EPA-600/3-76-060
August 1976
Ecological Research Series
FATE AND BIOLOGICAL EFFECTS OF
MERCURY INTRODUCED INTO
ARTIFICIAL STREAMS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Athens, Georgia 30601
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-060
August 1976
FATE AND BIOLOGICAL EFFECTS OF
MERCURY INTRODUCED INTO ARTIFICIAL STREAMS
by
Henry J. Kania, Robert L. Knight, and Robert J. Beyers
Savannah River Ecology Laboratory
Institute of Ecology
University of Georgia
Athens, Georgia 30601
Grant No. R. 800510
Project Officer
Harvey W. Holm
Environmental Systems Branch
Environmental Research Laboratory
Athens, Georgia 30601
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS, GEORGIA 30601
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DISCLAIMER
This report has been reviewed by the Environmental
Research Laboratory, U.S. Environmental Protection Agency,
and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement
or recommendation for use.
ii
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ABSTRACT
Mercuric ion was continuously input to artificial stream
channels to provide water concentrations of 0.01, 1.0 and
5.0 yg/1. Channel components were periodically sampled for
total mercury analyses. The effects of mercury on the algal
components of the periphytic communities and on the benthic
insects were determined. The sampling program continued
one full year after mercury inputs were stopped.
Approximately 15% of the added mercury was removed from the
water. The community components acquired very high
concentrations of mercury, although in most cases the
levels in these were not a linear function of the water
levels. Concentrations in invertebrates decreased most
rapidly after mercury inputs were stopped while the sediment
levels decreased most slowly.
Periphytic algae showed several treatment responses including
total inhibition, inhibition of certain life stages, possible
stimulation of certain life stages and morphological
alterations. One species was found only in the treated
channels and disappeared when mercury inputs were stopped.
The diversity and evenness of benthic insect communities
were affected by mercury treatment.
This report was submitted in fulfillment of Project Number
R800510 by the University of Georgia under the sponsorship
of the Environmental Protection Agency. Work was completed
as of April 1, 1975.
111
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CONTENTS
Sections Page
I Introduction 1
II Conclusions 4
III Recommendations 6
IV Experimental Design and Site Description 7
V Methods 16
VI Results and Discussion 34
VII References 115
VIII Appendix 123
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FIGURES
No. - Page
1 Artificial streams 8
2 Limestone-filled treatment system , 10
3 Average change in channel water-hardness 11
between limestone changes
4 Mercury recovery system 13
5 Air sampling system 18
6 Continuous flow chamber system 32
7 Total mercury levels in channel and effluent 35
water
8 Diurnal radiomercury uptake curve, June, 1972 37
9 Diurnal radiomercury uptake curve, August, 1972 38
10 Diurnal mercury uptake in treated channels on 39
January 22-23, 1974
11 Proportion of mercury associated with particulates 41
in water samples
12 Bacterial populations in channel water 42
13 Average mercury releases at the air-water interface 44
of the treated channels
14 Diurnal mercury releases on January 22-23, 1975, 46
at the air-water interface of channels receiving
an input of 1.0 yg/1
15 Diurnal mercury releases on January 22-23, 1975, 47
at the air-water interface of channels receiving
an input of 5.0 ug/1
16 Average mercury concentrations in the organic 49
portion of the sediments of the control channels
and channels receiving an input of 1.0 ug/1
VI
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Page
17 Average mercury concentrations in the organic 50
portion of the sediments of channels receiving
inputs of 1.0 and 5.0 yg/1
18 Average mercury concentrations in particulate 52
matter leaving the treated channels
19 Average mercury concentration in periphyton 53
removed from the PVC walls of the control
channels and channels receiving an input of
0.01 yg/1
20 Average mercury concentrations in periphyton 55
removed from the PVC walls of channels receiving
inputs of 1.0 and 5.0 yg/1
21 Average mercury concentrations in periphyton 56
removed from plexiglass plates in channels
receiving inputs of 1.0 and 5.0 yg/1
22 Average mercury concentrations in roots of Juncus 59
diffusissimus removed from channels receiving
inputs of 1.0 and 5.0 yg/1
23 Average mercury concentrations in leaves of Juncus 60
diffusissimus removed from channels receiving
inputs of 1.0 and 5.0 yg/1
24 Average mercury concentrations in damselfly nymphs 62
collected from channels receiving inputs of 1.0
and 5.0 yg/1
25 Mercury concentrations acquired by corixids 64
confined to flowing water containing 5.0 yg/1 Hg++
26 Mercury concentrations in mosquitofish removed 66
during the first year from channels receiving an
input of 1.0 yg/1
27 Mercury concentrations in mosquitofish removed 68
during the first year from the control channels
and the channels receiving an input of 0.01 yg/1
28 Mercury concentrations in mosquitofish removed 70
from treated channels during the last 21 months
of the project
Vll
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Page
29 Change in mercury concentrations in mosquitofish 72
moved from a contaminated area to clear water
30 Biomass of periphyton scraped from PVC strips 78
suspended in the channels
31 Biomass of periphyton scraped from channel walls 79
32 Calculated volume of periphytic algae scraped 81
from the channel walls
33 Diversity and evenness of periphytic algae 82
collected from the channel walls
34 Biomass of periphyton scraped from vertical 83
plexiglass plates
35 Calculated volume of periphytic algae scraped 85
from vertical plexiglass plates
36 Diversity and evenness of periphytic algae 86
collected from vertical plexiglass plates
37 Calculated volume of algae colonizing microscope 87
slides during mercury inputs (started October 31,
1973)
38 Calculated volume of algae colonizing microscope 88
slides during mercury inputs (started December 7,
1973)
39 Calculated volume of algae colonizing microscope 89
slides after mercury inputs were stopped (started
January 30, 1974)
40 Calculated volume of algae colonizing microscope 90
slides after mercury inputs were stopped (started
April 25, 1975)
41 Density of Oedogonium sp. on channel walls before 94
and after mercury inputs were stopped
42 Density of Oedogonium sp.on plexiglass plates 95
before and after mercury inputs were stopped
43 Density of Stigeoclonium elongatum on plexiglass 96
plates before and after mercury inputs were
stopped
VI11
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Page
44 Density of StigeocIonium elongatum on channel 98
walls before and after mercury inputs were stopped
45 Geminella turfosa 100
46 Density of Cosmarium aspherosporum on plexiglass 102
plates before and after mercury inputs were
stopped
47 Density of Cosmarium aspherosporum on channel 103
walls before and after mercury inputs were
stopped
48 Density of Spondylosium planum on plexiglass 105
plates before and after mercury inputs were
stopped
49 Density of Spondylosium planum on channel walls 106
before and after mercury inputs were stopped
50 Total macrophyte biomass in channels during the 109
final year of the project
51 Densities of insects collected from benthic 111
dishes before and after mercury inputs were
stopped
52 Diversity and evenness of insects collected from 112
benthic dishes before and after mercury inputs
were stopped
IX
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TABLES
No. Page
1 Well water quality 12
2 Limestone composition 14
3 Distribution of Juncus diffusissimus on April 2, 58
1973, in the artificial streams
4 Organisms collected from the artificial streams, 76
February 11-16, 1975
5 Incidence of abnormal Geminella turfosa cells on 101
glass slides during and after mercury input
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ACKNOWLEDGMENTS
The support and assistance of the personnel of the EPA's
Environmental Research Laboratory and the
University of Georgia's Savannah River Ecology Laboratory
is gratefully acknowledged. Special recognition is due
Dr. Harvey Holm of the EPA, who patiently provided
competent technical and administrative direction throughout
the project, and Mrs. Vicki Buley of the University of
Georgia, who has willingly participated in every aspect
of the program, from the tedious analytical work to the
typing and editing of manuscripts.
XI
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SECTION I
INTRODUCTION
The toxic effects of Hg and its compounds on both plants
and animals have been known for a long time. It is only
within the last twenty-five years, however, that the extent
of man's uses of this element, and the actual and potential
problems resulting from his uses, have been recognized.
The history of Hg, its chemical and physical properties,
sources, uses, and toxicological effects as related to
current problems have been reviewed in a number of recent
books (Jones, 1971; D'ltri, 1972; Friberg and Vostal,
1972; Hartung and Dinman, 1972), dissertations (Curtis,
1974; Ferens, 1974), and review papers (Nelson, 1971;
Wallace et al., 1971; Peakall and Lovett, 1972;
Putman, 1972; Saha, 1972; Lepple, 1973).
At present, high-level environmental releases of Hg from
point sources such as chlor-alkali plants and paper pulp
operations have been virtually eliminated. Releases due
to agricultural usage of organomercurials have also been
greatly reduced. It is unlikely that we will see another
Minimata Bay type incident, although the poisoning of man
by the misuse of grain treated with mercurials remains a
possibility. The two major problems still facing man with
respect to mercury are: (1) what to do about the areas
already contaminated by large releases in the past and,
(2) what are to be the consequences of the continuing large-
scale diffuse releases of Hg from the burning of fossil
fuels and the discarding and incineration of Hg-containing
products.
Since the recognition that Hg releases of any kind may
result in ecosystem alterations and even poisoning of man
himself, a great deal of research dealing with this metal
has been initiated. The majority of the studies have been
surveys or experiments of short duration involving
relatively high Hg levels and/or very simple biological
systems, or performed in existing contaminated areas without
adequate controls and replication. These studies have
demonstrated that:
(1) Mercury is universally distributed and occurs in
small amounts in all components of the biosphere. The
inability to find Hg in a sample is probably a result of
the analytical techniques used.
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(2) There is a natural biogeochemical cycle in which
Hg circulates between living organisms and the environment.
Inputs to the natural cycle include releases from the
earth's crust by the weathering of Hg-bearing rocks,
volatilization of Hg from covered deposits, leaching by
ground water, and releases associated with volcanic activity
and thermal springs.
(3) Mercury can exist in three general forms (organic
compounds, inorganic compounds and free metal) which differ
greatly in their physical and chemical properties and,
therefore, their behavior in biological systems. These
forms have different accumulation and clearing rates, sites
and physiological effects on living organisms. All possible
conversions between forms can and do take place in natural
systems.
(4) Man adds to the Hg load of the environment in
several ways. Point source releases have caused localized
problems including human deaths. Agricultural use of
mercurials has resulted in wildlife losses. Although point
source and agricultural releases have been almost entirely
eliminated in the past few years, contaminated aquatic areas
still exist and fish from these areas remain unfit for
human consumption. There continue to be extremely large
releases of Hg wherever fossil fuels are utilized.
(5) From the standpoint of human health, methyl Hg
compounds are the most important because this is the form
that tends to accumulate in food organisms. Methyl Hg is
almost completely absorbed in the gut of mammals, is very
slowly eliminated, has a great effect on the central
nervous system at low levels, and easily passes through
placenta! membranes.
(6) Relatively low levels of all forms of Hg may be
toxic to aquatic organisms. The toxicity is a function
of species and water quality.
In spite of the recent surge of Hg-related research, several
important questions have not been fully answered. The site
of methylation of the Hg that occurs in fish as methyl Hg
has not been clearly demonstrated. The effects of long-term
exposures of biological systems to slightly elevated Hg
levels have not been adequately investigated. The rates of
recovery to be expected from contaminated systems are not
clearly defined. And the maximum levels that will not cause
environmental alterations or the contamination of human food
resources have not been determined. The research program
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reported here was directed to the last three points as
pertaining to aquatic systems. More specifically, the
artificial stream research program had the followinq
general goals: (a) to determine the fate of Hg introduced
on a continuous basis at low levels into biologically
complex aquatic communities over a long period of time,
(b) to determine the fate of Hg remaining in the systems
after the inputs were stopped, (c) to assess the impact of
the added Hg on the communities in the systems and, (d) to
compare results obtained from the artificial streams with
data from other sources in order to evaluate the artificial
streams as a potential tool for the future routine screening
of environmental pollutants.
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SECTION II
CONCLUSIONS
1. Water levels of only 0.01 yg/1 Hg++ above ambient
resulted in Hg levels significantly greater than
background in components of the communities that
developed in the artificial streams.
2. At 1.0 and 5.0 yg/1 Hg++, very high Hg concentrations
accumulated in the biota and sediments of the treated
channels although, except for insects and macrophytes,
the relationship between water levels and the levels
measured in the community components was not linear.
3. In a simple food chain (periphyton-midge larvae-
mosquitofish) no biomagnification was observed.
4. At dosing levels of 1.0 and 5.0 yg/1, approximately
15% of the Hg was removed from the water during the
two-hour retention time of the channels. Approximately
33% of the Hg leaving the 1.0 yg/1 channels and 21%
of that leaving the 5.0 yg/1 channels was attached
to microscopic particulates. No diurnal Hg uptake
patterns were observed, indicating uptake was by
passive adsorption in the channels.
5. The major loss of fixed Hg from the channels was with
exported macroscopic matter. These losses were erratic
and very much affected by weather conditions.
6. A loss of elemental Hg from the channels at the air-
water interface was observed and this loss rate was
linearly related to water concentrations of Hg and
not to sediment levels. The release of Hg from the
channels appeared to be related to the diurnal
photosynthetic activities of channel communities.
The maximum releases measured were always less than
1% of the Hg present in the water.
7. After Hg inputs were stopped, there were major
differences between the clearing rates of Hg from
the various channel components. The sediments showed
greatest Hg retention (T% = 7.85 years, C.I.95 =
5.6-13.0 years in 1.0 yg/1 channels, T% = 8.67 years,
C.I.g5 = 6.11-15.0 years in 5.0 yg/1 channels), and
an odonate nymph the least (T% ^ 15 days). One year
after the Hg inputs were stopped, all system components
analyzed still had elevated levels relative to the
controls.
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8. During the project period the channel communities were
continuously changing, and no steady-state systems
developed. There was continual downstream expansion
of the macrophyte populations, a constant rearrangement
of insect and algal populations, and a continuous
influx of new plant and animal species into the channels,
9. Periphyton colonization was retarded in the Hg-treated
channels with respect to controls although greater
biomasses eventually developed in these channels along
with decreased species diversities.
10. Periphytic algae were affected by Hg treatment in a
variety of ways including:
(a) inhibition during all parts of the life history.
(b) inhibition during the reproductive and stimulation
during the vegetative part of the life history.
(c) morphological alteration without a significant
effect on growth, and
(d) complete restriction of one species to treated
channels with the greatest densities occurring
at the highest Hg concentration.
Short-term studies using continuous-flow chambers
confirmed the growth retardation observed for some
of the stream algae under more controlled conditions.
11. The most abundant macrophyte, Juncus diffusissimus,
showed a negative response to Hg treatment.
12. Insect densities showed no consistent responses to
the Hg concentrations tested; however, the diversity
and evenness of the benthic insect communities were
significantly lowered at the highest treatment level.
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SECTION III
RECOMMENDATIONS
1. The present drinking water standards regarding Hg
should be carefully reviewed since currently permitted
levels can cause ecosystem changes, and also result in
fish with concentrations too high for human consumption.
2. Additional long-term experimental studies with
replicable complex microcosms should be done with both
Hg and other environmental contaminants. Treatment
levels should be realistically low, and a variety of
environmental conditions (geographical areas) utilized.
Interactions between contaminants should be considered.
3. Modifications of the artificial stream facility used
in this project should be made so as to decrease the
variability between channels treated alike.
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SECTION IV
EXPERIMENTAL DESIGN AND SITE DESCRIPTION
EXPERIMENTAL DESIGN
The basic experimental design was to input water of constant
quality at a constant rate into six artificial stream
systems. After biological communities became established
in the channels, fish were to be added, followed shortly
by the continuous introduction of Hg++ to establish water
levels of 0.01 and 1.0 yg Hg/1. The Hg inputs were to be
continued for one full year, during which time samples of
water, lining material, biota and sediments were to be
taken and analyzed for total Hg content. During this same
period, the structures of select portions of the channel
communities were to be determined by detailed species
counts of periphytic algae and benthic insects. Estimates
of community productivity and respiration were to be made
from the continuous monitoring of upstream-downstream
changes in dissolved oxygen and pH. All sampling programs
were to be continued for one additional year after the Hg
inputs were terminated.
The experimental program deviated from the original plans
as work progressed. Early data indicated certain sampling
programs were not producing useable information and these
were discontinued. Changes in sampling techniques were
made necessary by changes in the biological communities.
Analytical procedures were simplified to allow for more
efficient handling of samples. And the original Hg dosing
schedule was changed when it became evident that the
0.01 yg/1 water concentrations were not providing
contamination levels sufficiently high to permit the
study of the clearing of Hg from the channels receiving
this input.
ARTIFICIAL STREAM FACILITY
The artificial stream complex is an Environmental Protection
Agency (EPA) facility located on the Energy Research and
Development Administration's (ERDA) Savannah River Plant
(SRP), a 507-km2 reserve that includes portions of Aiken,
Barnwell and Allendale Counties in South Carolina. The
six concrete Block channels (Figure 1) are 91.5 m long,
0.61 m wide and 0.31 m deep with concrete block pools
(1.5 m X 3.0 m X 0.92 m) at both ends of each stream.
The channels were constructed on a level concrete slab
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FIGURE 1, ARTIFICIAL STREAMS, IN THIS PHOTOGRAPH TAKEN
FROM THE HEAD POOLS, CHANNEL 1 IS ON THE RIGHT AND CHANNEL 6
IS ON THE LEFT, THE PLASTIC STRIPS AND FISH-HOLDING CAGES
CAN BE SEEN,
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which was once a building foundation. For this study, the
pools and channels were lined with a 0.05-cm-thick black
polyvinyl chloride (PVC) film.
CHANNEL WATER AND SUBSTRATE
Water for the channels is pumped from a well located near
the facility. The chemical composition of this water/
based on analyses made over a 10-year period by personnel
of the E. I. duPont Corporation, is presented in Table 1.
During the period of this study, the average pH of the
well water was 4.6 rather than the .5.3 shown in Table 1.
The water was treated at the stream site by passage
through limestone-filled tubes (Figure 2) in order to
decrease the free C02 content and increase the hardness
and pH. The limestone, obtained at the beginning of the
project from a quarry in Rome, Georgia, was changed
approximately every 30 days. An analysis of the
limestone as provided by the supplier is given in Table 2.
Figure 3 shows the average hardness of the water input to
the pools at the head of the channels as a function of time.
In addition to this time variation in water quality between
limestone changes, there were measurable differences
between channels during the latter part of the project.
This was due to leaks that developed in the head pools,
which made it necessary to input water at different rates
into these pools in order to maintain desired flows into
the channels.
Periodic measurements of dissolved organic matter, nitrate
nitrogen, orthophosphate, conductivity, pH, temperature
and dissolved oxygen were made on samples of the influent
water. The dissolved organic carbon levels were always less
than 0.5 mg/1, the nitrate-N levels averaged 38 ug/1
(Ferens, 1974), and the orthophosphate levels averaged 76
Vig/1 (Ferens, 1974) . The conductivity decreased from
approximately 30 to 22 ymho/cm and the pH decreased from
6 to 5 during the period between limestone changes. The
average temperature of the incoming water varied seasonally
but only slightly about the mean of 23°C. Water entering
the channels was generally saturated with respect to
dissolved oxygen.
The rate of water entering each channel was controlled by
a manual valve located just before the limestone-filled
treatment tubes. Flows were checked daily against
gradations on a V-notch weir separating the pool at the
head of each channel from the channel itself.
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FIGURE 2, LIMESTONE-FILLED TREATMENT SYSTEM,
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5 10 15 20 25
TIME AFTER LIMESTONE CHANGE, days
30
FIGURE 3, AVERAGE CHANGE IN CHANNEL WATER HARDNESS BETWEEN LIMESTONE CHANGES,
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Table 1. WELL WATER QUALITY 1963-1973
(After Ferens, 1974)
Analysis
N
X
cv
Temp
PH
Cond
TDS
DO
Hardness (EDTA)
Mg++
Ca++
K+
Na+
Fe+++
Cu++
HC03-
ci-
504 =
SiO2
4
13
11
8
5
12
8
7
7
10
10
8
8
8
8
9
22.8°C
5.3
19.7 umho/cm
13.1 mg/1
7.1
6.56 (CaCo3)
1.87
0.63
0.47
2.48
0.04
0.09
0.39
2.59
1.90
7.82
15%
7
18
42
18
67
88
108
17
115
57
71
149
79
71
70
In September of 1971, washed builders sand was distributed
in the channels to a uniform depth of 0.05 m and the water
input started. Flows of 95 1/min into each channel, as
measured by V-notch weirs between the head pool and channels,
were maintained until February, 1975, except for several
short intervals of reduced flow due to water pump failures.
Stainless steel end plates between the channels and tail
pools were adjusted to maintain a water depth of 20 cm over
the sand. With these settings, mean retention time of the
water in the channels was 2 hr and the average water
velocity was 0.013 m/sec.
Water leaving the treated channels was passed through beds
of shredded rubber, (Figure 4) a waste product of
commercial tire recapping operations. Results of water
analyses made on a number of different occasions show
that at least 50% of the Hg leaving the channels was taken
up by the rubber particles. Laboratory uptake studies
have shown that a much higher removal of Hg is possible if
contact between the water and rubber is complete (Kania and
Beyers, 1974). The use of shredded rubber for Hg removal
12
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H
•••
FIGURE L\, MERCURY RECOVERY SYSTEM, EFFLUENT WATER ENTERED POOLS FILLED WITH
SHREDDED RUBBER OBTAINED FROM A TIRE-RECAPPING PLANT,
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Table 2. LIMESTONE COMPOSITION
(Analysis provided by the quarry)
CaO
MgO
Si02
Fe2°3
A1203
Na2O
s
52.73%
1.70
3.42
0.25
0.33
0.10
Trace
was suggested by Edwin D. Russell of the E. I. duPont
Corporation.
CHANNEL BIOTA
The biological communities that became established in the
channels developed from organisms carried in by the wind
or by animals visiting the stream facility. A variety of
microorganisms was probably added with mosquitofish,
Gambusia affinis, that were introduced at two different
times in the channels.
Mosquitofish were seined from a nearby pond and were
acclimated for two weeks in artificial ponds receiving
water similar in quality to that input to the channels.
In April, 1972, 400 fish were introduced into each channel
and 40 into each of two 51-cm X 30.5-cm stainless-steel
screen cages suspended 10 m from both ends of each channel.
Fish were confined to the channels by coarse stainless-
steel screens. Caged fish were fed three times a week
with a commercial tropical fish food (Bi-0-Rell). In
May, 1973, all fish remaining in the channels and cages
were removed for analyses and new animals introduced,
again 400 into each channel, 40 into each cage.
MERCURY INPUT
Mercury was pumped continuously into the streams with a
four-channel, peristaltic tubing pump (Sage Instruments
Model 375). Solutions for each channel were made daily
with the concentrations adjusted to compensate for
14
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changes in pump characteristics. The concentrated
solutions from which the daily solutions were prepared
were made by dissolving dried reagent grade HgCl2 in
2% (vol/vol) HNC>3 to provide Hg++ levels of 0.1 mg/1,
10 mg/1, and 20 mg/1. Measured amounts of these stocks
were diluted with well water to 2 1 in a volumetric flask
and transferred to polyethylene bottles connected to the
input tubes of the pump. Based on the volume removed,
and the elapsed time between changes, a new concentration
for the input mixture was calculated and new solutions
prepared.
Mercury inputs to four of the six channels were begun on
May 15, 1972, so as to establish water concentrations of
Hg++ at 0.01 yg/1 above ambient in two channels (3 and 6
as counted left to right looking upstream), and 1.0 yg/1
in another two channels (2 and 5). Channels 1 and 4 were
used as controls. Since the six channels are structured
as three pairs (Figure 1), the dosing arrangement was
chosen so that there was a northern and a southern exposed
surface for each treatment.
On August 1, 1973, the input levels to channels 3 and 6
were increased to 5.0 yg/1. This was done because it
was obvious that the 0.01 yg/1 dosing levels were not
providing high enough Hg concentrations in the channel
systems to permit the study of the removal of this metal
after inputs were stopped. All Hg inputs were stopped
on January 29, 1974, although routine sampling continued
until February 10, 1975, when the study was terminated.
15
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SECTION V
METHODS
SAMPLING PROCEDURES
Water
Prior to August 1, 1973, only occasional water samples
were taken for Hg analyses, and then only from the channels
receiving an input of 1.0 yg/1. Mercury in the water
from the channels receiving inputs of 0.01 yg Hg/1 was
below detection limits of the techniques used. After
August 1, 1973, the date of the dosage change from 0.01
yg/1 to 5.0 yg/1, samples were taken routinely from two
locations in each channel (including controls) and also
from two regions of the effluent channel leaving the
stream facility. Sampling stations were located
approximately 6 m from the Hg input tubes at the heads
of the channels and 1 m from the downstream ends of the
channels. Samples of the effluent water leaving the tail
pools were taken just below where the individual outflows
joined into a common stream and also 100 m farther
downstream.
The samples were taken by submersing a nitric-acid-rinsed
250-ml Erlenmyer flask. The contents of the flask were
checked for visible particulates and a new sample taken
if particles were present. A clear sample was transferred
to a nitric-acid-rinsed polyethylene bottle containing
2.5 ml of concentrated HNOj.
Air
In the initial stages of the project, no attempt was made
to measure Hg losses from the channels at the air-water
interface. Based on reports by Fagerstrom and Jernelov
(1972) and Jernelov and Lann (1973), no losses were
expected from the acidic waters (pH 5-6) of the channels
because monomethyl Hg was supposed to be formed rather
than the more volatile dimethylmercury. A slight increase
in Hg levels of some components of the control channels,
however, indicated a transfer might be occurring from the
treated systems. The possibility of leaks was remote since
each channel was covered with a single continuous sheet of
PVC film and there was no pressure differential across
the wall separating adjacent channels. Therefore, the
possibility of air transfers was investigated.
16
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Six air sampling systems, as shown in Figure 5, were
constructed and the glass boxes, which covered 0-187 m2,
suspended at the midpoint of the channels by stainless-
steel straps. Air was drawn from under the boxes at the
rate of 0.47 1/s, dried with Mg(C104)2 and then passed
through gas-scrubbing bottles containing 100 ml of a 10%
(vol/vol) H2S04, 1% (wt/vol) KMn04 solution. On three
occasions, two scrubbing bottles were connected in series.
Analytical results showed that 98% of the Hg was collected
in the first scrubber.
To define the form of the Hg at the air-water interface,
traps consisting of silver-plated copper turnings were
placed between the drying tube and the gas scrubber
containing the acid permanganate. On three occasions,
adjacent boxes were placed in channel 6 (5.0 yg/1), one
with and one without the Ag-Cu trap and the air sampled
for a two- or three-hour period.
During routine sampling, air samples were taken in the
early afternoon on clear days.
Sediment
Several different techniques were used to obtain sediment
samples. Early in the project, a coring device, which
removed frozen cores 14 mm in diameter, was used to
determine the vertical distribution of Hg in the sediments.
The frozen cores were sectioned into three zones and
analyzed. This method of sampling was discontinued when
it became obvious that the Hg in the sediments was entirely
in the organic component present as a thin layer on top
of the sand.
As described in a following section, the processing of
pyrex dishes that had been placed in the channels for
invertebrate sampling resulted in a separation of the
organic portion of the sediments from the sand. Both of
these components were analyzed for Hg and the organic
content of the organic component was measured.
During the last year of the study, samples were taken with
a plexiglass tube 5.5 cm in diameter and 40 cm long. The
tube was pushed vertically through the sediments until it
touched the channel bottom. The material was retained in
the tube by hand as it was transferred to a container for
transportation. In the laboratory, the macrophytes were
removed and saved. The core material, free of plants,
was blended for several seconds and the suspended sediments
17
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AIR
INTAKE
TO -^
VACUUM PUMP
GLASS BOX
TRAP
FLOWMETER
DRYING
TUBE
GAS
SCRUBBER
Mg(CI04):
H.2S04
+
KMn04
FIGURE 5. AIR SAMPLING SYSTEM.
-------
and algae poured off from the heavier sand. The sand
was rinsed once with tap water and was combined with the
first suspension removed. The total volume of the resulting
suspension was measured, blended for one minute, and
subsampled for Hg analyses, organic matter determinations,
and algal counts.
Export
The original plans were such that the organic particulate
material leaving the channels would be collected
quantitatively by a series of screens and nets of
decreasing mesh and analyzed for both Hg and organic
content. It became obvious very soon after sampling
started that this method would not work because of the
almost immediate plugging of the screens by very large
amounts of a slime associated with the material moving
out of the channels, and another method for collecting
export was developed.
In the early stages of the project, virtually all of the
material moving downstream was floating. Therefore, two
stainless-steel screen skimmers were suspended in each
channel, one at the effluent end and one midway between
ends. The mats that formed behind the skimmers were
harvested quantitatively with a slightly submerged plastic
dishpan and transferred to a 4-1 graduated cylinder for
measurement of total volume. The contents of the graduated
cylinder were thoroughly mixed, a 200-ml volume removed
and blended for one minute, and two 10-ml subsamples
taken, one for organic content determination, one for Hg
analysis.
Periphyton
Channel Walls-
Shortly after the water input to channels was started in
September, 1971, 40 plastic strips (10 X 20 cm) of the same
PVC material lining the channels, were suspended at upstream
and downstream locations in each channel. These strips
were removed periodically and the periphyton was scraped
off into a small amount of water. Biomass determinations
and Hg analyses were made on samples of a blended suspension
of this material. Sampling of these plastic strips was
discontinued in October, 1973, because the large growth of
an emergent aquatic plant, Juncus diffusissimus, in the
channels interferred with the hanging strips to the extent
that they could no longer be considered representative of
the channel walls. A method was then devised so that a
known area of channel wall could be scraped and the periphyton
19
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analyzed as above. A plexiglass box with the top and one
side removed was placed tightly against the wall with the
top above the water line. The enclosed section of wall,
an area 47-mm wide and 125-mm deep, was scraped clean with
the flat end of a plastic stirring rod and the slurry was
transferred to a beaker by means of a hydrometer suction
tube. All samples were taken from the north-facing (shaded)
wall. These samples were treated the same as the plastic
strip samples except that a 5-ml aliquot was saved for
algal enumeration.
Plexiglass Plates-
Plexiglass plates measuring 10 X 10 X 0.63 cm were placed
in the channels in December, 1971, to provide substrates
for periphytic growth. Four trays carrying a total of 32
vertical plates (the sides were parallel to the direction
of stream flow) were placed at the head and tail of each of
the six channels. The same number of horizontal plates
were placed on the sand substrate interspersed with the
racks holding the vertical slides. During the first two
years, two plates of each orientation were removed monthly
for biomass determination and algal enumeration. In the
following year, one vertical plate was removed monthly from
each position in each stream for Hg analyses in addition to
the algal counts and biomass determinations. The horizontal
plates were abandoned after April, 1973, because of the
high variability between them (Ferens, 1974). Vacated
positions were refilled with new plates that were allowed
to colonize for at least six months before being removed.
During the last year of the study, all of the plates used
had been in the streams for at least one year.
Periphyt ic growth on the plates was carefully scraped with
a plastic stirring rod and washed with a small amount of
distilled water into a beaker. This sample was homogenized
for one minute in a Waring blender, the total volume was
determined, and measured subsamples were removed after
additional mixing for Hg analysis, biomass determination,
and algal counts.
Between July, 1972, and April, 1973, two clean plates were
placed each month at both positions (head and tail) in all
channels and harvested after one week of exposure as a
study of short-term productivity. These plates were
sampled as described above except that the entire
suspension was filtered for biomass determinations.
In June of 1974, five months after the mercury inputs had
been stopped, 10 clean plates were placed in channel 6
20
-------
(formerly 5 ug/1). Two plates were harvested biweekly over
a three month period and handled as described above.
Glass Slides-
Glass microscope slides (25 X 75 mm) were used to directly
study the initial colonization of periphytic algae in the
artificial streams. Slides were hand-washed with a mild
cleanser, thoroughly rinsed with deionized water, and
placed at upstream and downstream locations in each
channel on five occasions, two before the Hg inputs were
stopped (October 31, 1973 and December 7, 1973) and three
after the inputs were stopped (January 30, 1974, April 25,
1974 and October 23, 1974). Eight to ten slides were
suspended 7.6 cm above the sand substrate at each location
and oriented with their flat sides facing the water flow.
One slide was removed from each location in each channel
at three-to-seven-day intervals for at least one month,
the downstream side was wiped clean of periphyton and the
upstream side was counted as described below. On two
occasions (after 40 and 80 days of colonization), sets of
glass slides were scraped and rinsed with distilled water
and the biomass and Hg concentrations were determined.
Macrophytes
The initial project plans had made no provision for sampling
macrophytes. By spring of 1973, however, it became evident
that a macrophyte, Juncus diffusissimus, was to assume
increasing importance in the channel communities. Routine
sampling of this Juncus for Hg analyses was initiated in
June, 1973. Individual plants were removed by hand from
the channels, the roots were washed free of sand, the
leaves were washed free of periphyton and these two portions
separated. During the last year of the project,
quantitative estimates of the macrophyte standing crop
biomasses were made from plants removed with the large
core sediment samples previously described. These plants
were also used for Hg analyses.
Aquatic Insects
In November, 1971, 16 rectangular pyrex baking dishes
(15.7 X 26 X 4 cm) were filled with sand and placed in
upstream and downstream locations in each channel. One
dish from each position was removed monthly, the organisms
being retained by either a rectangular stainless-steel
screen box fitted inside the walls of the dish or by a
flat piece of screen held against the surface as the dish
was removed. The contents of the dishes were transferred
21
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to stainless-steel buckets, the dishes washed, filled with
sand and replaced in their former positions. Tap water
was added to the bucket, the contents stirred by hand and
the suspended material poured into white enamel pans from
which the living organisms were removed with forceps.
The procedure was repeated until nothing was found in two
consecutive rinsings. Insects removed were preserved in
70% or 80% ethanol until they could be identified and
counted. All dishes sampled had been in the channels
approximately one year before removal.
Aquatic insects were qualitatively sampled by sorting
through the detritus collected on the fish-retaining
screens at the tails of the streams and also by collecting
individuals emerging from the streams onto the plants or
into the air. Adult odonates laying eggs in the streams
were netted and preserved throughout the warmer months to
aid in the identification of nymphs.
Fish
From April, 1972, until January, 1973, the fish were
collected from the channels with small nets. This method
was inefficient because of the care required to prevent
disturbance of the wall and bottom communities. A small
portable shocker was constructed and utilized for the
remainder of the project. Fish were removed from the
screen cages with a small dip net.
ANALYTICAL PROCEDURES
General
All analyses were carried out on a Coleman MAS-50 Hg
Analyzer System modified by the addition of a digital
read-out and fused quartz windows on the absorption cell.
Standards were prepared prior to each set of analyses from
a 1.0 yg/ml solution made weekly from a commercially
available 1000 yg/ml standard (Harleco Corp., Philadelphia,
Pa.). Standard curves derived by the "standard additions"
method with fish and periphyton digestates did not differ
significantly from those derived by adding measured amounts
of Hg to 100 mis of a deionized H90 solution containing
5 ml concentrated 112804, 1 ml concentration HN03, and 1 ml
of a 6% (wt/vol) KMn04 solution. Therefore, the simple
acid-permanganate solution was used for the preparation
of standards.
Regardless of the material sampled, an acid-permanganate
solution resulted from the digestion procedure. This was
22
-------
reduced with a 1.5% (wt/vol) hydroxylamine hydrochloride
solution, further reduced with a 10% (wt/vol) SnCl2 in 20%
(vol/vol) HCL solution, and the resulting free Hg sparged
from solution by a recirculating current of air which
passed through a Mg(ClO4)2 drying tube into the UV absorption
cell of the Hg detector (Coleman, 1970). The maximum
absorbance reached was recorded. Analytical procedures
were checked by analyses made of Hg standards in gelatin
(Eastman Kodak Co., Rochester, N.Y.) and by a series of
recovery experiments with samples spiked both with inorganic
(Harleco) and methylmercury (Alfa Inorganics, Beverly, Mass.)
standards. In all cases recoveries ranged from 95-103%.
The few methylmercury analyses that are reported were
performed by personnel of the Environmental Protection
Agency's Southeast Environmenta.l Research Laboratory under
the direction of Dr. Harvey W. Holm using the technique of
Longbottom et al. (1972).
Water
Analyses were made of 100-ml aliquots of both filtered
(0.3 ym Gelman Type A glass fiber filters) and unfiltered
water. These samples were treated with 5 ml of concentrated
H2S04, 1 ml of concentrated HNO.,, and 1 ml of a 6% (wt/vol)
KMnO4 solution and allowed to stand at least 30 min before
being analyzed. Results from this technique were compared
against those obtained from a procedure which included the
addition of K2S2Og and a heat treatment. These extra steps
were found to be unnecessary for the channel water samples
although they may be required in some cases (EPA, 1971).
Filters tested for Hg were found to contain less than 0.002
yg of this metal. Filtration of 100 ml of 0.1 yg Hg/1
solutions of HgCl2 in 1% (vol/vol) HN03 showed that the
filters removed less than 1% of the dissolved Hg from
solution.
Air
The gas scrubbers containing the 100 ml of H2304-KMn04
solution used for extracting Hg from the air were carried
to the laboratory and the excess permanganate reduced by
the addition of crystaline hydroxylamine hydrochloride.
The contents of the scrubbers were then transferred to BOD
bottles and handled in the usual manner, starting with the
addition of SnCl2. In some cases 10-ml aliquots were used
instead of the entire sample when high levels were expected,
based on previous data. These were diluted to 100 ml with
deionized water. Samples (100 ml) of the H2S04-KMn04
23
-------
solution used in the sample scrubbers were analyzed so that
corrections could be made for the slight amount of Hg in
the reagents.
Sediment
The small (14-mm) cores removed in the early part of the
project were cut, while frozen, into three parts. The
well-defined organic layer, which ranged from 2-5 mm in
thickness, was removed and the remaining sand was divided
into two approximately equal portions. The three fractions
from each core were dried at 60°C overnight, and cooled in
a dessicator. Weighed subsamples were transferred to
100-ml volumetric flasks and treated with 5 ml of a 6%
(wt/vol) aqueous KMn04 solution. The flasks were covered
with marbles and autoclaved for 15 min at 121°C (EPA,
1974). After the flasks had cooled, approximately 80 ml
of deionized water was added. The flasks were allowed to
stand for several hours, and then brought to volume with
additional deionized water. The contents of the flasks
were transferred to BOD bottles for analysis. For the
quantification of organic content, weighed subsamples of
the dried core fractions were placed in 40-ml Vycor
evaporating dishes and ashed for approximately 18 hr at
400°C.
The sampling procedure for benthic insects effectively
separated the organic portion of the sediment in the pyrex
dishes from the sand. Virtually all of the organic matter
was transferred into the enamel pans with the wash water
used to remove the insects. This was saved and the
total volume measured when sampling was complete. The
water was then mixed to suspend settled matter and a
100-ml portion was removed -which was blended and subsampled
for Hg analysis and the determination of organic content.
Estimates of the organic matter contained, in the blended
samples were obtained in one of two ways. A 10-ml sample
of the homogenate was either filtered through a prepared
0.3 nm Gelman Type A glass fiber filter, or was placed in
a prepared 40-ml Vycor evaporating dish. Filters were
washed with deionized water, dried at 110°C, fired at
500°C for at least four hours, and dessicated until they
were removed for weighing prior to use. The same treatment
was applied to Vycor dishes except the dishes were scrubbed
with a mild abrasive (Bon-Ami) before being rinsed.
Samples on the filters or in the Vycor evaporating dishes
were dried to constant weight at 65°C, cooled and weighed.
24
-------
They were then ashed at 400°C overnight, cooled and
reweighed. The loss between the last two weighings was
considered to be an estimate of the ash-free dry weight
which was used to convert Hg readings to a relative (yg
H
-------
results were consistently higher by about 5%. This indicates
that the blending process may release soluble organic cell
constituents which pass through the filter.
The relationship between wet weight and ash-free dry weight
was determined on three occasions. Composite samples of
export material were collected, blended, centrifuged and
weighed amounts of the resulting mass placed in Vycor dishes
for drying and ashing. Data show that the weight loss on
ignition was (5.9%) of the wet weight which agrees well with
the value of 6.4% calculated from data presented by Sladecek
and Sladeckova (1963) for periphyton colonizing vertical
glass slides.
Periphyton
All periphyton sampling procedures resulted in aqueous
suspensions of the material scraped from the various
substrates. These suspensions were blended for 1 min and
treated in the same manner as described for the organic
portion of the sediments except that an additional
subsample was taken for algal counts. The volume taken
for digestion was a function of the density of the suspended
matter in the sample.
Macrophytes
The washed leaves and roots of the Juncus plants removed
from the channels were allowed to dry at room temperature
for 24-48 hr. The roots and stems were cut into small
pieces and weighed portions placed in 100-ml volumetric
flasks and treated with 5 ml of concentrated H^SO^ and 1 ml of
concentrated HNO3. The stoppered flasks were manipulated
so that all the plant material was in contact with the
acid, and then allowed to stand for 18-20 hr. Five ml of
6% KMnC>4 was then added and the flasks stoppered after the
initial frothing had stopped. The samples were allowed to
digest at room temperature for an additional
After this period, the volumes were adjusted to 100 ml
and the contents of the flask transferred to BOD bottles
for analysis. This procedure was sufficient to dissolve
and oxidize the plant tissues and no residues were visible
when the permanganate coloration was destroyed with
hydroxylamine hydrochloride during the analytical process.
Weighed and dried samples of roots and stems were oven
dried at 60°C for 18-20 hr, cooled in a dessicator, weighed,
ashed at 400°C, cooled and weighed again. The weight lost
during the ashing procedure was used to calculate the
26
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percent organic matter in the original air dried sample.
The figure was then used to calculate the weight of the
organic component of the plant samples analyzed for Hg.
Insects
Insects collected from the screens at the ends of the
channels were blotted dry, weighed, and placed in 100-ml
volumetric flasks. Five ml of concentrated H2S04 and 1 ml
of concentrated HNOs were added to the flasks which were
allowed to stand, with occasional shaking, until a clear
solution resulted. Five ml of a 6% (wt/vol) KMnC>4 solution
were then added to each flask. The flasks were stoppered
after initial frothing had ceased and allowed to stand for at
least 16 hr at room temperature. They were then filled to
100 ml with deionized water and transferred to BOD bottles
for analysis.
Fish
Mosquitofish were killed by submersion in hot water, blotted
dry, weighed and placed in 100-ml volumetric flasks. They
were then processed in the same manner as described for
insects. When the fish were large (> 0.5 g), twice the
usual amount of acids and KMn04 were added. Reagent blanks
were prepared in the same manner.
ALGAL COUNTS
Representative subsamples (approximately 5 ml)- of the
homogenates resulting from the processing of sediment cores,
channel walls and plexiglass plates were placed in 15 X 125-
mm tubes and refrigerated in the dark until counts could be
made. Usually samples were counted the same day they were
collected, however, a few were stored for as long as two days,
For counting, the samples were brought to room temperature,
resuspended by shaking the test tube at high speed with a
vortex type mixer (Cole-Palmer Super-Mixer) and a 50-ul
portion removed immediately with an Oxford micropipettor
fitted with a disposable tip. The portion was placed as a
drop on a clean microscope slide and spread evenly under
a 22 X 22-mm, #1 glass coverslip. The algal cells observed
in measured areas of the slide were identified and counted
at 400 X with a Nikon Model SKe phase-contrast microscope.
An attempt was made to identify and count a total of 200
cells from each slide although up to 1000 cells were
counted on very dense slides. On a few occasions, because
of the low numbers of organisms in the samples, counting
27
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was terminated at 100 cells. During the last part of the
study, a replicate count from another measured drop was
made for each sample.
All cells in colonies and filaments were counted individually.
Vegetative cells were counted if chloroplasts were present,
and tiny, highly motile flagellates were not counted unless
they were observed in resting stages.
Qualitative observations of blended and unblended periphytic
material indicated only one common algal species was altered
beyond recognition in the blending process (Eremosphaeria
viridis) while filamentous forms were commonly broken up
into single cells or small groups of cells permitting more
accurate enumeration.
Algal counts for individual species were converted to
relative values (cells/mm2 of substrate) for treatment
comparisons. To compare total populations, the volume of
each species was estimated from average measurements of the
cells (APHA, 1965) and the total cell volumes (in cubic
standard units, 1 CSU = 8000 mm3) per unit area (mm2) of
substrate compared between treatments. The total organic
content of periphyton attached to each substrate was
determined from dryed and ashed subsamples of the same
suspension on which the algal counts were made. Diversity
and evenness values were calculated from equations given
by Pielou (1969) for subsamples from large populations,
S
H=- E (n./N) log, (nt/N) , and
i=l 1 ^
E=H/log2 S
where S is the number of species, N is the total number of
cells counted, n^ is the number of cells in the it'1 species,
H is the Shannon Weaver diversity and E is the evenness.
Algae colonizing the glass microscope slides suspended in
the channels were counted directly. After a slide was
removed, the downstream surface was wiped clean and a
coverslip was placed on that end of the upstream surface
that had been deepest in the water. Again, a known area
was examined and the algal species identified and counted.
STATISTICAL TREATMENT
The amount of replication that was possible in sampling the
channel components was limited by the physical size of the
28
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channels, the long duration of the study, and the amount of
personnel time available. At some time during the project
period, however, at least one estimate was made of the
variability associated with each method and these
variability data have been presented in the appropriate
section.
In analyzing the large quantity of routine data collected,
the positional effects resulting from the difference between
upstream and downstream stations and the differences between
channels treated alike have been largely ignored. Separation
of the effects of the most important factors influencing
the results, Hg level and time, were made by a two-way
analysis of variance (ANOVA, fixed effects model) by means
of subroutines included in the Statistical Analysis System
(SAS) (Service, 1972). Correlations, regressions, and
basic calculations were done either with SAS subroutines
or with BASIC programs specifically written for the Nova
1200 computer associated with the stream facility.
RELATED STUDIES
General
During the project period, several short-term studies
relevant to the stream research were carried out. Some
of these were initiated to confirm channel results, others
were done to provide information not available from the
channels. The rationale for these short-term studies and
the methods used will be presented in the following
paragraphs. The results and conclusions will be discussed
in Section VII.
Bacterial Counts
Since the sensitivity of microorganisms to Hg salts is well
known, the initial project plans included daily sampling of
water for total bacterial counts. From the beginning of
June, 1972, until the middle of October, 1972, samples of
water were taken between noon and 2 PM daily in sterile
dilution bottles from head and tail positions in each
channel. The suspended bacteria were counted by serial
dilution pour plates made with tryptone glucose extract
agar. Because no differences were found between treatments ,
sampling was reduced to a weekly schedule in November,
1972, and discontinued completely at the end of the year.
The Hg levels in the channels were below those found to have
an effect on heterotrophic bacteria by Albright et al. (1972)
29
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Productivity Estimates
The original project also included plans for channel
productivity determinations based on upstream-downstream
measurements of dissolved oxygen (Odum, 1956) and pH
(Beyers, 1970). For this purpose, Schneider Instrument
Co. RM-15 water quality monitors were borrowed from the
EPA's Southeast Environmental Research Laboratory (SERL)
Athens, Georgia, and installed in small buildings located
at both ends of each channel and a system constructed which
switched the water input to the monitors from one channel
to another every 10 min. Dissolved oxygen, conductivity,
pH, and temperature were recorded by both monitors and the
monitor near the head of the channels also recorded solar
input. Plans to interface the monitors with a digital
data-processing system based on a Nova 1200 computer were
abandoned when it became apparent from the analog outputs
that there were no measurable differences, except with
respect to temperature, between the ends of the channels.
The water was generally saturated with oxygen at all times.
The maintenance and calibration of the monitors was
extremely time consuming and the fulltime monitoring of
water quality was discontinued.
During the period the monitors were in use, the standing
crop biomasses in the channels were relatively small. To
see if the lack of. usable results was simply a result of
the lack of biota, upstream-downstream measurements of
oxygen and pH were made on several occasions shortly before
the termination of the project. There were well-defined
differences between stations and it appears that
productivity measurements could be made in the channels
after sufficient growth has occurred.
Diurnal Uptake Patterns
There was, and still is, some question as to whether or
not the removal of Hg from water is mediated by the
physiological activities of organisms in aquatic systems.
In June and August of 1972, 25 mci of Hg-197 was input over
a 24-hr period into the channels maintained at 1.0 yg Hg/1
(2 and 5). The low-level channels set at 0.01 yg/1 could
not be used because the specific activity of commercially
available Hg-197 was too low. During the 24-hr input
period, and for several hours afterwards, 5-ml water
samples (unfiltered) were taken hourly from three locations
in both channels, from the effluent stream below the
shredded rubber Hg removal systems, and from a small pond
connecting with the effluent channels.
30
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All samples were counted in a deep-well, 5-cm Nal (11)
scintillation crystal coupled to a 400-channel pulse height
analyzer. Counting times were adjusted to maintain a
relative counting error of approximately 10%. The purpose
of taking the hourly water samples was to look for diurnal
patterns of Hg uptake by the channel communities as
determined by differences in activity between upstream
and downstream samples of the same water mass.
An intensive 26-hr survey was also done in January, 1974,
when all six channels were sampled for water at upstream
and downstream stations, and for Hg losses at the air-water
interface at the mid-point of the channels every 2 hr. Both
filtered and unfiltered water subsamples were analyzed for
Hg by the methods described previously.
Continuous-flow Chambers
To determine if the responses to Hg observed in the channels
could be verified under conditions of reduced interspecific
interactions, continuous-flow chambers (CFC) were designed
and built to test Hg uptake and growth responses of specific
channel organisms in a more-controlled fashion. The system
is illustrated in Figure 6. Water from the well feeding
the artificial streams was passed through a limestone
treatment similar to the system used for the channels and
passed through the plexiglass chambers to give a velocity
equal to that used in the channels (0.013 m/sec.) Mercuric
chloride was pumped into the input line of three of the
plexiglass chambers to provide a concentration of 5 yg
Hg/1 and the other three chambers served as controls.
The CFC system was first used to determine direct uptake
rates of Hg from water by mosquitofish and by a corixid
hemipteran (Hespercorixa sp.). For these uptake studies,
30 animals were placed in each chamber and 5 were removed
at each sampling time.
Attempts were made to isolate the dominant algal species
of the artificial streams to use in the CFC system.
Suspensions of channel periphyton were sprayed with a
spray-diffuser (Pringsheim, 1946) onto agar plates of Hold's
medium (Bischoff and Bold, 1963), modified by the elimination
of buffers and the addition of vitamins, or onto agar plates
of "Algal-Grow," a product of Carolina Biological Supply Co. ,
Burlington, N. C. Five species were isolated and maintained
on agar slants. Transfers to aqueous media were made to
develop seeding stocks for the CFC system.
31
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LIMESTONE
CHAMBERS
EFFLUENT
GLASS SLIDES
f
n r
n
T_rn r
ill
i rn r
d
SIDE VIEW
FIGURE 6. CONTINUOUS FLOW CHAMBER SYSTEM
-------
For the study of the effects of Hg on the algal species,
racks holding glass microscope slides perpendicular to the
direction of flow were installed in the chambers. Mixed or
unialgal cultures were poured into the two mixing tanks.
The colonization of the glass slides was observed, by the
methods previously described for the channel glass slides,
for a period of up to 27 days.
Biological Half-life of Mercury in Mosquitofish
After the Hg inputs to the channels were stopped, 200
Hg-contaminated mosquitofish were collected from the
effluent system of the channels. These fish were placed
in 200-1 tanks, 40 per tank, into which a constant flow
of fresh water was piped. The fish were fed with a
commercial fish food preparation (Bi-0-Rell) low in Hg
(0.20 ug/g dry wt.). Samples of 10 fish were removed
periodically and analyzed by the methods previously
described. The study was done to compare the rates of
Hg loss from contaminated fish transferred to clean systems
with those calculated from fish remaining in contaminated
systems (the stream channels). It was also done to compare
estimates of the biological half-life of Hg in mosquitofish
exposed over a long period with estimates based on fish
exposed for a short period to relatively high levels
(Huckabee and Blaylock, 1973).
33
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SECTION VI
RESULTS AND DISCUSSION
FATE OP MERCURY
General
There are several common ways of expressing analytical
results and problems can arise when units are not explicitly
stated (Olley, 1973). In the discussions that follow, Hg
concentrations in fish and insects are expressed on a wet-
weight basis. The concentrations in all other community
components, with the exception of the sand portion of the
sediment, are based on an ash-free dry weight. The ash
free dry weight is the weight lost during the ashing
process previously described-, and is considered to be an
estimate of the total organic content of the sample.
Results of analysis made on the sand component of the
sediment are based on an oven-dry weight, the usual method
for expressing sediment results.
Analytical results of export, periphyton, and organic
sediment samples, can be converted to a wet-weight basis
by multiplying the ash-free dry weight value by 0.06.
This conversion is necessary for making comparisons between
trophic levels.
Water
During the first 12 months of the project, the average
Hg levels in the channels., as calculated from the daily
flow and Hg input records, were 1.04 and 1.05 yg/1 in
channels 2 and 5 respectively, and 0.01 yg/1 in channels
3 and 6. Analyses of unfiltered water samples taken
approximately 10 m from the Hg input tubes in channels
2 and 5 support the calculated values for these channels.
A summary of Hg analyses of unfiltered water samples
collected between August 1, 1973 and January 29, 1974, is
presented in Figure 7. The data show that, during this
period, the input levels were slightly higher than desired
and also that the Hg concentrations in the water leaving
the channels were lower than those at the head end. The
average uptake in the 1.0-yg/l channels (2 and 5) was 17.5%
and in the 5.0-yg/l channels (3 and 6) was 16.8%. The
similarity in the relative uptake values at the two different
doses indicates there is no saturation of adsorption sites
34
-------
6r
4
cr
I 3
o
I 2
0
H3
H6
? }
T3
T6
H2
El
I
E2
I.O/m/l 5.0/^g/l
EFFLUENT
DITCH
SAMPLING LOCATION
FIGURE 7, TOTAL MERCURY LEVELS (X ± 2SE) IN CHANNEL AND EFFLUENT
WATER, SAMPLES WERE TAKEN FROM UPSTREAM (H) AND DOWNSTREAM (T)
POSITIONS IN EACH CHANNEL AND FROM TWO POSITIONS (EL E2) IN THE
EFFLUENT DITCH DRAINING THE CHANNEL SYSTEM,
35
-------
at the levels of Hg introduced. The relative uptake values
^™?Ch,_10Wer, than. those rePorted by Huckabee and Blaylock
(1973) who worked with a 100-m length of natural stream and
found that 75% of Hg added as Hg (1103)2 was removed. The
natural stream Huckabee and Blaylock (1973) studied was
much more turbulent than the artificial streams, and
probably supported a higher total biomass per unit area.
That turbulence might affect the uptake of Hg is shown by
the data presented in Figure 7 for the two positions in
the effluent stream. Although the distance between sampling
stations in this stream was about the same as between the
two channel stations, there was a greater relative uptake
of Hg (54.6%) from the effluent water, even though the
retention time of the water in this section was short. The
greater uptake might be explained by the high turbulence
in this region resulting in greater probability of contact
between a Hg atom and some portion of the stream wall. The
biological communities and lining (poured concrete) in the
effluent region, of course, were quite different from those
in the experimental channels and the possibility of
differential uptakes resulting from differences in community
structure and channel lining cannot be disregarded.
The results of the two Hg-197 diurnal uptake studies are
summarized in Figures 8 and 9. No uptake patterns are
evident, although the variability of the data is high,
probably due to the inclusion of particulates in the
sample. In the June, 1972, experiment, an average of
12.5% of the radiomercury was removed from the water.
In August, 1972, this value increased to 17.9%, probably as
a result of the increased biomass in the channels.
The results of the 26-hr intensive water sampling done in
January, 1974, are presented in Figure 10. The data show
that an average of 13.2% of the Hg added was retained in
the 1.0-yg/l channels and 15.6% in the 5.0-yg/l channels.
The bimodal uptake curve in both treatment levels is
difficult to relate to the biological activity in the
channels. A diurnal curve of the type described by Odum
(1956) for dissolved oxygen levels or by Beyers (1963,
1965) for carbon dioxide uptake and release in aquatic
systems, with a single maximum and minimum each 24-hr
period, would be expected if Hg uptake were related to
the diurnal photosynthetic or respiratory patterns of
the channel communities.
All data pertaining to uptake (Figures 7-10) show that a
constant proportion of the added Hg was removed regardless
36
-------
Q Stream 2
m Stream 5
-15
i i i i i i i i i i i i i i i i i
i i i i i
10
am
2
pm
6
pm
10
pm
TIME
2
am
6
am
10
am
FIGURE 8. DIURNAL RADIOMERCURY UPTAKE CURVE/ JUNE. 1972,
PLOTTED ARE THE DIFFERENCES BETWEEN RADIATION LEVELS IN
UPSTREAM WATER SAMPLES TAKEN AT THE INDICATED TIME AND
DOWNSTREAM SAMPLES TAKEN TWO HOURS LATER,
37
-------
20
K)
=3
o
o
UJ
o
z:
UJ
cr
LU
u_
u_
O
o -10
D Stream 2
am 5
,J — — —
10
am
2
pm
6
pm
10
pm
TIME
2
am
6
am
10
am
FIGURE 9, DIURNAL RADIOMERCURY UPTAKE CURVE, AUGUST, 1972,
PLOTTED ARE THE DIFFERENCES BETWEEN RADIATION LEVELS IN
UPSTREAM WATER SAMPLES TAKEN AT THE INDICATED TIME AND
DOWNSTREAM SAMPLES TAKEN TWO HOURS LATER,
38
-------
1.0 -
u
0.5
OJ
CJ>
X
0.0
I.O^g/l
5.0/ig/l
2 4 6 8 10 I2M 2 4 6 8 10 I2NOON
PM AM
TIME
FIGURE 10, DIURNAL MERCURY UPTAKE IN TREATED CHANNELS ON JANUARY 22-23, 1974,
PLOTTED ARE THE DIFFERENCES BETWEEN HG LEVELS IN UNFILTERED UPSTREAM SAMPLES
TAKEN AT THE INDICATED TIME AND DOWNSTREAM SAMPLES TAKEN TWO HOURS LATER,
-------
of dose, which indicates there was no saturation of potential
binding sites at Hg levels up to 5.0 ug/1- These results
differ from those reported for Chlorella by Matsui and
Gloyna (1972) who found that the higher the Hg concentration,
the greater the percent uptake by this alga. The difference
may be due to the complex nature of the channel communities
compared to unispecific Chlorella cultures.
Figures 8-10 show no clear pattern that would couple Hg
uptake with the metabolic activities of the channel
communities. These results, which indicate passive uptake,
are consistent with the findings of Hannerz (1968),
Glooschenko (1969) and Fujita and Hashizume (1972) who attribute
inorganic Hg uptake in algae to surface adsorption although
Hannan and Patouillet (1972) concluded that in the freshwater
algae they studied, Hg uptake could not be accounted for
by surface adsorption alone. Burkett (1975) also found
indications of processes other than passive adsorption in
the uptake of methylmeifcury 'by Cladophora, a green alga.
Data showing the fraction of the Hg in the water that was
attached to particulate material in the January, 1974,
intensive sampling are presented in Figure 11. In all
treated channels, the proportion of the total Hg removed
by filtration was greater at the downstream stations than
at the upstream stations. The percent of the total Hg
bound to particles was greater in the 1.0-yg/l channels
than in the 5.0-yg/l channels. This indicates a saturation
phenomenon with respect to the suspended particulates which
may be related to the number of bacteria in the channel
water. Figure 12 summarizes bacterial count data based on
results of daily samples taken between June 1, 1972, and
October 10, 1972. Counts made after October 10, 1972 fall
in the limits presented in Figure 12, even after the dose
increase of August 1, 1973. The constant number of bacteria
at a given point in all channels provide a fixed number of
attachment sites for Hg. If the number of sites were
limited, there would be a decreased relative uptake at
higher Hg levels. This is what was observed (Figure 12)
and, although the results are by no means conclusive, the
relationship between the increased number of bacteria at
the tail end of the channels and the greater proportion
of particulate-bound Hg at these stations strongly implicates
the suspended bacteria as adsorbing agents.
After the Hg inputs to the channels were terminated (9 AM,
January 29, 1974), water samples were taken hourly for 6 hr
at the two sample stations in each channel, and on several
occasions during the following month. The water levels
40
-------
c
. o
en V-
X a
JD
CD
O
I-
cr
< b
°- E
CD
50 i-
40
£ ~ 30
20
0
0
a
T5
H2
I
T3
T6
H5
H3
H6
I.O/ig/l 5.0/ig/l
SAMPLING LOCATION
FIGURE 11. PROPORTION OF HG ASSOCIATED WITH PARTICIPATES
IN WATER SAMPLES, PLOTTED ARE MEANS (± 2SE) BASED ON THE
13 SETS OF SAMPLES TAKEN DURING THE JANUARY,, 1974,
INTENSIVE SAMPLING PERIOD,
-------
Q)
JD
ce
LJ
H
O
<
CD
HOOr
1000
900
800
700
600
500
400
300
200
100
I
4 36 25 14 36 25
UPSTREAM DOWNSTREAM
CHANNEL NUMBER
FIGURE 12. BACTERIAL POPULATIONS IN CHANNEL WATER, RESULTS
(X ± 2SE) ARE BASED ON 130 DAILY COUNTS MADE FROM JUNE TO
OCTOBER OF 1974.
42
-------
decreased rapidly and were approximately 0.2 yg/1 (filtered
samples) in all treated channels by January 31, 1974. By
the end of February, 1974, all samples were below the
0.05 ug/1 detection limit of the procedure used, even
though the Hg levels in the channel periphyton and sediment
remained high.
Air
Quantitative data for Hg losses from the channels at the
air-water interface were not obtained until December, 1973.
No estimates exist, therefore, for losses from the two
channels that were receiving an Hg input of 0.01 yg/1.
The air-loss data, which are summarized in Figure 13, show
that the releases were highly variable, although related to
water concentration, and continued after the Hg inputs were
stopped. No losses were measured from the control channels.
There appears to be no logical explanation as to why the
losses from channel 5 were consistently low, or why the
losses decreased rapidly before the Hg inputs were stopped.
The data are insufficient to show any seasonal patterns.
The form of Hg lost at the air-water interface is mostly
elemental Hg. The results from two air sampling boxes
located next to each other in the same channel, one with
a silver trap in the line, showed that the filter removed
approximately 97% of the Hg before it reached the gas
scrubbing system. According to Braman and Johnson (1973) ,
silver does not adsorb dimethyl mercury, and will only
partially retain monomethyl mercury compounds, but will
quantitatively retain elemental Hg. The specific filters
used in this study were checked by inserting them in the
line of the Coleman MAS 50 leading into the absorption
cell, and then processing a 1-yg Hg standard. No increase
in absorption was noted indicating that none of the
elemental Hg released in the analytical process passed
through the filter. The loss of elemental Hg from aquatic
systems has been found in other studies (Fagerstrom and
Jernelov, 1972; Spangler et al., 1973; Parks, 1973; Holm
and Cox, 1974) and some of the possible mechanisms of its
formation have been investigated (Toribara et al., 1970;
Alberts et al., 1974; Wood, 1974; Bisogni and Lawrence, 1975).
During the air sampling periods, it appeared that the amount
of Hg collected in the traps was a function of the number
of bubbles rising from the bottom. Samples of the gas
rising from undisturbed sediments were collected and
immediately injected into the closed air system of the Hg
43
-------
0.9
o>
0.7
0.6
LU
CO
UJ
*
en
X
0.5
0.4
0.3
cr 0.2
an-
o.o
a— I.O^g/l
A—5.0/tg/l
FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
SAMPLING DATE 1974
FIGURE 13, AVERAGE MERCURY RELEASES AT THE AIR-WATER INTERFACE OF THE
TREATED CHANNELS.
-------
analyzer. This resulted in a significant absorption of the
UV beam, and, although no attempts were made to quantify the
data, it confirmed the presence of elemental Hg in the gas
bubbles. Samples of gas from the control channels were
checked and the results were negative. The loss of volatile
Hg species with bubbles released from sediments has been
suggested as a Hg release mechanism by Bothner and Carpenter
(1974).
There were no significant correlations between the air
losses and either the organic contents or the Hg content
of the sediments under the sampling boxes. Perhaps a
correlation would have been observed if some quantification
of the living portion of the sediment, especially the
bacterial components, could have been made at the same
time the air samples were taken. Bacteria are known to
be able to release elemental Hg from both inorganic and
organic compounds of this metal (Suzuki et al., 1968;
Matsumura et al., 1972; Furukawa and Tonomura, 1972),
although elemental Hg production can also occur in the
absence of bacteria (Alberts et al., 1974; Bisogni and
Lawrence, 1975).
Results of the intensive 24-hr air sampling program of
January 22-23, 1974, are shown in Figures 14 and 15. The
data again show that the releases were greater from the
high-level channels than from the low-level channels.
The rates of the releases appear to be related to the
water concentrations (5:1) rather than to the Hg levels in
the sediment on that date (1.6:1, Figure 17). This
implies that the reaction-producing elemental Hg was
occurring at the water-sediment interface rather than
deeper in the sediments.
The air release data shown in Figures 14 and 15 indicate
that there may be a diurnal release pattern with a maximum
occurring in the late afternoon. This diurnal pattern
would not be reflected in the water concentrations of Hg
because the maximum air losses observed, if extrapolated
to the entire channel, were always less than 1% of the
total Hg in the water.
Sediments
When the water inputs to the channels were started in
September, 1971, the bottom substrate consisted of a 10-
cm layer of washed builders sand. As time passed, a thin
mat community consisting of detritus, fungi, bacteria,
algae and small invertebrates formed on the sand. The
45
-------
0.2 r-
LJ
UJ
-J
UJ
cr
cr>
0.1
0.0
D-CHANNEL 2
•-CHANNEL 5
I
7
PM
I! I
7
AM
TIME
FIGURE 14, DIURNAL MERCURY RELEASES ON JANUARY 22-23, 1975, AT THE
AIR-WATER INTERFACE OF CHANNELS RECEIVING AN INPUT OF l,0yu.g/l.
THE AREAS SAMPLED WERE 0,186 M2,
-------
2.0
u
.5 -
LLJ ID
LJ
cr
CT>
cr 0.5
0.0
A—CHANNELS
A—CHANNELS
_L
I
I
I
9 II
3579 II I 357
PM AM
TIME
FIGURE 15, DIURNAL MERCURY RELEASES ON JANUARY 22-23, 1975, AT THE
AIR-WATER INTERFACE OF CHANNELS RECEIVING AN INPUT OF 5,0/lQ/l.
THE AREAS SAMPLED WERE 0,186 M2,
-------
thickness of this mat was always greatest near the heads of
the channels and became thinner toward the tails. Gradually
this mat became less structured as emergent aquatic plants
and larger' animals, especially amphibian larvae, became
established in the channels.
The sediment samples removed in the first part of the
project consisted of frozen cores which were sectioned to
give a vertical profile of Hg distribution. After several
sets of samples, it became evident that all of the Hg in
the cores was associated with the mat community covering
the sand. Sand samples from the cores, and also from the
benthic dishes used for invertebrate sampling, contained
Hg levels of less than 0.2 yg/g except when they were
contaminated with organic debris forced in by the coring
procedure. At no time during the project were significant
amounts of Hg found in the sand portion of the sediment.
This was not a result of the drying process used because
portions of mat were also dried and analyzed without a
significant loss of Hg. Because of the localization of Hg
in the organic layer on top of the sand, Hg values were
calculated as yg/g ash-free dry weight of organic matter
for comparisons between channels.
The results of analyses of the organic component of the
channel sediments are presented in Figure 16 for the
control and 0.01 yg/1 treatments, and in Figure 17 for
the 1.0 yg/1 and 5.0 yg/1 treatments. The data show that
even in the channels receiving Hg inputs of 0.01 yg/1,
there were elevated Hg levels in the sediments relative
to the controls. When the 0.01 yg/1 input was changed to
5.0 yg/1, there was a rapid increase in sediment levels
although the data are insufficient to show whether or not
an equilibrium level was reached. The sediment Hg levels
in all treated channels showed an increase four months
after the Hg inputs were stopped. There is no obvious
explanation for this although it appears to be more than
chance variability. There was no marked decrease in any
other components of the channel communities at the time
of the increase in the sediments.
Figures 16 and 17 clearly show the slow decrease in sediment
levels in the 13-month period following the cessation of
Hg input. The half-lives calculated from data collected
after May, 1974 were 7.85 years (€.1.95% = 5.62-13.0 years)
for the 1.0 yg/1 treatments and 8.67 years (0.1.95% =
6.11-15.0 years) for the 5.0 yg/1 treatments, assuming
first order kinetics. These half-lives are considerably
longer than the 1.3 years calculated by Bothner and
48
-------
20 r
vo
1!
2 15 -
IT
\~
•z.
LU
10
o
o
Ltl
Q
U
cn
0
Hg INPUT
INCREASED
Hg INPUT
OFF
O
CONTROL
•0.01
j i
SONDJFMAMJJASONDJ FMAMJJASONDJ
1972 1973 1974 1975
SAMPLING DATE
FIGURE 16. AVERAGE MERCURY CONCENTRATIONS IN THE ORGANIC PORTION OF THE SEDIMENTS OF
THE CONTROL CHANNELS AND CHANNELS RECEIVING AN INPUT OF 0,
-------
en
O
2000
CP
^ 1500
<
Ld
o
-z.
o
o
Ui
Q
UJ
1000
500
0
D-I.O/xg/l
Hg INPUT Hg INPUT
INCREASED OFF
0
N
D
M
A
J
A
0
N
D
M
A
M
A
0
N
D
1972
1973 1974
SAMPLING DATE
1975
FIGURE 17. AVERAGE MERCURY CONCENTRATIONS IN THE ORGANIC PORTION OF THE SEDIMENTS OF
CHANNELS RECEIVING INPUTS OF 1.0 AND 5.0^ g / | .
-------
Carpenter (1974) for losses from a contaminated marine
system but are similar to the values they calculated from
the data of other workers (Spangler et al., 1973).
Export
Figure 18 summarizes the Hg concentrations measured in the
particulate matter leaving the channels. Data for the
control channels are not presented because of the generally
low values (<4 yg/g ash-free dry weight) found in these
samples. Although Figure 18 shows the typical high
variability associated with the Hg analyses, several trends
are evident. The Hg levels in export from channels 3 and 6
rose dramatically in the three-week period following the
treatment changes in these channels from 0.01 to 5.0 yg/1.
Indeed, it appears that an equilibrium level of approximately
1800 ug/g was reached in this short'period of time. This
concentration was a factor of only 2-3 times that measured
in the 1.0 ug/1 channels, even though the water levels were
greater by a factor of 5.
Mercury concentrations were consistently higher in export
material than in the sediments (Figure 17) until Hg inputs
were stopped in January, 1974. Export levels then decreased
more rapidly than the sediments and were much lower when the
project was terminated. This indicates that the majority
of the exported particulate was not derived from the
sediments.
Periphyton
The most complete set of data for Hg levels in the channel
periphyton is for material removed from the PVC lining.
The results for the control channels and the channels
receiving an input of 0.01 yg/1 are presented in Figure 19.
Figure 19 shows that Hg levels in the periphyton exposed to
a water concentration of only 0.01 ug/1 were higher than in
samples from the control channels. It also shows that there
was an increase in Hg concentrations in the control
channels when the doses were changed in August of 1973,
and a decrease in the control concentrations after the Hg
inputs were stopped. Mercury was probably entering the
control channels at the air-water interface since.a series
of wall periphyton samples taken every 20 m along the length
of channel 1 (control) showed the Hg levels were about the
same along the entire length. If there were a leak between
adjacent channels, only areas downstream from the leak would
have been affected.
51
-------
Ul
2500
1
,~ 2000
o
h-
LU
O
O
O
500
1000
n: 500
h-
tr
o
o_
X
UJ
0
Hg INPUT Hg INPUT
INCREASED OFF
D - I.O/ig/l
o - 0.01/xg/l
- 5.0/xg/l
S 0 N D J F
M A M J
J ASOND JFMAMJ JASONDJ F
1972
1973
1974
975
SAMPLING DATE
FIGURE 18. AVERAGE MERCURY CONCENTRATIONS IN PARTICULATE MATTER LEAVING THE TREATED CHANNELS.
-------
ui
U)
Hg INPUT Hg INPUT
INCREASED OFF
O —
CONTROL
0.01/tg/l
1972
1973
SAMPLING DATE
1974
1975
FIGURE 19, AVERAGE MERCURY CONCENTRATION IN PERIPHYTON REMOVED FROM THE PVC WALLS OF
THE CONTROL CHANNELS AND CHANNELS RECEIVING AN INPUT OF 0,01/ig/l.
-------
Results of the Hg analyses of periphyton removed from the
PVC in the 1.0 and 5.0 yg/1 channels are shown in Figure 20.
The levels in the 1.0-yg/l channels reached a peak of 3000
yg/g after about seven months. They remained constant at
this level for about five months and then, for no obvious
reason, began to decrease until they reached 600 yg/g by
the time the Hg was turned off. During the last six months
of the project, the levels remained relatively constant at
150-200 yg/g. This was slightly lower than the concentration
measured in the export leaving the channels during this
same period (Figure 18) which indicates at least some
contribution to the export from the sediments.
Figure 20 shows that the periphyton Hg levels in the 5.0
yg/1 channels were very erratic. The high value observed
in October, 1973, which like all other points is an average
of four samples, appears to be a valid measurement since
all four samples were high. After this peak, the decrease
in levels in the 5.0-yg/l channels parallels the observations
from the 1.0-yg/1 channels, although the values reached just
prior to the end of the project were slightly higher.
Data for periphyton removed from the plexiglass plates are
summarized in Figure 21. Periphyton removed from the
plexiglass plates always had much higher Hg levels than
that scraped from the PVC walls. This difference was
maintained until the end of the project when periphyton
removed from the plexiglass plates had concentrations of
approximately 1000 yg/g and PVC periphyton had concentrations
of 200 yg/g. The highest Hg levels measured in plexiglass
plate samples (>8000 yg/g in January, 1974) were still
lower than those reported by Matida and Kumada (1969) for
attached algae samples taken from a contaminated river in
Japan. Concentration factors calculated from the maximum
periphyton levels measured in plexiglass plate samples
range from 1 X 104 to 3 X 104. This range is considerably
higher than the values reported by Bannerz (1968) for algae
from experimental ponds but is very similar to the range of
concentration factors reported by Fujita and Hashizume (1972)
for a freshwater diatom.
Although the water levels of Hg during the last year of the
project were below detection limits, periphyton colonizing
new plexiglass plates put into a channel formerly treated
with Hg levels of 5 yg/1 in June, 1974, acquired equilibrium
Hg concentrations of 45 yg/g after two months.
54
-------
Ul
Oi
o>
1: 3000
o
a:
LU
o
o
o
X
z
o
X
Q_
ir
UJ
Q_
2000
000
0
Hg INPUT Hg INPUT
INCREASED OFF
Q
A— 5.0 /tg/ 1
0
NDJFMAMJJASONDJ
F M A
M J J A S 0
N
D J
1972
1973
SAMPLING DATE
1974
1975
FIGURE 20, AVERAGE MERCURY CONCENTRATIONS IN PERIPHYTON REMOVED FROM THE PVC WALLS
OF CHANNELS RECEIVING INPUTS OF 1,0 AND 5.0/i.g/I.
-------
U1
en
9000 r
o
or
8000 -
7000 -
6000
LU
O 5000
"Z.
o
4000 -
en
3000 -
2000 -
1000 -
o:
LU
Q_
INPUT
OFF
MJJAS
1973
1974
SAMPLING DATE
1975
FIGURE 21. AVERAGE MERCURY CONCENTRATIONS IN PERIPHYTON REMOVED FROM PLEXIGLASS PLATES IN
CHANNELS RECEIVING INPUTS OF 1.0 AND 5.0 fJ.g/\.
-------
Macrophytes
In the first 18 months of channel operation, the bottom
communities were relatively simple with no rooted vegetation.
By the spring of 1973, however, a great number of small
rooted plants were noted, especially in the upstream portions
of the outer channels. Although there appeared to be two
distinct species, these were later identified as variants
of a single species, Juncus diffusissimus. The distribution
of this plant in the channels in April of 1973 is shown in
Table 3. Quite obviously, channel one received a much
greater input of seeds than the other channels. The upstream-
downstream gradient in the distribution pattern may be due
to the input of seeds to the pools above the channels.
Starting in June, 1973, samples of Juncus were periodically
analyzed for Hg content. Results of root analyses are
shown in Figure 22, results of leaf analyses are shown in
Figure 23. No data are presented for the control channels
or the channels receiving an input of 0.01 yg/1 -because the
values were so low. In all cases, when Hg was being input
to the channels, the levels in the leaves were higher than
in the roots. The root levels were significant, however,
indicating movement of Hg within the plants
since the sand in which the roots were growing had an
extremely low Hg content. The rapid increase in root
levels during the spring of 1974, several months after the
Hg inputs were stopped, also indicates internal shifting
of Hg. The Hg in the leaves of the Juncus appears to have
been tightly bound since there was little Hg loss from the
leaves for several months after the inputs were stopped.
The rapid decrease in leaf concentrations beginning in
May was probably due to dilution by growth and the inclusion
of newly grown plants, which had not been exposed to Hg, in
the samples.
During the latter part of the period when Hg was being input,
the levels in the Juncus leaves were in approximately the
same ratio (1:5) as the water levels. This is one of the
few components of the channel communities that showed this
relationship. At the termination of the project, Hg levels
in the roots and stems were approximately equal in both
treatments, and were declining very slowly.
Concentration factors calculated from the maximum Hg levels
reached in the stems during the project are on the order of
2.5 X 103. The conversion factor for estimating wet-weight
concentrations from the ash-free dry weight data was 0.13
and was derived experimentally from Juncus removed from the
57
-------
Table 3. DISTRIBUTION OP JUNCUS DIFPUSISSIMUS ON APRIL 2,
1973, IN THE ARTIFICIAL STREAMS
(number of plants/interval)
Channel number
1
Distance control
from input
weir, meters
0-7.5
7.5-15
15-22.5
22.5-30
30-37.5
37.5-45
45-52.5
52.5-60
60-67.5
67.5-75
75-82.5
82.5-90
Total
453
261
144
98
97
124
222
70
38
26
49
236
1818
2
1.0
yg/i
112
131
30
23
13
18
31
13
6
0
17
18
412
3
0.01
yg/i
30
8
8
3
3
11
20
3
0
3
2
5
96
4
control
170
39
16
10
11
34
33
6
2
7
2
31
361
5
1.0
yg/i
139
10
9
5
8
18
25
1
4
1
12
5
237
6
0.01
ug/i
294
40
20
11
5
13
16
3
2
5
5
9
423
channels. The concentration factors determined by Hannerz
(1968) for rooted aquatics seldom exceeded 100, although
Dietz (1972) calculated values > 430 for submerged
spermatophytes.
58
-------
VD
400 -
I
g
<
h- o» 300 -
LU
o
o»
o
1
200
100
0
Hg INPUT
INCREASED
Hg INPUT
OFF
1.0 jig/I
5.0
973
1974
SAMPLING DATE
1975
FIGURE 22, AVERAGE MERCURY CONCENTRATIONS IN ROOTS OF JUNCUS DIFFUSISSIMUS REMOVED
FROM CHANNELS RECEIVING INPUTS OF 1,0 AND 5,0 /xg/1.
-------
D —1.0/j.g/l
— 5.0 fig/1
Hg INPUT
INCREASED
975
SAMPLING DATE
FIGURE 23. AVERAGE MERCURY CONCENTRATIONS IN LEAVES OF JJJNCUS DlEfiiS.LS_si/iu.s REMOVED FROM CHANNELS
RECEIVING INPUTS OF 1.0 AND 5.0 fJ.Q/\.
-------
Aquatic Insects
Early in the study, the larger aquatic insects were
relatively rare in the channels and no attempt was made
to collect these on a routine basis. During the entire
project period, the organisms removed from the benthic
dishes were never available in sufficient numbers to
provide an adequate biomass for Hg analyses. Also, the
taxonomic problems associated with the groups most common
in the benthic samples made it impossible to identify and
enumerate these organisms without first preserving them.
The number of species and population densities of aquatic
insects increased as the rooted macrophytes became an
important channel component. During the last 14 months of
the project, the material collected in the fish-retaining
end screens was periodically searched for macroinvertebrates
for analysis. The organism for which most data were
collected was a damselfly of the genus Ischnura. The data
are summarized in Figure 24 in which each point represents
the average of two to nine analyses, each of an individual
nymph. The average coefficient of variation (CV)
associated with the results from the 1.0-yg/l channels was
46% and from the 5.0-yg/l channels, 40%. Results for
Ischnura collected from the control channels are not shown
because the numbers were typically very low (<0.1 yg/g)
and also variable (CV=116%). Figure 24 shows that the Hg
levels in this damselfly were probably a result of direct
water uptake. The concentrations in animals collected in
the two treatments were approximately in the same ratio as
the water levels (5:1) and these concentrations converged
rapidly to low levels after the Hg inputs were stopped.
During the Hg input period, another damselfly, Argia sp.,
was collected occasionally from all channels other than
those receiving an input of 5.0 yg/1- Based on a sample
of six animals collected from the 1.0-yg/l channels during
January, 1974, the mean Hg level of 12.8 yg/g (SD=5.7)
for this damselfly was about the same as that found in
Ischnura sp. taken from the same channels at about the same
time. Argia collected from the same channels in February
and March of 1974 had levels that ranged from 3-5 yg/g,
values very similar to those observed for Ischnura. In the
time interval between the Hg shut-off and the project
termination, Argia became established in channels 3 and 6
and at the end of the project, levels measured in this
species were 0.25, 1.7 and 2.2 yg/g respectively for
animals from the control, former 1.0-yg/1 and former-5.0
yg/1 channels. Levels in Ischnura at the end of the study
61
-------
NJ
Hg INPUT
OFF
D —1.0/z.g/l
A —5.0/xg/l
1974
SAMPLING DATE
1975
FIGURE 24. AVERAGE MERCURY CONCENTRATIONS IN DAMSELFLY NYMPHS (ISC.ENJJRA SP.) COLLECTED
FROM CHANNELS RECEIVING INPUTS OF 1.0 AND 5.0 {J.Q/\.
-------
were 0.09, 2.6 and 2.8 yg/g in these same channels, again
indicating the two species were quite similar with respect
to Hg uptake and elimination.
Analyses were made of other immature insects, including
several other species of odonates, dipterans, aquatic
lepidopterans and several species of adult coleopterans,
notonectids and corixids. Levels for other odonate nymph
species were about the same as those presented for Ischnura
and Argia. Values for the adult forms (notonectids"]!
coleopterans and corixids) were extremely variable,
probably as a result of movement of these groups in and
out of the channels. During the course of the project,
corixids and other adult insects were seen flying from
one channel to another. In January, 1974, corixids were
observed to be very common in the channels and 18 individuals
(Hesperocorixa sp.) were collected, three from each channel.
Mean values (x + SD) for the Hg concentrations in the three
treatments were 0.11 + 0.13 ug/g, 0.34 + 0.20 yg/g and
3.91 + 5.5 yg/g for the controls, 1.0 yg/1 and 5.0 yg/1
treatments, respectively.
In August, 1974, a large number of uncontaminated
Hesperocorixa were collected from a nearby pond. These
animals were placed in the continuous-flow chambers
previously described to determine their ability to take
up Hg directly from the water and to measure the
variability of their concentrating ability under controlled
conditions. The results are presented in Figure 25. The
relatively rapid uptake by the unfed animals indicates that
direct uptake by this species was of major importance in
the channels. The lower variability in the CFC-treated
animals in contrast to the channel organisms was probably
due to the fact that they were confined to a given treatment
and there was no exchange between treatments as took place
in the channels.
Midge larvae were an important link in the food web of the
channel communities, and were numerically the most abundant
invertebrates. Because of the small size of the individual
organisms, composite samples of a number of individuals
were made for Hg analyses. Average concentrations in midge
larvae at the time the Hg inputs were terminated ranged
from below detection limits in the control channels to
8 yg/g in the 1.0-yg/l channels and 20 yg/g in the 5.0-
yg/1 channels.
The concentrations of Hg found in the channel insects were
much lower than those found by Matida and Kumada (1969) in
63
-------
en
•»
^
O
H
cr
h-
LJ
O
O
O
CP
X
Q
X
cr
O
o
4.0 r
3.0
2.0
• —CONTROL
A—5.0/ig/l
I 23456789
EXPOSURE TIME, days
FIGURE 25, MERCURY CONCENTRATIONS (X ± 1SD) ACQUIRED BY CORIXIDS
(HESPERQCORIXA SP.) CONFINED TO FLOWING WATER CONTAINING 5,(
HG++. EACH POINT is BASED ON ANALYSES OF 10 ORGANISMS,
-------
a contaminated Japanese river although the concentration
factors that can be calculated from the channel results,
are much higher than those reported by Hannerz (1968) for
his pond experiments with
Fish
The background level of Hg in the mosquitofish collected
for this project, as determined in April, 1972, was 0.04 +
0.01 yg/g (X + 2SE, n=20) . This value is much lower than"
the 0.22 yg/g (wet weight) level in the experimental
mosquitofish used by Schoper (1974). The pond from which
the fish for this project were taken is located adjacent
to an installation on the SRP which consumes a large
quantity of coal. The large stack releases from this
installation do not appear to have affected the pond
system, although other components have not been checked.
During the first year of the project, a number of fish
escaped from both the channels and cages when the screens
at the ends of the channels plugged and water levels became
excessive. The reduced numbers of fish made sampling
difficult and also greatly limited the number of samples
that could be taken. It was for this reason that all
remaining fish were removed at the end of the first year
and new ones introduced.
The results of analyses of fish removed from the 1.0-yg/l
channels (2 and 5) between May, 1972, and May, 1973, are
presented in Figure 26. Fish in channel 2 acquired Hg
faster and to a greater extent than those in channel 5.
In May, 1973, ,the average Hg concentration in_the 21 fish
removed from channel 2 was 5.30 + 0.96 yg/g (X + 2SE) , in
the 62 fish removed from channel 5, 3.41 + 0.30 (X + 2SE) .
The means are significantly different (p < 0-01) based on
a t-test of the log transformed (to uncouple the variance
and mean) data. There is no apparent reason for the
differences between channels. The fish population in
channel 5 was greater than in channel 2 but the average
weight of the fish did not differ (0.52 g in channel 2,
0.57 g in channel 5) when the populations were removed in
May, 1973.
Figure 26 shows that the Hg levels in the mosquitofish
exposed to 1.0 yg/1 rose slowly over a 10- month period to
equilibrium levels of 4-7 yg/g. There may be a seasonal
cycle indicated, with a peak occurring during the winter
months, especially if the values for the fish collected
during the population removal in May, 1973, are included.
65
-------
CPi
? 8
LU
O
O
O
C7>
X
5
4
3
2
0
D — CHANNEL 2
• — CHANNEL 5
M
J
J
A
S
0
N
D
J
F
M
A
M
1972
SAMPLING DATE
1973
FIGURE 26. MERCURY CONCENTRATIONS IN MOSQUITOFISH (GAMBUSIA AFFINIS)
REMOVED DURING THE FIRST YEAR FROM THE CHANNELS RECEIVING AN INPUT OF
l.Oft.g/1. EACH POINT is AN AVERAGE BASED ON TWO ANALYSES OF
INDIVIDUAL FISH.
-------
A similar seasonal trend is indicated in Figure 27 which
summarizes the results of analyses of fish removed from the
control channels and those receiving an input of 0.01 yg/1
Hg. Figure 27 shows that at levels of even 0.01 yg/1/
mosquitofish accumulate Hg above control levels. The
increase in the control fish is probably due to air transfers
of Hg between channels although this may be a natural
seasonal phenomenon that has simply not been observed before.
Statistical tests of measurements made on the fish removed
from the control and 0.01-yg/1 channels show that there were
no significant differences in Hg levels and weights of fish
between channels treated alike, but that there was a
significant (p < 0.01) difference between the Hg _
concentrations in the control fish (0.072 +_ .009, X + 2SE,
n=34) and those removed from the 0.01-yg/l~channels ~
(0.179 + 0.007, X + 2SE, n=99).
The Hg levels in fish removed from the control channels
after 1 year were significantly (p < 0.01) higher than
those measured in the fish at the beginning of the project.
This may reflect a natural increase with age, or it may be
due to cross contamination between channels, as mentioned
above. The differences in population sizes between channels
can in no way be attributed to treatment since so many fish
escaped from the channels.
Linear correlation coefficients between the body weights
and the Hg concentrations in fish were calculated on a
channel basis for all fish sampled between May, 1972,
and May, 1973. Only in channel 6 was there a significant
(r=0.82, n=12, p < 0.01) correlation. Correlation
coefficients for the same variables were calculated with
data collected from the fish populations removed from the
channels in May, 1973. There was a significant negative
correlation (r=-.61, n=21, p < 0.01) for animals removed
from channel 2. There does not appear to be a neat,
consistant relationship between weight and Hg levels in
mosquitofish exposed to low levels of Hg over a long time
period. This is not consistent with the data reported by
Johnels et al. (1967), Johnels and Westermark (1970),
Fimreite et al. (1971), Knight and Herring (1972) and
Kelso and Frank (1974) although Scott and Armstrong (1972)
found all possible relationships (positive, negative, and
zero correlation coefficients) between Hg levels and fish
lengtl^ depending on what species they were considering.
At the end of one year of exposure, there was no difference
between the Hg levels in fish living in the channels and
those confined to cages and fed with a commercial tropical
67
-------
t .5
CO
O
H
<
o:
UJ
o
z
o
o
I
1
5
A
• —CONTROL
0—0.01
M
ASON DJFMA
M
1972 1973
SAMPLING DATE
FIGURE 27. MERCURY CONCENTRATIONS IN MOSQUITOFISH (fiAMflii&iA AFFINIS)
REMOVED DURING THE FIRST YEAR FROM THE CONTROL CHANNELS AND THE CHANNELS
RECEIVING AN INPUT OF 0.01/J.g/l. EACH POINT IS AN AVERAGE BASED ON
FOUR ANALYSES OF INDIVIDUAL FISH,
-------
fish food containing 0.20 + 0.02 yg/g Hg on a dry weight
basis. This indicates that direct uptake of Hg from the
water makes a significant contribution to the body burdens
of mosquitofish. Based on two sets of analyses on composite
samples of several fish, the portion of the total Hg present
as methyl mercury was 28% in mosquitofish exposed for one
year. This value is low compared to published results
(Kamps et al., 1972; Bache et al., 1971) probably because the
entire fish was digested for total Hg (including gut
contents) rather than only the muscle tissue of the fish.
Fish data collected the last 20 months of the project from
fish placed in the channels in June, 1973, are summarized
in Figure 28. Mercury levels in fish from the control
channels ranged from 0.005 to 0.17 yg/g. The data are
averaged for treatments since there were no significant
differences between channels treated alike during this
period.
Figure 28 shows that the fish in the 1.0-yg/l channels
acquired about the same Hg concentrations as the fish of the
previous year. This equilibrium level was reached more
quickly however, (6 months as opposed to 10), which
indicates that fish placed in a contaminated area
concentrated Hg faster than those placed in a clean area
that was then contaminated. This result implies that
direct uptake of Hg++ from the water is not the only method
by which mosquitofish acquire Hg. Food chain uptake may
be involved, although the conversion of Hg++ to elemental
Hg, a process already mentioned as occurring in the
channels, may also be important. Schoper (1974) found
that elemental Hg is more rapidly taken up by mosquitofish
than Hg++.
Figure 28 shows the rapid increase in Hg levels in fish
living in the channels when the concentrations were changed
from 0.01 yg/1 to 5.0 yg/1. The equilibrium level of
11-14 yg/g in the fish was reached in only four months.
Although the Hg concentrations in the water of the treated
channels were in the ratio of 5:1, the levels reached by
the fish were only 2:1. Hence the concentrations attained
by fish were not a simple linear function of water
concentrations.
The levels in all treated fish decreased to approximately
3 yg/g within three months after the Hg inputs were turned
off. These data are variable but indicate that biological
half-lives may be a function of the initial concentration
levels of Hg. From May, 1974, until the end of the project.
69
-------
en
9+
O
h-
cr
h-
LJ
O
O
O
en
D— 1.0 p.q/\
—.01 fj.q/\
L Hg INPUT
INCREASED
0
973
1974
SAMPLING DATE
1975
FIGURE 28. MERCURY CONCENTRATIONS IN MOSQUITOFISH (£AMM)SXA AEFJIUJS) REMOVED FROM THE
TREATED CHANNELS DURING THE LAST 21 MONTHS OF THE PROJECT. EACH POINT IS AN AVERAGE BASED
ON FOUR ANALYSES OF INDIVIDUAL FISH.
-------
there were no differences between fish exposed to the two
treatment levels. By September, 1974, an equilibrium was
reached in the contaminated fish at a level significantly
above that measured in the controls. This level was
maintained until the project was terminated in February,
1975. At that time the average concentration in fish from
the treated channels was approximately 0.4 ug/g. Only a
small fraction of the fish recovered at the end of the
project have been analyzed.
During the project period included in Figure 28, there was
successful mosquitofish reproduction in the channels.
The increased community complexity that developed provided
the necessary cover for young animals. Fish collected
during this interval (June, 1973-March, 1975), might have
been born in the channels. This was not true for fish
collected the first year.
A single set of methyl mercury analyses was made on
composite samples of fish removed from the channels in
August, 1973. Although the total Hg concentrations ranged
as high as 5.6 yg/g, the portion of Hg present as methyl
mercury was on the order of 10%, considerably lower than
the 28% value calculated for fish removed from the channel
after a full year. This suggests that the proportion of
total Hg present in fish as methyl mercury increases with
exposure time. This result was also observed by Bache et al.
(1971) who found that the proportion of methyl mercury to
total Hg increased with age in lake trout.
Data obtained from the half-life study (Figure 29) show
that the Hg is removed from mosquitofish rapidly when the
animals are transferred to clean water. The best estimate
of the biological half life, based on the data, is 13.5
days. (C.I.95% = 9.9-23.1) based on a least squares fit
assuming first order kinetics. This half-life, obtained
from fish that had been in a contaminated environment their
entire lives, is considerably lower than the 60-day half
life reported by Huckabee and Blaylock (1973) for
mosquitofish contaminated by a short-term direct exposure.
Schoper (1974) calculated a half-life of 45 days for Hg in
mosquitofish but her data are limited and she presents no
confidence interval for her estimate.
BIOLOGICAL RESPONSES
Introduction
During the 41-month project period, the biological
communities that developed in the artificial streams
71
-------
10 20 30 40 50 60 70
TIME IN Hg-FREE WATER, days
80
FIGURE 29, CHANGE IN MERCURY CONCENTRATIONS IN MOSQUITOFISH (GAMBUSIA AFFINIS)
MOVED FROM A CONTAMINATED AREA TO CLEAR WATER, EACH POINT IS BASED ON 10
ANALYSES, EACH OF AN INDIVIDUAL FISH,
-------
were undergoing continuous changes and no equilibrium
systems evolved. There was a continual downstream
expansion of the macrophyte populations, a constant
rearrangement of insect and algal -populations, and a
continuous influx of new plant and animal species into
the channels. It is within this constantly changing
pattern that the effects of Hg must be considered. In
the statistical analyses of the data, time-treatment
interactions often appeared as significant factors in
comparisons between treatments.
In the initial planning, it was assumed that the only
factor that would vary among the six channels would be
the concentration of Hg++ in the water. There were
several deviations from this ideal situation, however,
resulting from the physical structure and positioning
of the channels. These included: (1) differential inputs
of seed material resulting from the orientation of the
channels with respect to the prevailing winds, (2)
differential disturbances by vertebrates, including
mammals and amphibians, which were largely confined to the
outermost channels, and (3) the input of dissimilar water
qualities which was a result of the different flow rates
through the limestone water treatment systems made necessary
by the development of major leaks in several of the head
pools. These factors contributed greatly to the variability
of the data and decreased the sensitivity of the statistical
procedures to reject a null hypothesis of "no effect of Hg"
on the various biological parameters studied. Nevertheless,
there were effects observed that can be related to the
input of Hg.
Ferens (1974) summarizes the data regarding the effects of
Hg on the benthic and periphytic communities in the
channels prior to the dose increases in August, 1973.
Even at concentrations of 0-01 and 1.0 Ug/1/ levels not
uncommon in natural waters (Wershaw, 1970; Jenne, 1972),
she found small but significant reductions in algal
number, biomass, and diversity, although the number of
species affected was small, and there was no indication
that these effects were transferred to consumer trophic
levels.
Taxonomy
Taxonomic efforts were concentrated on the algae, vascular
plants, and insects in the channels. Early in the study,
for the identification of algae, a number of samples were
sent to Dr. L. A. Whitford at the North Carolina State
73
-------
University. As new species were found, they were identified
by keys and descriptions presented in Huber-Pestalozzi
(1938, 1941, 1955), Prescott (1962, 1970), Ralfs (1848),
Smith (1950) , Tiffany and Britton (1952) , and Whitford and
Schumacher (1969) . Vascular plants were identified by keys
in Radford et al. (1968). The identity of the dominant
plant, Juneus diffusissimus was confirmed by Dr. J. Coffee
of Queens College, Charlotte, N. C. Most of the insects
were identified as to genus by reference to keys in Pennak
(1953) and Edmondson (1959) . Certain dipterans were
further identified by Beck (1968) and Mason (1973) and
vertebrates were keyed with Blair et al. (1957).
A complete species list of identified channel biota is
presented in the Appendix. In addition to these organisms,
a variety of bacteria, fungi, protozoa, nematodes, rotifers,
tardigrads, oligochaetes, cladocerans, ostracods, copepods,
and aquatic mites was also observed during the study.
This assemblage of organisms that colonized the streams,
including at least 50 species of algae, 5 species of vascular
plants, and 61 species of insects, approached a natural
ecosystem in complexity and possibilities of interaction.
In their review of artificial stream research, Warren and
Davis (1971) stress the relationship between the complexity
of artificial stream systems and the conclusions that can
be extrapolated from them to natural systems. Because of
the duration of the Hg study, the complexity of the
biological communities that evolved, and the similarity
of the water to natural streams in this region, data
obtained from the artificial streams should be directly
applicable to soft-water streams of the southeastern
United States and in a more general way to all aquatic
systems.
During the study, with the exception of amphibians, no
attempt was made to limit recruitment of new species into
the channels. Frog egg masses were removed because these
were found only in the outermost pairs of channels. When
it became obvious that some eggs had been missed (September,
1973) , an attempt was made to place an equal number of eggs
(Rana pipiens) in each channel. Based on later observations,
it appears that few, if any, of the eggs placed in Hg-treated
channels hatched. This was probably a direct result of the
Hg treatment. Birge et al.(1974) found that levels of 10
Vtg/1 of inorganic Hg were 100% effective in preventing the
development of Rana pipiens embryos when applied during the
cleavage of biastula stage. During the period after the Hg
inputs were stopped, amphibian eggs successfully completed
development in all channels and an unknown number of tadpoles
of at least three species (see Appendix) were observed.
74
-------
At the end of the project, an attempt was made to
quantitatively remove all vertebrates and larger
invertebrates from the channels. The results of these
efforts are summarized in Table 4. These results were
somewhat unexpected in that the great differences that
existed between channels with respect to fish, snail and
tadpole densities were never obvious from the routine
sampling programs. There is no question but that the
density differences in these animals, which appear to be
totally unrelated to Hg treatment, affected other channel
components. These effects contribute to the variability
of the data and make it even more difficult to determine
responses due to Hg treatment.
All of the algae identified in the artificial streams
during the course of this study could be described as
tychoplanktonic, attached or benthic algae characteristic
of soft, acidic waters of low-current velocity. The algal
community was clearly dominated by members of Chlorophyta
(green algae) with desmids and filamentous forms the most
abundant. Diatoms were very poorly represented with only
four species observed and only two of these occurring
regularly. The diatoms were not limited in the streams
by a lack of silicon in the water (Table 1), as the level
observed was well above the silicon concentration in nearby
diatom-dominated systems, but rather from a combination of
adverse environmental parameters such as the lack of shading,
the very slow current velocity, and the year-round water
temperatures above 20°C. The Cyanophyta or blue-greens
were never dominant either, but one species, Merismopedia
punctata, was present in almost every sample taken.
Filtering of large volumes of water from the channels
indicated that truly planktonic algae were not common
although Dictyosphaerium pulchellum was occasionally seen.
Community Responses-
Of the various substrates in the streams that were
regularly sampled, the walls contained the oldest and
least-disturbed communities of periphyton. Figure 30
summarizes the biomass results obtained from the PVC strips
during the first part of the study. Figure 31 shows the
biomass levels of the samples from the stream walls during
the latter part of study. The apparent abrupt increase in
wall biomass between October, 1973, and January, 1974, with
respect to the 5-yg/l channels may have been a result of
the change in sampling technique rather than a real increase
75
-------
Table 4. ORGANISMS COLLECTED FROM THE ARITIFICIAL STREAMS,
FEBRUARY 11-16, 1975
Organism
(* indicates
a new species
on this date)
Odonata :
Argia sp.
Ischnura sp.
Erythrodiplax
miniscula
Pantala hymenea
*Ladona deplanta
*Celithemis
fasciata
*Tetragoneuria
semiaquea
*Plathemis lydia
*Pacydiplax
longipennis
1
Channel number
4253
control
430
7
162
10
3
11
7
1
637
131
187
3
12
110
6
i.o yg/l 5.0 yg/i
545 591 465
101 3L8 314
858 451 297
4
14
12 89 84
1
2
53
296
47
8
Ephemeroptera:
Caenis sp.
Callibaetis sp.
26
14
4
18
4
68
3
15
5
Hemiptera:
*Ranatra sp.
Pelocoris femoratus^
Hesperocorixa spp.
21
44
2
14
40
31
44
20
46
9
22
2
154
Megaloptera:
*Chauliodes sp.
14
Diptera:
Tanypodinae
Chironominae
pupae
*Tipula sp.
Helius sp.
Dasyhelea sp.
10
53
6
6
4
1
3
186
12
27
2
6
4
6
3
1
1
140
1
2
2
76
-------
Table 4 (continued)
Channel Number
1 4 253
Control
Organism
1.0
b.U yig/r
Lepidoptera:
Parapoynx sp.
*Synclita sp.
3
1
13
8
32
19
23
34
2
3
3
Coleoptera:
Berosus spp.
Coptotomus sp.
* Trop i sternus II
*Tropisternus #2
Hydocanthus sp.
Hydroporus sp.
*Agabus fl
*Agabus #2
*Enochrus #1
*Enochrus #2
Laccophilus sp.
4
2
2
1
44
9
6
1
13
1
18
6
1
62
9
1
1
33
2
1
50
1
24
7
11
15
1
1
38
6
4
9
1
3
Others:
snails
(Phy^sa and Lymnea)
Rana spp .
Gambusia affinis
Total Insects
Insects/meter 2
£ insect species
H - insects
E - insects
Grand Total
# species
H
E
30
167
858
15.4
25
2.58
0.56
1055
27
2.91
0.61
405
30
246
1204
21.6
20
2.31
0.53
1886
23
2.84
0.63
4
284
217
2001
35.9
24
2.56
0.56
2506
27
2.98
0.63
262
489
172
1750
31.4
24
2.70
0.59
2673
27
3.20
0.67
129
148
1453
26.1
21
2.73
0.62
1730
23
3.08
0.68
1077
629
76
628
11.3
15
2.28
0.58
2410
18
2.28
0.55
77
-------
00
CM
E
CP
**
CO
CO
o
DQ 3
-z.
O
cr
LU
Q_
0
•-CONTROL
• -I.O/ig/l
O-.OI/ig/l
Hg INPUT
INCREASED
A
0 I N
D
M
A
M
0
1972 1973
SAMPLING DATE
FIGURE 30. BIOMASS OF PERIPHYTON SCRAPED FROM PVC STRIPS
SUSPENDED IN THE CHANNELS.
-------
vo
Hg INPUT OFF
•—CONTROL
D — I.O/ig/l
A S 0 N D
1974
SAMPLING DATE
1975
FIGURE 31. BIOMASS OF PERIPHYTON SCRAPED FROM CHANNEL WALLS.
-------
during this time interval. There was a certain amount of
loss from the PVC strips when they were removed from the
channels and these losses were relatively greater when the
growth was heavy, as it was in the 5.0-yg/l channels.
The periphytic biomass in all channels fluctuated similarly
during the summer of 1972 probably due to environmental
changes, but in September, channels 3 and 6 (0.01 yg/1 at
that time) did not follow the biomass reductions that
occurred in the other streams, indicating a possible
stimulation in growth at low-Hg levels. After the Hg
levels in channels 3 and 6 were increased to 5.0 yg/1 in
August, 1973, the walls of these channels continued to
support higher periphytic biomasses than in the other
channels. Figure 31 illustrates a direct correlation
between the Hg concentration in the streams and the standing
crops of periphyton inhabiting the walls, with a subsequent
equalization in biomass levels after the input was shut off.
A stimulation of algal growth at a Hg concentration as high
as 50 pg/1 has been reported (Matsui and Gloyna, 1972).
Figure 32 shows that the algal component of the wall
periphytic communities, as summarized by total cell volumes,
followed the same general pattern as shown in the biomass
data. In October, 1974, the total algal volume was greater
in the control channels than in- the treated, although this
pattern is not repeated in the biomass data of the same
date. This indicates that, at least at that sampling time,
the non-algal component of the periphyton, either living
or dead, made up a greater percentage of the organic matter
on the walls of the Hg-treated channels.
Figure 33 summarizes the calculated diversity and evenness
values for these algal components of the periphytic
communities in time. The control and low-level streams were
quite similar with respect to these parameters (as they were
in biomass and algal volume), and the high-level streams
initially had decreased diversity and evenness values. An
analysis of variance (ANOVA) of the data collected while Hg
was still being input to the channels showed there was a
significant treatment effect (p < .05) on H, and highly
significant effects (p < .01) on E and cell volume.
Although biomass values of periphyton collected from
vertical plexiglass plates were quite variable (Figure 34),
certain trends are evident. During 1972, the biomass
levels on this substrate had slightly lower values than
were found on the PVC strips. Channels 3 and 6 again
diverged from the other channels in the fall of 1972 with
80
-------
Hg INPUT OFF
00
I973
I975
I974
SAMPLING DATE
FIGURE 32, CALCULATED VOLUME OF PERIPHYTIC ALGAE SCRAPED FROM THE CHANNEL WALLS,
-------
00
NJ
CO °'8
00 07
LU u-'
^ 0.6
UJ 0.5
UJ 0.4
0.3
02.5
2.0
CO
tr 1.5
LU
Q
I
IX
1.0
I 1
Hg INPUT OFF
I I
I I
D J F ! M
A
M J J
0
N D
1973
1974
SAMPLING DATE
1975
FIGURE 33. DIVERSITY AND EVENNESS OF PERIPHYTIC ALGAE COLLECTED FROM THE CHANNEL WALLS.
-------
00
Ul
e
\
en
C/)
CO
o
00
X
Q_
LT
LU
Q.
»—CONTROL
.01/z.g/l
I.O/ig/l
5.0/tg/l
Hg INPUT Hg INPUT
INCREASED OFF
M
A
M
J
J
A
S
0
N
D
J
F
M
A
M
N
D
J
F
M
A
M
J
J
A
S
0
N
D
J
0
SAMPLING DATE
FIGURE 34, BIOMASS OF PERIPHYTON SCRAPED FROM VERTICAL PLEXIGLASS PLATES,
1975
-------
higher biomass values. This pattern continued after the Hg
input levels were increased in August, 1973, until the
winter of 1974, 11 months after the Hg was shut off. At
the end of the project, all channels were similar. The
graph summarizing algal cell volumes for the plexiglass
plates (Figure 35) shows that the increase in biomass
observed in streams 3 and 6 was partially the result of
increased algal growth at that time. Figure 36 shows
that algal diversity and evenness of periphyUc samples
removed from plexiglass plates were lowered by Hg treatment
during the period Hg was input to the streams. These
differences were significant (ANOVA, p < 0.01).
The results obtained from the first two glass slide
experiments are summarized in Figures 37 and 38 in terms
of the calculated algal volumes. These two experiments
were carried out while Hg was still being added to the
channels. Both graphs demonstrate the significant delay
in the colonization of microscope slides by algae in the
5.0-yg/l channels and the similarity between the control
and 1.0-yg/l channels.
In both experiments, the diversity and evenness of the algal
component of the periphytj.c communities were significantly
(ANOVA, p < 0.01) affected by the treatment with the highest
diversities in the high-level streams. On a small number
of slides from these experiments examined after 40 and 80
days of colonization, the trend towards lowered algal
biomass with higher Hg exposure had disappeared and even
reversed, with the highest algal densities occurring on
the slides from the 5.0-yg/l channels. The observed
increases in algal biomass in the high-level streams were
not accompanied with any decreases in diversity or evenness.
The initial retardation of algal growth on microscope slides
placed in the 5.0-yg/l channels disappeared in the glass
slide experiments carried out after the inputs of Hg were
stopped. Figure 39 illustrates algal volume changes on
slides placed in the channels one day after the Hg was
turned off. There were no significant treatment effects
found for these data with respect to cell volume, diversity
or evenness (ANOVA, p > 0.05). Figure 40 summarizes the
data from a glass slide colonization experiment started •
three months after inputs were stopped. While the former
control and low-level channels were similar in cell volumes
and diversities throughout the study, the former high-level
channels developed larger cell volumes and lower diversity
values.
84
-------
Hg INPUT OFF
00
Ul
CM
E
CO
O _
t
CO
UJ
Q
UJ
S
ID
o
N
D
TIT-
M
A
M
J
J
A
S
0
N
D
J
1973
1974
SAMPLING DATE
1975
FIGURE 35. CALCULATED VOLUME OF PERIPHYTIC ALGAE SCRAPED FROM VERTICAL
PLEXIGLASS PLATES,
-------
co
CO
UJ
UJ
LU
.8
.7
.6
.5
.4
.3
Hg INPUT OFF
I I I I
J I
J I L I
CO
CTl
CD
O
2.5
>-- 2.0
co
LU 1.5
Q
IX 1.0
1973
•-CONTROL
Q-I.O/tg/l
N|D|J|F|M|A|M|j|j|A|StO|N|D|J
1974
SAMPLING DATE
1975
FIGURE 36, DIVERSITY AND EVENNESS OF PERIPHYTIC ALGAE COLLECTED FROM VERTICAL
PLEXIGLASS PLATES,
-------
CJ
CD
£ 20
15
£- 10
CO
UJ
Q K
ID
O
r>
Ld
0
0
>—CONTROL
k—5.0/ig/l
5 10 15 20 25 30
COLONIZATION TIME,days
35
FIGURE 37, CALCULATED VOLUME OF ALGAE COLONIZING MICROSCOPE
SLIDES DURING MERCURY INPUTS (STARTED OCTOBER 31, 1973).
-------
25
20
15
10
0
• — CONTROL
Q—I.O/ig/l
A — 5.0/ig/l
5 10 15 20 25 30
COLONIZATION TIME, days
35
FIGURE 38, CALCULATED VOLUME OF ALGAE COLONIZING MICROSCOPE
SLIDES DURING MERCURY INPUTS (STARTED DECEMBER 7', 1973),
-------
00
10
CVI
25
E
e
en
o
Z
LU
Q
UJ
O
. 15
10
0
—CONTROL
—I.O/^g/l
5 10 15 20 25 30
COLONIZATION TIME, days
35
FIGURE 39, CALCULATED VOLUME OF ALGAE COLONIZING MICROSCOPE
SLIDES AFTER MERCURY INPUTS WERE STOPPED (STARTED JANUARY 30,
1974),
-------
CM
E
E
60
50
40
30
I 20
o
UJ
o
>
10
0
CONTROL
I.O/ig/l
5.0f«.g/l
5 10 15 20 25 30
COLONIZATION TIME, days
35
FIGURE 40, CALCULATED VOLUME OF ALGAE COLONIZING MICROSCOPE
SLIDES AFTER MERCURY INPUTS WERE STOPPED (STARTED APRIL 25, 1975),
-------
The final glass slide colonization experiment was begun
in October, 1974, nine months after Hg inputs were stopped.
During this experiment, the control and former 5.0 pg/1
channels were very similar, and the algal volumes in the
former 1.0-yg/l channels were consistently lower. There
were no significant differences between the treatments
with respect to diversity and evenness.
In August, 1974, experiments were initiated in the
continuous flow chambers to check some of the results
observed in the channels. In the first of three experiments
with algae, mixed cultures containing Oedogonium sp.,
Stigeoclonium elongatum, Chlamydomonas sp., Oscillatoria
geminata, and Microspora quadrata, species isolated from
the artificial streams, were used as seeding material.
Three boxes received a Hg input of 5.0 yg/1 and three were
used as controls. Colonization on these glass slides was
observed for 10 days and was found to be dominated by the
Oscillatoria geminata and Chlamydomonas sp. with very few
§.• elongatum or Oedogonium sp. and no M. quadrata observed
on the slides. After 10 days, the mean number of cells/mm^
was 1256 + 357 (X + SE) for the controls and 10.0 +3.6
(X + SE) for the treated chambers. The colonization of
both 0. geminata and Chlamydomonas sp. was inhibited in
the Hg-treated chambers. The limited number of Chlamydomonas
sp. that were observed were immobile in palmelloid groups
that did not stir while the slides were being counted. When
the Hg was cut off in these boxes, these cells were observed
to develop flagella and start swimming as had been
continuously observed on slides from the control chambers.
Although the M. quadrata did not develop on the glass slides
in any of the~*chambers, lush growth of this filamentous
green algae did develop in the outflow holes of the control
chambers, while very little growth occurred in this position
in the Hg chambers.
In a single-species experiment with S_. elongatum, very little
growth was obtained over a 25-day period in the treated
chambers while this and other contaminating species grew
profusely in the control chambers. After the Hg input was
discontinued, five clean slides were placed in each chamber.
The colonization of S. elongatum was observed and found not
to differ between the chambers. These experiments indicated
a very pronounced retardation of growth of certain algae at
a Hg concentration of 5.0 yg/1, and confirm the trends seen
in the artificial streams.
The observations from the artificial substrates and the
system presented above can be summarized as follows. While
91
-------
the Hg was input into the 5.0 yg/1 channels, there was an
initial inhibition of algal growth which disappeared after
about 40 days and was replaced by increased cell numbers
and periphytic biomasses compared to the controls. After
the Hg inputs were shut off, the initial inhibition
previously found was nonexistent but the trend towards
higher biomasses in the former 5.0-yg/1 channels continued
for about nine months. The algal population dynamics in
the 1.0-yg/l channels showed very little modification as
compared to the controls throughout the study.
Individual Responses-
Thirty-one of the algal species listed in the Appendix were
found at one time or another in all of the six channels.
Although the other species were not observed in all channels,
with one exception (Microthamnion strictissimum), their
absence was not related to Hg treatments. M. strictissimum
was found only in the four treated channels. The species
that appeared to be absent from one or more streams were
rare wherever they were found. Two exceptions were
Navicula notha,a motile diatom that was very abundant in
channel 6 but never seen in channel 1, and Spirogyra sp.,
which became very common in the tail area of channel 1 but
never was found in channels 3, 5, and 6. No.definite
reasons can be presented to explain the localized abundances
of these algae, but they probably illustrate differences
between the biota of the channels caused by a combination of
random events and possibly Hg treatments.
Detailed cell counts of algae from the various substrates
indicated that certain species were important in creating
biomass differences between the treatments. A few species
of algae were found to be quite sensitive to the low Hg
levels used, and one species exhibited morphological
alterations apparently attributable to Hg concentrations.
Oedogonium sp. - This green alga was present throughout the
study and, although a few cells which may have been oogonia
were observed, it was never observed in a true fruiting
condition and identification as to species was not possible.
Dr. L. Whitford of N. C. State University tentatively called
it 0. reinschii because of the lack of fruiting filaments
and the presence of some diamond-shaped cells he observed.
However, continuous examination of this species during the
last year of the study indicated that it was smaller
(usually 3.5 ym in diameter) than the typical O. reinschii
(5-11 ym) and fit very closely the description of sterile
filaments of 0. pusilium.
92
-------
Initial colonization of this species on glass microscope
slides was rapid in the control and 1.0 yg/1 channels but
significantly slower in the 5.0-yg/1 channels during the
two experiments carried out when Hg was being input. In
two of three colonization experiments performed after the
Hg input was shut off there was no significant difference
between the former treatments. In the third experiment,
a significantly greater growth was observed in the former
1.0-yg/l streams.
The occurrence of this species on the long-term substrates
is summarized in Figures 41 and 42. This species was much
more abundant in the high-level streams than in the low-level
streams or in the controls during the input of Hg, but
during the year of recovery this dominance disappeared on
both the walls and plexiglass plates. No seasonal variation
was observed for the populations in the two control channels
where this species remained at a density of about 300
cells/mm^ throughout the year.
These data indicate that the reproductive and vegetative
portions of the life cycle of Oedogonium sp. were
differentially affected by low Hg levels. In spite of the
large population densities in the Hg-treated channels, the
slower colonization of this species on glass slides indicates
that its reproduction was affected by the Hg treatment.
Since no fruiting filaments were observed in any of the
channels, reproduction was asexual by means of zoospores.
The zoospores, or zoospore production, therefore, must have
been sensitive to the Hg levels in the water although the
growth of vegetative filaments was stimulated.
StigeocIonium elongatum - This species was found to be very
sensitive to Hg. During the first two years, a significant
growth stimulation on plexiglass plates was observed in the
0.01-yg/1 channels and a slight reduction in abundance was
found in the 1.0-yg/l channels as compared to the controls.
When the Hg concentrations in channels 3 and 6 were increased
to 5.0 yg/1, the numbers of cells of S. elongatum were
significantly reduced. Averages (cells/mm?)based on 14
counts from each treatment while Hg was being input were
46 +12 (X + SE) for the controls, 25+7 for the 1.0-yg/l
and 11+5 for the 5.0-yg/1 levels.
Figure 43 summarizes plexiglass plate data from the last
year of the project. After the Hg was shut off, all of the
streams went through similar seasonal fluctuations with
summer maxima lasting for about 4-5 months. During this
latter period, S. elongatum was much more common in
93
-------
cvj
E
E
CD
o
O)
z:
LJ
Q
LJ
O
O
O
c
If)
I
Hg INPUT OFF
CONTROL
D
J|F
M
A
M
J
J
A
S
0
N
D
J
1973
1974
SAMPLING DATE
1975
FIGURE 41, DENSITY OF QEDOGONIUM SP, ON CHANNEL WALLS BEFORE AND AFTER
MERCURY INPUTS WERE STOPPED,
-------
>D
> ^*
E
Hg INPUT OFF
o
CONTROL
5-Oftg/l
N
D
J
F
M
A
M
J
J
A
S
0
N
L>
J
1973
1974
SAMPLING DATE
1975
FIGURE 42, DENSITY OF QEDQGONIUM SP, ON PLEXIGLASS PLATES BEFORE AND AFTER
MERCURY INPUTS WERE STOPPED,
-------
Hg INPUT OFF
vo
N
D
J
F
M
A
M ! J
J
A
S
0
N
D
J
1973
1974
SAMPLING DATE
1975
FIGURE 43. DENSITY OF STIGEQCLONIUM ELQNGATUM ON PLEXIGLASS PLATES
BEFORE AND AFTER MERCURY INPUTS WERE STOPPED.
-------
channels 3 and 6 than in the other channels. At the end of
the project, its density on plexiglass plates was similar
in all channels.
Counts for this species from wall samples are summarized
in Figure 44. The results are variable but indicate that
S^. elongatum was most abundant in the 5.0-pg/l streams.
During the year after the Hg input was shut off, there was
much less variation in the numbers of £3. elongatum cells
on this substrate than was seen on the plexiglass plates.
This may have been due to the shaded location of the
southwest-facing wall used for sampling.
Data for this species from the glass slides showed
significant differences in cell densities due to treatment
in the first two colonization experiments (ANOVA, p < 0.01)
with reduced numbers in the Hg-treated channels. After
the Hg was shut off, this reduction in initial colonization
disappeared with a trend towards greater densities on the
slides in the former Hg streams.
SL elongatum was isolated and tested under more controlled
conditions with the CFC system. In the experiment in which
the Hg concentrations were held relatively constant at
5.0 yg/1, the number of cells of this species was
significantly reduced in the treated chambers (p < 0.01)
compared to the controls. When the Hg input was shut off
and clean slides were placed in the chambers, S^. elongatum
colonized slides in all chambers at similar rates.
Oscillatoria geminata - During the first summer of the study,
this species was found to comprise numerically more than 10%
of the algae inhabiting plexiglass plates in the streams,
but during the next two years it was greatly reduced in
abundance in the plexiglass plate and channel wall samples.
It appears that 0. geminata is well adapted for initial
colonization and rapid growth on a new substrate but is
unable to compete at later stages of succession.
This species exhibited a highly significant negative response
to Hg treatment during the two glass slide experiments
carried out just prior to the shut-off of mercury
(ANOVA, p < 0.01). In the glass slide experiments after the
Hg input was stopped, this treatment effect disappeared and
0. geminata was found to be equally distributed in the
streams. In two experiments with the CFC system this
species was found to grow profusely in the controls and
not to grow at all on the slides in the 5.0-yg/l chambers.
97
-------
Hg INPUT OFF
00
CM
e
.£
o
*•»
cn
•ZL
LU
Q
LJ
O
D
J
F
M
A
M
J
J
A
S
0
N
D
J
1973
1974
SAMPLING DATE
1975
FIGURE 44, DENSITY OF STIGEQCLONIUM ELONGATUM ON CHANNEL WALLS
BEFORE AND AFTER MERCURY INPUTS WERE STOPPED,
-------
Chromulina pseudonebulosa - Although C. pseudonebulosa is
flagellated, it was most often found on glass slides in a
resting condition. During the early part of the Hg project,
this species was observed to be most abundant on the
plexiglass plates in the 1.0-yg/l channels (high level at
that time) (Ferens, 1974); however, it did not appear to
be common on this substrate in later samples. This species
showed a significantly lowered colonization rate on glass
slides in streams receiving a Hg input. In the glass
slide experiment started one day after the Hg input was
turned off, greatest colonization was found on the slides
in the former Hg channels. In the last two microscope
slide experiments this species was rare and showed no
differences in growth between channels.
Geminella turfosa - There is no record of this species
being observed in North America, but the relatively
abundant filaments of narrow cells with spiral chloroplasts
(Figure 45) resemble in every way the excellent description
of Gloeotila turfosa in Sweden (Skuja, 1956). This species
was later transferred to the genus Geminella (Ramanathan,
1964) and will be referred to by that name.
No significant density differences were seen between
treatments for this species for plexiglass plates, walls,
or glass microscope slides. There were, however,
morphological changes in cell shape that were apparently
caused by Hg treatment levels. Figure 45 illustrates
normal and various abnormal cells observed on glass
microscope slides during the input of Hg. Abnormal cells
were often only slightly swollen but some appeared as
spheres or branched cells. The numbers of abnormal cells
observed during routine counts of glass microscope slides,
both before and after the Hg was shut off, were recorded.
Table 5 lists the relative abundance of abnormal cells
observed for the various treatments and indicates a highly
significant difference between the 5.0-yg/l treatment and
controls and also between the two treatments while Hg was
on. After the Hg inputs were discontinued, the differences
disappeared and by the time of the last glass slide
experiment (October, 1974) no more abnormal cells were
observed. This species was never isolated into pure culture
so laboratory experiments were not conducted. Working with
three marine algae in axenic cultures, Nuzzi (1972) also
found morphological abnormalities associated with Hg at
concentrations in the growth medium equal to those in the
artificial streams.
99
-------
FIGURE 45, QEMINELLA IUREQSA, A. NORMAL FILAMENT SHOWING
LOCATION OF SHEATH, B, VARIOUS ABNORMAL CELLS OBSERVED IN
MERCURY CHANNELS,
100
-------
Table 5. INCIDENCE OF ABNORMAL GEMINELLA TURFOSA CELLS ON
GLASS SLIDES DURING AND AFTER MERCURY INPUT
(% OF TOTAL GEMINELLA CELLS)
During Mercury Input
Treatment
X + 2SE
n
Control
0.19 + .24
21
1.0 yg/1
1.16 + 1.01
24
5.0 ug/1
45.99 + 10.
35
40
After Mercury Input
Previous Treatment
Control
X + 2SE
n
0.42 +
17
.84
1.0
5.43
yg/i
+ 5.42
21
5.
3.84
0
+
22
yg/i
4.12
Microthamnion strictissimum - This branched green alga was
unique because it was found to be common in the four
Hg-treated streams, reaching its highest abundances on
slides placed in the 5.0-yg/1 channels, and was never seen
in the control channels. This species was observed only
during the winter of 1973 and showed a significant positive
response to the Hg input level. These observations suggest
that M. strictissimum might be an indicator of the presence
of mercuric ions in low concentrations. This species was
not isolated because it quickly disappeared after the Hg
inputs were stopped.
Other species - Several other species of algae were more
dense on the plexiglass plates and walls in the treated
channels, and contributed to the higher total biomasses
measured in these channels. Cosmarium aspherosporum, a
small placoderm desmid that was common in all channels,•
was most dense in the controls (Figure 46 and 47) .
101
-------
Hg INPUT OFF
o
KJ
1973
1974
SAMPLING DATE
^^ - PLEXIGLASS PLATES BEFORE
-------
o
to
CM
E
Hg INPUT OFF
C/)
o
l-
UJ
Q
_J
UJ
O
CONTROL
°T
21
.0/xg/l
D
J
F
M
A
M
J
J
A
S
0
N
D
J
1973
1974
SAMPLING DATE
1975
FIGURE l\7, DENSITY OF COSMARiUM ASPHERQSPQRUM ON CHANNEL WALLS BEFORE
AND AFTER MERCURY INPUTS WERE STOPPED,
-------
The density differences disappeared during the last year of
the project. Spondylosium planum, a filamentous desmid, was
also most dense in the 5.0-yg/l treatments and least dense
in the controls while the Hg inputs were on (Figure 48 and
49). This pattern continued after the Hg inputs were
terminated. In the short-term glass slide colonization
experiments, both of these desmids showed greater growth
in the treated channels with the highest numbers occurring
in the 1.0-yg/l treatments.
Another species that was more dense on the plexiglass
plates and walls of the treated channels was Merismopedia
punctata, a blue-green alga of common occurrence. In the
short-term colonization studies, however, this species
showed no significant response to Hg levels.
Past studies concerning toxicity of Hg compounds to algae
have all been short term in nature. These studies have
shown various levels of toxicity by this metal to chlorophyll
production (Hannan and Patouillet, 1972), primary production
(Harriss et al., 1970; Knauer and Martin, 1972), and cell
numbers (Ukeles, 1962; Nuzzi, 1972; Ben-Bassat, et al.,
1972 and Matsui and Gloyna, 1972). Working with three
species of marine phytoplankton, Nuzzi (1972) demonstrated
reduced growth of all three species at a Hg concentration
of 5.0 ng/1 as HgCl2- Knauer and Martin (1972) also found
this same concentration to be inhibitory to marine
phytoplankton. Our short term studies using the CFC
system also showed this reduction in the initial growth
of several fresh-water algae at a low concentration of
HgCl2; however, the study in the artificial streams
demonstrated that an initial inhibition of growth may not
always affect the long-term accumulation of a species.
The initial slow colonization seen in the Hg-treated
channels seemed to be a result of a lowering of the
reproductive potential of the algae and was related to
the level of Hg in the water. The lower colonization rates
were found in spite of denser periphytic communities on
the older substrates in the treated channels, which should
have provided larger seeding stocks of colonizers relative
to the control channels. Apparently the cells that actually
colonized a bare substrate such as a microscope slide were
less common or less successful in the Hg channels. For
some of the most abundant species such as Oedogonium or
Stigeoclonium these would have been zoospores and for
others they would have been merely drifting cells or
fragmented filaments (Cosmarium spp., Oscillatoria, and
Merismopedia).
104
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o
Ul
CVJ
E
o
CO
z
LU
Q
Ld
O
Hg INPUT OFF
CONTROL
N
D
J
F
M
A
M
J
J
A
S
0
N
D
J
1973
1974
SAMPLING DATE
1975
FIGURE 48, DENSITY OF SPONDYLOSIUM PLANUM ON PLEXIGLASS PLATES BEFORE
AND AFTER MERCURY INPUTS WERE STOPPED,
-------
CM
E
E
o
LU
O
LU
O
Hg INPUT OFF
CONTROL
1973
1974
SAMPLING DATE
1975
FIGURE 49, DENSITY OF SPONDYLOSIUM PLANU^ ON CHANNEL WALLS BEFORE
AND AFTER MERCURY INPUTS WERE STOPPED,
-------
The greater algal populations that were found in the
Hg-treated channels may have resulted from nutrient level
differences between channels, or they may have resulted
from differences in consumer densities (including parasites
and pathogens).
With respect to nutrients, the input of nitrogen with the
Hg solutions never exceeded 0.5 ug/1, which represented an
increase of 1.3% over background. This amount is probably
not sufficient to cause the observed differences between
control and treated channels. Nutrient stripping by
macrophytes, especially in the dense stands that formed
just below the input weirs of the channels, might have been
an important factor. These macrophytic stands were much
more dense in the control channels than in the treated,
especially just below the Hg-input tubes. An enrichment
of the water caused by death and decay of organisms coming
into the channels from the head pools when these organisms
were subjected to Hg inputs may have been a factor.
Williams and Mount (1965) concluded that the higher
periphytic biomasses they observed in Zn-treated channels
were due to this mechanism. Unfortunately, personnel, time
and equipment were not available for routine water analyses.
Little can be said with respect to consumer densities in
the channels and their effects on periphytic growth. It
has already been stated that tadpoles were established
in the control channels long before they were observed
in the treated. Dickman (1968) has shown that tadpoles
enclosed in wooden cages severely reduced the standing
crop of algae in proportion to the number of individuals
present. Tadpoles may have been partially responsible
for the reduced periphytic levels measured in the control
channels, although this was not the only factor since glass
slides suspended in the channels showed the same trends
as the walls and plexiglass plates, and these glass slides
were not grazed by tadpoles.
From the available information it is impossible to tell
if the ruling influence on the differential development
of algae in the streams was directly due to nutrient
differences, grazing differences, a combination of these
influences or some other factor dependent on Hg concentration.
Macrophytes
The vascular plants that became established in the channels
were all typical of shallow ponds or very slow-moving water.
107
-------
By the end of the study, Juncus diffusissimus and Utricularia
biflora/ were common in all six channels, although their
densities were quite different between channels. The other
three species of vascular plants found (see Appendix) were
represented by very small numbers of individuals and seemed
to be randomly distributed among the streams.
Early colonization and long-term biomass accumulation in
the channels by vascular plants was dominated by Juncus
diffusissimus, a species of rush of common occurrence in
wet habitats of the southeastern coastal plain of North
America. This plant was first observed in the streams
nearly a year after water input was begun and by 20
months was well established in all of the channels (Table 3).
It appears that the outside channels (especially channel 1)
received higher inputs of seeds, and also that this plant
was much more common at the heads of the streams than
downstream. This increased abundance in the upper reaches
of the channels became increasingly evident during the next
20 months until it was necessary to remove some plants
to allow water to flow downstream.
Attempts to quantify the Juncus in the channels were not
made until February, 1974. At that time the control
channels were found to average 134 g/m^ ash-free dry weight
and the 1.0-and 5.0-ug/l channels averaged 60.3 and 62.5
g/m^ respectively. The extremely poor growth of Juncus
immediately downstream from the Hg input tubes showed that
this plant was affected by Hg. Data collected during the
last year of the project (with no Hg input) are summarized
in Figure 50. There is no obvious explanation for the
decrease in standing crop biomass of Juncus in the control
channels except that this might be a normal successional
trend, not seen in the treated channels because of the
effects of the Hg.
The second most common plant by the end of the study was
Utricularia biflora which bloomed during the spring of
1974. This species was not found to be abundant in any of
the streams until after the Hg was shut off. Since no
differences were observed between the control and treated
channels, this appears to be another successional phenomenon.
The other two species of flowering plants present, Typha
latifolia and Callitriche heterophylla, were not observed
to flower or fruit during the study.
Insects
All of the species of insects collected from the artificial
streams were characteristic of sluggish streams or shallow,
108
-------
o
vo
ElSOh
en
++
en
CO
O
QD
100
< 50
_l
Q_
0
• — CONTROL
a— i.o/^g/l
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
NOV
DEC
JAN
1974
SAMPLING DATE
1975
FIGURE 50, TOTAL MACROPHYTE BIOMASS (INCLUDING STEMS AND ROOTS) IN CHANNELS
DURING THE FINAL YEAR OF THE PROJECT,
-------
pond-type habitats of the southeastern United States. New
species were continuously discovered in benthic dish samples
throughout the three years of study indicating that
colonization continued throughout this period. After 19
months of study, only 15 different species had been collected
(Ferens, 1974). After 40 months of colonization, these
original 15 species were still present but rare, and a total
of 61 species had been encountered. Fifteen of these were
first collected when the streams were cleaned out in
February 1975.
The original species colonizing the streams were mostly
midge larvae with one dragonfly, Pantala hymenea, present
after the first year. By the end of two years another
dragonfly, Erythrodiplax minuscula, and a damselfly,
Ischnura sp., were present in addition to several new
midge species. The first beetle larvae and adults were
collected in January 1974, 29 months after input of
water was started. At this same time the first true bugs,
Hesperocorixa sp., were also collected. Four months later
in May, 1974, the first tricopterans were found and in
August 1974 the first aquatic lepidopterans were observed.
As mentioned earlier, several species, including a helgrammite,
a waterscorpion, several species of dragonflies and beetles
and another lepidopteran, were not observed until the streams
were cleaned out in February 1975.
During the initial part of the study when input concentrations
were 0.01 and 1.0 pg/1, no significant differences in mean
numbers and relative abundances of dominant species of
midges were found (Ferens, 1974). Small but significant
(p < 0.05) increases in diversity and evenness values were
found for downstream samples from the 1.0-yg/l streams as
compared to the controls; however, diversity values were
very low during this time (about 1.0) because of the
initially slow rate of colonization by insects.
Benthic dish data collected after the Hg inputs to channels
3 and 6 were increased to 5.0 yg/1 in August, until the Hg
was shut off six months later, indicated that insect
densities were greatest in the 1.0-pg/l streams and very
similar between the controls and the 5.0-yg/l streams
(Figure 51). Fluctuations in these densities were observed
to be similar between treatments for the year following the
Hg shut-off, but statistical analysis showed that the
density of insects was significantly lower (ANOVA, p < 0.01)
in the previously treated streams. Diversity and evenness
values for the benthic dish samples collected during this
last year (Figure 52) were found to be significantly higher
110
-------
Hg INPUT OFF
CVJ
c>
*^>^*^xv^,,^xlr*^5*'^'^
CONTROL!!
'"'"
H
CO
I-
O
LU
CO
o
D
J|F
M
A
M
J
J
A
S
0
N
n
J
1973
1974
SAMPLING DATE
1975
FIGURE 51, DENSITIES OF INSECTS COLLECTED FROM BENTHIC DISHES BEFORE
AND AFTER MERCURY INPUTS WERE STOPPED,
-------
1.0
co 0.9
UJ
z 0.8
g 0.7
0.6
Ld
0.5
Hg INPUT OFF
CD
O
Q
i
IX
2.5
t 1.5
co
o:
LU
1.0
—CONTROL
D
M
M
A
0
N
1973
1974
SAMPLING DATE
D
1975
FIGURE
BEFORE
52, DIVERSITY AND EVENNESS OF INSECTS COLLECTED FROM BENTHIC DISHES
AND AFTER MERCURY INPUTS WERE STOPPED,
-------
in the control streams than in the 5.0 yg/1 channels
(Students t, p. < 0.05). This trend of lower diversity
values for higher treatments was almost gone at the end
of the summer after the Hg had been shut off.
Using ANOVA methods, three species of insects were found
to be negatively affected by treatment levels in the
streams. These were a damselfly, Argia sp., that reached
considerable densities by the end of the study; a tiny
mayfly, Callibaetis sp.; and one midge species,
Paralauterborniella elachisa. Another mayfly, Caenis sp.,
was not collected in channels 3 or 6 during the input of
Hg and this may have been related to the treatment.
Three species of chironomids (Pseudochironomus sp.,
Ablabesmyia ornata, and Psectrocladius sp.) and one
ceratopogonid (Dasyhelea sp.) were most abundant in the
1.0-yg/l streams.
When the water was turned off in February 1975, an
attempt was made to remove the entire populations of the
larger insects, amphibians, molluscs, and mosquitofish in
the streams. The water level in the channels was lowered
to about two inches above the sand. Starting at the tails,
an 8.5-m stream section was enclosed with two screens, and
ten passes were made with a 3-mm mesh .dip net. The lower
screen was then moved up 17 m thereby enclosing the next
section. This was repeated until the entire 100 m length
of each stream had been sampled. The material collected
by this method was carefully examined in the laboratory
and all animals were removed, sorted and counted.
Representative organisms from each group were frozen for
Hg analyses. Table 4 summarizes the data from this final
sampling. The insect densities shown in Table 4 are much
lower than those calculated from benthic dish data (Figure
51). This is because the sampling technique used did not
collect midge larvae, the most common benthic insects.
Table 4 shows that the greatest densities of larger insects
occurred in channels 2 and 5, the 1.0-yg/l treatments, and
the lowest densities in channels 1 and 6 (the first a
control, the second a 5.0-yg/1 treatment). There appears to
be no relationship between treatment levels and diversity,
density or evenness values.
Of the parameters measured for insects, diversity values
appeared to be most sensitive to Hg levels with an initial
increase in diversity for the 1.0-yg/l streams and an
eventual lowering of the diversities corresponding to
higher treatment levels. No significant differences were
seen for insect densities while the Hg was on. In general
113
-------
the streams appeared to be variable with respect to insect
numbers in a manner unrelated to treatment levels. This
variation was probably the result of the finite number
of non-random visits by egg-laying adults. None of the
common species of insects occurring in the streams
demonstrated a conclusive direct or indirect reaction to
Hg levels, although possible inhibitory effects were seen
for four less-abundant species.
Warnick and Bell (1969), studying the acute toxicity of
various metals to three insect species, found Hg in the
inorganic form to be lethal at 2.0 mg/1 for all of the
species tested. Of the three species, the mayfly was
found to be most sensitive to the various metals. In
our streams both mayfly species present showed a possible
inhibitory response to a Hg concentration as low as 5.0
pg/1; however, this highest concentration tested has a
very small effect on the total insect community.
114
-------
SECTION VII
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122
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SECTION VIII
APPENDIX
BIOTA OBSERVED IN THE ARTIFICIAL STREAMS
Phylum Chlorophyta
Sub-Phylum Chlorophyceae
Order Volvocales
Family Chlamydomonadaceae
Chlamydomonas sp.
Order Tetrasporales
Family Gloeocystaceae
Gloeocystis vesiculosa Naegeli
Family Chaetochloridaceae
Porochloris filamentorum Pascher
Order Chlorococcales
Family Chlorococcaceae
Chlorococcum humicola (Naegeli) Rabenhorst
Desmatractum bipyramidatum (Chodat) Pascher
Family Oocystaceae
Eremosphaera viridis DeBary
Oocystis sp.
Family Scenedesmaceae
Scenedesmus acutiformis Schroeder
Family Coccomyxaceae
Dispora crucigenioides Printz
Order Ulotrichales
Family Ulotrichaceae
Hormidium subtile (Koetzing) Heering
Geminella turfosa (Skuja) Ramanathan
Family Microsporaceae
Microspora pachyderma (Wille) Lagerheim
M. quadrata Hazen
Order Chaetophorales
Family Chaetophoraceae
Microthamnion strictissimum Rabenhorst
Stigeoclonium elongatum (Hassall) Kuetzing
Order Oedogoniales
Family Oedogoniaceae
Oedogonium sp. (probably 0. pusillum Kirchner)
Order Zygnematales
Family Zygnemataceae
Mougeotia sp. (5 micrometer width)
Mougeotfa sp. (10 micrometer width)
Spirogyra sp.
Family Mesotaeniaceae
Cylindrocystis brebissonii var. minor West and West
Mesotaenium macrococcum (Kuetzing) West and West
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Family Desmidiaceae
Arthrodesmus incus (Brebisson) Hassall
Cosmarium asphaerosporum Nordstedt
C. pseudoconnatum var. ornatum Allorge
C. laeve var. septentrionale Wille
C. undulatum var. minutum Wittrock
C. viride var. minor West
Penium spinospermum Joshua
Shaerozosma excavata Ralfs
Spondylosium planum West and West
Phylum Euglenophyta
Order Euglenales
Family Euglenaceae
Euglena mutabilis Schmitz
Phylum Pyrrophyta
Class Dinophyceae
Order Dinokontae
Family Glenodiniaceae
Glenodinium sp.
Phylum Chrysophyta
Sub-Phylum Xanthophyceae
Order Rhizochloridales
Family Rhizochloridaceae
Stipitococcus vasiformis Tiffany
Order Mischococcales
Family Pleurochloridaceae
Chlorocloster minimus Pascher
Family Sciadaceae
Ophiocytium deserturn var. minor Prescott
Sub-Phylum Chrysophyceae
Order Rhizochrysidales
Family Rhizochrysidaceae
Rhizochrysis sp.
Family Stylococcaceae
Lagynion macrotrachelum (Stokes) Pascher
Order Chromulinales
Family Chromulinaceae
Chromulina pseudonebulosa Pascher
Order Ochromonadales
Family Ochromonadaceae
Ochromonas sp.
Family Dinobryaceae
Epipyxis sp.
Sub-Phylum Bacillariophyceae
Order Pennales
Family Eunotiaceae
Eunotia tenella (Grun.) Cleve
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Family Naviculaceae
Navicula notha Wallace
Pinnularia abaugensis (Pant.) Ross
Family Cymbellaceae
Cymbella sp.
Phyltim Cyanophyta
Order Chroococcales
Family Chroococcaceae
Merismopedia punctata Meyer
Order Oscillatoriales
Family Oscillatoriaceae
Osciliatoria geminata Meneghini
0. meslini Fremy
P.- splendida Greville
Order Nostocales
Family Nostocaceae
Anabaenopsis sp.
Family Rivulariaceae
Calothrix parietina (Naegeli) Thuret
Phylum Spermophyta
Class Monocotyledoneae
Order Alismatales
Family Typhaceae
Typha latifolia Linnaeus
Order Liliales
Family Juncaceae
Juneus diffusissimus Buckley
Order Graminales
Family Poaceae
Panicum sp.
Class Dicotyledoneae
Order Geraniales
Family Callitrichaceae
Callitriche heterophylla Pursh
Order Scrophulariales
Family Lentibulariaceae
Utricularia biflora Lamarck
Phylum Arthropoda
Class Arachnoidea
Order Araneae
Family Pisauridae
Dolimedes sexpunctatus Hentz
Family Lycosidae
Lycosa helluo Walckenaer
Class Insecta
Order Odonata
Family Coenagrionidae
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Argia sp.
Ischnura sp.
Family Libellulidae
Celithemis fasciata
Epicordulia sp.
Erythrodxplax minus'cula
Ladona deplanta
Pacydiplax longipennis
Pantala. hymenea
Plathemis lydia
Tetragoneuria semiaquea^
Family Aeschnidae
Anax junius
Order Coleoptera
Family Dytiscidae
Agabus spp.
Coptotomus sp.
Hydrocanthus sp.
Kydroporus spp.
Hydrovatus sp.
Laccophilus sp.
Suphisellus sp.
Family Hydrophilidae
Berosus spp.
Enochrus spp.
Tropisternus spp.
Family Gyrinidae
Gyrinus sp.
Order Ephemeroptera
Family Baetidae
Caenis sp.
Callibaetis sp.
Order Trichoptera
Family Leptoceridae
Oecetis sp.
Triaenodes sp.
Order Hemiptera
Family Veliidae
Microvelia sp.
Velia sp.
Family Notonectidae
Notonecta indica
Family Navcoridae
Pelocoris femoratus
Family Gerridae
Gerris sp.
Family Nepidae
Ranatra sp.
Family Corixidae
Hesperocorixa spp.
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Order Lepidoptera
Family Pyralidae
Parapoynx sp.
Synclita sp.
Order Megaloptera
Family Corydalidae
Chauliodes sp.
Order Diptera
Family Tipulidae
Tipula sp.
Helius sp.
Family Chironomidae
Subfamily Chironominae
Chironomus spp.
Cladotanytarsus sp.
Cryptochironomus sp.
Dicrotendipes sp.
Microtendipes sp.
Parachironomus sp.
Paralauterborniella elachista
P. nigrohalterale
Polypedilum sp.
Pseudochironomus sp.
Rheotanytarsus sp.
Tanytarsus sp.
Subfamily Tanypodinae
Ablabesmyia ornata
A. peleensis
Larsia sp.
Zavrelimyia sp.
Subfamily Orthocladiinae
Cardiocladius
Cricotopus sp.
Psectrocladius sp.
Family Ceratopogonidae
Subfamily Dasyheleinae
Dasyhelea sp.
Subfamily Heleinae
Alluandomyia sp.
Palpomyia tibialis
Phylum Mollusca
Class Gastropoda
Family Lymnaeidae
Lymnea sp.
Family Physidae
Physa sp.
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Phylum Chordata
Class Teleostorai
Order Cyprinodontiformes
Family Poeciliidae
Gambusia affinis (Baird and Girard)
Class Amphibia
Order Anura
Family Bufonidae
Bufo terrestris (Bonnaterre)
Family Ranidae
Rana catesbeiana Shaw
R. pipiens Schreber
Class Aves
Order Coraciiformes
Family Alcedinidae
Megaceryle aIcyon (Linnaeus)
Class Mammalia
Order Carnivora
Family Procyonidae
Procyon lotor (Linnaeus)
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
. REPORT NO.
EPA-600/3-76-060
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Fate and Biological Effects of Mercury
Introduced into Artificial Streams
5. REPORT DATE
August 1976 (Issuing date
6. PERFORMING ORGANIZATION CODE
J. AUTHOR(S)
Henry J. Kania, Rbbert L. Knight, and Robert
J. Beyers
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Savannah River Ecology Laboratory
Institute of Ecology
University of Georgia
Athens, GA 30601
10. PROGRAM ELEMENT NO.
1BA023
11. CONTRACT/GRANT NO.
R800510
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Athens, GA 30601
13. TYPE OF REPORT AND PERIOD COVERED
Final report
14. SPONSORING AGENCY CQDE
EPA/ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Mercuric ion was continuously input to artificial stream channels to
provide water concentrations of 0.01, 1.0, and 5.0 yg/1. Channel com-
ponents were periodically sampled for total mercury analyses. The
effects of mercury on the algal components of the periphyton communities
and on the benthic insects were determined. The sampling program con-
tinued one full year after mercury inputs were stopped.
Approximately 15% of the added mercury was removed from the water.
The community components acquired very high concentrations of mercury,
although in most cases the levels in these were not a linear function of
the water levels. Concentrations in invertebrates decreased most
rapidly after mercury inputs were stopped while the sediment levels
decreased most slowly.
Periphytic algae showed several treatment responses including total
inhibition, inhibition of certain life stages, possible stimulation of
certain life stages and morphological alterations. One species was
found only in the treated channels and disappeared when mercury inputs
were stopped.
The diversity and evenness of benthic inject communities were
affected by mercury treatment. i
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
Streams, Aquatic animals, Food chains
Purification, Water pollution,
Aquatic microbiology-algae, sediments
invertebrates, Fishes
Heavy metals,
Mercury, mercuric io
Elemental mercury,
Microcosms, Fate,
Transformations,
Fish Food Organisms
Periphyton
6F
8. DISTRIBUTION STATEMENT
"formal distribution
19. SECURITY CLASS (This Report/
UNCLASSIFIED
21. .NO
141
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
129
*U.S. GOVERNMENT PRINTING OFFICE: 1976-657-695/5497
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