUS HARRISI!
M-0585 25°C
27
23
CD 23
** 91
QT
o 19
0 0
CD
< 2
g >:
UJ
>
CO
V
<
o
0 B Q
E' B
B
0 0
<
P. 17
rzr
Qr 0b Sq
B <=*bi
C AC 10 I Clppm C AC 10 I O.lppm C AC 10 I O.lppm
5%. 20%. 35%.
Figure 21-2. Time of Development for Zoeae and Megalopa of
Rhithropanopeus Harrisii Maintained at 25°C, 5 ppt, 20 ppt and
35 ppt when Exposed to Concentrations of MONO-585.
NOTE: C-controI; AC-acetone control: 10, 1, 0.1 ppm-dilutions of
MONO-585. The vertical column represents the range and the horizontal line
the mean.
325
-------
CALLINECTES SAPIDUS
M- 0585 25 °C
|
>
o:
3
W
o
tE
100
90
00 80
<
K 70
O
60
2 50
OT 40
a.
o 30
-I
< 20
CD
W 10
o
0 0 0 0
c to i ait
5%.
C 10 I O.lppm
20%.
C 10 I Qlppm
35%.
Figure 21-3. Survival of Megalopa of Callinectes Sapidus
Maintained at 25°C, 5 ppt, 20 ppt, and 35 ppt, when Exposed to
Dilutions of MONO-585.
NOTE: C-control: 10, 1, 0.1 ppm-dilutions of MONO-585. The vertical column
represents the range and the horizontal line the mean.
CALLINECTES SAPIDUS
M-0585 25 ®C
DAYS OF DEVELOPMENT
m 14
a)
< 12
or
o to
O 8
« 6
S 4
* 2
(S
UJ
2
fl fia n
0 0 0 0 0 0
C 10 1 Qlppm C 10 1 O.lppm C 10 1 O.lppm
5%. 20%. 35%.
Figure 21-4. Time Required for Metamorphosis of Megalopa of
Callinectes Sapidus Maintained at 25°C, 5 ppt, 20 ppt, and 35 ppt,
when Exposed to Concentrations of MONO-585.
NOTE: C-control: 10, 1, 0.1 ppm-dilutions of MONO-585. The vertical column
represents the range and the horizontal line the mean.
326
-------
CALLINECTES SAPIDUS
M-0585 - CYCLIC TEMPERATURES
PERCENT SURVIVAL
100
90
80
70
60
0
- 50
1
a 40
30
20
10
0
1
I
1
1
!
1
1
!
1
1
I
1
18%.
25%.
35%.
1,
C 1 PPM QlPPM C 1 PPM QIPPM c i PPM 0.1 PPM
20-25° C 25-30'C 30"35*C
Figure 21-5. Survival of Megalopa of Callinectes Sapidus
Maintained at Three Cycles of Temperature (20-25°C, 25-30°€,
and 30-35°C), Three Salinities (15 ppt, 25 ppt, and 35 ppt)
when Exposed to Two Dilutions of MONO-585.
-------
callinectes sapious
M- 0585 - CYCLIC TEMPERATURES
hI-
B t
I
1,1
I 111 flit ell D.Jfll
I fit" ::»
I
.Jlk.
I 1
a
fl.
y 12—i
' )
I
I
15%.
25%.
Ippm
20-25'C
C ippm Olp,
25"30*C
9-
i- flV fl_
Figure 21-6. Time Required for Metamorphosis of Megalopa of
Callinectes Sapidus (M-1C) and Subsequent Juvenile Molts
(1C-2C, 2C-3C, 3C-4C, and 4C-5C) when Maintained at
Three Cycles of Temperature (20-25°Cf 25-30°C,
and 30-35°C), Three Salinities (15 ppt, 25 ppt, and 35 ppt)
when Exposed to Two Concentrations of MONO-585.
NOTE: The vertical column represents the range and the horizontal line the
mean.
328
-------
Once the megalopa have metamorphosed to juvenile crabs, there is no
significant effect on survival during the subsequent four juvenile molts due to
either salinity or the presence of 1.0 ppm or 0.1 ppm MONO-585 (Figure
21-6). The time required to complete the individual molts (first crab to second
crab, second crab to third crab, third crab to fourth crab, and fourth crab to
fifth crab) varies considerably in all experimental series (Figure 21-6). As might
be expected, these same intervals were considerably reduced when the
megalopa and juvenile crabs were maintained in the cyclic temperatures of
25-30°C and 30-35°C (Figure 21-6).
Effect of Methoprene on Development
Neither the megalopa nor the early juvenile crabs of C. sapidus
demonstrated significant changes in survival when exposed to 0.1 or 0.01 ppm
dilutions of methoprene combined with salinities of 15, 25 and 35 ppt, and
maintained in cyclic temperatures of 25-30°C or 30-35°C (Figure 21-7). In the
reduced temperature cycle of 20-25°C, however, survival was reduced from
20-25 percent in the presence of 0.1 ppm methoprene, in all salinities. Juvenile
crab stages, one, two, three, and four, however, did not show any reduction in
survival when maintained in these same combinations. In the same
combinations of cyclic temperature, salinity, and methoprene, there is no
apparent change in time required for completion of the megalopa stage or in
the interval periods observed for the first and subsequent crab stages (Figure
21-8).
DISCUSSION
The relatively few studies to date on the effect of insect growth regulators
on marine Crustacea have demonstrated that one may expect a variety of
effects, depending upon the species and the chemical compound itself. Gomez
et al (9) and Ramenofsky et al (13) found that hydroprene (Altozar^) caused
premature metamorphosis of larvae of the barnacle B. galeatus while a second
mimic, methoprene (Altosid^) had no effect on the time of metamorphosis,
nor did it prevent settling when a proper substrate was available.
Two other analogs, farnesyl methyl ether (FME) and ethyl, 10, 1 l-epoxy-3,
7, 10, 1 l-tetramethyl-2-cis-trans-6-cis-trans-dodeca-dienoate (Ro-8-4314) were
shown by Tighe-Ford (1977) to interfere with the development of Elminius
modestus larvae, with the effect apparently related to the state of physiological
development of larvae at the time of exposure. Costlow (4) described the
effects of methoprene (Altosid^) on larvae of the estuarine mudcrab,
Rhithropanopeus harrisii (Gould) and indicated that 1.0 ppm resulted in total
mortality of the larval stages, usually within the first two days of hatching. If
the larvae were maintained in salinities as low as 5 ppt, survival within the
329
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CALLINECTES SAPIDUS
ZR-515 - CYCLIC TEMPERATURE
pg||||||||||j
rWm^m
C 0.1 ppm 01 ppm C Olppm Olppm C Olppm Olppm
20-25 *C 25-30 °C 30-35 °C
—
r—
pg^um
r—
¦—-
f—
f—
—
—
—
| —
P—
¦
f—
P—
i i
~r
in
l l
1
aMj«
100
O 80
. 60
O
ro 40
20
i
o o o o o
O CD <0 9 N
os-oz
o o o o o
O ® to + w
02-DI
o o o o o
O (D (0 t N
Dl-W
IVAIAHflS !N30«3d
Figure 21-7. Survival of Megalopa and Early Juvenile Crabs of
Callinectes Sapidus Maintained at Three Cycles of Temperature
(20-25°C, 25-30°C, and 30-35°C), Three Salinities
(15 ppt, 25 ppt, and 35 ppt) when Exposed to Two Dilutions
of Methoprene.
330
-------
Figure 21-8. Time Required for Metamorphosis of Megalopa of
Callinectes Sapidus (M-1C) and Intermolt Periods of Subsequent
Juvenile Stages (1C-2C, 2C-3C, and 3C-4C) when Maintained at
Three Cycles of Temperature (20-25°C, 25-30°C, and 30-35°C),
Three Salinities (15 ppt, 25 ppt, and 35 ppt) when Exposed to
Two Dilutions of Methoprene.
NOTE: The vertical column represents the range and the horizontal line the
mean.
331
-------
megalopa stage was reduced in concentrations of 0.01 ppm and 0.000L
methoprene, but there was no significant effect on the survival for larvae
maintained in higher salinities. The developmental time was not significantly
altered by the two lower concentrations of the compound and super-numerary
larval stages were not observed in any of the experimental series. There was
evidence that the early megalopa stage represented a period of extreme
sensitivity to environmental stress in any form, including the presence of 0.1
ppm methoprene when combined with 5 ppt or 35 ppt (Costlow, 1977).
Christiansen, Costlow, and Monroe (1) reported a significant reduction in
survival of zoeal larvae of Rhithropanopeus harrisii with increasing
concentrations of methoprene (Altosid^: ZR-515) and further observed an
increase in the duration of zoeal stages as the concentration of methoprene was
increased, irrespective of changes in temperature or salinity. Below 0.1 ppm,
methoprene did not inhibit metamorphosis. The work with a second
compound, hydroprene (Altozar^: ZR-512) also resulted in a significant
reduction of survival of larvae of Rhithropanopeus harrisii, and the first stage
larvae appear to be the most sensitive stage within the four zoeae and one
megalopa. Metamorphosis to the first crab stage was not inhibited at
concentrations of 0.5 ppm or lower.
An additional study on the way in which a third compound, MONO-585,
affected the response of larvae of R. harrisii to light, indicated that both
swimming speed and phototaxis were altered by the presence of this compound
at sublethal concentrations (8). Further information on how this general group
of compounds may affect a variety of physiological and developmental
processes in marine crustacean larvae, however, is needed to determine if the
effects observed by previous authors are limited to the relatively few
compounds and few species which have heretofore been studied.
From the present study it would appear that the compound MONO-585 is
not as toxic as methoprene. Although at the concentration of 10 ppm,
MONO-585 was lethal to larval stages of R. harrisii at salinities 5, 20 and 35
ppt, 1 ppm of this compound was only lethal when it was combined with a
salinity known to represent a stress condition to the developing larval stages (5
ppt). In similar studies on the effect of methoprene on the development of R.
harrisii (4) concentrations of 0.01 ppm and 0.0001 ppm methoprene resulted
in a reduction in survival of larvae at a salinity of 5 ppt but did not
significantly affect survival of larvae maintained in the higher salinities. A
concentration of 0.1 ppm MONO-585 had no obvious effect on survival in any
of the experimental salinities. As with experiments on methoprene, duration of
the four zoeal stages and one megalopa of R. harrisii was not affected by the
lower concentrations of MONO-585.
332
-------
Previous studies on survival and length of life of megalopa of Callinectes
sapidus (3) have indicated that they will withstand a wide range of salinity and
temperature and display remarkably uniform survival in all but the lower
salinities (5-10 ppt). In the present experiment, survival of the control series at
20 and 35 ppt was similar to that recorded for previous studies, but the
reduction in survival of the 10 ppm and 1 ppm MONO-585 clearly indicate the
toxicity of this compound, to the late larval stages of the commercial crab
(Figure 21-3). Total mortality was observed when the larvae were exposed to
10 ppm regardless of the salinity, and at 1 ppm, survival was reduced to
approximately 40 percent, while Survival at 0.1 ppm resulted in a slight
reduction in survival relative to the control series (Figure 21-3). As with/?.
harrisii (4), there was no significant reduction in time required for
metamorphosis, regardless of the concentration of MONO-585 (Figure 21-4).
Earlier studies on the effect of cyclic temperatures, as opposed to constant
temperatures, on the survival of larvae of the mud-crab Rhithropanopeus
harrisii (6,16) indicated that at one particular five degree cycle of temperature,
30-35°C, a significantly higher survival could be expected relative to that
observed in a constant temperature of either 30°C or 35°C. In the present
study with megalopa of .Callinectes sapidus, the only obvious effect on survival
in three cycles of temperature combined with three salinities and two
concentrations of MONO-585, was also associated with the high cycle of
temperature, 30-35°C (Figure 21-5). Although there was no significant
reduction in survival at 15 or 25 ppt combined with 1.0 ppm MONO-585,
megalopa maintained in a salinity of 35 ppt, 1.0 ppm MONO-585, showed a
significant reduction in survival at a cycle of 20-25°C and at 25-30°C. When
the megalopa were maintained at 1.0 ppm MONO-585, 30-35°C, survival was
considerably increased but, as with the study on larvae of R. harrisii, there is at
present no obvious explanation as to how this high cycle of temperature
contributes to an increase in survival of the larval stages.
Very little information is available on the way in which early juvenile stages
of any crab respond to natural environmental conditions or artificial
compounds which may be present within the water. From the present study it
would appear that the intermolt period for the first four juvenile crabs may
exhibit considerable variability, but this variability cannot be attributed to
either salinity, temperature, or insofar as this experiment is concenrned, the
presence of sublethal concentrations of either MONO-585 or methoprene
(Figures 21-5 and 21-7).
A broad range of questions remains concerning the physiological response of
many crustacean larvae and adults to the juvenile hormone mimics and insect
growth regulators. Nothing is known as yet as to how these compounds may be
incorporated within the animal, or the way in which they may further alter
333
-------
behavioral or locomotory patterns. While the short-term effects on
development of two species of Decapoda are described in this paper, nothing is
known of the way in which long-term exposure to sublethal concentrations
through a number of successive generations may contribute to mutagenic
effects. Within the realm of the chemistry of these compounds, a number of
questions also remain unanswered. Several authors have described the rate at
which insect growth regulators degenerate within certain natural and artificial
environments (16, 18) but none of these studies have investigated the
degradation rate in either an estuarine or a marine environment. Most of the
research which has been conducted thus far has concentrated on the effects of
the intact compound, and no data appear to be available on either the
breakdown products which may occur under estuarine conditions, their fate in
the natural marine environment, or the way in which they may affect
developmental processes of marine invertebrate animals.
Although it is clear that the juvenile hormone mimics and insect growth
regulators may offer great potential as replacements for many of the more
persistent pesticides, it seems equally clear that a considerable amount of
research remains to be done to assure their proper use within the estuarine and
coastal environments.
ACKNOWLEDGMENTS
We are grateful to Zoecon Corporation^ Palo Alto, California, for providing
the pure compound methoprene (Altosid ) and to the Monsanto Corporation,
St. Louis, Missouri, for supplying MONO-585 for experimental use. This
research was supported by a grant (R-803838-01-0) from the Environmental
Protection Agency.
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336
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SOME SUGGESTIONS FOR THE
COLLECTION AND ANALYSIS OF
MARINE ENVIRONMENTAL DATA
Saul B. Saila, Martin A.M. Hyman and Ernesto Lorda
Marine Experiment Station
Graduate School of Oceanography
University of Rhode Island
Kingston, Rhode Island 02881
ABSTRACT
Some problems of environmental monitoring and baseline studies are briefly
introduced. Use of a simple linear model is illustrated by an example which relates to
determining where to establish monitoring stations along a cross-sectional area of a
hypothetical estuary. A scheme for data collection and a general analysis of variance
table is provided for this particular model. The methodology for analysis of a
specific data set gathered at one station at regular time intervals is outlined in detail.
INTRODUCTION
There seems to be no limit to the demands for more and more data
concerning the problems of prediction and protection of the marine and
estuarine environments. From the volume of data being generated in some
studies, the ultimate goal in monitoring and baseline establishment appears to
be to measure everything, everywhere, continuously. It should be recognized
that even if this virtually infinite amount of data were gathered, there is no
guarantee that it would lead to complete understanding or predictive inferences
from a given system. Thus, any environmental monitoring or baseline study
should be practical and feasible within reasonable time and cost constraints.
However, the data gathered must be accurate, pertinent to the problems at
hand, concise and purposefully collected.
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It is evident from the above that proper sampling of the marine environment
is an important step in monitoring and impact assessment. For example, even
though analytical methods for estimating certain environmental parameters
may be highly accurate and precise, if the sample being analyzed is not
representative, the data resulting from the analysis is relatively worthless.
A sampling program for any environmental monitoring or baseline study
must consider explicitly the following items: a) the number of samples
required; b) sampling frequency; c) parameters to be measured; and
d) sampling locations. These items are premised on some accepted definition of
the level of perturbation or impact which is ecologically significant. It is
recognized that a complete environmental assessment program encompasses a
relatively comprehensive characterization (physical, chemical, biological) of a
system, and includes determination of the potential impacts of pollutants or
environmental changes on human health and ecological systems. Lucas (8) and
Eberhart (5) have provided a review of some of the difficulties in assessing
impacts and have proposed some models as bases for taking and analyzing
environmental data. It is our belief that programs with more limited objectives
of characterizing existing conditions or identifying previously defined impacts
or changes can be developed as subsets of larger environmental assessment and
monitoring programs. It is our objective to briefly describe some aspects of the
design and analysis of such experiments to answer some specific questions on
sampling with particular reference to ichthyoplankton.
Most biotic elements of the environment are highly variable and
everchanging. They must be sampled with sufficient intensity to determine the
course of such changes in time and space. Empirical evidence to date
concerning ichthyoplankton, as well as juvenile and adult fishes, suggests that
intensive sampling over time and space may be necessary to detect reasonable
changes in these populations.
STATISTICAL METHODOLOGY
There have been two general kinds of statistical methodology applied to
environmental impact analysis and monitoring. They are linear model analysis
and time series analysis.
Time series analysis will not be considered in detail in this report. However,
it will be briefly mentioned. In time-series analysis, the correlation of a
response variable to past observations is taken into account in the formulation
of a statistical model. Statistical time-series analysis has been treated in several
excellent books including: Anderson (1), Box and Jenkins (2), and Nelson (9).
The treatment of the methodology of time-series is comprehensive in these
references, and the reader is referred to them for details. Our limited
338
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understanding of the methodology suggests that relatively large amounts of
data gathered at frequent and evenly spread sampling intervals are highly
desirable for this methodology to be effective in most instances.
The general linear model analysis is described at various levels of detail in
several statistics books, such as Cochran and Cox (3), Davies (4), Federer (6),
Kempthorne (7), Sheffe (10), and Snedecor and Cochran (11). This linear
model approach includes analysis of variance and regression analysis. In this
approach variations in a response variable measured over time and space are
decomposed into assignable sources of variations and these variations are
assumed to be additive. Tests of significance, such as F-tests or variance ratio
tests to determine the change in the mean value of some variable from several
sample events, are based on certain assumptions such as a normal probability
density and independent and homogeneous variance (10). Data from samples
taken over time frequently do not conform to these assumptions.
Nonstationary elements, such as seasonal or diurnal, and tidal components are
often present, and the data may be highly correlated in time.
In the linear model approach time, space and sampling locations, along with
replications, become a part of a planned experimental design. As a means for
considering spatial and temporal variability in the linear model, the spatial and
temporal distributions of biota (i.e., ichthyoplankton) are treated as a sum of
responses due to assignable sources of factor levels. In addition,
transformations of the response variable are sometimes used to achieve
homogeneity of variances. Finally, because one can expect certain physical and
biological data to be correlated, these relationships can be effectively utilized
by carrying out multivariate analyses of variance and covariance analyses.
In carrying out the linear model approach to monitoring and impact
assessment, the method involves formulating hypotheses or linear contrasts for
carrying out the statistical tests. Among these contrasts one tests for main
effects due to a defined factor and the interaction of factors of interest.
SCHEME FOR ESTABLISHING SAMPLING STATIONS
We will illustrate the use of a simple linear model by an example which
relates to deciding where to establish monitoring stations along a
cross-sectional area of an estuary which is of interest. For simplicity we will
assume that cost constraints limit the number of samples to about 50. Our
prior knowledge of the problem suggests that we should be concerned about
the depth distribution of the given organism (say winter flounder larvae).
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Assume that triplicate determinations of a given larval species at five locations
along the cross-section of an estuary have been made at three depth levels. Since
each station location has three replicate samples at each depth, the classifications
"depths" and "locations" are completely crossed, while the triplicate determinations
provide three replications for each combination of depth and location. In a
tabulation of the data there would be three columns corresponding to depths, five
rows corresponding to the five locations, arid 15 cells each containing three replicate
observations.
In the general case there are Xjja observations, i = rows, j = l,...,q
columns, a = l,...,i) replicates. The model for the analysis is assumed to be .
xija = V + £ + + xij + 6ija "
In the above model, n is the mean, | represents row effects (location
variability), and t)j represents column effects (depth variability). The
interaction effects Ay represent any variations which may be peculiar to a
particular combination of station and depth, and the effects 8^ are normally
distributed random components with average value zero for each ij.
The general analysis of variance table (Table 22-1) for this particular model
is shown. From this table the partitioning of the degrees of freedom can be
determined as well as the appropriate tests of significance determined. Two
points can be made concerning this model. They are: 1) the variation between
sample locations can be estimated, and 2) the systematic variations between
sample locations can be eliminated from the study of other effects, i.e. depth.
SINGLE STATION EXPERIMENT BACKGROUND
A single sample site was chosen in the West Passage of Narragansett Bay, Rhode
Island to examine within station variability at a water depth of approximately 50
feet. At the time of this report, all of the detailed analyses of the data have not been
completed. However, some indication of the nature of the data and the approach to
be utilized are presented herein.
Sampling was conducted on four days, hourly for 24 hour periods. During each
hour p iired 60 cm Bongo net hauls (505 (i mesh) were made. One tow was taken in
the direction of the tide and the other against it. A data set consisting of 275 samples
taken on 28 June, 1 July and 7 July 1976 are being used for detailed analysis. Oblique
tows of standardized length and duration were made in all cases.
The data collected were separated into two categories: 1) Physical data; and 2)
species counts. The species counts were further separated into three classes:
ichthyoplankton eggs, ichthyoplankton larvae, and zooplankton.
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Table 22-1. General Analysis of Variance Table for First Stage Monitoring Station Selection
(Basis for Table 22-1 Layout)
Source of
Estimate
Sum of Squares
df
Aver. Value of Variance
Estimate
Between rows
WW-"2
P-1
a2 + r?( 1 -gffX2 +VE2
Between columns
Sj = 7?qS|(xj. -*x)2
q-1
02 + r?(1-^-)ax2 + r?paT?2
Interaction
Sij^ijlXij-^.-x.j +^2
(P-1) (q-1)
°2 + T?ax2
Replicates
2
^a(ij) ~ ^ija'xija ~~ xij'
N-Pq
a2
TOTAL
s-2ii
N-1
In the above table:
Xj. = row mean, x. j = column mean;
~xjj = class mean;
P and Q refer to population sizes, large with respect to q in this case.
-------
For each sample, the number, hour, tow direction, wind speed, surface
temperature, mean water column temperature (weighted), bottom temperature,
water level function, light intensity, tidal factor, inside volume, outside volume, time
of first oblique tow, time between tows and time of second oblique tow were
recorded.
SINGLE STATION ANALYSIS
The expected sources of variability in these data are categorized and summarized
as follows:
Major Source
Effect on Density Data
Seasonal
Time of Day
Net Differences
Changes in abundance over the three
sampling days
Changes in abundance over a 24 hour
period due to tidal cycle and/or
water temperature, and diurnal cycle
Differences between the two samples
from nets A and B
Avoidance
Changes in abundance over a range of
tow velocities. Individual species
responses may be light dependent
as well
It was found from an analysis of the physical data that mean tow velocities from
north and south tows (with or against tidal currents) were significantly different.
Therefore it will be necessary to compare overall mean densities from north and
south tows. In order to do this it is first necessary to detrend the data by means of a
polynomial function, and test for the significance of the residuals from various
detrending functions. After detrending the residuals can be used to test for the effect
of tow direction alone.
It should be noted that if a harmonic response to time is demonstrable the
response of the system variables to time cannot be effectively measured by means of
a linear model.
The 275 valid observations in the data set can be classified according to certain
mean effects as follows:
N = (ns) x (np) •: (N^ ) * (r) - m = 275
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where:
rig =3 levels of seasonal effort (3 days)
nD =2 levels of diurnal effort (day-night)
nN = 2 levels of net effect (2 nets)
r = 24 replicates (2 * 12) from 2 tow directions and 12 hours within
each of the 2 levels of the diurnal effect
m =13 missing samples
Due to uniform environmental conditions over the three sampling days the SD
(seasonal - diurnal) interaction is not considered of interest. The second order
interaction, SND (seasonal - diurnal - nets) is considered unrealistic and dropped
from consideration. The general linear model then reduces to:
Yjjkl = yU + Si+Dj+N|< + SNjk + DNjk + Ejjkl
The missing terms SDjj and SNDjjk are pooled with the random error term E jjy
According to the above model, the partitioning of the available degrees of freedom
(N - 1 = 274) for testing results in the univariate analyses of variance Table shown as
Table 22.2. The high sensitivity of the test is guaranteed by the large number of
degrees of freedom available for the error term (W = 267). This analysis of variance
model may be applied to several response variables, including eggs, larvae and
zooplankton.
In addition to linearizing the periodic responses to diurnal and tidal cycles, their
variance components may be removed prior to testing for these responses known to
be linearly correlated with time. The approach will involve spectral analysis and
periodic regression.
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Table 22-2. ANOVA Table for the Analysis of the Variance Due To Selected Main
Effects and Interactions, Showing the Partitioning of
Degrees of Freedom for Testing.
SOURCES OF VARIANCE AND INTERACTIONS
Seasonal (Between Days) S
Diurnal (Between Day and Night) D
Nets (Between Net A and Net B). .N
Interaction (Days) * (Nets) SN
Interaction (Day - Night) * (Nets) DN
LEVELS
d.f.
3
2
= 3 -1
2
1
: 2 -1
2
1
2 -1
2
{3 -1)
(2 -1)
1
(2 -1)
(2 -1)
267*
274
Observations: N = 275 estimates of the density of each species of fish eggs, fish larvae and zooplankton.
•The breakdown of d.f. for the "within" or error term is:
W = (r - 1) (3) (2) (2) + sd + sdn - m = 267
where r = 24 replicates; m = 13 missing observations; and sd = 2 and sdn = 2 are the
d.f. corresponding to the two interactions pooled with the error term.
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REFERENCES
1. Anderson, T. W. 1971. The Statistical Analysis of Time Series. John Wiley
and Sons, Inc., New York.
2. Box, G. E. P. and G. M. Jenkins. 1970. Time Series Analysis, Forecasting
and Control. Holden-Day, Inc.
3. Cochran, W. G. and G. M. Cox. 1957. Experimental Design. John Wiley and
Sons, Inc., New York, xiv + 617 pp.
4. Davies, 0. L. (ed.) 1954. Design and Analysis of Industrial Experiments.
Oliver and Boyd, Edinburgh.
5. Eberhardt, L. L. 1976. Quantitative Ecology of Impact Evaluation. J.
Environmental Mgt. 4:27-70.
6. Federer, W. T. 1955. Experimental Design Theory and Application. The
MacMillan Co., .New York.
7. Kempthorne, 0.1952. The Design and Analysis of Experiments.
8. Lucas, H. L. 1976. Some Statistical Aspects of Assessing Environmental
impact. In: Proc. Conf. on the Biological Significance of Environmental
Impacts, K. K. Sharma, J. D. Buffington, J. T. McFadden (eds.),
NR-CONF-002. pp. 295-306.
9. Nelson, C. R. 1973. Applied Time Series Analysis for Managerial
Forecasting. Holden-Day, Inc., San Francisco.
10. Sheffe, H. 1959. Analysis of Variance. John Wiley and Sons, Inc., New
York.
11. Snedecor, G. W. and W. G. Cochran. 1967. Statistical Methods 6th Edition.
The Iowa State University Press, Ames, Iowa.
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KANEOHE BAY: NUTRIENT MASS BALANCE,
SEWAGE DIVERSION, AND
ECOSYSTEM RESPONSES
Stephen V. Smith
Hawaii Institute of Marine Biology
University of Hawaii
P.O. Box 1346
Kaneohe, Hawaii 96744
ABSTRACT
Kaneohe Bay, Hawaii, is a coral reef/estuary ecosystem presently subjected
to stresses from sewage discharge and runoff. The sewage discharge is scheduled
to be diverted from the bay. This "relaxation" of sewage stress will be a major
ecosystem perturbation: the termination of a chronic stress which has been
imposed, with increasing intensity, on the bay over the past two decades. We
are treating this sewage diversion event as a controlled experiment designed to
ascertain ecosystem responses to such environmental perturbation. The
experiment is being performed by means of time-series field monitoring,
discrete field studies, and laboratory experiments.
The stream runoff imposes short-term, catastrophic stress from fresh water
and sediment influx. The sewage accounts for about 90 percent of the
land-derived nutrient delivery to the bay, thus imposing an influence which
stimulates biological activity.
The sediments in the bay have been a major repository for nutrients
discharged into the bay; nutrient release from the sediments has been, and will
continue to be, a significant process affecting the ecosystem. When the sewage
stress is relaxed, planktonic responses to that event will be more rapid than
benthic responses, both because the plankton are immediately responsive to
the point-source sewage discharge, and because of characteristic high biomass,
efficient nutrient cycling, and limited mobility of benthic organisms.
INTRODUCTION
Kaneohe Bay is a coral reef and estuary complex on the northeast
(windward) coast of Oahu, Hawaii (Figure 23-1). The bay was once renowned
as one of the most beautiful coral reef ecosystems in Hawaii. The reef
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Figure 23-1. Index Map of Kaneohe Bay, Oahu, Hawaii,
Showing the Locations of the Three Sectors of the Bay.
community has deteriorated, and the waters have become more turbid in
response to human perturbations. Chief among these perturbations have been
domestic sewage discharge and stream runoff. Both processes have been closely
related to the tenfold increase of the human population in the watershed over
the past three decades. Banner (2) and Smith (8) have summarized the
historical conditions leading to the present environmental status of the bay.
The present stress regime is about to be drastically modified by diversion of
the sewage discharge to a site removed from Kaneohe Bay. This paper
discusses, from the bias of my own mass-balance approach to ecosystem
analysis, interim results of a team investigation designed to ascertain ecosystem
responses of Kaneohe Bay to the relaxation of sewage stress, and to derive
predictive ability therefrom. The data, and many of the ideas presented here,
are properly credited to other members of the research team. '
1. Working group leaders are: S. V. Smith (chemistry), E. Laws
(phytoplankton), J. Hirota (zooplankton), R. E. Brock (benthos), P. L.
Jokiel (microcosms). Principal cooperators from the Naval Ocean Systems
Center are E. C. Evans III, J. G. Grovhoug, and R. S. Henderson.
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The investigation combines field monitoring with field, microcosm, and
laboratory experiments. The spatial distribution of variables in the bay is
relatively well established; we have been gathering time-series data in the bay
since early 1976. The outfall is due to be diverted shortly after this is written
(November 1977), and we anticipate continued collection of time-series data
for at least one year after the diversion.
PROJECT DESIGN
Kaneohe Bay is relatively well-described spatially, and methods by which
chemical and biological characteristics of marine environments are measured
are reasonably standardized. Therefore the analytical details of the study do
not need discussion at this juncture. Let us instead examine the conceptual
approach to this analysis.
Sewage discharge presently imposes a large and well-documented loading of
biologically active materials on the ecosystem. The change of that discharge
volume with time is known, and the termination date of the discharge will be a
discrete, well-defined event. The discharged materials alter the water
composition near the discharge sites, become incorporated into the food web,
cycle within the ecosystem, and flush from the ecosystem. In addition to
biostimulatory responses from the fertilization of the ecosystem, there may be
responses from the loading of plant and/or animal toxins on the system. When
the discharge terminates, there will be ecosystem responses as both direct and
indirect ramifications of the sewage diversion.
There are three main components to the present study.
1. Routine field sampling, to document the sequence of chemical, plant,
and animal changes through time. This sequence may be divided into "before
diversion" and "after diversion" periods which may be compared as two
distinct statistical populations of data, each of which may show seasonal or
other temporal variations. The frequency of the time-series sampling is largely
dependent upon the assumed or documented time scale of variability. For
example, some water composition variables are measured one or more times
per week, while characteristics of the benthos are measured every two months.
Important adjuncts , to the routine monitoring are the utilization of available
Kaneohe Bay field data antecedent to our own, and use of data from
environments which may be comparable to Kaneohe Bay with respect to some
(but not all) of the natural and artificial ecosystem characteristics.
2. Field studies, designed to answer specific questions about the ecosystem.
These studies may also establish time sequences and spatial variations in the
348
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bay, but they are not undertaken as ongoing routine monitoring. These
measurements are being made before diversion and, to the extent necessary,
will be repeated after the diversion. The sampling design is modified to answer
specific questions. For example: What are the relationships among variables
obviously related to water clarity? While an answer can, to some extent, be
extracted from routine sampling, it is more satisfactorily addressed by sampling
along strong water clarity gradients which may or may not coincide with the
routine sampling stations.
3. Laboratory experiments, also designed to answer specific questions
about the ecosystem. Particular responses of communities within Kaneohe Bay
are best addressed by controlled laboratory experiments. These experiments
vary in volumetric scale from batch phytoplankton cultures in 500 ml flasks, to
flow-through microcosm tanks which are 500 liters or larger in volume. The
questions addressed in these simplified, but controlled laboratory experiments,
cannot be easily answered under natural, and largely uncontrolled field
conditions. Of course, the largest of the controlled experiments is the bay
itself, a "reaction vessel" with a water volume in excess of 200 million m^. The
time scales of these experiments vary from a few days in the flasks, to months
in the microcosms, and several years in the field.
In this presentation, I do not explicitly separate these various research
components. Rather, I synthesize the components into our present view of
total ecosystem characteristics and predicted responses to sewage diversion.
This exercise is, of necessity, a preliminary analysis of our ongoing study.
MAJOR ECOSYSTEM CHANGES IN THE
PAST TWO DECADES
The impact of runoff on Kaneohe Bay is largely in the form of short-term
"catastrophic events." In the past 17 years, there have been three years with
monthly rainfall in excess of 75 centimeters within the Kaneohe watershed
(Figure 23-2). In terms of water delivery to the bay, May 1965 represented an
extreme: most of the rain fell in a 2-day period and was followed by rapid
runoff. A freshwater lens from that storm killed corals and other reef
organisms on the fringing reef and nearshore patch reefs to a depth of up to 1.5
meters (1). The reef flats are less than 1 meter deep, so such a destructive
"freshwater kill" virtually decimated the stenohaline marine organisms of the
reef flats and upper portion of the reef slopes. Below about 2 meters the
organisms were relatively unaffected.
Sediment loading associated with runoff has two general effects on the
ecosystem, one as the material is deposited, the other as the material is in the
water column. Deposition smothers reef organisms and lowers the availability
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60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
YEARS
Figure 23-2. Monthly Rainfall Since 1960 at a Selected Rain
Gauge in the Kaneohe Bay Watershed.
of hard substratum for settlement; particulate material in the water column
lowers light and interferes with feeding mechanisms.
Nutrient loading from sewage discharge has increased about sixfold since
1963 (Figure 23-3). This increase is consistent with the previously cited rate of
human population increase in the Kaneohe watershed. Virtually the entire
nutrient load delivered to Kaneohe Bay is stripped from the water by biological
uptake. There have been several obvious responses to the increased nutrient
loading. Benthic algae are locally abundant on the reef flats and compete
successfully with the corals for space on the reef slopes (2). The zone of
present algal dominance on the reef slopes corresponds with the zone left
undamaged by the 1965 freshwater kill. Phytoplankton standing crop and
productivity are elevated above pre-loading levels; included in this high
standing crop are frequent plankton "blooms" (4, plus our own data). Various
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OLj <-
60 62 64 66 68 70 72 74 76
YEARS
Figure 23-3. Volume of sewage discharge into Kaneohe Bay since
1960.
NOTE: The Kaneohe and Marine Corps discharge enters the southeast sector,
whereas the Ahuimanu discharge flows into a stream which drains into the
northwest sector.
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detritivores are favored directly by organic loading associated with the sewage
input, and indirectly by the products of inorganic nutrient loading (3, plus
additional project data).
The relative impact of streams, rainfall, and sewage on freshwater and
nutrient delivery on Kaneohe Bay can be established by a simple budgetary
analysis. Sewage contributes a minimal amount of freshwater (Table 23-1);
however, sewage accounts for most of the nitrogen and phosphorus delivery
(Table 23-2).
The bay may thus be seen to be sporadically perturbed by fresh water and
associated sediment delivery, and chronically perturbed by nutrient delivery.
This latter perturbation, which has increased dramatically over the past two
decades, will be terminated.
Table 23-1. Spatial Distribution of Water Inputs to Kaneohe Bay
(millions of m^/month)
Process
Runoff +
Rain-
Sector
groundwater
evap.
Sewage
Total
Southeast
2.6
-0.7
0.5
2.4
Central
1.0
-0.8
0.0
0.2
Northwest
5.0
-1.6
0.0
3.4
Total
8.6
-3.1
0.5
6.0
Table 23-2. Total Loading of "New" Nutrients on Southeast
Sector (thousands of moles/day)
Process
Nutrient Sewage Streams Total
Nitrogen 30.5 4.2 34.7
Phosphorus 3.4 0.4 3.8
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NUTRIENT FLUXES
Figures 23-4 and 23-5 illustrate data for several water quality variables at
three diverse locations in Kaneohe Bay. "OF" is within about 100 meters of
the major sewer outfall; "S" is in the southeast sector of Kaneohe Bay; and
"C" is in the central sector. Samples have also been taken from the northwest
sector, where values are similar to those of the central sector.
In order for net exchange of material to occur between two water masses, a
concentration gradient of that material is required. An estimate of the net mass
flux between two well-mixed source waters may be obtained as the exchange
volume times the concentration difference between those water masses. The
concentration differences illustrated by Figure 23-4 qualitatively demonstrate
OF
S
C
O.F
S
I
0.0
I
0.0
P0,
2.0
NH.
2.0
4.0
OF.
0.0
OF -
S-
c -
0.0
dissolved
organic P
0.5
i.o
NO:
0.5
1.0
O.F -
dissolved
organic N
0.0 5.0 10.0
ALL UNITS ARE mmole/m3
Figure 23-4. Concentration of Selected Water Quality Variables
Near the Kaneohe Outfall (OF), Near the Middle of the
Southeast Sector (S), and Near the Middle of the Central
Sector (C).
NOTE: The bars represent the mean values ± one standard error unit.
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OF ¦
S-
c ¦
c
OF -
S
C
PN
50
100
chlorophyll a
OF •
S ¦
c ¦
OF
s
c
POC
250
500
microzooplankton
dry weight
250
500
OF
S
C
macrozooplankton
dry weight
0 100 200
ALL UNITS ARE mg/m3
Figure 23-5. Concentration of Selected Water Quality Variables.
NOTE: Symbols explained on Figure 23-4.
the following points: (1) there is net phosphorus dispersal from the outfall to
the southeast sector, and from the southeast sector to the central sector; and
(2) nitrogen flux occurs from the outfall to the southeast sector, but
insignificant from there to the central sector; and (3) there are also fluxes of
the various particulate materials (Figure 23-5).
Quantitatively, just how significant are these fluxes? The mean residence
time of water in the southeast sector of Kaneohe Bay has been variously
estimated but is apparently about 20 days (10). The volume of the southeast
sector is about 80 x 10^ m^, so the daily exchange volume is about 4x10^
m^/day. Table 23-3 summarizes nitrogen and phosphorus fluxes calculated
from the concentration gradients and exchange volume, and compares these
fluxes with input rates of "new" nitrogen and phosphorus. There is a dramatic
imbalance between stream plus sewage input to the southeast sector, and
advective output from that sector. The calculation is not entirely accurate,
because some material is known to pulse from the southeast sector in a
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Table 23-3. New Nutrient Input Versus Oceanic Advection
from the Southeast Sector (+ is in) (thousands of moles/day)
Process
Nutrient
Sewage
Streams
Advection
Imbalance
Fixed inorg. N
Dissolved org. ISI
Particulate N
Total N
+ 15,8
+13.5
+ 1.2
+30.5
+2.0
+1.0
+ 1.2
+4.2
-0.6
-3.6
-4.5
-8.7
+26.0
Inorg. P
Dissolved org, P
Particulate P
Total P
+ 2.6
+ 0.7
+ 0.1
+ 3.4
+0.2
+0.1
+.01
+0.4
-1.0
-0.3
-0.5
-1.8
+ 2.0
low-density plume which flows northwestward from the sewer outfall.
Nevertheless, either this simple mixing model underestimates advective losses
by two-three fold, or there are additional budgetary terms to be considered.
The budget can be further amplified. There is uptake of nutrients by
planktonic and bentliic algae, and subsequent cycling of these particulate
materials within the food web. There is fallout of particulate organic material
to the lagoon floor and nutrient release from the lagoon floor back into the
water column. We have obtained nutrient release rates, pthered over one year
by using 1-meter diameter Plexiglas hemispheres as in situ incubation
chambers, and we can solve for fallout by difference between nutrient inputs
and outputs (Table 23-4). The advective flux of nutrients from the southeast
sector equals 30-50 percent of the nutrient inputs from terrigenous sources.
The fallout of particulate nitrogen substantially exceeds terrigenous nitrogen
inputs to the southeast sector. The high nitrogen fallout is maintained by rapid
nitrogen release from the sediments. Particulate phosphorus fallout is also high,
although it does not quite exceed terrigenous inputs. As with nitrogen, the
rapid phosphorus fallout is maintained in large part by nutrient release from
the sediments. The steps from stream plus sewage input to particulate fallout
to release from the sediments show a progressive increase in the N".P ratio
(9-*14^<-l8). Material advected from the southeast sector is proportionally low
in nitrogen (N:P ^ 5), largely reflecting the virtually complete uptake of
dissolved inorganic nitrogen from the water.
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Table 23-4. Total Nutrient Budget for the Southeast Sector
(+ is in) (thousands of moles/day)
Process
Sediment
Nutrient Sewage Streams Advection Release Fallout
Fixed inorg. N
+15.8
+2.0
-0.6
+14.4
0.0
Dissolved org. N
+13.5
+1.0
-3.6
0.0(3)
0.0
Particulate N
+ 1.2(1)
+1.2(2)
-4.5
0.0(4)
-40.4
Total N
+30.5
+4.2
-8.7
+14.4
-40.4
Inorg. P
+ 2.5
+0.2
-1.0
+ 0.8
0.0
Dissolved org. P
+ 0.7
+0.1
-0.3
0.0
0.0
Particulate P
+ 0.1
+0.1
-0.5(5)
0.9
- 2.8
Total P
+ 3.4
+0.4
-1.8
+ 0.8
- 2.8
(1) Particulate N & P calculated as follows. Steinhilper (9) gives sewage Part.
N ^ 1 g/m^; flow is 18 x 10^ m^/day; assume N:P= 10:1.
(2) Particulate N & P in streams from stream carbon by Steinhilper (9), plus
assumption of C:N:P= 100:15:1.5.
(3) Our limited data plus Hartwig's (5) data show dissolved org. N and P
flux from sediment is small.
(4) Sediment resuspension of organic material excluded from calculation.
(5) Assume particulate N:P= 10:1.
The lagoon sediments are the repository for most of the "new," or
terrigenous, nutrients which have been delivered to the southeast sector of
Kaneohe Bay. There is a substantial cycling of nutrients between that
repository and the water column, with surprisingly little loss (especially of
nitrogen). This situation has been recognized on the basis of a water-column
nutrient budget. We do not yet have enough sediment nutrient data to establish
a quantitatively defensible sediment nutrient budget, but the nutrient level in
the sediment lends qualitative support to the assertion.
356
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PREDICTED BIOLOGICAL RESPONSES TO SEWAGE
DIVERSION
The post-sewage diversion delivery of land-derived nutrients to the bay will
decrease to about 10 percent of the present delivery (Table 23-2). The
sediment reservoir will temporarily continue to release nutrients, but that
reservoir must eventually be depleted as a fraction of the released material is
constantly lost to advection. The sediment nutrient release to the water
column is diffuse; while that release is sufficient'to sustain a high total standing
crop of plankton, that standing crop will not be as locally concentrated as the
crop presently sustained by the point-source sewage input (Figure 23-5,
chlorophyll).
The central and northwest sectors of the bay presently have 14C produc-
tivity rates of about 5 mg c m~3 hr"1, in comparison with about 9 mg C m~3
hr-1 in the southeast sector and 24 mg Cm"3 hr1 near the sewer outfall.
There is relatively little inorganic nutrient export from the southeast sector
to the other sectors (Table 23-4), so those sectors are not directly affected by
the sewage. They are indirectly affected, because particulate material
produced from the sewage nutrients is swept from the southeast sector and
is sedimented in the other sectors, where it then releases nutrients back to
the water column. Without the sewage point-source "new" nutrient input to
the southeast sector, phytoplankton productivity there will stabilize near
that of the other sectors. Except in the immediate vicinity of the present
sewage plume, actual planktonic biomass decrease associated with the
diversion should be small. Compositional shifts of both phytoplankton and
zooplankton will undoubtedly occur, but we do not anticipate a significant
change in the number of species present. We do anticipate a decrease in the
abundance of certain meroplankton (e.g., barnacle larvae).
The benthos will also respond to sewage diversion, but more slowly than the
plankton. The relatively large biomass, longevity, and relative immobility of
the benthic organisms provide a substantial nutrient pool which is not as
efficiently removed from the system as are suspended and dissolved materials.
Some of the filter-feeding benthic animals, particularly those immediately
within the sewage plume, will not survive lowered food availability. Biomass
may gradually drop, but plant-animal symbioses and other relatively "tight"
pathways of nutrient cycling within the benthos community are mutualistic
strategies which will tend to preserve the status quo. Efficient internal cycling
of phosphorus has been demonstrated in shallow reef benthos communities
elsewhere (6,7). Nitrogen is not as efficiently retained as phosphorus (11);
however, experiments we have performed suggest that, given adequate
phosphorus reserves, the reef benthos community can rely on nitrogen fixation
and nitrification to supplement fixed nitrogen losses (see also 12, 13).
357
-------
Eventually, discrete events will disrupt portions of the benthos community.
Strong onshore winds rip the benthic algae loose from the bottom, and some of
that material is swept from the system. The filter feeding animals and other
detritivores will largely survive until they are killed by fresh water and/or
sediment inputs, although some will starve from lowered planktonic food
availability.
The benthos community of the southeast sector is dramatically different
from the reef community which was once found there, although historical data
are insufficient to document the gain or loss of taxa. Corals, which once
dominated the reefs there, as elsewhere in the bay, survive as isolated
specimens. Benthic algal biomass locally exceeds pre-sewage biomass and shows
large temporal fluctuation. The reef community structure has been obliterated.
Benthos recovery will be back towards a coral-dominated, low-algal biomass
community only if there is adequate substratum for coral settlement; if the
periods between the interruptions by freshwater runoff are sufficient for
community succession to proceed to the successful recruitment of corals; and
if sediment nutrient release cannot maintain the high algal biomass. Banner (1)
reported some coral recovery, in areas not otherwise significantly stressed,
within three years of the 1965 "freshwater kill" previously mentioned. A
return to coral dominance, if it ever occurs, will probably take one or more
decades; shorter-term recovery patterns should indicate the direction of
environmental rebound.
SUMMARY
1. The present biological structure of Kaneohe Bay may be related to the
combination of catastrophic lethal events (runoff) and chronic biological
stimulation (sewage discharge).
2. The nutrient deposition as particulate materials in bay sediments and
subsequent release from those sediments is an important and previously
undocumented part of the internal nutrient cycle within the bay. This efficient
cycle allows very little nutrient loss from the bay and comprises an
instantaneous nutrient delivery to the water column comparable in magnitude
to the sewage input. Of course, the sediment release contrasts with the sewage
input in being a diffuse, rather than a point-source, delivery of nutrients to the
water column.
3. The planktonic portion of the biota can respond rapidly to alteration of
environmental regimes, by virtue of advective exchange with more nearly
oligotrophic waters in the absence of the point-source sewage discharge. The
plankton of the bay retain relatively minor vestiges of the 1965 freshwater kill.
The plankton of the southeast sector should shift rapidly to a post-sewer
358
-------
composition and activity. Some qualitative aspects of the plankton community
— namely the composition of the meroplankton — will have longer term
residual characteristics.
4. As key components of the benthos change, their planktonic larvae
should do likewise (e.g., barnacle nauplii, which dominate some plankton
tows). The style of benthos succession after the 1965 freshwater kill has been
influenced by sewage loading, towards a high plant and animal biomass,
filter-feeding, and detritivore community. There will be a lag in the benthos
response to sewage diversion. The lag will last until catastrophic events disrupt
the long-term inertia maintained by the high biomass, limited mobility, and
mutualism of material cycling among the benthic organisms.
5. This relatively simple examination of mass balance, hydrography, and
trophic structure provides a useful basis for predicting responses of the
Kaneohe Bay ecosystem to sewage diversion. As we test the predictions by
post-diversion observations and continued experiments, we will be able to
refine and generalize our predictive ability further.
ACKNOWLEDGEMENTS
This study is funded by U. S. Environmental Protection Agency grant
R803983 and by the Hawaii Marine Affairs Coordinator. The Sewers Division
of the City and County of Honolulu has provided information for the study.
The investigation is being undertaken by the Hawaii Institute of Marine
Biology in cooperation with the Naval Ocean Systems Center. I thank the
working group leaders and other investigators for this cooperation in this team
endeavor. Hawaii Institute of Marine Biology Contribution Number 533.
REFERENCES
1. Banner, A. -H. 1968. A Freshwater "Kill" on the Coral Reefs of Hawaii.
Hawaii Inst. Mar. Biol. Tech. Rep. 15:1-29.
2. Banner, A. H. 1974. Kaneohe Bay, Hawaii: Urban Pollution and a Coral
Reef Ecosystem. In: Proc. 2nd Int. Coral Reef Symp. (Brisbane)
2:685-702.
3. Brock, R. E., and J. H. Brock, (in press). A Method for Quantitatively
Assessing the Infaunal Community Residing in Coral Rock. Limnol.
Oceanogr.
359
-------
4. Caperon. J.. S. A. Cattell. and G. S. Krasnick. 1971. Phytoplankton
Kinetics in a Subtropical Estuary: Eutrophication. Limnol. Oceanogr.
16:599-601.
5. Hartwig, E. 0. (in press). The Impact of Nitrogen and Phosphorus Release
from a Siliceous Sediment on the Overlying Water. Proc. 3rd Int. Est. Res.
Conf. (Oct. 7-9, 1975) Galveston.
6. Pilson, \1. E. Q.. and S. B. Betzer. 1973. Phosphorus Flux Across a Coral
Reef. Ecol. 54:581-588.
7. Pomeroy, L. R., M. E. Q. Pilson. and W. J. Wiebe. 1974. Tracer Studies of
the Exchange of Phosphorus Between Reef Water and Organisms on the
Windward Reef of Eniwetok Atoll. In: Proc. 2nd Int. Coral Reef Symp.
(Brisbane) 2:87-96.
8. Smith, S. V. 1977. Kaneohe Bay: A Preliminary Report on the Responses
of a Coral Reef/Estuary Ecosystem to Relaxation of Sewage Stress. In:
Proc. 3rd Int. Coral Reef Symp. (Miami) 2:578-583.
9. Steinhilper. F. A. 1970. Particulate Organic Matter in Kaneohe Bay. Oahu.
Hawaii. Hawaii Inst. Mar. Biol. Tech. Rep. 22:1-53.
10. Sunn, Low, Tom, and Hara, Inc. 1976. Kaneohe Bay Data Evaluation
Study. Report Prepared for the U. S. Army Corps of Engineers. 7
separately Numbered Chapters Plus Appendices.
11. Webb, K. L., W. D. Dupaul, W. J. Wiebe, W. Sottile, and R. E. Johannes.
1975. Enewetak (Eniwetok) Atoll: Aspects of the Nitrogen Cycle on a
Coral Reef. Limnol. Oceanog. 20:198-210.
12. Webb, K. L., and W. J. Wiebe. 1975. Nitrification of a Coral Reef. Can. J.
Microbiol. 21:1427-1431.
13. Wiebe, W. J., R. E. Johannes, and K. L. Webb. 1975. Nitrogen Fixation in a
Coral Reef Community. Science 188:251-259.
360
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REPLICABILITY OF MERL MICROCOSMS:
INITIAL OBSERVATIONS
Michael E. Q. Pilson,
Candace A. Oviatt, Gabriel A. Vargo and Sandra L. Vargo
Graduate School of Oceanography
University of Rhode Island
Kingston, Rhode Island 02881
ABSTRACT
Nine microcosms at the Marine Ecosystems Research Laboratory (MERL)
were run in replicate during the fall of 1976. Each microcosm tank is 5.5 m
high and 1.8 m in diameter, contains 13 m^ of water and 0.8 m^ of sediment,
and sits outdoors exposed to ambient light. Water and sediment were from
Narragansett Bay. Water from the bay was run through the tanks at a rate of
330 ml per minute, resulting in a turnover time of about 27 days.
In this paper a set of the data collected during the first four months of
operation is examined to discover the extent to which the microcosms
replicated or diverged from each other and from the bay. Total chlorophyll a,
nutrients, counts of individual phytoplankton species, and some other
observations show that while there was considerable variability among the
tanks at any given time, their overall behavior in the major features of bloom
dynamics and species succession was consistent with that observed in the field.
INTRODUCTION
The development of marine microcosms has accelerated in recent years,
due to an increased interest in investigating the properties of complex
ecological systems, in understanding the effects of pollutants or other
pertubations on such systems, and in using microcosms to carry out
biogeochemical experiments (13) (3) (8) (17) (16) (10) (9) (14). The
imposition of artificial boundaries and the limitation in size inevitably cause
microcosms to differ from the natural systems they model. Nevertheless, the
need to carry out controlled experiments on systems which represent a higher
level of organization than cultures of single species has encouraged various
attempts to pursue microcosm research.
361
-------
At present there are no accepted criteria by which it is possible to establish
whether a microcosm behaves in a way similar to the natural system it is
designed to mimic, or to judge whether its behaviour is satisfactory for use as
an experimental tool. A major concern should be with replicability, but one
difficulty here is that nature herself is highly variable, and it is not easy to
properly frame the tests to be applied.
In general, we suggest that if the enclosed ecosystems maintain similar
species composition and diversity, if the metabolic rates in the systems and the
major chemical fluxes and transformations are within the range of variability of
the natural systems, and if the statistical behaviour of the systems is similar to
that of the natural system, then it is reasonable to conclude that the major
biological activities are carried on in similar ways. If so, one may have some
confidence that the enclosed ecosystems are useful experimental tools.
In this paper we analyse a portion of the data obtained during the first four
months of running the microcosms at the Marine Ecosystems Research
Laboratory, to examine their replicability with respect to each other and to
Narragansett Bay.
FACILITY AND PROCEDURES
Narragansett Bay
Since the microcosms to be described were designed in part to act as a
model of Narragansett Bay, a brief introductory description of this bay is
presented here.
Narragansett Bay is about 40 km long by 18 km wide, oriented N-S with
the mouth opening into Rhode Island Sound (Figure 24-1). The presence of
islands causes a complex tidal current regime and some isolation of regions.
Small fresh water inputs result in a weak salinity gradient from the mouth (31
o/oo) to the northern end ("V20 o/oo). The water column is generally well
mixed, although slight stratification occurs at times. The annual temperature
range is from -1°C to about 25°C. Sediments are generally a mixture of silt and
clay, although sand is found in some locations. Tidal currents resuspend
flocculent bottom sediments in the bay, which has an average depth of about 8
m. The turnover time of the bay, based on a hydraulic model (USACE 1959)
and a numerical hydrodynamic model (6) (7) is about 30 days.
Phytoplankton populations in Narragansett Bay are characterized by a
winter-spring diatom bloom, followed by multiple blooms of flagellates,
diatoms, and micro-flagellates in the summer. There is considerable
year-to-year variation in the occurrence and timing of the various blooms.
362
-------
Figure 24-1. A Map of Narragansett Bay Showing the Location of
the 13 Stations Sampled During the 1972-73 Survey, the Benthic
Station and the Location of MERL.
363
-------
Zooplankton of the bay are dominated by two species of Acartia which
switch dominance depending on season. They are generally present in greatest
biomass in late spring. During summer they are heavily grazed by larval fish,
menhaden and ctenophores. The benthos of the bay consists mostly of
heterotrophic soft bottom communities with Mediomastis sp. and Nucula sp.
dominating numerically. Several areas of the bay have communities dominated
by amphipods; where coarser sediments occur, large bivalves such as Mercenaria
mercenaria and Pitar morrhuana may provide the most biomass.
A eutrophication gradient exists in Narragansett Bay due to sewage inputs
from the Providence River (about 380,000 m^/day). However, the lower bay is
relatively clean and the water quality excellent. Average primary productivity
at one station in the Bay, mostly due to phytoplankton, has been estimated to
be 308 g C/yr (4) of which 45 percent may be consumed by the benthos (12).
Narragansett Bay, as well as much of the marine coastal waters of the
northeast coast of the United States, is characterized by ecosystems in which
most of the photosynthesis is carried out by phytoplankton, but in which the
benthos plays an important part in the total cycling of energy and nutrients.
The microcosm tanks were designed to maintain ecosystems functioning in a
similar manner. The stirrers were designed to direct turbulent energy onto the
sediments, thus effecting a resuspension of flocculent material. The tanks are
exposed to natural sunlight, and their temperature regime follows that of the
bay within a few degrees.
Description of microcosms
A brief description of the facility was presented by Pilson et al. (1977).
Twelve fiberglass tanks are set up outdoors on land adjacent to a laboratory
building. Figure 24-2 and Table 24-1 provide information on the tanks and
some physical characteristics of the systems. All piping to the tanks is PVC or
fiberglass, and water is pumped from a pier 30 m offshore by a diaphragm
pump that appears to be non-destructive to plankton.
Sediment in the microcosms is held in fiberglass containers in the bottom
of each tank. The containers were filled with sediment collected north of
Conanicut Island (near "benthic station," Figure 24-1). An attempt was made
to place the sediment in the right orientation in the containers, but inevitably
considerable mixing occurred. Nevertheless, the major features of the benthic
community in the tanks were similar to those in the bay during the period of
the experiment (F. Grassle, personal communication).
Nine of the tanks were first filled during August, 1976, and maintained on
a flow-through regime (330 ml/min) giving a turnover time of about 27 days.
364
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5.5 m
If
he
A
1.83m
Figure 24-2. Diagram of One of the MERL Tanks.
NOTE: Each fiberglass tank is insulated and has three flanged ports on the side
and one drainage port. The sediment container, also of fiberglass, contains
about 30 cm of sediment. The tanks are filled through a port on the side and
water exits from about 1 m below the surface through a level-control stand
pipe. The depth of water is about 5 m. The mixer moves vertically through an
excursion of about 60 cm with a frequency which is variable but is now set at
about 5 cycles per minute.
Table 24-1. Characteristics of MERL microcosms
Tank interior diameter, 1.83 m
Tank interior height, 5.49 m
O
Surface area, water, 2.63 m
Depth of water, 4.98 m
Volume of water, net, 13.0 m
Salinity, 30 o/oo
Mass of water, 13.3 tons
O
Area sediment, 2.52 m
Depth of sediment, 0.30 m
O
Volume of sediment, 0.756 m
Mass of sediment (wet), 1.10 tons
Mass of sediment (dry), 0.568 tons
365
-------
Except for some difficulties associated with the initial operations, the nine
tanks were run identically during the first four months in order to assess the
replicability of the systems. Some of the data obtained during this time are
used in this paper to test the similarity of the tanks to each other and to
Narragansett Bay.
Biological measurements used in comparisons
Table 24-2 gives a list of the data used in the analyses to be presented here.
A brief description of the analytical procedures follows.
Nutrient analyses were performed using Technicon AutoAnalyser
procedures somewhat modified for our purposes. Chlorophyll a was
determined by the fluorometric method of Holm-Hansen et al. (1965).
Phytoplankton were counted on 1-ml aliquots from a pooled sample (3
depths pooled) from each MERL tank or from single samples taken at the end
of the dock or in the bay. Generally the samples were counted live using a
Table 24-2. List of Measurements Yielding Data Used
for Intercomparisons; Weekly Sampling During
August-December 1976 Unless Noted
MERL
Narragansett Bay*
9 microcosms
a
b c
ammonia
X
X
x —
nitrate "l nitrite
X
X
x —
silicate
X
X
x —
phosphate
X
X
x —
chlorophyll a
X
X
x —
phytoplankton species counts
X
X
X X
zooplankton biomass
X
X
X X
*Narragansett Bay
a. Data from a year-long biweekly survey made in 1972-73 at 13 stations
(surface and bottom).
b. Input water to MERL microcosms, sampled from end of dock near intake
line, close in time to weekly sampling of the tanks.
c. Three stations (S. Quonset, S. Patience Island, and Ohio Ledge area)
sampled weekly from September to November 1976, from Durbin and
Durbin (unpublished data).
366
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Sedgwick-Rafter cell, but when this was not possible counts were done within a
few days of collection on samples preserved with Lugol's iodine.
Zooplankton biomass was measured on split fractions of a pooled sample
of two 1-m^ net tows from each tank. The sub-samples were rinsed with
deionized water, lyophilized and weighed.
The greatest part of the total data set consisted of phytoplankton counts.
About 74 species or species categories were identified in the tanks (Table
24-3), but those that appeared five times or less were eliminated from the
correspondence analysis (2). The remaining 54 species are identified in Table
24-3 by a number in brackets following the species name. Generally only 3 to
20 species were found at one time in any individual count.
RESULTS
When the microcosms were started in mid-August, 1976, the
phytoplankton concentration in the Bay was decreasing after a very dense
bloom (Figure 24-3). Concentrations continued to decrease until the end of
August, and thereafter stayed low until the middle of November when another
bloom began. The MERL microcosms followed a similar course, with the
second bloom beginning somewhat earlier in some of the tanks than in the Bay.
The values for chlorophyll a in water samples taken from the end of the GSO
dock in all cases fell within the range of values plotted for the MERL
microcosms.
Figure 24-4 shows a plot of the number of species of phytoplankton
counted in samples from the MERL microcosms, from the end of the dock and
from three other stations. In nearly every case the number of species in samples
from the dock lies within the range of total species reported for the tanks.
Occasionally the values for the other three stations in the Bay lie outside the
range in the tanks, but the variation appears random and the data sets do not
appear to be separable.
Indices of diversity and similarity were calculated using the phytoplankton
species counts for the period noted. The Shannon index of diversity (Pielou
1969), which takes account of the abundance of each species, was calculated
for each of the tanks and the dock for each of the weekly samples (Table
24-4). The mean value for the dock samples was higher than for the tank
samples, indicating a somewhat greater phytoplankton diversity in the adjacent
bay than in the tanks.
367
-------
Table 24-3. List of Phytoplankton Species or Categories
to which the Counts in Samples from the MERL Tanks
have been Assigned
Diatoms
1. Asterionella japonica (1)
2. Attheya decora (2)
3. Biddulphia aurita
4. Ceratulina bergonii (3)
5. Chaetoceros affinis (4)
6. Chaetoceros compressus (5)
7. Chaetoceros costatus
8. Chaetoceros curvisetum (6)
9. Chaetoceros danicus (7)
10. Chaetoceros decipiens (8)
11. Chaetoceros didymus
12. Chaetoceros gracilis (9)
13. Chaetoceros lorenzianus (10)
14. Chaetoceros subtilus v. abnormis (11)
15. Chaetoceros perpusillus (12)
16. Chaetoceros sp. (solitary, small) (13)
17. Coretheron hystrix (14)
18. Coscinodiscus concinnus
19. Coscinodiscus spp. (15)
20. Coscinosira polychorda
21. Dactyliosoleri mediterraneus
22. Detonula confervacea (16)
23. Ditylum brightwelli (17)
24. Eucampia zoodiacus (18)
Flagellates
49. Amphidinium spp. (38)
50. Dinobryon spp. (39)
51. Dinophysis sp. (40)
52. Distephaneus speculum
53. Ebriasp. (41)
54. Eutreptia sp.
55. Glenodinium sp.
56. Exuviaella baltica
57. Exuviaella sp.
58. Gymnodinium simplex (42)
59. Gymnodinium sp. 1
60. Gymnodinium spp. (43)
61. Gyrodinium sp. (44)
25. Guinardia flaccida
26. Lauderia borealis (19)
27. Leptocylindrus danicus (20)
28. Leptocylindrus minimus (21)
29. Melosira nummoloides
30. Nitzschia closterium (22)
31. Nitzschia longissima (23)
32. Nitzschia seriata (24)
33. Phaeodactylum tricornutum (25)
34. Rhizosolenia delicatula (26)
35. Rhizosolenia fragilissima (27)
36. Rhizosolenia hebetata
37. Rhizosolenia setigera (28)
38. Skeletonema costatum (29)
39. Stephanopyxis turris
40. Thalassionema nitzschioides (30)
41. Thalassiosira aestivalis (31)
42. Thalassiosira decipiens
43. Thalassiosira nordenskioeldii (32)
44. Thalassiosira rotula (33)
45. Thalassiosira sp. (solitary cells) (34)
46. Thalassiosira sp. (unidentified) (35)
47. Thalassiothrix frauenfeldii (36)
48. m pennates (37)
62. Masartia rotundatum (45)
63. Olisthodiscus luteus
64. Oltmanziella sp.
65. Peridinium steinii
66. Peridinium triquetrum (46)
67. Peridinium trochoideum (47)
68. Peridinium spp. (48)
69. Prasinocladus sp. (49)
70. Prorocentrum redfieldii (50)
71. Prorocentrum scutellum (51)
72. Prorocentrum triangulatum (52)
73. Pyramimonas torta (53)
74. ju flagellates (54)
368
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m
>-
X
Q.
O
£T
O
_l
X
o
>-
<
CD
So
o
o
10 I—
o ,
o° <0
8 2]
"5"
-------
Figure 24-4. Species of Phytoplankton
NOTE: Solid lines indicate the range of the number of species of
phytoplankton counted in samples from the MERL microcosms. The large
opaque circles indicate the number of species from the dock samples. Three
small dots connected by a vertical line indicate the number of phytoplankton
species for three stations in upper Narragansett Bay (Durbin and Durbin,
unpublished data).
In order to assess the similarity in types of species in the community a
similarity index (13a) was calculated for each weekly data set. This index (S =
2C/(A+B)) is a measure of the number of species in common between two
samples. Calculations were made for each tank in comparison with the dock
samples, and for some tanks in comparison with each other. Some of these
indices are shown in Table 24-4. In general, the inter-tank similarity indices
were about the same magnitude as the bay-tank indices.
370
-------
Table 24-4. Comparisons of Phytoplankton in the Microcosms and in
Narragansett Bay at the GSO Dock During
the Replicability Experiment.
NOTE: Similarity indices for the comparison of bay with all microcosms and
for microcosm 3 with all other microcosms. Shannon index of diversity (H) for
the microcosms and the bay.
Similarity Index (S) Shannon Index (H)
Bay-Tank Tank3-Tanks
Comparison Comparison Tank Bay
Date
Mean
Range
Mean
Range
Mean
Aug.
16
0.379
0.267-0.485
0.608
0.533-0.710
0.263
0.248
30
0.289
0.143- 0.944
0.617
0.500-0.800
0.021
0.270
Sept
13
0.442
0.267-0.640
0.445
0.261-0.593
0.247
0.270
20
0.325
0.111-0.552
0.398
0.222-0.552
0.327
0.424
27
0.185
0.074-0.333
0.328
0.231-0.581
0.288
0.093
Oct.
12
0.373
0.200-0.500
0.171
-
18
0.262
0.133-0.429
0.308
0.125-0.571
0.048
0.520
27
0.286
0.190-0.545
0.458
0.316-0.667
0.209
0.391
Nov.
8
0.584
0.421-0.696
0.683
0.452-0.824
0.524
0.695
15
0.642
0.571-0.727
0.612
0.516-0.769
0.441
0.525
22
0.584
0.457-0.667
0.671
0.545-0.824
0.586
0.795
29
0.639
0.208-0.757
0.678
0.345-0.762
0.471
0.769
Dec.
6
0.660
0.467-0.788
0.669
0.588-0.757
0.558
0.810
14
0.549
0.483-0.628
0.584
0.455-0.714
0.592
0.933
22
0.530
0.414-0.648
0.584
0.455-0.709
0.599
0.807
28
0.620
0.480-0.743
0.536
0.428-0.667
0.607
0.890
Mean
0.465
0.535
0.372
0.563
Std. Dev.
±0.163
±0.130
±0.201
±0.270
While we do not have concurrent data on nutrients and chlorophyll from
more than one station in Narragansett Bay, the results of a year-long survey at
13 stations in the bay taken in 1972-73 are available. Data from three of these
in the lower west passage of the bay are shown in Figure 24-5. The data set
from the bay is in most respects similar to that from the MERL microcosms.
Chlorophyll concentrations indicated a bloom in November in both the tanks
and the bay which did not occur in 1972. Ammonia concentrations in the
microcosms tended to be higher than in the bay, but mean values generally fell
371
-------
_ORDr
-M v0\! n
v-v*
y* v ¥
v* v
w
W V X
w V-
V V*1*
yxap
¦7»
¦^E7
acw
V». A' -•£
V- V -¦-
"*? 5* ~ V •
ik?
f
NQZ t-NO'S
.03 i4.<;o ,
-CJSK^v
V w*
v.gF-
JR :'L
A;D ^.C«3
fV'Vx
W< S?--
w
-*7
7V< V
**!?-
-OW
w
V-••***
V
-.y
v
Figure 24-5. Data Collected from Surface (x) and Bottom Water (A) at Three Stations in the Lower West
Passage of Narragansett Bay, During a Survey Carried out in 1972-73, are Compared with Mean Values
from the Nine MERL Microcosms (0) During August 17 to December 31, 1976.
-------
within the range of variability in the bay. Phosphate concentrations in the
microcosms tended to be higher than in the bay during the latter part of
September and October. Comparison between the chlorophyll a graphs in
Figure 24-5 and in Figure 24-3 indicates that the timing of the phytoplankton
blooms may have been different in the two years. Nevertheless, the overall
behaviour of the tanks and the bay is difficult to distinguish. Figure 24-5 also
gives some indication of the patchiness and other variability apparent in
Narragansett Bay.
The variability and scatter of the magnitudes of individual measurements
both in the bay and in the tanks show that it is difficult to distinguish tank
behaviour from bay behaviour by using comparisons of single variables. In
addition, various of the single variables are correlated, making statistical
analysis of single variables less rigorous. Accordingly, multivariate statistical
comparisons were attempted.
A stepwise discriminant analysis (11) was performed on a weekly data set
from the microcosms and bay input water collected from August 17 to
December 6, 1976> to observe the replicability of the microcosms. The first
two axes of this analysis explained 84 percent of the variation in the data set
(Figure 24-6). The first five variables in the order of their importance
(nitrate-nitrite, phosphate, ammonia, silicate and zooplankton) accounted for
99 percent of the variation explained in the first two axes (Table 24-5).
Chlorophyll concentration explained so little of the variation (less than 1
percent) that the analysis did not include it. Generally microcosms 1,5,6,7, and
8 were more similar to each other while microcosms 2,3,4, and 9 and bay input
water showed a greater individuality. If the microcosms were exactly similar,
10 percent of each group would be classified into itself and each of the other
nine groups. In fact, microcosm 3 classified 47 percent to itself, microcosm 9
classified 53 percent to itself and bay classified 44 percent to itself, indicating
that these microcosms and the bay had the most individuality (Table 24-5).
The individuality of microcosms 3,9 and bay, as indicated mainly by
differences in nutrient concentrations in the discriminant analysis, was not
reflected in phytoplankton species as analyzed by correspondence analysis (2)
(Figure 24-7). The nine microcosms were not distinguishably different in their
species composition from August to December. Initially all microcosms and the
bay were tightly clustered on the lower right hand side of Figure 24-7. A
bloom in November was reflected in a greater variability in microcosm location
and species location (left hand side of Figure 24-7), but at no time was there a
: characteristic species or species group which caused clearly different
microcosm location.
373
-------
2
84% VARIATION EXPLAINED
CVJ
I 0
<
?68,
BAY
¦2 -I 0
AXIS I
Figure 24-6. A Stepwise Discriminant Analysis of Data on
Concentrations of Zooplankton Biomass, Chlorophyll, Ammonia,
Nitrate Plus Nitrite, Phosphate and Silicate in 9 MERL Microcosms
and Bay Input Water from August 17 to December 6,1976,
was Used to Prepare this Plot of Centroids in Reduced Space.
NOTE: Detailed information on the Analysis is presented in Table 24-5.
A comparison of microcosm data and bay data allows us to determine
whether the greater variability of some of the microcosms makes them truly
different from the bay. A second discriminant analysis was preformed with the
same microcosm data set (nutrients, phytoplankton, zooplankton), and a
similar set from the 1972-73 bay survey at 13 stations around the bay for the
same period of the year (Figure 24-1, Table 24-2). In Figure 24-8, the bay
stations are found positioned in a roughly linear arrangement, parallel to the
first axis, which corresponds approximately to the eutrophication gradient in
Narragansett Bay. Stations at the clean mouth of the bay and in the deeper east
passage are positioned to the right. Stations in the west passage to the upper
bay are progressively positioned to the left with the Providence River on the
extreme left. All the microcosms, including those which exhibit more divergent
behaviour, are positioned in mid-bay locations. Microcosm 2 appears
374
-------
Table 24-5. Stepwise Discriminant Analysis of the 9 MERL Tanks and
Bay Input Water from August 17 to December 6,1976
Percent
Predicted Group Membership
Actual
Number of
Group
1
2
3
4
5
6
7
8
9
Bay
Weeks
1
6.7
0
26.7
13.3
6.7
13.3
13.3
0
13.3
6.7
15
2
0
33
20.0
6.7
0
13.3
0
13.3
6.7
6.7
15
3
6.7
6.7
46.7
26.7
0
0
0
0
6.7
6.7
15
4
0
7.1
2&6
28.6
0
0
0
0
7.1
28.6
14
5
0
13.3
13.3
20.0
20.0
6.7
13.3
0
6.7
6.7
15
6
0
6.7
0
0
26.7
20.0
6.7
0
13.3
26.7
15
7
6.7
0
0
6.7
13.3
13.3
20.0
0
13.3
26.7
15
8
0
6.7
6.7
20.0
13.3
13.3
13.3
0
13.3
13.3
15
9
0
0
0
0
6.7
13.3
13.3
0
53.3
13.3
15
Bay
6.3
0
0
iae
0
0
12.5
0
18.8
43.8
16
Percent of group cases correctly classified 27.3%. Variable not used: Chlorophyll. Total variation explained:
99%. Variables: 1 - NO3-NO2 (62% variation) 2 — P04 (23% variation) 3 — NH^ (7% variation) 4 — SiO^ (6%
variation) 5 — Zoop. (1% variation).
-------
3.0
20 -
cvi
<0
- 1.0
X
<
-1.0
-1.0
PHYTOPLANKTON '00'
MERL TANKS 0
BAY INPUT WATER •
37% VARIATION EXPLAINED
'31'
4
44'
'20'24'
6 j^22 8 '47^0', „
6 '33,13,2 '23'53 2S
'38' "
- V°\-
26l2 4.90 '42' '22
r
'25'
'37'
1.0
2.0
AXIS I
Figure 24-7. Correspondence Analysis on 49 Phytoplankton Species
('00') in 9 MERL Microcosms (0) and Bay Input Water (•) from
September 14, to December 6, 1976.
NOTE: In some cases sample locations (microcosms) are closely adjacent and
not all of the 10 samples per microcosm are plotted. All variable locations
(phytoplankton species) are plotted as accurately as possible. Microcosm
location and phytoplankton species location were closely adjacent on the lower
right hand side of the figure until the first part of November when blooms
occurred in the bay and in the microcosms. The arrows indicate divergence
from non-bloom conditions to bloom conditions in November when variability
in species composition and microcosm behavior became greater.
376
-------
X
<
85% VARIATION EXPLAINED
MOUTH
EAST PASSAGE
MOUTH
WEST PASSAGE SOUTH - ROSE ISLAND
• •
DUTCH ISLAND
PROVIDENCE
RIVER
CONIMICUT
# OHIO
LEDGE# SOUTH-
MOUTH # PATIENCE My HOPE31
EAST GREENWICH 5 BRIDGE/7
EAST-PRUDENCE 4/
6'; '5' MERL
2
OUONSET
POINT
SOUTH-EAST PRUOENCE
TANKS
A X
0
I S
Figure 24-8. A Discriminant Analysis of Data from the 9 MERL
Microcosms and the 13 Bay Stations Using the Variables:
Chlorophyll, Zooplankton, Ammonia, Nitrate Plus Nitrite,
Phosphate and Silicate.
NOTE: MERL data from August 17 to December 6, 1976. Bay data from
1972-1973 survey. For detailed information on the analysis see Table 24-6.
significantly different from microcosms 3, 5, 9, and microcosm 3 is
significantly different from microcosm 9, but the bay stations "Dutch Island,"
"Southeast Prudence," "East Prudence" and "Mt. Hope Bridge," are not
significantly different from any of the microcosms (Table 24-6). Other mid-bay
stations are different from some of the microcosms, but no more so than they
are with regard to each other.
These intercomparisons between the MERL microcosms and Narragansett
Bay show that the MERL microcosms diverged surprisingly little from stations
in the bay or from other microcosms, with regard to concentrations of
nutrients, chlorophyll, zooplankton biomass and phytoplankton species
composition during the replicability experiment.
377
-------
Table 24-6. A Discriminant Analysis of 9 MERL Microcosms and 13 Bay Stations Using
Variables Chlorophyll, Zooplankton, Ammonia, Nitrate Plus Nitrite, Phosphate and Silicate from
August 17 to December 6,1976 and 1972 Respectively
PERCENT PREDICTED GROUP MEMBERSHIP
From
Group
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
~ 1
9
18
0
0
9
9
18
9
18
0
0
0
0
0
0
9
0
0
0
0
0
0
2
0
73
9
0
0
9
0
0
0
0
0
0
0
0
0
9
0
0
0
0
0
0
3
0
10**
SO
0
20
0
0
0
0
0
0
0
0
0
0
0
0
10
0
0
0
10
4
0
33
0
0
11
11
0
0
22
0
0
0
0
11
0
0
0
11
0
0
0
0
5
9
r#
36
0
27
0
0
0
0
0
0
0
0
9
0
0
0
9
0
0
0
0
6
0
22
11
11
11
22
11
0
0
0
0
0
0
11
0
0
0
0
0
0
0
0
7
13
13
13
0
13
13
0
0
25
0
0
0
0
0
0
0
0
13
0
0
0
0
8
0
0
0
0
25
25
13
25
0
0
0
0
0
13
0
0
0
0
0
0
0
0
_ 9
11
11**
0**
0
11
11
0
0
44
0
0
0
0
0
0
0
0
11
0
0
0
0
"10
0**
0**
0**
0**
0**
0*.
0«.
0**
0**
71
0
29
0
0
0
0
0
0
0
0
0
0
11
0**
0**
0**
0**
0**
0**
0**
0**
14**
43
29
0
0
0
14
0
0
0
0
0
0
0
12
0**
14* *
0**
0
0**
14#*
0**
0**
14**
14**
14
14
0
0
0
14
0
0
0
0
0
0
13
0**
0"
0**
0
0**
0
0**
0**
29* *
0**
14
29
14
0
0
0
0
0
0
0
14
0
14
0
0**
0**
0
0**
0
0
0
29
0**
0"
29
29
0
0
14
0
0
0
0
0
0
15
14
43
0**
0
0**
0
0
0
0
0'*
14"*
0
0
14
0
14
0
0
0
0
0
0
16
0
0
0
0
29
0
0
0
0
0**
0"
14*"
0*'
0
0
43
0
0
14
0
0
0
1?
0
0"
43
0
0
0
0
0
0
0"
0**
0**
0**
0**
0**
29
29
0
0
0
0
0
18
0"
0**
0**
0*"
0**
0**
0**
0**
0**
0"*
0**
0**
0**
0**
0"
0
0
88
14
0
0
0
19
0
Q**
43
0
0
0
0
0
o».
0**
0**
0"
0'*
0**
0**
0
14
14
29
0
0
0
20
14
0
29
0
14
0
0
0
0
0**
0**
0**
0**
0
0
14
0
0
0
14
0
14
21
14
0
0
0
0
29
0
0
0
0**
0"
0
0
29
14
0
0
o#*
0*
14
0
_22
29
0
0
0
0
0
0
0
14
0**
0**
0
14
14
0
0
0
0**
0
0
14
14
* * groups different at .01 level 28% correctly classified
LEGEND: 1—9 MERL microcosms, 10 Providence River, 11 Conimicut, 12 Ohio Ledge, 13 Month Greenwich, 14 S. Patience, 15Quonset, 16 Dutch Is., 17
Mouth W. Passage, 18 Mouth E. Passage, 19 S. Rose Is., 20 S. E. Prudence, 21 E. Prudence, Potters Cove, 22 Mt. Hope Bridge.
-------
DISCUSSION
These initial observations provide us with insight into the problems of
running microcosms in such a way that they are analogous to some natural
system. The natural system itself is highly variable and difficult to define,
except within broad limits. Generally, for most of the variables measured, the
values from the MERL microcosms fell within the ranges observed for adjacent
Narragansett Bay. We have no evidence that the major features of
phytoplankton and nutrient dynamics were different from Narragansett Bay.
This lends support to the hope that the MERL microcosms will be useful
experimental systems in which investigations will produce results transferable
to comparable open, natural systems.
An exception to the generalizations above was the zoo plankton abundance.
The biomass of zooplankton in the MERL microcosms (Figure 24-9) was
somewhat less than in the bay, especially towards the end of the time period
considered. This factor is responsible for the microcosms lying somewhat
outside the fields for the bay data shown in Figures 24-6 and 24-8, because the
1972-73 bay survey also returned somewhat higher zooplankton biomass
concentrations than were found in the tanks. We believe that the tendency
towards low zooplankton biomass was due to an artifact associated with the
delivery of water to the tanks and that this problem will be rectified by
subsequent changes to the plumbing. It is therefore premature now to dwell on
the nature of the differences in zooplankton.
The general behaviour of the nutrient and the phytoplankton data sets
(and, to a lesser extent, that of the zooplankton) was most reassuring. No wild
excursions occurred. The variability in the microcosms was generally similar to
that in mid-bay stations, and the species abundances were generally similar,
taking the data as a whole. On the other hand, the quantitative variability of
the tanks between themselves (as may be inferred by examination of the ranges
shown in Figure 24-3) violates our usual perception of the way in which
experimental systems should behave. We expect them to replicate well, so that
experiments can be performed and the results have good statistical validity.
The difficulty with replication of nature is that nature herself is highly variable.
Working with such systems requires that large data sets be obtained, and that
multivariate statistical techniques be applied to reduce these correlated data
sets to manageable formats for analysis.
A possible way of assessing microcosm and natural system behaviour and
developing a criterion for comparison is to calculate the generalized distance
between data sets (Blackith and Reyment 1971). The assumptions of
homogeneity, multivariate normality and linear correlation between variables
must be met for such a technique to be rigorously applied but, as with all
379
-------
320
280
240
200
I 60
to
s
e>
2
lij
S
>
K
O
o
X 120
z
<
£L
O
S 8°
40
• 443
s I \
_4 / \
\ / \
\ / \
\ / \
\ \ / \
\ \ / \
\ \ / \
_\ \ i \
\ \ / \
• \ * / \
\ t / \
\ \ i \
\ \ 1 \
•
\ \ / \
— \ 1 / \
\ \ 1 \
\ i /i \
\x / 1 \
\ M ' 1 \
VI f \ \
— \r 1 1 \
* ^ / 1 \ ^
% / t
\ i \ \
\ / \
*
h
m
J i
w i T
V t \
7 > *
1 1
V 1 /
\ 5
'\ > /
\ * —~—
\ 1
\ /\ 1 f
H • /\\
t y
1 /
\ • V
y— / \ \ /
\ / \ \\ /
\/ - \ V v / y
• N/
S.
I I ^""l
\ /-•>
—J
8/15
9/4
9/24
10/14 11/3 11/23 12/13
Figure 24-9. Zooplankton Biomass in the MERL Tanks and
in the Bay, During the Fall of 1976.
NOTE: Solid lines give the range of data from three stations occupied by
Durbin and Durbin tc.f. Figure 24-4). The solid circles give data from the GSO
dock. Dashed lines give the range of data from the nine MERL tanks.
380
-------
multivariate techniques, some interpretations may be explored even when the
assumptions are not met. Generalized distances are determined in discriminant
analysis and the maximum distances for the microcosms and the bay for the
first axis in Figure 24-8 and Table 24-6 are shown in Table 24-7. These distances
are much smaller among the replicated microcosms than among bay stations. It
seems feasible to consider that standard generalized distances exist for natural
systems, for specific variable sets, which might be compared to generalized
distances which result during experiments on perturbation and subsequent
recovery in microcosms.
Table 24-7. Maximum Generalized Distances and
Normalized Distances Among the Microcosms and Among
Bay Stations from August to December 1976 and 1972
Respectively, from the Discriminant Analysis Shown in
Figure 24-7 and Table 24-6.
Normalized
max max
Microcosms:
2 vs 3 0.9 3
2 vs 5 0.7 2
2 vs 9 0.3 1
3 vs 9 0.6 2
Bay Stations:
Prov. River vs mouth E. Passage 4.4 14
Ohio Ledge vs mouth E. Passage 3.0 9
Ohio Ledge vs mouth W. Passage 1.9 6
CONCLUSIONS
The nine MERL microcosms operated during the 4-month replicability
study were generally as similar to each other as they were to adjacent areas of
Narragansett Bay using nutrient and phytoplankton data sets for comparison.
Zooplankton abundance in the MERL microcosms was somewhat low but
this was probably caused by an artifact that can be removed.
Multivariate statistical techniques seem essential to the comparison of the
large and heterogeneous data sets generated in such studies as this.
381
-------
The similarity in the behaviour of the microcosms and Narragansett Bay
gives some confidence that these systems will be good experimental tools for
ecological and biogeochemical experiements.
ACKNOWLEDGMENTS
This work was supported by grant No. R803902020 from the
Environmental Protection Agency. We thank Ann and Ted Durbin for allowing
us to use their phytoplankton counts and zooplankton biomass data in Figures
24-4 and 24-9, and Scott Nixon for helpful discussion.
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In EPA 600/9/77-012, 563 pp.
17. Smetacek, V., B. Von Bodungen, K. Von Brockel and B. Zeitzschel. 1976.
The plankton tower. II. Release of Nutrients from Sediments due to
Changes in the Density of Bottom Water. Mar. Biol. 34:373-378.
18. Strickland, J. D. H. and T. R. Parsons. 1972. A Practical Handbook of
Seawater Analysis. Fish. Res. Board of Can. Bull. 167, 2nd Edition. 310
pp.
19. U. S. Army Corps of Engineers. 1959. Contamination Dispersion in
Estuaries. Narragansett Bay, Hydraulic Model Investigation. U. S. Army
Eng. Waterways Exp. Sta. Misc. Paper 2-332, Report 2, Vicksburg, MI.
383
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TURBULENT MIXING IN MARINE MICROCOSMS-
SOME RELATIVE MEASURES AND ECOLOGICAL
CONSEQUENCES
Scott W. Nixon, Candace A. Oviatt and Betty A. Buckley
Graduate School of Oceanography
University of Rhode Island
Kingston, R.I. 02881
ABSTRACT
The effect of turbulent water motion on pelagic organisms has seldom been
studied. Nevertheless, a consideration of the theory of turbulent energy flux as
well as the few bits of empirical data which do exist suggest that it may be a
factor of some importance for marine plankton, and that turbulence may
influence the growth, metabolism, and behavior of pelagic species as well as
their spatial distribution. This paper reports the results of a series of turbulence
experiments carried out over an annual cycle using small (150 1) laboratory
microcosms designed as analogues of Narragansett Bay, R.I. (U.S.A.).
Turbulence levels in the mocrocosms and in the natural system were
characterized using conventional (neighbor diffusivity, vertical eddy diffusivity,
energy flux) parameters as well as a number of relative measures of water
mixing (dye dissipation, CaSO^ dissolution rate, gas exchange coefficients).
The response of phytoplankton and zooplankton populations to varying
turbulence levels was dramatic during warmer months, but absent or unclear in
winter. The results suggest that while phytoplankton may be stimulated by
higher turbulence levels, at least in warmer water, the response of the
zooplankton is quite the opposite during these periods. It is not clear if the
response of the phytoplankton reflects a decline in grazing pressure or a real
enhancement of growth. The problem is complex and deserves considerable
further study both in the field and in the laboratory.
INTRODUCTION
".. . diffusion is confusion. Only Maxwell's Demon
really knows what's going on."
Akira Okubo (1971),
Horizontal and Vertical Mixing in the Sea
384
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A Problem of Size and Scale
The increasing use of relatively small experimental ecosystems or
microcosms in marine research has raised a number of interesting questions
related to the importance of size or scale in natural, as well as experimental
systems. In pelagic marine environments, one of these questions for which we
have very little relevant information is the importance of turbulence in the
water. The relationship between turbulence and scale was first formalized by
Richardson (1926), who distinguished between classical Fickian diffusion (in
which scale is not a factor) and the mixing that is characteristic of turbulent
fluids such as the sea. While the nature of turbulence is extremely complex,
Richardson's concept of turbulent energy passing through a series of
progressively smaller eddies from the wavelength at which it is put into the
fluid until it is ultimately dissipated in viscosity has continued to prove
valuable in studying the mixing of marine waters (Okubo 1971). In natural
systems, turbulent energy is added at a rather large scale by winds, tides, and
major currents. Since none of these is usually effective in microcosm tanks or
bags, some artificial means of introducing turbulent energy at smaller scale may
be required to develop pelagic ecosystems that are credible experimental
analogs of the "real world" (Perez et al 1977).
There are at least two aspects to the turbulence problem, one involving the
actual distribution of organisms, particles, or dissolved constituents in the
water - the problem of patchiness (see Steele 1974), and the other involving
the metabolic or behavioral responses of organisms to water turbulence. The
study of plankton patches usually concerns water masses on a scale larger than
the largest microcosms yet developed (1300 m^, 10 m dia.; see Menzel and
Case 1977), and it is generally conceded that this aspect of the ecology of
marine waters is not well represented in microcosm experiments. The
importance of this omission is not yet known. While there have been numerous
studies which have documented the response of sessile plants and animals to
the special case of turbulence in flowing water (Fox et al 1935; Kerswill 1949;
Whitford 1960; Jaag and Ambiihl 1963; Whitford and Suchumacker 1964;
Mclntire 1966; Westland 1967; Nixon and Oviatt 1971), the responses of
pelagic organisms to small scale turbulent energy have received much less
attention.
With the exception of an older qualitative study of the morphological
response of Daphnia to water motion by Brooks (1947), the recent work by
Pasciak and Gavis (1975) on the relationship between turbulence and nutrient
uptake by phytoplankton, and our own studies on marine plankton in
laboratory microcosms (Perez et al 1977), the effect of water turbulence on
the growth and metabolism of planktonic organisms is almost totally unknown.
While this situation is largely a result of the difficulties involved in measuring
385
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turbulent energy levels in the laboratory or in the field, it may also reflect the
feeling of many ecologists that the small size of plankton generally places them
below the size scale at which turbulence is "felt". Both of these considerations,
along with technical difficulties, have caused almost all marine microcosm
studies to neglect turbulence as a factor in their experimental design. In the
simplest terms, the justification appears to have been that since turbulence is
difficult to measure, hard to mimic, and of unknown importance, it was
reasonable to avoid the problem of deciding on how to include it in
microcosms. There is a certain amount of appeal to this argument, especially
since there are so many other problems to be resolved in developing a
microcosm. However, the evidence in the papers cited above, as well as the
experience of anyone who has tried to culture or maintain phytoplankton and
zooplankton in the laboratory, suggest that turbulence is an important
consideration in pelagic systems. Our earlier experiments with turbulence in
marine microcosms also indicated that the scaling of mixing energy in
laboratory tanks can dramatically influence the results of phytoplankton and
zooplankton growth studies in the microcosms (Perez et al 1977). The
argument about plankton being too small to "feel" turbulence is also
questionable.
Turbulence
Following Richardson (1926), Richardson and Stommel (1948), Stommel
(1949), Batchelor (1950) and others, the flow of turbulent energy from large
scale motion is passed down through successively smaller eddies until it is
dissipated in viscosity. Above a certain size, the energy content of eddies is
solely a function of their size (k) and the rate of energy flux (e) through the
system. Below this critical size, defined by
KtT
where v = the kinematic viscosity
e = the energy flux per unit mass
k = upper limit of the Kolmogoroff viscous zone
viscous forces become important and the energy content decays more rapidly
with decreasing size as energy is dissipated. In order to give some feeling for the
scale involved, it is possible to estimate k for the West Passage of Narragansett
Bay using a value for the energy dissipation of 4.3 x 10^ ergs sec"' (Levine
and Kenyon 1975) and an approximate volume of 7.2 x 10^ m^. The result
suggests that k is on the order of 0.06 cm. While this is larger than individual
phytoplankton cells found in these waters (<0.01 cm), it is about the size of
386
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many of the diatom chains found in the bay (~0.05-0.1 cm) and smaller than
the dominant zooplankton. Moreover, even below this size, the effect of
turbulent energy persists. As the time averaged flux of energy through larger
eddies is increased, the viscous shear in the Kolmogoroff zone will also increase
and this increase in shear will be felt even at the very small scales seen by the
plankton. Moreover, as the energy flux through larger eddies increases, the
upper size limit of the Kolmogoroff zone will decrease, so that larger plankton
will begin to experience direct turbulent effects.
In addition to simple mechanical effects, such as the disruption of feeding
or copulation by zooplankton, this turbulent energy flux at small scale may
influence the plankton (or other particles) in at least two ways. Around any
given cell of size 2, there will exist a thin laminar boundary layer in which
Fickian or molecular diffusion must be relied upon to transport dissolved gases,
nutrients, waste products, etc. Since molecular diffusion is much slower than
turbulent diffusion, this is often the rate limiting step in exchange processes
between the cell and the surrounding medium. As the turbulent energy flux in
the medium increases, however, the water just outside of the boundary layer is
renewed more rapidly, with the renewal rate being proportional to:
This increase in renewal rate tends to maximize the concentration gradient
across the laminar boundary layers and, thus, the diffusion of materials across
the layer. In addition, the increase in renewal rate by turbulent velocity will
also decrease the thickness of the boundary layer itself, since the boundary
layer thickness is proportional to:
Again, the reduced thickness of the laminar layer will increase the exchange
rate of materials between the particle and the medium.
While the cascade of turbulent energy through successively smaller eddies
has been studied frequently in the sea (Okubo 1971), the emphasis in the field
has generally centered on measurements of eddies larger than 10 m. The nature
of the turbulent energy spectrum in small experimental ecosystems has only
recently begun to receive attention (Boyce 1974, Steele et al 1977, Gust
1
(3)
Z
387
-------
1977). While the results of Gust's study are restricted to the specific flexible
chambers and conditions of his measurements, they show quite convincingly
that it is possible to obtain a small scale turbulent spectrum in a chamber that
is similar to that found in the surrounding coastal waters. Unfortunately,
almost all of these turbulence measurements were made in a metabolic
chamber used with benthic algae rather than in plankton studies, and no
biological data were included.
This paper reports the results of a number of turbulence experiments carried
out at different times of the year using coupled benthic-pelagic microcosms
designed as analoges of Narragansett Bay, R.I. While some of the data from
experiments conducted during the spring have been reported previously (Perez
et al 1977), we have now carried out identical studies during winter and
summer months. In addition, we have explored in this paper a number of
techniques for characterizing the turbulent mixing levels in the microcosms and
compared them to the dissolution rate measurements used previously (Oviatt et
al 1977, Perez et al 1977). Finally, we have attempted to carry out
experiments to test our earlier conclusion that phytoplankton and zooplankton
respond independently to different turbulence levels. The impression that the
phytoplankton and zooplankton were not coupled in their response to
turbulence was based on indirect evidence (Perez et al 1977) and we felt it
desirable to test this conclusion directly by adjusting the levels of zooplankton
in replicate tanks and observing whether concommitant but opposite changes
would occur in the phytoplankton.
METHODS
The Microcosms
The microcosms used in these experiments have been described in detail in
earlier papers (Perez et al 1977; Oviatt et al 1977 and in press). Each
microcosm consisted of a 166 liter plastic tank containing 150 1 of water (0.7
m deep) collected by bucket from the lower West Passage of Narragansett Bay.
This area of the bay shows a well mixed water column about 8 m deep with
salinity between 28-3 l0'oo throughout the year. Characteristics of the bay have
been described in some detail by Kremer and Nixon (1978). The microcosms
were maintained in a running sea water bath in the laboratory near field
temperatures and illuminated for the appropriate natural photoperiod by
Westinghouse Cool White fluorescent lights. The response of the microcosms to
light input is complex and the choice of a value for any particular experiment
is difficult (Nixon et al, in press). The experiments described here were carried
out at 5-25 ly/day, values considerably below the average light energy found in
the water column of the bay.
388
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Each microcosm also contained an opaque plastic box (167 cm ) of intact
bay sediment and associated benthos. This size produced the same sediment
surface to water volume ratio as found in the bay. Water from the pelagic phase
of the microcosm was moved through the box and over the sediment by
vacuum pump so as not to damage the plankton. The inside walls of the tanks
were cleaned regularly to prevent fouling, and organic matter settling on the
bottoms of the tanks was collected and placed in the sediment Soxes. In all of
the turbulence experiments, it is important to note that the artificial nature of
the "bottom" community isolated it from the turbulent energy of the water,
except as the benthos might respond to changes in the plankton. However,
even in tanks that were unstirred, the benthic box pumps provided some very
gentle circulation for the pelagic community, since the flow rate used was
capable of putting 150 liters of water through the box about three times each
day. Additional mixing was contributed by the approximately daily wall
cleaning and by the addition of 10 liters of bay water to each tank three times
each week. The latter was maintained so that the microcosms functioned as
open systems with a flushing rate similar to that of Narragansett Bay.
Different turbulence levels were imposed on the microcosms by leaving
them unstirred except for the benthic pump and cleaning operations or by
mixing them with plastic mesh paddles of 0.14 re? or 0.07 m^ area. The
opening size of the plastic grid in the paddles was 1.2 cm x 1.2 cm. The paddles
were driven at 32 rpm by an electric motor connected to all the paddle shafts
by a chain, thus producing identical rotation rates in all of the tanks. Each
paddle was rotated in one direction for 30 sec., then stopped for 6 sec., then
reversed for 30 sec. in a continuous cycle.
Turbulence Measurements
Vertical Eddy Diffusivity
We attempted to obtain a variety of both relative and absolute
measurements of turbulent mixing in the microcosms and in Narragansett Bay.
In some cases, such as the estimation of a vertical eddy diffusivity, the
techniques used were conventional. A small amount (~2 ml) of Rhodamine-WT
dye was dissolved in sea water (1:100) and released "instantaneously" at
mid-depth in the microcosms. Near-surface and near-bottom water samples
were then collected at short intervals (2-5 min.) and the concentration of dye
determined fluorometrically. The rate-of-change in concentration in both sets
of samples was virtually identical, indicating that the tanks were mixed
uniformly up and down, and that the vertical eddy diffusivity could be
estimated by
389
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z2
Dv =
2t
(4)
9 1
Dy = vertical eddy diffusivity, cm sec
Z = distance between relative point and measurement
point (1/2 depth in this case), cm
t = time for the concentration to asymptote, sec
The average value of this parameter for Narragansett Bay has been computed
by Hess (1976) using a detailed numerical hydrodynamic model.
The Horizontal Turbulent Field
Determination of the horizontal turbulent component was more difficult.
At one extreme, we measured the time it took for small dye patches (0.5 ml of
1:100 dye in seawater) to disperse in the microcosms and in the bay under a
range of conditions. This approach was simple, rapid, and with enough
replication and a constant observer, it gave a good (low variance) relative
measure of horizontal mixing rates. Unfortunately, it is also a bit subjective
and qualitative and cannot be expressed directly as a standard hydrodynamic
parameter.
In an attempt to overcome these limitations, we have also obtained
measurements of neighbor diffusivity (Richardson, 1926; Stommel, 1948) and
the flux of turbulent energy along two arbitrary perpendicular coordinates
(Batchelor, 1950) using the relative motions of pairs of small floats with a
range of distances separating them. The measurements were made by releasing
several dozen floats and then photographing them from a fixed position at
short time intervals. The size of the floats used and the length of the time
interval were varied somewhat according to the scale of the turbulent eddies of
interest. In the West Passage of Narragansett Bay, larger scale mixing (1-25 m)
was studied using colored balloons filled with fresh water so that they floated
just beneath the surface. These floats were dispersed from a small boat and
photographed every few minutes from a high bridge. Smaller scale eddies
(1-200 cm) were studied in the field using small (~0.5 cm) colored plastic
beads that were released from the end of a pole off the stern of a small boat
and photographed every few seconds using a 16 mm movie camera operated
from the flying bridge of the boat. The beads and the movie camera were also
used in the microcosms. In all cases, floating rods of standard length were
included in each photograph to give an accurate scale.
After they were developed, the films of the floats were put through a
microfiche reader for enlargement. Large numbers of pairs of floats were
390
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selected at various distances of separation to be followed from frame to frame
over time. In each frame the scalar distance between the floats as well as their
separation along two perpendicular vectors (x and v) was obtained. These data
were then analyzed using the relationship given by Batchelor (1950).
fix-," - fix
e =
2x,
2t 11 + 1/3 ¦
£o"
312
(5)
where iQ is the initial scalar distance between floats
is the initial distance between the floats
projected on the x axis
f is the distance between the floats after some
x.*>
- time, t. projected on the x axis
e is the rate of turbulent energy flux
The same operation was carried out for the 'y' vector which should yield a
similar value if the turbulent field is isotropic. Unfortunately, it is extremely
difficult to extend the analysis to a third dimension and it is not practical to
use the method to explore the horizontal turbulent field below the surface.
Additional Relative Turbulence Measures
As discussed earlier, the flux of turbulent energy influences the rate at
which materials may be exchanged across a laminar boundary layer. This
suggests that the dissolution rate of a solid substance placed in the water may,
at least to some degree, be a function of the turbulent energy of the fluid. The
importance of the turbulent effect should be greatest for materials that are
near saturation in sea water. After some exploration, we have found that the
mineral gypsum (CaSO^) is particularly well suited for this purpose. It is easily
obtained, inexpensive, and a large number of uniform pieces can be cut from a
single rough block. The dissolution rate is influenced somewhat by temperature
and salinity, but these relationships are easily established in the laboratory in
order to compare measurements made under different conditions. Since the
rate of weight loss is also a function of size, we have found it best to use
standard pieces of gypsum measuring ~2.5 x 1.8 x 0.7 cm with an initial
weight of about 6-8 gms. Blocks of this size are suitable for making
measurements of weight loss over periods ranging from about 6-24 his.
Replication appears to be quite good and duplicate blocks are hung off a fixed
or free floating line to obtain a vertical profile of dissolution rate.
Unfortunately, however, the CaSO^ dissolution rate may provide only a
relative measure of turbulent energy and it is not clear if it is possible to relate
391
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it to any more conventional physical measurements. It is also not clear how
one might separate vertical and horizontal components of mixing using the
blocks.
An additional indirect estimate of mixing in the microcosms and in the field
was obtained by measuring the diffusion coefficient for oxygen across the
aii-water interface. The measurements were made using a small floating plastic-
dome from which virtually all of the oxygen was displaced by nitrogen gas. The
partial pressure of oxygen in the dome and in the water was then monitored
over time and the flux of oxygen from the water into the dome calculated.
Since the flux is a product of the gradient in partial pressure and the diffusion
coefficient, it was possible to obtain the coefficient from such a data set.
Measurements with domes containing turbulent or still air have confirmed that
the diffusion coefficient is largely a function of turbulence in the liquid phase,
and that the effect of wind is felt through its influence on water mixing.
RESULTS
Turbulence Levels Obtained
The results of the various turbulence measurements lead us to be
particularly sympathetic to Okubo's lament that "diffusion is confusion." In
spite of, or perhaps because of the fact that a large number of floating pair
observations were made in Narragansett Bay and in the microcosms, there was
a very large amount of scatter in these data. As a result, the calculation of
neighbor diffusivity (F) and energy flux (e) was subject to a large uncertainty
and there is some question about how meaningful the numbers may be. While
the values of F tended to decrease approximately according to the 4/3 law
(Richardson 1926; Stommel 1948), it also appeared that e had a tendency to
fall off with size. The latter result is disturbing since the theoretical framework
for the computation suggests that e should be constant at steadystate in the
inertial range between the size at which energy is put into the system and the
viscous zone in which it is dissipated.
Functional regressions (Ricker 1973) relating F to scale are given below. In
general, both the neighbor diffusivity and the energy flux indicated that the
turbulence levels in the microcosms with one paddle were appreciably higher
than found in the bay or in the mocrocosms with one half paddle and no
paddle (Table 25-1). The F Values did not show a great difference between the
half paddle microcosms and the bay and the very large variance associated with
the calculation of e in the half paddle tanks made it difficult to see any clear
differences in terms of energy flux.
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Table 25-1. Estimates of Turbulent Energy
Dissipation Rates in the Experimental Microcosms
and in Some Natural Marine Waters
2 -3
e. cm sec
Microcosms with one paddle 1.44
Microcosms with half paddle 0.03
Microcosms with no paddle 0
Narragansett Bay, West Passage 0.05, 0.07, 0.11,
measured at different times 0.17, 1.0
Narragansett Bay, West Passage
estimated from tidal currents,
(Levine and Kenyon, 1975) 0.21
Irish Sea, estimated from tidal
currents (Taylor, 1919) 0.08
N.W. Pacific coastal water, U.S.A.
surface 30m, calcualted from
changes in microstructure
(P.W. Nasmyth in Gregg 1973) 0.02
Open sea, surface mixed
layer (50 m) (Gregg 1973) 0.002
2 1
Functional Regressions for F, cm sec
West Passage F = 0.007 £1.89 (6)
Microcosms with one paddle F = 0.108 £1.38
with half paddle F = 0.013 £1.73
The simpler measurements of horizontal dye patch dispersion suggested that
while the one paddle microcosms were considerably more turbulent than those
with half a paddle, both were more rapidly mixed than the bay (Table 25-2).
This same trend with respect to differences between the whole paddle and half
paddle microcosms was also shown by the weight loss of gypsum blocks and
the gas exchange measurements (Table 25-2). However, both of these
parameters indicated substantially higher mixing rates in the bay than in any of
393
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Table 25-Z Relative Turbulence Measurements
in the Experimental Microcosms and in Narragansett Bay
Vertical Eddy Diffusivity
0 1
K, cm sec
Microcosms with one paddle 3.6 - 3.8
Microcosms with half paddle 2.2 • 3.8
Microcosms with no paddle -» 0
Narragansett Bay, mean for the
West Passage (Hess 1976) 5
Rates of Horizontal Dye Patch Dispersion
Time to Disperse, sec
Microcosms with one paddle (N=7) 5.4 ±1.2
Microcosms with half paddle (N=7) 8.4 ± 1.2
Microcosms with no paddle (N=1) » 900
Narragansett Bay, West Passage
day 1 (N=10) 18.0 ± 5 4
day 2 (N=6) 13.4±1.9
day 3 (N=6) 17.5+2.5
CaSO^ Dissolution Rate
Weight Loss, % hr"1
Microcosms with one paddle 1.83 ± 0.20
Microcosms with half paddle 0.73 ± 0.36
Microcosms with no paddle 0.27 ± 0.05
Narragansett Bay, West Passage, 3.30 ± 0.60
mean for vertical profiles 4.39 ± 1.10
O2 Diffusion Coefficient at the Air-Water Interface
2 1 1
K, fiM m hr atm
Microcosms with one paddle 27
Microcosms with half paddle 8
Microcosms with no paddle 2
Narragansett Bay, West Passage
calm day 30
windy day 137
windy day 125
394
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the microcosms. To some extent this may reflect the fact that the floating pair
measurements and dye patches respond to the horizontal turbulent field and
the gypsum and gas exchange are also influenced by vertical motion. The
rotating plastic paddles appeared to add a lot of horizontal mixing energy to
the tanks, but the vertical eddy diffusivity in the microcosms was lower than
Hess (1976) calculated for Narragansett Bay (Table 25-1).
While none of these measurements allows us to make a very convincing
absolute comparison of turbulent energy in the microcosms with that of the
bay, it does seem clear that the full paddle, half paddle, no paddle
configuration provided quite different turbulent water regimes in the
microcosms. Since the input of turbulent energy to Narragansett Bay must Vary
considerably during the tidal cycle and from day-to-day according to the
winds, it seems reasonable that the natural pelagic community may well
experience all of the turbulent conditions used in the microcosms. For
comparative purposes, it is interesting to note that all of the methods used for
measuring turbulence except the determination of neighbor diffusivity and
energy flux (e) indicated that even the full paddle configuration was low
relative to the bay.
Response of the Plankton
The first turbulence experiment was carried out during the month of April
when water temperatures in the microcosms ranged from 8 to 12°C. The
standing crop of phytoplankton as indicated by chl a increased dramatically in
the one paddle and half paddle treatments compared with the unstirred tanks
(Figure 25-1). A number of cursory analyses of water samples did not indicate
that there were any major shifts in species composition in the different tanks.
However, there were also marked and significant differences (Perez et al 1977)
among treatments in the numbers ofAcartia clausi, the dominant zooplankton
in the microcosms and in the bay (Figure 25-2). While the rapid increase in
phytoplankton in the one paddle tanks began almost immediately, Acartia
nauplii did not really start to decline until after 10 days. In fact, a portion of
the decline in nauplii between 10 and 16 days was simply due to growth of the
animals into juveniles (Figure 25-2). An analysis of covariance was performed
to establish whether the changes in phytoplankton density could be attributed
to changes in zooplankton density the covariate, total grazers was found to be
non-significant. This meant that the inverse relationship expressed by
zooplankton and phytoplankton to water turbulence was due to a direct
pattern than the indirect effect of water turbulence. In fact, an analysis of
covariance on the mean algal standing crop during the experiment indicated
that interactions with the total numbers of grazers in the microcosms (the
covariate) was not significant (Perez et al 1977). It is possible, however, that
the zooplankton present did not feed as effectively in the more turbulent
395
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DAYS AFTER 5 APRIL
Figure 25-1. Chlorophyll Concentrations (In Vivo Fluorescence)
in the Microcosms and in the Lower West Passage of Narragansett
Bay During the First Turbulence Experiment Begun April 5,1976.
NOTE: Data points are the mean of duplicate tanks.
tanks. It is striking that the zooplankton standing crop with one paddle was
very similar to that found during the same period in the bay, while the
phytoplankton populations in those tanks reached levels 3 times greater than
found in the bay with similar zooplankton numbers. Conversely, the low and
relatively constant phytoplankton standing crops in the unstirred microcosms
were almost identical to that found in the bay, but much larger zooplankton
populations were sustained, at least for 15 days, in the microcosms. It may be
that the plankton in the microcosms escaped a significant grazing and/or
predation pressure that was important in setting the standing crop maintained
in the field. This experiment was repeated during May with virtually the same
results.
The next turbulence experiment was not begun until December, when water
temperatures ranged from 1 to 6°C and the standing crops of phytoplankton
were low. The experiment was designed to explore not only the effect of
turbulence, but also the interactions of turbulence with light and nutrient
enrichment. Again, the standing crop of phytoplankton was significantly (0.05
level) higher in tanks mixed with a full paddle, though the effect was not as
dramatic as in the earlier runs (Figure 25-3). The response to light was not
significant at the 0.05 level but was significant at the 0.10 level. The response
to turbulence was highly significant (greater than the 0.01 level) (Figure 254).
396
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Figure 25-2. Numbers of Adult, Juvenile, and Nauplii
Acartia clausi in the Microcosms and in the Lower West Passage
of IMarragansett Bay during the First Turbulence Experiment.
NOTE: Data points are the mean of duplicate tanks.
397
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Figure 25-3. Phytoplankton Cell Counts in the Microcosms and
in the Lower West Passage of Narragansett Bay During the
Turbulence-Light Interaction Experiment Begun December 9,1976.
NOTE: Data points are the mean of duplicate tanks.
398
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8
6 -
to
O 4
E 2
O 0
I- 8
X.
2
<
a!6
o
h-
X 4
Q.
1/2 PADDLE
NO PADDLE
0 5 10 15 , 20
LIGHT, ly day-1
16 ly
/ 9 \y/
— /
1
1 1
0 >/2 I
MIXING, No. of Paddles
Figure 25-4. Average Phytoplankton Population Levels During
the 34 Day Turbulence-Light Experiment.
NOTE: Data points are the mean of duplicate tanks.
399
-------
The interaction of light and turbulence was not significant nor was the effect
of ammonia enrichment. However the ammonia addition brought the
concentration in the tanks from ~3juM to ~6/uM, so that the plankton were
never seriously nutrient limited. It was also interesting that there was no
response of the phytoplankton in the unmixed microcosms to increased light,
while there was a clear increase in the stirred tank populations with higher light
levels (Figure 25-4). The numbers of zooplankton, again dominated by A.
clausi, were very low throughout the experiment (Nauplii ~10/L; juveniles
%5/L) and no dramatic differences among treatments developed. However, the
mean numbers of nauplii and juveniles observed during the experiment were
higher in the tanks with no paddle and lowest in the tanks with one paddle.
Analysis of the data showed that this difference in the means was statistically
significant (a:0.05) and that there was no significant interaction of nauplii,
juveniles, or adults with light intensity. There was no statistically significant
difference in the mean number of adults in the different turbulence levels.
In order to find out if turbulence had a direct stimulating effect on
phytoplankton, two experiments were carried out during January and
February in which an attempt was made to remove zooplankton from some of
the microcosms by filtering the water through a #20 (80 n) net. This was
effective in reducing the zooplankton levels by about 70 percent in the first
experiment and by about 90 percent in the second. In addition, light levels
were increased from 6 ly/day during the January run to 16 ly/day in February
and ammonia was added to all tanks at the start of the second experiment in an
attempt to stimulate vigorous phytoplankton growth. Temperatures ranged
from 0-0.5°C during the first experiment and from 0-3°C during the second.
The results of the first experiment showed no significant effect of
turbulence on the numbers of phytoplankton or zooplankton in the
microcosms (Figures 25-5 and 25-6). The lack of turbulence effect on the
phytoplankton was observed in tanks with and essentially without zooplankton
(Figure 25-5). It is interesting to note that the variation in zooplankton
numbers by a factor of about 3.5 had no significant effect on the levels of
phytoplankton, probably due to low temperatures and therefore reduced
grazing rates.
When the experiment was repeated a month later with higher light and
nutrients, a phytoplankton bloom was produced during the first week in all of
the microcosms (Figure 25-7). During this period there did not appear to be
any effect of the turbulence on phytoplankton growth either with or without
zooplankton. Moreover, the grazing pressure of the small number of A. clausi
in the unfiltered water (~15 animals/L) at these low temperatures had little or
no effect on the bloom. However, the bloom declined much more slowly in the
unstirred microcosms, so that after 10-15 days the standing crops in the
400
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15
_ 10 -
WITH Z00PLANKT0N
A
- BAY
-o NO PADDLE
-• 1 PADDLE
I L
io -
5 —
0 5 10 15 20
DAYS AFTER 19 JANUARY
Figure 25-5. Phytoplankton Cell Counts in the Microcosms and
in the Lower West Passage of Narragansett Bay During the
January 1977 Turbulence Experiment.
NOTE: Data points are the mean of duplicate tanks.
401
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DAYS AFTER 19 JANUARY
Figure 25-6. Numbers of Acartia clausi nauplii and Juveniles
in the Unfiltered Microcosms and in the Lower West Passage of
Narragansett Bay During the January Turbulence Experiment.
NOTE: Data points are the mean of duplicate tanks.
402
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I
Figure 25-7. Phytoplankton Cell Counts in the Microcosms and
in the Lower West Passage of Narragansett Bay During the
February 1977 Turbulence Experiment.
NOTE: Data points are the mean of duplicate tanks.
403
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turbulent tanks were markedly lower both with and without zooplankton
(Figure 25-7). Statistical analysis of the mean numbers of cells in each
treatment during the experiment showed that the average standing crop of
phytoplankton was significantly higher (a = 0.05) in the unstirred tanks. Again,
this is clearly the reverse of the pattern found in the first three experiments,
but repeats the trend suggested by the January run (Figure 25-5). There were
no significant differences in the new numbers of zooplankton between the two
turbulence levels during this experiment, with both showing small populations
that fluctuated between about 5-15 animals per liter. This is the first
experiment in which the phytoplankton showed a significant response to
turbulence (albeit opposite to that found previously) but zooplankton numbers
did not. Again, it is interesting to note that an almost 9 fold increase in
zooplankton numbers did not result in any statistically significant decline in
the numbers of phytoplankton.
We attempted to repeat the zooplankton removal experiment during July of
the following summer with water temperatures between 19-20.5°C. However,
the hatching and development rate of zooplankton eggs and nauplii is so rapid
at the higher temperatures that it was virtually impossible to reduce the
numbers of zooplankton very much by the filtration method used.
Nevertheless, the results were interesting. The pattern found in the first three
experiments emerged once again, with phytoplankton growth clearly enhanced
by the turbulent mixing and zooplankton surpressed (Figures 25-8 and 25-9).
A final experiment was carried out during August in which the interaction
of turbulence and water turnover rate in the microcosms was explored. Water
temperatures varied between 19-21°C. While there was no significant effect of
turnover rate on the plankton, the same statistically significant stimulation of
phytoplankton growth was found in the stirred microcosms where zooplankton
significantly declined by a factor of 2-3 (Figure 25-10). The ten-fold increase in
phytoplankton associated with somewhat more than a halving of the
zooplankton in the turbulent microcosms may reflect the tight coupling of
these two compartments that has been suggested in numerical simulations of
the summer plankton (Kremer and Nixon 1978). This result contrasts with our
earlier experiments carried out at lower temperatures in which significant
reductions in zooplankton numbers had no significant effect on the mean
phytoplankton standing crop.
DISCUSSION
The Importance of Turbulence
It seems clear that the presence or absence of turbulent mixing in the
microcosms had a significant influence on the abundance of phytoplankton
404
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Figure 25-8. Phytoplankton Cell Counts in the Microcosms and
in the Lower West Passap of Narragansett Bay During the
July 1977 Turbulence Experiment.
NOTE: Zooplankton numbers in the filtered tanks (80/u net) were only slightly
lower than in the unfiltered (see Figure 25-9). Data points are the means of dup-
licate tanks.
405
-------
o
<
_l
Q_
O
O
Nl
O
h-
0 5 10
DAYS AFTER
Figure 25-9. Total Zooplankton Counts in the Microcosms and in
the Lower West Passage of Narragansett Bay During the
July 1977 Turbulence Experiment.
NOTE: Data points are the mean of duplicate tanks.
406
-------
I
— BAY
DAY TURNOVER
NO PADDLE
ONE PADDLE
DAY TURNOVER
NO PADDLE
ONE PADDLE
0 5 10 15 20 25
DAYS AFTER 3 AUGUST
Figure 25-10. Counts of Phytoplankton and Total Zooplankton
in the Microcosms and in the Lower West Passage of Narragansett
Bay During the August 1977 Turbulence Experiment.
NOTE: Data points are the mean of duplicate tanks.
407
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and zooplankton during the warmer months. Unfortunately, the results of the
experiments do not make it clear if the turbulence effect is felt directly by
both populations or if the enhancement of phytoplankton growth is the result
of lower zooplankton grazing pressure in the more turbulent tanks. The lack of
a significant turbulence effect on phytoplankton during the colder months may
result from the fact that the phytoplankton and zooplankton virtually do not
interact at low temperatures when feeding rates and excretion approach zero
(Heinle and Vargo, 1978). During the warmer months there is evidence from
some of our other microcosm experiments that the zooplankton are more
effective at cropping down phytoplankton than a 60 percent artificial level of
cropping imposed biweekly (Oviatt et al in press). In some cases, such as the
April run, it appeared that the lower grazing pressure might be due to less
effective zooplankton feeding as well as to a higher zooplankton mortality in
the well mixed microcosms. At this point, however, it is still not clear if this
increased zooplankton mortality was the result of a real physiological or
behavioral response to the turbulent field or if it was a simple mechanical
artifact resulting from the manner in which turbulence was generated.
Not only is the physical basis of turbulence confusing, but, at least at this
point, so are its ecological consequences. The experiments described here are
among the first ever reported on this problem, and it is not surprising that so
much remains obscure. The results demonstrate the potential significance of
turbulence as an ecological factor in pelagic systems and illustrate the
importance of carrying out relatively long term (15-30 day) experiments at
different times of the year, or at least at different temperatures, when studying
the problem. It is also important to explore different ways of generating
turbulence as well as the effects of its intensity in experimental ecosystems.
ACKNOWLEDGEMENTS
We are grateful to Ken Perez, Peter Murphy and Don Winslowof the U.S.
Environmental Protection Agency, Narragansett, R.I., for their help in
maintaining the microcosms and in the collection of plankton data. Mark
Wimbush, Randy Watts, Diego Alonso and Michael Pilson of the Graduate
School of Oceanography at the University of Rhode Island struggled with us
over the matter of turbulence and its measurement and meaning. Patrick
Roques and Dana Kester at the Graduate School of Oceanography contributed
to the gas exchange measurements. The research was supported in part by grant
No. R-803143 from the U.S.-E.P.A. to the University of Rhode Island.
408
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