&EFA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Narragansett Rl 02882
Research and Development
EPA-600/S3-81-049 Oct. 1981
Project Summary
Nutritional Requirements of
Marine Larval and
Juvenile Fish
Kenneth L Simpson, Paul S. Schauer, Cynthia R. Seidel, and Leslie M.
Richardson
This project summary describes and
assesses the results of a cooperative
research effort to evaluate a variety of
diets and diet regimes for the labora-
tory culture of a marine larval fish. The
Atlantic silverside (Menidia menidia)
was chosen for this study on the basis
of its regional availability, ease of
handling in the laboratory, and its use
as a bioassay organism. A major aim of
the study was to develop a diet that
would provide good growth and sur-
vival and provide a test fish similar to
its wild counterpart.
Initially, data were gathered on the
composition of the silverside egg and
wild fish, plankton, brine shrimp and
various commercial and modified
diets. Following an analysis of the diet
components, some improvements
were made in the artificial diets. Later
studies concentrated on the effect of
modified diets in relation to the feed-
ing of brine shrimp nauplii.
Silversides cultured on artificial
diets generally had poor growth and
survival rates. Freeze-dried brine
shrimp [Artemis] nauplii and ground-
up adult silversides also failed to
promote good growth and survival. A
superior diet was provided by live, 3-
day-old brine shrimp nauplii. How-
ever, a combination diet of brine
shrimp and artificial diets provided
, growth and survival more similar to
the "all live" brine shrimp diet, even
when the live diet was fed only every
eighth day.
Biochemical data were compared to
reported requirement levels for essen-
tial amino acids and essential fatty
acids in marine fish, and it was con-
cluded that all diets seemed adequate
in these two components. These data
also indicated that fish accepted both
artificial and live diets equally. The
characteristics of the storage of
dietary amino acids and dietary fatty
acids in silversides was assessed.
Although the reason for the live diet's
nutritive superiority remains unclear,
several recommendations are
presented.
This Project Summary was develop-
ed by EPA's Environmental Research
Laboratory. Narragansett. Rl, to an-
nounce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
The purpose of this study was to
formulate .an artificial diet for Atlantic
silversides (Menidia menidia) such that
this fish could be cultured as a repre-
sentative test organism. Toward this
end, a comprehensive project was
undertaken with the close cooperation
of the Tunison1, URI2/ and EPA
laboratories3.
'United States Fish and Wildlife Service. Tunison
Laboratory of Fish Nutrition. Contend, NY 13045.
'Department of Food Science & Food Technology.
Nutrition and Dietetics. University of Rhode Is/and.
Kingston. Rl 02881.
'United States Environmental Protection Agency,
Environmental Research Laboratory, Narragan-
sett, Rl 02882.
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Atlantic silversides are euryhaline
fish of importance as forage for piscivor-
ous fish including such valuable
commercial species as striped bass,
bluefish, cod, haddock, mackerel, and
summer flounder. All life history stages
of silversides are found in the estuaries
along the Atlantic coast. The natural
diet of silversides includes algae, detri-
tus and zooplankton, such as megalopa
larvae, copepods, and mysids.
The purpose of the University of
Rhode Island's part of the study was to
provide biochemical evaluations of the
diets and the cultured organisms. The
diets were formulated to specifications
worked out by URI, EPA and Tunison
laboratory personnel. The diets came
from feeds formulated by the research
group and by others (Tables 1A and 1B).
Composition of the artificial diets and
the artificial semi-purified diets are
shown in Tables 2 and 3.
Initial studies concentrated on analy-
sis of the diet components and the wild
silverside eggs and adults. Later studies
concentrated on the effect of modified
diets in relation to the feeding of brine
shrimp nauplii.
An additional diet, the Conklin
modified lobster diet (CMLD) was
included in only experiment 1 and was
only evaluated from a fatty acid point of
view (Schauer and Simpson, 1978).
Therefore, it was not included in the
general diet treatment, although the
performance of this diet will be included
in the fatty acid results of this year's
feeding trials. The CMLD diet has been
proposed as a reference standard for
nutritional studies of invertebrates.
Test fish in experiment' 1 were 23
days old at the beginning of the experi-
ment (17.6 ± 0.59 mg; 15.5 mm) and
they were fed the various diets for 24
days. In experiment 2, fish were 59 days
old (92.8 ± 0.85 mg; 25.8 mm) at the
onset of the study and were fed the
various diets for 50 days.
Discussion and Results
The joint research project between
the URI and the EPA to determine some
nutritional requirements of the Atlantic
silverside was begun in July 1975 and
concluded in January 1979. Experi-
ments were conducted in the summer of
each research year and the samples
were analyzed during the off-season.
Pre-Diet Testing Analyses
Much of the first year's work con-
sisted of the amino acid analyses of
various life stages of winter flounder
(Pseudopleuronectes americanus) and
a number of natural food organisms
known to be consumed during the early
life stages of wild marine fish. The basic
reason for the initial sample analyses
was to elucidate the amino acid pattern
of a number of marine organisms so that
an artificial diet of suitable quality could
be formulated.
Young winter flounder adults (16-1E
cm) varied little in amino acid contem
from the juveniles (4-5 cm). Plankton
collected from Narragansett Bay
showed marked variations in amino acid
profiles. It was assumed that the levels
of amino acids in the winter flounder
eggs were optimum for growth. Based
upon the resemblance of the plankton
amino acid profile to that of the winter
Table 1A. Treatments in 1977*
Treatment/Diet Source and Description
1, Artificial, Conklin
modified lobster
diet
2. Artemia salina
nauplii, live
3. Starved
4. Artificial, HPM-1
(pellet form)
5. Artificial, MF-1
(flake form)
University of California Bodega Marine Lab diet 76C. Ground to
coarse powder passing through a 600-/jm mesh sieve.
Nauplii hatched from San Francisco Bay brine shrimp, Artemia
salina, eggs and harvested 72 hours after immersion of eggs in
filtered seawater of 20-22°C and 31% salinity. Size approxi-
mately 140-fjm x 400 fjm.
Unfed.
Cort/and modified practical diet with soy oil and fish oil. Ground
to coarse powder passing through a 600-um mesh sieve. >
Closed formula prawn diet. Dr. S. Meyers, Louisiana State
University. Ground to a coarse powder passing through a 600-
fjm mesh sieve.
"For further description see Bengtson et a/., 1978.
Table 1B. Description of Diets Used in 1977
Treatment/Diet Description
1
2
3
4
Combination diet. Artificialpra wn flake diet MF-1 given for 3
consecutive days, live Artemia salina 3-day nauplii substi-
tuted on the fourth day.
Live Artemia salina 3-day nauplii given every day.
Unfed.
Combination diet. Artificial salmonid flake diet HPM-1 and
live Artemia salina 3-day nauplii given on alternating days.
Combination diet. Artificial salmonid flake diet HPM-1 given
for 3 consecutive days, live Artemia salina 3-day nauplii
substituted on the fourth day.
Combination diet. Artificial salmonid flake diet HPM-1 given
for 7 consecutive days, live Artemia salina 3-day nauplii
substituted on the eighth day.
4
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flounder egg, the plankton appeared to
be an excellent diet and was included as
a diet in study year 1976.
Feeding Studies
The initial study year was comprised
of two separate investigations. One
study was undertaken to analyze the
effect of a continuous live brine shrimp
diet on larval 60+ day-old juveniles. The
second study was initiated to evaluate a
practical commercial hatchery diet,
Cortland 1 (Tunison), which was
formulated with respect to the nutri-
tional requirements of a freshwater
fish, the Atlantic salmon fry.
The second year of the project (1976)
centered mainly on the chemical and
biochemical aspects of the diets fed and
Table 2. Composition of Cortland
(Tunison) Diet (HPM-1F)
Percent
Ingredient Composition
Herring Meal 37.14
Soybean meal, 49%
protein 9.28
Corn gluten meal, 60% 9.28
Wheat middlings,
standard 8.36
Brewer's dried yeast 4.64
Dried whey 4.64
Dried condensed fish
solubles 4.64
Meat and bone meal 4.64
Soybean oil, table grade * 4.64
Fish oil 4.64
Mineral mixture^ 0.37
Vitamin mixture^ 0.56
Choline chloride, 70% 0.002
Niacinamide 0.02
Starch, Instant Clear
Jel, national 1.86
Lecithin 1.86
Cholesterol 0.19
Kelgin (HV) alginate 2.32
Sodium
hexametaphosphate 0.93
'The soybean oil, fish oil, lecithin and cholesterol
were blended and added topremix simultaneously.
t Mixture provided the folio wing minerals fin gm/kg
of diet): magnesium sulfate, 1.78: potassium
iodate.0.0076; sodium selenite, 0.00027; zinc
sulfate. 0.4; ferrous sulfate, 0.3; cupric sulfate,
0 33; and manganous sulfate. 0.889.
^Mixture provided 13.800 IU Vitamin A palmitate;
5.500 IU Vitamin Da 100 IU Vitamin £, as dl-a-
tocopheryl acetate, and the folio wing amounts (mg)
of other vitamins per kg of diet: Vitamin K as
Menadione dimethylpyriminidol bisulfite, 30;
^hiamin-HCL, 28; d-biotin, 8; folic acid, 20; Vitamin
, 0.003, mositol, 691; and ethoxyquin, 207.
the analysis of the fish cultured on these
diets. The diets consisted of wild plank-
ton, brine shrimp nauplii (fresh and
freeze-dried), freeze-dried silversides,
Tetra Marin, and four other artificial
diets. The latter four diets incorporated
changes suggested by the first year's
trials (e.g., use of a marine oil and the
incorporation of a semi-purified diet
with and without an amino acid supple-
ment).
The diets fed in the two experiments
for the 1977 feeding trials are listed in
Tables 1A and 18, and included the live,
unfed 3-day-old brine shrimp nauplii,
the Tunison flake diet (Table 2) and the
Meyers prawn diet 1023-77-1 (Table
3). The other treatments in the two
experiments consisted of feeding
regimes of artificial diets with brine
shrimp nauplii at variable intervals. An
additional diet, the CMLD (Table 4) was
included in only experiment 1 (Table
1A).
The growth and survival data of silver-
sides in 1977 experiments 1 and 2 are
presented in Table 5 and Figure 1, re-
spectively. In experiment 1, survival
was best on the live brine shrimp diet,
slightly less on the combination diet and
poor on all artificial diets. Growth of the
brine shrimp (only) fed fish was signifi-
cantly better than in the combination
diet group, which in turn was signifi-
7able 3. Composition of the Meyers
Prawn Diet*-\
Percent
Ingredient Composition
Shrimp meal (South) 15
Fish meal 14
Fish protein concentrate 15
Soy protein 15
Yeast protein 14
Rice gel 5
Whey 2
Linolenic acid 0.5
Fish oil (menhaden) 4
Cod liver oil 1
Soy oil 2
Cholesterol 0.1
Vitamin mix 2
Fish solubles 5
Lecithin 2
Kelgin 2.5
Sodium
hexametaphosphate 1
"of. I.B. Tarshis (1978).
^Proximate analysis- protein, 53.1%; fat, 11 7%;
fiber, 4.7%; ash, 14.0%; carbohydrate, 16.5%(dry
weight basis).
cantly better than any of the artificial
diets.
The Conklin diet produced a survival
rate of 78.7%, although growth was the
poorest of all diet fed groups (22.8 ± 1.8;
17.3 mm).
In experiment 2, Figure 1, survival
was equally high in three of the diet
groups. These were brine shrimp alone,
Meyers diet + brine shrimp every 4th
day, and Tunison diet + brine shrimp
every 4th day. Statistically, growth was
best, and similar, on brine shrimp as a
single diet source and Tunison diet +
brine shrimp every 2nd day.
In the diet studies and in the combina-
tion live and artificial diet studies, silver-
sides actively consumed all diets which
seems to eliminate leaching as a major
cause of nutritional incompleteness of
artificial diets or freeze-dried brine
shrimp. If a critical nutrient was leached
out, feeding brine shrimp every 8th day
would give results similar to feeding an
artificial diet alone, which was not the
case. The reasonable growth and sur-
vival which resulted from feeding brine
shrimp every 8th day also tends to
eliminate a digestive enzyme as the key
growth factor since an enzyme would
not likely remain active over an 8-day
Table 4. Composition of the Conklin
Modified Lobster Diet
Ingredient
Vitamin free casein
Cellulose
Wheat gluten
Brewer's Yeast
Lipid mix S*
Corn starch
Albumin
Salt mix. BTM-\
Vitamin mix D§
Choline chloride
Thiamin
Cholesterol
Rovimix E
Vitamin C
Percent
Composition
30.00
16.10
15.00
15.00
6.00
5.00
4.00
3.00
2.00
1.00
2.00
1.00
0.20
0.20
"Corn oil, 2 03%; cod liver oil, 3.96%; and ethoxy-
qum* 0.01%.
Salt mixture in % of mix: CaCOa, 2.10%; Ca(POt)z
73.5%, citric acid, 0.21%; 2Cu2C HsOi-SHtO.
0.05%; FeCsHSOj-SHzO. 0.56%; MgO, 2.50%; Kl,
0.001%. KtHPOi 8.10%; KiSOt. 6.80%; NaCI,
3.06%; NatHPOt-2HzO, 21.4%; 7/V3 (CoWsCMj-
2H20, 0.13%; NaF, 0.02%; and CoC/2, 0.02%.
^Vitamin mixture in % of mix: thiamin hydrochlo-
ride, 0.32%; nboflavin, 0.72%; niacinam/de,
256%, biotin, 0.008%. Ca-Pantothenate, 1.44%.
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Table 5. Summary of Results From Three Experiments to Evaluate Effects of Diet on Survival and Growth of the Atlantic
Silverside
Treatment
Experiment Diet Diet1
1977-H 1 Conklin modified lobster 'diet
Artemia nauplii
HPM-1 pellet
HPM-1 flake
MF-1
Combination Artemia & MF-1
Amt. Fed.
% Wet Wt.
of Fish
10
10
10
10
10
10
Duration
(Days)
23
23
23
23
23
23
Age
of
Fish
24
24
24
24
24
24
Survival
(%)
79
93
47
43
45
88
Initial
Wt.
(mg)
17.8
17.8
17.8
17.8
17.5
17.8
Final
Wt.
(mg)
22.6
103.6
43.3
40.5
34.4
91.0
Gain %
Initial
Wt.
27.0
482.0
143.3
127.5
92.7
411.2
' Three replicates per treatment were provided Statistical analyses given in individual references noted elsewhere
500 \—
400
I
.c
<5
300
•2> 200
I
700
0
1 2 4
Figure 1. Interval between Artemia feedings (days).
period. Freeze drying would not sub-
stantially destroy digestive enzymes,
amino acids or fatty acid components of
the brine shrimp, yet feeding freeze-
dried brine shrimp gave results equally
as poor as feeding an artificial diet
alone. It appears unlikely from our
present knowledge, therefore, that the
live brine shrimp is providing a digestive
enzyme, amino acid or fatty acid to
silversides that is not being provided by
the artificial diets or freeze-dried brine
shrimp.
Biochemical analyses demonstrated
that the various test diets provided the
majority of the 10essential amino acids
in quantities at, or above those noted as
adequate. The whole body amino acid
profiles for every essential amino acid of
all cultured fish were in excess of the
minimum requirement levels. The dif-
ferences observed in growth and
survival of silversides cannot be attri-
buted directly to amino acid deficiencies
of the diet.
It was shown that a combination of
the Meyers + brine shrimp diet pro-
duced a fatty acid spectrum in silver-
sides which was nearly identical to the
average spectra of each single 'source
diet (Meyers alone; brine shrimp alone).
Two important differences were evident
between the cultured and wild fish
lipids. The first was that the cultured
fish contained a greater amount of the
16 and 18 carbon fatty acids, whereas
the wild fish contained more of the 20-
22 carbon polyunsaturated faty acids
(PUFA). The second major difference,
was that total lipid levels were substan-
tially higher in the cultured group than
in the wild fish group.
Live brine shrimp have provided some
added requirement for maintaining
good survival. With this increased sur-
vival, the nutritional requirements of
marine larval fish can now be more
accurately evaluated by feeding modi-
fied artificial diets. It would appear that
brine shrimp cannot be totally elimi-
nated from the dietary regime of the
larval fish, but they can be more con-
servatively utilized.
It is thus possible to supplement the
fatty acid content of the brine shrimp so
that the resulting fish does survive,
grow and more closely resemble the
natural (wild) fish than do fish grown on
only the live brine shrimp nauplii. This
can be done without having to maintain
the fish on a total live diet.
Conclusions
The intent of this project, started in
1975, was to provide an artificial diet
that would give good growth and surviv-
al of test fish. Generally speaking, all of
the artificial diets—commerciaf or pre-
pared, flaked or pelleted, and freeze-
dried silversides or brine shrimp—gave
poor survival and/or growth. Only li
brine shrimp nauplii, or a combination'
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of an artificial diet with brine shrimp
nauplii, gave acceptable survival and
growth. Nevertheless, brine shrimp
feedings resulted in a test organism that
did not resemble its wild counterpart in
biochemical composition. However, a
combination feeding of an artificial diet
and brine shrimp gave good survival and
growth and appeared to correct some of
the deficiencies of brine shrimp alone.
While the silversides were not com-
pletely "weaned" from the expensive
(time and cost) brine shrimp, at least it
was minimized.
Because of the importance of Anemia
nauplii in feeding laboratory fish the
authors and the EPA group embarked on
a study of brine shrimp cysts. At a
conference at Szymbark, Poland, in
September 1977, where some of these
artificial diet data were presented, an
International Study of Anemia (ISA)
group was formed. This group was
formed in response to the obvious need
to study the properties of brine shrimp,
in general, and some commercial
strains, in particular. The same close
cooperative effort between the EPA and
URI laboratories in the first project con-
tinued into the second. The comparative
studies on the five geographical strains
of brine shrimp were presented at the
1st International Symposium on
Anemia (1979, Corpus Christi, TX) in
seven papers from the two laboratories.
The results showed clearly that there is
wide variation in the brine shrimps'
ability to support the life and growth of
test organisms and also a variation in
their chemical composition. The cause
of the variability-of biological value of
brine shrimp is the subject of further
investigation between the two labora-
tories.
Kenneth L. Simpson, Paul S. Schauer. Cynthia R. Seidel, and Leslie M.
Richardson are with the Department of Food Science and Nutrition. University
of Rhode Island, Kingston, Rl 02881.
Allan D. Beck is the EPA Project Officer (see below).
The complete report, entitled "Nutritional Requirements of Marine Larval and
Juvenile Fish," (Order No. PB 81 -248 130; Cost: $8.00, subject to change) will
be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
South Ferry Road
Narragansett, Rl 02882
U S GOVERNMENT PRINTING OFFICE; 1981 — 559-017/7370
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