WATER
QUALITY
LABORATORY
SOUTH FERRY ROAD • NARRAQANSETT, R.I.
-------
ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL MARINE WATER QUALITY LABORATORY
SEMI-ANNUAL REPORT - JANUARY-JUNE, 1974
AN ASSOCIATE LABORATORY OF
NATIONAL ENVIRONMENTAL RESEARCH CENTER. CORVALLIS, OREGON
-------
This report does not constitute publication but is for information
only. All data must be considered provisional.
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
NATIONAL MARINE WATER QUALITY LABORATORY
SOUTH FERRY ROAD
NARRAGANSETT RHODE ISLAND 02882
1 July 1974
The National Marine WateA Quality Laboratory seems to be on a never-
ending treadmill--with new problem; new priorities; and challenging
crises that always seem to require an answer yesterday'.
The Ocean Dumping Act and the. '72 WateA Pollution Control Amendments
demand technical assistance, a.4 well ah a large r&sexirch effort ftorn
oua facility. In the. past months we have completed a thorough review
of oxygen and tmpenatuAe criteAia for development of water quality
standards. This has been a majoA effort, involving one-third oj$
the laboratory. HoweveA, in addition to producing new proposed
criteria; the effort resulted in white papers on oxygen and tempeAatuAe
cAiteAia at well as a re-definition of research needs within the
laboratory pAogAam.
Oua ocean dumping research is moving along actively with excellent
extramural as well as well-coordinated inhouse programs combining
both laboratory and field woAk. Oua field reseaAch has alAeady proven
worthwhile in helping to develop permits and monitoring programs for
existing offshore dumping sited.
The oil program, though small in numbers of people doing inhoase reseaAch,
is a viable and strong AeseaAch effort that is presently delineating
the i{ate and e^ec£4 oh oil in the maAine environment. With the onslaught
of Outer-Continental-Shelf drilling, deep water ports and new coastal
oil refineAies, we (Jeel this program will be given greater emphasis by
the Agency under the new energy initiatives.
Heavy metals research is in full geaA attempting to determine the e^ect
of metals on marine organisms. At present we are developing a large
matrix with metals on one axis and effect of marine organisms along
another axis. This matrix will act as a screening process to demonstrate
areas in which we have little toxica logical information as well as those
organisms for which more research is needed.
A major portion of oua research here at the National Marine Water Quality
Laboratory is the determination of tolerable stress levels foA individual
organisms and populations of organisms in the marine environment. In the
past twelve months we have developed several new standard bioassay
methods which will go into the Standard Methods Manual and are presently
being applied to ocean dumping permits. We believe that bioassays are
not only critical for determining water quality criteria but are becoming
an integral part of the management decision process in ocean dumping
thermal effluent guidelines and hopefully in the permit program of the
Agency. The standardization of these methods and their sophistication
is a major goal of oua laboratory at this time.
xilO s Ur
'A*
4< oooll0
-------
The past six months have. been the time foA manked change in the.
labonatoAy. We aAe still Matting down within oua facility on the.
edge o(J UaAAagansett Bay, AeconfiguAing oua wet lab to suit the needs
ami AeseaAch pnionities. We aAe setting up new flow-through bioassay
system and foA the fiAst time aAe conducting long-tenm, cliAontc tests
on the effect ojf pollutants on maAine organisms. We aAe still main-
taining oua baAge at Point Judith and hopefully we will te/uninate oua
openation theAe upon the completion of oua new wet laboAatoAy within
the next two yeaAS. Oua staff Is still spAead throughout southeAn
Rhode Island In thAee locations with AeseaAch and suppoAt facilities
AequiAed in all aAeas. We cue coping with this situation as best we
can and looking foAuxxAd to the day when we can all be consolidated in
one facility with oua new building pAognam heAe on the edge of
UaAAagansett Bay.
Foa those who have questions about oua pAogiam, oua pAogAess OA oua
pAoblems, please feel fAee to call me, oa oua team leadens, who aAe
directing the AeseaAch in each of theiA nespective aneas. Even
betteA, if you aAe in the axea, please feel fAee to dAop by UaAAagansett
and enjoy oua hospitality as well as discussing oua science and oua
pAoblems. If you have suggestions, cniticisms, oa aAe just a fAiend,
please feel fAee to call.
SinceAeZy,
, Etic V, Schneiden
VinectoA
EVS'-dgg
-------
DIRECTOR
Eric D Schneider
789-3361
GRANT & CONTRACT COORDINATOR
C. S. Hegre
789-7741
?T OAS SAY METHODS TFAM
^n^ile, Teara Leader
TECHNICAL OPERATIONS
Jan C. Prager
789-7761
SCIENTIFIC COORDINATOR
Donald K. Phelps
789-7731
ADMINISTRATIVE OFFICER
789-9751
FACILITIES
James H. Wood
783-0567
OCEAN DISPOSAL TEAM
Gerald G. Pesch,
789-1272
Team Leader
METALS TOXICITY TEAM
Earl H. Davey, Team Leader
789-3346
MARINE CULTURE TEAM
Alan D. Beck, Team Leader
789-6465
SYNERGISMS & RESPONSE PARAMETERS TEAM
Don Miller, Team Leader
789-6465
OILS TEAM I
Peter F. Rogerson, Team Leader.
789-7604
ECOSYSTEMS ANALYSES TEAM
Kenneth T. Perez, Team Leader
789-7711
RECREATIONAL WATER CRITERIA
Victor Cabelli, Team Leader
789-7721 *
COMMERCIAL PHONE NUMBERS LISTED ABOVE
FTS 401/847-8175 and 8176
*401/528-4371
7/74 pmv
-------
DIRECTOR
Eric D. Schneider
Dot It C Clrtrd, See
GRANT & CONTRACT COORDINATOR
C. S. Hegre
Roseann Gamache, Librarian
TECHNICAL OPERATIONS
Jan C Prager
SCIENTIFIC COORDINATOR
Donald K Phelps
ADMINISTRATIVE OFFICER
Claire Geremia
Mary S. Malcolm, Admla. Clerk
Catherine A. Leavene, Sec.
FACILITIES
James H. Wood
Denlse A. McNulty, Sec.
Sauadra Sarrlngton, Froc. Clk.
Douglas B. Adams, Supply Clerk
Ullliaa J. Egan, Laborer
Robs L. Johnson, Eng. Tech.
Thomas Callanan, Fac. Eng. Foreman
George Cottrell, Fac. Malnt Mech.
Maurice E Hlnes, Plumber
James Duff, Bldg. Maine. Man
Edward Weber, Bldg. Malnt. Man
BIOASSAY METHODS TEAM
John H. Gentile, Team Leader
Gerald E Zarooglan, Res. Chemist
Carol ,G. Pesch, Microbiologist
John A. Cardln, Biol. Aide
Suzanne SoBnovskl, Biol. Lab. Tech.
Mary E. Johnson, Biol. Lab. Tech.
OCEAN DISPOSAL IT AM
Gerald G Pesch, Team Leader
Richard J. Blasco, Res. Microbiol.
Bruce H. Reynolds, Res. Aq Biol.
METALS TCKTCTTY TFAM
Earl H. Davey, Team Leader
Ronald Elsler, Res Aq. Biol
Marcla M Barry, Biol Lab. Tech
Albert E. Soper, Phys. Scl. Tech
Gregory Telek, Chemist
Richard L. Lapan, Phys. Scl. Tech.
MARINE CULTURE TEAM
Alan D. Beck, Team Leader
Paul P. Yevich, Res Team Leader
Linda A Ferraro, Biol Lab. Tech
Raymond L Highland, Electronics Tech.
William Giles, Biological Aid
C. A. Barezcz, Biol. Aide
Raymond Hennelty, Biol. Aide
SYHERCIS?tS & RESPONSE PARAMETERS TEAM
Don Miller, Team Leader
Eugene H. Jackim, Res Chemist
Richard L. Steele, Res. Aq Biol.
George E. Morrison, Res. Aq Biol.
Juan Gonzalez, Res. Aq. Biol.
Sue Cheer, Biologist
Richard A Voyer, Res Aq. Biol.
Neal F. Lackie, Biol Lab. Tech
Frank A. Osterman, Biol. Lab Tech.
Dlanne E. Everlch, Biol. Lab. Tech.
OILS TEAM
Peter F Rogerson, Team Leader
George Gardner, Res. Aq Biol.
ECOSYSTEMS ANALYSES TEAM
Kenneth T Peres, Team Leader
Neal Goldberg, Computer Sys. Analyst
RECREATIO"AL WATER CRTTTRIA
Victor Cabclli, Team Lcuder
Gertrude Calande, Sec
Alfred Dufour, Microbiologist
Maurice Levin, Microbiologist
Edley Strickland, Biol. Tech.
Donald Wlnslov, Biol. Tech.
Cynthia Thomas, Microbiologist
William Watklns, Microbiologist
-------
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
communities, trophic levels, and feeding types are selected so as to
permit evaluation of the widest possible impact. While the emphasis of
this program has been on short-term techniques, we have expanded to
include whole life cycle with a strong emphasis on the use of sensi-
tive early life history stages. Our program utilizes species with
short life history which permits evaluation and comparison of short-
term and reproductive effects which are necessary for projections on
population viability.
The assay program is also strongly directed toward the problems of
bioaccumulation and the significance of organisms body burden to
both public health standards and the effect on reproduction and pop-
ulation integrity. More recently we are evaluating mixed effluents
and their effect on planktonic communities (particularly ocean disposal).
Bioassays designs are 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 phyto-
plankton, zooplankton, fish larvae, and polychaetous annelids (ROAP 21 AKK-
-1-
-------
BIOASSAY METHODS TEAM
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 10); and
(3) evaluate the performance of long-term bioassay systems, (ROAP 21AKK,
Task 12).
ROAP 21 AKK, Task 07 - Develop and Evaluate Short-Term Bioassay Techniques
Phytoplankton
The short-term algal bioassay program concentrated on using mixed
wastes and modifications of methodology. The primary emphasis was
comparison of two synthetic seawaters; the commercially available
Instant Ocean and the modified Kester's medium. We had already
established that there was considerable difference in the response of
Thalassiosia pseudonana to heavy metals in the two media under identical
nutritional conditions. The following results focus on toxicity of
mixed metals, sodium dodecyl sulfate, and an unidentified waste material
that is being dumped offshore. Table 1 represents partial data from
these studies.
Table 1. Comparison of growth rates (k) of Thalassiosia pseudonana
to sodium dodecyl sulfate and a mixture of heavy metals.
Cone, mgs SDS/1 Kesters Instant Ocean
zero 3.54 2.10
0.5 3.18 1.56
1.0 1.82 0.29
2.5 0.17 0.16
Dilution of Mixed Metals
control 3.11 3.35
10, 2.98 3.45
10j 2.89 2.83
10 0.86 2.25
undiluted 0.25 0.11
-2-
-------
BIOASSAY METHODS TEAM
3
Studies on unidentified mixed wastes indicated that in at 10 dilutions
A
complete inhibition of growth was noted in both media. At 10 dilutions
there was a 50% greater depression in growth rate in Instant Ocean
than in Kester's, which was almost identical to the control. Studies
of Skeletonema costatum sensitivity to mixed metals indicate that in
Kester's medium only the undiluted mixture was inhibitory to growth.
At 10^" dilution Skeletonema showed no decrease in growth rate whereas
Thalassiosira was 60% inhibited.
During the period covered in this report a manuscript was prepared on
marine phytoplankton bioassay procedures for publication by Marine
Science and Technology. This was a product of their symposium on
Marine Bioassays. In addition, a review and comments on the Marine
Algal Assay Procedure for Standard Methods was submitted to Dr. Tarzwell.
Ichthyoplankton
Short-term static bioassays on newly hatched winter flounder (Pseudo-
pleuronectes americanus) ware performed on copper, mercury, zinc, cadmium,
nickel, and cyanide. These studies were designed to develop levels that
could be used for chronic exposure studies but more importantly to
standardize the methodology. In this series 240 larvae were used as
controls with only 15 deaths after 96 hrs (6.2%). This lends consider-
able support to the feasibility of using early life history stages of
economically important species as bioassay organisms. This methodology
is in draft form, and under the guidance of J. Cardin was used success-
fully by TRIGOM, Inc. Marine.
-3-
-------
50
1.0
03
40
OB
*— 0-7
0.6 O
Q.30
Q.
— 0.5
"O
"O
20
o
0.3
0.2
5.0
o
25
Time - Days
The LC-50 isopleths for Fundulus heteroclitiis larvae chronically exposed to
Cd+2 (0) and Cu+2 (•).
-4-
-------
BIOASSAY METHODS TEAM
Using the larvae of Fundulus heteroclitus, two-week chronic exposures
+2 +2
to Cu and Cd were successfully performed (Fig. 1). The data
indicates incipient lethal limit to 50% of the population to be 0.15
+2 +2
mgs Cu and 1.0 mgs Cd /l. Adult Fundulus have 96 hr LC^qS of 10
mgs Cu+^/l and 55 mgs CD+^/.l.
This graphically illustrates the difference in sensitivity between life
stages and the further success of using larval forms.
+2 +2 +2
Long-term continuous exposure of Fundulus larvae to Cu , Cd , and Hg
are underway to further evaluate exposure systems, test species response,
and wet lab facilities.
Polychaetes
Validation of culture and bioassay procedures prepared by Donald Reish,
EPA Grant 800962, was started. Specimens of Capitella capitata were
received on February 25, 1974. Static cultures of this species were
set up according to Reish's procedure manual. Certain aspects of the
technique require modification.
In addition to the culture techniques, a procedure for bioassays was
also evaluated. Two 96 hr static bioassays were conducted on adult
+2
Capitella using Cd as the toxicant. Responses obtained were within
the range reported by Reish.
A polychaete short-term bioassay procedure being prepared for Standard
Methods (14 ed.) was reviewed. A revised format was suggested to
Dr. Tarzwell.
-5-
-------
BIOASSAY METHODS TEAM
A prototype culture system employing flowing filtered seawater was
set up as an altertiative to the static system. This system is pre-
sently being tested to determine if it will support the complete life
cycle of this species.
A manuscript of Cd^"^ uptake in the food for Fundulus heteroclitus
was completed and reviewed by Drs. G. Pesch and J. Gentile. A few
additional experiments were suggested and subsequently conducted at
the Rhode Island Nuclear Science Center. The revised manuscript has
been prepared and submitted to the Fisheries Research Board of Canada
for publication.
Zooplankton
Our program involving development of culture and bioassay systems for
Indigenous marine calanoid copepods (Acartia tonsa) was adapted to
the cogeneric winter form, Acartia clausi. The culture system, when
operated at 10°C, worked well permitting us to perform bioassays on
adults of this species of uniform physiology. Material examined in-
cluded six heavy metals and cyanide. The 96 hr LC,.Q,g are tabulated
as mgs/1.
Species/Toxicant Hg+2 Cu+2 Cd+2 Zn+2 Ni+2 Cr+6 KCN
Acartia clausi 0.011 0.046 0.23 1.4 2.85 6.5 0.096
A long-term bioassay of cadmium using Acartia clausi shows dramatically
the effects of increased exposure on adult survival (LC50; mgs/1).
Exposure Time (Hours)
96 168 192 216 240
Acartia clausi 0.23 0.058 0.0085 0.0045 0.002
-6-
-------
BIOASSAY METHODS TEAM
In a continuing effort to design bioassays to reflect in situ
exposure patterns a series of mixed effluents that are being ocean
dumped were studied. Since there are potentially both immediate and
protracted impacts from this type jof operation, responses of the test
organism were evaluated at 1, 4, 8, 12, and 24 hours post exposure and
then at 24 hr intervals for 96 hours. Data from this type of assay
has direct application in determining a dumping rate which coupled to
expected dispersion rate will reduce immediate impact. The longer
exposures permit evaluation of potential long-term effects.
In conjunction with our Ocean Disposal Team, analyses of zooplankton
populations were performed on two stations in a dump area off the
mid-Atlantic states (Table 1).
A culture of the marine rotifer, Brachionus plicatilis, was established
in cooperation with the Culture and Holding Team. This organism
is used as a primary food source for many fish larvae. We are also
looking into the possibility of using it as a bioassay organism.
ROAP 21 AKK. Task Oil
This task focuses on establishment of design criteria and fabrication
of long-term exposure systems.
Design criteria and a prototype exposure system for lobster larvae
and semi-microorganisms (100-1000 micron) have been completed. This
system permits the examination of organisms microscopically when
necessary by removal of individual chambers from the exposure tank.
-7-
-------
TABLE 1.
Summary Sheet for Ocean Dumping Data
T°W SPECIES z o£ tQW
Station 14
Oct. surface
Station 17
Oct. surface
Grice & Hart
Sept. oblique
Bigelow & Sears
Oct. oblique
Psuedocalanus minutus elongatus
Temora turbinata
Centropages typlcus
*Salps
Pseudocalanus nunutus elongatus
Temora turbinata
Temora longicornis
Oncaea venusta
Centropages typicus
Centropages typlcus
Centropages typicus
Paracalanus
Calanus
Pseudocalanus
Metrldia lucens
39.7
32.3
19.6
37.7
20.1
19.0
8.0
4.5
50.0
14.0
12.0
7.0
4.0
2.0
*Salps are indicative of warm and/or oceanic waters
none in Station 17 sample.
There were
-8-
-------
BIOASSAY METHODS TEAM
In addition the chambers can be readily mounted into a variety of
troughs and tanks where macroorganisms are being exposed.
Exposure systems for larval, juvenile, and adult fish have been designed
and are being tested. In addition we plan to participate in an inter-
laboratory comparison of a bioassay system designed for the the sheeps-
head minnow, Cyprinodon varieagatus. This system was designed at Gulf
Breeze Laboratory and permits the complete life history to be evaluated
in the laboratory.
A continuous flow culture system with automatic feeding capabilities
was built for the calanoid copepods. Initial studies with Acartia
tonsa achieved excellent growth and survival. This system is now being
modified for whole life cycle bioassay studies.
ROAP 21 AKK. Task 12 - Evaluate Performance of Long-Term Bioassay Systems
Lonft Term Exposure of Oyster to Cadmium
A study was started on November 12, 1973 to determine the kenetics
+2
of bioaccumulation of Cd in oysters (Crassostrea virginica) during winter
and summer conditions; also, to determine if the cadmium accumulated
by the oysters has any effect on spawning and fecundity.
+2
The oysters were exposed to 5 and 15 ppb Cd in fiberglass troughs
using the continuous-flow seawater system in the wet lab. Ambient sea-
water temperature and salinity were maintained throughout the exposure
period. These oysters, along with controls, were sampled bi-weekly and
the total soft parts were prepared for Atomic Absorption Spectrometry
and analyzed for cadmium and copper.
-9-
-------
BIOASSAY METHODS TEAM
The 20 weeks winter exposure period ended on April 10, 1974. Throughout
the studies, samples have been examined histopathologically.
Exposure levels are monitored by using the Chelex technique of Dr.
Davey.
Oysters continued to concentrate Cd in spite of diminished pumping
and feeding during the cold seawater temperatures of the winter months.
Before exposure, cadmium levels in the oyster meats were 2.72 ppb wet
wt, whereas at the end of the 20 week exposure period, a mean Cd level
of 9.65 ppb was obtained in the wet meats of oysters exposed to 15 ppb
+2
C and 2.82 ppb for the controls. This trend is similar to the one
+2
found in a previous study where oysters were exposed to 10 ppb Cd
under similar conditions.
Histopathologic examination has shown that the reproductive tract of
the Cd exposed animals is slower in maturing than the oysters exposed
+2
to 5 ppb Cd and the controls.
There oysters are being analyzed for copper also, since they are
laden with copper when harvested. Therefore, we want to determine if
Cu is depurated in clean water and if Cd competes with Cu for sites
within the tissues. It appears at the time that Cu is not depurating,
since the controls at the onset of exposure contained a mean Cu concen-
tration of 198 ppm per whole wet meat and after 20 weeks the oysters
+2
exposed to 16 ppb Cd contained 203 ppm Cu; whereas, the controls
contained 116 ppm. This data is preliminary and the variation in copper
amongst the oysters is great; therefore, even though the data appears to
show that some depuration occurs, when analyzed statistically there might
-10-
-------
BIOASSAY METHODS TEAM
be no significant difference between the values.
The spring-summer phase of the long-term exposure of oysters to 5 and
+2
15 ppb Cd is continuing. Samples are being taken weekly during
this phase of the study, since it was shown in a previous study that
greater metal uptake occurs during this period thereby allowing the
kenetics to be studies more precisely.
During the course of this study, a natural set of barnacles (Balanus
balanus) and mussel (Mytilus edulis) occurred. It was interesting to
observe that the cadmium levels in the troughs appeared to have no
effect on setting and growth on the mussel; whereas a heavier set of
+2
Balanus appeared to occur in the troughs containing 5 ppb Cd than in
+2
the control troughs. But at 15 ppb Cd the set of Balanus was less than
controls.
+2
In conjunction with the long-term exposure to Cd , fecundity studies
are being performed at the time of spawning.
ROAP 21 AKK, Task 014 - Grant 802871 - The Development of Continuous
Flow Systems Suitable for Bioassay of Larval
Arthropods and Test Measures of Response for
Variance and Reliability - University of
South Carolina, Belle Baruch Inst., Winona
Vernberg, Principal Investigator
The first six months of this grant have been devoted to developing
a flow-through system and response delineation of Paleomonetes. This
system has permitted the successful rearing from hatching to adult.
A duplicate system is presently being assembled, and the environ-
mental chamber will be operational within one month.
-11-
-------
BIOASSAY METHODS TEAM
Studies on Paleomonetes in static systems indicate that toxicity
3
was size dependent and sex independent. Data were collected on a 3
factorial matrix of temperature and salinity with and without cadmium.
Metabolic studies (oxygen consumption rates) on control and experimental
groups of males and females. Results indicate differential response
between exposed and control males but not among females.
Preliminary screening of various behavior patterns (feeding, locometor
activity, predation vulnerability) was carried out to find indicators
which were suitably sensitive to sublethal levels of cadmium.
Technical Assistance
Considerable effort was expended on data gathering for Temperature Cri-
teria. This effectively consumed approximately one month of two
technicians' time. In addition, intensive rounds of meetings on
this subject involving senior staff further cut into research and this
team leader's functions.
Peripheral involvement in affidavit preparations for hearings and revision
of Blue Book Criteria were further responsible for another month of
team leader effort.
ROAP preparation and NERC program review completed extra-curricular
activities.
A comparison of the sensitivity of Artemia salina to indigenous marine
species is being prepared for Region II permit program. This involved
-12-
-------
BIOASSAY METHODS TEAM
the performance of numerable bioassays on Artemla, fish larvae, copepods,
and phytoplankton.
A series of bioassays on winter flounder larvae and Acartia tonsa were
performed to evaluate cyanide toxicity. This data was requested by
Dr. Rogerson for an affidavit on cyanide toxicity in the marine environ-
ment .
Conferences/Documents
1. Drs. Gentile and Cheer participated in EPA workshop at ATP, April
22-24, at Ada, Oklahoma. Seminar and demonstration of techniques
and applications were presented.
2. Carol Pesch completed draft manuscript on the uptake of Cd115
via food by Fundulus heteroclitus and revised polychaete bioassay
procedure for Standard Methods.
. 3. Methodology for fish larval bioassays was prepared and used successfully
by TRIGOM. John Cardin visited their laboratory and consulted on
the use of this procedure.
4. Drs. Zaroogian and Cheer have completed manuscript on cadmium
accumulation in oysters.
5. Dr. Zaroogian attended National Shellfish Sanitation Workshop,
New Orleans. Standard setting procedures for analyzing and
monitoring shellfish for metals.
6. Dr. Zaroogian attended a chemistry task force of the NSSA to prepare
a handbook on standard methods for analyzing shellfish for metals.
Gloucester Pt., Virginia.
7. Suzanne Sosnowski prepared a manuscript on the quantitative and qual-
itative distribution of zooplankton at Region II ocean dumpsite.
Technical Report.
8. Dr. Gentile, S. Sosnowski, and John Cardin prepared zooplankton
bioassay procedure for Standard Methods and MST-Bioassay Symposium.
9. Dr. Gentile and M. Johnson prepared a manuscript on phytoplankton
bioassay procedures for MST-Bioassay Symposium.
-13-
-------
BIOASSAY METHODS TEAM
10. Dr. Gentile attended NERC-Corvallis conference in March.
11. Dr. Gentile and S. Sosnowski visited Dr. Heinle at Solomon's
Island, Virginia to collect Acartia tonsa and discuss problems
of copepod culture.
12. Dr. Gentile, N. Lackie, and S. Cheer prepared a draft manuscript
on the effects of entrainment on microzooplankton.
13. Dr. Gentile prepared a report on entrainment studies of
phytoplankton for Martin-Marietta.
-14-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
What is the long-term effect of a pollutant on a population? Is growth or
reproduction retarded, or are behavioral responses associated with feeding
or avoidance of prey destroyed? The development of biological parameters to
evaluate the effects of sublethal stress is one responsibility of this
research team. The other charge is to elucidate how a contaminant may effect
an organism under the full range of its natural environmental conditions.
The estuarine environment, for example, characteristically experiences wide
fluctuations of a number of environmental factors. Annually the temperature
may range over some 20°C; daily it can fluctuate over 4°C. Estuarine waters
vary from nearly fresh to full oceanic salinities. High turbidity is a
dominant feature during periods of storm or high runoff from upland regions.
Oxygen is naturally depleted in poorly circulated waters during the summer.
How do such conditions influence the rate of uptake of a contaminant by
organisms or effects its toxicity following exposure? These two questions
are also primary concerns of this team.
I. Response Parameter Development
A. Development of Methods to Evaluate Nutritional and Physiological
Condition in Laboratory Held Animals (RIAP 21 AKF):
TASK 002 - Establish collection, culture, holding and rearing techniques,
preliminary ecological requirements, including minimum, optimum,
and maximum range for temperature, dissolved oxygen, salinity,
turbidity and nutritional requirements for successfully cultured
organisms in support of this ROAP and 21-BBG, 16-AAT, 21-AKK
and 16-AAV.
TASK 021 - Develop and modify technology necessary for verifying physiological
state, nutritional state, histopathology, and recent history.
The nutritional state of an animal reflects a basic response to his total
environment. Liver stores indicate a balance between energy availability and
demand. A chronic response to stress may result in a greater demand than
energy availability warrants, resulting in decreased nutritional stores.
-15-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Measurement of energy stores may then be appropriate as a response parameter
to long-term environmental stress. Similarly, growth rate may express degree
of chronic stress experienced by a test animal.
This project was initiated to evaluate several methods for monitoring
nutritional stores as well as growth rate for use as response parameters to
measure effects of chronic stress. An immediate spin-off from this effort
is to provide methods for the Culture Team to evaluate suitability of labora-
tory holding systems to meet the physiological requirements of experimental
material. We must be able to measure how similar our experimental organisms
are to populations in the field as well as separate, and eventually minimize
effects of laboratory holding from those of the experimental treatment.
Two species were used for the initial studies: a fish, Atlantic silversides,
(Menidia menldia) and the surf clam (Spisula solidissima). These species
have been identified by our laboratory as having high potential for water
quality research and represent contrasting life modes (sedentary V£. active
swimmers) and feeding types (filter feeders \re. graptorial feeders). Animals
were held in the laboratory under markedly different feeding regimes selected
to provide clear nutritional differences. Groups were subsampled periodically
over a 30- or 60-day period and compared with freshly collected field animals.
1. Evaluation of Nutritional Laboratory Holding Conditions for Atlantic
Silversides.
Analyses were conducted by E. Jackim and S. Cheer on nearly 300 Atlantic
silversides (Menidia menidia) representing two laboratory holding regimes
(fed and non-fed) plus freshly collected field animals. The simple determina-
tion of liver to body wet weight ratios correlated well with liver
-16-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
glycogen values (Fig. 1). This weight ratio is advanced as one useful
indicator of nutritional stores and can be employed routinely to evaluate
whether the fish's energy needs are being met or exceeded. In this study,
total lipids, protein and water content of the livers were also determined
for the three groups of fish. While t hese components also reflected
shifts in direct relation to the amount of food available, the correlation
was not consistently was not consistently as close as with glycogen.
Suitability of the total environment, to maintain an animal's physiological
condition is indicated in part by growth. Nucleic acid ratios can be
related to tissue synthesis and hence to growth. RNA and DNA levels for
muscle tissue were determined during this study with Menidia. Field popula-
tions showed slightly greater growth than the laboratory group which
received food, even though the latter had greater stores of liver glycogen.
Laboratory starved fish showed very little growth by the RNA:DNA method,
as expected. Determination of the DNA:RNA ratio as a measure of short-term
growth is suggested as an additional parameter to determine whether the
physiological needs of an organism are being met. This ratio can be
employed as a criterion to evaluate stress effects of a contaminant on growth
during a bioassay. The new ethidium bromide fluorometric assay for RNA
and DNA (Prasad, e£ al, 1972) used in the present study was dependable,
straight-forward, and relatively rapid. RNA and DNA values for ten fish
can be determined in under 2 hours.
2. Evaluation of Nutritional Condition in Laboratory Held Surf Clams.
A study to develop response parameters and to evaluate the effects of
laboratory holding of the surf clam, Spisula solidissima, was conducted by
-17-
-------
4.6
4.0
FED
H
^ 3.0
>-
Q
O
CD
tr
LU
>
N=I6
H
C0NTR0L
LJ
O
cr
LU
Q_
UNFED
14,15
DAY
22
28,29 34
Fig. la. Liver to body weight ratios for field control Co), fed (a),
and unfed (&) fish,
-18-
-------
600
LlI
k-
o
£ 450
ct>
e
~z.
LlI
O
O
^ 300-
FED
_J
O
N= II
150
CONTROL
90
30
UNFED
7,8
14,15
22
28,29
34
DAY
Fig. lb Liver glycogen per unit protein for field control, fed, and
unfed fish.
-19-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
G. Morrison, N. Lackie, S. Cheer, and E. Jackim. Clams were held in
three feeding regimes (starved, natural unfiltered seawater, and supple-
mentarily fed with Skelatonema costaturn) for sixty days. Groups were
compared with a field population at twenty day intervals. The parameters
considered were shell length/body mass dry weight ratio, glycogen/protein
ratio, DNA/RNA ratio, lipid content, general histology, physiological
indices (Gill cilia beat frequency), and analysis of fecal matter for possible
accumulation of the F/2 algal medium enrichments.
A mean weight loss of 7 to 22% among laboratory animals was evident by day
20, compared with freshly collected field animals. By day 40, clear
differences in weight were apparent between the three contrasting feeding
regimes in the laboratory. These differences, expressed by using shell
length/body mass dry weight ratio, are detailed in Figure 2. Histological
examination showed field animals to be in a more advanced reproductive
condition than laboratory organisms. Gonadal differences between and
within laboratory treatments were also evident, with the reproductive
tract more advanced in the better fed clams. Laboratory held animals
which received supplementary feeding were nearly comparable to field
animals in extent of digestive cell granulation in the digestive di-
verticula. This was not true for those feeding from natural seawater or
those maintained in filtered seawater.
Dry weight of the visceral mass of a bivalve, expressed in relation to
the animal's shell length was the best criteria found during this study
for general evaluation of biological condition. Histological
determination of reproductive condition is suggested as a good method
-20-
-------
i H3 y I
i ye
1 I /27
2/^ I 1
n
F-L.O I
r-i_o i
¦l_D I
a
r^_
4J
<0
jC
60
•H
0)
3
M
P
.c
CO
IS
e
fVi.
l/i
r-
l/»
ri
q
3
NsS.
Ne 1 SB.Ne I IZI.Ne 1 CS.NJcS.
Me I GO.NeS. Nle 1 B.Nb I 0.
Ne I CS Me 1 03.Ne 1 E5.M®
FIGURE 2. Shell length/viscera dry weight ratios (mn/gm) of surf clams (Spisula
solidissimal) - Maintained with three different feeding regimes and
samples at three week intervals. Laboratory holding began 10/18.
FLD = Freshly collected Field animals; 1 = supplimentary fed;
2 = unfiltered seawater; 3 = filtered sea water.
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
to evaluate overall condition of laboratory held bivalves relative to field
populations, at least during the fall and winter when this study was
conducted. Histological assessment of the digestive diverticula is of less
value, as it only indicates that food has been ingested and taken up by
the digestive cells. Sections of the diverticulum provide little indication
of the long-term quantitative or qualitative adequacy of the laboratory
diet.
Analysis of fecal matter showed accumulations up to 50% greater levels
of the media enrichment metals (Zn, Co, Fe, Cu, Mn, and Mo) in those organisms
receiving natural non-filtered seawater; compared to metal level in feces of
clams receiving supplementary feeding. The reason for these differences
in trace metal accumulation is not clear at present. Variations in total
body weight or histological condition with feeding regime were not re-
flected in any of the biochemical analysis of total visceral and muscle
glycogen, lipid, protein and nucleic acid content in the nearly 300 animals
analyzed. It should be noted, however, that activity was thermally depressed
during the period of this study. While quantitative nutritional indices,
such as glycogen content, did not provide any indication of condition during
this study, they might prove useful during warmer seasons when bivalves are
more metabolically active.
B. Feasibility Studies to Evaluate Swimming Behavior in Larval and Juvenile
Fish to Indicate Sublethal Stress (ROAP 21 AKK:
Task 08 - Develop and validate short-term and long-term methods for
bioassay of the effects of metals on carnivorous, herbivorous and
plankton feeding fish, bivalve molluscs, gastropod molluscs, rooted
aquatic plants, attached, algae, arthropods, burrowing worms,
corals, communities of benthic microflora and naturally occurring
communities of the above (being implemented by ROAP 21 AKK 1B1022).
-22-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
1. Responses of Schooling Fish to Toxicants
A study of the sublethal effects of toxicants on small schooling fishes
is being conducted by K. Koltes. The experimental system consists of
four circular chambers, 2 1/2' in diameter by 6 1/2" deep, each with a
12" opening to a central chamber. Free movement can occur between
chambers. A grid on the white plexiglass bottom facilitates description
of school position. A one-way mirror located above the tanks permits
observation by the experimenter without detection. The experimental system
is housed in a sound-proof room, with light photoperiod and water temperature
controlled. Filtered seawater enters the delivery stand pipe at the far
side of each peripheral chamber and flows through the tank to a drain in
the central chamber. Toxicants are metered by a peristaltic pump into the
system. Circulating pumps create sufficient current to disperse the
toxicant uniformly within a few minutes and minimizes transition time
between the initial control period to introduction of toxicants.
Initial observations with small schools of juvenile silversides,
Menidia menidia, indicate detection of 15 ppb cadmium by a breakdown
in the schooling bond and reduction of swimming speed. Based on these
preliminary studies, the above two parameters are being used to measure
toxicant induced stress using a matrix in the Markovian process. The
focus will now be to determine threshold concentrations required to elicit
these responses for a variety of toxicants.
2. Development of an Experimental Chamber and Video System to Study
Swimming Behavior of Larval Fishes
A closed-circuit video system has been set up by Dianne Everich to study
locomotion in larval fish exposed to various toxicants and environmental
stress. Preliminary recordings, using juvenile Fundulus heteroclitus,
-23-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
have aided in identifying equipment problems and to elucidate quantifiable
behavior. Swimming speed and distance, rate of turning, time spent in
specific areas of the tank, and percentage of time resting are examples
of easily quantifiable behavior. An environment-control box has been con-
structed to house the video camera and test aquaria to reduce noise
level and control water temperature and photoperiod. A search of the
literature on behavior in larval and post-larval fish is continuing.
C. Extramural Research Supporting Response Parameters Development
1. ROAP 21 AKF, Task 027
Develop and mcdify technology necessary for verifying physiological
state, histological history, and environmental history through research
grant (Neomysis americana, Acartia tonsa, Uca Pugnax, Menldia menidia).
Grant to Dr. S. Barbar, Lehigh University (D. Miller, Project Officer)
Upon completion of the first year's work, research emphasis has shifted
to center on one of the initial four study species, the mysid shrimp
(Neomysis americana). Initial stresses to be studied are temperature
and cadmium. Sites of uptake and accumulation of the metal will be
determined using the electron probe. Effects of these stresses, at sublethal
levels, will be evaluated from the behavioral standpoint.
2. ROAP 21 AKF, Task 035 - Determine effects of temperature and current
on the survival and behavior of larval marine and estuarine fishes
801032.
Grant to Dr. R. Stevenson, University of Miami (D. Miller, Project Officer)
Techniques to rear larval marine fishes in the laboratory have been perfected
and multiple current chambers are developed to test larval swimming stamina
under various thermal regimes. Larvae evaluated to date by this response
parameter include anchovy (Anchoa mitchilli) and sea bream (Archosaugus
rhomboidalis).
-24-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
3. 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).
This project has developed capabilities for holding winter flounder for
extended periods in laboratory recirculating seawater aquaria. This
report period saw the perfection of an acrylamide gel technique for separating
fish serum proteins. Several methods for measuring 0£ transport were also
adopted from the Maine Medical Center and a series of O2 dissociation
curves for winter flounder blood generated. The hematological study
was extended to obtain baseline patterns and establish seasonal changes
over a years time. Other accomplishments were to perfect a pH/gas analysis
technique for winter flounder blood and develop a flounder metabolism
chamber. Two manuscripts reporting on the hematological and 02 dissociation
work have been completed.
II. Environmental Effects
The responses of organisms to contrasting environmental regimes was studied
following cadmium exposure for the common mummichog (Fundulus heteroclitus)
and the surf clam (Spisula solidIssima). Also, the requirements of a dominent
estuarLne macroalga for an array of environmental factors has been elucidated.
Each of these studies illustrate how certain environmental factors can
appreciably Influence organism response to a test pollutant. Such facts
must be taken into account in designing and conducting bioassays, in using
bioassay results to develop water quality criteria or In writing discharge
permits.
-25-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
A. Influence of Nutritional Condition on Metal Uptake and Toxicity in
Marine Animals
ROAP 16 ATT, Task 008 - Develop and validate short and long-term bioassay
methods for metals effects on carnivorous, herbivorous, and plankton-
feeding fishes, bivalve and gastropod molluscs, rooted aquatics, crustaceans,
worms, corals, benthic communities of microflora and naturally occurring
communities of above (being implemented by ROAP 21 AKK, 1BA022).
In order to determine how laboratory feeding affects uptake and toxicity
of a metal, cadmium was added to one set of experimental tanks during
the nutrition evaluation study of the surf clam, Spisula solidissima.
Exposure was for 34 days at an initial concentration of 0.74 mg/1 cadmium
chloride. Dosed animals were examined for accumulation of Cd and F/2
algal media enrichment metals in both the body and fecal matter, the effects
of temperature stress on gill ciliary activity and their general histological
condition.
Cd-exposed animals provided with unfiltered seawater as their only food
source exhibited more extensive and more frequent histopathological
changes in the digestive diverticula, red gland, and auricle than did
the supplementary fed group. All cadmium-exposed animals showed some
necrosis and sloughing of the kidney mucosa with amebocytic infilteration
in the cells of the glandular mass of the kidney. The histopathologies
of the red gland and auricle have not been seen before in molluscs exposed
to cadmium and their significance is not known. Interestingly, animals
sacrificed immediately after the 34-day cadmium exposure showed less
pathology of the digestive diverticula than animals maintained in non-
contaminated seawater for a 30-day post-exposure period. This points to
the importance of considering latent effects of a toxicant.
-26-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Levels of the F/2 enrichment metals found In the bodies and feces were
generally lower in all cadmium exposed animals (fed and non-fed) com-
pared to the non-Cd exposed controls. Yet, regarding cadmium accumulation
per se, there were no significant differences in cadmium concentration
in the total viscera or in the fecal matter between fed and non-fed
animals. Also, there were no clear differences in gill ciliary
activity between laboratory treatments of Cd-dosed v£ non-dosed animals,
or when compared to field animals, when heat stressed at 33°C.
Development of gut and kidney histopathologies was the principle conse-
quence of cadmium poisoning in this study. Food availability was found to be
an improtant variable influencing development of these histopathologies
in bivalves. Further research is required to determine how and why feeding
influences toxicity to cadmium and other contaminants. Is this relation-
ship between food and Cd induced histopathologies peculiar to filter
feeding organisms, such as the bivalves, or might this response occur
generally with poor nutritional condition in graptorial feeders as well?
Further clarification of these points has considerable relevance to all
long-term bioassay work.
B. Influence of Temperature, Salinity, and Dissolved Oxygen on Metal
Toxicity in Marine Animals.
ROAP 16 ATT, Task Oil - Conduct state-of-art short-term bioassays of metals
toxicity using mixtures of single elements to fish, arthropods, molluscs,
and phytoplankton; specifically to identify synergisms or antagonisms be-
tween metals and effects of physiochemical regime on short-term responses.
Provide interim estimates of safe levels for various life stages. Identify
levels and water quality parameters to be used in long-term tests. Fishes:
Menidia, Fundulus, Pseudopleuronectes; crustaceans: Palaemonetes, Acartia;
Phytoplankton: Skelotonema, Thalassiosira.
-27-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
1. Importance of Uptake Route on Cadmium Toxicity in Fish
When mummichogs (Fundulus heteroclitus) are exposed to cadmium in seawater^
the initial and most pronounced lesions occur in intestines. Only incon-
sistent pathology is evident in gills. Concentration of the metal is found
to be greatest in visceral tissues. The fact that the gills are little
affected is also attested to by tolerance of these fish to reducted
dissolved oxygen to about 4 mg/1 within 96 hours at salinities between 10
and 32 °/oo. In contrast, responses of freshwater fishes exposed to acutely
toxic concentrations of heavy metals include widespread necrosis of gill epi-
thelium, decreased resistance to poisoning at reduced levels of dissolved
oxygen, and concentration of metal in gills.
Such dissimilar patterns of response between freshwater fishes when exposed
to various metals and mummichogs subjected to cadmium in seawater suggest
different avenues of entry by metals. This is possibly associated with
contrasting osmoregulatory mechanisms of marine and freshwater fishes.
Water balance, for example, in the latter group of fishes involves inflow
of water through semipermeable gill and oral tissues, with little water
ingested. Marine and estuarine fishes, in contrast, drink water to compensate
for the dehydrating effects of the medium in which they live.
If this hypothesis is accurate, one would assume a species capable of living
both in fresh and full strength seawater will also show these same contrasting
responses to a metal. This question was tested with the mmranichog, which is a
holeuryhaline osmoregulator and capable of acclimating to freshwater.
Specific effects evaluated were tolerance to reduce dissolved oxygen
-28-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
stress and the nature of hlstopathologies resulting from short-term
exposure to cadmium while this fish was maintained in freshwater.
Two 96 hour acute-toxicity bioassays were conducted at 20°C based on
a completely randomized block design with a factorial arrangement of
treatments (4 cadmium concentrations and 3 dissolved oxygen levels).
Each of 12 treatments was replicated twice with 5 fish exposed per
replication.
Results after 96 hour exposure periods showed a decrease in time to
achieve 50% mortality with decreasing ambient oxygen concentration.
Mean 96-hour TL-50's were 3.0, 1.9. and 1.3 mg Cd/1 at dissolved
oxygen levels of 8.5, 4.1, and 2.3 mg/1, respectively. Analyses of
variance indicate responses of mummichogs were significantly (P <0.01)
altered by concentrations of cadmitun and dissolved oxygen as well as
by time of exposure. Analyses also show that interactions between
cadmium and dissolved oxygen and between cadmium and time significantly
influenced responses as well.
Significance of cadmium, dissolved oxygen, and time is also evident
graphically (Fig. 3). Response trends clearly show rate and extent of
mortality to be a function of the independent variables studies. For
example, a maximum mortality of 45% occurred only after 96 hour among
mummichogs at 2.9 mg Cd/1 and 8.5 mg DO/1. In comparison, at the same
cadmium concentration and 2.3 mg DO/1, 80% mortality.developed within
48 hours and 100% after 72 hours. At 4.1 mg DO/1, an intermediate
dissolved oxygen concentration, response by mummichogs was also intermediate.
This general scheme is also apparent at lesser cadmium concentrations.
-29-
-------
I
OJ
0
1
too
2 3mg DO /liter
24 48 72 96
HRS Exposed
4 I mg 0 0/ liter
72 96
HRS Exposed
k 60 -
8 5 mg 00/ liter
2 4 48 72 96
HRS Exposed
FIGURE 3: Mortality among mummichog, Fundulus heteroclitus. as a function of
time and cadmium concentration at 3 levels of dissolved oxygen.
Test conducted at 19 C using a dilution water with an alkalinity
of 58 mg/1 as CaCO^.
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
No histopathology was evident in any specimen surviving the 96-hr.
exposure. However, results of a static bioassay conducted subsequent
to the main series of tests showed mummichogs subjected to 28 mg/1
cadmium had necrosis and sloughing of the gill mucosa and buccal cavity.
This developed within 6 1/2 hours of exposure. Of particular interest
was the absence of intestinal lesions in these fish, while these are
commonly found in seawater mummichogs exposed to similar levels of cadmium.
The gill damage observed in mummichogs dosed in freshwater may explain
the marked reduction in tolerance of this group to low dissolved oxygen.
In conclusion we find responses of mummichogs subjected to cadmium in
freshwater and in seawater differ with regards to the sites of histological
lesions associated with exposure and in tolerance in these fish to reduced
dissolved oxygen concentration. In terms of these two responses, fresh-
water dosed mummichogs are similar to other freshwater fishes. This lends
support to our initial hypothesis that response syndrome to metals in fish
is associated with water uptake as related to osmotic-concentration
gradients. These results also emphasize the need for caution in attempting
to extrapolate toxicity data from freshwater to seawater fish.
C. Environmental Requirements of Macroalgae
ROAP 21 AKF, Task 002 — Establish collection, culture, holding, and rearing
techniques, preliminary ecological requirements, including minimum, optimum,
and maximum range for temperature, dissolved oxygen, salinity, turbidity,
and nutritional requirements for successfully cultured organisms in support
of this ROAP and of 21 BBG, 16 AAT, 21 AKK, and 16 AAV.
ROAP 21 AKF, Task 030 - Development of biological criteria in support of
legal standards for temperature.
ROAP 21 AKF, Task 032 - Development of biological criteria in support of
legal standards for salinity.
LIBRARY / EPA
-31- flat'onnl Environmental Flcsoailll Cantor
?.GO S \V 33th Street
Corvalba, Oiegoo 97330
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
ROAP 21 AKF, Task 034 - Development of biological criteria in support of
legal standards for light, turbidity, pH, (X^.
Environmental requirements for an estuarine pest alga, Codium fragile,
were elucidated for several factors during this report period. Con-
clusions from multifactorial studies suggest the following conditions
for optimum growth: temperature, 15-17°C; salinity, 25-35 °/oo; light
250-400 f.c. Above 23°C and below 10°C, there was very little growth.
No improvement in growth over filtered seawater as a media for laboraotry
culture has been found. Media enrichment tends to make the plant grow
as a dissociated filamentous mass. Earlier work on growth requirements for
Codium fragile and Enteromorpha compressa are summarized in a paper to
be published in the proceedings of the EPA Bio-stimulation and Nutrient
Assessment Workship, October 1973.
A review of Recent Trends in Research in the Macroalgae is being written
By R. Steele. Topics to be reviewed include taxonomy, ecology, life history,
culture, physiology, histology, and effects of pollution on macroalgae.
Published studies on pollution effects are scarce; two papers are known for
temperature tolerances of macroalage, only one on effects of heavy metlas.
Marine eutrophication studies deal with overgrowth or destruction of species
by enrichment, although often these only pertain in highly polluted waters.
D. Extramural Research Supporting Environmental Effects Studies
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)
1. All five study species (Homarus americanus, Cancer irroratus,
-32-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Paleomenetes pugio, Pagurus longicarpus. and Rithopanopeus harrisii)
have successfully cultured in the laboratory and optimal temperature
and salinity conditions determined for the larvae. Larval tolerance
to reduce dissolved oxygen and influence of temperature on larval metabolic
pathways has been documented for two species to date. Studies of larval
development and survival under a cyclic temperature regime have also
been initiated, with data now on hand for the American lobster.
ROAP 16 ATT, Task 007 - Grant for study of toxicity of metals to larval
arthropods 801305
Grant to Dr. J.D. Costlow, Duke University (D. Miller, Project Officer)
2. An analytical capability for mercury has been developed with a
detection limit of 0.1 ppb. The behavior of mercury at 20 ppb in
culture dishes or Erlenmeyer flasks, which involved logarithmic loss
in concentration with time from the liquid seawater phase, was docu-
mented. Lethal levels of mercury were established for larvae of three
xanthid crabs under optimal temperature and salinity conditions. Ef-
fects of sublethal dose levels on development rate, morphology, and
number of larval stages was detailed for Rithropanopeus harrissi.
Stress effects of mercury are more apparent when salinity and tem-
perature conditions are also at stress levels. Effects of mercury dosing
on lactate dehydrogenase activity in larvae and first crabs is being
explored, but without conclusive results at present. Weight loss of
approximately 25% in the first crab stage is reported for 10 ppb dosed
animals.
III. Development of Water Quality Criteria for Environmental Factors
A. Development of Marine Temperature Criteria and Assessment of Total
-33-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Power Plant Impact on the Estuary
ROAP 21 AKF, Task 030 - Development of biological criteria in support of legal
standards for temperature.
New marine temperature criteria were developed by a laboratory task force
which reviewed thermal effects literature for all major marine taxa including
bacteria, micro-and macroalgae, micro-and macroinvertebrates, and fish.
Over two hundred published papers, technical reports, thesis, and manuscripts
were carefully analyzed for quality and applicability to thermal criteria.
Thermal effects data were applied to the specific biotic regions represented
by the study. Elevated temperatures which proved limiting were compared
to annual temperature curves from each region. These data show the problem
of thermal addition is most acute in the summer, where naturally occurring
extremes can exceed the thermal tolerance of some organisms indigenous to
a region. Accordingly, an incremental rise of only 1.1°C above ambient is
recommended for receiving waters during the summer, until ceiling temperatures
are reached. Maxima and true daily mean ceilings have been recommended for
waters of the East Coast cold temperate zone south of Long Island, for the
East and Gulf Coast warm temperate zone and the tropics. There are insufficient
published data to develop general ceilings for the remaining regions, but their
establishment on a case-by-case basis is suggested. The recommended permissible
incremental rise above ambient for the fall, winter, and spring is 2.2°C. This
will assure maintenance of an annual regime which approaches the historical
norm under which organisms have evolved In order to maintain thermal hetero-
geneity, natural diurnal temperature fluctuations should also be maintained
outside the mixing zone in both amplitude and frequency.
-34-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
Recommendations have also been made for implementing these thermal
criteria. Specifically, when criteria for heated water discharge are applied
to power generating stations, it is imperative to evaluate the cooling water
process as a unit, considering the potential impact of water intake and
inner-plant circulation as well as the discharge per se. In some instances,
temperature standards have been met during warm months by increasing the
volume of water pumped. It is now apparent that high mortalities of plankton
and small fish can occur during inner-plant passage at estuarine sites,
particularly during the summer. Unless the pumps used in dilution pumping
are specifically designed to minimize damage to pumped organisms, this
practice would not be a suitable measure to protect the environment.
A review has been completed of entrainment studies conducted at 14 operating
power plants located at marine sites. While the cause of entrainment damage
is commonly multiple, mechanical damage appears to be responsible for a very
significant portion of the kill during the life of a power plant. Mechanical
stresses are experienced continually whenever cooling water is pumped.
Papers have recently been published which relate mechanical forces alone to
70-80% of the kill of entrained juvenile fishes and copepods. At this labor-
atory, Gentile and Lackie determined pumping effects to be the primary
contributor to phytoplankton and zooplandton mortality. Prior to the pumps,
average mortality was 10%; after the pumps 50%; after condenser passage, 60%.
Some fish larvae are sensitive to mechanical effects to the extent that most
individuals cannot survive sampling at the intake. These include Anchoa
mitchili (bay anchovy), Alosa pseudoharengus (alewife), and Alosa aestivalas
(blueback herring). It is reasonable to expect these species to also be severely
-35-
-------
ENVIRONMENTAL EFFECTS AND RESPONSE PARAMETERS TEAM
effected by mechanical stresses of plant passage.
Plant passage of planktonic reproductive stages (meroplankton) and small
non-screenable fishes of all life history stages appear to present the most
serious potential for adverse ecological impact. This is particularly a
problem in estuaries and has serious implications for many commercial fisheries.
It is estimated that 63% of the U.S. Atlantic commercial catch of fishes and
invertebrates consists of estuarine dependent species. Meroplankton is
a major component of the estuarine ecosystem, where about 70% of the fauna is
estimated to have planktotrophlc eggs and larvae.
Predicting the consequences of plant passage kill for the ecosystem, even
when it is massive, exceeds current capabilities of the science. One problem
is difficulty of discriminating between natural variation and man-induced
changes in complex and open biological systems. It is unlikely that a single
power plant on a sizable body of water could alone have an appreciable Impact
on a population. However, where multiple units exist at one site, or several
plants exist in one portion of an estuary, a critical level of larval mortality
could occur. A population is particularly vulnerably if its numbers have been
previously reduced by other causes, natural or man-induced.
Hence, it is recommended that thermal criteria be implemented in a fashion
which not only protects the environment adjacent to a discharge point, but
which also protects the ecosystem from potential adverse impact by efforts to
modify or treat a heated discharge, such as may occur with augmentation
pumping.
-36-
-------
ECOSYSTEMS ANALYSES TEAM
When a particular type and level of stressor(s) is applied to a total
system, the system will change. The major problem confronting E.P.A.
is, how much and what type of change is acceptable. Our strategy
in answering this question is that any degree or type of systems change
is acceptable provided the relaxation of the stressor(s) results in the
complete recovery of the system back to its "original state" in a
"reasonable period of time". The latter is arbitrary while the former
in terms of its structural and functional properties can be defined.
Thus, our tactic to the above problem is to delimit the boundaries of
the ecosystem as a function of different stressors and disturbances.
The major and rather obvious difficulty in experimentally manipulating
most total system is their size i.e., it is usually impossible to (1)
control physical and chemical inputs and (2) impose disturbances such
as species changes, nutrient concentrations, etc. in the field. One
approach to this problem is to miniaturize the system, the microcosm.
Such miniaturization is feasible for a marine ecosystem such as Narragansett
Bay because (1) most of the biotic components are microscopic thus,
providing a measure of numerical as well as functional responses in a short
period of time, (2) the macroscopic forms can be experimentally simulated
and (3) it appears simple i.e., the elements of the system consist of a
pelagic phase interacting with a benthic phase. We hope to describe
the functional relationship between changes in these resiliency boundaries
for total marine microcosms and (1) different levels and patterns of
-37_
-------
ECOSYSTEMS ANALYSES TEAM
temporal change in artificial sewerage and (2) structural and functional
biotic manipulations. While we are still in the construction phase,
the experimental details of the total systems parameters and the actual
experimental microcosms to be used in the study are presented below.
The experimental microcosms used will consist of a benthic and pelagic
phase both of whom are interconnected physically and, therefore, are
capable of interacting with one another (see Figure 2). The size of the
2
system will be 150 liters and 167 cm for the pelagic volume and benthic
surface area, respectively. The surface to volume ratio used is equivalent
to that found in Narragansett Bay. Scraping of the walls of the pelagic
tanks will prevent the fowling and settling of benthic animals and, thus,
the only surface area provided will be that in the benthic chamber.
Other physical and chemical conditions of the microcosms such as water
turnover (15 days), temperature, average light intensity and quality
for the whole water column, salinity, dissolved oxygen, water exchange
between the benthic and pelagic phases, etc. will be made equivalent or as
close as we can come to that found in the bay or our best guess if no
data exists. The microcosms will be open in the sence that natural
complete assemblages of planktonic organisms from Narragansett Bay will
be added daily (and, of course, a similar volume will be removed).
Because light is effectively extinguished on the bottom of Narragansett
Bay, all benthic chambers will be placed in the dark. Replicate benthic
communities for each pelagic tank will consist of a prefiltered mixture
of mud and small benthic organisms; large (hand contable) organisms such
as Nepthys and Yoldia, two dominant (structurally and functionally)
-38-
-------
ECOSYSTEMS ANALYSES TEAM
benthic species will be added to the mixture in known but naturally
found densities/unit bottom area. Fish larvae are possibly going to be
excluded from the microcosms because (1) their natural densities are very
low (5 x 10 -3 larvae/1 in West Passage of Narragansett Bay) and, therefore,
their functional role minimal (note that their numerical response in terms
of this studies time frame is infinitly large) and (2) their elimination
and/or control will decrease the within treatment variability.
The biotic variables to be measured will be the classical structural
(size and shape frequency histograms, diversity, etc) and functional
(metabolism, nutrient fluxes, etc) properties of ecological systems.
The latter are pigment ratios, carbon/nitrogen (storage to structure
relationships) for different trophic and size categories, ATP (living
to dead), DOC, nutrient and energy (1^C) fluxes, primary productivity and
respiratory metabolism of the benthic and pelagic phases. It should
be emphasized that one of the objectives of this study is to determine
which of the above well discussed variables are at all indicative of
changes in the systems resiliency boundaries.
Our first prototypes necessary for testing conceptual plumbing, tank
design, experimental manipulations and monitoring are almost complete and
will be running before the end of this month. Most of the above tech-
nologies required to measure the above have been set up or developed.
Although, there are details which must be determined for the particle
counter and DOC.
Statistical & Mathematical Support:
Statistical and mathematical support through the computing center has
-39-
-------
ECOSYSTEMS ANALYSES TEAM
grown considerably both in time (Fig. 1) and team diversity (Table 1)
since its initiation in November to present. Although it is not possible
for us to asses the helpfulness of the computing facilities, we can
certainly say that they are being used.
-40-
-------
Table 1. NMWQL team use of computing facilities since last semi-
annual report, 12/1/73.
JOB TYPE TEAM
Metals
Bloassay
Ocean
Dump
Syner-
gisms
Systems
eco.
Phantom
(unknown)
Atomic
Absorption
65
—
36
—
—
—
Chemical
Equilibrium
31
—
—
—
—
—
Statistics
18
70
35
30
25
10
Graphics
—
15
—
20
30
—
Other
(Storet, Special
Programs, etc.)
—
4
—
25
30
20
Utilities
Unknown
30
4
10
10
25
135
Approximate number of jobs run on the Data 100 & Techtronics Terminals
-41-
-------
MACHINE
UTILIZATION
(CPU + I/O)
47.5
* Q
Nov.
73
Instilation of
computing facilities
TIME(months)
Fig- 1 Temporal changes in computer
use at N.M.W.Q.L.
-42-
-------
Average Seasonal
Light Quality &
Quantity
Narraganaett
Bay Water
Pelagic
Tank
O
V;
Fig. 2 Experimental Microcosm
^Exhaust
Benthie
Tank
Muc
[Held at Ambient
Seasonal Water
Temp.
-------
MARINE CULTURE TEAM
The Culture Team provides an array of experimental animals for use in specific
research projects of other NMWQL teams as a service function - Organisms are
provided through:
1. Field collections.
2. Laboratory culture
3. Procurement from outside sources
Though service-oriented, a modest research effort is directed to culturing
early life stages of fishes and invertebrates. Emphasis is on physical
systems for culturing and nutritional requirements. Physiological condition
indices developed by the Response Parameters group are applied to characterize
status of experimental animals as a "quality control" function. For research
program management, tasks accomplsihed by this team are identified under
ROAP 21AKF "Ecological Requirements for the Protection of Estuarine and Marine
Life."
ROAP 21AKF - Task 02 Collections
Large numbers of experimental organisms are provided through field collection
efforts. Following is a listing for the past fiscal year.
Species Collection Nol
trlpa ~ collected
Salmo salar, Atlantic salmon
1
230
Fundulus heteroclitus, mummlchog
28
6,000
Pseudopleuronectes amerlcanus, winter flounder
S
466
Alosa pseudoharengus, alewife
2
111
Menidia nenldla. Atlantic sllversides
25
13,735
Cynoscion recalls, veakflsh
2
50
tfassa obsoleta, mud snail
1
500
Mercenana irercenaria, quahog
16
9,200
Hva arenaria, soft shell clam
13
920
Crassostrea virpinica, oyster
4
1,000
Arctlca icelandica, mahogany clam
4
925
-Araooecten lrradlans lrradlans, bay scallop
3
2,100
Cancer lrroratus, rock crab
4
470
Homarus ameneanus, lobster
3
24
Pagurus longicarpus, hermit crab
2
500
Enterooorpha lntestlnalls
1
3 gal.
Plankton (mixed tows)
5
TOTALS
m
36.321+
-44-
-------
MARINE CULTURE TEAM
Increasing demand for early life stages of fishes and invertebrates
has been met by a major expansion of in-house culture capabilities and an
informal cooperation agreement with the neighboring NMFS - NOAA laboratory.
Fertilized egges were supplied:
SPECIES
Winter flounder, Pseudopleuronectes
americanus
Scup, Stenotomus chrysops
Atlantic silversides, Menidia menidia
Mummichog, Fundulus heteroclitus
SOURCE USER
NMWQL & Ecosystems group
NOAA Bioassay Methods - Culture
NOAA Bioassay Methods
NMWQL Bioassay Methods
NMWQL Bioassay Methods
The fish larval culture lab is now operational in space convenient
to the wet laboratory. The companion invertebrate larval culture
facility will be completed shortly. Numerous phytoplankton species are
under culture by personnel in the Bioassay Methods and Multiple Effects
teams.
Fish Culture
Current fish culture work has concentrated on the Atlantic silversides,
Menidia menidia which spawns in local water from late May to early July.
Emphasis has been placed on: (1) characterizing the condition of adult
sqawners, (2) refinement and development of techniques for laboratory
rearing of eggs and larvae, (3) establish of optimal conditions for
incubation of fertilized eggs, (4) study and characterization of the normal
embryological development from fertilization through hatching, and (5)
supplying fertilized eggs to other teams for experimental purposes (Bioassay
Methods and Response Parameters teams), (6) Larval rearing and related
nutritional studies. The most significant area of progress involves larval
rearing. Three groups of several hundred larvae are being maintained
-45-
-------
MARINE CULTURE TEAM
temperature controlled closed systems developed by Dianne Everlch,
(Fig. 1). Three size groups of larvae are being rearing on a diet of
Artemia mauplii and finely ground "Biorell" flake food. Survival has
been 99% and growth appears normal as compared to field populations
although this is a preliminary observation subject to verification
as the season progresses.
Preliminary nutritional studies on larval Menidia are being accomplished
by Linda Ferraro. Several groups of larvae are being maintained and
comparasions are being made on larvae reared on (1) the diet of brine
shrimp mauplii and Biorell, &(2) Marine rotifers, Brachionus plicatilis
Survival and growth of the several groups will be assessed as the season
progresses. Field collections will provide baseline data on growth.
The winter flounder (Pseudopleuronectes americanus) culture effort was
accomplished at research barge facilities by Dianne Everich. Three
groups of fertilized eggs, approx. 7000 total, were delivered for subsequent
experimental use. Future plans call for relocation of this effect to
Narragansett Wet Lab and expansion of the availability period for obtaining
eggs and larvae through use of temperature controlled facilities.
Further expansion of culture activities to other representative species
of marine fish awaits the control sea water temperature system now
under construction in the Narragansett Wet Lab.
ROAP 21AKF Task
Invertebrate culture
Culture methodology of the marine polychaete Capitella capitata developed
-46-
-------
?ISH REARlAie APPARATUS
•k
T£MP.
&MT90L.
&
V PO
j
HATCHING
TAR
COOUH6
cons
AQUARIUM
FILTER
REARING
TANK
r/SOB£: /
-47-
-------
— MARINE CULTURE TEAM
by Dr. Donald Riesh under EPA grant 800962 is being validated by
Carol Pesch. The nolvchaete is under culture in Narraeansett lab facilities
and several generations have been produced, see Figures 2, 3, and 4. A
full assessment and verification of the methods manual submitted by
Dr. Riesh will be completed in the next semi-annual report.
21AKF Task 039 Production of larval lobsters.
The methods manual, detailing methods and techniques for year—round
production of larval lobsters, has been completed. Delivery is expected
in the next quarter. Mimi Johnson has been assigned to accomplish in-house
culture and validate the manual. This effort will be initiated upon receipt
of the manual.
21AKF Tasks 03, 04, 05, 06, 43 - Research Grants & Contracts
Extramural resources are utilized to provide advanced methods and
techniques in culture of marine species not yet dealt with in-house.
When planned new wet lab facilities become a reality, the necessary
knowledge will be available for immediate application. Species covered
include fishes: Mullet
Striped bass
Invertebrates: American lobster
East Coast Polychaetes
West Coast Polychaetes
Plants: Phytoplankton - 150 spe,cies
21AKF Task 02E - Nutrition
Nutritional studies with the Atlantic silverside have received the focus
of attention. Previous studies involved development of maintenance diet
for M. raenidia adults and determining optimal (in terms of normal physiology
-48-
-------
:&&-\'•>;?# . ¦:*?.; ./
fc®- • •MSS?' --£® i-
s«ate>ay
-------
C* CAfjur/j y 1 GA^iUMA. &L0A//rs
TZZL
C> £A/3jrr/jY i£'£4&z&A_ &£0*//rs
COLT*** JXsrrs*
-50-
-------
MARINE CULTURE TEAM
and stress response) diet for juvenile silversides. Local feeding
experiments were previously described.
The present goal is to develop an artificial diet, varying with life
state are required, capable of providing all necessary nutrients for
"normal" growth, survival, physiology of test organisms as compared
to field populations. Hopefully, by standardizing culture methodology
and developing quality controlled artificial diets, stress response of
experimental animals from different culture populations will be similar
within defineable limits. When repeating bioassays with the same toxicants
studies with varying levels and/or different toxicants will be more
closely comparable.
Experimental approach: (Figure 5).
ROAP 21AKF Task 13 — Design of Marine Environmental Simulation System
The project was completed by Ocean Data Equipment Corp. of East Providence,
R.I. under contract EPA 68-03-0277. The purpose was to define preliminary
design concept and system characteristics to ensure compatability with
physical structure and supporting systems of the proposed new NMWQL
wet laboratory.
The system, Figure 1, has been broken down into three logical pieces,
the Data Subsystem, the Controller Subsystem, and the Physical Subsystem.
The Data Subsystem is the portion of the system that interacts with the
external data world. It accepts real time data, Includes two tape recorders,
one for historic data playback and one for recording data, and has provision
for the synthesis of data. Real time data, historic data or synthetic data
-51-
-------
FIG. 5. EXPERIMENTAL APPROACH - NUTRITIONAL REQUIREMENTS OF MARINE FISH LARVAE
(Modified from Lasker N.M.F.S. LaJolla, Calif.)
a.
1.
NUTRITIONAL CHEMISTRY
DEFINE PRECISE PROPORTIONS OF
PROTEIN, AMINO ACIDS, FATTT .
ACIDS, MINERALS, VITAMINS OF
WHOLE ORGANISMS
1
In
to
I
2. DEVELOP ARTIFICIAL
DIET WITH DEFINED
CONSTITUENTS
3a,
STUDIES TO DETERMINE
'ESSENTIAL INGREDIENTS
)
^ «*»
DETERMINE ESSENTIAL
ENVIRONMENT REQUIREA
MENT
TEMP.
D.O.
SALINITY
LIGHT
ETC.
FEEDING TRIALS
COMPARE:
1) SURVIVAL
2) CROWTH
3) BIOCHEMISTRY
4) STRESS RESPONSE
5) OTHER PARAMETERS
IN LAB AND FIELD
POPULATIONS
LABORATORY
CULTURE & HOLDING
LARVAE
JUVENILE
DETHHMINE OPTIMAI DIET (FOR
EXPh KIHENTAL ORCA'.ISMS) EN-
CAPSULATED MICROTARTICALS
(EMP'e)
ADULTS
5.
EXf.
USB
-------
*f AL
t.-r
co
MODtn
I
U1
w
(jrccp5 Historic
data
fUfr
Ul(
DAT *.
algorithms
m
Hf'n'cijy
opt»«r» ir n i rif ric
iNrtHri. [ o < i a
DATA SUOSyST£M
.StTPOitvT
DATA
MANUAL
~1
I
I
FINAL CONTROL
tirncwrs
CONTROL
ALGORITHMS
- • -—-a-—
~—
S F M S> 0 R
" -»(Acr
INTtRfACf CAHUS
HZh
crwsops
Q—
HZ>
HZh
COMTROlLER SUBSySTfMS
emmc v1*??!?
*8WAf
V*T ik fRfSH 8AV
PHYSICAL
SUOSySTEri
_HC AT
-COOL
-U&rtT
_G f* j
e*p«z €%Pm3
FIGURE £
MARINE
ENVIRONMENTAL
SIMUL A T I ON
SySTEM
-------
MARINE CULTURE TEAM
may be modified by offsets or coefficients within the data subsystem.
Finally the subsystem presents setpoint data to the controller subsystem.
The data subsystem will be the normal location for the interaction with the
human operator and the location at which he will select types of data,
offsets, alarm limits, etc.
The controller subsystem will provide the control algorithms for the
system. It will include the sensors, final control elements and the
electronics to implement the algorithms and drive the final control
elements. Setpoint data will come from the data subsystem. Provision
will be made for manual setting of setpoint data in the event of a data
subsystem malfunction.
The physical subsystem actually carries the water and is composed of the
tanks, pipes, etc. Parameter values and flow are controlled by the valves
of the controller subsystem.
The final report on Contract 68-03-0277, Design of a Marine Environmental
Simulation System, has been completed, submitted, and is under review for
publication as an EPA research report.
Histological Group
The histological group accomplished a number of projects for research teams:
16AAV-T-B1 Chronic cadmium, oysters Dr. Zaroogian
21AKF-2F Nutrition study, Menidia Culture, Ferraro
21AKF-2F Holding study, salmon Culture, Giles
21BBG Ocean dumping, many species Ocean Dumping, Pesch
16AAV-T-B2 Acute metal exposure to fish larvae Bioassay Methods, Cardin
21AKF-T Menidia Response Parameters,
Koltes
16AAT-11 Dissolved oxygen & cadmium, Fundulus Dick Voyer
21AKF-2A-4 Baseline histology, soft shell clam Mr. Yevich
16AAT-12 Chronic cadmium, oysters Dr. Zaroogian
Technical Main Oil Spill, soft shell clams Mr. Yevich
assistance
-54-
-------
MARINE CULTURE TEAM
21BBG
Special
project
Pfizer waste, Spisula & lobster Ocean Dumping, Pesch
Tetracycline, crabs & Menidia Linda Meiklejohn
Two major projects were reported on (1) Histopathologic findings in
oysters exposed to cadmium, and (2) Effects of cadmium exposure and
nutritional regime on histological state of the surf clam. Detailed
findings on these and other projects are to be found elsewhere in
reports on research by the various NMWQL teams.
Marine Culture Team Non-ROAP Activities
Development of recommendations for EPA's marine temperature criteria
was a major effort in the past six months. Team members involved were
Linda Ferraro, William Giles, Ray Hennekey and Ray Highland. Team leader,
A1 Beck, spend greater than 50% of available time and provided the major
input for determining the recommended criteria relative to marine fishes.
Also developed, were recommended guidelines for other aspects of thermal
related problems including effects of intake structures, biocides, plant
passage (entrainment) and effluent discharges other than heat. Dr. Miller
and A1 Beck will be preparing a monograph threating all aspects of using
marine waters for cooling purposes. This is treated in more detail in
another section of this report.
Manuscripts and Publications
1. 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. (in press)
Ferraro, L.A., R.E. Wolke and P.P. Yevich. 1974. Effects of water-
borne dimethylnitrosamine (DMN) on Fundulus heteroclltus (L.)
{Pisces: Cyprinodontidae) I. Acute effects, (in preparation)
Ferraro, L.A., P.P. Yevich and R.E. Wolke. 1974. Effects of water-
borne dimethylnitrosamine (DMN) on Fundulus heteroclltus (L.)
-55-
-------
MARINE CULTURE TEAM
(Pisces: Cyprinodontidae) II. Chronic effects, (in preparation)
Jackim, E., S. Cheer and L. Ferraro.
nutritional status, (in press)
1974. Evaluation of fish
5. Gonzalez, J.G., P.P. Yevich, J.H. Gentile and N.F. Lackie. 1974.
Problems associated with culture of marine copepods. (in press)
6. Miller, D.C. and A.D. Beck. 1974. Development and application of
criteria for marine cooling waters. Proc. IAEA/ECE Symposium
on the Physical and Biological Effects on the environment of
cooling systems and Thermal discharges at nuclear power stations
Oslo, Norway, August 26-30, 1974. (in press)
Presentations and Workshops
Ferraro, Linda A.
Yevich, Paul P,
May 27-30, 1974 attended one-week workshop in
Flourescence Microscopy held in Massachusetts.
(1) Jan. 21, 1974 Augusta, Maine, cross-examination in
the Eastport Oil Refinery investigation.
(2) Jan. 30, 1974, Univ. of S. Fla., Inst, of Mar. Sci.,
presented seminar on histopathologic effects of oils
and metals on molluscs.
(3) Jan. 31-Feb. 3, 1974 U. of S. Fla. conference/workshop
on marine environmental implications of offshore
drilling.
(4) March 6-7, 1974 Boston, Mass. attended seminar entitled
"To assess the present and future marine and coastal
research needs of the mid-Atlantic bight."
(5) March 12, 1974 Guest speaker, Adv. Parasitology
seminar Univ. of Rhode Island, directed by Dr. Hyland,
Zoo. dept.
-5(r
-------
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 in-
formation necessary for the appropriate selection of new dump sites,
including photographic records, hydrographic data, bioassays and
determination of baseline levels of a variety of trace materials in
the biological and physical-chemical environments. It will establish
criteria for monitoring sites and adjacent areas for ecological com-
promise, establish routes, rates and mechanism for recycling of materials
from disposal sites through biological uptake as well as by physical and
chemical processes, and will establish standard bioassay technology.
ROAP 21 BBG, Task 1 - Ocean Monitoring Survey—Philadelphia-DuPont Dumpsites
A Request for Proposal (RFP) was issued cooperatively with Region III
for a background survey of sediments and benthic organisms in an area of
the Mid-Atlantic Bight encompassing the DuPont acid waste and Phila-
delphia sewage sludge dumpsites. Prime emphasis is to track the extent
that dumped contaminants have spread across the bottom environment from
the areas of discharge by means of measurements of several metals in
benthic macrobiota and sediments. There were six responses. These
were reviewed by Region III and NMWQL and recommendations were for-
warded to Washington, D.C. on June 7.
OCEAN DISPOSAL TEAM
ROAP 21 BBG, Task 2 - The Movement of Trace Materials from Sewage Sludge—
Rates, Routes, and Mechanisms into the Biota
We have negotiated an interagency agreement with Dr. Frank Lowman of
the NEC Nuclear Science Center at Mayaguez, Puerto Rico. His group will
-57-
-------
OCEAN DISPOSAL TEAM
study contaminant fluxes from sewage sludge including uptake and transfer
by biota. Flow-through systems will be used. Prime emphasis will
be 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 has been awarded
a grant to assess contaminant impacts in historical context. Various
radioisotope determinations will be used to date sediment layers in
cores taken from strategic locations selected, along the east coast.
Correlating contaminant concentrations and age of each layer will
permit drawing time — concentration curves for the past. It is hoped
that this Information will permit the prediction of future trends.
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 provided standard bioassays and advice
on monitoring programs for the following ocean disposal permits:
Pfizer wastes (Region I): N.L. Industries acid wastes (Region II);
DuPont acid wastes (Region III); and Shell Oil wastes (Region VI).
ROAP 21 BBG. Task 5 - Standard Site Monitoring Methods
To develop site monitoring methods three representative sites were
chosen early in this program. These include a dredge spoil site (Fig.
1, Providence River spoils dumped in Rhode Island Sound), and industrial
wastes site (Fig. 2, DuPont acid wastes dumped 45 miles off Delaware
Bay), and a sewage sludge site (Fig. 2, Philadelphia sludge dumped near
-58-
-------
OCEAN DISPOSAL TEAM
the DuPont site). All three sites were visited during the period
covered by this report.
At the Rhode Island Sound dredge spoil site, a clear demonstration of
impact is developing. Preliminary water chemistry indicates high Cd and
Zn values over the spoil. Tissue analysis of the clam, Arctica islandica,
a ubiquitous species of commercial importance, revealed higher metal
residue levels than found at either of the other sites (Table 1). His-
topathological observations demonstrated kedney-damaging concretions
in clams found as far as five miles from the site. Tissue damage was
greater in clams found closer to the site. The chemical nature of
these kidney concretions is being determined by Dr. Rogerson of this
laboratory. Crabs caught on the spoil had abnormally eroded and dis-
colored carapaces.
Samples of Arctica islandica are being processed for metal residues in
soft tissues. These bivalves were collected at almost all stations
depicted in Figure 1. Ten animals per station are being analyzed in-
dividually for 13 metals, Fe, Cu, Pb, Zn, V, Ti, Al, Mn, Cd, Ag, Co, Ni,
and Cr. Sediment samples from many of these same stations are being
analyzed for the same metals. Additional metals may be examined as the
need arises. Future work will include extensive water, pore water, and
sediment chemistry in order to elucidate the problem of routes and
rates of transfer of biologically available metals.
A contract with Sheldon Pratt at the Graduate School of Oceanography,
University of Rhode Island, will quantify the extent and nature of
Impact on the benthic community structure at and near the spoil site.
-59-
-------
OCEAN DISPOSAL TEAM
A final report should be forthcoming shortly.
Research continues in the Delaware dumpsite area. Figure 2 describes
the path of the "Ides" cruise, carried out on March 11-15, 1974 in
which 13 new stations were visited and an abundance of benthic organisms
were collected (Table 2). These sites offer a unique opportunity to
test the concept of analytically "finger printing" a waste material.
Large volumes of waste are dumped at both sites approximately three
times weekly. Both wastes may be distinguished by their metalic content,
(Table 3). In our monitoring efforts we attempted to follow the fate
of each waste by following particular metal "tags". For example,
the DuPont waste contains 98% of the vanadium while the Philadelphia
sludge contains 96% of the cadmium dumped at both sites. Metal analysis
of the water, sediments and biota in areas adjacent to the dump sites
should trace the fate of each waste. Our initial efforts considered
the sediments and shellfish in the dumpsites and surrounding areas.
Samples collected during the "Fatch" Cruise, carried out on November 5-10,
1973 at the Delaware sites, have been processed during the first quarter
of this calenderer year. These included four species of mollusc and one
echinoderm. Data are reported only for Arctica islandica, a mollusc,
because it was represented at almost all stations.
Of the 16 stations visited, 14 yielded the ocean quahaug, Arctica is-
landica. Three of these, stations A, C, and H, yielded only one indi-
vidual, with 11 stations yielding from 3 to 13 individuals. These were
subsequently analyzed by atomic absorption spectrophotometry for total
body levies of the 13 metals listed previously. The mean concentration
-60-
-------
OCEAN DISPOSAL TEAM
for each of the 14 stations are presented in Table 4. An analysis of
variance was performed on replicate Arctica samples for each of the 13
metals. Results are reported in Table 5.
Statistical groupings of stations with high vanadium and cadmium
concentrations are plotted in figures 3 and 4. These distribution
patterns reflect the expected source and provide confidence in the
"fingerprinting" approach to following the fate of individual wastes.
Samples collected on a Spring 74 cruise (IDES) should provide a
conclusive test of this monitoring tool.
Some inferential evidence of impact on shellfish may be obtained by
quantifying distribution patterns of dead shell. Figures 5 and 6
represent measures obtained in a half hour dredge haul at each station
during the Spring 74 cruise (IDES). These patterns provide initial
evidence of impact by the DuPont waste. In-situ bioassays should provide
a conclusive test of the impact.
Conclusions
In summary, conclusions drawn from a statistical treatment of the
analytical results show: 1) Metals disposed of at sea do accumulate
in the tissue of Arctica islandica in quantities roughly proportional
to the amounts dumped, i.e. in no case did the area of highest measured
accumulation fail to correspond to the dumpsite with the greater load of
the respective metal. 2) Certain metals, particularly vanadium and
cadmium, are dumped in such quantities in one of the two sites that they
may be used as tracers for that dumpsite. 3) The metals are distributed
predominately in the direction of the prevailing currents over an under-
-bl-
-------
OCEAN DISPOSAL TEAM
minedly large area. 4) For materials having nearly the same density as
seawater and dumped in a dispersal rather than a concentrated pattern,
the concept of a discrete dumpsite is inappropriate and misleading since
the effects measured are in no way limited to the area bounded by the
dumpsites proper. The present sample grid is not large enough to determine
the extent of the impacted area.
ROAP 21 BBG. Task 6 - Dredge Spoils and Sewage Sludge in the Trace Metal
Budget of Estuarine and Coastal Waters
A grant to develop field methods for assessing metal fluxes from natural
sediments has been awarded to James Simpson of Columbia University
(G. Pesch, Project Officer). Simpson proposes to attack the following
specifically:
(1) Define the flux of Cu, Pb, Zn, Cd, Mn, and Fe supplied to the New
York Bight by the Hudson River in the dissolved and particulate
phases;
(2) Determine the sediment binding capacity for these elements and
their partition coefficients between dissolved and particulate
phase as a function of sediment type and salinity;
(3) Determine the chemistry of the interstitial waters in estuarine
and dumping ground sediments to establish the potential for dif-
fusion of heavy metals from the sediments to the overlying water;
(4) Estimate the rate at which material is released from the sediments
to the water column.
ROAP 21 BBG. Task 7 - Pathways and Effects of Human Pathogens in
Marine Ecosystems
This will be Implemented in-house by ROAP 21 BEY (Pathways and Ef-
-62-
-------
OCEAN DISPOSAL TEAM
fects of Human Pathogens in Marine Ecosystems), Task 27 (Development
of a Model to Predict Dispersion of Human Pathogens from Ocean Out-
falls and Sludge Dumpsites) (V. Cabelli). The scope of the project
will include the study of abundance and propagation of human and
marine pathogens or of indicator organisms that present a direct or
indirect threat to man and marine resources. Funds are pending and
the research is still in the planning stage.
ROAP 21 BBG, Task 14 - The Problems of Ocean Dumping — Stability and
Resiliency in Experimental Ecosystems Exposed
to Constant and Time-Varying Stresses
A grant has been awarded to Dr. Scott Nixon of the Graduate School of
Oceanography, University of Rhode Island, to develop a mathematical
model to assess the effects of time dependent and time independent
sewage aludge stress on marine plankton systems (K. Perez, Project
Officer).
ROAP 21 BBG, Task 15 - Influence of Dredge Spoils and Sediment Pollu-
tion on Trace Metal Assimilation by Organisms
A grant has been awarded to Dr. Michael Bender of the Graduate School
of Oceanography, University of Rhode Island, to develop methods to
assess contaminant fluxes between sediments and overlying water in
controlled laboratory simulations of ocean dumpsites.
TECHNICIAL ASSISTANCE
During the period Jan-June 1974 the Ocean Disposal Team responded
to assistance requests from all the coastal Regional Offices.
Papers published
Pesch, G. 1974. Protein polymorphisms in the hard clams Mercenaria
mercenaria and Mercenaria campechiensis. Biol. Bull; 146:393-403.
-63-
-------
FIGURE 1
W '^iWSL \
u^:
.' . J rVTv
{ - 0 c • "
( oJV ^ ^ V.
NEWPO
Ml'3o'
( <4c> -v •
<• '\>^ry ,. r / J{ ... - ,
\/-«w \fe ^ '•'A : x •
,'-» , 1 '(V) ' v
>( ^ i ' <-—> •}>) / OS^> t
,rt' V. '.V s 1 ,' ¦ *\ ¦ -'
/ //^
r~l \ /«? • '
»M S. W
/ \" ¦ ¦>•¦¦•¦ i
DREDGE SPOIL SITE
V
o • ^
(
:Ull\
/uOP* >
SO'
>. PT. JUDITH , <->;W % , Lvl? v*°/°x ^ • •
! (rv7 'I > rv< / ' Vn 'A " , S v
o - ' ¦, / 3«) r' . \l^r^/;, ;r _ X „ •>•
-tV**
". 1
> ¦
'¦
/V.
^ >
/ /
.*'7,
'/
¦'> I V
? '
'/
r'i
V
v
~v
"7 .:"
>3
yi_
v
^ \
''IV> 7/:^
v. ?U j>yJ/
r'
r
\f
"!*/, / ^ • ' V I —> / ' V .y/ /
7r^¦ .i ^ ' /v^x " i.
V
Pc
- -1
'J'A^rJ/ - . >x A ~ ? '
x
af ^ \ N
i/'S-'s~*'9i?¦ -» \x.
6
_9.ii/5"
r
* I I ,, . /AX
'
-------
FIGURE 2
* :
.. !
w s — /- 73
\.S
.1 .
I - .•
")(«
»? I>
x
C's
2i
J.»j» r\s " T/'"ov, .
rn ¦ / i
f . - *>'-, » \
V ' ' - 71 'J
^>V y \
'—:7c7::;7,, "•"rk-'-.fo « \
/ / ,
' / ao'/fy-
.' / ,D \
> vsi . DuTV.,1;
f- "¦.
17 / h Vr- " / "k ,J
a_ ; \ • * '21 I \ \ / 31 N* 30
.7 ,? |4/»**/>OW ?'/,\i 2,T*L23 f '\, V %27 / /
'V "V ' . , <3\ , 71 Jo \/.„ \
16 |S
'V-T-rj&fe'.
• O ¦• - , Ti 21 "
•V • * -/»"«.,
m\I'.8 ^:a\; \ 21/
13 VI8 K .21 - -"'
, 3,°
'-..M >' jj
rr » *>j fr^'i
— \y s J1).} *"
' I*
, .^<;' '
V">i 1 vV •*¦>
,!. -' ii« • ^
1 - p /
'ia ^— / » fJ !.
,» » V. 20
17 '* / \
„/ IS
. " /
! 'V' A 21
- Y IV^ - »' \ M / T
/'[ ~;t?v n\>. aV^'/ •
IS \ / \7
13
12 ' *
15 ^90 10 ' /
P'
^ 18 ' 31
20 £ /'^A "l /
14 ^ / -n*S~
r/jj' NsM / ^ '? \
'?3 ^ \ l« / 19 / »
*
f
13
18
2b
/5*\ • ..V-i ai-. '
i.b ' '^'
7
i»< 21 2S -
.'v '
\». /
'> • ..£ " " J. A
/ 17 / /'j / \
70 .« 18 »J Jr /„ V>
a 5 1IV- / , " N
^ y I *'/
^ -1 s ' Cf5 1 ^ . j 1 *~
-H, " 1 v. r -<. '8
. LC -° i>
t&r !i/ ^
I S t3 x
/2f * 'C P-*
\ I, — 6
— /
' \
*«~' CD '
•
i it
>s /
/
X'' '
^ ^ /
" «9 / 64
V
s
h9l
I
*\ 4'j7
; /
v/ »e
/ ,
"v.
•l fl*8"
J '
\ '
57
jm %-
, \
/ rs"
NM \
- «r*i
•70.
Tr"^;
ji__3fr'pe>'
5 .,4 19 ?'*'
I» / ^3 70
r? **'
^r>»—24 ae
?)
/•»
id
~
1
?j
» n
?i 79
%
" ^ L > - j". ''¦*« ' •»
/-» « "%.\ l ,' * *v'
" \7 /»
». /„i "» « ,j\ "/
v_\ 31
ti IT
:v :7
133
7.*9 ii.
a?/
/ ¦" \ 59 •*/
-------
STATION LOCATIONS
SCALC n statutc MILES
Fig. 3. Homogenous Subset of High Vanadium Concentrations (ppm dry vt)
in Arctica lslandica from Fall, 1973 Cruise.
4
-------
STATION LOCATIONS
SCALE N STATUTt MILES
Fig. A. Homogeneous Subset of High Cadmium Concentrations (ppm dry wt)
in Arctica islandica from Fall, 1973 Cruise.
-------
XI
Figure 5
DISTRIBUTION OF DEAD SHELL (unit = buckets) IDES CRUISE
-------
3$ 30 A/
/O
Figure 6
DISTRIBUTION OF ARCTICA CLAPPERS - IDES CRUISE
-------
Table 1. Metal Concentrations ppm Dry Weight
8
Newport
16
Dumpslte
18
20
Arctica
A
Dupont
C G
Phila
E
14
Dupont*
Waste
Phila**
Sludee
Mercenaria
Newport
9 1
Phelps
r x> n d
n . j i > 4
Fe
1086
1033
873
811
631
624
610
386
204
58800
21500
132
117
rrlncle et al
90 - 830
Cu
12.0
11.3
10.1
10.8
4.4
4.2
5.2
6.5
6.6
4.0
1890
18.9
17.4
38.7
23.2
10 - 165
Cr
8,6
6.4
6.8
6.1
2.4
3.5
3.7
3.8
2.9
36.5
1720
4.2
3.2
12.7
4.1
1.9 - 58.0
A1
15l"
248
212
198
62.0
29.0
11.6
820
24900
48.6
43.2
Ag
3.6
3.7
4.5
3.8
0.9
0.7
1.3
1.9
1.5
0.15
52.6
5.3
4.8
1.3
2.7
Mn
25.1
21.6
17.5
19.6
28.3
22.4
17.9
9.9
3.4
1120
1850
22.4
19.4
63.7
84.8
7.0 - 297
Pb
10.2
10.2
9.4
8.7
3.8
4.4
5.2
7.4
5.4
13.1
3290
4.8
4.4
4.9
5.1
1.0 - 75.0
Co
1.5
1.2
1.5
1.4
0.5
2.8
2.8
2.0
1.6
10.6
199
2.2
2.6
4.9
4.4
1.0 - 2.0
Ni
14.5
11.6
15.7
15.9
24.3
11.5
19.5
9.4
948
17.7
25.3
23.7
21.2
V
A.8
5.2
4.1
4.6
4.7
2.9
3.7
2.5
2.1
171
50.6
2.3
1.5
Cd
2.8
2.2
2.6
2.3
1.3
1.6
2.0
2.7
4.2
0.26
141
0.8
0.7
0.7
0.7
1.0 - 7.3
Zn
Ti
103
94.2
103
119
41.3
39.0
54.6
79.0
83.4
36.8
7590
124
143
174
186
115 - 402
2030
133
* wet weight
** dry weight
-------
23
21
29
27
30
24
19
14
25
F
C
A
2
D
8
B
17
9
26
E
Table 2. Ides Samples
Area
Arctica
Pecten
Polinices
Arctica
Clappers
Sediment
N
NE
Dupont
Between-E
E of Dupont
NNE
S
NE
S of Phila
ENE
Between-EE
SE
SE of Phila
Dupont
N of Dupont
Phila
Between
Phila
Dupont
SW
W of Phila
SW
Phila
Between-W
6
1
6
5
21
1
3
4
15
11
6
4
2
1
6
16
18
3
2
10
3
7
2
5
4
1
5
3
30
7
7
2
1/2 bu
5
3
A
16
2
9
3
6
12
1/2 bu
3
1
6
1
2
4
4
9
none
none
72
none
none
2
none
6
1
2
none
none
1
6
1
10
11
91
15
7
6
12
-------
Table 3. Annual Input Delaware Dumpsites
1,072,000,000 kg/yr 584,500,000
Specific Metal Input
Fe 53,400,000 kg/yr 946,000
Cu 3,634 " 83,150
Cr 33,160 " 75,670
A1 745,000 " 1,094,000
Ag 136 " 2,314
Mn 1,017,500 " 81,400
Pb 11,900 " 145,000
Co 9,630 " 8,740
Ni 8,540 " 41,655
V 155,400 " 2,226
Cd 236 " 6,200
Zn 33,400 " 334,000
Ti 1,844,000 " 5,851
-------
Table 4. Mean Metal Concentrations in ppm Dry Weight in ArctJca
islandlca from the Delaware Dumpsites Collected During
"Fetch" Cruise.
Station N Fe Cu V Cr Cd A1 Mn Pb Zn Ni Ar Co Ti
A
1
631
4.3
4.7
2.3
1.3
—
28.3
3.8
41
—
0.9
0.5
1.3
C
1
624
4.2
2.9
3.5
1.6
—
24.2
4.4
39
—
0.7
2.8
4.8
D
6
226
6.4
2.5
2.6
2.2
22
7.0
5.3
84
17.7
1.9
0.8
1.8
E
10
337
6.1
2.5
3.8
2.6
29
9.1
7.8
75
11.4
1.8
2.2
1.3
G
8
504
5.1
3.4
3.8
1.9
70
14.2
5.7
52
24.3
1.4
2.6
4.8
H
1
293
7.4
3.2
6.3
3.9
—
7.6
8.4
141
—
0.4
2.1
1.3
1
12
523
9.7
2.1
2.5
2.6
30
6.8
5.7
104
10.8
2.3
2.1
2
4
231
7.2
2.9
2.5
2.6
22
6.1
4.9
101
9.7
2.2
1.8
5
3
213
4.6
2.4
2.9
3.5
31
7.0
7.6
73
12.1
1.4
1.0
8
5
397
6.3
2.1
2.6
2.2
35
6.7
6.2
101
12.6
2.0
X i 1
1.2
9
10
305
6.1
4.0
4.3
2.1
37
8.8
5.5
66
8.8
2.0
¦L _L
1.5
11
6
340
5.5
2.6
3.4
2.8
20
8.4
6.4
88
14.5
1.6
1.2
1.1
14
3
192
6.3
2.1
2.9
3.9
12
3.3
5.2
111
19.5
2.3
1.2
1.7
17
8
338
5.3
3.4
3.0
3.4
25
9.0
6.0
70
8.8
2.1
0.8
2.5
-------
Table 5. Analysis of Variance of Metal Concentrations
in Arctica lslandica from "Fetch" Cruise.
Metal
F Ratio
Significance Level
Vanadium
5.77
<.1%
Chromium
3.73
<.1%
Cadmium
3.49
.5%
Aluminum
2.95
.5%
Titanium
2.70
1%
Manganese
2.25
5%
Lead
2.20
5%
Zinc
1.24
>20%
Nickel
1.22
>20%
Copper
1.05
insig.
Cobalt
0.96
insig.
Iron
0.48
insig.
Silver
0.31
insig.
-------
METALS TOXICITY TEAM
The Metals Project is devoted to realistically assessing metals as a
problem in the marine environments. Field methods for determining
metal levels in water, sediments, pore water, and biota are being de-
veloped 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 (in-
cluding 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
-64-
-------
METALS TOXICITY TEAM
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.
ROAP APPROACH
I. Construct matrix of toxicity data for metals and aquatic biota
available from literature, using in-house research grants, and
other sources. 16 AAT, all Tasks except 018
II. Formulate and implement biogeochemical field and laboratory
investigations for marine waters impacted by high metal use
point sources. 16AAT-Task 018
ROAP 16 AAT - Task 018:
Field investigations of highly Impacted metals areas.
I. The Quonset Point, Rhode Island, Naval Air Rework Facility (NARF)
A. Purpose: In August 1973 an extensive field sampling program
was formulated by the Metals Toxicity Group. The NARF at the
Quonset Point Naval Air Station was Judged, based on its years
of metal stripping and plating activity, to be a significant
point source metal outfall to lower Narragansett Bay, R.I..
A comprehensive examination of the marine environment in the
vicinity of NARF waste discharges had a two-fold purpose.
1. A field gathering effort which encompassed metal analyses
of the water column, benthic infauna, sediment and in-
terstitial water in order to determine whether or not the
potential ecological impact of this high metal discharge
-65-
-------
METALS TOXICITY TEAM
could be characterized and qualified.
2. To evaluate, invent and improve upon field collection and
analytical procedures which in turn could be utilized for
more efficient field studies of other potentially metals
impacted areas throughout the U.S.
B. Methods Evaluation
1. Biota: Quantitative measurements for the purposes of
species diversity interpretation and body burden metal
analyses of infauna can be accomplished with a Foster
anchor dredge. The larger bivalue mollusks can be best
collected using a "rocking chair" dredge; however, since
this dredge is not qualitative it can only be used for
qualitative sampling.
2. Sediment: Future analyses of sediments for trace elements
depends upon the completion of a series of tests which will
compare the precision and accuracy of present methods of
drying, ashing and analyzing sediments.
Changes from present methods should include (a) A switch to
metal free coring apparatus (b) The measurement of pH, Eh
and/or sulfide in sediments by institu sampling or extraction
of intersitial water, (c) Grain size and organic carbon
analyses since they are key parameters in interpreting
species diversity and mobility of metals.
3. Water Column: Methods have been developed and described in
our last semi-annual report for sampling near bottom water for
-66-
-------
METALS TOXICITY TEAM
dissolved oxygen pH, salinity and dissolved trace metals,
(pg 24 and 25).
4. Pore water: Evaluation of the effects of pressure, time,
temperature, and aeration on cationic mobility in
sediment cores must be made before further work is done
with pore water. Until these problems are resolved, near
bottom water samples are recommended.
RESULTS
1. SEDIMENT
Field work at the Quonset Point Naval Air Rework Facility (NARF), located
centrally on Narragansett Bay, R.I. was accomplished on four one day cruises.
The first two cruises (Sept 9 and 20, 1973) involved dredging of sediment
and benthic fauna (bottom dwelling animals) for analysis by neutron
activation. Sediment was sampled to a depth of 10 cm by this technique.
Preliminary examination of the dredged sediments for total Ag, Co, Cr, Fe
and Mil by neutron activation analysis, revealed no aberrant values as
compared with previous sediment analyses for that general geographic
location.
Gravity coring to a depth £ 50 cm was carried out on the third and fourth
cruises to NARF's 17 stations. Replicate sediment cores were depth
fractioned in the following manner: 0-5, 5-15, and 15+ cm. Following
hydraulic squeezing for the removal of interstitial water, the depth
fractions for all stations were analyzed by neutron activation and atomic
absorption.
Surficial sediments, 0-5 cm depth, exhibit a general trend for all acid
-67-
-------
METALS TOXICITY TEAM
soluble metals examined by atomic absorption (Ag, Cd, Co, Cr, Cu, Fe, Mn,
Ni, Pb, and Zn). Concentrations of the above elements are generally
greater (2-6x) in the vicinity of the waste discharge than at other
stations in central Narragansett Bay. Like Cr (see figure 1), Cd, Pb,
Mn and Zn show particular enhancement (2.5 vs. 0.1, 80 vs. 20, 300 vs. 1C0 and
170 vs. 50 ppm dry weight respectively) at the immediate discharge area
than at stations of greater distance. Neutron activation analyses, where
analysis is possible for the same elements, show the same concentration
patterns as the atomic absorption results.
2. Biota
Six key species, three polychete worms and three bivalved mollusks,
were predominant in dredged samples from the first two cruises. These are
Nephtys incisa, Glycera dlbranchiata and Ninoe nigripes. The mollusks
including Ensis direcus, Yoldia limatula, and Pitar morrhlana. Analyses of
Nephtyst Glycera, Ninoe, Ensis and Yoldia by neutron activation are nearly
completed. Atomic absorption analyses of the larger Pitar have been completed
for the metals Ag, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn.
Body burden analyses of Pitar morrhuana, the hard shell "widgeon" clam,
exhibit elevated levels of the metals Pb, Fe, Mn, and Cr, (see figure 2),
at stations near the sewage and run-off outfalls as compared with other
Narragansett Bay stations. Although the clam was not found at stations in
the immediate vicinity of the discharge pipes, it was common to 12 of the
17 stations. The following average values are expressed as yg/gm dry weight
for Pitar flesh.
-68-
-------
METALS TOXICITY TEAM
Pb
Fe
Mn
Cr
near
sewage
discharge
26
800
160
17
mid bay
stations
15
300
60
5
3. H^O Results were reported on in the previous semi-annual report.
June - December 1973 pgs 24 and 25 also 4. Pore H^0 was reported on page
26.
II Cmega Pond, Rhode Island
A preliminary set of samples were taken in the Seekonk Fiver above and
below the entrance of Omega Pond, the Providence River and the Upper end
of Narragansett Bay. In general, these samples were characterized by low
pH (6.4 to 7.5), low dissolved oxygen (0.6 to 6.2 mg/1), low salinity
(0.42 to 27.3%) and high dissolved trace metal values. The Narragansett
upper bay stations (Sabin Point and Seekonk River) appeared to be heavily
impacted with respect to trace metals when compared to middle bay stations
(Quonset Point and NMWQL) or the mouth of Narragansett Bay in proximity
of the Brenton Ligh Tower (Table 1).
III. Mr. Thomas Bickford, a graduate student from Brown University working
for a Masters degree in Geochemistry, has started a project to establish
whether or not chromium can be used as a flag for organic pollution from
sewage treatment plants in the area. The problem is most critical immediately
following a heavy rainfall (1 inch in 24 hours) when the sewage treatment
plants overflow, sending a large slug of untreated sewage into the bay.
This condition occurrs most frequently during the spring thaw and runoff.
Often during this period the State Health Department if forced to temporarily
-69-
-------
METALS TOXICITY TEAM
suspend shellfish harvesting in the northern portion of the bay due to
the increased pollution levels. Their monitoring method consists of
taking surface water samples and performing both total and fecal coliform
determinations. This technique does not adequately reflect near-bottom
conditions, where the particulate material resulting from the overflow
is moving. Previous work here at the lab has demonstrated that Cr is
associated with particulate material in the bay environment.
The project area includes the portion of Narragansett Bay north of
Prudence Island and the southern reaches of the Providence and Warren Rivers.
A grid of seston (material which has settled during a known time period
from the water column into collecting bottles placed one meter above the
sediment surface) collectors are to be set out in the study area.
The sampling will be carried out throughout the rest of this year and into
next spring during the period of normally high runoff associated with the
spring thaw and rains. Samples will be collected at 7, 14, and 21 day
intervals. They will be processed to separate the organic from the inorganic
fraction and these fractions analyzed via atomic absorption spectroscopy
to determine Cr levels. It is hoped that increased Cr levels, associated
with the organic pollution, can be coorelated with the spring closure periods.
IV. Ecological impact of Point Source Metals Discharges in Marine Ecosystems:
Corpus Christi, Texas.
Marine waters of Corpus Christi, Texas and environs are heavily impacted
by wastes from smelteries, petrochemical and other operations. Anomalously
high levels of Zn and Cd in CC waters are documented in the open technical
-70-
-------
METALS TOXICITY TEAM
literature. OYSTERS (C. vlrginlca) collected from Corpus Christ! Harbor in
April were analyzed for Cd, Fe, Cu, and Pb. Comparisons of mean values
with those of oysters from New England of similar size and weight is listed
below. All values are in mg element/kg wet wt., and are based on at least
six animals.
TEXAS
NEW ENGLAND
Zinc
3271
2324
Copper
20
103
Cadmium
0.50
1.21
Lead
0.92
0.07
Iron
33
16
Results of this preliminary study are difficult to interpret; however, inter-
action and suppression effects appear likely. It is recommended that
collection of additional shellfish and other organisms from Corpus Christi
Harbor and environs be implemented and that laboratory studies with metals
mixtures approximating field values vs. shellfish be initiated.
ROAP 16AAT Task 031:
METALS BIBLIOGRAPHY
Dr. Eisler and student aide Mary Jane Wapner are preparing Volume 2
of "Annotated Bibliography on Biological Effects of Metals in Aquatic
Environments." Approximately 400 technical articles of recent vintage
in this subject area have thus far been assembled, abstracted, typed, and
indexed. A target date of December 1974 has been set for the first
complete draft.
A matrix of metals versus marine organism response is being developed from
the above bibliography, other sources of literature and in- house laboratory
-71-
-------
METALS TOXICITY TEAM
experiments which should clearly Indicate the present state of knowledge
and future areas of research.
Manuscripts:
Davey, E.W., M.J. Morgan, and S.J. Erlckson. 1973. A Biological
Measurement of the Copper Complexation Capacity of Seawater. Litnnol.
and Oceanog. 18(6): 993-997.
Eisler, R. 1974. Radiocadmium exchange with seawater by Fundulus
heteroclitus (L.) (Pisces: Cyprinodontidae). Journal of Fish
Biology, Vol. 6, 601-612.
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 Verner, S.S. (ed). Proceedings of a seminar
on methodology for monitoring the marine environment, U.S. EPA
Report, in press.
Johannes, R.E G. Telk....and others. 1972. The Metabolism of
Some Coral Reef Communities: A Team Study of Nutrient and Energy
Flux at Eniwetok. Bioscience. 22(9): 541-543
Marshall, N., A. Durban, R. Gerber, and G. Telek. 1974. Observations
on particulate and dissolved organic matter in coral reef areas.
Marine Biology. In Press.
Presentations:
Davey, E.W. and A. Soper. Apparatus for the In-Situ Concentration of
Trace Metals from Seawater. ASLO Meetings, Seattle, Washington,
June 24-28, 1974.
Eisler, R. Biological effects of petrochemicals to Red Sea teleosts.
American Fisheries Society, McAfee, N.J. Feb. 24-27, 1974.
-72-
-------
METALS TOXICITY TEAM
Eisler, R. Presented testimony on role of cadmium and mercury in
marine ecosystems. EPA effluent standard guideline hearings,
Washington, D.C. April-May 1974.
Eisler, R. EPA NERC-Corvallis Metals Program Review, presented at
Gleneden Beach, Oregon, March 10-14, 1974.
Telek, G. Vertical Distribution of Zinc, Manganese, and Iron in
Narragansett Bay, Rhode Island. ASLO Meetings, Seattle, Wash.,
June 24-28, 1974.
Miscellaneous:
Dr. Davey, attended a Chemical Equilibria Workshop in Corvallis,
Oregon, March 27-29, 1974.
Dr. Eisler was appointed chairman, Water Pollution Abatement Committee,
NE. section, American Fisheries Society.
16AAT. Task 025
Grants:
Title: Bioaccumulation of Heavy Metals by Littoral and Pelagic Marine
Organisms.
Investigator: Dr. John Martin, Moss Landing Marine Laboratories
Moss Landing, California 95039
#802350
Project Officer: Dr. Ronald Eisler
Progress Period 12/17/73 - 6/17/74
Highlights:
Dr. Martin reported at the AAAS meetings in San Francisco on March 1 regarding
levels of cadmium in sea otter kidney; however, no definite link has been
-73-
-------
METALS TOXICITY TEAM
made between renal Cd residues and otter mortality. Markedly high Cd
concentrations were also found in Baja California plankton and are believed
to be of anthropogenci orgins. Further analysis of squid hepatic tissues for
the elements Ag and Cu reveal significant enhancement in inshore species vs.
mid Pacific Ocean species.
Other findings have included a surprising excessive selenium to mercury
ratio in aborted sea lion featuses versus normally born pups. Selenium,
an element of known toxicity, has been reported in recent literature of
having beneficial effects in prevention of Hg poisoning. However, the
reverse, whether Hg prevents Se toxicity, is yet to be examined.
-74-
-------
?!•»• rrz* rfzy ?r? 2
•it
tooo
coo
2000
PRIMARY 94
SCWACC
OUT fALL
P°MARY )
PUN-orr ft/#5
•7
• 0
• ©
41* >4
• »
1ARRAGANSETT
i BAY
• 11
CONANICUT ISLAND
• to
Microgram Cr/gram dry weight surficial sediment (0-5 cm. depth)
FIGURE 1
-------
Tl*H'
Tf25'
rrz#
rrjy
4i* a r
*•36'
4IM5'
41* 31'
mass
0*VTSVILL€
¦ C TCR3 *
l{>00 20OO
PRIMARY
SCWACE
OUTFALL
01^T PRIMARY )
Y
RUK-Or F,, fc'
SICONOARt
NAQRAGANS€7T
BAY
CONANlCuT (5CAMO
4i*5r
— 4i*5C
— 4C3V
— 41*54'
— 41*13'
rrzt' WW 7i#24' rray
Microgram Cr/gram dry weight flesh Pitar morrhuana, "widgeon clam"
FIGURE 2
7i*22
-------
OILS TEAM
ROAP - 16AAV
After completion of the report on the effects of waste motor oil, we
moved directly into planning and preparing for our overall oils program.
This program utilizes both in-house and out-house resources. It is
separable into three broad categories, encompassing the scope of the
problems of oils in the marine environment. These three areas are the fate
and effects of:
1. Spilled oil,
2. Discharges of the water-soluble-fractions of oils,
and
3. The long-term effects of oil-contaminated sediments.
We recognize this is somewhat arbitrary, as these categories are not
strictly separable, but rather do interact.
1. Spilled oil: Much of our program on the fate and effects
of spilled oil will be conducted extrmurally thru closely-monitored
grant and contract activity. We have a large ongoing contract with
Mississippi State University to study the fate and effects of Empire
Mix crude oil spilled in a simulated estuarine ecosystem. This is a
diverse multidisciplinary study, examining the effects of the oil
from many points of view. In addition to detailed chemical analysis
of the fate of the oil, long-term affects will be measured by looking
at mortality, pathology, population changes, behavioral effects, enyymatic
changes, and reproduction. To date, acute laboratory studies have been
performed with shrimp, mullet, oysters, and marsh gasses as well as
photo and zooplankton. The large-scale simulated ecosystems have been
-75-
-------
OILS TEAM
constructed and stocked, including a tidal simulation system. Long-term
field studies are set to go momentarily.
Another contract, to study the effects of spilled oil in an estuarine
environment has just been negotiated with the Virginia Institute
of Marine Science. For this study, they are to spill //2 fuel oil at
initial concentrations of 250 and 500 ppm in blocked off sections of an
estuary containing clams and oysters. Detailed chemical analysis of the
water, sediments, and organisms will be performed; including mass spectro-
metric identification of individual compounds from the oil. This will be
conducted through an inter-agency agreement with the NASA-Langly Air Force
Base Facility. In addition, living samples of clams and oysters will
be shipped to our laboratory for subsequent histological examination.
In future years, our plans call for additional contracted studies of
controlled spills in other geographic areas, as well as studies including
effects of oils on surface microlayer biota, the fate of dispersed oil,
the tar ball problem in the north Atlantic, and the mechanisms of oil
transport into sediments.
The majority of regulations promulgated by the agency so far have
implied that oils are materials which form visible sheens or deposits.
This point of view ignores water-soluble fractions and oil contaminated
sediment problems. Our program is examining both of these areas. The
goal is to set defensible criteria. Much of this work will be done
utilizing our ln-house bioassay and chemical analysis expertise. For
the water-solubles study, we are testing an oil-water separation device
to produce a constant flow of the water-soluble fraction from //2 oil.
(Figure 1). _7fi_
-------
FIGURE I
SEAWATER
OIL
WATER SCLUELE FRACTION TO TEST
DRAIN
-77-
-------
OILS TEAM
This toxicant will be mixed with clean seawater to achieve the desired
hydrocarbon concentration, which can be measured by CCl^ extraction
and infrared absorbance measurements. A variety of organisms and their
life stages will be exposed in long-term flowing water bioassays.
Ballast treatment plants take oily ballast and bilge waters from
tankers and pass them thru an oil-water separator. The water fraction
is dumped into receiving waters. In order to evaluate this potential
problem with realistic and meaningful bioassays, the fate of these
materials in estuaries and coastal areas has to be determined. For
example, if the hydrocarbons were to be rapidly absorbed into the sedi-
ments, then a chronic bioassay on a free-swimming fish is not realistic.
Likewise, if the compounds were to stay in the water column, a deposit-
feeding polycheate worm is not the bioassay species of choice.
To gather an information base, we have excuted an interagency agree-
ment with the Navy to examine the distribution of hydrocarbons around
a ballast treatment facility. This should allow us to model our bioassay
results in the real environment.
Long-term goals include work in taste and odor criteria as response
parameters for commercially important species, and expanding our horizons
to Include other discharges, such as petrochemical, refinery discharges,
and drilling brines. Some will be done on an extramural basis.
The third major area of the oil pollution problem is the little known
area of the fate and effects of oil-contaminated sediments. It
is in this area where many of the chronic biological effects occur.
-78-
-------
OILS TEAM
Bacterial action can degrade hydrocarbons in sediments, but the rates
are slow and the more toxic aromatic fractions are degraded most slowly.
We wish to find out the rates of release into the water column, the rates
of degradation under different conditions, the rates of transport with
and through the sediments, and finally the biological effects of these
materials. Eventually, we plan to arrive at concentration values
which may not be exceeded in sediments in order to protect sensitive
organisms. These values can then be compared with environmental samples
to delineate the hydrocarbon Impact zones in sediments. In addition,
these numbers will help generate ocean disposal criteria for dredge
spoils and sewerage sludge which contain oil residues.
We have constructed a set of tanks to contaminate clean sand in a simulated
6pill of #2 fuel oil, (Figure 2). We are observing effects of soft-shell
clams living in the sand over an extended period of time. In addition
a variety of organisms are exposed to the effluent from the contaminated
sediment tank in a lower tank. The clams were chosen in order to
corroberate previous field observation of cancer in these animals which
is related to a spill of if 2 fuel oil at Sear sport Maine. We are also
planning to observe the fate and distribution of the oil by chemical
analysis of the water, sediments and organisms.
We have spent much of the last several months working on the analytical
techniques to perform these analyses. The water analysis appears to be
quite straightforward at the concentration ranges between 0.1 and 10 ppm.
One simply extracts a water sample with CCl^, and measures the strength
of the infrared absorption at 2930 cm This frequency corresponds to
-79-
-------
FIGURE 2
SEAWATER
OIL
CLAMS (in4"sand)
FISH OR
¦ LOBSTER
MUSSELS
- -> DRAIN
-80-
-------
OILS TEAM
the C-H stretch, and gives a good estimate of the total hydrocarbon content
of the sample. For more detailed analysis, one can turn to a gas chro-
matograph (GC) or ultimately to our gas chromatograph-mass spectrometer
(GC/MS) combination.
More sophisticated techniques are required when analyzing for hydrocarbons
in organisms and sediment. For these, the most difficult problems lie
in the sample preparation and purification steps. The basic procedure
is well known. One exhaustively extracts the sample with an organic
solvent and then treats the extract with strong base to soponify the
lipids into acids and alcohols. These are then separated from the non-
saponifiable hydrocarbons by liquid-liquid extraction, and the hydro-
carbons separated by column chromatography. The sample is then ready for
GC or GC/MS analysis.
Our sample handling problems have revolved about removing water from
homogenized animal tissue in order to get complete extraction. Our best
results have been with centrlfugation. An initial spin separates most of
the water which can then be poured off and saved. The residue is then
homogenized with methanol and spun again. This removes most of the
remaining water. This is then repeated twice using benzene, with a
refluxing step added to ensure removal of all lipophilic material.
All extracts are then combined and refluxed with KOH for saponification.
Following extraction to remove the saponified products, the straight-
chain hydrocarbons are eluted from activated silica gel with petroleum
ether. An elution with 15% benzene in petroleum either removes substituted
naphthalenses from the column, and an elution with 35% benzene removes
-SI-
-------
OILS TEAM
tricyclic aromatics such as anthracene. The whole procedure is still
being checked for losses, contamination, and interference, but out pre-
liminary indications are good. We expect the same procedure to work
with sediments, but a sulfur-removal step will be necessary for some
samples.
-82-
-------
RECREATIONAL WATER QUALITY CRITERIA TEAM
Recreational Water Quality Criteria Program (ROAP 21 BEZ) has as its
primary objective the development of health effects, water quality
criteria for marine recreational waters. A secondary objective of this
program, and one for which funding has not been received to date, is the
development of the data base required for the translation of target
area criteria (such as those for recreation and shellfish growing areas)
into effluent standards and design criteria for ocean outfalls.
The first phase of the program to develop criteria for marine recreational
waters consisted of a pretest of the epidemiological and available
microbiological methodology and verification of the suitability of the
test and control beaches tentatively selected. After screening a number
of salt water beaches in the vicinity of large urban population centers,
those in the vicinity of 20th Street, Coney Island, and 67th Street,
Rockaways, in New York City were selected as the "barely acceptable"
and "relatively unpolluted" beaches respectively. In order to avoid
"human volunteer experiments" or the experimental bias which could occur
by conducting the trials at beaches which local authorities post as unsafe,
the test beach must be one which is "barely acceptable" according to existing
criteria and standards; thereby the initial trails are, in fact, a test
of those standards.
The second phase of the research compared the rates of illness at the
"relatively unpolluted" beach and a "barely acceptable" test beach using
study populations large enough for significant results. Periods of peak
pollution and maximum bather density were selected. The methods used were
refinements of the techniques evaluated during the previous summer's pretest.
-83-
-------
RECREATIONAL WATER QUALITY CRITERIA TEAM
A comparison of the "barely acceptable" and "relatively unpolluted"
beaches In terms of densities of these microorganisms quantltated during
the pretest Is given In Table 1. In addition, quantitation of Salmonella
species Indicated a significant (95%) difference between the two beaches
but a very low overall density (maximum recovery: 45 organisms/10,000 L).
As a result, Salmonella quantitation was not Included In the second phase.
During the second phase (June-September, 1974) Clostridium perfrlngens,
Staphylococcus aureus, Staphylococcus epidermidis, and Vibrio parahemoly-
ticus densities were obtained and Coprosterol levels were measured.
Analysis of the epidemiological data from the pretest permitted calculation
of the number of interviews required to insure statistical reliability
in the data to be obtained and establish in the interview routine. The
sequence of events for each interview is given in Table 2. It was calcul-
ated that approximately 12,000 interviews involving 42,000 individuals
would be required.
The pretest data did produce two interesting observations associated
with the choice of the test and control beaches. One was based on a
comparison of the E^. coli and Enterobacter ratios (Table 2) from which
it can be seen that the very low levies of pollution (as measured by coliforms)
reaching the Rockaways beach are not a further dilution of those at Coney
Island. Rather, the data suggest an Immediate source of raw fecal wastes.
Furthermore, the Salmonella recoveries obtained by the Salmonella high-
volume sampling (S-HVS) method do reflect the disparity in the total
coliform densities at the two beaches. This should not affect the choice
of the Rockaways as the control beach. It does, however, point out the
value of examining the distribution of coliform biotypes in determining
-84-
-------
RECREATIONAL WATER QUALITY CRITERIA TEAM
sources of pollution.
The second relates to the analysis by trial (day) of the illness rates
at Coney Island beach. The trials (days) were ordered according to the
mean coliform or fecal coliform density and segregated into four groups
of two trials each. The illness rates for the four groups were determined
by type (respiratory, gastrointestinal and other) for swimmers and the
non swimming controls. This analysis revealed no increase in illness
associated with swimming during the "high" as compared to the "low"
pollution days. On the other hand, there was a strong suggestion that
the incidence of gastrointestinal illness among swimmers, when corrected
for the rate among non swimmers, was higher at the "barely acceptable"
(Coney Island) than at the "relatively unpolluted" (Rockaways) beach.
Since this was a pretest of methodology the number< of individuals queried
was too small to draw any firm conclusions relative to water quality
criteria.
The microbiological effort for Phase 2 comprized approximately 240
samples and 1500 analyses. In addition, in excess of 10,000 interviews
were obtained. The data from these trials is being analyzed.
The pretest results and outline of the entire Recreational Water Criteria
Program were presented in a briefing conducted for EPA personnel from all
the regional offices and the Office of Environmental Sciences. A similar
presentation was conducted for a joint meeting of the New York City and
Nassau County Health Departments in New York City and a third time for
Drs. Moore, (England); Mood, (U.S.); Keckes, (Yugoslavia); Batiston, (Argen-
-85-
-------
RECREATIONAL WATER QUALITY CRITERIA TEAM
tine): and Brlsou, (France) who visited the laboratory under the auspices
of W.H.O.
Many of the techniques developed during the preliminary microbiological
studies and a summary of the pretest have been published.
Papers Published
1. Levin, M.A., Fischer, J.R., and Cabelli, V.J. A High Volume Sampling
Procedure, Appl. Microbiol., September, 1974.
2. Cabelli, V.J., F.T. Brezenski, A.P. Dufour, and M.A. Levin. Micro-
biological Methods for Monitoring Marine Waters for Possible Health
Effects, Proc. Sem. for Monitoring the Marine Environment. S. Verner
(ed), Seattle, Wash. (Publication date not known).
3. Levin, M.A., Fischer, J.R., and Cabelli, V.J. Quantitative Large-
Volume Sampling Technique, Abst. Amer. Cos. Microbiol., May 1974,
p. 34.
4. Thomas, C.D., Watkins, W.D., and Cabelli, V.J. Quantitation of
Vibrio parahemolyticus from Estuarine and Coastal Marine Water, Abst.
Amer. Soc. Microbiol, May 1974, p. 65.
5. Cabelli, V.J., M.A. Levin, A.P. Dufour, and L.J. McCabe.
The Development of Criteria for Recreational Waters. Int'l.
Symposium on Discharge of Sewage from Sea Outfalls, paper #7,
London, August, 1974.
6. Marcus, A., Riccio, J., Levin, M., Cabelli, V., and Dufour, A.
Evaluation of Microbiological Parameters for Dynamic Modeling of
Lake Eutrophication, ASM, Conn. Valley Branch, Fall, 1974.
Five manuscripts have been submitted for review.
1. Dufour, A.P., and Cabelli, V.J. A Membrane Filter Procedure for
Enumerating the Coliform Group and its Component Genera in Seawater.
2. Dufour, A.P., and Cabelli, V.J. Characteristics of Klebsiella
from Textile Finishing Plant Effluents.
3. Huntley, B.E., Jones, A.C., and Cabelli, V.J. Klebsiella
Densities in Waters Receiving Wood Pulp Effluents.
4. Levin, M.A., Fischer, J.R., and Cabelli, V.J. Membrane Filter Technique
for Enumeration of Group D streptococci in Marine Waters.
5. Cabelli, V.J., Kennedy, H., and Levin, M.A. Pseudomonas aeruginosa -
Fecal Coliform Relationship in Estuarine and Fresh Recreational Waters.
-86-
-------
TABLE 1
Companion 01 lot: mean indicator .111;! pathogen demine*
(per 100mlj.>t :!>_¦ luieiy .'.cj.-ptjb'e' anJ "roljfvj!» unpjl! t;J'
K- iches
Coney Hand
Rockasva> s
Orijanismfs)
Method
(20th St)
(67th St)
Total coltforrm
mC
9S3"
39 8
T*xal coliiorms
mC
165-
21 5
E. coh
mC
174*
24-8
Klebsiella
mC
122"
137
Enterobacter
mC
530*
IM
Fecal streptococci
mSD
91 2
21-8
P aeruginosa
mPA
30 4*
6-5
.-J. hydrophila
mA
25 3
26 5
* Significantly different at 95 per cent confidence level
TABLE 2
Sequence of events for epidemiological-microbiological trials
Day of week Day number
Activity
Function
Saturday 1 Beach interview, water sampling
Sunday 2 Same as Saturday
Monday 3 Reminder letter
Monday 10 Phone or mail interview
(a) Obtain name, address, phone, etc.
(b) Reject pre-trial midweek swimmers
(c) Query on beach activity
(d) Assay of water samples
As above
(a) Provide name of physician
(b) Reminder to note illness
(a) Obtain illness information
(b) Reject post-trial midweek swimmers
(c) Obtain demographic information
TABLE 3
E. colilEnterobacter ratios at Coney Island (20th St.) and Rockawayj (67tn St.)
beaches by trial
Trial dire
Mean recovery (per 100 ml)
E. co!ij Enter. ratio
Coney Island
Rockaways
Coney
Inland
Roekawa>s
E. coh
Ei.ier
E. coh
Enter.
14 July
59
162
13
5
0 35
26
22 July
197
¦524
16
7
0-61
2-3
28 July
79
226
24
4
0 35
60
29 July
S3
239
20
27
0 37
0 74
11 \vi
I0OO
69 9 S
12
2
0 16
60
12 Aug
4J3
4-^7
17
1 1
0':
1 2
13 Aus.
240
279
41
16
0 36
2 6
19
7S
2-il>
56
14
031
d0
Median ratio*
0 35
26
-87-
-------
TECHNICAL ASSISTANCE
The bulk of our non-research efforts from January through June have concerned
writing guidelines for administration of the 1972 Water Bill Amendments,
Section 316,in saltwater environments. This portion of Thermal Regulation
deals with the situation in which an applicant for a discharge permit wishes
to demonstrate that applicable regulations are more stringent than necessary
to protect the balanced indigenous biotic community. In conjunction with
drafting sefctions on 316(a) guidelines for the EPA task force, we have been
drafting a document on marine temperature criteria to be used in setting
water quality standards. These two major efforts have required a decrease
in the number of individual responses to specific Regional requests for tech-
nical assistance. However, we hope that broad guidance manuals when in use
will provide much of the information for which we have been asked on a case
by case basis.
Marine Temperature Criteria;
Requirements and deadlines set by the 1972 Water Bill Amendments have
provided on opportunity to re-examine temperature criteria for marine
waters last set by the National Technical Advisory Committee's "Green Book"
published in 1968. We searched the literature for thousands of hours,
compiling thermal tolerances of a spectrum of marine plants and animals—
all life history stages—and were rewarded with hundreds of references
containing valuable field and laboratory data. These were tabulated according
to biogeographlc region, species, life history stage, and thermal response
(acclimation temperature, upper lethal limit, growth optimum, optimum for
reproduction, lower lethal limit, etc).
-88-
-------
TECHNICAL ASSISTANCE
From these temperature response data and a knowledge of the abundance,
distribution, and reproductive requirements of marine species, we constructed
a series of regional thermal recommendations. These concerned protection of
intertidal zones of passage, benthic (bottom) communities, and entrainable
juvenile fishes and invertebrates.
Studying cooling water effects in the field and in the literature demonstrates
that this industrial water use can not be regulated on thermal criteria alone.
Mechanical, chemical, and hydrodynamic factors need be considered together
with thermal ones. Further, bureaucratic divisions of Intake from discharge
effects are not very meaningful to a biota that needs protection—which
requires our careful consideration in developing thermal regulatory language.
Clear caveats must warn state and federal permits personnel as well as indus-
trial cooling water users that environmental protection can not be assured
by temperature regulation alone. In fact, for certain sites increased
temperature may be preferable to increased volume of cooling water.
Administration of PL92-500, Section 316(a):
Section 316 of PL92-500 has caused considerable confusion among permit per-
sonnel, industrial cooling water users, and EPA headquarters personnel charged
with guiding regional application of this exception to thermal standards.
Consequently, NERC-Corvallis laboratory personnel, together with headquarters
personnel representing OEGC and OWP have drafted a guidance manual which was
submitted to Regional offices and Industry for comment.
-89-
-------
TECHNICAL ASSISTANCE
NMWQL contributed manuscript sections concerning criteria for thermal
exceptions in the marine environment. The challenge was to draft guidelines
for use by non-biologists in writing alternative permit limitations on thermal
discharge that will assure "protection of a balanced indigenous population of
fish, shellfish, and wildlife in or on the body of water." Further constraints
on our assignment were (1) that the end result be applicable to either existing
or proposed cooling water use, (2) that the environmental constraints on the
applicant s activities be enforceable, (3) that the demonstration of "assurance"
required of the applicant be reasonable in time-and resources required, (A)
that appropriate levels of demonstration be available to the applicant based
upon both existing and proposed discharge considerations and environmental
values of the receiving water body, and (5) that we not re-invent the
environmental impact statement through misplaced enthusiasm to achieve
scientific completeness rather than managerial adequacy.
Several hundred hours spent in reading, meetings, and discussion with EPA
and academic experts in marine ecology resulted in guidance manual sections
on marine mixing zones, variance in ecological measurement parameters, and
matrices on important thermal limits of marine species to be protected.
Among thermal limits considered were those for growth, reproduction, avoidance,
upper survival limits, lower survival limits, and lethal temperatures. Another
matrix matched species representing various important ecological roles
(primary producers, secondary consumers, habitat formers, migrants, top
carnivores, etc) against thermally associated stresses encountered in industrial
cooling waters (impingement, entrapment, entrainment, pump damage, scouring,
velocity stress, etc). The goal was to pack important managerial information
-90-
-------
TECHNICAL ASSISTANCE
Into a succinct format useful both to applicants and Regional permits personnel.
Assistance with Regional Ocean Dumping Permit Problems:
Regional differences are emerging among types of ocean disposal problems for
which our assistance has been requested. Region I faces dredge spoil and
mycelial waste disposal decisions; Regions II and III are concerned with massive
volumes of sewage sludge and two types of iron-acid waste from Ti02 manufacture,
Region IV's ocean disposal problem areas are metals wastes, Region VI has
requested assistance with design of bioassays requested of permit applicants
having complex industrial wastes to dispose of offshore, and Region X required
assistance with a peculiar problem of disposal of large quantities of salt
in shallow waters on the north continental shelf of Alaska.
NMWQL research programs on ocean dumping have taken direction from recurring
requests for technical assistance. Thus, questions concerning the fate and
effects of polluted dredged spoils and iron-acid waste from TiO^ manufacture
are discussed in detail under the activities of the ocean dumping research
team. These are high-volume sources.
The lesser volume, one-time requests are no less interesting however—
and it is possible that some may have far-reaching environmental impacts.
Reviewing literature concerning regional assistance, it is evident that few
attempts to trace dumped materials have yielded direct evidence of their
ultimate fate in the sea and biological accumulation or effects. Assistance
to Regional Permits Programs has helped NMWQL staff to improve field sampling
techniques and apply bioassays needed to solve these problems. Another year's
research may see us in position to provide a general guidance document. Some
-91-
-------
TECHNICAL ASSISTANCE
examples of regional requests for assistance are listed below:
Region III: Public hearing on ocean disposal permit applications submitted
by DuPont (acid wastes) and the City of Philadelphia (sewage sludge).
G. Pesch attended and served on the review panel considering the permits.
Region I: Public hearing on ocean disposal permit application submitted
by Pfizer Pharmaceutical of Groton, Conn. NMWQL staff members G. Pesch,
B. Reynolds, and M. Levin attended the hearing to provide technical advice
to the Regional staff.
Region II: A conference between Reg. II personnel and N.L. Industries
was attended by G. Pesch. N.L. applied for a special permit to dispose of
titanium dioxide wastes at sea. Dr. Pesch provided technical assistance in
evaluating N.L.'s application.
Region III: G. Pesch met with Reg. Ill personnel in Philadelphia to evaluate
responses to an RFP issued jointly by NMWQL and Reg. Ill to conduct a base-
line survey of the Mid-Atlantic Bight in the vicinity of the Delaware
shelf dumpsltes.
Region It In response to a request for comments on a permit application
for New England Power Company to begin disposal of coal ash at sea, B. Reynolds
and G. Pesch supplied information on alternatives and possible marine disposal.
U.S. Navy: A proposal was received from the Naval Ordinance Laboratory for
a study on the environmental impact of explosions. Comments on this
proposal were forwarded by B. Reynolds through Dr. D.K. Phelps.
Assistance with Power Plant Permit Problems:
The first flurry of section 316(a) public hearings brought several requests
for expert testimony and evaluation of applicant's demonstrations of environ-
mental protection assurance. Regions I, IV, and IX have requested various
levels of NMWQL involvement in specific power plant permit activities.
Kahe Generating Station, Region IX: Dr. Prager met in San Francisco with
Region IX Permits Program personnel to review the Hawaiian Electric Company
application and supporting data for thermal discharge effluent limitation
waiver under Section 316(a). Together with Region IX personnel, a permit
was drafted requiring some modification of discharge location In order to
allow corals prevalent in the area to recolonize coral heads destroyed by
-92-
-------
TECHNICAL ASSISTANCE
present discharge from existing units. A 316(a) hearing on this application
is scheduled for fall, after an NMWQL-University of Hawaii study team has an
opportunity to make field observations.
Pilgrim Power Plant, Region It Dr. Steele attended a meeting with personnel
from Boston Edison Co. and Region I to discuss necessary proofs for the
applicant's request for a 316(a) waiver hearing. Quality and quantity of
the applicant's available information was discussed. Guidelines were pro-
vided to the applicant for additional studies needed to obtain a 316(a)
waiver in this special case. Region X attorneys had met earlier in the
week with Drs. Steele and Prager to discuss strategy for this discharge
permit.
Newington, N.H. Power Plant, Region I: NMWQL personnel met with Region I
Power Plant Siting and Operations Committee to discuss the status of
available information on this facility's request for a 316(a) waiver.
Questions concerning adequacy of the Companies' available geological data
were partially resolved. Regional strategy concerning allowable power plant
chlorination practices was also discussed with Dr. Gentile.
Proposed Charlestown, R.I. Nuclear Plant Site, Region I: Dr. Prager met twice
with a group of concerned citizens in Charlestown, R.I. to discuss environ-
mental aspects of New England Electric Co.'s proposed nuclear power generating
facility. A two unit nuclear plant is proposed for the Charlestown Naval
Air Station, a training facility recently given over to G.S.A. by the Navy.
The public group, concerned about aspects of resource conservation and nuclear
safety, was advised on how and from whom to obtain the information needed
for townspeople to contribute to the decision process.
-93-
-------
TECHNICAL ASSISTANCE
Anclote Power Plant Public Hearing, Region IV: Dr. Prager, together with
personnel of National Park Service (DOI) and NOAA, performed a third
annual underwater site survey of the proposed discharge and intake areas of
the Anclote, Florida plant. Following this Dr. Prager presented testimony
concerning needed modifications in the plan for cooling water use proposed
by Florida Power Corp. at a 316(a) Public Hearing on the matter held at
Clearwater, Fla.
-94-
-------
T.F GREEN AIRPORT
1 EAST GREENWICH
1^— DIVISION ST EXIT/
RT.4
INTERSTATE 95-*.
RT. 2
RT. 2
RT4
RT. I
RT. IA
King's Ini
BLINKER
| I SOUTH | FERRY RD.
[ / |A F.P4.
r—-T narragansett
BRIDGETOWN RD. ) LAB
U.R.I.
RT. 138
FAIRGROUNDS
RD.^ /
LIBERTY LN.
ENVIRONMENTAL
PROTECTION AGENCY,
WEST KINGSTON
RT
JERUSALEM
SNUG HARBOR
EXIT \
SUCCOTASH RD.
E.PA. RESEARCH
BARGE ~
------- |