IMPAQ - IMProvement of AQuaculture high quality fish fry production.
How to increase the reliability of copepods as live prey in fish farms:
DYNAMIC Status report April 2013
1
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 two projects below will focus on semi-extensive
copepod production culture for larval fish in two different locations.
Maximus is a fish farm in Jutland, northern Denmark, where turbot larvae are fed upon live feed from
the yolk stage till metamorphosis (up to 5 weeks, depending on temperature). At this point the fish are
brought indoor in the intensive aquaculture system where they are fed formulated feed (pellets) and
sold when they reach weight of minimum 4g. Our study is focusing on the first part of the fish
development where the diet is the predominant factor in the success of fish production. Two campaigns
(3 month in 2011 and 1 month in 2013) were performed and some of the results are showed in the
following document.
The second outdoor system is located in southern Taiwan. In there, farmers cultivate mostly copepods
in shallow brackish ponds at high density and then sell them to local fish hatchery farms as live feed.
This detailed study is the first of its kind to document basic parameters and also secondary productivity
of these tropical ponds. A full scale project is scheduled to start April 2013 and last for 1.5 year.
Both systems consider local copepod species and the results of the projects aim to benefit the farmers
that hopefully will get recommendations from IMPAQ to achieve a better copepod and fish production
during the production season in the future.
2
SEMI-EXTENSIVE AQUACULTURE SYSTEM IN DENMARK
MAXIMUS 2011
The aim of the project was to describe all trophic levels including phytoplankton, zooplankton and fish
larvae with respect to species assemblages, densities, secondary production, which all translates into
the resulting fitness of fish (Turbot) larvae. Additionally fingerprinting by phytoplankton pigment
profiles, organism amino acids and fatty acids profiles were provided. After the fish were weaned in
Maximus’s indoor facilities they were sorted and quality assessed. The final survival and quality of the
fry was related to their early life in the semi-extensive enclosure ecosystem characteristics.
The investigation took place on a land based fish farm located at the estuary Limfjorden, Dragstrup
Vig, in Denmark (N 56.8; E 8.5). Three successive production cycles of Scophthalmus maximus
(Turbot) larvae were monitored in the periods from 20st May to 13th September 2011. Triplicate
outdoor open top concrete tanks (280m3, ɸ10m) were filled with pre-filtered water from the estuary
(Hydrotech HDF 2007-2H drum filter, fitted with 50µm screens). Copepods were filtered (UNIK
Rotating Wheel Filter Type 1200) from a seeding tank and added to the triplicate production tanks 2 to
5 days prior to addition of yolk sack turbot larvae. The turbot larvae used in all three production cycles
were all obtained from Stolt Sea Farm in Norway. The stocking procedures were that fish larvae
arrived less than 48 hours after hatching at yolk sack stage. Upon arrival they were immediately gently
oxygenated, this to keep them in suspension. Next day the fish larvae were acclimatized to production
tank conditions by slowly adding tank water to their transport boxes, this process took half a day,
where after they were enumerated and released into the production tanks.
The tanks were initiated by using bay water from a nearby estuary that experiences decreasing nitrogen
loading from spring to fall (Fig. 1). A maximal chl a peak developed upon initiating a production cycle,
but showed decreasing peak values from spring to fall concurring with the nutrient concentration in the
bay water, indicating the available nutrient loading was the main governor. During the 3 production
cycles studied, we observed different phytoplankton community developments with an overall
decreasing peak of chl a value as the productive season progressed. We measured a maximal chl a
concentration of 19 µg chl a L-1 in the 1st production cycle, a maximal peak values of ca. 7 µg chl a L-1
in the 2nd cycle and hardly any chlorophyll peak beyond the initial chl a was observed in production
cycle 3 (Fig. 2). Resembling the decreasing of DIN and chl a concentrations towards the end of the
production season, we also saw a similar decrease in zooplankton biomass towards the end of the 3rd
production cycle (Fig.3).
3
Nutrients (Fig.1) and phytoplankton biomass (Fig.2)
3,0
a
2,5
-1
15
DIP (µmol L )
DIN & Si (µmol L-1)
production cycle 1
20
2,0
10
1,5
1,0
5
0,5
0
16/05
23/05
30/05
06/06
13/06
0,0
20/06
date
Water added to the tanks
production cycle 2
b
3,0
2,5
-1
15
DIP (µmol L )
DIN & Si (µmol L-1)
20
2,0
10
1,5
1,0
5
0,5
0
25/07 29/07 02/08 06/08 10/08 14/08
0,0
date
Nutrients added to the tanks
production cycle 3
c
3,0
2,5
-1
15
DIP (µmol L )
DIN & Si (µmol L-1)
20
2,0
10
1,5
1,0
5
0,5
0
17/08
0,0
21/08
25/08
29/08
02/09
date
The tanks were seeded with an increasing numbers of copepods during the season. However, we
observed opposite and decreasing nauplii abundances from spring to fall in the rearing tanks and a
species succession. Acartia species were taken over during the season by Centropages hamatus (Fig. 3,
pie graphs). Parallel laboratory incubations conducted during the production cycles indicated low but
similar egg production rates of copepods collected from the tanks (statistical analyses to perform). We
suggest that the decreased nauplii abundance is the product of the combined effect of changes in
copepod composition and change in the underlying quality of the phytoplankton food. The change in
copepod species composition may also increase grazing on eggs and even cannibalism on nauplii and
copepodites.
4
All lower trophic levels lead to a direct influence on turbot larval survival and growth: the effort
revealed limited success rate for turbot fry during late summer when the fish farmers follow standard
procedures. We therefore speculate that the reduced phytoplankton availability and the temperature
increase during late spring and early summer promotes the change from Acartia spp towards
Centrophages hamatus and increase nauplii mortality over the production cycle. In the rearing tanks
the copepod nauplii biomass was decreasing over the production season pointing at an increasing stress
of the copepod community recruitment. We explain this by nitrogen depletion of the bay water used to
inoculate the production cycle that changed the food quality: it decreased the absolute concentration of
nutrients and essential amino acids that affected nauplii growth and sustainable secondary production.
Moreover, the change copepod species composition from the filter feeding Acartia spp. to the
cannibalistic and more raptorial copepod Centropages hamatus over the production season most likely
caused further stress on the secondary production and biomass development among copepods.
Fig. 3. Secondary production (Copepods abundance)
Adult L-1
Copepodite L-1
nauplii L-1
Eggs L-1
Production cycle 1
Abundance [Ind. L-1]
400
Centrop
ages
hamatu
s
300
200
Acartia
spp
100
0
23/05
30/05
13/06
20/06
Production cycle 2
400
Abundance [ind. L-1]
06/06
Acartia
spp.
300
Centrop
ages
hamatu
s
200
100
0
23/07
27/07
31/07
04/08
08/08
12/08
5
Production cycle 3
Acartia
spp.
300
200
Centrop
ages
hamatu
s
100
0
20/08
24/08
28/08
01/09
05/09
EGG PRODUCTION
40
-1
Production cycle 1
Acartia spp.
Centropages hamatus
Egg production [Egg Female ]
-1
Egg production [Egg Female ]
40
30
20
10
0
Production cycle 2
30
20
10
0
28-05-2011
01-06-2011
05-06-2011
09-06-2011
13-06-2011
28-07-2011
Time
Production cycle 3
Acartia spp.
Centropages hamatus
30
20
10
0
22-08-2011
26-08-2011
30-08-2011
01-08-2011
05-08-2011
Time
40
Egg production [Egg Female-1]
Abundance [Ind L-1]
400
03-09-2011
07-09-2011
Time
6
09-08-2011
13-08-2011
percentage
survivals
As a consequence, larval survival decreased from an appropriate level of 18% in the first campaign in
early summer, to just 4% during the last campaign in late summer. The number of defect individuals
increased over the season: 3.5% of the fish presented malformation during the first production cycle,
2.4% in the second and 9.6% in the third one. When investigating the turbot larvae’s stomach content
the youngest turbot larvae examined in all three campaigns always had a high number of nauplii and
only a few copepodites and never any adult present in the gut content. The abundance of prey is much
more determining for the survival of the fish larvae compared to the differences between the copepod
strains. Supporting this statement is also that there is not found a big difference in the consumed prey
between the different production cycles when investigating the fish larvae’s stomach content. When
fish larvae are in low density in the tanks from their stomach content we observed that they had
consumed a significantly higher amount of prey compared to the larvae in tanks where the competition
for prey were much higher. This further show that the system, is top down controlled, since the turbot
larvae controls the zooplankton population.
25
20
15
10
5
0
larvae survival
18%
12%
Early summer
High summer
4%
Late summer
CONCLUSIONS
There are several governing parameters such as irradiance, temperature and nutrients that could have
created the different phytoplankton food web patterns observed, that ultimately cascaded in the reduced
nauplii development observed during production cycle 2 and production cycle 3 that caused low fish
survival. There is no doubt that decreasing temperatures affects the metabolic processes in the food
web but the most obvious candidates for the decreased turbot productivity is inorganic nitrogen (see
Jakobsen et al. in prep. and Jepsen et al. in prep.). Copepods nauplii in fact, have high demands of
amino acids for somatic growth and need a nitrogen rich diet to increase their body biomass. We
therefore speculate that the reduced phytoplankton availability and the temperature increase during late
spring and early summer affected the zooplankton community. The decreasing survival throughout the
productive season most likely is due to the inorganic nitrogen limitation that influenced the
phytoplankton availability in late summer, which was observed to translate into food shortage for the
turbot larvae. We suggest that the adjustment in the seeding phytoplankton community by maintaining
constantly higher Chl a levels > 15 µg Chl a L-1 before copepods being added to the rearing tanks.
7
MAXIMUS 2012
Based on results obtained during our 2011 campaign at Maximus we located the critical period for the
fish larvae in late summer. During this period low survival of fish larvae was observed with a higher
percentage of fish that presented malformation. The main discovery was the insufficient availability of
live feed caused by the limited amount of inorganic nitrogen that could not sustain the primary
production as food for zooplankton. For that reason a manipulation experiment was performed in 280
m3 open top tanks in late summer 2012 (middle august to early September) to increase productivity of
life feed. Two times 3 tanks were spiked with inorganic Nitrogen (full and pulse dose) and 3 tanks were
used as controls just with filtered bay water. The initial NO3 concentration of the full dose treatment
was 85 µmol/L while in the pulsed dose treatment the same amount was divided in 3 doses and added
weekly during the campaign.
We hypothesize that a higher availability of nutrients during the entire production cycle, in particular
nitrogen in the form of NO3, will affect the food chain providing a higher production of live feed used
to forage turbot larvae in the aquaculture system. The final goal is in fact to increase the survival and
development of the turbot larvae Scophthalmus maximus where the crucial factor is the appropriate diet
especially at the end of the productive season (see Jepsen et al.in prep.).
Inorganic nutrients, phytoplankton and zooplankton abundance and composition were monitored. Fatty
acids (FA) of all major trophic levels in the pelagic food web were analysed. This effort was
established to set up budgets for energy and matter from phytoplankton to zooplankton to measure
what was available in terms of copepod food and energy for fish larvae.
When looking at the dissolved inorganic nitrogen (DIN) it is clear that it is available during the entire
cycle in the full treatment (Fig.1a), but it decrease after 6 days in the pulsed dose (Fig.1b) where it was
necessary to spike with more inorganic nitrogen. The control (Fig.1c) show insufficient levels of
nitrogen to sustain a necessary primary production (Fig. 2). The concentration of DIN on the contrary
in the full dose and pulsed dose treatment could sustain the phytoplankton population for the entire
production cycle (Fig. 2).
Figure 1. Dissolved Inorganic Nitrogen concentrations in the three treatments. a) full dose; b) pulsed dose, c) control
8
Chl a
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
Pulsed
Control
0
5
10
pH
Pulsed
Control
pH
9.0
8.0
5
10
15
Days
Days
15
20
20
25
25
O2 in surface
Full
10.0
0
Full
Full
22.0
19.0
16.0
13.0
10.0
7.0
O2 (mg L-1)
Chlorophyll a (µg
L-1)
The nutrient addition caused phytoplankton biomass to increase: chlorophyll a was less than 3.8 µg L-1
in the control enclosures, while it increased up to 50 µg L-1 in enclosures where nitrogen was added and
was above 10 µg L-1 during the entire cycle (Fig.2). Therefore, a very high primary production is
established as showed by high values of pH and Oxygen (Fig. 3-4).
Pulsed
Control
0
5
10
15
20
25
Days
As a consequence, the abundance of zooplankton, especially in the full dose, increased significantly
(Fig. 5). Additionally the productivity and fecundity of the copepods in the full dose treatments was
higher than the pulsed one or the control (Fig. 6). We also observed a shift in species composition: the
most abundant species at the beginning of the experiment, Centropages hamatus, was taken over by the
Acartia species at the end of the campaign. Some experiments are in progress to explore this fact: it is
possible that Acartia spp. present late delayed hatching eggs that hatched in conditions occurred during
our sampling program. Acartia egg production is also higher than Centropages at the end of the study,
showing a better performance of Acartia as compared to Centropages.
9
Pulsed
300
200
100
0
300
Individuals/L
Individuals/L
Full
Eggs
Nauplii
Copepodites
200
Eggs
100
Nauplii
Copepodites
0
8/13/2012
Copepods
Copepods
time
time
Individuals/L
Control
300
200
100
0
Eggs
Nauplii
Copepodites
Copepods
time
Egg production
60.0
eggs/female/day
50.0
40.0
centropages full
30.0
centropages pulse
20.0
centropages control
10.0
acartia full
0.0
acrtia control
-10.0
Days
10
During the manipulation experiment we observed high mortalities of the fish. We can speculate that
high pH might have damaged them. Other aspects have to be taken in consideration such as pathogens
(vira or bacteria) or the incapability of the fish to find and catch the zooplankton due to high turbidity.
Analysis of phytoplankton shows no occurrence of toxic algae. In conclusion we can assume that
inorganic nitrogen controls the productivity of the classical food chain and increase the phyto- and
zooplankton biomass, along with the fecundity of the copepods. Copepods increased their fatty acid
composition in the full dose indicating that the higher availability of phytoplankton influence the
quality of the zooplankton.
The aim of the project was to reach a really high density of zooplankton available for the fish in the
tanks. We fulfill this task increasing also the nutritional value of the copepods that is important for the
fish larvae. Higher quality of live feed will sustain fish growth and give higher survivals.
Unfortunately, due to the high pH measured we observed high fish mortalities; for that reason we
suggest disposing of nutrient enriched green-water tanks to produce copepods for life feed in
aquaculture in designated tanks and use the stock for feeding fish larvae to enhance the survival and
development of turbot fry. This will establish a big stock of copepods that can be used at whatever time
is needed during the entire production season of the turbot larvae. It is even possible that size
fractionation of these copepod assemblages with rotating disk filters will enable the farmer to isolate
relevant copepod stages for specific purposes.
RECOMMENDATIONS AND FUTURE EFFORT:
Since we are still missing some valuable data, we cannot conclude definitively which is the best
condition to have a copepod system at high density. We can anticipate that spiking with nitrogen is one
solution to have more copepods available that have higher fatty acids than the natural sources. We
suggest to separate copepods production and fish production due to the pH but we cannot exclude other
interactions in the system itself that might affect the fish larvae.
Experiments on hatching success from copepods eggs collected from the bottom of the production
tanks are in progress; these results show the presence of delayed hatching eggs and explain the
condition under which they hatched. Results of fatty acids analysis from the campaign will show which
species between Acartia and Centropages is more appropriate for live feed for turbot larvae.
When all these data will be available we will have the idea of the appropriate food for fish larvae, we
will have data on environmental condition that increase productivity and fitness of copepods to produce
in the future a high quality system in aquaculture to produce a high quality live feed.
Future experiments can be performed under lower concentration of nutrients to better be able to keep
the pH below deleterious levels or with the same conditions as in 2012 but with separated copepodsfish production tanks to observe the growth and survival of the fish larvae. Experiments with different
kind of food items for copepods could be investigated to gain a even better profile of FA essential for
fish larvae.
11
SEMI-EXTENSIVE COPEPODS AQUACULTURE PONDS IN TAIWAN 2012
The study, performed in summer 2012, in collaboration between RUC and NTOU, describes a semiextensive aquaculture system for mass rearing of copepods by studying three (1 hectare large - 1 meter
deep) earth ponds in southern Taiwan. A one month comprehensive monitoring of abiotic factors (pH,
oxygen, light, salinity and temperature), inorganic nutrients, phytoplankton and zooplankton
composition of the ponds was performed to obtain a better understanding of the interactions between
the trophic levels in the pelagic. The most common copepod species, Pseudodiaptomus annandalei, is
somewhat under studied in the field despite its importance in natural ecosystems and it is already
recognized for its application to aquaculture as feed for shrimps and fish larvae. We observed its entire
habitat to comprehend it and try to improve, in the future, its productivity and quality in these pond
systems: a higher availability of P. annandalei will provide an even more fruitful use of this copepod in
the local aquaculture as life feed for fish larvae, mainly grouper. Future studies will include a seasonal
description of the ponds (1.5 year study from April 2013) to further understand population dynamics
and which factors influence the productivity of copepods during the year, leading to recommendations
to increase copepod pond productivity. Hand in hand with in situ campaigns, laboratory physiological
experiments will be performed to discover the tolerance of the prevailing copepod species to
temperature, pH, anoxia, and to food availability and also the expected presence of resting eggs in the
ponds and their hatching characteristics.
The research pilot study was conducted from June 19 to July 13 (25 days), in Donggang, southern
Taiwan (22º28’57.78N ;120º26’06.71E) where 3 copepod rearing ponds were visited. These ponds are
situated close to the estuary and have existed for more than 10 years where the farmers have cultivated
fish, shrimps and copepods with the same approach until now. The ponds are ~ 1 meter deep, filled by
coastal brackish water (salinity up to 15) and represented by an area of 1 hectare each. The ponds are
not closed systems and therefore are exposed to unstable condition like exchanging of water coming
from other ponds, stirring of paddle wheel at intermittence that allow interchange of oxygen, weather
conditions such seasonal rain or heavy rain from typhoon and high evaporation due to sun irradiation.
In the ponds few fish or shrimps are also cultivated but the most production is about copepods. Chicken
manure or other kind of oil fish food is used for fish-shrimp feeding in the ponds.
The abiotic measurements were done in situ every day or second day using Hobo loggers and Oxygard
probes while every second day samples of water for nutrients, phytoplankton and zooplankton were
collected. Analyses of part of the samples were done during our stay at Tungkang Biotechnology
Research Center, Fisheries Research Institute in Taiwan and the rest of the samples were analyzed in
Denmark at Roskilde University (RUC) or AaU (Århus University) due to limited time in Taiwan.
The following paragraph describes some of the results we analyzed so far, statistical analyses has to be
done and few more data has to be analyzed or calculated.
12
During the sampling period temperature was above 30 ⁰C, pH around 8-9, salinity below 15 PSU and
saturation of oxygen was present during day time (Fig.1). Oxygen probes measured the level of oxygen
in the ponds continuously showing values below 2 mg/L (severe hypoxia) during night time (Fig.2). As
explained before, the parameters measured in the ponds follow environmental conditions and
manipulations from the fish farmers: an example is showed by the salinity and temperature change in
Fig.1. During the sampling period in fact we came across 2 typhoons that obviously affect the water
pond. An example of manipulation can be seen in the nutrients pattern in Fig.3: Dissolved Inorganic
Nitrogen (DIN) and Silicate decreased significantly after day 4; at the same time we observed change
in the pond water depth, a signal that strongly suggest there was a water exchange between ponds or
from the estuary. The same pattern will be showed by the presence of the crustacean daphnia
Diaphanosoma in the zooplankton composition (Fig.6).
Figure 1.Average data of abiotic factor of the three ponds are shown with standard deviation.
13
Figure 2.Continuous value of oxygen recorded during the sampling period in the three ponds.
Figure 3.Representation of Inorganic nutrients as average value between ponds. Standard deviation is showed.
14
Changes in the silicate concentration in fig. 3 are also the results of mineralization after the diatom
bloom (Fig. 4). During the entire campaign the quantity of phytoplankton available in the ponds was
extremely high (value of Chl a in average 92.5 ± 36.6 STD). It is clear though that most of the
phytoplankton is represented by nano and picoplankton and that the food available for adult copepods
might not be sufficient: diatoms, dinoflagellates and green algae are most likely at the appropriate size
to be cached and assimilated by the copepods and their abundance is low as compared to the entire
amount of pico algae which are not retainable by the copepods (Fig.4 and Fig. 5).
Phytoplankton biomass
18000
16000
14000
[µg C/L]
12000
10000
diatoms
blue-green algae
green algae
dinoflagellates
flagellates
pico-nano algae
ciliates
8000
6000
4000
2000
0
18-06-12
24-06-12
30-06-12
06-07-12
12-07-12
Sampling Days
Figure 4.Phytoplankton biomass in the ponds during the entire campaign (average of the three ponds).
Chlorophyll a (size fraction)
140
> 15 µm
concentration (µg/L)
120
< 15 µm
100
80
60
40
20
0
18-06-12
24-06-12
30-06-12
06-07-12
12-07-12
Sampling days
Figure 5. Chl a above (food for advanced copepodites and adults) and below 15 µm (food for copepod nauplii)
in the ponds during the entire campaign (average of the three ponds).
15
We also described the zooplankton abundance during the whole period: the main copepod species was
Pseudodiaptomus annandalei that represented more than 70% in average of the entire zooplankton
composition (Fig. 6). Few other copepod species such as Oithona sp. and Apocyclop royi were
recorded. In particular cases a high amount of rotifers was noted.
Zooplankton
250
Abundance (ind/L)
200
150
P. annandalei
Oithona
Apocyclop
Acartia
Harpacticoids
Temora
Diaphanosoma
Rotifers
Others
100
50
0
12-06-12
18-06-12
24-06-12
30-06-12
06-07-12
12-07-12
Sampling days
Figure 6. Zooplankton composition from 50 µm net in individuals/L.
To discover the physiological response of the main copepod species of the physical-chemical
conditions in the ponds we measure the specific growth rate and thereby the secondary productivity of
P. annandalei in the laboratory through growth incubation experiments. Results demonstrate a value of
0.9 d-1 ± 3 (STD) (Fig. 7) which is extremely high compared to temperate species. We can state that
tropical temperature have a high response to the metabolism of this species. However, the overall
production of copepods in the ponds is by the farmers described to have been decreasing during the last
10 years with no obvious reasons. Apart from the food availability that cannot explain entirely the
relatively low abundance of copepods, we discovered that during night time the oxygen level in the
ponds can reach as low levels as 1-2 mg/L which potentially can be lethal or detrimental for the growth
of some species of copepods. Presence of hydrogen sulphide in the bottom sediments of the ponds can
affect the survival of the zooplankton.
Studies on the fatty acids (FA) of phytoplankton and zooplankton are in progress: preliminary data
shows that Pseudodiaptomus annandalei FA composition is different from their diet. They probably
assimilate FA from the environment (oil from the chicken manure and fish oil) or can synthetize some
FA from their precursor. Other studies have to be performed to confirm this hypothesis.
16
Figure 7. Specific growth rate of nauplii.
CONCLUSIONS AND FUTURE STUDIES
From these preliminary results we can conclude that the most abundant copepods species in the ponds
during summer period is Pseudodiaptomus annandalei. Their tolerance to variation in salinity and
oxygen is essential in this kind of aquaculture ponds and the high temperature provides for them a fast
growth. Their FA composition show high amount of unsaturated chains, and DHA/EPA ratio is higher
than 2. This species is, therefore, of high nutritional value and can be used as live feed for grouper
larvae.
The copepod density measured in the ponds during summer was not the highest recorded for these
farms. Therefore a seasonal study will be performed starting from April 2013. Four field campaigns
will be achieved to demonstrate which abiotic and biotic factor is the main problem or the main yield
for this species. The environmental conditions and the food availability for this species will be
described; composition of zooplankton and species interactions will be studied to have a complete
annual production cycle from the fish farmers’ harvest.
Laboratory experiments will be performed to incorporate physiological knowledge of the key
components into observations and measurements from the field studies: temperature and pH tolerance
levels will be provided. Oxygen consumption and tolerance investigations will be completed to map
their resistance to low level of oxygen. Feeding experiments will be achieved to investigate if the
amount of phytoplankton available is sufficient and to eventually demonstrate their ability to synthetize
new FA from their precursor or if they gain dissolved FA from the environment.
17