LABORATORY
South Ferry Road
Narragansett, R.I. 02882
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ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL MARINE WATER QUALITY LABORATORY
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SEMI-ANNUAL REPORT * July-December 1971*
AN ASSOCIATE LABORATORY OF
NATIONAL ENVIRONMENTAL RESEARCH CENTER. CORVALLIS. OREGON
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This report does not constitute publication but is for information
only. All data must be considered provisional.
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
NATIONAL MARINE WATER QUALITY LABORATORY
SOUTH FERRY ROAD
NARRAGANSETT. RHODE ISLAND 02882
April 15, 197 5
Enclosed is the Semi-Annual Report &nom the National Marine WateA Quality
LaboratoAy for June 1974 to Jaiiuary 1975. I regreX. the. 3-month delay in
getting this report out. for final publication, but as you wilt see, it is
a thorough compendium of our research activities during the. 6-month peAiod.
As I have, stated eaAlieA, the research activities and 6ci.enti.iic programs
at the National Marine Water QualiXy Laboratory ajie Ivighty varied. (tie
ate constantly consented with new problem which demand unique and
innovative solutions. Qua. approach to these complex problems is matuAing
and the data that we are producing will serve both EPA program and regional
ollic.es as well as the scientific community.
The purpose of these reports is severalfold'- first, they allow oua scientific
investigate is to go thAough a tlxorough review of their woAk and formulate
theiA thoughts and woAds with diagrams and graphs in preparation for formal
publication in peer-reviewed articles, for EPA publications, oa for cAttenia
development and for legaJl cases.
Secondly, it allows oua customers (those mentioned in the above paAagAaph)
to take a preliminary look at oua data., oua experimental methods, and oua
scientific approach. In publishing such a AepoAt as this, we open ourselves
to ouA cAJLtA.cs as well as oua proponents, as we attempt to discuss in an
open forum the pros and cons, the scope and failures oft the woAk we do in
this facility.
Spring is on the veAge of breaking here in Rhode Island, and the forthcoming
months will be exciting times for oua laboratory. The planned 20, 000 it2
o.ddilion to oua existing facility is moving along on schedule with the
coopehjotian and hard worJk of manj. We look forward to a ground breaking
and initiation of construction sometime in midsummer.
OtheA changes are on the hoAizon--such as the reorganizcution oi EPA's
research and development program.
Our past relationships with MERC, CorvaJUUs, and our Washington counterparts
have been productive and fruitful experiences. We look foAioard to the new
directions suggested by EPA's new managment structure and will work
dilligently as a laboratory to fulfill the plans that they have proposed.
It is oua wish, as well as that oh the new management structure in Washington,
that oua laboratory research programs proceed without interruption.
^1° 3'4^
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-1-
To those ofa you who have. the opportunity to visit us in NaAAaganseXt in
the. ^ollowitig month6, we welcome you. 1 ^eeZ that the. tnae spiAit,
capability and capacity o($ this laboAatoiy it, but ob&enved by meeting with
the. individual scientists, technicians and slLppoAt pe/isonnel who caAAy out:
ouA wik he/iz.
I encouAag e you to visit with us, neviw and cAitiquz oua pfiogAams and
suggest new solutions and experimental app>ioach&6 to oua pwblms. I
you cannot viiit us, £eel fcee to call ok mite. Qua phone numbeAS and a
map o<{ ouA facility axe included at the end oj$ this K
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DIRECTOR
Eric D. Schneider
Doris G Clrard, Sec
(TRANT & CONTRACT COORDINATOR
C. S. Hegre
TECHNICAL
OPERATIONS
Jan C.
Prager
DEPUTY DIRECTOR
Donald K. Phelps
Roseann Gauchfl t Librarian
Hary J. Malcolm, Admin. Tech.
Catherine A. Leavene, Sec.
ADMINISTRATIVE OFFICER
Claire D. Geremia
FACILITIES
James H. Wood
Denlse A. HcNulty, Sec
Ross L. Johnson, Eng. Tech.
Vayoe R. Davis, Res. Aq Biol.
Douglas
H.
Adams, Suppl> Clork
Wllliasi
J.
Egan, LC. Vech. Oper,
Saundre
A.
Barrington, Proc. Clerk
Thomas Callanan, Lab. Maint. Foreman
George Cottrell, Lab. Maint. Man
Maurice E. Hines, Lab. Maint. Man
Edward Weber, Lab. Maint. Man
BIOASSAY MFntODS TEAM
John H. Gentile, Team Leader
Gerald C. Zaroogian, Res. Chemist
Carol G. Pesch, Res. Aq. Biol.
John A.. Card in, Res. Aq- Biol.
Suzanne Sosnovski, Res. Aq. Biol.
Mary V. Johnson, Biol. Lab. Tech.
OCEAN DISPOSAL TEAM
Gerald G Pesch, Team Leader
Richard J Blasco, Res. Microbiol.
Bruce H. Reynolds, Res. Aq Biol
HETAIS TOXICITY TEAM
Earl W. Davey, Team Leader
Ronald Eisler, Res. Aq. Biol.
Marcia M. Barry, Bio. Lab. Tech.
Albert E. Soper, Phys. Sci. Tech.
MARINE CULTURE TEAM
Allan 0. Beck, Team Leader
Paul P. Yevich, Res. Biologist
Jay C. Sinnett, ELcc. Engineer
Linda A. Ferrcro, Res. Aq. Biol.
Raymond L. Highland, Electronics Tech.
William Giles, Bio. Lab Tech
C. A. Barszcz, Biol. Tech.
Raymond Hennckey, Res. Aq. Biol.
WFRGISMS ft RrSPONSC PARAMETERS TEAM
Don Miller, Icnm leader
Eugene H Jackim, Res. Chemist
Richard L. Steele, Res. Aq. Biol.
Ccoige E. Morrison, Res. Aq. Biol.
Juan Gonzalez, Res. Aq. Biol.
Richard A. Voycr, Res. Aq. Biol
Neal F. Lackic, Bio. Lab. Tech.
Frank A. Ostcrman, Bio. Lab. Tech.
Dianne E. Evcrich, Res. Aq. Biol.
OILS TEAM
Peter F. Rogerson, Team Leader
George Cardncr, Res. Aq. Biol.
Sue Cheer, Res. Chemist
Gregory Telek, Chemist
Richard L. I^pnn, Environmentalist
ECOSYSTEMS ANALYSES TEAM
Kenneth T. Perez, Team Leader
Ncal Goldberg, Computer Programmer
RFCR1 ATI0\AL I'AThR CRITERIA
Victor Cabelli, Team Leader
Gertrude Calandc, Sec.
Alfred Dufour, Microbiologist
Morris Levin, Microbiologist
Edlcy Strickland, Bio. Lab. Tech.
Donald Winslow, Bio. Lab. Tech.
Cynthia Thomas, Microbiologist
William watkins, Microbiologist
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BIOASSAY METHODS TEAM
The Bioassay Team is responsible for the development of bioassay
systems and techniques which permit the interaction of test organism
and pollutant in a manner closely approximating their natural en-
counter. The program has and will continue to focus on developing
methodology for a wide variety of estuarine species of both ecolog-
ical and commercial importance. Species representing different com-
munities, trophic levels, and feeding types are selected so as to
permit evaluation of the widest possible impact. Previous emphasis
of this program has been on short-term techniques; however, now we
have expanded to include all life stages with a strong emphasis on
the use of eggs and larvae. Our program utilizes species with short
life histories. This permits evaluation and comparison of short-
term as well as reproductive effects which are necessary for projec-
tions on population viability.
New trends in the bioassay program include pollutant bioaccumulation
and its significance to both public health standards and possible
effects on reproduction and population integrity. Most recently we
have started to evaluate mixed effluents and their effect on plank-
tonic communities. Bioassay designs are being developed that more
closely reflect the actual exposure profile and thus permit a more
realistic estimate of impact.
This report will cover three major tasks in our work plan:
(1) the development and evaluation of short-term bioassay techniques
for phytoplankton, zooplankton, fish larvae, and polychaetous anne-
lid larvae (ROAP 21 AKK - Task 07); (2) the establishment of design
criteria for long-term exposure systems and the fabrication and
testing of these systems for compatibility with all life history
stages (ROAP 21 AKK - Task 11); and (3) evaluate the performance of
long-term bioassay systems (ROAP 21 AKK - Task 12).
During the period covered in this report we have completed a
series of short-term bioassays on metals in cooperation with the
1
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BIOASSAY METHODS TEAM
Metals Team (ROAP 16 AAT). The data from these studies will appear
in the following sections.
ROAP 21 AKK - Task 07 — Develop and Evaluate Short-Term Bioassay
Techniques
Phytoplankton
Short-term bioassays were performed on both individual metals
and in two instances on mixed waste materials. Individually tested
metals included silver, chromium, nickel, lead, arsenic, cobalt, and
selenium. Mixed wastes included arsenic waste and antimony wastes.
Cyclotella nana (Thalassiosia pseudonana' and Skeletonema costatum
were tested at 30 o/oo salinity and 20°C. Figures la and lb are a
summarization of this information. The graphs show the EC-50 (growth
rate) response for a variety of test materials. The graph (courtesy
of Neal Goldberg) shows that the mixed wastes are considerably less
toxic than the individual metals. However, not having a compositional
analysis it is difficult to evaluate the toxicity of the metal itself.
It is interesting to note that there were some obvious differences
in sensitivity between the test species. Copper was considerably
more toxic to C_. nana while Su costatum was far more sensitive to
silver. Thus when waste materials are being evaluated for their
potential environmental impact it is necessary to look at more than
one species whenever possible.
Of further interest is the mild stimulation of growth rate re-
corded for the metal selenium at concentrations as high as 33 ppm.
Preliminary studies of selenium toxicity indicate Acartia tonsa
is sensitive to selenium at concentrations below 1.0 ppm. If further
studies warrant, a series of food chain studies involving Skeletonema
costatum and Acartia tonsa will be initiated.
In order to streamline data reduction from algal assays a com-
puter program was developed by Neal Goldberg of our Ecosystems
Analysis Team. This program will accept either cell density or bio-
mass input for each observation period and exposure level and cal-
2
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Fig. la Comparison of EC-50's for growth rate of Cyclotella nana
exposed to individual heavy metals, sodium lauryl sulfate
(SLS) and arsenic (ARW) and antimony (ATW) wastes.
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Fig. lb Comparison of EC-50's for growth rate of Skeletonema costatum
exposed to individual heavy metals, sodium lauryl sulfate (SLS)
and arsenic (ARW) and antimony (ATW) wastes.
4
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BIOASSAY METHODS TEAM
culate growth rates, correct for coincidence when appropriate, and
plot the complete growth response curves. The data is stored on
tape from which hard copies are made. An example of the data presen-
tation capabilities of this program is shown in Figure 2. The table
presents cell density/ml of culture with the growth rate for each
incremental observation period in parenthesis. This capability has
reduced our data reduction time in half.
ROAP 21 AKK Task 07
Lobster Larvae
Lobster larval assays were not emphasized this year due to
problems associated with collection and more importantly holding.
Laboratory facilities are now approaching the stage where ovigerous
females can be maintained in a controlled temperature environment
prior to hatching. Hatched larvae will be held in kriesels and used
for short-term bioassays. A series of exposure chambers have been
designed and fabricated and are currently being evaluated using
other microcrustaceans and fish larvae as the test species.
A methods manual describing the year-round production of lobster
larvae was prepared by Schlesser et al. under contract to EPA. A
detailed critique was prepared and returned to the authors for
revision. As a result of revisional problems and the availability
of materials we will be unable to initiate validation of the metho-
dology for several weeks.
In preparation for the validation, we have obtained a permit
to collect ovigerous female lobsters from the Rhode Island Depart-
ment of Natural Resources. Presently, the lobsters are being held
in communal tanks at ambient seawater temperature since late Sep-
tember. The divided holding and brood tanks have been manufactured
and we are awaiting delivery. Upon receipt of these tanks, we will
initiate a program of gradually increasing the temperature of the
seawater to accelerate maturation and hatching. The procedures
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BIOASSAY METHODS TEAM
we will follow will be those described in the mannual. We have
ordered the necessary kriesels for culture of the ensuing larvae.
ROAP 21 AKK - Task 07
Zooplankton
An attempt was made to evaluate the feasibility of using mixed
indigenous zooplankton populations in routine bioassays. This type
of assay is potentially useful in monitoring the effects of ocean
dumping to offshore species which have not been cultured through a
whole life cycle. A feasibility experiment was performed to evaluate
this potential assay technique. We found that the high degree of
expertise required in the design, performance, species identification
and interpretation prohibit this assay from being used routinely.
Further, there was evidence of interspecific predation which further
complicated interpretation. The use of natural indigenous populations
in bioassays becomes feasible only when essentially "monospecific"
populations exist.
A culture status report on the availability of zooplankton
species was completed and submitted to the Culture and Holding Team
(ROAP 21 AKF).
At the request of the Metals Team (ROAP 16 AAT) information on
the toxicity of heavy metals to zooplankton was compiled from our
laboratory research program. The data consists of short-term bioassays
on the species and metals listed below:
METALS MATRIX FOR COPEPODS
TEST ORGANISM 96 Hr. LC^ (in mg/1) of Toxicant
+2
„ +2
„ +2
r, +2
+6
„ .+2
Cd
Cu
Hg
Zn
Cr
Ni
Acartia tonsa
.320
.023
.060
.400
7.20
.620
Pseudodiaptomus coronatus
1.50
.140
.070
1.75
7.40
12.0
Eurytemora affinis
1.00
.500
.080
1.30
8.00
Tigriopus japonicus
4.30
.880
.115
2.70
22.5
4.60
Acartia clausi
.230
.046
.011
1.40
6.5
2.85
7
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BIOASSAY METHODS TEAM
In conjunction with the Metals Team (ROAP 16 AAT) a series of
static bioassays were performed on Acartia tonsa from field tows.
Results show that antimony waste has a very high oxygen demand.
After twenty-four hours exposure, the dissolved oxygen levels were reduced
to 1.5 ppm at 1.0% and .33% waste. This probably contributed to the
100% mortality noted at these levels. In an effort to rule out the
dissolved oxygen effect, an air-water life system was devised to
maintain the dissolved oxygen at 5.1 ppm in the .33% level for
twenty-four hours. Mortality at -^3% was about 35% with aeration, compared
to 100% in unaerated cultures. Sixty percent mortality was observed
a£ter 72 hours. These mortalities were not related to dissolved
oxygen levels (D.O. >5.1 ppm) (Fig. 3). The arsenic waste material
was twenty times more toxic to Acartia clausi than the antimony
waste. Of particular interest were a series of assays on the
+2
toxicity of Ag . This metal has in the past received very little
attention at this laboratory. The indications are that it is of
+2 +2
comparable toxicity with Hg and Cu to the genus Acartia. We
anticipate examining the chronic effects of this metal to Acartia
tonsa.
The continuous flow culture system for calanoid copepods is
now successfully rearing the generation from individual Acartia
tonsa isolated from an October field collection. Automatic continuous
feeding has been replaced by manual batch feeding with improved re-
sults. The continuous flow head tank is now heated providing 18°C
filtered seawater to the culture system. Initial attempts to set
up nine replicate continuous cultures of Acartia tonsa were compli-
cated by repeated contamination with harpacticoid copepods that
developed from eggs that passed thru the filter system. Work is now
in progress on the installation of a multi-media seawater filteration
system. This should allieviate problems with contamination. Experi-
ments are now in progress to evaluate the feasibility of
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Fi§• 3 Response of Acartia tonsa to antimony waste.
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BIOASSAY METHODS TEAM
using populations of Acartia tonsa for long-term bioassays. We are
presently defining the degree of replicate variance we can expect
from nine unstressed populations. Each replicate was started with
20 pairs of males & females and will run for 60 days. Qualitative
and quantitative population characterization will then be performed
on each replicate.
As part of developing assay parameters for such long-term
bioassays (Task 11), studies on Acartia fecundity are in progress.
The difficulty in using this parameter relates to its sensitive and
positive correlation to nutrition. An initial study comparing
females exposed and unexposed to cadmium, while not statistically
significant, did show a definite decrease in fecundity in exposed
females. However, handling and sampling problems remain to be
resolved before consistent and reproducible information can be
expected from this technique in an assay.
ROAP 21 AKK - Task 07
Polychaetes
A prototype culture system using flowing filtered seawater was
set up. This system will support the complete life cycle of C_.
capitata if temperature control is available to maintain a water
temperature warm enough for reproduction. The flow-through system
is definitely superior to static cultures. This fall the static
cultures became grossly contaminated with numerous cilliates,
harpacticoid copepods etc.,and the remaining polychaetes looked
unhealthy. The flow-through cultures were only slightly contaminated,
and the polychaetes remained active and healthy. The reasons for the
mortalities and unhealthy appearance in the static cultures are
unknown.
A prototype flow-through chamber for exposing £. capitata to
toxicants was devised. The chamber has not yet been tested.
Three more polychaetes were added to the culturing efforts. A
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BIOASSAY METHODS TEAM
clone of local Capitella capitata collected from Charlestown Pond was
established. A culture of polychaetes, tentatively identified as
Polydora ligni, was started from planktonic larvae collected from
Narragansett Bay. Ctenodrllus serratus, a minute polychaete which
commonly reproduces by transverse fission, was brought from California
by Donald Reish on December 6, 1974.
Short-term static screening bioassays of five metals were con-
ducted on polychaete larvae (tentatively identified as Polydora ligni)
collected from Narragansett Bay and Narrow River in November and
December. No mortalities were seen with selenium or chromium at
10 ppm which was the highest concentration used. LC-50 values for
the other metals are as follows:
Metal 96 hr 192 hr
Ag 187 ppb 96 ppb
Hg 54 ppb
AsQ^ 5.6 ppm 2.8 ppm
Concentrations for silver were verified by atomic absorption
analysis. The mecury bioassay involved the daily changing of media
and toxicant redosing.
ROAP 21 AKK - Task 12—Evaluate Performance of Long-Term
Bioassay Systems
Long-term Exposure of Oysters to Sublethal Levels of Cadmium.
A study was started in November, 1973 to determine the kinetics
of bioaccumulation of cadmium in oysters (Crassostrea virginica)
during winter and summer conditions. In addition to the bioaccumu-
lation of cadmium we wanted to determine 1) if any effect of accu-
mulated cadmium on fecundity and/or viability of larvae occurs; 2) if
depuration does indeed occur after termination of the cadmium dosing;
and 3) if any histopathological damage could be detected. Since the
oysters are laden with copper upon harvest, the copper content of the
oysters was studied in conjunction with the cadmium study. Our in-
tent here is to determine whether copper is depurated by the oysters
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BIOASSAY METHODS TEAM
in seawater containing ambient levels of copper and if cadmium
competes with copper for sites within the tissues.
Throughout the duration of the study, we are always in constant
search for the occurrence of some phenomenon or criterion that
would allow us to predict a detrimental effect to oysters or a
population when monitoring oysters exposed to levels of cadmium in
seawater not normally found in their natural environment.
A) Bioaccumulation - The oysters were continually exposed to
5 and 15 ppb cadmium in fiberglass troughs using a continuous-flow
seawater system in the wet lab. Ambient seawater temperature and
salinity were maintained throughout the entire study. The exposed
oysters along with controls were sampled bi-weekly for the first
24 weeks and weekly for the remaining 16 weeks during which time
the water temperature was higher resulting in greater amounts of
seawater passing over the gills. The total soft parts of the oyster
are removed from the shell, prepared for atomic absorption spectrometry
and analyzed for cadmium and copper.
Cadmium addition was terminated after 40 weeks. Levels of cad-
mium in the troughs were monitored weekly using the Chelex resin
technique developed by Dr. Davey of this laboratory.
Before exposure, cadmium levels in the total soft parts of the
oysters was 2.72 ppm wet weight; whereas at the end of 40 weeks
exposure to 5 and 15 ppb cadmium, mean cadmium levels of 13.57 ppm
and 33.34 ppm were found respectively. After 40 weeks exposure to
non-contaminated seawater the control oysters had a mean cadmium
level of 1.69 ppm in the total soft parts.
Both exposed and control animals appeared healthy by gross exam-
ination. Also, the exposed oysters produced as much new shell growth
as the controls. Some oysters in each group added as much as 1.8
cm new growth during the study.
B) Histopathology - Oysters were sampled for histopathological
12
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BIOASSAY METHODS TEAM
examination concurrent with samples for bioaccumulation. Histopatho-
logical examination has shown that the reproductive tract of the
oysters exposed to 15 ppb cadmium is slower in maturing than that of
the oysters exposed to 5 ppb cadmium and the controls.
C) Depuration - After 40 weeks the addition of cadmium to the
experimental troughs was terminated. The initial samples of oysters
was taken 1 month after termination of exposure to cadmium and suc-
ceeding samples have been taken bi-weekly. The oysters are prepared
for atomic absorption spectrometry and analyzed for cadmium and
copper.
The depuration phase of the study is still in progress.
At this time it appears that neither cadmium nor copper is
depurated by the oyster. However, it must be emphasized that this
data is preliminary and the variation in copper and cadmium amongst
the oysters within a sample is substantial; therefore, we cannot be
certain as to exactly what is happening until the study is terminated
and the data is analyzed statistically.
D) Viability of Gametes - Oysters were induced to spawn and the
larvae from oysters exposed continuously to 5 and 15 ppb cadmium and
from control oysters were incubated for 72 hours at 20°C in 22 o/oo
seawater.
This phase of the study was performed to determine whether gam-
etes from oysters exposed to cadmium are as viable as gametes from
oysters not exposed to cadmium and to determine if the male and/or
female gametes are effected differentially by cadmium. Also, we wanted
to determine if the larvae from gametes of cadmium-laden oysters were
as viable as those from controls.
If an effect on the gametes occurs when exposed to cadmium then
this quite conceivably could have a detrimental effect on a natural
oyster population.
In this study we encountered a problem not experienced before
in that the oysters exposed to 15 ppb cadmium could not be induced
13
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BIOASSAY METHODS TEAM
to spawn as readily as those exposed to 5 ppb cadmium and the controls.
Therefore it was extremely difficult to synchronize spawning in order
to get gametes to make the various crosses.
It appeared that the gonads took longer to mature in the oysters
exposed to 15 ppb cadmium. Then when they did mature and started to
spawn there was a shorter interval of time between the onset of
spawning and when they were spawned out as compared to controls and
the oysters exposed to 5 ppb cadmium. We observed in a similar study,
where oysters were exposed to 10 ppb cadmium continuously for 40 weeks,
that the control oysters spawned out sooner naturally than those
exposed to cadmium. However, in this case we encountered no difficulty
in synchronization of inducement to spawn.
Larvae produced from gametes from exposed parents showed normal
development to the straight hinge stage. However, at the 48 hr obser-
vation time up to 53% of these straight hinge larvae were dead, as evidenced
by empty shells. This did not occur when either of the gametes came
from control oysters. This phenomenon was observed regardless of
whether the larvae were incubated in cadmium-laden seawater (5 or 15
ppb Cd) or clean water.
Also, it is important to note that the gametes from one oyster
in each treatment were used in the appropriate cross fertilizations.
E) Viability of Larvae - Larvae from oysters exposed continuously
to 5 and 15 ppb cadmium for as long as 36 weeks were incubated in
natural seawater with and without cadmium (5 and 15 ppb) for 4 weeks
to determine if normal embryological development occurs under these
separate conditions. These larvae were fed Isochrysis galbana every
3 days.
Larvae from control oysters grew better in seawater containing
5 ppb cadmium than in natural seawater and that containing 15 ppb
cadmium.
Less mortality occurred in seawater containing 15 ppb cadmium
than in seawater containing 5 ppb cadmium or no cadmium adds. Per-
14
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BIOASSAY METHODS TEAM
haps the cadmium is exerting its bacteriostatic or fungistatic pro-
perties on the microflora.
The highest mortality amongst the larval treatments or crosses
was observed when female gametes from oysters exposed to 15 ppb cad-
mium were crossed with male gametes from control oysters and reared
in natural seawater with no cadmium adds. A 90% mortality occurred
with this cross and treatment whereas a 40% mortality was observed when
both gametes came from control oysters and reared in natural seawater.
A mortality of 75% occurred with the cross of female gametes from
oysters exposed to 5 ppb cadmium with male gametes from control oysters
and reared in natural seawater. Also interesting is the fact that these
larvae measured the largest of any other treatment (122u).
Long-term Exposure of Quahaugs to Sublethal Levels of Cadmium.
A study was started in December to determine the kinetics of bio-
accumulation of cadmium in the mahogany quahaug (Arctica islandica).
In addition to the bioaccumulation we are monitoring the toxicological,
biological, and histopathological effects of cadmium on this animal.
The same system which was used for the oyster study above is
being used for this study. The addition of 10 cm of sand to the
troughs to permit the quahaugs to burrow was the only alteration.
The quahaugs are being exposed to 5 and 15 ppb continuously and the
exposure should continue for 12-15 weeks. The quahaugs are sampled
bi-weekly to determine cadmium body burdens and for histopathological
examination. The total soft parts are used for atomic absorption
spectrometry of Cd.
The study is proceeding well as was anticipated. At the time
of the next report we should have completed this study.
Publication
Drs. Zaroogian and Cheer prepared a manuscript on cadmium accu-
mulation in oysters.
15
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BIOASSAY METHODS TEAM
ROAP 21 AKK - Task 07
Ichthioplankton
Short-term static bioassays were performed using the Atlantic
silverside (Menidia menidia). The four-spined stickleback (Apeletes
quadracus) and embryonic summer flounder (Paralicthys dentatus)
as test species. Bioassays were conducted to quantify the toxicity of
silver, arsenite, selenium, chromium, lead, and zinc. Several other
metals (barium, vanadium, beryllium, etc) were omitted from study
either because of their insolubility or their potential hazard to
laboratory personnel.
Results of these assays are summarized in Table 1 and generally
represent a single assay. While repetition of these assays can be
expected to refine the data, present results permit ranking of the
tested meta]s as highly toxic (Ag, Hg, Cu); moderately toxic (Cd, As,
Se, Cr+^) and low toxicity (Ba, Zn, Pb). It is interesting to note
that if we were to use other bioassay data from this report somewhat
different patterns would emerge in terms of ranking the toxicity of
these metals. This re-inforces our contention that until more data
is available which will allow us greater predictability, an array
of species should be tested whenever possible. Future studies will
focus on long-term exposures to one or two of the above metals in
an effort to obtain data on incipient lethal levels, rates of bio-
accumulation and potential effects on reproductive success.
In cooperation with the Culture Team, we have been trying to
expand our larval assay capabilities. In our geographical area, the
potential exists for working with flounder larvae for 9 months of the
year. For the first time we had the opportunity to study the summer
flounder larvae, Paralicthys dentatus which were successfully spawned
by Grace MacPhee of our staff. Unlike the winter flounder larvae,
this species was extremely sensitive to handling and temperature
variation. We did perform a very limited number of assays on both
16
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BIOASSAY METHODS TEAM
the eggs and larvae for comparative purposes. Both the handling and
temperature control problems previously mentioned will be resolved
so that a more definitive study can be performed during the next
spawning season.
Short-term static bioassays were performed on two mixed indus-
trial wastes. One waste was classified as an arsenic waste while the
second was not identified chemically. The results of these assays,
summarized in Table 2, indicate a relatively low toxicity compared
to heavy metals. However, long-term chronic exposures that concen-
trate on bioaccumulation, reproductive, growth and other sub-lethal
parameters must be evaluated before the potential environmental
impact of these wastes can be ascertained. Further since these wastes
are in a dilute aqueous state the concentration of an active ingrediant
is also diluted. Without proper chemical characterization the true
hazard of these materials is difficult to assess.
A considerable amount of biological testing for ocean disposal
permits involves the use of the brine shrimp, Artemia salina. While
offering many obvious advantages relating to ease of storage, hatching,
and holding, assays using this organism are subject to a wide range
of criticisms. Principal among them are: it is not an species indigi-
nous to estuarine and coastal waters (except San Francisco Bay); it
has, at best, limited ecological importance and significance in
marine food webs. Since most of our assay work involves indigenous
marine species, we assembled some comparative short-term assay data
(Table 3 ). It is obvious that there is wide variation between
organisms when compared to one toxicant and between various toxicants
on the same species. This brief comparison points out that Artemia
salina can not be used as the sole indicator of toxicity. For that
matter, no one organism is always more toxic than any other. Yet
there are those that display a consistently high sensitivity to many
compounds and it is these species that are currently being recommended
17
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BIOASSAY METHODS TEAM
for marine assays. They include the phytoplankton, the zooplankton
Acartia tonsa and Acartia clausi; the oyster larvae (Crassostrea
virginica), and fish and larvae from Menidia menidia and the flounder
larvae (Pseudopleuronectes). The polychaete worms are best suited
for long-term studies involving toxicant accumulation and mobilization.
At the request of Linda Ferraro of the Culture Team, acute
bioassays were conducted on five groups of Menidia menidia. The
first group was field animals, a second was held in natural unfiltered
seawater, and the remaining three were held in filtered seawater and
exposed to different dietary regimes. The unfed group showed widespread
mortality at all toxicant concentrations and were extremely sensitive
to handling. Interestingly sensitivity to cadmium of each group of
diet fish did not differ significantly from field animals (Table h ).
Interpretion is complicated by the presence of two distinct size groupings-
3.5 cm and 7.8 cm fish. When examined with respect to each of these
size groups the lab diet fish exhibit more tolerance to cadmium, (Table 5 ).
There is the possibility that field animals were stressed from collecting
which might account for their greater susceptibility. This study will
be repeated using larger numbers of same size organisms but exposed
continuously to the toxicant permitting evaluation of an incipient
lethal concentration.
Four continuous exposure systems have been fabricated by R. John-
son and are being tested. Initially this system will be used to pro-
vide short-term continuous exposures for comparison to the statics.
Chronic long-term exposures on toxicants chosen with regard to their
potential as marine pollutants, detectability by current analytical
capabilities, and low hazard human risk will be initiated this spring.
The embryo's and larvae of winter flounder, and atlantic silverside;
lobster larvae, and other species will be studied in this system.
This system has been designed for maximum flexibility and may be used
for a wide range of organisms, singly or in combination.
18
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BIOASSAY METHODS TEAM
TABLE 1
Menidia menidia
juveniles 3-5 cm
Apletes quadracus
48 hr LC50
96 hr LC50
48 hr
96 hr LC50
Silver
.43 ppm
.43 ppm
.55 ppm
. 55 ppm
Mercury
.66
.072 ppm
1.2 ppm
.45 ppm
Copper
Arsenite
17 ppm
16.5 ppm
17.5 ppm
16 ppm
Selenium
17 ppm
14.5 ppm
41 ppm
18.5 ppm
Cadmium
22 ppm
10.5 ppm
Chromium4^
greater
than 30 ppm
19 ppm
greater
than 30 ppm
greater
than 30 ppm
Barium
Lead
greater
than 100 ppn
greater
than 100 ppm
greater
than 100 ppr
greater
1 than 100 ppm
Zinc
— — —
Table 2: Toxicity of two industrial waste products to various
organisms
Arsenic Waste Mixed Effluent
Menidia Menidia juv. 96 hr TL^q
cm' Greater than 300 ppm
Apeltes quadracus 144 hr TL50
Greater than 300 ppm
Paralichtys dentatus 120 hr TLjq
embryos
Greater than 30 ppm
Fundulus heteroclitus 96 hr TL50
larvae (newly hatched) 260 ppm
Artemia salina 96 hr TL5Q
580 ppm
19
-------
Table 3: Comparative Toxicity of Artemia
Values are 96 hr LC-50 in mgs/1
SPECIES SLS
Artemia salina 2.3
Phytoplankton
Thalassiosira pseudonana 1.5
Skeletonema costatum 2.0
Zooplankton
Acartia tonsa
Acartia clausi 0.2
Eurytemora affinis 5.0
Pseudodiaptomus coronatus 0. 2
Tigriopus japonicus 5.8
Tisbe furcata 1.2
Molluscs
Crassostrea virginica 1.0
(larvae)
Polychaetes
Neanthes arenaceodentata
Capitella capitata
Ophryotrocha
Fishes
Fundulus heteroclitus 4. 5
(adult)
Fundulus heteroclitus
(larvae)
Pseudopleuronectes americanus
(larvae)
Menidia menidia (larvae)
Apeltes quadracus (adult)
Menidia menidia (juvenile)
salina with Indigenous Marine Organisms. Tabular
CU+2 Cd+2 Hg+*" KCN Effluent
2.2 51.0 0.6 0.06 580
0.015 0.10 0.015 100
0.10 0.33 0.03
0.023 0.32 0.06 0.09 120
0.064 0.52 0.04
0.50 1.0 0.08
0.14 1.5 0.07
0.88 4.3 0.12
0.10 3.8 0.006
0.30 12.0 0.03
0.20 7.5
0.40
10.0 55.0
0.64 11.0 .16 260
0.22 .75 0.48
1.2 0.12
.45
10.5 .072
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BIOASSAY METHODS TEAM
Table 4: Toxicity of Cadmium to Various Groups of Menidia menidia
(3-8 cm) 96 hr LC-50 Values.
Unfed Group Undeterminable. High mortalities
at all levels
Diet I 10 ppm
Diet II 12 ppm
Diet III 9 ppm
Field Population 10 ppm
Table 5: Toxicity of Cadmium to Various Groups of Menidia menidia
Size 48 hr 96 hr LC^q 48 hr *6 nr
3-5 cm 17.5 ppm 11 ppm greater than 35 ppm
30 ppm
7-8 cm 4.7 ppm 17 ppm
21
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BIOASSAY METHODS TEAM
Dr. Gentile and Mrs. Johnson prepared a manuscript on phyto-
plankton bioassay methodology for the ocean dumping manual.
Dr. Gentile, S. Sosnowski, and J. Cardin prepared a manuscript
on zooplankton bioassay methodology for the ocean dumping manual.
Dr. Gentile represented the NMWQL at a conference on Ocean
Disposal Bioassay Methods, July 6-8 at Atlanta, Ga.
Dr. Gentile presented a paper "Power Plants and Estuaries" at
the 104th Annual Meeting of the American Fisheries Society in Hono-
lulu, September 9-11.
Drs. Gentile and Cheer presented a workshop at ATP methodology
and application to power plants at NFIC, Denver, Colorado, October
5-8, 1974.
22
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
The research activities of this team currently include considera-
tions of the effects of pollutants on organisms in a dynamic environ-
ment. Some environmental factors can influence rates of toxicant
uptake by organisms. Elevated temperature, for example, which will
increase both metabolic and whole organism activity, can also result
in increased uptake of metals. Low salinity can have the same effect.
We are interested in defining patterns and quantifying the changes in
uptake rate following environmental alterations. We are also inter-
ested in understanding the mechanisms underlying observed changes in
uptake in order to ascertain whether they are applicable for a variety
of metals and a diversity of organisms. In considering environmental
interactions, we should also know the normal adaptive range of our
principle experimental organisms regarding temperature, oxygen and
salinity. This can be elucidated in a preliminary fashion by multi-
variate studies which determine the tolerance limits for each of these
factors, singly and in combination.
The team is also concerned with methods development in order to
identify and measure the effects of sublethal stress on organisms.
This question is currently being addressed at several levels of bio-
logical organization—from schooling behavior of fishes to subcellular
indicators, such as ATP analysis to indicate viability of the bacterial
flora of marine sediments.
I. Environmental Effects Studies
A. Influence of Temperature, Salinity and Substrate on Cadmium
Uptake by Marine Organisms (ROAP 21 AKF)
Task 067—Determine the influence of environmental and physiological
variables on uptake of specific pollutants.
A project was designed and conducted by E. Jackim, R. Steele, G.
Morrison and F. Osterman to elucidate the effects of selected environ-
mental factors on uptake of a metal. This project will also provide back-
ground information contributing to development of bioassay systems
23
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
which more closely resemble the field situation.
Radio-cadmium (Cd-109) was used as a tracer to evaluate
the effects of temperature, salinity, and presence of sediment on
uptake rates of this metal by acclimated and non-acclimated organisms
in a static system. Stacking dishes containing the desired organisms
and/or bottom substrate were placed in 40 liter aquaria containing
20 liters of filtered seawater of known salinities. These were
held in light and temperature controlled boxes with a magnetic
stirrer slowly circulating water. Sufficient cadmium was added
throughout the run to each tank to maintain a concentration of 20
micrograms/liter. Likewise, sufficient tracer (Cd-109) was added to
maintain the radioactivity to about 300 CPM/ml.
Nine species of animals and eight macroalgae were utilized in
preliminary studies (Table 1). The * indicates four animals and three
macroalgae which were selected for further study.
Table I - Experimental Organisms
Yoldia limatula
Common Name
*Mya arenaria
soft shell clam
*Mytilus edulis
bay mussel
*Mulinia lateralis (/
coot clam
*Nucula proxima
pea clam
Podarka obscura
small polychaete
Strongylocentrotus drobach-
common sea urchin
lensxs
Menidia menidia
silverside fish
Palaemonetes pugio
grass shrimp
Laminaria saccarina
kelp
Grinellia americana
red alga
*Codium fragile
antler weed
Rhodomenia palmata
red alga
Ulva lactuca
sea lettuce
Enteromorpha intestinalis
sea lettuce
*Ascophyllum nodosum
sea wrack
*Fucus vesiculatus
rock weed
The first series of studies was conducted without any sediment
in the test container. Rates of cadmium accumulation by seven species
24
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
of macroalgae and invertebrates, when exposed at 20°C and 20% salinity,
are illustrated in Fig. I. The bay mussel, Mytilus edulis, had the
greatest and most rapid uptake of cadmium at all temperature/salinity
combinations. None of the three filter feeding bivalves appeared to
have reached equilibrium. The deposit feeding pelecypod, Nucula
proxima, accumulated relatively little Cd and was the only animal that
appeared to reach a state of equilibrium with respect to cadmium
concentration. The macroalgae tested are not major accumulators of
the metal when compared to Mytilus. Ulva uptake reached an equili-
brium concentration of Cd in 14 days. The maximum exposure period
was 26 days, although water quality in the static experimental systems
deteriorated somewhat in runs of this duration. A biological filter
will be used in future work to assure good water quality throughout
the study.
All bivalves, (Mya, Mulinia, Nucula and Mytilus) exposed to
cadmium showed greater accumulation rates of the metal at the lower
salinity (20 °/oo) (Table 2). Cd uptake in the macroalgae, Codium
fragile, Ascophyllum nodosum, and Fucus vesiculatus also followed this
pattern. Temperature had a direct effect on cadmium accumulation rates
in the four molluscan bivalves, with greater uptake at the higher
temperature (20cC). In contrast macroalgae showed no appreciable
difference in cadmium uptake between 10 and 20°C. Table 2 summarizes
the Q;lq values for Cd uptake observed for each species between 10 and
20°C in both 20 and 32 °/oo salinity. The possibility of temperature/
salinity interaction affecting cadmium accumulation are presently
being evaluated.
Table 2: Cadmium Uptake QlO for Marine Organisms Exposed at 10 and
and 20°C. Quotient represents mean uptake over three
successive weeks. N=10 for the animals.
Species 20 °/oo 32 °/oo
Mya 1.88
Mytilus 1.17 1.66
25
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Mulinia
Nucula
1.23
2.30
2.56
1.83
1.89
1.22
1.68
Palaemonetes pugio
Codium
Ascophyllum
Fucus
1.94
1.65
2.31
1.21
The influence of temperature and salinity acclimation on cadmium
uptake was initiated to determine whether previous environmental
history affects uptake. Experimental organisms were acclimated at
two salinities (20 and 32 °/oo) and two temperatures (10 and 20°C)
for two weeks. These organisms were then placed in cadmium-dosed
water at seven temperature and salinity combinations and the cadmium
«-
accumulation recorded. Acclimation did not appreciably alter the
pattern or rates of cadmium uptake in Mya, Mulinia, and Codium.
Acute transfer of Mytilus edulis from 20°C acclimation temperature
to 10°C caused only 15^ initial reduction in cadmium uptake relative
to mussels acclimated and exposed at 10°C.
The interaction between sediment, test organisms, and cadmium
added to the water was also explored. It is recognized that the
behavior of burrowing organisms in a container without any sediment
could be quite atypical relative to that of the field. This could
influence metal uptake in a number of ways. Further, marine sediments
are recognized to act as a reservoir of heavy metals which can be
released by natural processes which alter the oxidized state of
sediment surface layers. This was demonstrated in the laboratory by
stirring cadmium-contaminated natural silty-clay sediments in an
aerated beaker for 36 days. Approximately 26% of the bound cadmium
was released back into the water.
Natural sediments were found to be highly variable with regard
to cadmium uptake. Over 50 cores and sediment dredgings from Narra-
gansett Bay and open coastal beaches were examined. The observed up-
take ranged from 4.4 mg Gd/gm dry sediment in dark reducing sediments
26
-------
^ 50
TJ
• ULVA
NUCULA
PALAEMONETES
TT~"Tc ODIUM
6 10 14 18 22 26
EXPOSURE TIME (DAYS)
FIGURE 1
27
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
to less than 1 mg Cd/gm in beach sand. Because of this variability,
a standard synthetic sediment was made up which would have a predic-
table cadmium uptake capacity as well as some compatability for ben-
thic organisms. After testing many mixtures, one consisting of dia-
tomaceous earth containing 6% FeS was selected. FeS was included
because of its presence in natural bottom sediments and its demon-
strated affinity for cadmium.
Experiments with natural, artificial, and no sediment showed
about 25% less uptake of Cd in Mulinia and Mya (filter feeding bi-
valves) when held in either sediment. This work is being continued
with other types of organisms.
B. Effect of Cadmium on Juvenile Stages of Macroalgae (ROAP
21 AKF):
Task 069—Determine the influence of multiple environmental stress
on marine organisms previously exposed to specific pollutants.
Little is known of the sensitivity of the early stages of
macroalgae to pollutant stress. There is the potential of greater
sensitivity of eggs and young sporophytes. Also such material is
attractive as research material as a large number of specimens,
all of the same developmental stage and representing similar genetic
makeup are available for study.
Research has been undertaken to study the effect of toxicants,
in this case Cd, on viability of eggs and sporophytes of Fucus
vesiculatus. Techniques are being developed to elucidate which
parts of plants collected in the field are most likely to contain
fertile gametes. It appears that a turgor shock is necessary for
expulsion of the eggs and sperm from their receptacles. The best
method yet found for optimum production of gametes is initial wetting
with distilled or deionized water, air drying for 15-45 minutes, and
then reimmersion into seawater.
Optimum conditions are being determined for growth of zygotes
28
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
and juvenile stages as a preliminary to determining the effects of
stressful conditions and toxic substances.
C. Temperature, Dissolved Oxygen and Salinity Requirements
of a Marine Amphipod (ROAP 21 AKF):
Task 070—Development of biological criteria in support of legal
standards for dissolved oxygen, temperature, and salinity, singly
and in combination.
Mr. Neal Lackie is conducting a factorial study on the marine
amphipod, Jassa falcata. The 24-hr. TLM was determined for a wide
range of dissolved oxygen (D.O.), temperature and salinity conditions.
Animals were acclimated to 20°C + 1 and 30 °/oo salinity + 2 for one
week in flowing seawater. One hundred animals were run at each
experimental combination in a static system which controlls temper-
ature and D.O. Results are presented graphically for temperature vs.
D.O. (Fig. 2) and temperature vs. salinity (Fig. 3). The mortality
curve for most stress conditions is sharply sigmoid, which suggests
death results from a single threshold phenomon. Temperature is
limiting at 28 to 29°C when oxygen and salinity are at normal
levels (i.e. D.O. at saturation and 32%). Further, there is no
reduction of this limiting temperature when D.O. was reduced to
6 or 4 ppm, or salinity reduced to 20%. This suggests these lower
oxygen and salinity levels do not exert any additional stress.
When salinities and D.O. were reduced further, reduced thermal tol-
erance did result showing the effect of multiple stresses.
Two flow-through experimental systems controlling D.O. and
temperature are being developed and tested for future factorial
studies with small invertebrates. These systems will permit obser-
vation of motility, which will be used to indicate sublethal stress.
Two different electronic systems are being considered to quantify
motility.
29
-------
5% 10% 21 %
M
O
uj c
o w
w
to
o
IT)
sz
C\J
>-
<
h-
£T
O
Jassa falcata
EXPOSED TO REDUCED
OXYGEN LEVELS
SALINITY 32 %o
TEMPERATURE VARIABLE
.3 PPM
2.5 PPM
TE
M
PE
RATURE °C
-------
o
a
100
5 °/c
oo
>-
en
o
o
m
^r
OJ
SALINITY vs TEMPERATURE
Jassa falcata
10 °/c
OO
15 °/c
OO
1
,20 %o
1
8 10 12 14 16 18 20 22 24 26 28 30 32
TEMPERATURE °C
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
D. Influence of Reduced Dissolved Oxygen and Salinity on
Growth in Marine Fishes (ROAP 21 AKF):
Task 070—Development of biological criteria in support of legal
standards for dissolved oxygen, temperature, and salinity, singly
and in combination.
A number of investigators have shown various physiological pro-
cesses of fishes to be highly sensitive to alterations in environmen-
tal conditions. For instance, rates of growth of laboratory-held
fishes is impaired by decrease in D.O. concentrations to levels
only slightly below saturation. Reduction in growth rates have
also been correlated with changes in levels of salinity beyond the
"optimal" range of the variable. Effects of each of these two
factors on growth of fishes have been studied independently of one
another. Surprisingly, no information is available to describe the
impact of both factors acting in concert despite the influence of
each on energy expenditures associated with osmoregulation. Conse-
quently a project was initiated by Dick Voyer to study the combined
effects of D.O. and salinity on growth patterns of marine fish.
The working hypothesis of the project is that maximal growth occurs
at a salinity isotonic with blood of the fish since minimum osmotic
work is required at that point.
The experimental system developed for the project is presented
schematically in Fig. 4. Basically, the system can provide several
levels of salinity by combining of seawater and tap water in varying
proportions, resulting in final solutions of seawater at 10, 20 and
30 °/oo salinities. Overall three contrasting D.O. concentrations
for each of the salinities are provided by bubbling nitrogen gas
upward against the flow of water downward through stripping columns.
The system was initially tested with juvenile Atlantic silver-
sides (Menidia menidia). A total of 540 fish were exposed to 9 D.O.
and salinity combinations, with three replicates per level. Test
32
-------
OJ
U)
FRESH - SEAWATER
MIXING BOX
TAP WATER
v-i-Q
-CH
II
5=5
JT
JT
kN2
*2
OVERFLOW
overflow
JT-
CARTRIOGE FILTER
^c=H
RESERVOIR
WATER PUMP-/
DRAIN TU8E
STRIPPING COLUMN
c~
SEAWATER
MULTIMEDIA
FILTER
^STANOPIPE
*2
WATER
DISTRIBUTOR
CHAMBER
~~~~~~~~~~~~~~~~~~~~~~~~~~~
EXPOSURE
CHAMBER
i ii ii iii ii ii ii ii ii n
DO T
DO IE
DOZE |
DO I
D03E
002E |
DOZ
oon
oom
SALINITY X
SALINITY X
SALINITY 2ZC
FIGURE 4, Schematic of experimental apparatus
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
levels of salinity were 10, 20, and 30%; for D.O., 4.0, 5.5 and 7.7
mg/1. Fish were sampled at regular intervals over a 52-day test
period, with 9 fish randomly selected from each replicate and liver-
body weight ratios, RNA-DNA ratios, and the relationship between
body weight and body length of each fish estimated. Analytical
assistance was provided by Drs. S. Cheer and E. Jackim.
No clear-cut effects of D.O. and salinity stress were dis-
cerned at these exposure levels. Both liver-bddy weight ratios
and RNA/DNA ratios were highly variable and inconclusive. Body
length-body weight relationships of all fish remained constant
throughout the test. Mathematically the curve can be described by
the equation, y=2.53x-3.49. It was expected alteration in growth
patterns would be reflected in difference in slope coefficient and
ordinate intercepts.
Randomness of test results signifies that, if RNA-DNA and liver-
body weight ratios are to be used as valid response parameters, large
sample sizes (larger than 9 in case of silversides), will be necessary.
However, in studies based on factorial designs, such as the one reported
here, large samples may pose a problem in view of extensive replicates
required in this type of study.
A cooperative study with the Culture Team on effects of salinity
and D.O. concentration on development of eggs and larvae of winter
flounder was subsequently initiated using this experimental system. The
general purpose of the study was to assess effects of various combin-
ations of D.O. and salinity on hatchibility, growth and survival of
selected stages of winter flounder. Results will be of value in
establishing oxygen-salinity optima for culturing the species and in
elucidating oxygen-salinity requirements of the species.
A continuous flow system was developed and attached to the oil-
exposure system managed by the Oils Team to study effects of continuously
renewed oil solutions on embryological material, larvae and small
34
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
invertebrates. Embryos of the winter flounder (Environmental Effects
Team) and polychaete worms (Bioassay Team) are presently being
exposed. With regard to the flounder embryos, response parameters
selected for study include hatchability of embryos, larval size, and
yolk sac volume.
Plans for a system permitting delivery of toxicant solutions
on a continuous basis to test chambers were provided to the Bioassay
Methods Team. The apparatus outlined is now being put to use.
E. Effects of Power Plant Thermal Discharges in Tropical
Waters (ROAP 21 AKF, Task 070).
Dr. Juan Gonzalez is conducting research to evaluate the
effects of thermal discharges on biota of the south coast of Puerto
Rico. Field studies have been initiated at a newly operating power-
plant at Guayanilla. The thermal plume is being mapped in the cove
affected, and the hydrography and biota of this immediate region
described. The copepod, Acartia tonsa is found in the heated water,
but it appears to be maintained there primarily by recruitment
from adjacent coastal waters. During summer months, cove temperatures
exceed maximum levels permitting growth and reproduction.
One of the most common macro-invertebrates inhabiting the anoxic
mud of the mangrove zone in this region is the mud clam, Phacoides
pectinatus, a mollusc used for food by local fishermen. This bi-
valve is found in the mud at a depth of between 25 to 45 cm. During
the course of this research the temperature in their habitat has
been between 25.4°C to 29.5°C; the oxygen concentration has ranged
from 0 to 1.9 ml/1; pH values have varied from 6.8 to 7.5; and
salinity from 31.2 to 35.4 °/oo. The physical and chemical character-
istics of the water overlying the mud are different to those in the
mud proper (Table 3). For instance, oxygen is in higher concen-
tration and salinity is lower. However, we suspect water temperature
35
-------
Table 3 Physical and chemical factors associated
with the mangrove-mud environment in
Parguera, Puerto Rico
November December January
Water
Mud
Tube
Water
Mud
Tube
Water
Mud
Tube
Water Mud
Tube
ml/1
O2
2.6
1 .5
2.5
0.2
2.6
0.1
3.8
0
Ph
7.8
7.5
7.8
7.9
7.4
8.0
7.9
7.3
7.9
7.8
7.9
+°c
29.5
29.5
29.5
27.4
27.5
27.0
26.8
24
26
5%o
31
35.4
30.8
31 .2
32
32.5
34
35
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
to be more variable throughout a 24 hr period based on a concurrent
temperature survey being undertaken in another, but similar, area.
Another benthic species being studied now is the mangrove
oyster Crassostrea rhizophorae. This species, although strongly
associated with mangroves, has a habitat different from that of the
mud clam. Crassostrea lives attached to mangrove roots at the
intertidal zone. It is a filter feeder and, therefore, is not
directly dependent on the detritus coming from mangroves. However,
it depends on recycled nutrients from the mud which keep phyto-
plankton populations available.
Temperature has been monitored hourly over a 24-hour period
monthly at 3 coastal stations since last November. Station 1
(Fig. 5) is on a reef that gets exposed during low tide. Conse-
quently, when lows occur at night during the winter, the temperature
decreases to values close to that of air temperature. The lowest
temperature recorded during this period was 21.8°C. When low tides
occur during day time in the summer the temperature raises to values
above air temperature due to the effects of radiation. The highest
temperature recorded last summer during one of the 24-hour stations
was 31.5°C in one of the shallower spots; this was in March 1974.
Reports of higher temperature have been published elsewhere by
Glynn (1968). The diurnal temperature range of the reef water is
often as great as 5 to 7°C.
Station 2 is located off the influence of reefs and mangroves.
There the temperature is more stable throughout the 24-hr period.
Temperature has ranged between 24.9 in the winter to 29.4 in the
summer.
Station 3 at the mangrove islet. Here the temperature oscillates
considerably during the 24-hr period and throughout the year. The
temperature has ranged from 24.9 in the winter to 30.8 during the
summer. Oysters are exposed to such a range of water temperatures,
37
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FIGURE 5. Area in southwest coast of Puerto Rico where 24 hr stations are occupied once a month
03'30" 03'00"- - 02'30" 02*00" 0t'30*
Lg Pcrauara
Cayo
\Boyo
Reef
man
>ve
Tor
r /'/\
/ 50 I
Ahogado
•rya
Lt7 Goto
-50
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
as well as being exposed to a wide range of air temperatures during
low tides.
One of the results of high temperatures on the reefs is the
"browning" of Thalassia plants, to the point that only the "rhizomes"
are left. Many sea urchins, including Lytechinus variegatus and
Tripneustes esculentus are killed during summer low tide periods.
Another sea urchin, Diadema antillarum "walks out" of these conditions
during day time.
The thermal tolerance limits of two bivalves common to this
coastal zone were determined in the laboratory.
1. The clam, Phacoides pectinatus, was collected and main-
tained in holding "cages" near the mangrove forest located close to
the marine field station of the University of Puerto Rico. Subse-
quently, three or four dozen clams were brought to the laboratory
and held in a tank with sufficient aeration. A thick concentration
of Chlorella sp was dispensed to each tank for food. Temperature
in the tanks varied from 19-21°C during a holding period of one
week.
The upper thermal tolerance for this clam was examined by
acute exposure of 20° + 1 acclimated animals to 28 and 35°C. Only
25% mortality occurred in the 28° group within five days. At 35°C,
the TL50 occurred in 62 hours and all clams died by 129 hours.
Mean shell length of these experimental animals was 6.38 cm. Another
group of smaller clams (5.6 cm mean shell length) exposed to 35°C,
had a only slightly shorter TL50 of 60 hours. At 40°C, TL5g=3.5 hrs;
at 43°C, TL5g=2.5 and 3 hours in two replicate runs.
2. Similarly, over 200 oysters, (Crassostrea rhizophorea)
were picked from the roots of the red mangrove and held in wire
"baskets" in the mangrove forest near the marine station. Several
dozen were brought to the laboratory and held in a room at a temper-
ature of 19-20°C. Aeration was provided and Monochrysis lutheri
39
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
was supplied as a food source.
Thermal tolerance studies were followed the same routine as
for mud clam. Oysters acutely exposed to 35°C showed a TL5Q at
72 hrs of exposure. At 40°C, the TL5Q occurred in 35 hours; at 43°C,
at 2 3/4 hours.
This work will be repeated after acclimating the animals to
temperatures comparable to those of their habitat in order to de-
termine if they are able to compensate (shift physiologically) to
higher temperatures.
F. Extramural Research Supporting Environmental Effects Studies
1. ROAP 21 AKF, Task 031 - Develop biological criteria for
NE crustacea in support of legal standards for temperature 800981.
Grant to Dr. A.N. Sastry, University of Rhode Island—Effect
of Thermal Pollution on Pelagic Larvae of Crustacea (D. Miller,
Project Officer)
Larvae of five benthic crustacea from New England region, Homarus
americanus, Cancer irroratus, Palaemonetes pugio, Rhithropanopeus
harissi and Pagurus pollicaris have been cultured and their best
temperature and salinity conditions for survival have been determined.
The optimum combination for larval development of each species are:
30 °/oo and 15 and 20°C, H. americanus (spring); 30 °/oo and 15°C,
C. irroratus (spring); 30-35 °/oo and 25°C, P. pugio; 25 0/00
and 25°C, II. harissi and 30 °/oo and 25°C, P_. pollicaris. The
optimum combination for larvae of the two subtidal species shifts
for summer hatches increasing from 10-20°C to 25° in H. americanus
and 15° to 20°C for C. irroratus. Detailed studies on the develop-
ment and survival of larvae of C. irroratus and H. americanus under
cyclic temperature regime are in progress.
Tolerance of larvae to acute temperature and low dissolved
oxygen has been determined for C^. irroratus, H. americanus and
V_. pugio. Larvae of estuarine species proved to be more tolerant
40
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
than those of subtidal species. Variation in the tolerance of
different larval stages within each species was also observed.
Metabolic-temperature response patterns have also been determined for
C_. irroratus and P_. pugio larval stages. Larvae of the subtidal
species are relatively stenothermal and showed significant shifts
in the pattern of response during development. The relatively
eurythermal larvae of estuarine species showed no such shift in
their metabolic temperature response patterns although the upper
temperature limits have shifted to more normal temperature (30-35°C)
for the later stages.
ROAP 21 AKF, Task 035—Determine effects of temperature and
current on survival and behavior of larval marine and estuarine fishes.
#R-801032, Grant to Dr. R. Stevenson, Univ. of Miami (Fla.).
Testing of larval fishes occupied the period from June through
December, 1974. A total of 450 Archosargus rhomboidalis (sea bream)
and Cynoscion nebulosus (spotted trout) were tested for their ability
to swim for one hour against current velocities of 1.0, 2.5, and 5.0
centimeters/second. Tests were run at ambient temperatures and four
degrees above (26 and 30° for Archosargus and 28 and 32° for Cynoscion).
These species were tested during the period that they were naturally
spawning in the area. Larvae were sacrificed, dried, and weighed
after testing.
As yet, there are insufficient data to make statistically valid
statements about the effects of temperature and current on the swimming
ability of larvae. Attempts to relate the data in a number of ways
have not yet shown significant trends. Analyses by length classes
and weight suffer because not enough data points are present in each
class. The data show high variability both within and among classes
of larvae. Although rearing methods were standardized in so far as
possible, large batch differences were seen in swimming stamina of
the larvae. It appears that such culture problems must be resolved
41
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
before swimming stamina can be used effectively as a method to evaluate
sublethal stress.
ROAP 16 AAT, Task 007—Grant for study of toxicity of metals in
larval arthropods 801305
Grant to Dr. J.D. Costlow, Jr. Duke University (D. Miller,
Project Officer)
Efforts this period have continued to consider which stages
in the larval development of the xanthid crab, Rhithropanopeus
harrisii, are the most sensitive to exposure to sublethal doses of
mercuric chloride. The response to mercury of the megalopae from
different mother crabs proves to be replicable. However, a consid-
erable degree of variability has been noticed in the response to
mercury of the more sensitive zoeal stages with various genotypes.
Further replications, in which descreet developmental stages were
subjected to several different sublethal levels of mercury, suggest
that the ecdysis period between the fourth zoeal stage and the mega-
lopae represents the more sensitive time in the development of the
crab to mercury exposure.
Work on the protein complements of control and mercury exposed
larval crabs has been continued. No differences have yet been found
in control and experimental animals at exposure levels lower than
those producing obvious morphological or viability changes.
Study began on the effects of mercury on the oxygen-transporting
protein, hemocyanin. The oxygen-binding properties of hemocyanins
from Callinectes sapidus and Limulus polyphemus as a function of pH
were measured. The effects of varying amounts of sodium chloride
on the oxygen affinity are also being explored. Initial observations
indicate that sodium chloride will have a significant regulatory
effect and that chloride ions are the active component. The effects
of varying amounts of mercury will be studied next.
42
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
II. Response Parameter Development
A. The Sub-Lethal Effects of Toxicants on the Schooling Behavior
in Juvenile Menidia menidia (ROAP 21 AKF): Task 072
Experiments to determine the sub-lethal effects of toxicants
on the behavior in the silverside, Menidia menidia, were continued
through this fall by K. Koltes Robinson. The experimental system,
fully described in the previous semi-annual report, consists of four
circular chambers, 2 1/2 feet in diameter by 6 1/2 inches deep, each
with a 12 inch opening to a central chamber.
After investigating the effects of 5 sub-lethal levels of
cadmium on group behavior in M. menidia during early summer, difficulties
arose in determining base-line behavior in subsequent studies.
Schooling bonds attenuated during control studies which had not
occurred with schools used during the winter and spring. This could
be attributed to one or both of the following factors. There may
be a seasonal variability in the schooling bond, similar to that
found in birds, operates in these fish. This is manifest by a
tight, organized school during the colder months giving way to a
loosely structured aggregation during the warmer months. Breder
(1959) and Atz (1953) have reported the occurrence of this seasonal
variation in other schooling fish. Breakdown of schooling bonds can
also occur with prolonged laboratory holding, as reported by McFarland
and Moss (1970) with northern anchovies. Yet this was not observed
in a school of M. menidia held for an extensive period during the
winter months, lending support to the idea of a seasonal variability.
None the less, the experimental procedure for the present study was
altered to accommodate the possibility of a laboratory artifact.
Thus, during the later summer months as the school became increasingly
more dispersed, experimental fish were held not more than 48 hours
after collection. The period for behavioral accommodation for a
school after transfer into the experimental system was also shortened
43
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
to two hours. This appeared sufficient for the fright response to
disappear. If the fish were not swimming normally through the tanks,
a longer acclimation period was allowed.
Data is collected according to a Markovian Chain whereby a
measurement of the position of the school is recorded every 10
seconds. Since the nature of the schooling bond is of particular
concern, a "school" is defined as not exceeding 1/2 the area of any
one tank. A normal school is usually well within these limits.
The same fish are used for the control and experimental run, each of
which is of 30 minutes duration. A short interval between these two
runs is required for the system to come to equilibrium with the
toxicant - about 10 minutes. Transition probabilities are then
computed for each matrix and the two matrices compared. At least
five replicates at each concentration level are run. At the
completion of an experiment, a sample of water1is withdrawn from
each chamber and the toxicant concentration is verified on a Perkin-
Elmer Atomic Absorption Spectrophotometer. The fish are removed
and the entire system is cleaned with a 5% solution of nitric acid
followed by rinsing with de-ionized water.
Studies of 5 sub-lethal levels of cadmium (15 ppb - 1 ppm)
and 5 levels of copper (50 ppb - 1 ppm) have been completed, but the
statistical analysis of this data is still in progress. Preliminary
results have indicated detection of at least 30 ppb cadmium by
schooling;'fish. In contrast, Menidia appeared less sensitive to
copper, with 100 ppb Cu required to produce a mild stress response.
Generally, Cd caused the school to be dispersed in about 10
minutes after introduction of the toxicant. Individual swimming speed
also slowed. These studies were conducted in the colder months
when normal responses to stress situations, such as fright, resulted
in tightening the school. Hence, breakdown of the schooling bond
upon exposure to cadmium is considered an atypical behavior. School
44
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
response to copper was of a different nature. Rather than a disorganized
slowing of movement, the fish responded by increasing their speed in
what appears to be exploratory behavior. This response would clearly
be of adaptive value in that it would contribute to the fish leaving
a contaminated area more rapidly by orthokinetic analysis. Cadmium
on the other hand, appears to elicit a non-adaptive response. These
studies with cadmium and copper should be repeated during the same
season to consider whether seasonal variability in schooling could
have influenced the contrasting behaviors observed upon exposure
to these two metals.
B. Development of a method for studying the behavioral re-
sponses of larval and juvenile fish to toxicants (ROAP 21 AKF):
Task 021—Develop and modify technology necessary for verifying
physiological state, nutritional state, histopathology, and recent
history.
Task 072—Select parameters for detection of response on chronic ex-
posure, changes in which could alter ability to compete and survive
in an ecosystem. Develop and evaluate techniques for measuring res-
ponse of selected organisms.
Experiments were conducted by Dianne Everich to develop a
method for assessing the effect of cadmium on the swimming behavior
of juvenile Menidia menidia. Swimming speed was measured while fish
were exposed to 2 sublethal cadmium concentrations: 10 and 100 ppb.
Controls were in filtered sea water. Fish were transferred from the
rearing tanks into 4 circular polyethylene tanks, 5 fish per tank.
Each experimental tank had a grid of 2 cm squares drawn on the bottom
and contained 1 liter of toxicant solution in filtered sea water.
The tanks were housed in an environmental-control box to reduce noise
and control water temperature and photoperiod. Observations were
made directly or with a closed-circuit video system 1 hour, 24 hours
and 48 hours after transfer of fish to the observation tanks.
45
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Cadmium dosing was initiated at the time of transfer. Swimming
speed was estimated by counting the number of 2 cm grid lines
crossed during a 15 to 30 sec. time period.
The results of two preliminary experiments are summarized in
Table 4 and Figure 6 and 7. Fish size was similar between test
groups of each run, but not between runs. This precludes comparison
of these two experiments, for size variations lead to marked differ-
ences in mean swimming rate. Differences in swimming speed of Cd
exposed fish vs. controls was tested for significance with "Students"
t-test. In each experiment, there was no significant difference
between controls and 10 ppb Cd fish after 24 or 48 hours. Fish
exposed to 100 ppb did show a reduction in swimming speed. In
Run #1, there was about 32% reduction after 24 and 48 hours. In
//2, a 17% reduction was observed after 24 hours exposure; 22% by 48
hours.
C. ATP Analysis Methodology (ROAP 21 AKF): Select parameters
for detection of response on chronic exposure, changes in which could
alter ability to compete and survive in an ecosystem. Develop and
evaluate techniques for measuring response of selected organisms.
Dr. Sue Cheer has been conducting an extensive literature
search concerning the applications and improved methodologies for
ATP analysis. The major emphasis of this search was on potential
techniques for the analysis of ATP in marine sediments. A biblio-
graphy of relevant publications is now available. This survey is
being continued. In addition, experiments were carried out to verify
that some organisms (algae) are subject to lysis and subsequent loss
of ATP when improperly sampled. Also, it was verified that room
temperature, pre-extraction thawing of quick-frozen (dry ice or
liquid nitrogen) filtered field samples resulted in no loss of ATP
for at least five minutes. Findings from these studies and the
current literature review were utilized in an ATP Techniques Short
46
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TABLE 4. Swimming speed (cm/sec) of juvenile Menidia menidia
exposed to cadmium (mean + standard error).
Exposure Time (hrs)
Experiment Cd"1"4" Concentration - ^7
1
control
0.67
+
0.05
0.72
+
0.04
0.93
+
0.06
10 ppb
0.80
+
0.06
0.81
+
0.04
0.88
+
0.04
100 ppb
0.61
+
0.13
0.49
+
0.04
0.62
+
0.08
2
control
0.73
+
0.05
0.66
+
0.03
0.82
+
0.03
10 ppb
0.02
+
0.07
0.69
+
0.03
0.76
+
0.05
100 ppb
0.86
+
0.05
0.53
+
0.02
0.64
+
0.02
47
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1.6
1.4
3 1.2
•o
FIGURE 6
Sukmm-tng Speed o£ MenicUa menidia
exposed to Cadmium
tj 1.0
CJ
Cj
Q.
«0
§» 0.8
to
0. 6
10 ppb
corUAol
0.4
lOOppb
0.2
0
—4
49
12
24
HOURS
36
48
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FIGURE 7
Suo-unin-Lng Speed ol MznicLui m2.yu.du1
expoizd to Cadmium
JO ppbli--..
100 ppb—p' >».
12
24
HOURS
lb
4S
49
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Course taught in Denver (NFIC) by Drs. Cheer and Gentile in November.
D. Extramural Research Supporting Response Parameter Develop-
ment.
1. ROAP 21 AKF, Task 027—Develop and modify technology
necessary for verifying physiological state, histological history,
and environmental history through research grant (Neomysis americana,
Acartia tonsa, Pea pugnax, Menidia menidia). Grant to Dr. S. Barbar,
Lehigh University (D. Miller, Project Officer).
All work is now focused on the effects of temperature and
cadmium exposure, singly and in combination, on the mysid shrimp,
Neomysis americana. Research has progressed in three areas of mysid
biology: natural history, laboratory maintenance and culture, and
adult histology. Biweekly field sampling for a full year has
contributed information on such aspects of population dynamics as
abundance, size distribution, sex ratios and reproductive conditions.
Hourly collections over complete tidal cycles has contributed infor-
mation on tidal migrations. Problems of extreme sensitiviey to
mechanical damage in transport and handling were overcome. In the
laboratory, best survival was obtained in continuous flow trays.
Studies to elucidate a good laboratory feeding regime and optimal
temperature conditions are underway.
Histological studies of serial sections of the mysid were
undertaken to provide an understanding of normal structural details.
Three methods of fixation have been explored for electron probe
analysis in order to locate and quantify cadmium uptake by the mysids.
Other methods to be employed to localize cadmium is histochemistry
and autoradiography.
2. ROAP 21 AKF, Task 024—Development of biomedical procedures
for characterizing physiological state of test organisms 800831.
Grant to Dr. Donald Horton, TRIMGOM, Portland, Maine (E. Jackim,
Project Officer).
50
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Studies relating hematological changes to temperature changes
in winter flounder were completed. Several differences in the
hematology and cardiac response between naturally summer acclimatized
(c.a. 15°C) fish and 20°C stressed fish were apparent.
Whole animal respiratory responses of fish exposed to Cd
are also being assessed.
Electrophoretic studies on fish blood are being developed
to the point at which quantification of individual proteins
can be realized.
3. Task 040—Vital microscope development, software modific-
ation. Grant to Dr. J.O.B. Greaves, Southeastern Massachusetts
University, North Darmouth, Mass., "The Development of an Inter-
active System to Study Sub-lethal Effects of Pollutants on the
Behavior of Organisms." (D. Miller, Project Officer.)
This project is for development of a system for the acquisition,
analysis and display of behavioral data from both micro-and macro-
forms. The system includes a video-to-digital processer to convert
images of organisms to a reduced information form readable by a
digital computer. This device is now being interfaced to other
system components, which include a 16-bit minicomputer. Software
development, which is progressing as scheduled, is in FORTRAN to
provide a system which is as machine independent as possible
to enhance system exportability to other laboratories.
III. Team Publications:
Jackim, E. 1974. Enzyme Responses to Metals in Fish. In
"Pollution and Physiology of Marine Organisms" Edited by John
and Winona Vernberg. Academic Press, N.Y.
Miller, Don Curtis and Allen D. Beck, 1975. Development
and applications of criteria for marine cooling waters. In:
Symposium On The Physical And Biological Effects On The
Environment Of Cooling Systems And Thermal Discharges At
Nuclear Power Stations, Int'l Atomic Energy Agency, (in press).
51
7BRARY / EPA
tonal Environmental Jtesaage& CSgafay
JSW 35thStress
'nrvalhs, Oregon 97339
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ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Miller, Don C., Workshop Participant for "Behavioral Bioassays",
in Marine Bioassays, Workshop Proceedings, Marine Technology
Society, 1974.
52
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ECOSYSTEMS ANALYSES TEAM
We are attempting to define the limits a total system can be
disturbed such that when the disturbance or stressor is relaxed the
system will return back to its original structural and functional state.
The approach being used is the microcosm method i.e., a miniature
of the real world ecosystem is stimulated physically in the laboratory.
Such a miniaturization is possible for a marine system because (1)
most of the biotic components are small and (2) it appears simple
i.e., there are only two major elements interacting together, a
benthic phase and a pelagic phase. The types of stressors and
disturbances used in the study are artificial sewerage and various
biotic manipulations (e.g., species removals, additions, etc).
To date, we have made one "dry run" to test (1) how well our
physical system mimics the real world and (2) how the biotic portion
of the system responded. It was found that number of modifications
in the system had to be made and that original computations for
simulating certain conditions were incorrect. Some of the modifi-
cations, computations and results of this first run are described
below.
Initially, the imposed light levels and spectra chosen for the
microcosms were based upon the following conditions and assumptions:
(1) conditions -
a. average depth of Narragansett Bay is 9 meters
b. almost complete oxygen water saturation of the bottom
layer and only 1-2°C difference between top and bottom
c. given extinction coefficients (k) and mean daily and
monthly surface radiation levels (Im)
(2) objective-wanted average intensity and spectral composition
found in Narragansett Bay for assume mixing between top and bottom
is complete and fairly rapid.
53
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ECOSYSTEMS ANALYSES TEAM
b. rate of exchange between top and bottom and, therefore,
the rate of light exposure is much less than the
rate of change in light intensity with season.
c. rate of change in light intensity over 9 meters of
water has no biotic effect.
Using the above, we computed the daily average light intensity/
month, 1^ with the formulation Im=(Im/kz) (l-e~'cz). The seasonal
values for k=1.0, a predetermined extinction coefficient, are
presented in Figure 1. We did not have extinction coefficients
for different intensities/wave length so that it was not possible
to make the same computation for the average spectral composition
in a uniformly mixed 9 meter water column as done for light inten-
sity. Instead, we calculated the depth (»Im) at which lm would
occur in a 9 meter water column and then compared the known spectra
for coastal waters at that depth to the light spectra used on the microcosms
(Fig. 2). Since the longer wave lengths and ultraviolet components
would be attenuated rapidly and absorbed in the surface, respectively,
the "cool-white flourescent" bulbs were quite satisfactory. We,
therefore, assumed that the average spectral composition in a uni-
formly mixed 9 meter water column was the same as the spectral
composition at the depth computed above for the average light
intensity, Im. It was found that such a light regime was too high.
A major bloom occurred seven days after the water was initially added
to the 12 microcosms. The resulting die-off was too great an organic
load for the benthic system to handle which brought about anaerobic
conditions. Direct measures are now being made to correct this problem.
All the microcosms were mixed equally with a rotating plastic
shaft. After comparing the dissolution rates of plaster-of paris
blocks and "sour-ball" candy in Narragansett Bay to three of our
microcosms, it was found that the former was approximately two times
higher than the latter. The necessity for obtaining equivalent
mixing rates other than for water column motion is that gas diffusion
54
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44 4
AVERAGE
RADIATION.
29 3
14.
TIME (MONTHS)
FIG. 1. Average seasonal light Intensities in a 9 meter water column.
55
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FIG. 2 Spectral composition for coastal waters and Sylvania cool
white fluorescent bulbs.
*
100
O
M
V,
o
o
o
S-S
Q
W
M
H
O
z
50
cool white
fluorescent»
bulbs
3 meters for coastal
waters
400
550
WAVELENGTH
700
56
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ECOSYSTEMS ANALYSES TEAM
coefficients must be similar so as to insure realism in the total
metabolism measurements. A new system of stirring is being designed
to produce a more turbulent mixing.
The stressor used in the study, an artificial sewerage has been
produced. However, the form,species and quantity of metals to be
added to the sewage has not been decided due to a lack of data. We
have recently sampled two local sewerage inputs for metals and expect
the results soon.
ROAP 21 AKF/029—A Demonstration of Sublethal Effect Due to
Pollution Stressors on Corals and Associated Biota. J.E. Bardach
and S.V. Smith, University of Hawaii.
This study is working with total but somewhat manipulated
tropical marine microcosms. The microcosms were exposed to different
stresses such as salinity temperature, nutrients and species
manipulations and the resulting response of total metabolism and
structure was observed. General relationships and conclusions
are forthcoming.
57
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MARINE CULTURE TEAM
A major expansion in fish culture capabilities occurred during
the past half year. Grace MacPhee, Fish Physiologist, GS-9, joined
the staff. She brought with her, considerable expertise in flounder
culture.
Considerable study was given to indigenous fish species,
amenable to laboratory culture and potentially acceptable subjects
for experimental projects of the various NMWQL research teams. A
supply of fish embryos and larvae is desired on a year-round basis.
Because of limited wet lab space, and no large capacity sea water
temperature control systems, it is not possible to provide any one single
species throughout the year. However, working within ambient water
temperatures an array of species can be provided during the annual cycle,
with embryos and larvae of one or two different species continuously
available. To achieve this, the following representative important
species were selected for in-house culture to provide the necessary
experimental organisms in the early life stages:
Pseudopleuronectes americanus December-March
(winter flounder)
Limanda ferruginea April-June
(Yellowtail flounder)
Menidia menidia May-July
(Atlantic silversides)
Tautogolabrus adspersus May-August
(summer flounder)
Implementing this yearly plan, Grace MacPhee successfully
spawned and cultured embryos and larvae of the summer flounder.
Substantial numbers were provided for experimental use to several
NMWQL teams. This was followed by culture of the winter flounder,
still in progress.
(cunner)
Scopthalmus aquosa
(windowpane flounder)
Paralichthys dentatus
May-August
October-December
58
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MARINE CULTURE TEAM
Other Marine Culture Team projects are in the area of character-
izing condition of animals provided for experimental use, nutrition
studies, invertebrate larvae culture and development of field
collection techniques. These, and other subjects are detailed in
the following pages.
For research management purposes, tasks accomplished by this
team are identified under ROAP 21 AKF "Ecological Requirements for
the Protection of Estuarine and Marine Life". This report is
organized by tasks under this ROAP.
ROAP 21 AKF - Task 02—-Collections
Collections: January 1, 1974 thru December 31, 1974
TABLE 1 - COLLECTIONS
Fish:
Menidia menidia,
(Atlantic silversides)
Fundulus heteroclitus,
(mummichog)
Gasterosteus aculeatus,
(3-spine stickleback)
Pseudopleuronectes americanus,
(winter flounder)
Alosa pseudoharengus,
(Alewife)
Paralichthys dentatus,
(gravid adults)
Crustaceans:
Homarus americanus,
(lobster)
Carangon septemspinosa,
(sand shrimp)
Paleomonetes vulgarus,
(grass shrimp)
Shrimp spp
Cancer irroratus,
(rock crab)
No. Collected
24,285
1,825
2,000
226
120
12
12
200
4,210
1,000
82
No. Collections
19
10
2
5
2
1
1
5
1
3
59
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MARINE CULTURE TEAM
Shellfish:
Nassarius obsoleta,
(mud snail)
Artica icelandica,
(ocean quahaug)
1,000
2
550
3
Crassostrea virginica,
(oyster)
4 bu.
3
Mya arenaria,
(clam)
Mercenaria mercenaria,
(quahaug)
Argopecten irradians irradians
(scallop)
765
15
1,500 plus
7
400
TOTAL 80
*Purchased from local supplier
Worthy of special notice were the success of Menidia collections.
Unprecedented low mortalities resulted from use of new handling
techniques in the field and in transport. Considering the suscep-
tibility of this species to handling, the techniques used could be
easily adapted to other delicate fishes thus expanding our total
capabilities for new species.
Collection Equipment: Several new seines were purchased this
year. They incorporate a unique "deep bag", designed by Ray Hennekey.
This has proved most effective and of considerable help in making
better handling-transfer techniques possible.
A new fish-sorting device is in the design-construction stage.
Hopefully, this device will replace more traditional sorters because
of its capability to sort delicate species with minimal handling
and damage. The return of Menidia in sizeable numbers will allow for
final testing and modification of the device in the spring.
A "German" style "otter" trawl has been procured and is used
for benthic fish and invertebrate collections from the 45 foot
chartered fishing vessel.
60
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MARINE CULTURE TEAM
Fish Culture - Task 02G2(a)
Laboratory cultured embryos and larvae were supplied for
experimental use:
SPECIES
Winter flounder, Pseudopleuronectes
americanus
Summer flounder, Paralichthys
dentatus
Atlantic Silversides, Menidia menidia
USER
Bioassay, Oil, Synergisms
Bioassay, Oil
Bioassay, Oil, Response
Parameters
Major NMWQL fish culture activities in addition to supply
included (1) development and application of materials, methods,
and techniques for summer flounder, Paralichthys dentatus embryo
and larvae culture and (2) preparation for and collection of gravid
adults for winter-spring spawning and culture of Winter flounder,
Pseudopleuronectes americanus.
Summer Flounder Culture (by Grace MacPhee)
Holding and maintenance of adult summer flounder was accom-
plished as follows. Adults were collected in Narragansett Bay from
several stations in the West Passage, during July 29-October 18, 1974.
Fish were maintained in 250 gallon tanks in a flow through raw sea-
water system at ambient temperature. The fish were fed live quahogs
silversides, raummichogs, and sheepshead minnows.
Hormone injections to induce spawning were started on 10/15/74,
and the first results were obtained 10/16/74 when two males produced
milt. The first eggs were produced 10/22/74. A spontaneous tank
spawning took place 10/29/74 with very low fertilization resulting
(0.5%), presumably because the males produced very little milt.
Table I. gives dates of successful stripping, amounts of eggs produced,
and per cent fertilization.
61
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MARINE CULTURE TEAM
TABLE I
Date Amount of Eggs
% Fertilization
No. of fish Spawning
11/3/74 50,000
11/10/74 80,000
11/17/74 100,000
11/24/74 10,000
65%
80%
80%
95%
1 female, 2 males
1 female, 2 males
2 females, 2 males
1 female, 2 males
In all, seven fish produced sexual products (3 males, 4 females).
The other fish were probably immature females. At present it is
impossible to sex summer flounder externally.
The breeding stock was returned to original collection sites in
Narragansett Bay on 11/26/74 because lack of water temperature
control made it impossible to keep them any longer.
Some difficulties encountered in maintaining the fish in the
present seawater system were: outbreaks of disease, parasitic in-
vasions, and crowding due to small tank size. Fish which were
injured in the trawl developed vibriosis and fin rot. The disease
was diagnosed by William Watkins of the Recreational Water Quality
Team at Dr. Richard Wolke's (Animal Pathologist, University of Rhode
Isalnd) laboratory (see Linda Ferraro's section for further comments).
The fish were treated with the drug Furanace, by bathing for an
hour in a concentration of 10 g/ml water. Noticable improvement was
shown within t-hree days. Fin rot was also a problem. It occurred
in trawl damaged fish and also in intact fish after a month in
captivity. Topical treatment with malachite green 2% solution and
malachite green-formalin solution did not produce noticible improve-
ment. This was probably because histological grade malachite green
was used. This contains zinc which prevents the dye from working.
Collection Injury was significantly reduced. Allan Beck devised
a sorting tray for use on the boat,with fishes collected by otter trawl.
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MARINE CULTURE TEAM
The net was emptied into a large shallow fiberglass tank 4'x8'xl' which
was filled with water. The fish were not out of the water except when
hoisted over the side. They did not contact the deck during sorting and
consequently received minimal physical abuse and damage.
Summer flounder eggs and larvae were supplied to the bioassay
team for heavy metal toxicity studies. Some larvae were supplied
to the response parameters team to see if their behavior could
be monitored by remote T.V. camera.
TABLE 2
People supplied Number of eggs Number of Larvae Dates
Gentile & Cardin 4,500 600 11/5-11/26
(Bioassay team)
Everich 6* 11/20
(Response parameters)
* To evaluate behavioral video recording system.
The rate of development of summer flounder eggs was dependent
on water temperature. Because there is no water temperature
control, quantitative data was not available but estimates of
developmental time were obtained (Figure a). An important differ-
ence to note is the delay between embryonic Phase II (Stages taken
from Smith, W.G. and M.P. Fahay. 1970. Description of eggs and
larvae of the summer flounder Paralichthys dentatus.(Bur. of Sport
Fisheries & Wildlife Res. Dept. 75)
Further data on summer flounder Is detailed in MacPhee, Grace, K.,
1975. Synopsis of biological data on the summer flounder. Fisheries
Bulletin, NOAA. National Marine Fisheries Service Publ. In press.
Invertebrate culture—R0AP 21AKF—Task 2G3
Culture facilities have been designed and fabricated in the
Narragansett wet lab by Bill Giles. Previously this culture effort
was accomplished at the Research Barge, Jerusalem, by George Morrison.
All future invertebrate culture will now be at Narragansett.
63
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0 Average of 9°C
X Average of 11°C
0 Average of 15°C
LAG STAGES OF
EGG & LARVA
EYE PIGMENT - t X 0
HATCH - • X 0
PHASE II •
GASTRULA - *X0
BLASTULA -
J I I I
5 10 15 20 DAYS
FIGURE A. DEVELOPMENT TIME-TEMPERATURE - SUMMER FLOUNDER
64
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MARINE CULTURE TEAM
Presently available facilities will support the culture of
the following invertebrates:
marine rotifer - Brachionus plicatilus
brine shrimp - Artemia salina
barnacle - Balanus improvisus, spp.
mussel - Mytilus edulis
coot clam - Mulinia lateralis
scallop - Argopecten irradians irradians
oyster - Crassostrea virginicus
quahaug - Mercenaria mercenaria
ROAP 21 AKF - Task 2G4—Culture of marine polychaete Capitella
capitata
The culture methodology provided by Dr. Donald Reish under EPA
Grant 800962 was validated in NMUQL facilities by Carol Pesch. The
organism is now available for experimental use. Some difficulty was
encountered in transferring Dr. Reish's successful methodology to
NMWQL facilities. Ms. Pesch prepared a written evaluation of the
methods manual provided. The main criticism was lack of specificity
in the routine daily physical procedures involved in culture
maintenance. The manuals should be sufficiently detailed to permit
successful culture by a competent biological technician with no
previous experience in polychaete culture. A culture and bioassay
methods manual is due to be published and will include input from
Ms. Pesch and other members of the Bioassay Methods Team.
Task 2G4—Validation of EPA Grant 800962 polychaete culture
methodology is considered substantially complete.
ROAP 21 AKF—Task 09—Production of Larval Lobsters
The American Lobster (Homarus americanus) culture methods
manual was provided under EPA Grant 802494 by Dr. Schleser, Univ.
of California, Davis. The Marine Culture Team has neither the
facilities nor personnel, so validation of culture methodology
65
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MARINE CULTURE TEAM
combined with bioassay experiments will be accomplished by personnel
of the Bioassay Methods Team and progress detailed in another section
of this report.
Nutrition—21AKF—Task 2E-By Leslie Richardson
The determination of the nutritional chemistry of winter flounder
(Pseudopleuronectes americanus) is now being carried out by Leslie
Richardson. The emphasis is presently on the precise proportions of
proteins and the amino acid profiles for the eggs, yolk-sac larvae,
post larvae, juveniles and adults of the flounder. Potential foods
for the various life stages are also being analyzed beginning with
wild plankton tows from the Narragansett bay.
The accompanying flow sheet Fie. B describes the laboratory approach
to determine the nutritional characteristics. The results are
expressed on a moisture-free and ash-free basis. The nitrogen
characterization determines the percentage recovery of the total
nitorgen of each sample as free amino acid nitrogen. Both acid and
alkaline hydrolyses are carried out on the samples for complete
amino acid profiles. Tryptophan and cystine assays are included.
Analyses for mineral and fatty acid content are being incorporated
into the total laboratory design.
66
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FIGURE B. NUTRITIONAL CHEMISTRY ANALYSES
SAMPLE
(Lyophilized, ground)
ASH
MOISTURE
(Chibnall et al. '43) (Chibnall et al. 43)
TCA PRECIPITATION
(Schmidt & Thannhauser '45)
MINERALS
(Atomic Absorption)
residue
upernatant
ov
INSOLUBLE PROTEINS
FATTY ACIDS
(Schultz & Quinn '7
PEPTIDES & FREE AMINO ACIDS
NITROGEN
CHARACTERIZATION
/ \
TOTAL N
(Lowry et al. '51)
ACID HYDROLYSIS
(Moore & Stein '63,
Hirs '67)
ALKALINE HYDROLYSIS
(Noltmann et al. '62,
Miller '67, Spies &
Chambers '49)
AMIDE N NH3-N
(Wilcox '67) (Wilcox '67)
-------
Figure B
BIBLIOGRAPHY
MARINE CULTURE TEAM
1. Chibnall, A.C., Rees, M.W. and E.F. Williams. 1943. The total
nitrogen content of eggs albumin and other proteins. Biochem.
J. (37)354-9.
2. Hirs, C.H.W. 1967. Automatic computation of amino acid analyzer
data. In Enzyme Structure: Methods in Enzymology (S.P. Colovick
and N.O. Kaplan, editors in chief, C.H.W. Hirs, editor), Vol. XI
Academic Press, New York. '
3. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and R.J. Randall. 1951
Protein measurement with the Folin phenol reagent. J Biol
Chem. (193)265-75.
4. Miller, E.L. 1967. Determination of the tryptophan content of
feedstuffs with particular reference to cereals. J. Sci Fd
Agric. (18)381-6.
5. Moore, S. and W.H. Stein. 1963. Chromatographic determination
of amino acids by use of automatic recording equipment. In Enzyme
Structure: Methods in Enzymology (S.P. Colowick and N.O. Kaplan,
editors), Vol. VI, Academic Press, New York.
6. Noltmann, E.A., Mahowald, T.A., and S.A. Kuby. 1962. Studies on
adenosine triphosphate transphosphorylase. J. Biol. Chem
(237)1146-54.
7. Schmidt, G. and S.J. Thannhauser. 1945. A method for the
determination of desoxyribonucleic acid, ribonucleic acid, and
phosphoproteins in animal tissue. J. Biol. Chem. (161)83-9.
8. Schultz, D.M. and J.F. Quinn. 1972. Fatty acids In surface
particulate matter from the North Atlantic. J. Fish. Res Bd
Can.(29)1482-6.
9. Spies, J.R. and D.C. Chambers. 1949. Chemical determination of
tryptophan in proteins. Analyt. Chem. (21)1249-66.
10. Wilcox, P.E. 1967. Determination of amide residues by chemical
methods. In Enzyme Structure: Methods in Enzymology(S.P. Colowick
and N.O. Kaplan, editors in chief, C.H.W. Hirs, editor), Vol. XI
Academic Press, New York.
68
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MARINE CULTURE TEAM
Similar studies will be conducted on Atlantic silversides,
Menidia menidia.
As previously stated (NMWQL Semi-Annual Report January 1, 1974
thru June 30, 1974) the goal of nutrition studies is to develop a
series of artificial diets or natural diets which can be qualitatively
and quanitatively standardized, and which are capable of providing
the necessary nutrients at the various life stages of the organism.
Ideally, the developed diets should promote normal growth, survival,
and physiology of test organisms as compared to field populations.
Genetic variability between various fish of the same species will
result in a range of "normal" values for selected condition indices.
Hopefully, optimal laboratory culture and nutritional regimes will
provide experimental animals within the normal range. Until this
is achieveable emphasis will be on standardizing, if not normalizing,
condition of test organisms.
Results of the past six months nutritional studies with Atlantic
Silversides, accomplished by Linda Ferraro are as follows:
Menidia menidia Nutrition Experiments—Feeding Study No. 2
By Linda A. Ferraro—Marine Culture Team, NMWQL, January, 1975
I. INTRODUCTION
Preliminary nutrition experiments were conducted one year ago
in autumn, 1973, on Atlantic silversides, Menidia menidia, in which
laboratory fed and unfed test animals were subjected to histological
examination and biochemical analysis for comparison with field-
colelcted Menidia. The object of this work was two-fold: (1) to test
a simplified method for ascertaining biochemically the nutritional
status of the animals, and (2) to give baseline data on how closely
lab-fed fish approximated the condition of field animals. Compara-
tive evaluation parameters included laboratory survival, histological
examination, hepatosomatic index (HSI or liver-to-body weight ratio),
and biochemical analysis for glycogen, lipid and protein synthesis
69
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MARINE CULTURE TEAM
(RNA:NDA ratio). The experiment was conducted in an open, raw-sea-
water system at ambient temperatures (18°-15°C), and the test diet
consisted of 6 g. Biorell flake food and 16 g. thawed Artemia salina
brine shrimp per 100 fish per day. Data from the 1973 experiment
indicated the biochemical methods to be sensitive and suitable,
and that histological examination supported the biochemical findings.
The results indicated that the starved group approximated the 1973
field animals more closely than the laboratory fed group, while the
laboratory fed group showed significant increased in liver glycogen
and HSI with time. The conclusions drawn at that time were that
histological and biochemical methods used for analysis were sensitive
enough to detect nutritional differences even after one week of
laboratory holding. It was demonstrated that the simple use of
HSI alone paralleled or reflected the glycogen balance quite
closely, and thus could be used as a reliable indicator of nutritional
status with respect to energy storage capacity.
Since the laboratory fed animals received a generous diet
initially calculated to supply them with as much as they would eat,
the possibility of overfeeding vs. dietary imbalance may have been
a factor in the"analytical differences they displayed from the field
animals, coupled with their reduced energy expenditure due to lack
of activity required for predator-avoidance and food search. It
was not established whether the high glycogen contents of laboratory
fed fish were due to dietary imbalance (excess caloric content) or
merely due to overfeeding and/or lack of exercise comparable with
that of field animals. In addition, despite analytical differences,
no data was generated indicating whether or not laboratory fed fish
respond the same as field animals to conditions of stress and/or
toxicant exposure. No comparative bioassays were conducted. And
lastly, there was no certainty that the field "controls" were truly
representative of all field Menidia. Therefore, it was desirable
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MARINE CULTURE TEAM
to repeat and modify the experiment, both to confirm the reliability
of the analytical methods and to resolve the questions left unanswered
in the preceding experiment.
The purposes of the 1974 nutrition status experiment were fourfold:
(1) to validate the reliability of the HSI as an indicator of
nutritional (energy) state of the fish, at the same time comparing
the condition of Menidia collected in 1974 vs. 1973, (2) to determine
whether a reduced level of feeding of the same (1973) lab diet would
more closely approximate field conditions, (3) to test a higher
protein diet to see if a possible dietary imbalance was responsible
for biochemical differences found previously, and (4) to utilize
a portion of the test groups for comparative bioassays to determine
differences in response to a toxicant, in effect assessing the effects
of different laboratory diets on the fishes' performances in a
bioassay, as compared with the responses of newly-collected field
fishes.
The ultimate objective of this work is the development of a
capability to hold Menidia menidia of varying sizes for extended
periods and culture from egg to egg. Such a capability would lend
itself to the conductance of long-term chronic bioassays, also
requiring long periods of laboratory holding and feeding.
II. MATERIALS AND METHODS
The 1974 experiment was conducted at the same time of year
(October) as the 1973 experiment, to insure some comparability of
data between the two year-populations. All experimental work was
conducted in aerated, open-system, filtered seawater round aquaria
of 230-liter capacity. Uniform flow rates were established by means
of a constant diameter inflow opening of one (1) mm. The entire
experiment was conducted at ambient temperatures ranging from 16.4°C
to 9.0°C.
The initial collection of Menidia menidia, from which all
71
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MARINE CULTURE TEAM
experimental fish were taken was made on October 11, 1974.
Approximately 1,000 juvenile and adult Menidia were collected by
beach seine from Narrow River, Middlebridge, Rhode Island and
held overnight in a large round tank of 578 liter capacity.
Immediately upon collection, 15 fish were placed in Dietrich's
fixative and furnished to Mr. Paul P. Yevich for histological
examination, and 25 fish were submitted for body length and weight
determinations, dissection, liver weight determinations, HSI
calculations, and subsequent freezing for later biochemical analy-
sis, if warranted. This aspect of the research was accomplished
with the cooperation and advisement of biochemist, Dr. Sue Cheer,
who participated in this research during the 1973 experiment.
Both initial histological and analytical samplings comprised the
initial field "control" data groups, which were used as an in-
dicator of the fishes' condition prior to being placed on laboratory
diet regimens.
On the following day, October 12, 1974, (Day 1 of the experiment),
800 Menidia were randomly distributed into eight 230-liter round
aquaria (100 animals per tank). Both large and small fish were
distributed into each tank. Each tank was assigned to one of four
experimental diet groups, resulting in two replicates, A and B,
or 200 fish, per diet. Experimental feeding groups are listed
according to the schedule presented in Table 1. Feeding began
on Day 1. The time of feeding and amounts fed refer to food
administered per tank of 100 fish. Biorell and pulverized prior
to feeding to ensure a particle size palatable to the fish.
Frozen brine shrimp (Artemia salina) were fed after thawing.
72
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MARINE CULTURE TEAM
Table
1:
Daily Feeding Regimen — Menidia menidia 1974
Comparative
Feeding Study
Group I
Group II
Group III
Group IV
Time
(=
= 1973 diet)
(Half-1973)
(high-Protein)
(unfed)
A.M.
6
g. Biorell
3 g. Biorell
15 g. Artemia
0
P.M.
16
g Artemia
8 g. Artemia
15 g. Artemia
0
All tanks were cleaned at weekly intervals using a siphon
modified at the intake end with the attachment of a plastic
bristle vacuum-cleaner brush head, for dislodging and removing
sedimented particulate from the sides and bottom of each tank.
Tanks were monitored daily for the following parameters: water
temperature, fish behavioral aberrations, moribundity and mortal-
ities. Dead animals were removed immediately upon discovery and
preserved in Dietrich's fixative for histopathological examination.
Dead fish which had whitish gills and cloudy eyes were not pre-
served, as these criteria were considered evidence of advanced
post-mortem autolysis, making histological examination fruitless.
This was usually seen in conjunction with deaths occurring over-
night .
During the course of the experiment, three samplings of each
diet group, including equal numbers of A and B tanks, were taken
for comparison with field fish collected on similar dates. These
samples were submitted for biochemical, HSI and histological
analysis. Sampling dates corresponsed with one week, three
weeks and six-and-a-half weeks of laboratory feeding. Both
large and small Menidia were included as equally-distributed as
possible in these samplings. With the exception of Group IV
(Unfed) animals, all of which had succumbed to starvation by
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MARINE CULTURE TEAM
Day 46, all diet groups were represented, along with field-
collected fish, at each sampling interval. Following the final
sampling on Day 46, the experiment was officially terminated.
Comparative acute cadmium toxicity bioassays were conducted
on three separate groups of newly-collected field Menidia, followed
by bioassays on each of the four feeding groups, to ascertain
comparative responses attributable to nutritional status. Ninety
(90) Menidia were exposed in each bioassay, 15 controls plus 15
exposed to each of five concentrations of cadmium. Concentrations
used were 30, 10, 3, 1 and 0.3 PPM of cadmium, and the bioassay
was conducted using filtered seawater at 14°C. Both large and small
fish were represented in the bioassays in all cases, both field
and experimental. The bioassay was conducted by Mr. John A. Cardin
of the Bioassay Methods Team.
A timetable of all fish sampling and bioassay assignments is
presented in Table 2. Large and small fish are indicated as L
and S, respectively, when measured. Animals with a total body
weight of 0.14-0.80 g were classified small, and those whose body
weight fell between 1.21-4.78 g were designated large. Total
body length measurements corresponded well and supported such .a
size classification.
The fish were collected from a different location (Narrow
River, Middlebridge, Rhode Island), which proved to be the only
feasible site which provided enough Menidia at this time
of year. The two size groups were intermixed in all field
collections made to provide animals for this experiment.
Statistical analysis of the data, when compared with respect to
fish size, revealed significant differences in the nutritional
condition of the animals. These differences are reflected both
in HSI and in the cadmium bioassay responses, appearing in both
field and lab-fed animals.
74
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Table 2. Timetable of Sampling Data for 1974 Menidia menidia Comparative Feeding Study — Numbers of
Fish Sampled.
Date Day Analysis Field I II III IV
10-11-74
0
Histology
HSI
15
25
(L)
(L)
—
—
—
—
10-17-74
6
Histology
HSI
10
25
(5L,
10S)
10
• 15
(14L, IS)
10
15
(12L, 3S)
10
15
(8L, 7S)
9
15
10-24-74
13
Cd bioassay
Cd bioassay
90
90
(L)
(S)
—
—
—
—
10-31-74
20
Cd bioassay
Histology
HSI
90
10
20
(S)
(10L,
10S)
10
15
(7L, 8S)
10
15
(9L, 6S)
10
15
(8L, 7S)
10
15
11-18-74
38
Cd bioassay
—
90
(L+S)
—
—
90
11-19-74
39
Cd bioassay
—
—
90
(L+S)
—
—
11-20-74
40
Cd bioassay
—
—
—
90
(L+S)
—
11-25-74
45
Histology
HSI
11
20
(10L,
10S)
—
—
—
—
11-26-74
46
Histology
HSI
—
10
15
(10L, 5S)
10
15
(10L, 5S)
3
14
(10L, 4S)
—
90 (L+S)
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MARINE CULTURE TEAM
III. RESULTS
A. Laboratory Feeding — Gross Observations and Pathology
During the first week of laboratory feeding, a number of
deaths occurred amont all diet groups from 5 to 7% of total number
by group. Their distribution was evenly scattered throughout
all eight tanks. The lest of these mortalities occurred on Day
7, after which no further mortalities occurred in Groups I, II and
III. These initial deaths were attributed to stress factors
due to collection, transfer and failure to acclimate. Interestingly,
all of these were fish of the larger size range, suggesting that
larger Menidia are more sensitive to handling than smaller ones.
A consistent pattern of gross lesions occurred in these fish,
generally preceded by a uniform body hypermelanism, usually visible
the day before the animal was found dead or moribund. These lesions
consisted of fin necrosis, commencing with the caudal fin and
progressing anteriorly, sometimes including dorsal and pectoral
fins. Ventral and opercular subcutaneous ecchymoses were often
present in conjunction with the fin lesions.
Vibrio spp. in an opportunistic pathogen sometimes present
in the raw sea water supply pumped from Narragansett Bay. There
may have been a possibility that the more severely stressed Menidia
were more susceptible to the invasiveness of Vibrio anguillarum,
resulting in their ultimate death by vibriosis. Since Vibrio is
invasive through a dermal route of entry, slight handling abrasions
would favor such an infection. However, no bacteriological
isolations were made on affected Menidia to recover the organism,
a positive diagnosis of death by vibriosis can not be made, but
it is a logical supposition.
Following the initial mortalities, which totaled 48, no
further mortalities occurred during the entire experimental period
in any of the lab-fed groups, I, II and III. Behavioral observations
revealed them to be active and healthy-looking, with retention of
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MARINE CULTURE TEAM
schooling behavior. No gross lesions of any kind were observed.
Both large and small fish appeared equally healthy on visual in-
spection .
Beginning on Day 31, the Group IV fish, which were unfed,
began a gradual, consistent pattern of mortality, displaying
characteristic gross signs of piscine starvation. These signs
included a general appearance of emaciation, a sunken-in antero-
dorsal region, and a pinched, jaundiced abdomen. Concurrent with
these signs of malnutrition were hypermelanism and fin necrosis.
Ulcerative lesions were absent.
A distinct behavioral change was noted in the Group IV
animals by the fourth week. Schooling behavior of the fish had
disappeared, even after attempts to deliberately startle them.
The animals swam slowly and sluggishly in random directions, and
some were observed to "rest'1 on the bottom at times, until prodded.
It was observed that the larger Menidia were the first to
die, followed by the smaller ones. Termination of the experiment
on Day 46 was mandated by the virtual complete mortality of Group IV
animals. A total of 47 Menidia in Group IV died during this period.
Table 3: Mortality and Temperature Daya — 1974 Menidia menidia
Feeding Study (October 12, 1974 to November 26, 1974).
DIET GROUPS
Ambient
Seawater
Day Temperature I II III IV
1 15.0°C — — — 1
2 15.0 5 2 1
3 14.0 5 6 6 5
4 16.0 13 1 2
5 16.4 2 1 — 2
6 15.5 — 1 2 1
7 15.0 1
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MARINE CULTURE TEAM
TABLE 3 (Cont'd)
Ambient
Seawater
Day Temperature
GROUP DIETS
II III
IV
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
14.5°C
14.0
13.5
15.0
13.5
13.7
12.5
12.0
12.5
12.0
13.0
14.5
15.4
15.5
12.5
12.5
13.0
13.0
12.8
12.5
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
1*
3
1
5
6
3
78
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MARINE CULTURE TEAM
TABLE 3 (cont'd)
Ambient
Seawater
Day Temperature
DIET GROUPS
II III
IV
36 11.5°C
37 11.0
38 11.8
39 10.8
40 10.8
41 11.0
42 10.8
43 9.5
44 9.0
45 9.2
46 9.0
Cumulative Mortality 14
Time of''Occurrence Week 1
*This Group III animal was found dead next to an unidentified
sharp metal object on the tank bottom. A sharply-defined, acute,
peduncular, ulcerative lesion, accompanied by swelling, tissue
edema and hemorrhage (ecchymosis) supported the diagnosis of
accidental death resulting from peduncular concussion, ruling out
a nutritional or infectious etiology.
B. Hepatosomatic Index (HSI) and Other Fish Measurements
The results of the 1973 experiment indicated that for that
population of Menidia, four parameters measured gave a sensitive
and consistent indication of nutritional status, these being total
body weight, liver weight, HSI and liver glycogen determination.
In 1973, these parameters followed the same pattern and could be
correlated directly with the animal's mode of feeding. Laboratory-
79
14
5
4
2
13 10 59
Week 1 Week 1 Week 1 (12)
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MARINE CULTURE TEAM
fed Menidla showed considerably higher measurements for all four
parameters with time, followed by field-collected fish and then
laboratory-unfed fish. Surprisingly, among the 1973 animals, the
lab-unfed fish approximated the field- collected fish more closely
than the lab-fed fish.
Measurements conducted on the 1974 fish showed quite the
opposite results among field and lab-fed fish, both in overall
comparative dietary evaluations, as well as stress response patterns
apparently correlated with the size of the fish studied. Table 4
lists the mean measurements and HSI values for the 1974 field and
experimental Menidia. A graphical plotting of field animal total
body weights vs. time, comparing 1973 and 1974 field Menidia, is
presented in Figure 1. These data demonstrate the three distinctly
separate aize groups represented over the course of the two studies.
It can be seen that the 1973 field Menidia were of an intermediate
size range from the two Menidia sizes collected in 1974. Figure 2
is a graphical plotting of 1973 and 1974 field Menidia liver
weights vs. time. Figure 3 represents comparative 1973 and 1974 liver-
to-body weight ratios (HSI) vs. time, and it demonstrates that
both sizes of Menidia collected in 1974 had much higher HSI
values than the 1973 Menidia. The 1974 HSI values for large and
small fish were both increasing gradually with time, compared to
those of the 1973 fish, whose HSI values remained relatively
static. All the 1974 field and experimental animal and 1973 field
animal HSI values are plotted similarly in Figure 4. Relatively
higher HSI values are noted in all groups among the smaller fish,
when contrasted to those of the larger fish. It is also apparent
that, when both 1973 and 1974 field-collected Menidia are considered,
the HSI's of all three experimental diet groups fall within the
range of the HSI values exhibited by the field Menidia (excluding
the unfed groups, particularly the large animals, whose HSI values
80
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Table 4. Mean Measurements and IISI Values — 1974 Menidia menidia
Feeding Study — 6^ Weeks' Duration
Time
(Weeks)
Sample
Total Body Ugts(g.)
Large Small
¦
Liver I'gts. (mg. )
Large Small
HSI(%)
Large Small
0
Field control
2.87
—
46.9
1.66
—
1
Field control
3.18
0.39
60.8
18.0
1.93
4.57
Diet I
2.66
—
42.6
—
1.66
—
Diet II
2.53
0.49
34.8
15.7
1.38
3.26
Diet III
2.75
0.43
42.0
14.0
1.56
3.25
\
t
Gp. IV Unfed
2.27
0.46
31.1
11.1
1.37
2.46
- —— —-
•
3
1
Field control
2.18
0.61
66.7
32.5
3.07
5.33
Diet I
3.05
0.48
54.1
22.0
1.84
4.50
Diet II
2.88
0.42
41.3
15.8
1.44
3.79
Diet III
2.78
0.34
63.0
13.8
2.26
3.86
\
t
Gp. IV Unfed
2.71
0.28
29.2
5.1
1.06
1.89
C
)J5
Field control
2.84
0.44
114.0
25.8
4.05
5.81
Diet I
!
3.05
0.61
50.3
31.1
1.64
3.37
Diet II
2.75
0.41
31.5
13.9
1.14
3.37
Diet III
2.74
0.55
67.1
27.1
2.48
4.93
Gp. IV Unfed
1
All dead by this t:
!
A
\
V
7
81
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FIGURE 1. 1973/74 MEAN TOTAL BODY WEIGHTS
4.0
3.5
3.0
2.5
oo
ro
2.0
1.5
1.0
0.5
DAY
1974 Large
Field Controls
'• —
• 1973 Field Controls
•
1974 Small
Field Controls
-------
Mg
180
170
160
150
140
130
120
110
110
90
80
70
60
50
40
30
20
10
DAY
FIGURE 2. FIELD CONTROLS 1973 and 1974 - MEAN LIVER WEIGHTS (mg)
1974 Large
Field Controls
__ — 9
1974 Small
Field•Controls
-------
RATIO (%)
9
8
7
6
5
00
¦C~
4
3
2
1
DAY
FIGURE 3. FIELD CONTROLS 1973, 1974
^ ^ tiiit ^
14
21
1974 Small
Field Controls
1974 Large
Field Controls
® 1973 Field Controls
J-L
28
35
42
45 46
49
-------
7
6
5
4
3
2
1
8
6
4
2
FIGURE 4. ALL 1974, 1973 FIELD CONTROLS (MENIDIA) - MEAN HSI (LIVER/BODY WEIGHT RATIOS)
_ — O
— ^ °
o-
Sm Field Cont.
'74
o—o
Diet I Sm
0-~ a
Diet III Sm
A- —A
Diet II Sm
Lg Field Cont.
'74
Diet III Lg
)( -)(
Gp IV Unfed Sm
Diet I Lg
'73 Field Cont.
Diet II Lg
I 1 I GpfIV Unf6d Lg
14
21
28
35
42 45 46 49
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MARINE CULTURE TEAM
would continually decline with starvation).
The striking differences in field animals HSI's for 1973 and
1974 may be readily contrasted in Table 5 and indicate rather
marked variation in nutritional status of field animals alone,
manifested both in different year-values and among different
sizes of Menidia. With such a marked variation in the naturally
occurring field animal HSI, it is difficult to assess exactly
the nature of a standard HSI range for laboratory-fed animals,
since some field animals could conceivably be undernourished in
their natural habitat. This condition appears probable for the
1973 field Menidia.
TABLE 5: Mean Liver: Body Weight Ratios (HSI) for 1973 and 1974
Field Animals — 1974 Menidia menidia Feeding Study
Time 1974
(weeks) 1973 Small Large
1 1.6 4.57 1.93
2 1.3
3 1.8 5.33 3.07
4 1.5
6-1/2 — 5.81 4.05
1973 Week 0
1974 Week 0 -
C. Acute Cadmium Toxicity Bioassays
Detailed results of the acute cadmium toxicity bioassays are
reported elsewhere by Mr. John A. Cardin of the Bioassay Methods
Team.
D. Histological Examination
The results of the histological samplings are pending completion
of these examinations by Mr. Paul P. Yevich of the Histopathology
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MARINE CULTURE TEAM
Unit.
IV. DISCUSSION
The results of this study are incomplete at this time, but
several points merit discussion, and some conclusions may be drawn
on the basis of what is presently known.
The initial feeding study observations indicate that a period
of seven days elapsed before handling mortalities ceased, suggesting
a one-week acclimation period is desirable prior to experimentation
with Menidia. In addition, the larger-sized Menidia appear more
sensitive to handling stress than the smaller Menidia. This
finding is supported by Cardin's personal observations during the
cadmium bioassay, in which he found the larger Menidia to be less
adaptable to the experimental conditions, among both field and
control fishes.
The use of HSI values as a measurement for condition shows a
striking range among field animals alone. Reference to Figure 3
reveals a field variation in mean HSI ranging from a low point of
1.6 in 1973 to 5.81 in 1974, with smaller Menidia exhibiting
consistently higher HSI values than larger Menidia, both in the
field and under experimental conditions. Comparative HSI values
depicted in Figure 4 indicate all laboratory-fed groups to fall well
within this range. It has yet to be established definitively which
HSI indicates a negative energy status, but it would appear to
be in the range of 1.0 or thereabouts.
Acute cadmium toxicity bioassays were conducted by John Cardin
of the Bioassay Methods Team. Results of his work, pertinent to
culture, include (1) varying response differences, particularly
among different sizes of Menidia, (2) laboratory-fed and field
fish were comparable in responses, (3) the group of larger Menidia
appeared to be more sensitive, whether laboratory-fed or field, and
(4) within a particular size grouping, field fish were more
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MARINE CULTURE TEAM
sensitive than laboratory-fed fish. The Bioassay Methods Team
plans further bioassays to correct for possible handling mortalities.
Current findings indicate this may have influenced toxicity
results. While the laboratory-fed fish had been laboratory-
acclimated for approximately one month prior to their bioassay,
the field-collected Menidia used were bioasaayed virtually
immediately upon collection. Therefore, what appears to be a
lower resistance to cadmium among field fish may in fact be a
manifestation of failure to acclimate. It is proposed to correct
for this by holding both field and laboratory-fed fish without
feeding for a period of seven days prior to their bioassay, to
eliminate acclimation stress among the field populations tested.
The results of the histological examinations may indicate
subtler differences in condition among laboratory-fed and field
animals, particularly among different diet groups. Hopefully
they will be supportive of the conditions implied by the comparative
HSI values, particularly with respect to liver and pancreas morph-
ology, as well as the occurrence of visceral fat.
Electronics Group
Jay Sinnett, Electronics Engineer, GS-12 joined the NMWQL
staff in November. An Electronics Group was formed and Ray High-
land assigned with Jay as Electronics Technician.
Major duties of this group include (1) maintenance and repair
of existing electronic equipment in the laboratory and (2) design
and construction of specialized equipment for unique applications
in biological experimentation. This includes:
21AKF Task 02—Invertebrate culture-Temperature control unit
21AKF Task 12—Behavioral studies—Automated positioning
21AKF Task 61—Microcosm studies—Multiple bath temperature controller
Other projects in progress include:
21AKF Task 02G(2) Winter flounder culture - Automated light control
and dimmer switch
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MARINE CULTURE TEAM
21AKF Task 72—Behavioral studies—Light control integrated
with TV camera positioning.
21AKF Task 19— Contract 14-12-872
Through both contract and in-house work, a capability is
progressing for unmanned detection and telemetering of certain
marine parameters to a laboratory minicomputer, which in turn
will be capable of controlling these same parameters in experi-
mental tanks at a remote location. The progress during November
and December included emplacement of additional sensors for Tem-
perature, Dissolved Oxygen, conductivity, pH, and an experimental
sensor for turbidity.
Histology Group
The Histology Group accomplished a number of projects
for various research teams:
16AAT-12 Chronic cadmium exposure oysters Bioassay
21AKF 2F Nutrition study-Menidia (In progress) Culture
21BBG Ocean dumping Ocean Disposal
16AAT-11 Dissolved oxygen and cadmium effects Synergisms
16AAV Chronic oil exposure Oils
Two major reports were (1) Report to State of Maine on
Histopathological Findings in Soft Shell Clams From Searsport, Me.
Oil Spill site, (2) Histological Findings in Ocean Quahaugs From
Newport, R.I. Ocean Disposal Site. Results of these histological
findings are included elsewhere in reports of projects by the lead
research team involved.
Marine Culture Team, Non ROAP Activities
Allan Beck covered the subject area of impingement and
entrainment of organisms in power plant cooling water systems
at an EPA lawyers' seminar organized by Dr. Prager and held at
NMWQL, Narragansett. Subject area treated was directed towards
biological considerations in 316(a) & (b) guidelines to implement
provisions of water law PL 92-500.
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MARINE CULTURE TEAM
Several publications resulted from technical assistance to
EPA Headquarters, recommending marine water temperature require-
ments and guidelines for enforcing 316(a) and (b) provisions of
PL 92-500. These are listed in the following sections.
Manuscripts and Publications
MARINE CULTURE TEAM
Beck, A.D. and D.C. Miller. 1974. Analysis of inner plant
passage of estuarine biota. Proc. ASCE Power Div. Spec.
Conf. Boulder, Colo. August 12-14, 1974. pp 199-226.
Beck, A.D. and N.F. Lackie. 1974. Effects of passing marine animals
through power plant cooling water systems. Presented as Symp.
Effects of Nuclear Power Plants on the Ecosystem. Am. Fish Soc.
Ann. mtg. Honolulu, Hawaii. Sept. 7-11, 1974.
Miller, D.C. and A.D. Beck. 1974. Development and application of
criteria for marine cooling waters. Proc. IAEA/ECE Symp. Phy-
sical and Biological Effects on the Environment of Cooling
Systems and Thermal Discharges at Nuclear Power Stations Oslo,
Norway, August 26-30, 1974.
MacPhee, G.K. 1975. Synopsis of biological data on the summer
flounder, Paralichthys dentatus. Fisheries Bulletin. NOAA,
National Marine Fisheries Service. In press.
HISTOLOGICAL GROUP
Rinaldo, Ronald G. and Paul Yevich P. 1974. Black Spot Gill
Syndrome of the Northern Shrimp Pandalus borealis. Journal of
Invertebrate Pathology, Vol. 24, pp 224-233.
Gardner, George, Paul Yevich, Margaret James, J.C. Prager; 1974.
The Microscopic Perils of Marine Pollution. Underwater Naturalist,
Vol. 8, #4. pp 15-19.
Betzer, Susan B. and Paul Yevich. 1974. Copper Toxicity in
Busycon Canaliculatum L. The Biological Bulletin.
Voyer, R.A., Paul Yevich, and Carolyn Barszcz. Alterations in the
Response Pattern of Cadmium-exposed Mummichogs Fundulus heteroclitus
_L. (Submitted for publication to Water Research)
Publications and Workshops
Allan Beck - October 4. Presentation of talk Biolofiical Effects of
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MARINE CULTURE TEAM
Power Plants on the Marine Environment. Interstate Seafood Seminar
Atlantic Beach, N.C.
Paul P. Yevich - July 10 Mr. Yevich went to Washington, D.C. to
give a paper entitled Ovarian tumors in the quahog and mesenchymal
tumors in the softshell clam at the Interagency Collaborative Group
on Environmental Carcinogenesis.
Sept. 20 Mr. Yevich gave a Seminar on The histopathologic effects
of water pollutants on marine life to the Scientists at the Edgewood
Arsenal in Edgewood, Maryland.
Sept. 23 Mr. Yevich was in Tampa, Florida for a pre-hearing meeting
in the Belcher Oil Co. proposed refinery and tanker terminal
complex.
Dec. 3-4 Congerence/Workshop, Marine environmental implications of
off-shore drilling in the Baltimore Canyon region of the Mid-Atlantic
coast, at the Center for Adult Education, University of Maryland,
College Park, M.D.
Dec. 5-7 Attended the Eighth Advanced Seminar in Clinical Ecology,
Denver, Colorado to give a talk on The histological effects of oil
pollutants on marine life.
Dec. 9 Gave a lecture at Woodward Hall, University of Rhode Island,
to Dr. Tarzwell's class on pollution.
•Dec. 17 Sat in on a grant proposal by Rita Colwell at EPA head-
quarters, Washington, D.C.
Dec. 17 Consulted with pathologists at the Armed Forces Institute
of Pathology, Washington, D.C. about soft shell tumor slides.
Dec. 18 Attended the meeting of the Working Group on Aquatic
Carcinogens, National Institutes of Health, Bethesda, M.D.
Aug. 26-30 Carolyn Barszcz attended the American Society of
Clinical Pathologists workshop on Histological Topics in
Chicago, ILL. Aug. 26-30.
91
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METALS TOXICITY TEAM
The Metals Project is devoted to realistically assessing metals
as a problem in the marine environments. Field methods for deter-
mining metal levels in water, sediments, pore water, and biota are
being developed and standardized for investigation of problem areas
as they are identified. A matrix of existing toxicity and body burden
data using animal species (including various life stages) as one
axis and metals (including various species, chemical states, and
modes of application) as the other is being created as a secondary
goal by the Metals Project. The matrix will define the data base
currently available for criteria decision making; it will point out
information gaps in animals, their life stages, and metals and their
various states, thereby defining needed research goals; and it will
provide a basis -for comparing metal levels and their modes of
application in laboratory toxicity studies with levels and pathways
defined in metal-problem-areas in the natural environment.
Metal levels and their natural "modes of application" (chemical
states and combinations in seawater and food chains) derived from
field studies will be applied to laboratory bioassays through close
coordination with the Bioassay Systems Project. The results of this
approach will be to assess, broaden, and validate the data base
needed for metal criteria decision making.
ROAP 16-AAT (Previously 21-AKF - Task 21, 23): Criteria for heavy
metals to protect estuarine and marine life.
ROAP OUTPUT: To determine the sources, fate and effects of heavy
metals discharged into estuarine and coastal areas in order to
recommend maximum allowable concentrations of metals that are not
hazardous to marine biota or man via marine food chains.
I. ROAP Approach 16 AAT, All Tasks except 018: Construct matrix of
toxicity data for metals and aquatic biota available from literature,
using in-house research grants and other sources.
A. Metals Matrix
A matrix of existing toxicity and body burden data using
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METALS TOXICITY TEAM
marine species (including various life stages) as one axis and
metals (including various forms, chemical states and modes of
application) as the other has been formulated by Marcia Barry,
George Hartson, and Earl Davey. The matrix to date consists of
information from the Metals Bibliography, Water Quality Criteria
1972 prepared by the National Academy of Sciences, in-house bio-
assays and experiments and more recent literature. We are now in
the process of attempting to computerize this data base and
continuously update this information with the help and cooperation
of Don Worley and Joe Wilson of Data Systems Division, Office of
Administration, US-EPA, RTP, N.C.
The metals matrix indicates that there is information on
only 36 elements out of a possible 104. Of these, only 18 have
toxicity data listed and of the 18, only Hg is sufficiently docu-
mented to perhaps formulate good criteria. Consequently, the metals
matrix is now being utilized to suggest metals and their respective
forms to be applied to in-house testing by the Bioassay Team.
B. Metals Bibliography
Dr. Eisler and student aide Maryjane Wapner are preparing
Volume 2 of "Annotated Bibliography on Biological Effects of Metals
on Aquatic Environments". A total of 725 references have been
collected on toxicological, physiological and metabolic influence
of stable and radiolabeled chemical species of metals to marine,
estuarine, and freshwater fauna and flora. References are annotated
and each is indexed by metal, taxa, and by author in cumulative
indices which encompass the present report and the initial report
in this series (published as EPA Report R3-73-007). The manuscript
is now complete and a contract has been let for final typing. A
target date of April 1 has been set for submission of the completed
work to the Director, NMWQL. Publication will be in the US EPA
Ecological Report Series.
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METALS TOXICITY TEAM
II. ROAP Approach 16 AAT - Task 018: Formulate and implement
biogeochemical laboratory and field investigations for marine waters
impacted by high metal use point sources.
A. Biogeochemical impact of Quonset Point Naval Air Rework
Facility (NARF) on Narragansett Bay, Rhode Island.
All marine samples taken during the autumn of 1973 from NARF
have finally been completely analyzed for selected heavy metal
concentrations by atomic absorption and/or neutron activation
analysis. Statistical and graphical analysis beyond the preliminary
observations summarized in the January-June 1974 semi-annual report
are currently being prepared for publication. Highlights of our
results are as follows:
1. Concentrations of Ag, Cd, Cr, Cu, Ni, Pb and Zn in acid-
soluble components of sediments were highest at stations nearest the
NARF discharge outfalls and lowest at the more distant stations.
Some metals are distributed homogenously throughout the 50 cm core
(Cd, Co, Fe, Mn), but all others concentrated in the upper (0-5 cm)
sediment component.
2. Highest values recorded for interstitial waters from the
study are in ug11 (ppb) 7048 for Mn, 2351 for Zn, 559 for Fe, 55 for
Pb, 46 for Hi, 44 for Cu, and <1.0 for Cd. Content of Mn, Zn,
and Cu in extracted pore waters were significantly higher in upper
(0-5 cm) than lower fractions; Pb, Ni and Fe were distributed
homogenously.
3. Concentrations of selected metals in yg/2. (ppb), from
bottom waters ranged from 0.1 to 0.38 for Cd, 0.10 to 0.41 for Co,
2 to 5 for Pb, 0.13 to 1.94 for Cu, 0.82 to 3.88 for Zn, 0.14 to
5.81 for Fe, 0.75 to 7.96 for Mn, and 1.07 to 9.08 for Ni. These
bottom water values were 1 to 3 orders of magnitude lower than similar
data for interstitial sediment water and 3 to 7 orders of magnitude
lower when compared to NARF sediment data.
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METALS TOXICITY TEAM
4. Data on elemental composition of biota from the NARF
study area are now complete. These data will be analyzed and
interpreted in terms of the surrounding sediment and water data.
In connection with the NARF study, Mr. Greg Telek reports
neutron activation analysis has been completely automated with the
application of automatic sample changers coupled either to a tape deck
or disc for data storage. Data reduction is done by a computer
program which corrects for flux variations, decay and gives the
final results in ppm for five elements. The program can be readily
altered to determine any number of elements.
Two separate systems are available for the counting of two
samples simultaneously on a continuous 24 hour basis. Therefore,
as many as 96 sediment samples or 48 biota samples could be analyzed
daily for at least 7 elements (assuming 1 hour count for biota and
1/2 hour for sediment).
The interferences involved in Zn determinations in sediment
have been determined. Sc, Ta, and Europium have interfering peaks
whose primary peaks can be used to subtract their respective
additions to the Zn peak. An experiment design utilizing Eu, Sc,
Ta spikes of orchard leaves has been formulated for evaluating
the accuracy of eliminating the interference of these spikes in Zn
determinations.
A manual for neutron activation analysis is being completed.
B. Mr. Bickford, a part-time employee of the NMWQL and a
geochemistry Master of Science candidate at Brown University, is
continuing on a project to establish whether chromium can be used
as a flag for organic pollution from sewage treatment plants in
Narragansett Bay. He has completed a series of analyses on seston
(material which settles out of the water column) samples which were
found to contain 5-12% organic matter and a carbon to nitrogen ratio
of 6 to 8 irrespective of where the samples were collected in the
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METALS TOXICITY TEAM
bay. Total chromium analyses of the seston indicated a clear gradient
with values ranging from 60 ppm dry weight in the relatively clean
lower bay to 350 ppm in the highly impacted upper bay. A peroxide
(H202) digestion, which only oxidizes organic matter in the samples,
was found to release 30% of the chromium from lower bay samples,
60% from mid-bay and 90% from upper bay samples. These results
tentatively indicate that Cr could be used as a tracer of organic
pollution from upper to lower bay stations.
In a related project, Mr. Bickford has developed a technique
to partition between the plus-three and plus-six redox states of
chromium in seawater. Plus-three chromium is taken up on the
chelating exchange resin, Chelex-100; whereas, plus six chromium is
taken up on tin (SnCl2) impregnated Chelex 100. The efficiency of
the resin uptake and elution was verified to be 99% by using the
chromium radio-isotopes. 51CrCl3 and Na2 51CrO^.
Seawater samples were also taken at Mr. Bickford's stations
by E. Davey and A. Soper for dissolved and particulate trace metal
analysis of eight metals of both 0.5 m from the surface and 10 cm
from the bottom. In general, all metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb
and Zn) particulate and/or dissolved forms, were found to decrease
with distance from the highly impacted upper end of Narragansett
Bay.
EPA Grant No. R804000-01-1
Title: History of heavy metal pollution in estuaries
Investigator: Dr. Edward D. Goldberg, Scripps Institute of
Oceanography, LaJolla, California
Earl Davey, Richard Lapan, and Tom Bickford of the NMWQL participated
with Eric Gamble and Kenneth Bruhland of Scripps Institute of Oceano-
graphy in the selection and collection of sediment box cores from
Narragansett Bay during the last week in August 1974. Dr. Goldberg,
in a recent quarterly report, stated that they have completed Pb210
geochronology on one Narragansett Bay core which showed a sedimentation
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METALS TOXICITY TEAM
rate of about 1 cm/year and an accumulation of shells that coincides
with the 1954 hurricane. Analysis with depth were completed for
cobalt and silver which show that silver is accumulating at faster
rates now than 50 years ago while there were no enhanced cobalt fluxes
to the sediment. They are now in the process of accumulating data
on plutonium and other heavy metals.
Manuscripts
Eisler, R. 1973. Latent effects of Iranian crude oil and a chemical
oil dispersant on Red Sea molluscs. Israel Journal of Zoology
22:97-105.
Eisler, R., G.W. Kissil, and Y. Cohen. 1974. Recent studies on
biological effects of crude oils and oil dispersant mixtures to
Red Sea macrofauna. In Proceedings of Seminar on Methodology for
Monitoring the Marine Environment held in Seattle, Washington,
Oct. 1973. U.S. EPA Report 600/4-74-004, pages 156-179.
Phelps, D.K., G. Telek and R.L. Lapan, Jr. 1973. Assessment of
Heavy Metal Distribution within the food web. Proceedings of
the second Int'l Symposium on Marine Pollution, San Remo, Italy.
Presentations
Davey, E.W. and A. Soper. Apparatus for the In-situ concentration
of trace metals from seawater. Symposium in Analytical Methods in
Oceanography sponsored by the Analytical Division of the American
Chemical Society. Atlantic City, New Jersey, Sept. 8-13, 1974.
Portions of the paper submitted will be published in the A.C.S.
Advances in Chemistry series. Other parts have been submitted for
publication to Limnology and Oceanography.
Miscellaneous
Dr. Ronald Eisler ahs been invited to present "Toxic, sublethal,
and latent effects of petroleum to Red Sea macrofauna" at the 1975
International Conference on Prevention and Control of Oil Pollution,
jointly sponsored by EPA, API, and the USCG. The proceedings of the
conference will be published.
97
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METALS TOXICITY TEAM
Dr. Eisler received a letter of commendation from A.C. Trakow-
ski, Asst. Administrator for Research and Development, for his
participation in the Section 307 (a) hearings of PL 92-500, involving
water quality criteria for mercury, cadmium, and other metals.
Dr. Eisler participated in an EPA Technical Workshop held
at Gulf Breeze, Florida, August 6-8, and assisted in the preparation
of the report "Physical, Chemical and Biological Considerations with
Respect to the Dupont Permit for Ocean Dumping of Industrial Wastes".
Dr. Davey participated in a pre-hearing on the possible ocean
dumping of arsenical-wastes in EPA Region II by Whitmoyer Laboratories
subsidary of Rohm and Haas at EPA Headquarters in Washington, D.C.
on Sept. 5, 1974.
98
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OCEAN DISPOSAL TEAM
ROAP 21 BBG - Ecological Assessment of Ocean Dumping
This ROAP is designed to establish criteria for the survey
and ecological assessment of existing ocean disposal sites as well
as provide the information necessary for the appropriate selection
of new dump sites. Emphasis is on establishing criteria for monitoring
sites and adjacent areas for ecological compromise, investigating
routes, rates and mechanisms for recycling of materials from dis-
posal sites through biological uptake as well as by physical and
chemical processes, establishing standard bioassay technology.
ROAP 21 BBG, Task 2 - The Movement of Trace Materials from Sewage
Sludge—Rates, Routes, and Mechanisms into the Biota
This study is being implemented by an interagency agreement
with Dr. Frank Lowman of the AEC Nuclear Science Center at Mayaquez,
Puerto Rico. Contaminant fluxes from secondary sewage sludge including
uptake and transfer by biota are being studied in flow-through
seawater systems. This study is proceeding. Secondary sludge
from New York City treatment plant is being used. Prime emphasis
is on metals, but hydrocarbons and pesticides will be considered
also.
ROAP 21 BBG, Task 3 - History of Metal Pollution in Estuaries
Dr. E. Goldberg of Scripps Institution of Oceanography is
studying contaminant impacts in historical context. Dr. Earl Davey
is project officer and he reports on this grant in the Metals Team
section.
ROAP 21 BBG, Tasks 4, 8 - Bioassay Methods Development
These tasks are being implemented by ROAP 21 AKK (Techniques
for Water Quality Criteria Development), Task 115 (Bioassay Methods
for Ocean Dumping). Dr. Gentile's team cooperated with Dr. Tom Duke,
Director of the EPA Gulf Breeze Environmental Research Laboratory,
to compile a manual of standard bioassay techniques for the ocean
dumping permit program. Dr. Gentile was involved directly with
99
-------
OCEAN DISPOSAL TEAM
several Regional permit actions, most notably Region Ill's permit to
Dupont for dumping acid wastes.
ROAP 21 BBG, Task 5 - Standard Site Monitoring Methods
Three representative sites are being studied to develop site
monitoring methods. These include a dredge spoil site, an industrial
wastes site, and a sewage sludge site.
The industrial wastes (Dupont acid wastes) and sewage sludge
(Philadelphia) sites offer a unique opportunity to test the concept
of analytically "finger printing" a waste material (Fig. 1). Both
wastes may be distinguished by their metallic content. In the last
semi-annual report we reported data from a monitoring cruise made in
the fall of 1973. We found that metals were accumulating in the
mahogany clams, Arctica islandica, in and around both dump sites.
The metals accumulating were those being dumped at one or the other
sites. The distributional patterns of accumulation reflected site
sources. The concept of using metal "tags" or "tracers" to follow
the fate of dumped wastes was substantiated. In this report we
include data from a monitoring cruise made in the spring of 1974.
Metal levels were measured in mahogany clams, Arctica islandica
(Table 1) and sea scallops, Placopecten magellanicus (Table 2).
These data conclusively confirm our initial findings. Figures 2
thru 7 illustrate patterns of accumulation of vanadium, cadmium,
and zinc in both species of shellfish found in the vicinity of these
dump sites. These metals serve as useful tracers for the dumped
materials. Four metals characteristic of the Dupont waste (Fe, Mn,
V, and Ti) and five metals characteristic of the Philadelphia
sludge (Cu, Ag, Ni, Cd and Zn) showed statistically significant
distribution patterns in the spring 74 data (Tables 3 & 4). Some
of these tracers displayed elevated concentrations in areas extending
from the dumpsites to the limits of the area covered by the spring
cruise. Vanadium levels in the tissues of the clam, Arctica is-
landica, and the scallop, Placopecten magellanicus. clearly show
100
-------
OCEAN DISPOSAL TEAM
this pattern (Figures 2 & 3). Presumably these animals were exposed
to Dupont wastes. Prevailing currents sweep wastes southwest from
the dumpsite. Significantly, vanadium tissue levels remain ele-
vated at the farthest station southwest of the Dupont site. The
spring 74 cruise was not extensive enough to permit delineation of
the area influenced by Dupont's wastes. A future cruise will extend
the sampling area in an effort to delineate the total area of effect.
ROAP 21 BBG, Task 6 - Dredge Spoils and Sewage Sludge in the Trace
Metal Budget of Estuarine and Coastal Waters
Dr. James Simpson of Columbia University has a grant to develop
field methods for assessing metal fluxes from natural sediments.
Simpson's approach has been "to build up the components which are
necessary to make trace metal budget calculations, based largely on
field measurements in the Hudson Estuary and adjacent coastal
waters." Initially he "employed natural tracers which have less
complicated chemical pathways than trace metals, to establish some
of the parameters needed to compute trace metal fluxes from sediments."
These tracers include radon , methane, chloride, Ra and Ra228.
These measures are being correlated with water column and sediment
metal concentrations in the estuary and bight areas.
ROAP 21 BBG, Task 14 - The Problems of Ocean Dumping — Stability
and Resiliency in Experimental Ecosystems Exposed to Constant and
Time-Varying Stresses
This task is being implemented by a grant awarded to Dr.
Scott Nixon of the Graduate School of Oceanography, University
of Rhode Island. This project represents a cooperative effort
between Dr. Kenneth Perez of this lab and Dr. Nixon. Dr. Perez
reports details of this study in his section of this semi-annual
report.
ROAP 21 BBG, Task 15 - Influence of Dredge Spoils and Sediment
Pollution on Trace Metal Assimilation by Organisms
101
-------
OCEAN DISPOSAL TEAM
Dr. Michael Bender of the Graduate School of Oceanography,
University of Rhode Island has a grant to develop methods to assess
metal fluxes between sediments and overlying water in controlled
laboratory simulations of ocean dumpsites. At this point emphasis
is on analyzing pore waters, for metals content. As the expertise
develops the complexity of the system studied will be increased
by including macrobiota.
Technical Assistance
During the period July - December 1974 the Ocean Disposal
Team responded to assistance requests from Regions I, II, III, IV,
VI, and X. We would like to acknowledge particularly Dr. Jan Prager's
assistance with our efforts to support Region III. He served on
review panels for two important public hearings on ocean "permit
applications.
Publications
Lear, Donald W. and Gerald G. Pesch. Effects of Ocean Disposal
Activities on Mid-Continental Shelf Environment off Delaware
and Maryland. Environmental Protection Agency publication
series number 903/9-75-015. January 1975.
102
-------
riGURE 1
a p-.*— a g— Ti ion/
MtltM ur OIUU1
70*
75*
40*
GREAT COG
H&RBOR INIET
BALTIMORE
DELAWARE
BAY
WASHINGTON DC
HENLOPCN
74* 25' 74' 15
REM080TH
BEACH
IVOIAN RIVER
inlct
38 35'
30' 30*
^8*25
OCEAN
City
38 20
74* 20' 74* 10*
CHINCOTCAGUE
inlCT
PC CHARLES
CAPE HENRY
77*
76*
75"
74'
SCALE IN miles
10 20 iO 40 50
103
-------
OPERATION " IDES"
ARCTIC A
AETAL CONCENTRATION
VANADlUi'/,
ppm c!ry wt
7.9
©II
811.2
©13.5
©21.5
®I9.8
•14.7
• 5.5
©19.5
9.4
21.4
38* 00
104
-------
riGURE
©17.5
• 14.7
OPERATION "IDES"
SCALLOP
4ETAL CONCENTRATION
VANADIUM
ppm dry wf
©27.7
®31.2
R "D'
•40.7
044
•23.1
®48.6
21.3
©58.7
38" 00
105
-------
FIGURE
©2
OPERATION "IDES"
ARCTICA
METAL CONCENTRATION
CADMIUM
ppm dry wt
©1.6
• 2.8
<91.6
©I.S
©2.4
• 2.4
®4.0
©2.0
•1.7
5*
38* 00
106
-------
•10.1
OPERATION "IDES"
SCALLOP
METAL CONCENTRATION
CADMIUM
ppm dry wt
37.5
011.4
• 10.8
R "D'
OI4.7
©19.5
alO.y
©12.0
•15
®22
24.9
•10.8
55.8
107
-------
IGURE
OPERATION "IDES"
ARCTICA
ETAL CONCENTRATION
ZINC
ppm dry w\
117.0
128.0
|90.5
©107.0
38' 30
®92
®lll.0
069.35,
©88,
©92.Q.
• 101.0
o90.
100.0
38* 00
108
-------
©32
OPERATION "IDES"
SCALLOP
ETAL CONCENTRATION
ZINC
ppm dry wt
®38
«46
«4I
127
•58
OI08
118
oil
©73
elOl
©99
0116
53
38' 00
1U9
-------
Table 1
OPERATION "IDES"
Metal Concentrations in Arctica
ppm/dry wt.
Sta/Reps.
V
Zn
Cd
Ag
Cu
Cr
Mn
Co
Ni
Ti
A1
Fe
Pb
2
2)
14.66
128.5
2.52
1.53
9.55
4.13
9.63
1 .09
11.59
6.55
103.4
438.6
6.11
9
10)
26.46
114.4
1.73
2.33
9.73
3.99
19.89
1.07
6.63
5.20
125.4
354.7
3.61
14
4)
8.18
92.1
2.78
1.11
7.04
2.95
9.39
1.17
13.36
2.54
40.6
264.3
6.08
17
10)
19.49
100.7
CO
0.85
7.89
3.65
14.03
0.83
6.77
3.53
38.0
320.0
4.92
20'
5)
10.95
127.7
1.77
0.76
9.67
4.92
26.09
1.32
9.56
3.49
60.1
562.1
6.47
P 22
/—s
10)
CO
(NJ
111.0
1.62
0.76
10.36
5.04
13.83
1.12
6.47
6.35
162.5
469.6
3.09
w
24
3)
16.20
104.5
2.36
0.69
8.02
4.09
5.20
0.87
9.11
1.62
36.3
142.9
2.41
25
10)
5.46
92.0
4.01
1.41
8.34
5.26
7.98
0.68
10.80
2.40
103.5
281.0
5.33
26
2)
21.44
90.0
^1
CO
0.58
8.85
5.17
10.96
0.57
4.21
4.79
87.6
290.6
3.44
27
10)
9.41
100.1
2.46
1.09
6.59
4.80
10.07
1.01
10.61
2.96
85.0
317.5
5.34
28
6)
7.47
152.4
2.08
1.77
11.31
5.67
16.95
1.56
11.73
3.01
48.6
261.3
3.57
29
5)
8.38
107.5
1.91
0.66
7.63
4.03
17.98
1 .23
8.85
3.36
65.0
288.5
4.14
A
4)
7.93
117.2
1.77
0.62
9.79
4.77
11.14
1.34
8.34
7.09
112.0
417.1
4.21
B
5)
13.46
107.3
1.61
0.73
9.55
6.07
22.37
1.06
7.79
20.38
341.1
562.4
4.51
-------
OPERATION "IDES"
Metal Concentrations!in Arctica (continued)
ppm/dry wt.
Sta/Reps. V Zn Cd Ag Cu Cr Mn Co Ni Ti A1 Fe Pb_
C ( 6) 11.23 90.5 1.48 0.83 8.85 5.18 12.85 0.72 7.35 7.20 121.6 480.4 5.89
E (7) 19.78 88.2 2.04 0.67 8.60 4.21 11.15 0.79 5.26 6.98 161.0 449.8 4.24
F (10) 5.86 89.4 2.41 0.65 6.64 4.02 10.61 0.95 10.53 8.77 183.5 492.3 3.54
-------
Table 2
OPERATION "IDES"
Metal Concentretions in Scallops
ppm/dry wt.
Sta/Reps
V
Cd
Zn
Ni
Cu
Cr
Ag
Ti
Mn
A1
Fe
Pb
Co
28
( 3)
14.66
10.06
31.59
1.10
4.79
1.28
0.46
7.35
17.11
144.7
575 7
1 .22
0.33
18
( 8)
27.69
11.44
45.52
1.91
5.16
2.76
0.71
17.27
41 .76
505.9
1409.7
2.45
0.65
20
( 2)
24.99
10.80
42.03
1 .65
5.63
2.01
1 .11
16.30
22.72
312.5
682.4
1.64
0.78
21
( 4)
31.22
14.21
40.50
2.06
5.25
2.90
0.64
8.68
29.26
202.8
1180.8
2.06
0.43
23
( 6)
30.75
19.55
57.77
4.34
8.30
2.08
1 .24
9.26
11.95
139.1
250.9
11.15
0.66
27
( 4)
21.29
55.88
53.43
1.35
6.28
3.26
0.43
9.19
22.83
204.1
1110.0
6.91
0.54
19
( 3)
17.93
37.51
38.50
1.06
4.27
2.85
0.39
4.98
32.17
151 .9
564.8
1.84
0.48
24
(10)
40.15
22.56
73.00
1.97
6.14
1.17
0.43
5.48
18.48
155.0
291 .1
1.54
0.58
25
( 2)
23.08
25.96
98.59
4.19
6.73
0.89
1 .13
6.21
12.86
126.5
243.4
3.73
0.76
30
( 3)
17.53
19.60
107.48
1.03
4.01
1.42
0.50
8.47
30.36
228.8
314.1
1.69
0.38
F
(10)
26.66
24.87
100.70
2.99
6.81
4.56
0.81
26.22
63.21
903.9
852.9
3.96
1.02
1 4
( 8)
27.13
59.28
95.90
1.89
6.69
3.12
0.48
12.00
32.76
617.1
299.3
1.89
0.65
2
( 5)
52.16
10.95
118.03
14.67
12.66
2.66
9.08
5.02
16.11
92.4
190.5
2.45
0.52
B
(10)
43.60
11.98
113.32
7.14
8.83
2.49
3.76
6.34
27.29
183.8
380.1
6.48
0.35
17
( 9)
48.56
15.12
121.71
6.00
8.81
4.19
2.15
10.97
38.77
774.9
342.1
3.99
0.41
26
( 5)
58.65
10.79
117.95
13.65
9.50
6.88
4.93
1.06
13.01
78.2
285.9
5.78
0.69
-------
OPERATION "IDES"
Metal Concentrations in Scallops (continued)
ppm/Gry wt.
Sta/Reps. V Cd Zn Ni Cu Cr Ag Ti Mn A1 Fe Pb Co
9 ( 3) 50.55 15.60 116.33 2.08 7.54 2.64 0.45 4.53 24.26 91.9 260.7 2.52 0.29
E (10) 44.12 11.08 108.47 2.64 6.31 3.31 0.58 6.03 28.81 200.5 400.1 2.80 0.48
22 (10) 40.65 14.70 127.35 6.33 8.63 4.05 2.96 6.82 26.88 166.3 249.8 1.83 0.45
M
M
LO
-------
Table 3
Analysis of Variance Results for Metal Levels
in Arctica islandica Collected in Spring 1974
in Vicinity of Delaware Dumpsites
Metal
F level
Significance
Vanadium
33.54
.01
Zinc
6.34
.01
Cadmium
5.12
.01
Silver
3.07
.01
Copper
3.02
.01
Chromium
2.80
.01
Manganese
2.74
.01
Cobalt
2.25
.01
Nickel
1.95
.05
Titanium
1.92
.05
Aluminum
1.62
—
Iron
1.36
—
Lead
1.29
-------
Table 4
OPERATION "IDES"
Metals in Scallops
ppm/dry wt.
Metal F ratio Significance
V 11.885 .01
Cd 6.896 .01
Zn 5.376 .01
Ni 5.300 .01
Cu 4.194 .01
Cr 3.130 .01
Ag 3.096 .01
Ti 2.832 .01
Mn 2.347 .01
A1 2.088 .05
Fe 1.787 .05
Pb 1.434
Co 1.117
115
-------
OILS TEAM
ROAP 16-AAV
The mission of the Oils Team is to determine the fate and
effects of oils and petrochemicals in the marine environment.
Our goals are to generate the data base from which defensible cri-
teria can be written to protect the integrity of the marine ecosystem.
To accomplish this, we have divided the problem into three somewhat
arbitrary categories. These are the fate and effects of:
I. Spilled oil
II. The water-soluble fractions of oils
III. Oil-contaminated sediments
I. Spilled Oil
A. Field Work
Our program on the fate and effects of whole oil spilled in
the marine environment continues to be primarily a grant and con-
tract activity. A large contract with Mississippi State University
on the fate and effects of crude oil added to a gulf coast estuarine
environment is in its last year. A set of simulated ecosystem
ponds has been constructed to study these effects in a field sit-
uation. Two ponds have been treated with Empire Mix crude oil and
two have been maintained as controls. Over the previous two years,
a series of laboratory exposures of oil to various organisms has
been conducted. Various response parameters have been observed.
The results of these laboratory observations are now being applied
to the ponds in order to observe as many responses to the oil as
possible. Some preliminary oil effects that have been noted
are shifts in planktonic populations and increased incidence of
disease in fish. These effects are being observed throughout the
year to elucidate the chronic effects as well as the fate of the
oil in these ponds.
We are also studying the effects of ft2 fuel oil on clam
(Mercenaria mercenaria) and oyster (Crassostrea virginica) thru a
contract with the Virginia Institute of Marine Science. The animals
116
-------
OILS TEAM
will be exposed to #2 fuel oil as in a spill situation in the field
for a period of six months, with the effects on the animals being
observed by histological examination. In addition, chemical
analysis of the animals, as well as sediments and water, will be
performed by gas chromatography and gas chromatography/mass spectro-
metry. This will give us some information on the fate of #2 fuel
oil in the environment along with telling us how much #2 oil the
animals were actually exposed to and how much they incorporated
into their tissues. We had planned to conduct these studies this
fall, but a number of problems delayed our spilling the oil until
nearly December, which was too late in the season to initiate the
study. The exposure is now set to start in the spring as soon as
the water warms up.
B. Laboratory Bioassays
Our recent in-house bioassay work also has involved No. 2 fuel
oil as the toxicant. We exposed the soft-shelled clam, Mya arenaria,
for five months in the system shown in Figure 1. The exposure was
on a continuous flow basis at ambient temperatures. An amount of
whole oil equivalent to 10 ppm was metered into the top tank. This
resulted in accommodated oil levels at mid-depth of between 0.1
and 0.2 ppm as measured by infrared absorbance. While the detailed
histological examination of these animals is still underway, pre-
liminary analysis shows the development of some lesions in the
digestive diverticula.
During this same time we also exposed several other species
to the No. 2 fuel oil effluent from the primary exposure chamber of
the clam studies. Secondary exposures were conducted in two cylin-
drical fiberglass containers connected in series. Mid-water fuel
oil concentrations were fairly constant at 1.8 and 1.5 ppm in the
two tanks. Lobster (Homarus americanus) and Scup (Stenotomus
chrysops) were exposed in the first tank, while mussels (Mytilus
117
-------
FIGURE I
SEA WATER
OIL
CLAMS (in4"sand)
r—
>
LOBSTER
FISH OR
MUSSELS
- -> DRAIN
118
-------
OILS TEAM
edulis) and scallop (Argopecten Irradlans) were exposed in the second
tank. Animals from these exposures are currently under-going histo-
logical evaluation. Dramatic behavioral responses were evoked by
these exposures, including the absolute inability of the lobster to
feed. Further studies with the system are planned.
A series of acute static bioassays of 96 hours duration were per-
formed using No. 2 fuel oil as the toxicant and Atlantic silverside
(Henidia menidia). shrimp (Palemonates pugio), and scallop (Argopecten
irradians) as the test species. These tests were performed using
standard methods in one gallon glass jars for comparison with the
results obtained in continuous-flow systems. The results are shown
in Table I.
TABLE I
Species
Temp. °C
48 Hr LC-50, ppm
96 Hr LC-50, ppm
Adult Atlantic Silversides
13
1000
370
4 cm
18
>5000
5000
Shrimp (Palaemonates)
13
180
70
11 M
18
>340
340
Scallop
18
>500
II. The Water-Soluble Fractions of Oils
A. Field Effects
Our program to determine the fate and effects of the water-
soluble (or accomodated) fractions of oil is also progressing with
both in-house and out-house efforts. Discharges of these fractions
have been largely ignored in the past since the major regulatory
emphasis has been on the prevention of visible sheens. Although
aesthetic considerations associated with sheens are important,
it is also very important to control the potentially more toxic
water soluble fractions. Our in-house effort is directed towards
119
-------
OILS TEAM
the biological effects of the water-soluble fractions, while the
fate of these fractions in the environment is being addressed thru
an interagency agreement with the Navy. Our study with the Navy
addresses the problems of the fate of dissolved and dispersed
hydrocarbons in the effluent from ballast water processing plants.
Such plants function primarily as oil-water separation facilities
with the discharged water still containing all of the water accommo-
dated fractions. The Navy is sampling water and sediments in the
vicinity of several different ballast treatment plants to determine
the extent of the plume in the water column and the rates of accumu-
lation in the sediments. These data can then be correlated with the
biological effects data that we are generating in the laboratory
to delineate the magnitude of the impact of such a plant on an environ-
ment .
B. Biological Effects of Water-Soluble Fractions of Oils
The purpose of this phase of the oils program is to determine
the biological effects of the water-soluble-fractions of oils. An
in-house continuous-flow bioassay has been designed to expose a
variety of organisms and their life stages to various concentrations
of the water-soluble-fractions on No. 2 fuel oil on a long-term
basis.
The apparatus consists of an oil-water separation chamber
(Figure 2) wherein a constant inflow of oil and unfiltered sea
water (~32% salinity and ambient temperature) is circulated over a
series of baffles and mixed. The insoluble oil slick is skimmed
from the surface and coJlected in a waste chamber, while the water-
soluble-fraction of the oil is distributed to the various dosing
tanks (Figure 3). The 55 gallon dosing tanks include a control
and three experimentals, one containing 0.1 ppm fuel oil, another
containing 1.0 ppm fuel oil, and a third containing 10 ppm fuel
oil. The expected concentrations of oil within each of the
120
-------
FIGURE 2
SEAWATER
OIL
WATER SOLUBLE FRACTION TO TEST
DRAIN
121
-------
Figure 3
Continuous Flow Dosing Apparatus
Oil Film
Oil & H_0 Sep,
ation Cnamber
Water Soluble
Oil Fractions
lOil Oil
Oil
} R Waste Oil
jy Collection
CIA Chamber
II
10 ppn
j^ontro]
Drain
Drain
Drain
Oil
Reservoir
I IV
jj.l PPnj
K\M
Constant Pump
Head
Tank
L.0 ppm
Drain
Drain
-------
OILS TEAM
experimental tanks are controlled by a metered flow of oil and
water (total rate of flow within each tank=3£/min), and are verified
on a daily basis by infrared spectrophotometry. The characteristics
of the water with respect to temperature, dissolved oxygen, pH,
and salinity ara also Jeternined on a routine basis.
The test organisms exposed thus far have included adult grass
shrimp, Palaemonetes pugio, juvenile quahogs, Mercenaria mercenaria,
adult bay scallops, Argopecten irradians irradians, adult mud snails,
Nassarius obsoletus, embryonic winter flounder, Pseudopleuronectes
americanus, and adult stages of the marine polychaete, Capitella
capitata.
Several parameters of response are being employed to
evaluate the chronic and sublethal effects of the toxicant to the
above organisms; for example, with the Mercenaria. Palaemonetes.
Argopecten, and Capitella we are evaluating the animals histological
condition. Furthermore, an attempt is being made to determine effects
of fuel oil on the hatchability of rinter flounder (Pseudopleuro-
nectes americanus) eggs, and larval development. In addition, an
attempt is being made to evaluate the effects of fuel oil on the
growth rate of palaemonetes by comparing the ratio of RNA to DNA in
muscle tissue.
Behavioral studies are also being conducted on several of the
test species. For example, daily observations are being noted on
respiratory ventilation and feeding rate in the molluscs, Mercenaria
and Argopecten, and on feeding activity, orientation, and general
stamina of Palaemonetes. Further, the chemotactic response of
Nassarius in the presence of water-soluble //2 fuel oil is being
evaluated. We are also attempting to see whether or not fuel oil
inhibits an escape response of the scallop, Argopecten. to the pre-
datory starfish, Asterias forbesi.
123
-------
OILS TEAM
Although our primary objectives are to evaluate sublethal
and chronic effects of the oil, we have also kept a daily record
of mortality throughout the exposure period (see Table II and
Figures 4 and 5). The data reveal that the highest concentration,
10.6 ppm, is extremely toxic to the test organisms, causing 100%
mortality of Palaemonetes within 6 days, Argopecten within 5 days,
and Mercenaria within 20 days. The next lower concentration, 0.56
ppm, has caused a gradual increase in mortality of Palaemonetes,
which has reached 32% thus far (i.e. after 72 exposure days).
Finally, we are analyzing the exposed test organisms for
uptake of petroleum hydrocarbons. We are using gas chromatography
to measure the amount of hydrocarbons taken up by the organisms,
and using our gas chromatograph-mass spectrometer to verify the
identity of the individual hydrocarbons. The sample preparation
techniques for these analyses were described in the last semi-annual
report (Jan-June, 1974; ROAP 16-AAV). These residue values will
then be compared with the biological response data from the bio-
assays to determine at what body-burden level of hydrocarbons
the biological effects manifest themselves. We can then compare these
body-burden levels with those of various field populations exposed
to oil contamination to predict possible biological effects in the
field populations.
TABLE II
Summary of Percentage Mortality in Response to the Water-Soluble
Fraction of No. 2 Fuel Oil to Date (1/16/75) (72 Exposure Days
Unless Other Wise Noted)
Expected Actual Cone.
Concentration (by I.R.) Palaemonetes Argopecten Mercenaria
Control 0.006 + 0.006 ppm 0.0 3.0(37 Days) 0.0
0.1 ppm 0.069 + 0.029 ppm 1.0 0.0(37 Days) 0.0
1.0 ppm 0.56 + 0.13 ppm 32.0* 10.0(37 Days) 0.0
10.0 ppm 10.6 +1.3 ppm 100.0* 100.0(5 Days)* 100.0 (20
(6 days) days)
*STARTED VALUES INDICATE SIGNIFICANT MORTALITIES FOR WHICH CURVES HAVE
BEEN GENERATED
A
124
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Figure 4
Exposure Time V£ Mortality
for
Several Species in 10.6 ppm Water Soluble Fraction of No. 2 Fuel Oil
100
90
80
70
60
Palaemonetes
Argopectin
50
40
Mercenaria
30
20
10
10
Exposure Time (Days)
-------
Figure S_
Exposure Time V£ Mortality
for
Palaemonates Pugio in 0.69 ppm Water Soluble Fraction of No. 2 Fuel Oil
100
90
80
70
60
50
U0
30
20
10
10
40
20
30
60
70
80
50
Exposure Time (Days)
-------
OILS TEAM
It is also interesting to compare the mortality figures
generated in the acute static bioassays to those from the continuous
flow bioassay. It took less than two days for over 50% of both
the grass shrimp and the scallops to die when exposed to 10 ppm of
the water-soluble fraction of No. 2 fuel oil on a continuous-flow
basis at 14°C. On the other hand, the static exposures were much
less lethal. As can be seen from Table 1, the shrimp were roughly
20 times less sensitive and the scallops more than 50 times less
sensitive in the static exposure system.
Some of the difference is of course due to the differences
in the toxicants-we are comparing the water-soluble fraction of No. 2
fuel oil to the whole oil. In addition, one might expect some
differences between continuous flow and static exposure for any
toxicant. However, the magnitude of these differences requires further
consideration because of the possibilities that criteria could be
written and standards set on the basis of static exposures. One
possible explanation of these large differences the typical use
of aeration in static exposures. Petroleum hydrocarbons can readily
partition between air and seawater, with the partition coefficient
being heavily weighted towards the air. Thus, the aeration may
effectively strip out the toxicant during the test period. In
addition, the exposure concentrations reported in the two kinds of
bioassays are not based on equivalent measurements. The exposure
concentrations reported for most flow-thru bioassays are values that
have actually been measured in the water column. This is not the
case for most static bioassays, where the reported values are usually
based only on the amount of toxicant added to the jar, not the
amount that the test organisms are actually exposed to in the water
column. The use of static bioassays is justifiable when only relative
toxicity values are desired either for a variety of oils or a variety
of organisms. However, if one is attempting to establish water
127
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OILS TEAM
quality standards for the protection of an ecosystem then it is
necessary to use flow-thru bioassays to establish the permissible
levels.
III. Oil Contaminated Sediments
The third phase of our oils program deals with the chronic
long-term effects of oil-contaminated sediments. The biological
effects of this form of pollution have yet to be studied in detail,
partly because the analytical methodology to measure low levels of
petroleum hydrocarbons in sediments is still under development. We have
a grant with Woods Hole to fund Farrington's continuing efforts to improve
this methodology. In addition, we have a grant with URI to study the
distribution of hydrocarbons in sediments and organisms in the vic-
inity of the Newport R.I. dredge spoil disposal site. This site
received the dredge material from the Providence river about five
years ago, and the material is known to have been contaminated with
hydrocarbons. We are investigating whether the hydrocarbons have
been mobilized and whether they have become incorporated into the
surrounding biota. We suspect that hydrocarbons have become incor-
porated into the surrounding biota because of histological findings
of masses of black particulate matter in the kidneys of Artica islan-
dica taken from around the dumpsite.
final project in this area is a project funded jointly with
NOAA to investigate the behavior of oil placed on the bottom in
a tropical environment. This is being conducted by Dames and Moore,
using saturation dives from an underwater habitat in the Bahamas.
Oil is being mixed with several sinking agents and placed on the
bottom. The oil can then be observed extensively during the dive,
and its fate and gross effects within the area noted.
Analytical Services
During the past few months it has become obvious that the various
people conducting routine analyses in our lab should be coordinated into
a single unit. A major benefit from such a move is to generate a
128
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OILS TEAM
viable quality control program to enhance defensibility of data
generated by the lab. An additional benefit is that each team in the
lab will not need a person trained in each technique or kind of analysis
that the team requires to accomplish goals. We are currently offering
heavy metals analysis by atomic absorption or neutron activation .analysis,
ATP analysis, and hydrocarbon analyses by infrared or gas chromato-
graphy and mass spectrometry. In addition, we plant to offer CHN
and total organic carbon analyses on solid samples as well as
micronutrients by Technicon auto-analyzer as soon as possible.
We have progressed the farthest with the metals analysis.
We currently run all of the routine determinations of metals in
sediments and tissues for the laboratory. We have been using nitric
acid digestion techniques and feel that we have control over the
tissue level determinations, but still have some unanswered questions
with the sediment digestions. Our tissue determination methods have
been checked using the National Bureau of Standards bovine liver as
well as the Food and Drug Administration round robin oyster sample.
Our results are summarized in Table III and graphed in Figures 6
and 7. We feel the agreement shown is quite satisfactory, although
we seem to be a bit low on the iron in the liver sample. The NBS
values are their provisionally certified values, but the FDA values
are simply the averages from a variety of different labs. All
results are in a yg per gram (ug/g) dry weight basis, with the
range, standard error, and standard deviation shown on the graphs
where applicable.
These are just our first attempts at ensuring the quality of
our analyses. The problems are many, especially with the sediment
samples because there are no available NBS-type reference materials.
In addition, one has to consider that an added spike may not act
in the same manner as the metal that may be tied up in complexes
within the sample. Also, the natural variation within replicate
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OILS TEAM
samples especially for organisms is very high, so that a large
number of replications must be compared to find statistically valid
differences. We are currently formulating plans to deal with these
problems.
130
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TABLE III
Metals Analysis of NBS Bovine Liver and FDA Oysters
NBS Bovine, ug/g FDA Oysters Mg/g
METAL NBS NMWQL FDA* NMWQL
Cd 0.27+0.04 0.33+0.03 2.06+0.33 1.64+0.03
Cu 193+ 10 188+2 24.8 +2.6 20.7 +0.2
Fe 270 +20 239+4
Mn 10.3 + 1.0 10.8 +0.3
Pb 0.34+ 0.08 0.43+0.12 0.54+0.34 0.61+0.01
Zn 130 + 10 134 +2 583 + 86 523 + 1
* Value given is average of 15 laboratories
131
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-------
RECREATIONAL WATER QUALITY CRITERIA TEAM
ROAP 21 BEZ - Development of Criteria for Marine Recreational Waters
A statistically significant increase in the incidence of gas-
trointestinal symptoms among swimmers relative to non-swimming beach-
goers was observed at the Coney Island ("barely acceptable") but not
at the Rockaway beach ("relatively unpolluted"). Such was not the case
with respiratory or "other" symptoms nor with the symptom severity
index. However, the severity index (stayed home, stayed in bed, visited
a physician) was higher among swimmers than among non-swimmers at the
Coney Island beach. These findings were derived from about 8000 use-
able responses to illness inquiries obtained in the course of eight,
Phase II, epidemiological^microbiological trials (in only 6 of the
trials were enough responses obtained for individual trial analysis)
conducted at the two beaches in the vicinity of New York City. Al-
though the differential (swimmers less non-swimmers) gastrointestinal
symptom rate at Coney Island was less than half that observed during
the previous summer (pretest), because of the larger number of
individuals queried (N), this essential observation from the previous
summer's study was confirmed*.
The decreased rate of gastrointestinal symptoms relative to that
obtained the previous summer appears to coincide with decreases in
the mean densities of IS. coli, K1 ebsiella, fecal streptococci and
Aeromonas hydrophila. The mean densities of the various potential
indicators in the water are shown in Table 1. Coprostanol assays
1 Cabelli, V.J., M.A. Levin, A.P. Dufour and L.J. McCabe. The Deve-
lopment of Criteria for Recreational Waters. International Sym-
posium on Discharge of Sewage from Sea Outfalls. London, 28 August
1974.
134
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RECREATIONAL WATER QUALITY CRITERIA TEAM
are still being performed. Although the analysis of the data from the
individual trials has not been completed, the rates of gastrointestinal
symptoms appears to correlate best with JE. coli densities in the water.
This was observed from the analysis of the data from individual trials
during Phase II and of the overall rates for the two series of trials
(pretest and Phase II) at the barely acceptable and relatively un-
polluted beaches.
Further analyses of the data are in progress, including inter-
actions such as severity versus type of symptoms and analysis by trial.
Confirmation of significant differences in gastrointestinal symptom-
atology among swimmers relative to non-swimmers at the Coney Island
beach does attest to the applicability of experimental design. However,
it would appear that these rather low attack rates fall into the lower
portion of the indicator-illness curve described by Cabelli and McCabe
C1974)2 and that, in the absence of any significant differences in
"severe" symptoms as defined herein, more polluted beaches must be
sought in order to describe the full nature of said curve. The analysis
of the data by trial should provide some insight whether temporal vari-
ability in indicator rates, which occur as a consequence of rainfall
with combined sewage systems and of other factors, is associated with
changes in the rate of gastrointestinal disturbances among swimmers.
Thereby, changes at a given beach as well as beaches on a pollution
gradient could be used in defining the entire indicator-illness res-
ponse curve.
An analysis of the confirmation frequencies for Clostridium
perfringens as obtained from the mCP method indicated that additional
in situ biochemical tests would be required if the picking of
colonies for confirmation is to be avoided. An additional in situ
test incorporating three biochemical characteristics was developed,
^ Cabelli, V.J. and L.J. McCabe. Recreational Water Quality Cri-
teria News of Environmental Research in Cincinnati. November 11,
1974.
135
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RECREATIONAL WATER QUALITY CRITERIA TEAM
and the procedure thusly modified is under evaluation. Similarly,
two additional Iji situ biochemical tests have been incorporated into
the mVP method for the quantification of Vibrio parahemolyticus.
The evaluation of the modified procedure is in progress; and the
precision and specificity of the modified procedure appears to be
within acceptable limits.
The search for additional specific inhibitors which will
increase the selectivity (an increase in the quantity of water
which can be examined without overgrowth due to background micro-
organisms) of the mC and mA procedures for differential coliforms
and Aeromonas hydrophila respectively is continuing.
The capability to quantify f-2 coliphage has been obtained.
These RNA, "tailless" phage are reported to be more resistant that
T phage and, at least, of comparable resistance to polio I as regards
chlorine and other environmental effects. A number of workers
have suggested that such phage may be an appropriate indicator for
enteroviruses. Said procedure will be incorporated into the exam-
ination of bathing water during next summer's trials.
A lab study comparing the survival in seawater of coliform
biotypes, Aeromonas hydrophila, Pseudomonas aeruginosa and fecal
streptococci, as influenced by the source of the organisms (sewage
versus pure cultures), temperature (4 versus 20 C) and the concen-
tration of sewage (0-50%), has been completed. A manuscript des-
cribing the findings has been sent forth for review.
136
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Table 1. Comparison of log mean indicator and pathogen densities (per 100
ml) at the "barely acceptable" and "relatively unpolluted" beaches
Organisms
Method
Recovery oer 100 ml at
Coney I si. Rock.
C.I./Rock.
Total coliforms
MPN
1213
43.2
28.1
Total coliforms
mC
549
14.6
37.6
Fecal coliforms
MPN
545
28.4
20.0
E. coli
mC
15.3
2.4
6.4
Klebsiella
mC
59.2
3.5
16.9
Enterobecter-Ci trobacter
mC
434
6.6
65.8
Fecal streptococci
mSD
16.4
3.5
4.7
C. perfrinqens
mCP
18.2
12.6
1.5
P. aeruginosa
mPA
45.8
3.1
14.8
A. h.ydrophila
mA
9.6
4.9
2.0
Total staphlococci
mSA
243
178
1.4
S. aureus
mSA
104
69.2
1.5
137
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RECREATIONAL WATER QUALITY CRITERIA TEAM
Papers Accepted for Publication
1. Dufour, A.P., and Cabelli, V.J. A Membrane Filter Procedure
for Enumerating the Coliform Group and its Component Genera
in Sea-Water. Appl. Microbiol.
2. A.P. Dufour and V.J. Cabelli. Comparison of membrane filter
brands for recovery of the coliform group. Presented at the
ASTM Symposium on Recovery of Indicator Organisms Employing
Membrane Filters. January 1975. Ft. Lauderdale, Fla.
Manuscripts submitted for review
1. Dufour, A.P., and V.J. Cabelli. Survival of Indicator Micro-
organisms in Sewage and Seawater.
»
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TECHNICAL ASSISTANCE
Non-research efforts from June through December in support of
Regional office activities and Headquarters needs required nearly
16% of our resources. Major categories of activities supported
continue to be industrial cooling water use and ocean disposal
of wastes. These is an emerging trend toward greater participation
of individual research scientists in Regional and Headquarters
responsibilities. At present, this has not exceeded 15% of our
efforts, but projections indicate that the coming press of Public
and Adjudicatory Permit Hearings among seacoast regions, most of
which concern PL 92-500 Section 316(a) applications, can consume up
to 25% of our total resources and involve over 60% of our senior
scientific staff. We view this as a short range expediency which
can have long range consequences damaging EPA's marine research capa-
bility.
We have initiated a program to systematize our technical
assistance efforts, expending some additional resources on efficient
dissemination of our knowledge to Regional permits program personnel.
In addition, we have requested and received coordinating functions
from Headquarters staff in setting priorities and negotiating
assignments among Regional Offices requesting our help. Our hope
is to continue to meet all technical assistance requests from
Regions, States, private citizens groups, other agencies and
Headquarters without diverting more than 15% of our research
resources from their primary mission. Meanwhile, our series of
"Bio-legal workshops" is well underway and training individual
Regional personnel through involvement in research and technical
assistance is continuing, along with close coordination with
Headquarters OR&D's Office of Program Integration and OEGC's office
of Water Enforcement.
First Bio-legal Workshop:
Section 316(a) of the 1972 Water Bill Amendments has been the
source of considerable controversy - and litigation is now in
139
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TECHNICAL ASSISTANCE
progress over its proper administration by EPA. Deputy Assistant
Administrator for Water Enforcement, Richard Johnson requested
that NMWQL initiate a program to inform Regional attorneys who
would be administering Section 316(a) permits about common biological
phenomena associated with industrial cooling water use. Together
with personnel from our sister institution, the National Water
Quality Laboratory (Duluth, Minnisota) we planned and presented a
workshop on the Biology of Power Plants.
One attorney from each Regional Office attended, along with
two Headquarters OEGC attorneys who would be coordinating all
Section 316 hearings for EPA. Workshop staff consisted of biologists
from Duluth, Headquarters, and Narragansett. Curriculum included
a day of illustrated lectures, a tour of an operating power plant
and discussions with company personnel, a mock 316 hearing
including direct testimony and cross examinations, and a water tour
of proposed power plant sites. Biological issues of legal importance
were emphasized (e.g. the kinds, firmness, and limitations of
biological evidence) along with legal techniques in dealing with
biological evidence. The workshop staff benefited from the ex-
posure to these issues as much (or more) than did the participants,
who requested that a second such workshop be conducted after the
mass of Section 316 Hearings began in the spring.
Headquarters Planning Functions:
Several senior staff scientists have been involved in a research
planning effort aimed at administration of special Presidential
programs for evaluating environmental impact of energy exploration
and exploitation. NMWQL has worked closely with the staffs of the
Office of Planning and Evaluation, and OR&D's Office of Program
Integration as well as "Sub-sector groups from NERC-Corvallis.
Major tasks included identification, prioritization, and cost
analysis of energy-relates environmental research. In addition to
numerous meetings held in Washington, Denver, Dallas and Corvallis,
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TECHNICAL ASSISTANCE
NMWQL staff consulted together in seminar fashion to be certain
that all marine impacts were included and given appropriate priority
rating by field personnel.
Surprisingly, staff felt that for certain categories of
energy exploration and exploitation activity, research funds might
be spent most wisely on impact-preventative technology rather than
ecological damage prediction or assessment. These specific recommen-
dations were passed on to appropriate sub-sector groups for tech-
nology development research.
Office of Planning and Evaluation personnel met at Narragansett
with staff members to discuss specific issues of general industrial
siting and thermal impact on estuarine and open coastal environments.
Matters of project priority, levels of funding, and appropriate
governmental and non-governmental sources of research expertise
formed the nucleus of discussion.
Dr. Schneider formed and consulted with an ad hoc panel of
University experts on energy-related impact on the marine environ-
ment. This panel has actively developed research priorities and
identified sources of expertise as well - paralleling intramural
efforts and adding to the conceptual basis of Project Independence's
environmental efforts.
Headquarters Regulatory Functions:
1. U.W.A.G. Hearings on PL92-500, Section 316
Drs. Schneider, Miller, Perez and Prager participated in a
Public Hearing held in Washington which had been requested by Edison
Electrical Institutes Utility Water Action Group (UWAG). Hearings
concerned evaluation of Section 316 decisions based upon ecological
and economic factors. NMWQL participants were involved in testimony
explaining the technical basis of EPA's Draft Technical Guidance
Manual on Section 316(a)-providing anecdotal evidence for the sound-
ness and necessity of several suggested methods of environmental
141
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TECHNICAL ASSISTANCE
evaluation. We also participated in discussions of UWAG's proposed
environmental and economic modeling systems.
2. Draft Technical Guidance Manual on Section 316(a)
Considerable NMWQL resources were expended on the EPA effort
to complete this technical guidance manual. In addition to
refining and re-writing several sections of earlier drafts, a new
section concerning criteria for decision at each level of proof was
drafted and submitted to headquarters for approval. The entire
manual was reviewed by several NMWQL senior staff members, and
editorial comments communicated to Office of Planning and Evaluation
personnel were discussed in a series of meetings held at Headquarters
and at Narragansett.
This effort demonstrated that a high level of communications
among field-personnel and headquarters personnel representing OEGC,
OPE, and OR&D was not only possible, but highly useful in developing
regulatory documents. NMWQL staff was very pleased by the degree
to which their technical knowledge industrial cooling water effects
on marine biota could be translated into regulatory guidelines, and
by the close cooperation of OEGC and OP&E personnel.
3. Standards and Regulations Information System
NMWQL staff members participated in a two day seminar at R.T.P.
held to outline"Scientific and Technical Assessment Report" document
development. Office of Program Integration Deputy Assistant Admin-
istrator John Buckley described the system of STAR report preparation
and uses to which such documents may be put. No STAR assignments
have been received by NMWQL to date.
4. Toxic Effluents Standards:
Dr. Donald K. Phelps met with headquarters staff to assist in
the development of toxic effluents standards (mandated by PL92-500,
Sect. 307(a)) for the estuarine and marine environments.
Assistance to Regional Offices
Region X: Drs. Pesch and Prager reviewed an ocean dumping
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TECHNICAL ASSISTANCE
permit application concerning the disposal of large quantities
of salt off Prudhoe Bay, Alaska. An advisory was prepared for
the Region X Permits Office and submitted in favor of the one-time
disposal.
Region IX: An advisory was submitted to Region IX on Hawaiian
Electric Co.'s request for a Section 316(a) exemption for the Kahe
Power Plant. Drs. Gentile and Prager had visited the plant and
conducted an underwater spot check of effluent effects in September
at the request of Region IX and OEGC's Counsel for Administrative
Litigation. NMWQL staff advised granting the 316(a) exemption
under certain specified effluent limitations.
Region IV: Mr Paul P. Yevich traveled to Tampa, Florida to
attend pre-hearing meetings concerning construction of an offshore
oil terminal in the Gulf of Mexico and a refinery complex proposed
for St. Petersburg, Florida.
The Interagency Research Advisory Board for Crystal River
Nuclear Unit 3 of which Dr. Prager is a member met with Florida
Power Corp. and their consultants to discuss research performed in
support of Florida Power Corp. NPDS Permit Application. The
Board decided that data accumulation for that activity was
complete but that additional analysis would be required.
Drs. Prager and Eisler provided Region IV and South Carolina
Department of Industrial Water Control with further information on
Manoa Metals effluent permit questions for adjudicatory hearings.
Drs. Prager and Eisler assisted Region IV by providing additio-
nal information on Manoa Metals Permit Application for Public Hearing
Testimony.
Mr. Reynolds, Dr. Eisler, and Dr. Prager provided information on
the toxicity of antimony of marine life to permits personnel in
Region IV. Little information exists in the scientific literature,
but indications are that antimony is not among the highly dangerous
metals. Biological assays were recommended to be performed by the
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TECHNICAL ASSISTANCE
applicant before permit issuance.
Region III: Dr. G. Pesch and Bruce Reynolds acted as members
of the Region III Staff Panel and Dr. J.C. Prager acted as member
of the EPA Region III Hearing Board for a Public Hearing concerning
the City of Philadelphia's Ocean Dumping Permit for its sewage
sludge.
Region Ill's Permit personnel and staff and consulting scientists
representing E.I. DuPont de Nemours Co. met with NMWQL's Ocean
Dumping Team to discuss research strategy for the Edge Moor iron
acid waste ocean dumping permit. Field monitoring and laboratory
bioassays were designed.
At the request of Region III, Dr. G. Pesch testified on the
effects of E.I. DuPont de Nemours ocean dumping off the coast of
Delaware at Public Hearings held in Ocean City, Maryland. Mr.
Reynolds, who also attended the hearings, and Dr. Pesch had conducted
research cruises in the dumpsite area. Dr. Prager served on the
Region III hearing review panel.
Dr. G. Pesch and Messrs. G. Morrison and B. Reynolds traveled
to the U.S. Naval Academy, Annapolis, Maryland., for a meeting
which was jointly sponsored by NOAA and EPA concerning (1) the
recent submarine survey of the Dupont and Philadelphia Ocean
Dumping Sites located off Delaware; and (2) use of a submersible
as a research tool for monitoring dump sites. Messrs. Morrison and
Reynolds delivered short papers at the meeting.
Region II: Mr Eric Outwater, Dpty. Reg. Admin., Region II,
visited NMWQL to discuss testimony and strategy for Public Hearings
to be held in New York concerning the dumping at sea New York
sewage sludge.
Personnel from Region II's Permits Branch met with NMWQL staff
to discuss power plant permits on the Hudson River. The Oyster
Creek Plant on Barnegat Bay, New Jersey, was also a subject of
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TECHNICAL ASSISTANCE
discussion. Personnel from NMFS and DI attended the meeting to
add their knowledge of these specific power plants to EPA's
permit decisions.
Dr. G. Pesch, B. Reynolds and Paul Dix sailed on the sludge
ship Newtown Creek out of NYC observing a normal dump at the dump-
site in the New York Bight. After returning from the dumpsite, these
same people toured the Newton Creek Sewage Treatment Plant where
the sludge is processed. They observed the normal treatment process
and an experimental treatment process being developed.
Dr. G. Pesch, Bruce Reynolds and Paul Dix conducted an aerial
survey on dumping activities in lower New York Harbor. Sludge
dumping and iron acid waste dumping were observed and photographed.
Ms. Barbara Pastalove, biologist in the Thermal Regulatory
Branch of Region II's Permits Office, spent one week at NMWQL for
training in biological aspects of PL92-500, Section 316(a).
Region I: Dr. D.K. Phelps accompanied Dr. Warren Oldaker of
Region I to Boothbay Harbor, Maine to discuss an EPA Grant to the
State of Maine's Department of Marine Resources with Dr. John Hurst.
Mr. Gilbert Chase, Army Corps of Engineers visited NMWQL to
take part in a joint Agency (cooperative) diving program. NOAA,
Corps, EPA and University SCUBA divers studied transects across a
dredge spoil dumpsite off Newport, R.I. during 3 cruises last week.
Drs. Eric Schneider and D.K. Phelps met with Region I and
Army Corps of Engineers personnel at Regional Hdqtrs. regarding
dredged spoil disposal at sea.
Dr. D.K. Phelps attended the hearings held at the Federal
Court, Hartford, Connecticut, for an injunction against the Navy's
dredging of the Thames River and the dumping of the spoils in the
New London section of Long Island Sound.
Dr. Prager and Management Intern Sigmund A. Ustaszewski assisted
Region I in a meeting with Boston Edison Company concerning its 316(a)
demonstration for the Pilgrim Power Station.
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TECHNICAL ASSISTANCE
Drs. Davey, Eisler and Prager provided advice to Region I
Permits Branch on the disposition of a permit application from
Kerramerican Mine Co.'s to discharge heavy metal wastes into an
estuarine environment.
Dr. Prager and George Gardner reviewed comments by DOI & NOAA
on Millstone 316(a) Demonstration for Region I.
Mr. Gardner and Dr. Prager accompanied Region I EPA Permit
Program personnel to an interagency meeting held at Milford, Conn-
ecticut., NOAA laboratory to discuss NOAA and DOI objections to
Connecticut's conduct of a 316(a) Public Hearing concerning the
Millstone Nuclear Generating Plant.
Dr. D.K. Phelps, member of Interagency Research Advisory
Board on Ocean Dumping, attended the Board's meeting to discuss
Army Corps of Engineers problems with the New London Dump Site
in Boston, Massachusetts.
International Technical Assistance:
Dr. W.D. Oliff, Durban, S. Africa, visited NMWQL to discuss
recreational water methodology and other research programs.
Dr. Sotoaki Onishi, Mgr., Nuclear Power Survey Office, Electric
Power Development Co., Ltd., Tokyo, Japan, visited NMWQL to discuss
nuclear power plant information with staff members.
Assistance to the Private Sector:
Messrs. J. Fornes, J. Eckels and H. Palmer, representatives
from Westinghouse Ocean Systems, Annapolis, Md., visited NMWQL to
discuss applications of bioassay and possible EPA funding for their
research.
Dr. Hugo Freudenthal, V.P. H2M Corp., Melville, N.Y., visited
NMWQL to consult with staff members on EPA Water Quality criteria.
Members of the Manufacturing Chemists Association Water
Resources Committee visited NMWQL, and after a briefing on the program
were taken on a tour of the laboratory facilities.
146
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TECHNICAL ASSISTANCE
Drs. Richard Toner and George Mathiessen of Marine Research,
Inc., E. Wareham, Mass. visited NMWQL to discuss data requirements
of a 316(a) demonstration for the Brayton Point Thermal Electric
Plant with Drs. Miller, Beck, and Prager.
American Petroleum Institute personnel, Dr. N.K. Weaver, J.R.
Gould and R.E. Eckardt visited NMWQL to discuss oil pollution studies
with various staff members.
A Graduate Class in Community Planning from the University of
Rhode Island met with Dr. Prager at NMWQL to discuss power plant
siting problems and to observe NMWQL photographs of well and poorly
sited facilities.
Miscellaneous Short-Term Assistance:
Drs. J. Gentile and S. Cheer held a Workshop at ATP Techniques
in Denver, Colo, at the request of NFIC.
Capt. Willard M. Adams, FDA, visited NMWQL and consulted with
Dr. M.A. Levin and A.P. Dufour on enumerative technology.
Drs. D. Phelps and P. Rogerson traveled to Washington, D.C. to
discuss the Oil Section of the draft Water Quality Criteria document
with Mr. K. Mackenthun.
Mr. Joel Fisher of R&D Program Integration, Hdqtrs, visited
NMWQL to consult with staff members on the re-write of Chapter V
of the CEQ Report.
Dr. D.K. Phelps traveled to Washington to meet with Head-
quarters staff on Toxic Effluent Standards, Section 307(a) of PL92-
500.
Dr. Roy Irwin, John Christian, Jeff Goodman, and Diane Olsson
and Michelle Zarubica of EPA Hdqtrs., Dr. Wm. Brungs of the Duluth
Lab, and Bruce Tichenor, PNERL, visited NMWQL to draft a final
316(a) Guidance Manual with staff members.
Dr. Jan Prager attended a Hdqtrs OWP Meeting to assist R&D
personnel in outlining Agency efforts to fulfill Sec. 104(n) of
147
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TECHNICAL ASSISTANCE
PL92-500.
Miss Dreena Felton, Water Planning Div., Hdqtrs, visited
NMWQL to gather information to be used as references in the upcoming
suit against EPA by several power companies.
Dr. Schneider, Miller and Prager reviewed the Edison Electrical
Institute "Utility Water Act Group" comments on EPA's draft guidence
manual on Section 316(a), PL92-500. A public hearing on this
subject was held at headquarters at which these persons participated
in an EPA panel constituted to receive and answer comments from the
public and concerned industrial interests.
148
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Narragansett, Ri
NATIONAL MARINE WATER QUALITY LABORATORY
TELEPHONE LISTING AS OF APRIL 17, 1975
Commercial ns
OFFICE OF THE DIRECTOR
Eric D. Schneider, Director 401/789-3361 847-8175 or 76
Doris G. Girard, Secretary 401/789-7711 847-8175 or 76
ADMINISTRATIVE OFFICE
Claire D. Geremia, Admin. Officer 401/789-0784 847-8175 or 76
Douglas H. Adams, Supply Clerk 401/783-0534 528-4370 or 71
William J. Egan, Lt. Vech. Operator 401/783-0534 528-4370 or 71
Saundra A. Barrington, Procurement Clk 401/789-9751 847-8175 or 76
401/789-0784 847-8175.or 76
DEPUTY DIRECTOR
Donald K. Phelps 401/789-7731 847-8175 or 76
Ross L. Johnson, Engin. Tech 401/789-1693 847-8175 or 76
Wayne R. Davis, Res. Aq. Biologist 401/789-7711 847-8175 or 76
Denise A. McNulty, Secretary 401/789-7731 847-8175 or 76
GRANT & CONTRACT COORDINATOR
C. S. Hegre 401/789-7741 847-8175 or 76
Roseann Gamache, Librarian 401/789-9753 528-4370 or 71
Mary S. Malcolm, Admin. Tech 401/789-7741 847-8175 or 76
TECHNICAL OPERATIONS
Jan C. Prager 401/789-7761 847-8175 or 76
Catherine A. Leavene, Secretary 401/789-7761 847-8175 or 76
FACILITIES
James H. Wood, Engin. Tech 401/783-0567 528-4370 or 71
401/789-8294 847-8175 or 76
Ihomas Callanan, Lab. Maint. Foreman.. 401/789-1693 847-8175 or 76
401/789-8294 847-8175 or 76
Maurice E. Hines, Lab. Maint. Man 401/789-1693 847-8175 or 76
George Cottrell, Lab. Maint. Man 401/783-0567 528-4370 or 71
Edward Weber, Lab. Maint. Man. .401/783-2004
BIOASSAY METHODS TEAM
John H. Gentile, Team Leader 401/789-6163 847-8175 or 76
Gerald E. Zaroogian, Res. Chemist 401/789-3346 847-8175 or 76
Carol G. Pesch, Res. Aq. Biologist 401/789-1272 847-8175 or 76
John A. Cardin, Res. Aq. Biologist 401/789-6163 847-8175 or 76
Suzanne Sosnowski, Res. Aq. Biologist 401/789-6163 847-8175 or 76
Mary W. Johnson, Biol. Lab. Tech 401/789-6163 847-8175 or 76
-------
Commercial FTS
OCEAN DISPOSAL TEAM
Gerald G. Pesch, Team Leader 401/789-1272 847-8175 or 76
Richard J. Blasco, Res. Microbiologist 401/789-0825 847-8175 or 76
Bruce H. Reynolds, Res. Aq. Biologist 401/789-0825 847-8175 or 76
MARINE CULTURE TEAM
Allan D. Beck, Team Leader 401/789-6465 847-8175 or 76
Paul P. Yevich, Res. Biologist 401/789-1427 847-8175 or 76
Jay C. Sinnett, Elec. Engineer 401/789-7751 847-8175 or 76
Linda A. Ferraro, Res. Aq. Biologist 401/789-7216 847-8175 or 76
Raymond L. Highland, Elec. Tech 401/789-7751 847-8175 or 76
William Giles, Biol. Lab. Tech 401/789-7751 847-8175 or 76
Carolyn Barszcz, Biol. Tech 401/789-1427 847-8175 or 76
Raymond Hennekey, Res. Aq. Biologist 401/789-7751 847-8175 or 76
OILS TEAM
Peter F. Rogerson, Team Leader 401/789-7604 847-8175 or 76
George Gardner, Res. Aq. Biologist 401/789-7751 847-8175 or 76
Sue Cheer, Research Chemist 401/789-0043 847-8175 or 76
Gregory Telek, Chemist 401/789-7711 847-8175 or 76
Richard Lapan, Environmentalist 401/789-0825 847-8175 or 76
METALS TOXICITY TEAM
Earl H. Davey, Team Leader 401/789-3346 847-8175 or 76
Ronald Eisler, Res. Aq. Biologist 401/789-0825 847-8175 or 76
Marcia M. Barry, Biol. Lab. Tech 401/789-0825 847-8175 or 76
Albert E. Soper, Phys. Sci. Tech 401/789-8383 847-8175 or 76
SYNERGISMS & RESPONSE PARAMETERS TEAM
Don Miller, Team Leader 401/789-6465
Eugene H. Jackim, Res. Chemist 401/789-0043
Richard L. Steele, Res. Aq. Biologist 401/789-7750
George E. Morrison, Res. Aq. Biologist 401/789-0043
Juan Gonzalez, Res. Aq. Biologist PR 809/832-8244
Richard A. Voyer, Res. Aq. Biologist 401/783-2004
Neal F. Lackie, Biol. Lab. Tech 401/783-2004
Frank A. Osterman, Biol. Lab. Tech 401/789-7750 847-8175 or 76
Diarme E. Everich, Res. Aq. Biologist 401/789-7751 847-8175 or 76
847-8175 or 76
847-8175 or 76
847-8175 or 76
847-8175 or 76
ECOSYSTEMS ANALYSES TEAM
Kenneth T. Perez, Team Leader... ......401/789-0825 847-8175 or 76
Neal Goldberg, Computer Programmer 401/789-7711 847-8175 or 76
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Commercial
ETS
RECREATIONAL WATER CRITERIA
Victor Cabelli, Team Leader 401/789-7721 528-4370 or 7
Morris Levin, Microbiologist..401/783-0456 528-4370 or 7
401/783-0253 528-4370 or 7
Alfred Dufour, Microbiologist.. 401/783-0456 528-4370 or 7
401/783-0253 528-4370 or 7
Donald Winslow, Biol. Tech 401/783-0147 528-4370 or 7
William Watkins, Microbiologist ..401/783-0125 528-4370 or 7
Edley Strickland, Biol. Lab. Tech 401/783-0253 528-4370 or 7
Cynthia Thomas, Microbiologist 401/7.83-0125 528-4370 or 7
Gertrude Calande, Secretary 401/789-7721 528-4370 or 7
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