IMPAQ - IMProvement of AQuaculture high quality fish fry production.
How to intensify the production of copepods as live prey:
By Per M. Jepsen & Benni W. Hansen
DYNAMIC Status report April 2013
IMPAQ Indoor RAS manual
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Background:
IMPAQ is a multi-disciplinary Research Alliance that aims at developing a sustainable live
feed in terms of copepods, to supply Danish and International aquaculture fish hatcheries
with a feed item that can be used to produce high value fish larvae. The projects below will
focus on intensive copepod production for cold storage of eggs to supply live feed for marine
fish larvae world-wide. To supply a stable and reliable flow of cold stored eggs for marine fish
farms, it is important for the copepod production to establish the entire value. Further it is
important to describe the production cost, and compare it with current live feed market to
validate the economic feasibility.
For an intensive copepod egg production the value chain can be divided into three main links.
1. Algae production as food supply for the copepod culture
2. Copepod rearing facility with high densities and egg production
3. Economic evaluation of the production
Algae production
The algae production facility is located at Roskilde University. It is system based on input
MINH/BENNi
Copepod production
Copepod cultures can be divided into intensive and extensive systems – the latter are uncontrollable
and thus neglected here. Within intensive calanoid copepod systems, there are numerous
descriptions of laboratory cultures (< 100L). To our knowledge only a few intensive systems a
Dutch system, Sintef in Norway and a newly established system at RUC. The system at RUC is
based on Recirculated Aquaculture System [RAS] with technology and experience from the Danish
aquaculture sector (13) (16). Two other intensive systems exist, in Australia and Italy, but are not in
operation anymore. The copepod rearing facility is at RUC is an unique system designed in
cooperation between a Danish RAS supplier and RUCs scientific experience with rearing of
copepods.
Economic Feasibility of Intensive Copepod Production for Commercial Scale:
Experiment
A Lab
It is well-documented in the literature that using Copepods as a live-feed has numerous advantages
over the commonly used live-feed (Artemia and rotifers). For example, Copepods have a better
nutritional quality, increase survival rate, improve growth condition, reduce mal-pigmentation,
enhance development of key organs, rise success of restocking programs and allows breeding of
new species (Guillaume Drillet). However, the paradox is that they are not widely used and they
remain less available in the market. One of the reasons is economic feasibility and lack of
awareness. In this research, we are going to explore the economic feasibility of intensive copepod
production for commercial scale. Data from Roskilde University lab experiment are used. Cost
benefit analysis is employed (without incorporating the monetized values of benefits of copepod on
the entire food chain). We assumed that the benefits of the copepods on the entire food chain are
IMPAQ Indoor RAS manual
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reflected on their price. The result reveals that intensive copepod production for commercial scale
can actually payoff.
1. Aim of Algae production
The objective is to rear algae in a novel Photo Bio Reactor (PBR). The chosen alga is Rhodomonas
salina since it is proven to be the best feed for the calanoids copepods (Buttino et al. 2012). The
algae part is divided into two parts, one focusing on production and optimization of algae and
another on the link between algae and copepods, the feeding of algae to copepods.
Algae production and optimization: The optimal light conditions for algae culture will be
determined. The optimum is to prevent photo inhibition (at low alga concentration) and light
limitation (at high alga concentration). This is found by adjusting the culture light at different
intensities.
The optimal climate for culture conditions will be determined. PH will be measured and adjusted by
the addition of CO2 (sufficiently).
Determine the biochemical content of alga. To ensure that the algae have a high nutritional quality
for the copepods following parameters will be determined in the algae:
– Carbon content (CNA analyzer)
– Nitrogen content (CNA analyzer)
– Fatty acids (GC/MS)
– Amino acids (HPLC)
– Chlorophyll a content (Spectrophometry)
Determination of optimal feeding regime for copepods. Experiments will determine the
sedimentation of algae in culture systems, and the optimal feeding strategy for copepods.
Approach of the project
Two PBRs has been developed for intensive cultivation of R. salina. The PBR is equipped with
sensors and controlled by Programmable Logic Control (PLC) is used since it is a labour efficient
system. Different challenged in the project will be solved with everything from fully controlled
laboratory setups to big scale experiments in the total PBR included with interactions with the RAS.
The goal is to effectively utilize as much algae for copepod food as possible, with less possible
labour effort.
Algae cultures
Algae cultures are essential as food for copepods. High quality algae have to be available for the
copepods since the copepods fitness is highly dependent on the quality of food. The algae used in
IMPAQ are Rhodomonas salina, which in numerous studies has shown to be optimal for copepods
in terms of nutritional value and size (Berggreen et al. 1988; Hansen 1991). In the IMPAQ project
IMPAQ Indoor RAS manual
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three different methods are used to supply copepods with food. 1. Algae in bottles (Extensive), 2.
Algae in bags (Semi-intensive), and 3. An algae bioreactor (Intensive).
Extensive (bottles)
Extensive algae are reared in round bottomed bottles,
which together with atmospheric air help keeping algae in
suspension. Air supply is filtered with a GF/F 20mm
syringe filter before supplied to the algae with a 10mL
glass pipette. The algae are placed in front of LCD light
panels that supply light that enhances algae growth (LCD
Light Tubes). The light system can be adjusted to three
different light intensities for flexibility so other algae can
be grown. Every week a backup of the algae are stored
and kept for eventually culture crash situations. The
volume varies from 2 to 6L depend on the bottle size.
This gives a daily harvest of maximum 2L pr. bottle.
Figure 1 is Rhodomonas salina reared in both round
bottomed bottles and in blue cap bottles.
Semi-intensive (plastic bags)
Semi-intensive algae are cultivated in plastic bags for larger amount
of algae. The system is the same as for bottles except that the
production is in bags. In IMPAQ bags of 15L are routinely used for
up scaling of algae production. This gives a daily harvest of at least
5L pr. bag. The algae are kept in suspension with air bubbles, but
often more sedimentation of algae are experienced in bags
compared to bottles. Bags have the advantage that algae can be
cultivated for up till 1 month in the bag, and then the bag can be
discharged. Also if a bag crashes the bag can easily be emptied and
trashed. With bottles or bioreactors these has to be thoroughly
Figure 2 is a picture of a 15L algae bag installed in the IMPAQ
project. The reared algae are Rhodomonas salina.
cleaned before they can be reused. A bag is trashed and a new bag is installed, facilitating easy
management.
IMPAQ Indoor RAS manual
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Intensive (Bioreactors)
3
7
5
2
6
I
4
Figure 3 A conceptual flow diagram of the two algae bioreactors installed at RUC-ENSPAC
for use in the IMPAQ project.
The system consist of two individual sub-units, each sub-unit are completely differentiated from the
other. The system is integrated into the existing copepod RAS.
Water supply (1): To avoid
contamination of the algae cultures
the water quality must be at as high
standard as possible. The seawater
used for the algae bioreactors should
be kept free of organisms which
compete with the algae. Competitive
organisms include other types of
phytoplankton,
phytophagous
zooplankton and bacteria. Therefore,
the installed water supply is filtered
through
0.2µm
Millipore
finefiltration system, after the
sequential filters an UV system is
IMPAQ Indoor RAS manual
Fact box – Photo Bioreactor equipment:
2 x Glass cylinders (diameter 20 cm, height 150 cm; V ≈ 47 liter)
4 x 2 side by side LCD light. Dimmable to 3 intensities
Low = 1/3 intensity, Medium = ½ intensity and High = full intensity
1 X UV system: 16 watts to inactivate any microorganisms
2 x pH electrodes (SWAN)
2 x nutrient pumps (0.1 to 1.5 L/h)
2 x Supply pumps (0.1 to 1.5 L/h)
4 x Copepod feeding pumps (0.1 to 1.5 L/h)
Page 5
installed, delivering a dose of 240,000mv/cm² at a flow of 1.5L/h to ensure clean water for algal
growth.
Air supply (2): The second most likely source of contamination in algal cultures is the air supply.
Micro-organisms can multiply quickly within the supply system, particularly if it has condensation.
All air supplies into the algae room are filtered with a 0.2µm WHATMAN filter and are oil free.
The definition of air is atmospheric air, and not purified oxygen.
Temperature (3): Temperatures are regulated, since low temperatures will reduce algal growth
rates and therefore production, whilst higher temperatures will lead to an increased likelihood of
crashes and contamination within the system. The upper and lower thermal limits will be different
for different algal strains, and the systems sub-units are therefore flexible so more than one algal
strain can be produced simultaneously. Temperature changes should be gradual in terms of both air
and water to avoid the risk of shocking the algae. Additions of water should be of the same
temperature as that of the culture volume. The water is cooled through Roskilde University’s central
cooling system, and is externally supplied to the algae bioreactors with air, to prevent contact and a
source for contamination.
CO2 and pH (4): The system has an integrated Proportional–Integral–Derivative (PID) controlled
solenoid valve that doses CO2 into the algae cultures depending on the pH inside the individual
culture. The pH probe and PID control are connected and controlled by a Programmable Logic
Control system (PLC).
Algae collection and feeding (5): From the top of each algae bioreactor the produced algae will
flow into an algae collection tank. From this tank the algae can be pumped with four individual
pumps into the four copepod production tanks, thereby feeding the copepods.
Nutrient supply (6): Two pumps are installed, one to each bioreactor, to supply the bioreactors
with nutrients. The nutrient is prepared in sterile blue cap bottles and has no contact with the users,
preventing contamination. The nutrient recipe used is B1 media, solution A (inorganic nutrients)
mixed with solution C (vitamins) (P. J Hansen 1989). The B1 media can be supplied form the
pumps with 0.1 – 1.5 l/hr.
Light (7): Optimal growth can be achieved using continuous light at an intensity of up to 10,000
lux when using continuous, strong light within the Photosynthetic Active Radiation range. LCD
lights are installed that can be dimmed to three different intensities. The advantage with LCD light
is that they do not heat the water, and are cost effective. The two algae bioreactors can be
individually regulated.
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Experimens, results and recommendations
Here I need input from Minh and Claire’s work!
Light conditions for culture
Aims: Adjusting the light during the time to prevent inhibition (at low concentration) and light
limitation (at high concentration)
 Understanding the absorption and scattering
•
By the algae cells
-R.baltica will be cultured in batch in 5-10 L glass bottles to reach the highest densities (≈ 23 x 10 cells/mL)
6
-Make a dilution series of the algal culture
-Measure the light in the cultures with difference densities: 3x106, 2.5x106, 2x106, 1.5 x106,
1 x106, 0.5x106 cells/mL
-Corellation between the light absroption and the density of the algae (in the photobioreactor
the density of algae can reach much higher than 3x106cells/mL in fact we aim at 107 cells/ml)
•
By the glass material of the cylinders
•
Calculation to model the light condition in the photobioreactor (Søren LN)
Principle for culture conditions CO2 (pH)
Aims: Adjusting pH by the addition of CO2 (sufficiently)
Start the photobioreactor with the 10 L algae cultures at the mid-exponential growth phase.
•
At start:
-
the initial concentration of algae in the photobioreactor is app. 200 000 cells/mL.
-
Without CO2 supply
-
Record the density and pH of cultures
 Follow the growth of algae until they reach the stable phase
•
Start supplying CO2 to adjust the pH
-
1 cylinder at pH 8.0
-
1 cylinder at pH 7.5
-
Record density
IMPAQ Indoor RAS manual
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Measurements on algae: When we have the system up running and steady-state production is
obtained
•
Concentration of algae (Coulter coulter)
•
Size of algae (Coulter counter)
•
Biochemical content of algae
– Carbon content (CNA analyzer)
– Nitrogen content (CNA analyzer)
– Fatty acids (GC/MS)
– Amino acids (HPLC)
– Chlorophyll a content (Spectrophometry)
Algae feeding system
Specifc growth rate ug C L-1
Berggreen et al (1988) has shown that it is important to ensure feeding above 20,000 cell ml -1 when
the ambition is to obtain maximum specific growth and egg production tae of A. tonsa.
Berggreen et al. (1988)
0.5
0.4
0.3
0.2
0.1
0
0
5000
10000
15000
20000
25000
30000
35000
R. salina [Cell ml-1]
Figure 4 Graph modified from Berggreen et al. (1988). Specific growth rate as a
function of cell ml-1 of R. baltica.
To
ensure the feeding criteria described by Berggreen et al. (1988), a series of experiments estimated
the specific loss of algae from the tanks over time. The variables are coursed by algae sedimentation
and water exchange in the production tank. The tanks were induced with a known volume of
Rhodomonas salina to the copepod culture tanks, following concentrations above feed limitation.
Batch feeding: Initial high concentrations of algae were monitored over at least 24h. 24h is the
normal practice when feeding copepods in most reported cultures. There were NO copepods in the
tanks, so the only variable were in- outflow velocity into the tank. Not surprising did we loss less
IMPAQ Indoor RAS manual
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45
Batch feeding
40
35
Chlorophyll a µg L-1
algae at the static situation with no
inflow of water, this is thereby the
isolated effect of sedimentation of
algae we obtain. And when water
velocity of the inflow were increased
the loss of algae from the tank
increased. Thereby we from this figure
can estimate effects of sedimentation
(1% h-1), loss effects to the tank
outflow and the combined effect of
these. The conclusions are that batch
feeding is a good strategy whit no
water exchange, but with water
exchange it is recommended either to
increase the intensity of batches over a
30
25
0L hr-1
20
10L hr-1
15
40L hr-1
10
100L hr-1
5
300L hr-1
0
0
Continuous feeding: An experimnet
was setup with same experimental
procedure as with batch feeding in
regards algae initial concentrations,
time and measurement methods. In
regard of flow only 0, 10 and 40L h-1
were used since with higher flows the
loss rate of algae are to high,
especially if grazing is applied to the
equation. A exponetial model was
fitted to the batch feeding result for
the three choosen flows and the
equation were used to calculated the
compensatory amount of algae that
has to be added into the tank. An
initial dose of algae were added,
similar to the the one used in the
batch experiment, thereby obtaining
feed in excess for copepods.
Thereafter algae are continuesly
supplied into the tank with a
IMPAQ Indoor RAS manual
10
15
Time (h)
20
25
30
Figure 5. Results from the experiment with measured
values of chl a the loss to sedimentation and outflow of
water from the tank, at different tank outflow water
velocities.
40
Continues feeding
35
30
Chlorophyll a µg L-1
day, or apply continues feeding.
5
25
20
40L hr-1 inflow +
0,8L hr-1 pump
15
10
10L hr-1 inflow +
0,4L hr-1 pump
5
0
0
5
10
15
20
Time (h)
Figure 6 Continues feeding. A batch of algae spikes the
tank to initial high concentrations; hereafter
concentration levels are kept by continues inflow of
algae from a peristaltic pump.
Page 9
peristaltic pump set to a flowrate compensating for sedimentation and outflow of algae from the
tank, at the different flow rates.
The results showed that at the chosen flow rates it is indeed possible to compensate for the loss of
algae due to sedimentation and outflow of algae from the tank. This results in keeping a feed level
for copepods in excess. Conclusions are that with simple calculation and flow control, algae can be
supplied as food in excess for copepods.
Acartia tonsa has a specific ingestion rate of maximum 1.3 d-1 at the optimal growth rate 0.44 d-1.
Thereby the feed requirement is a total of ~1000 µg C L-1. The Chl a: Carbon ration for R. salina is
around 30. Thereby 34ug Chl a equals the dietary need for 1 copepods d-1.
𝑇𝑜𝑡𝑎𝑙 𝐼𝑛𝑔𝑒𝑠𝑡𝑒𝑑 𝑐ℎ𝑙 𝑎 𝑑−1 = 𝑛𝑏𝑟. 𝑜𝑓 𝐼𝑛𝑑. × 34 chl a
1
Equation
Equation 1 is used to calculate the daily need of feed for the copepod culture. When the total daily
chl a ingestion is known then it can be compensated by adjusting the inflow of feed from the
feeding pump. Combined with the different compensation rates shown on figure 6 then the ideal
copepod feeding scheme can be setup.
IMPAQ Indoor RAS manual
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Copepod rearing
1. Aim of Copepod rearing facilities
The objective is to combine RAS from known aquaculture systems and apply it to copepod cultures. And to
optimise the system design to a high biomass production system that can provide an economical competitive
live feed system. The main aims of the study are as following:




Obtain high copepod stocking densities
Obtain high egg productions
Apply labour efficient culture management
Develop and modify culture system
Approach of the project
A novel Recirculated Aquaculture System (RAS) has been developed and modified to counter the challenges
when cultivating calanoid copepods intensively. The calanoid copepod Acartia tonsa is the specie used, since
it is an excellent and well documented model organism. Earlier study with Acartia tonsa has shown that their
eggs can be stored for prolonged periods, and from the eggs, nauplii can be hatched and used for live feed to
marine fish larvae. This provides a two way system. Either the technology from this project can be directly
transferred to the marine fish farms (De-central), or a central egg production facility can supply eggs for live
feed to the marine fish farm (Central). So the farmer has the option to choose between central versus
decentralised production of copepods eggs, adding flexibility to the system. RAS equipped with sensors and
controlled by Programmable Logic Control (PLC) is used since it is a labour efficient system. Different
challenged in the project will be solved with everything from fully controlled laboratory setups to big scale
experiments in the total RAS.
The goal is to effectively have high intensive copepod cultures, still with a high egg production
yield, as cost effective as possible.
IMPAQ Indoor RAS manual
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Copepod culture design
Conceptual designs of the intensive copepod system custom build for the IMPAQ project.
Figure 7 conceptual drawing of the Recirculated Aquaculture System (RAS) installed at
RUC-ENSPAC for use in the IMPAQ project.
Description of RAS
Fact box - RAS equipment list:
In the Recirculated Aquaculture System at
RUC the water is pumped around in the
system in two loops. One loop is a cleaning
loop where the water is purified in different
steps.
Multi Function Bag Filters (5µm, enviro EBF009-7)
First the intake water to the system in
filtered through a 5µm bag filter, this water
enters into the 2m3 pump sump. From here
the water is pumped in a flow that can be
regulated from 300L h-1 to 3000 L h-1
depending on how dirty the water is, into
the protein skimmer were proteins are
removed from the water by injecting micro
bubbles of air into the protein skimmer.
Automatic cleaning of the protein skimmer
by freshwater are installed to flush out the
IMPAQ Indoor RAS manual
Pump sump with 2 x IWAKI MX250CVSE pumps.
1 x AQUAFLOTOR type AQ 300, Protein skimmer.
1 x Water Biofilter 4 chamber version ( RK
BioElements, area pr. m3 = 750m2, weight mass = 0,93
g/cm3).
1 x WUVX 40 – UV(WATER Aps 40W).
4 x 320L HDPE black Copepod tanks (ɸ = 760mm) with
an intake flow meter regulated from 10 to 300 L h-1.
4 x OxyGuard 420 Dissolved Oxygen probe.
1 x OxyGuard PH MANTA (0 – 14 pH).
1 x OxyGuard salinity probe.
Page 12
collected proteins. After the protein skimmer the water is by gravity running into the 1m3 fourchamber biofilter. The biofilter is filled with bio media. There is a down flow in the first biofilter
chamber, up flow in the second, down flow in the third and an up flow in the last chamber before
the water returns to the 2m3 pump-sump. Inside the pump sump both heating and chilling are
installed to regulate the temperature of the water.
The other flow loop in the system is pumping the water through another 5µm bag filter, a UV and
then into the copepod tanks. From the copepod tanks the dirty water flows back into the pump sump
were it re-enters the cleaning loop.
The design of a copepod tank has to facilitate easy access for culture management. Tanks
hydrodynamics has to be optimized to keep animals and eggs inside tanks, not disturbing grazing,
facilitate high copepod densities etc.
Copepod densities
A number of experimental trials have been performed and are in progress: Daily measures of the
population were monitored to find the culture density. An insert in the tank was installed with a
false bottom consisting of a 200 µm screen. The idea with a screen was to separate the target
species, the calanoid copepod adult Acartia tonsa from eggs and nauplii, preventing cannibalism
and easing egg cleaning (see figure 8).
Inflow
Acartia tonsa
Individuals (L-1)
Outflow
200
180
160
140
120
100
80
60
40
20
0
Nauplii(L-1):
Copepods(L-1):
07/Jan
Figure 10. Acartia tonsa eggs
harvested from the production
tanks.
17/Jan
22/Jan
Figure 9. Population development of Acartia tonsa inside the
200µm mesh insert of the copepod tank.
2000
Eggs tank-1
Figure 8. The design of a
copepod tank with a 200µm
mesh screen inserted.
12/Jan
Acartia tonsa eggs
1500
1000
500
0
IMPAQ Indoor RAS manual
07/Jan
11/Jan
15/Jan
Page 13
After 3 weeks the population in the insert declined to a minimum level and the experiment stopped.
The conclusion is that an insert prevent recruitment for the adult population and will function well
in a batch culture system, but for a continuous RAS it appears not to be the optimal solution.
Further the screen kept clocking with air bubbles and algae, and daily caretaking was necessary,
which is not practical in a low labour automatic copepod system. Also determining the copepod
population above and below the screen is impractical since two different not comparable methods
has to be used. For tank cleaning the screen has to be removed which is impractical and will
potentially increase mortality for the copepods since handling of copepods can enhance mortality
(Jepsen et al. 2007).
The total harvest of eggs from A. tonsa did not meet the expectation according to the number of
adults present in the production tank. Only 20% of expected eggs were harvested from the system.
Eggs were lost in the system and further experiments will optimise egg harvest from tanks.
To investigating the harvest of A. tonsa eggs from the bottom drain, we first monitored different out
flow´s from the bottom drain. It was quickly obvious that maximum flow was required, which we
also expected (data not shown). Therefore another experiment investigated the amount of water
harvested at maximum flow to yield most possible eggs from the bottom drain (see figure xx).
Eggs per Liter at different outflow volumes
400
350
300
Eggs (ind L-1)
250
200
150
100
50
0
0
1
2
3
4
5
6
7
8
Volume tapped from the tank (L)
Figure 11 Acartia tonsa eggs harvested as a function of litres of water flushed out of the tank
The bottom drain samples were collected in successive flushes but the first sample had a higher
quantity of eggs. From figure 11 can be seen that flushing more than a quick first flush do not
harvest a lot of extra eggs. We harvest the eggs near the bottom drain and draining a lot more water
from the tank will not increase our egg harvest from the tanks. Therefore we must apply other
methods to harvest eggs stocked other places in the tanks. Further experiment will investigate an
effective harvest system like e.g. applying a brush, mechanical filter etc.
IMPAQ Indoor RAS manual
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Egg harvest and egg outflow experiment.
To quatify the egg harvest during a day a copepod production tank were stocked with 300,000 eggs.
The water in- and outflow was 10 L/h, and the eggs lost to the outflow, and hatched inside the tank
together with harvested 24h after stocking with eggs were estimated.
The fate of the eggs with a in- and out flow of 10L h-1, and an initial stocking of 300,000 eggs is
presented in figure 12.
Inflow 10L h-1
Initiated with 300,000 eggs
Hatching success24h: 43.4 ± 6.4%
Internal lost:
122,000 eggs
Lost from Top outflow d-1
18,000 eggs d-1
44,000 nauplii d-1
Harvested from Bottom outflow d-1
30,000 eggs d-1
1,000 nauplii d-1
Figure
12 shows
fateexperiment
of additionwith
of 300000
eggsoutflow
with anofinand
10Loptimized
h-1.
The results
from the
an in- and
10L
h-1outflow
showedofthat,
harvest
method has to be applied. We experience that 122,000 eggs was unaccounted for, and these should
potentially still be available for harvest. Further we had a daily loss of 18,000 eggs and 44,000
nauplii from the top outflow, therefore pre-screening of the outflow has to be installed, in future
setups.
Copepod culture management
Critical elements
Table 1: Highlights of biological features of Acartia tonsa there usage for aquaculture, and
recommendations for intensive cultures of A. tonsa.
Biological features of
Usage for aquaculture
Wide temperature range from - 
1 to 32°C (Paffenhofer and
Stearns 1988; Chinnery and 
IMPAQ Indoor RAS manual
Recommendations for
Acartia tonsa cultures
Cultures can be adapted to local 
temperature conditions
Eggs can be cold stored
17 to 25°C, for
optimal
egg
production
and
development time
Page 15
Williams 2004; Hansen et al.
2010).
Wide salinity range 5 to 36 ‰, 
tolerate rapid salinity change
(Cervetto, Gaudy et al. 1999; 
Chinnery and Williams 2004;
Hojgaard, Jepsen et al. 2008;

Ohs, Rhyne et al. 2009)
Cultures can be adapted to local 
conditions
Salinity change can be used to
suppress invasive pathogenic and
other nuisance organisms
Abrupt salinity change can be 
used to store eggs
Egg production 0.4 to 55 eggs 
female-1
day-1
(Stottrup,
Richardson et al. 1986; Jepsen, 
Andersen et al. 2007; Medina
and Barata 2004; Peck and
Holste 2006; Drillet, Jepsen et
al. 2008)
High number of nauplii day-1 
from batch or continues cultures
Eggs can be harvested and cold
stored and used as back up of live
nauplii production
Light regimes for nauplii to 
adult from 0L:24D to 12L:12D
(Peck and Holste 2006) Light 
regimes for eggs 12L:12D

(Peck and Holste 2006)
Cost for artificial light above 
cultures can be saved
Darkness can be used to suppress
invasive organism
Some reports about light
influence
hatching
success
therefore recommended regime 
for eggs
UV-radiation can be used to
enhance copepod pigmentation,
and thereby visibility for
predator.
Suitable size ranges can be 
“constructed” for different types
of marine fish larvae
Optimal feed uptake
and
thereby
egg
production
in
darkness
(Stearns,
Tester et al. 1989)
UV-radiation
as
pigment manipulator
(Hansson 2000)
Maximum stocking density for 
batch cultures can be calculated
Cultures can be maintained with
biofilter technology
Keep concentrations
below 0.03 mg NH3
L-1 for nauplii and
below 0.4 mg NH3 L-1
for Adult A. tonsa.

Body size and somatic growth 
can
be
regulated
by
temperature
(Ambler 1985;
Chinnery and Williams 2004;
Hansen, Drillet et al. 2010)
NH4/NH3 levels from 0,03 to 
0,47 mg L-1, with no observed
effect on cultures (Sullivan and 
Ritacco 1985; Buttino 1994;
Jepsen et al. 2013)
IMPAQ Indoor RAS manual
From 30 to 36‰ for
cultures, less energy
used
for
osmoregulation.
(Lance, J., 1965).
For eggs storage
transfer from ambient
culture salinity to
milliQ
water
(Højgaard, Jepsen et
al. 2008)
20 egg female-1 day-1
should be minimum
production
expectations
Smaller
cephalothorax
size
with
higher
temperature (Hansen,
Drillet et al. 2010)
Page 16
Oxygen levels above 2.0mg O2 
L-1 (Marcus, Richmond et al. 
2004; Sullivan & Ritacco,

1985)

pH level from 7.7 to 9.5 
(Sullivan and Ritacco 1985;

Hansen et al. in prep.)

Fast generation time from 14 to 
19 days (Chinnery and 
Williams 2004; Drillet, Jepsen 
et al. 2008)

Egg storage for up till 1 year 
(Drillet, Iversen et al. 2006;
Stottrup, Bell et al. 1999)
Feed (Berggreen, Hansen et al. 
1988)

Can tolerate low levels of oxygen 
Pressurised O2 is not necessary
for maintaining cultures
Eggs are not negatively affected
by anoxia
Temperature difference and
oxygen is not a problem
Can survive a wide range of pH 
without any effect
pH can be regulated to maintain
other abiotic factors steady in
cultures
Eggs are not affected by high pH
Keep oxygen levels
above 2 mg O2 L-1.
Selection

Restocking of cultures not critical
Fast adaption to environmental
factors
Physiological plasticity
Valuable tool for storage and 
supply of nauplii to fish larvae
Selection of large
males will optimize
the female’s egg
production (Ceballos
and Kiørboe 2010)

Can enhance egg production
Can be used to biochemically
enrich copepods.
Rhodomonas salina in
excess 1000 µg C L-1
Correct
size
range,
good
biochemical
profile
Keep pH below 9.0 to
ensure no effect on
egg production, egg
hatching,
and
copepod (especially
nauplii) mortality
Maximum
storage
time at 5°C and
anoxia is 1 year
Density depend egg production
A key feature to
optimise the production
in the RAS is to
maximize
eggs
produced per individual.
Earlier studies have
shown that eggs of A.
tonsa can be stored for
up till one year, still
keeping a valuable
biochemical
profile
(Drillet et al. 2006).
IMPAQ Indoor RAS manual
Fact box - Acartia tonsa eggs definitions:
Subitaneous eggs = eggs that hatch within 72 hours from produced, at 17°C.
Quiescent eggs = subitaneous eggs that with a change of the physical
condition can be provoke into arrested development, and be awakened again
when conditions is returned to normal.
Diapause eggs = Eggs that has to go through a refractory phase before they
can hatch.
Delayed Hatching eggs = Eggs that are maternally determined to hatch at a
predetermined time point
Page 17
Total egg production [eggs L-1 d-1]
Quiescent A. tonsa eggs provide a product that can provide the aquaculture industry with a live feed
product, similar to Artemia (brine shrimp) cysts. Since eggs easily can be transported around the
globe and hatched and feed out to marine fish larvae. One solution to optimize mass production of A.
tonsa eggs is by increasing individual stocking density L-1. In the literature it seems that no one has
stocked calanoid copepods above 2000 ind.L-1 in their experimental facilities, therefore this upper
limitation was worthwhile to challenge if live feed production based on copepods shall be
commercially interesting. In the IMPAQ project we tested in a small scale laboratory set-up the egg
production of A. tonsa at densities ranging from 10 to >5000 ind. L-1 and followed the hatching rate
of the produced eggs. With the ultimate ambition to maximize the calanoid copepod densities in
intensive mass cultures for live feed, where the egg harvest of subitaneous eggs is optimized
without stimulating an eventual delayed hatching egg production.
14000
12000
10000
8000
6000
4000
2000
0
0
1000
2000
3000
4000
5000
6000
Densities [Ind. L-1]
Figure 13. Acartia tonsa total egg production harvested per litre of culture per day (Drillet et
al. in prep.).
In figure 12 a yield of 10000 eggs were achieved with densities around ~1000 individuals L-1. In
this experiment there was not applied any water exchange and thereby the result is the combined
effects of chemical and physical density upon egg production. This resulted in an accumulation of
inorganic nutrients excreted from the copepods as a function of time as shown in figure 13. The
conclusion is that with batch cultures with combined effects of density and inorganic nutrients the
maximum stocking density is ~1000 Ind. L-1.
IMPAQ Indoor RAS manual
Page 18
Figure 14 shows accumulation of TAN, NO3 and NO2 over time at different copepod stocking
densities (Drillet et al in prep.).
To investigate the effect of inorganic nutrients another study was setup (see table 2).
Table 2 from Jepsen et al (2013).
NOEC adult
[µgNH3 L-1]
7.5
477
1,789
170* 106
30
8.0
477
1,789
55*106
30
81
3.5*106
8.5
477
1,789
18*106
30
81
1.2*106
9.0
477
1,789
7*106
30
81
444,223
30
81
213,262
9.5
477
LOEC adult Adult density
[µgNH3 L-1] [Ind. L-1]
1,789
3.4*10
6
NOEC nauplii
[µgNH3 L-1]
LOEC
Nauplii density
nauplii
[Ind. L-1]
-1
[µgNH3 L ]
81
10.8*106
pH
The water quality study showed that the earlier observed maximum yield of eggs were only an
effect of density and not of inorganic nutrients. Although it verified that it is important to keep track
IMPAQ Indoor RAS manual
Page 19
of inorganic nutrients over time and with its pH dependent equilibrium, increased pH will result in
more toxic environment for the copepods. It is recommended that pH and inorganic nutrients
are monitored weekly, especially for batch cultures of copepods. For high intensive culture
this is a daily task for the farm manager, as what is normal practices in aquaculture facilities.
Economic evaluation
In this research, we are going to explore the economic feasibility of intensive copepod production
for commercial scale. Data from Roskilde University lab experiment are used. Cost benefit analysis
is employed (without incorporating the monetized values of benefits of copepod on the entire food
chain). We assumed that the benefits of the copepods on the entire food chain are reflected on their
price. The result reveals that intensive copepod production for commercial scale can actually
payoff.
Results
Input from Tenaw
Conclusions and future studies
From these preliminary studies we can conclude that algae production… input MINH/Claire
When feeding algae to copepods it is important to apply continues feeding instead of batch feeding.
It is indeed possible to counteract both algae sedimentation and water exchange from copepod
rearing tanks with simple calculations. The foundation for these calculations has been investigated
and ready to apply for intensive copepod cultures. In regard of densities a preliminary limit were
found of ~1000 ind. L-1. This will be further investigated in experiments removing effects of
inorganic nutrients together with volume effects. Literature study together with experiments found
the basis for cultures of the copepod Acartia tonsa and will be utilized for general recommendations
of the caretaking of this animal. Initial experiments in the RAS showed a loss of eggs, nauplii and
copepods from the production tanks. Therefore the RAS are modified with new design of outflow
together with optimised harvest methods, and future results will show the effort of these studies.
Economics… Tenaw.
IMPAQ Indoor RAS manual
Page 20