Lucia 13 Prfa

unuftp.
is Final Project 2013
THE EFFECT OF DISINFECTION ON SURVIVAL AND FEED

QUALITY OF ROTIFERS (BRACHIONUS PLICATILIS) AND
BRINE SHRIMP (ARTEMIA SALINA)
Lucia Matlale Mzimba

Department of Agriculture, Forestry and Fisheries
Aquaculture Research and Development
Private Bag X 2, Rogge Bay, 8012
Cape Town, South Africa
matlale.lucia@gmail.com
Agnar Steinarsson
Marine Research Institute
P. O. Box 1390, Skúlagata 4
Reykjavík, Iceland
agnar@hafro.is
ABSTRACT
Live feeds are used as an essential component during the larval stages of most marine
finfish species in aquaculture. The microbial accumulation in batch culture of live
feed has been identified as one of the major factors limiting the expansion of
commercial farming of aquatic species in many countries. The main focus of the
present study was to test the use of the antimicrobial agents bronopol (Pyceze) and
Sanocare ACE for the disinfection of enriched rotifers and brine shrimp (Artemia) in a
marine hatchery. The study also involved a feeding experiment with three different
feeds in rotifer batch culture (Rocult, yeast and algae paste). The study has
successfully tested disinfection protocols that were highly effective with regards to
survival and feed quality of rotifers and brine shrimp. The results indicate that the use
of 50 ppm bronopol for 2 hours is a very effective protocol for rotifer disinfection
with negligible lethal effects on rotifers. A combination of 100 ppm bronopol and 600
ppm Sanocare ACE for 22 hours was a very effective disinfection protocol for brine
shrimp with only moderate lethal effects on the brine shrimp. The commercial feed
Rocult was the best feed in the feeding experiment but an inexpensive yeast diet
proved almost as good.
This paper should be cited as:

Mzimba, L.M. 2014. The effect of disinfection on survival and feeding quality of rotifers (Brachionus
plicatilis) and brine shrimp (Artemia salina). United Nations University Fisheries Training
Programme, Iceland [final project]. http://www.unuftp.is/static/fellows/document/lucia13prf.pdf
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TABLE OF CONTENT
LIST OF TABLES ...................................................................................................... 3

LIST OF FIGURES .................................................................................................... 4
1 INTRODUCTION................................................................................................. 6
1.1 Background of marine larval culture in South Africa ..................................... 7
1.2 Rotifer culture ................................................................................................. 8
1.2.1 The culture conditions for rotifers ........................................................... 8
1.2.2 Feeding and enrichment of rotifers .......................................................... 8
1.2.3 Batch culture of rotifers ........................................................................... 9
1.2.4 Rotifer culture contamination .................................................................. 9
1.3 Brine shrimp culture ........................................................................................ 9
1.3.1 The culture methods for brine shrimp ...................................................... 9
1.3.2 The culture conditions for brine shrimp................................................. 10
1.3.3 Feeding or enrichment of brine shrimp .................................................. 10
1.3.4 Brine shrimp culture contaminations ..................................................... 11
1.4 Use of antimicrobial agents in live feed culture ............................................ 11
1.5 The objectives of the study............................................................................ 11
2 MATERIALS AND METHODS ...................................................................... 12
2.1 Location and dates of the experiments .......................................................... 12
2.2 Density determination ................................................................................... 12
2.3 Rotifer trials................................................................................................... 12
2.3.1 Feeding trial ........................................................................................... 12
2.3.2 Rotifer disinfection trial ......................................................................... 14
2.4 Brine shrimp trials ......................................................................................... 15
2.4.1 Brine shrimp disinfection trial ............................................................... 15
2.5 Microbial analysis ......................................................................................... 16
2.6 Water quality parameters .............................................................................. 17
2.7 Statistical analyses of data............................................................................. 17
3 RESULTS ........................................................................................................... 18
3.1 Feeding of rotifers ......................................................................................... 18
3.1.1 Effect of different feeds on population density of rotifers ..................... 18
3.1.2 Rotifer production and harvesting ......................................................... 18
3.1.3 Egg percentage ....................................................................................... 19
3.1.4 Specific population growth rates ........................................................... 20
3.1.5 Effect of water quality parameters ......................................................... 22
3.2 Disinfection of rotifers .................................................................................. 22
3.2.1 Effects of disinfection on rotifer densities ............................................. 22
3.3 Culture of brine shrimp ................................................................................. 24
3.3.1 Effect of disinfection on brine shrimp densities .................................... 24
3.3.2 Effect of disinfection on total microbial counts from brine shrimp
cultures 26
4 DISCUSSION ..................................................................................................... 28
ACKNOWLEDGEMENTS ..................................................................................... 30
REFERENCES .......................................................................................................... 31
Appendix: tables and figures ................................................................................... 35
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LIST OF TABLES
Table 1: The table below shows the different feeds, quantities and the ClorAm-X used
to mix with individual feed during the rotifer feeding trial. .................................. 13
Table 2: The quantities of disinfectant used for the rotifer disinfection trial. Pyceze
(50% bronopol) was used to disinfect rotifers at different concentrations and
durations as described below. Each group included a duplicate set of tanks. ....... 15
Table 3: The quantities of disinfectants used for brine shrimp disinfection trial.
Pyceze and/or Sanocare ACE were used at different concentrations to disinfect
brine shrimps at different time periods as outlined below. .................................... 16
Table 4: Population density changes (in %) during the rotifer disinfection trial. ........ 23
Table 5: Population density changes in brine shrimp disinfected with Sanocare ACE
(S) and Pyceze (P). ................................................................................................. 25
Table 6: Water quality parameters and rotifer population densities during the rotifer
feeding trial. ........................................................................................................... 35
Table 7: Water quality parameters recorded during the rotifer disinfection experiment.
................................................................................................................................ 35
Table 8: Population densities after two- and six hour treatment of rotifers during the
disinfection trial. .................................................................................................... 36
Table 9: Water quality parameters recorded during the disinfection trial of brine
shrimps. .................................................................................................................. 37
Table 10: Population densities counted before and after disinfection of brine shrimps
during the disinfection trial. The chemicals uded were Sanocare ACE (S) and
Pyceze (P). P200 and P400 mean 200 ppm and 400 ppm, respectively. ............... 38
Table 11: Total CFU counted after disinfection of rotifers with Pyceze (50%
bronopol) for 2 and 6 hours. .................................................................................. 39
Table 12: Total CFU counted after 10 and 22 hours of disinfection of brine shrimp
with Pyceze (50% bronopol) and Sanocare ACE. ................................................. 40
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LIST OF FIGURES
Figure 1: Female rotifer (Brachionus plicatilis) with one egg, from Korea-US-
Aquaculture 2014. .................................................................................................... 6
Figure 2: Brine shrimp nauplius (Artemia salina), from Delbos and Schwarz 2009. ... 6
Figure 3: Rotifer silos used for the feeding trial experimental set up, with triplicate
silos for each type of feed diet. The picture was taken by author at Stadur, January
2014........................................................................................................................ 13
Figure 4: The rotifer cylinders showing a 6 x 2 setup for the rotifer disinfection trial,
picture taken by author in January 2014. ............................................................... 15
Figure 5: Rotifer population densities counted on a daily basis based on different
feeds. Each data point represents the average population density of rotifers on
each day and the error bars represent the standard error of the mean. ................... 18
Figure 6: The average daily rotifer production from the three feeds in the rotifer
feeding trial. Each column represents the average daily rotifer production from
each feed diet. The error bars indicate the standard error of the mean. ................. 19
Figure 7: The percentage of pregnant rotifers (egg carriers) during the feeding trial.
Each column represents the daily mean egg percentage from each feed diet each
day. The error bars indicate the standard error of the mean. ................................. 19
Figure 8: Specific population growth rates of rotifers observed on a daily basis during
the feeding trial. The data points represent the daily average growth rates of
rotifers. The last day with algae paste was excluded from the graph due to a
culture crash. The error bars indicate the standard error of the mean. ................... 21
Figure 9: Average daily growth rates of rotifers during the entire feeding trial. The
last day with algae paste was excluded from the calculation due to a culture crash.
The error bars indicate the standard error of the mean. ......................................... 21
Figure 10: Rotifer population densities before and after treatment with Pyceze
(bronopol). The columns represents the normalized density of rotifers. The error
bars represent the standard error of the mean. ....................................................... 23
Figure 11: Total microbial counts (CFU per rotifer) after 2 and 6 hours of treatment in
the rotifer disinfection trial. Each data point indicates the number of colonies
counted per individual rotifer. The error bars represent the standard error of the
mean. ...................................................................................................................... 24
Figure 12: Population densities of brine shrimp disinfected with Sanocare ACE (S)
and Pyceze (P). P200 and P400 mean 200 ppm and 400 ppm Pyceze, respectively
(100 ppm and 200 ppm active bronopol, respectively). The error bars represent the
standard error of the mean. .................................................................................... 25
Figure 13: Total microbial counts (CFU per brine shrimp) in the brine shrimp
disinfected with Sanocare ACE (S) and Pyceze (P). P200 and P400 mean 200 ppm
and 400 ppm Pyceze, respectively (100 ppm and 200 ppm active bronopol,
respectively). The error bars represent the standard error of the mean.................. 26
Figure 14: An s-shaped tray used for rotifer- and brine shrimp density determination.
................................................................................................................................ 40
Figure 15: Sponge filters used for trapping suspended particles in the rotifer feeding
trial. ........................................................................................................................ 41
Figure 16: Appearance of rotifers under a 40X magnification using a light microscope
during population density counting. ...................................................................... 41
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Figure 17: Appearance of brine shrimp under a 40 X magnification in a light

microscope used during population density counting. ........................................... 41
Figure 18: Colonies observed from plates during microbial analyses of rotifers. ...... 42
Figure 19: Colonies and fungal growth observed from plates during the microbial
analysis of brine shrimp. ........................................................................................ 42
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1 INTRODUCTION
Live feeds are used as an essential component during larval stages of most marine
finfish species. The use of natural food such as marine rotifers Brachionus (figure 1)
and brine shrimp Artemia salina (figure 2) was pioneered by Takashi Ito in Japan and
since then live feed has been used as food for marine fish larvae (Korea-US-
Aquaculture 2014). However, the production of suitable high quality live feed is still
widely considered a serious bottleneck, limiting the expansion of commercial farming
of aquatic species in many countries. Global statistics show that the mass-culture of
many live feed species (such as plankton, brine shrimps, rotifers and marine worms)
in hatcheries and grow-out operations is still one of the challenges faced in
aquaculture (FAO 2012).
Figure 1: Female rotifer (Brachionus plicatilis) with one egg, from Korea-US-
Aquaculture 2014.
Figure 2: Brine shrimp nauplius (Artemia salina), from Delbos and Schwarz 2009.
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1.1 Background of marine larval culture in South Africa
South Africa is known to be one of the leading countries in abalone production.

However, marine finfish production is still underdeveloped. A number of indigenous
species, such as dusky kob (Argyrosomus japonicus), yellowtail (Seriola lalandi),
white steenbras (Lithognathus lithognathus) and white stumpnose (Rhabdosargus
globiceps) have been identified as suitable candidates for marine finfish aquaculture
in South Africa (Hecht 2000). All these species and in fact most marine finfish depend
on live feed during their larval stages.
The Department of Agriculture, Forestry and Fisheries: Directorate: Aquaculture

Research and Development have an aquaculture research facility based at the Sea
Point Aquarium in Cape Town, South Africa. One of the key objectives of the fish
genetics and breeding subsection under the Directorate, is to supply the aquaculture
industry with quality juveniles and first-class farming advice. The facility is presently
conducting ongoing projects on dusky kob in order to produce quality eggs, larvae
and juveniles to supply to local fish farms.
At present the facility uses females above 10 kg size with fecundity of about 1 million
eggs per 10 kg fish or 100 000 eggs per kg female. Four females are induced per
session and about 4-6 million eggs are spontaneously spawned between 32-72 hours
after spawning induction using hormones. About 70 % of spawned eggs (~3.5
million) are on average fertilized, of which 90-95 % hatch. However, only about 15 %
of post hatch larvae survive to the juvenile stage (complete metamorphosis and
weaned = 28 mm). Hatching temperature is set at 22 °C and after hatching the
temperature is increased to an optimum growth temperature of 25 °C (Collette et al.
2008). The initial hatching temperature is at the lower indicated setting to reduce
larval deformities which are experienced when eggs are exposed to higher
temperatures (>24 °C). The size of fertilized kob eggs is 0.9-1.0 mm in diameter. The
larvae feed on live food at a length of 2.8 mm and approximately 3-4 days post hatch.
In the hatchery the eggs are incubated at a density of 500 per litre. A newly hatched
larva measures on average about 2.3 mm in length and yolk sac diameter is
approximately 0.8 mm. Larvae are stocked at an initial density of 200 per litre.
Weaned juveniles (28-31 mm) are reared at a density of 50 fish per litre and then
grown to fingerlings (40 mm) at a density of 10 fish per litre. The nursery grows
fingerlings to viable growers at 50 g body mass and at a production density of 20
grams per litre or 20 kg per cubic meter (Collette 2007).
Dusky kob is commercially produced by four companies in South Africa. The annual
total productions of the four farms was 7,909 tons in 2011 (Department of
Agriculture, Forestry and Fisheries 2012). This species is fed with microalgae (e.g.
Nannochloropsis oculata, Isochrysis spp and Pavlova lutheri), rotifers (Brachionus
plicalitis) and brine shrimp (Artemia salina) during the larval stage in South Africa.
These live feed organisms can also be a primary source of pollutants entering the
culture system (Davis 1990). It is believed that an excessive microbial load from live
feed can cause massive mortalities of fish larvae since pathogens can be transferred
from the live feed to the fish. Therefore, feeding of the fish larvae with live feeds
represents a critical phase, especially considering the nutritional health of the fish.
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1.2 Rotifer culture
1.2.1 The culture conditions for rotifers
The rotifer Brachionus spp. is the first live feed organism that has demonstrated
acceptability for most marine species, and can typically be raised on a commercial
scale (Delbos and Schwarz 2009). Brachionus plicatilis (L-strain) is a marine rotifer
with a size range of 200-360 μm and is the most common live feed species in
aquaculture (Delbos and Schwarz 2009). The culture of Brachionus spp. is influenced
by various water quality parameters, namely temperature, dissolved oxygen (DO), pH
and salinity. Rotifers of the genus Brachionus require oxygen concentrations above
1.0 mg/L for their growth, though some can tolerate anaerobic or near-anaerobic
conditions for a short period of time. A salinity of 10-35 ppt and total ammonia
nitrogen of ≤ 5 ppm are also recommended for the culture of this species (Korea-US
Aquaculture 2014). They require a temperature range of 22 to 28 °C. The culture of
this live feed is common due to some positive biological characteristics that include:
small body size, round body shape, slow swimming speed, positive buoyancy, easy
enrichment, high reproduction rate and high density cultures (Korea-US Aquaculture
2014).
1.2.2 Feeding and enrichment of rotifers
Rotifers are enriched with specially formulated feed products in order to boost their
nutritional profile prior to feeding them to fish larvae (Delbos and Schwarz 2009).
Activated baker’s yeast has been used successfully as an inexpensive grow-out diet
when fed at approximately 0.5 g/million rotifers/day (Delbos and Schwarz 2009).
Baker’s yeast has a small particle size (5-7 µm) and high protein content and is an
acceptable diet for rotifers. When applied as a sole diet, it may support the mass
production of rotifers in non-axenic (non-sterile) culture conditions where micro-
organisms provide essential nutrients (Hirayama 1987). However, it is well known
that yeast-fed rotifers lack the essential fatty acids required for the proper
development and survival of several species of marine fish. Therefore, the yeast
usually needs to be supplemented with essential fatty acids and vitamins to suit the
larval requirements of the fish to be cultured (Hirayama and Satuito 1991).
Many commercial, artificial diets are available for feeding and enriching rotifers, such
as DHA Protein Selco, S.parkle, S.presso, and S.tream. Rocult is another available
off-the shelf dry rotifer diet belonging to the Larviva hatchery feed product line from
Biomar in Denmark. Rocult is a formulation based on living yeast and algae that is
high in protein and is nutritionally complete diet to grow rotifers in an optimal way.
The feed is delivered in dried form which makes it easier to use and the daily
production of rotifers is predictable and reliable (Larviva Biomar Hatchery 2014).
Multigain also belongs to Biomar’s Larviva product line and has been used as an
enrichment feed for rotifers and brine shrimp. It has been designed to provide live
feed with adequate nutritional composition. As a result, less sensitive and healthier
larvae are produced with reduced deformity levels (Laviva Biomar Hatchery 2014).
Concentrated algae paste is also widely used as a feed for growing rotifers.
RotiGrow Plus is produced by Reed Mariculture and is a mixture of five different
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microalgae, rich in highly unsaturated fatty acid (HUFA) and vitamins for rotifers
culture. RotiGrow Plus is a clean, high yield rotifer feed that maximizes pre-
enrichment levels of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and
arachidonic acid (ARA). It can also be used as a sole feed or in combination with
other enrichments, depending on the nutritional requirements of the fish larvae (Reed
Mariculture Inc. 2014).
1.2.3 Batch culture of rotifers
Over the years, several culture techniques have been used in the culture of rotifers.
Among those, batch culture is the most commonly used method in rotifer production
for marine fish hatcheries, probably because of its simplicity (Fukusho 1983, Nagata
and Hirata 1986 and Snell 1991). This culture technique consists of either the
maintenance of a constant culture volume with an increasing rotifer density or the
maintenance of a constant rotifer density by increasing the culture volume (Fukusho
1983, Nagata and Hirata 1986 and Snell 1991). Although batch culture is simple, the
most frequently reported problems associated with this system are: the
unpredictability of rotifer mass production, the difficulty to manage and harvest large
rotifer populations and the difficulty of producing clean rotifers that are free of
floccules (suspended particles) and safe from a microbial point of view (Dhert et al.
2001). Jung (2012) has discovered that in the mass culture of rotifers, there are many
factors affecting their population growth. Among them is the biological environment,
such as contamination with bacteria and other organisms in the culture tanks.
Copepods (Tigriopus japonicus, Acartia), ciliate protozoans (Euplotes, Vorticella and
Holosticha) often co-exist in the marine Brachionus rotifer culture.
1.2.4 Rotifer culture contamination
The use of live feed is believed to be a major contributor to high mortalities of fish
larvae as a result of contamination that may be traced to the live feed culture. Most
bacteria are not pathogenic to rotifers, however, their proliferation must be avoided
since their accumulation and transfer via the food chain can cause detrimental effects
to fish larvae (Dhert 1996). Bacteria are always associated with mass production of
rotifers and may cause an unexpected mortality or suppressed growth of rotifers. In
some other cases, bacteria may cause no harm to the rotifers but infected rotifers may
cause detrimental effects on fish larvae, resulting in poor survival and growth (Perez-
Benavente et al. 1989).
1.3 Brine shrimp culture
1.3.1 The culture methods for brine shrimp
Among the live feeds used in the larval culture of fish and shellfish, nauplii and
metanauplii of the brine shrimp (Artemia) constitute another widely used live prey
item. Brine shrimp belong to the phylum Arthropoda, class Crustacea, like Daphnia,
which is a copepod used as live food in the aquarium trade and for marine finfish and
crustacean larval culture (Treece 2000). The cysts of brine shrimps are made available
in storable cans and can be incubated for 24 hours in seawater. These cysts release
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free-swimming nauplii that can directly be fed as a live food source to the larvae of a
variety of marine as well as freshwater organisms, which makes them the most
convenient, least labour-intensive live food available for aquaculture (Stappen 1996).
Furthermore, the cultivation methods have gradually been optimized through the years
and made more cost effective. Modern hatcheries now routinely disinfect the brine
shrimp and use decapsulated cysts to reduce labor and microbial loads. Various brine
shrimp biomass preparates are also being applied in hatcheries, nurseries and
broodstock rearing (Stappen 1996). Decapsulation is an easy method that is used to
remove the outer layer of the shell prior to incubation of the cysts (Gilbert and Watson
2006). Aside from eliminating the shell, decapsulation sterilizes the cysts and can
increase hatch rates if done properly (Gilbert and Watson 2006). Sep-Art is a new
technology that provides a magnetic coating on brine shrimp cysts. According to the
producer, this new technology is highly efficient and allows a fast and complete
separation of nauplii and unhatched cysts (Ocean Nutrition 2014).
1.3.2 The culture conditions for brine shrimp
Both temperature and salinity significantly affect the survival and growth of brine
shrimp, with the effect of temperature being more pronounced. A broad range of
temperatures and salinities meet the requirements for >90 % survival. Strains from
thalassohaline (hypersaline) environments have an optimal temperature range of 20-
25 °C where mortalities are <10 %. Interaction between temperature and salinity is
limited; substantial differences in tolerance have been recorded at low salinities
(around 5 g l-1) and high temperatures (30-34 °C). At elevated temperatures the
survival of the Great Salt Lake (GSL) strain is significantly higher than for other
strains (Stappen 1996).
1.3.3 Feeding or enrichment of brine shrimp
The nutritional value of brine shrimp can be improved to suit the predator’s
requirements by feeding specific amounts of particulate or emulsified products that
are rich in highly unsaturated fatty acids (HUFA) to the nauplii. Application of this
method of bio-encapsulation, is also called enrichment or boosting. This method of
enriching brine shrimp has had a major impact on improved hatchery outputs, not
only in terms of survival and growth of many species of fish and crustaceans, but also
with regard to their quality that includes reduced incidence of malformations,
improved pigmentation and stress resistance (Stappen 1996). Like rotifers, brine
shrimp act as ideal carriers that carry nutrients to fish larvae. They do, however, need
to be enriched with nutrional diets that are essential for the growth and survival of fish
larvae. SELCO (Self-Emulsifying Lipid Concentrate) is an enrichment product that
was developed by "Artemia Systems NV". This product has been the benchmark
Artemia enrichment formula in the aquaculture industry. SELCO is rich in Highly
Unsaturated Fatty Acids (HUFA's) and contains particularly high levels of the
essential Omega-3 fatty acids, EPA and DHA. This diet has been shown to increase
both the survival and the growth of marine fish larvae according to Brine Shrimp
Direct (2014).
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1.3.4 Brine shrimp culture contaminations
It has been demonstrated that bacteria can have a beneficial effect during brine shrimp
culture (Verschuere et al. 1999). Studies also show that some bacterial strains may
improve the survival and growth rates of brine shrimp. Similarly, the culture of brine
shrimp under non-sterile conditions usually results in a higher biomass production
than that under sterile conditions (Verschuere et al. 1999). Diseases can be
problematic in batch culture systems of brine shrimp. For example, some species of
Vibrios have been identified as harmful organisms that may dominate the flora of
brine shrimps and rotifers (Munro et al. 1999). Reduction of this harmful bacterial
load on live food organisms is important for achieving good survival and growth of
fish larvae.
1.4 Use of antimicrobial agents in live feed culture
The microbial accumulation in batch culture of live feed has been identified as one of
the major biochemical factors affecting the culture of marine finfish and molluscs. A
number of methods have been developed for reducing the bacterial load of live feed,
e.g. UV irradiation or Pyceze treatment (Novartis 2014). Antibacterial substances
such as Pyceze (50% bronopol) have been reported to be effective against bacterial
proliferation over a wide pH range (Bryce et al. 1978). Sanocare ACE (Artemia
Condition Enhancer, INVE Aquaculture) has also been used as an antibacterial agent
and a water conditioner for improved brine shrimp quality. Sanocare ACE increases
the quality and vitality of hatched, concentrated and stored Artemia nauplii. It
improves water quality conditions during enrichment, stabilizes pH levels and avoids
foaming during hatching, enrichment and storage (INVE Aquaculture 2014). Sanocare
ACE was the first product in the aquaculture industry able to reduce the development
and transfer of putative pathogens associated with live food cultures. Sanocare ACE is
a self-emulsifying product that keeps bacterial levels in brine shrimp hatching water at
a very low level. The use of it has been found to reduce the Vibrio load up to 10,000-
100,000 fold (INVE Aquaculture 2014).
1.5 The objectives of the study
The main aim of this study was to determine suitable disinfection techniques for the
production of rotifers and brine shrimp for fish hatcheries. A vital aspect of the study
was to analyse the effect of disinfection on the viability of the prey themselves. A
secondary aim was to compare the suitability of different foods for rotifer batch
culture. Specific objectives of the experiment include:
A) To compare the use of different feeds (Rocult, yeast and algae paste) for rotifers to
respectively monitor their fertility and population growth.
B) To analyse the effect of Pyceze disinfection on rotifer viability and microbial load.
C) To analyse the effect of Pyceze and/or Sanocare ACE disinfection on brine shrimp
viability and microbial load.
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2 MATERIALS AND METHODS
2.1 Location and dates of the experiments
All experiments were performed at the Marine Research Institute (MRI) aquaculture
facility in Staður, in Grindavík Iceland from the 12th to the 22nd January 2014.
Rotifers, brine shrimps, feeds, equipment, chemicals, transport and assistance were
provided by the facility.
2.2 Density determination
The density of live feed was counted by obtaining samples (0.25 ml for rotifers and
0.5 ml for brine shrimp) from each experimental container. The sample was put in a
test tube and 1 drop of Lugol’s solution (5 % solution) added to kill and stain the
rotifers (Delbos and Schwarz 2008). The samples were then diluted with tap water
and placed on an s-shaped tray used for live feed counting (figure 13 in the appendix).
The tray was placed under a light microscope for counting of individual animals at 40
x magnification. The total density of prey animals was determined from the counting
as well as the percentage of egg-bearing rotifers. The counting was done by sampling
each individual tank and calculating the mean for each duplicate set.
2.3 Rotifer trials
2.3.1 Feeding trial
A 3 x 3 setup of 25 L black, conical tanks (silos) was prepared (figure 3) and the silos
inoculated with rotifers for the experiment. The silos were aerated with pure oxygen
to ensure a continuous supply of dissolved oxygen. The water temperature was kept
between 25-27 °C and the salinity was stable at 32 ppt. All the tanks were exposed to
light throughout the experiment. Sponge filters were submerged in the tanks to trap
both large pieces of suspended matter and protozoans. The sponges were washed with
high pressure water on a daily basis.
The trial was designed to compare the following three feed products:
A = Rocult, Producer: Biomar, DENMARK.
B = Semi dry yeast, Producer: Angel Yeast Co. Ltd, CHINA.
C = Algae paste (Rotigrow Plus), Producer: Reed Mariculture Inc, USA.
The initial stocking density was ≈1000 rotifers/ml in each silo. The three sets of
rotifer tanks were fed with different diets (A, B and C, respectively) for a period of
four days. Feeds A and B were supplied at 0.45-0.7 g/million rotifers/day, feed C was
supplied at 1.5 ml/million rotifers/day. The feeds were supplied in 8 equal rations per
day by automatic pumps at rate of 125 ml per 3 hours. The population density and egg
percentage were measured daily after feeding. About 4 litres of water were flushed
from the silos each morning to harvest rotifers and create space for the new feed.
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Figure 3: Rotifer silos used for the feeding trial experimental set up, with triplicate
silos for each type of feed diet. The picture was taken by author at Staður in January
2014.
The feeds were used in different quantities as shown in table 1 below.
Table 1: The table below shows the different feeds, quantities and the ClorAm-X used
to mix with individual feed during the rotifer feeding trial.
Days Feed type Feed Quantity (g) ClorAm-X (g)
Rocult 39 35,0
Yeast 39 35,0
1 Algae paste 87 ml 34,8
Rocult 48 19,2
Yeast 42 16,8
Rocult 42 16,8
Yeast 42 16,8
Rocult 60 24,0
Yeast 60 24,0
The density of rotifers was measured daily after feeding different diets for four days.
The specific growth rate was calculated using the equation described by Rombaut et
al. (1999):
μ= (lnNt−lnN0)/t,
Where: μ=specific growth rate, Nt =rotifer density after culture period t (individual’s
ml−1), N0=initial rotifer density (individuals ml−1), t=culture period (day).
Feeds A and B were prepared by mixing the total daily ration of feed mixed with 40
% ClorAm-X (90 % during first day) in about 300 ml warm water in a blender for 2
minutes. Feed C was mixed gently with ClorAm-X in 300 ml of seawater. ClorAm-X
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is a dry powder substance used as water conditioner for detoxification and removal of
ammonia, chlorine and chloramines in both fresh and salt water. All feed for each
triplicate set of silos was stocked in 3 L cylinders under constant aeration and
connected to the automatic pumps. Feeding was adjusted daily depending on the
population density counted.
2.3.2 Rotifer disinfection trial
The disinfection trial was performed in 6L transparent plastic cylinders. Six duplicate
sets (figure 4) of cylinders (6 x 2) were inoculated with rotifers obtained from the
master cultures at a stocking density of ≈ 2000 rotifers/ml in a total volume of 6L. The
cultures were all aerated with pure oxygen and enriched with Multigain (Biomar,
Denmark) at 0.2 g/million rotifers. Temperature and oxygen were measured in all
cylinders. Pyceze (50 % bronopol) was added at two different dosages for two
different time intervals as shown below (table 2). One set of cylinders served as a
control group and did not receive any Pyceze treatment. Pyceze is a clear fluid bath
additive that contains 50 % bronopol. Bronopol (2-bromo-2-nitropropane-1,3-diol) is
a common name for the active ingredient in a series of broad spectrum antimicrobial
agents and it is marketed by The Dow Chemical Company and its global affiliates
under several trade names. Pyceze is one of the products containing bronopol and it is
available as a white to off white crystalline solid powder. Pyceze is produced by
Novartis Animal Health UK Ltd as a bactericide and for treatment and control of
fungal infections (Novartis Animal Health 2014).
To prepare a 50 ppm bronopol concentration, 600 ml water were mixed with 0.6 ml
Pyceze (100 mg/L) and added to the 6 L cylinder containing about 5.4 L culture
water. The dosage of the chemical was halved (i.e. 0.3 ml Pyceze) to prepare the 25
ppm solution. The density was counted after 2 hours of sterilization and again after 6
hours. These time intervals were selected due to the recommended enrichment period
for rotifers by the suppliers. Rotifers are normally enriched for 2 - 6 hours with
Multigain (Larviva Biomar Hatchery 2014). To facilitate a direct comparison of
treatment effect the initial density was normalised to 2000/ml and subsequent
densities were adjusted accordingly. The normalisation factor (NF) was calculated
from actual density (AD) according to: NF = 2000/AD. Normalised density (ND) was
calculated from actual density (AD) according to: ND = AD x NF.
Samples were collected at the end of each time interval for microbial analysis.
Collection was done by filtering about 3 L of culture medium on a fine filter (64
micron mesh) and collecting the sludge in 10 ml glass bottles. The equipment was
sterilized before sampling from each cylinder. It was first washed in a Virkon-S
solution, a disinfectant chemical that kills about 99.99 % of numerous pathogens
including 31 bacterial strains, 58 viruses and six fungi with no evidence of resistance.
The equipment was then rinsed with seawater and finally washed in water containing
vinegar for the removal of excess chlorine. Seawater was used to wash the rotifers
collected on the mesh sieve to remove suspended solids on the cultures. The sludge
was frozen immediately (-20 °C) and taken for microbial analysis at Matís some days
later. Matís Ltd. Icelandic food and biotech R&D company is a government owned,
independent research company that pursues research and development aligned to the
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food and biotechnology industries as well as providing analytical testing service for
public and private authorities.
Figure 4: The rotifer cylinders showing a 6 x 2 setup for the rotifer disinfection trial,
picture taken by author in January 2014.
Pyceze (50 % bronopol) was used to disinfect rotifers at different concentrations and
durations as described below. Each group included a duplicate set of tanks.
Table 2: The quantities of disinfectant used for the rotifer disinfection trial.
Tank No Treatment Dosage applied Duration
1A Control 0 mg/L 2 hours
1B Control 0 mg/L 2 hours
1A Control 0 mg/L 6 hours
1B Control 0 mg/L 6 hours
2A Pyceze (50% bronopol) 50 mg/L (25 ppm) 2 hours
2B Pyceze (50% bronopol) 50 mg/L (25 ppm) 2 hours
2.4 Brine shrimp trials
2.4.1 Brine shrimp disinfection trial
The Artemia cysts used in the study originated from the Great Salt Lake (GSL) in
Utah (INVE Aquaculture 2014). Non-decapsulated cysts were inoculated at 500
cysts/ml as a part of the hatcheries routine production. Sanocare ACE (0.5 g/L) and
bronopol (0.1 g/L) were added to the inoculation to enhance water quality and reduce
microbial levels. After hatching, the nauplii were separated from the cysts and washed
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with clean seawater before being stocked into the experimental tanks. A set of six
duplicate (6 x 2) 6 L tanks (transparent, plastic cylinders) were inoculated with
Artemia nauplii from the hatched cysts at a stocking density of ≈ 300 nauplii/ml. All
groups were immediately enriched with A1 DHA Selco (INVE Aquaculture 2014) in
one 200 ppm ration. The anti-microbial agents were added immediately to all except
one duplicate set of tanks at different concentrations and different time intervals as
shown below (table 3). Samples were collected after 10 and 22 hours and stored in a
freezer (-20 °C). The time intervals for sampling were selected based on the
enrichment period of brine shrimp, which has been recommended to be 12–24 hours
(INVE Aquaculture 2014). Samples collection was done following the same method
as rotifers which is described under section 2.3.2.
Table 3: The quantities of disinfectants used for brine shrimp disinfection trial.
Pyceze and/or Sanocare ACE were used at different concentrations to disinfect brine
shrimps at different time periods.
Tank No Treatment Dosage applied Duration
1A Control 0 g/L 10 and 22 hours
1B Control 0 g/L 10 and 22 hours
2A Sanocare 0.6 g/L 10 and 22 hours
2B Sanocare 0.6 g/L 10 and 22 hours
3A Sanocare + Pyceze 0.6 g/L + 0.2 g/L 10 and 22 hours
3B Sanocare + Pyceze 0.6 g/L + 0.2 g/L 10 and 22 hours
4A Sanocare + Pyceze 0.6 g/L + 0.4 g/L 10 and 22 hours
4B Sanocare + Pyceze 0.6 g/L + 0.4 g/L 10 and 22 hours
5A Pyceze 0.2 g/L 10 and 22 hours
5B Pyceze 0.2 g/L 10 and 22 hours
6A Pyceze 0.4 g/L 10 and 22 hours
6B Pyceze 0.4 g/L 10 and 22 hours
2.5 Microbial analysis
The assessments were done by pipetting 1 ml of sample to carry out three dilutions
(101, 102,103) to be inoculated on plates. The dilutions were transferred to a petri dish
containing a Plate Count Agar (PCA, Biokar Diagnostic cod. BK 144HA, Beauvais,
France) with 2 % NaCl. This growth medium, or culture medium, is a liquid or gel
that is designed to support the growth of microorganisms or cells under specific
incubation environment. Another 1 ml sample was also inoculated in a plate media
(100) and all the plates were incubated for 72 hours at 30°C. After three days the
plates were counted for total colony forming units under a microscope (De Donno et
al. 2010).
The microbial counts were done by counting total number of colonies in each plate.
Only the best plates (with countable colonies) were selected for the microbial counts.
The 103 dilution plate was selected for this study since the other plates contained more
growth which was difficult to count. The total colonies counted on each plate were
multiplied by 1000 to account for the 103 dilution.
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2.6 Water quality parameters
Water temperature and dissolved oxygen were measured on a daily basis using an
electronic multimeter, while the salinity was measured using a digital handheld
refractometer on a daily basis.
2.7 Statistical analyses of data
Data sets are presented as mean ± standard error. The data was subjected to one-way
and two way analysis of variance (ANOVA) using Excel software in order to
determine the significant differences among treatment means at p < 0.05.
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3 RESULTS
3.1 Feeding of rotifers
3.1.1 Effect of different feeds on population density of rotifers
The initial density of rotifers in all the silos was ≈1000 rotifers per ml. Yeast
produced the highest population density on the second day but Rocult produced the
highest density from the third to the last day (figure 5). A significant difference was
found between the feeds (p = 0.002), 1-way ANOVA. No difference was found
between Rocult and yeast (p = 0.745) but algae paste was different from both Rocult
(p = 0.003) and yeast (p = 0.014), according to Tukey Test.
Figure 5: Rotifer population densities counted on a daily basis based on different

feeds. Each data point represents the average population density of rotifers on each
day and the error bars represent the standard error of the mean.
3.1.2 Rotifer production and harvesting
The production from the rotifer batch system is ultimately contributed from the
combined population increase and the daily harvest of rotifers. During the feeding
trial an average total of 24.3 million rotifers was harvested from each Rocult tank,
which resulted in the average total production of 42.6 million rotifers/tank or 0.43
million rotifers/L/day. An average total of 22.4 million rotifers was harvested from
each yeast tank, resulting in an average total production of 0.35 million rotifers/L/day.
An average total of 12.7 million rotifers was harvested from each algae tank, resulting
in an average total production of 0.17 million rotifers/L/day. The daily production of
rotifers was thus highest on the Rocult diet but lowest on the algae paste diet (figure
6). There was a significant difference found between Rocult and algae paste (p ˂ 0.05)
but no differences were observed between Rocult and yeast, or yeast and algae paste
(p ˃ 0.05), Fisher LSD.
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Figure 6: The average daily rotifer production from the three feeds in the rotifer
feeding trial. Each column represents the average daily rotifer production from each
feed diet. The error bars indicate the standard error of the mean.
3.1.3 Egg percentage
Figure 7 shows the development of the rotifer egg percentage during the feeding trial.
Figure 7: The percentage of pregnant rotifers (egg carriers) during the feeding trial.
Each column represents the daily mean egg percentage from each feed diet each day.
The error bars indicate the standard error of the mean.
The percentage of egg carrying rotifers fed with Rocult was relatively stable
throughout the experiment but increased by 22 % during the last day of the
experiment. The percentage of egg carrying rotifers fed with yeast was high initially
but decreased during the second day and remained relatively low and stable. The egg
percentage of algae paste fed rotifers was low and variable throughout the experiment
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(figure 7). The percentage of egg carrying rotifers was less than 25 % in all groups
throughout the experiments, while a 30 % percentage is considered desirable during
the culture of rotifers (Delbos et al. 2001). A significant difference was found
between feeds (p = 0.0001), 1-way ANOVA. No difference was found between
Rocult and yeast fed rotifers (p = 0.663) but algae paste was different to both Rocult
(p = 0.000) and yeast (p = 0.001), Tukey Test.
3.1.4 Specific population growth rates
The specific population growth rates (SPGR) during the feeding trial were calculated
on a daily basis and proved highly variable between days (figure 8). The SPGR of the
Rocult group varied between 14 % and 39% per day and averaged 25.9 % during the
feeding trial. The SPGR of the yeast group was only slightly inferior and averaged
22.6 % during the feeding trial. The algae paste group had negative growth rates on
the last two days, consistent with a culture crash and thus the last day was excluded
from calculations of SPGR. Still the average SPGR of this feed group was 0 % during
the four day period, so clearly this feed was not suitable as used in this trial. The
results indicate that Rocult was the best feed in the trial, with yeast as a close second.
The daily specific growth rates during the trial are shown in figure 8 and the average
SPGR’s during the entire trial are shown in figure 9. The growth rates were based on
the daily production levels (production = harvest + population increase) and
calculated using the formula described by Rombaut et al. (1999). There was a
significant difference between rotifer specific population growth rates with respect to
the type of feed diet (p = 0.04). The Tukey Test showed no significant differences
between Rocult and yeast (p = 0.985); Rocult and algae paste (p = 0.066) or rotifers
and algae paste (p = 0.075). However, the Fisher’s Least Significant Difference test
(LSD) showed a significant difference between Rocult and algae paste (p = 0.027);
yeast and algae paste (p = 0.030) but no significant difference between Rocult and
yeast (p = 0.871).
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Figure 8: Specific population growth rates of rotifers observed on a daily basis during
the feeding trial. The data points represent the daily average growth rates of rotifers.
The last day with algae paste was excluded from the graph due to a culture crash. The
error bars indicate the standard error of the mean.
40.0%
30.0%
Rotifer growth rate (%/day)
20.0%
10.0%
0.0%
Rocult Yeast Algae paste
-10.0% Feed diets
Figure 9: Average daily growth rates of rotifers during the entire feeding trial. The last
day with algae paste was excluded from the calculation due to a culture crash. The
error bars indicate the standard error of the mean.
A significant difference was found between the three feed types (p ˂ 0.05), one-way
ANOVA. The Tukey test further showed a significant difference between Rocult and
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algae paste (p ˂ 0.05) and between yeast and algae paste (p ˂ 0.05). However, there
was no difference observed between Rocult and yeast (p ˃ 0.05).
3.1.5 Effect of water quality parameters
Oxygen levels were found to be the most important contributing environmental factor
affecting the population density of rotifers during the culture period. A decline in
oxygen levels apparently caused a massive collapse (42-92 % mortality) in culture
tanks fed with algae paste on the fifth day as shown in table 4. Salinity is also
suspected to be another factor affecting the population growth of rotifers. It is
possible, although unlikely, that the gradual reduction in salinity levels may have
affected the population growth in tanks since all the feeds were mixed with fresh
water. An accidental overdosing of ClorAm-X into the algae paste tanks might also
have contributed to inferior water quality in those culture tanks.
3.2 Disinfection of rotifers
3.2.1 Effects of disinfection on rotifer densities
The population densities in all the experimental groups were not affected by a 2 hour
disinfection with Pyceze (bronopol), while a 6 hours treatment resulted in reduced
densities (figure 10). The initial density of the control groups was only slightly
reduced after 6 hours, but the density of the treatment groups showed a huge negative
effect between 2 and 6 hours of treatment. There was no significant difference
observed after 2 hours on neither the concentrations (p ˃ 0.05). However, there was a
slight difference between the control and 25 ppm (p = 0.05), also between the control
and 50 ppm (p = 0.05) at 6 hours according to Fisher (LSD). Treatment duration had a
stronger effect on the densities as shown after 6 hour of treatment. Even the lower
concentration (25 ppm) is too high for a 6 hour duration as 50 % losses are
unacceptable.
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Figure 10: Rotifer population densities before and after treatment with Pyceze
(bronopol). The columns represents the normalized density of rotifers. The error bars
represent the standard error of the mean.
Table 4 below shows that the control tanks had a density effect of only -2 % after 2
hours and -7 % after 6 hours of treatment. The densities in both the treatment groups
were almost unaffected after 2 hours of treatment but a massive decline of -51 and -58
% was observed after 6 hours of treatment in both groups.
Table 4: Population density changes (in %) during the rotifer disinfection trial.
Treatment 2 hours 6 hours
Control (no treatment) -2 % -7 %
25 ppm bronopol 0% -51 %
50 ppm bronopol 0% -58 %
3.2.2 Effect of disinfection on total microbial counts from rotifer cultures
The microbial load on individual rotifers was calculated based on the assumption of
300,000 rotifers/ml sample or 300 rotifers/µl sample (1000-fold dilution). The
microbial loads in CFU per rotifer are presented on a linear scale to quantify the
relative treatment effects. Figure 11 shows that the effectiveness of the disinfection
treatments was both duration- and concentration dependent and the total microbial
counts showed a decreasing mean trend with both concentration and duration in all the
groups. The microbial counts were found to be 1300 CFU/rotifer after the 2 hours and
1000 CFU/rotifer after 6 hours for the controls. The 25 ppm treatment showed about
890 CFU after 2 hours of treatment and about 660 CFU after 6 hours of treatment.
The 50 ppm concentration showed microbial counts of about 710 CFU after 2 hours
of treatment and about 520 CFU after 6 hours of treatment (table 11).
The microbial counts were generally about 25 % lower after the longer treatment and
also about 25 % lower from the higher concentration. This is not a large difference in
effectiveness and in view of the lethal effects of the 6 hour treatment, it can be
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concluded that the 2 hour at 50 ppm bronopol treatment was the best protocol tested.
This protocol reduced the microbial load per rotifer by 30-40 % as compared to
controls, without any negative effects on rotifer viability.
A one-way-ANOVA showed a significant effect of treatment duration and treatment

concentration (p = 0.016 and 0.003, respectively,). There was a difference observed
when comparing both 25 ppm and 50 ppm with controls (p = 0.039 and p = 0.030,
Tukey’s Test). However, there was no statistical difference observed between the 25
ppm and 50 ppm concentrations (p = 0.079). There was no significant effect of
treatment concentrations (p = 0.355) after 6 hours of treatments.
Figure 11: Total microbial counts (CFU per rotifer) after 2 and 6 hours of treatment in
the rotifer disinfection trial. Each data point indicates the number of colonies counted
per individual rotifer. The error bars represent the standard error of the mean.
3.3 Culture of brine shrimp
3.3.1 Effect of disinfection on brine shrimp densities
Figure 12 shows the population densities of brine shrimp before and after 10 and 22
hours of treatment with different combinations of Sanocare ACE (S) and Pyceze (P).
After 10 hours of treatment with S and/or P, the densities in all treatment groups were
significantly reduced by about 20 % (p < 0.05). The final density in group S was,
however, restored to the initial level after 22 hours, suggesting that the counting after
10 hours was inaccurate. The densities in control and S group were not significantly
affected after 22 hours (p ˃ 0.05), however, the controls were significantly different
from P200 and P400 after 22 hours (p ˂ 0.05). S+P200 and S+P400 were also
significantly different from P200 and P400 after 22 hours (p ˂ 0.05). The group
notations are explained in the caption of figure 12. It is important to keep in mind that
Pyceze (P) includes 50% active bronopol.
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Figure 12: Population densities of brine shrimp disinfected with Sanocare ACE (S)
and Pyceze (P). P200 and P400 mean 200 ppm and 400 ppm Pyceze, respectively
(100 ppm and 200 ppm active bronopol, respectively). The error bars represent the
standard error of the mean.
After 22 hours of treatment the densities of groups S+P200 and S+P400 were still
reduced by about 15-20 % from the initial level but the densities of both groups P200
and P400 had shown a massive decline. The density in group P200 had been reduced
by -71 %, while group P400 had crashed almost entirely (-96 %). This shows that the
combination of Pyceze and Sanocare ACE has a much milder effect on population
densities as compared to Pyceze only, which proved lethal after 22 hours of treatment.
A one-way ANOVA revealed a significant duration-dependent effect on population
densities before and after treatment with Pyceze and Sanocare ACE (p < 0.05). Table
5 shows a review of the relative density effects from the different treatments.
Table 5: Population density changes in brine shrimp disinfected with Sanocare ACE
(S) and Pyceze (P). P200 and P400 include 100 ppm and 200 ppm active bronopol,
respectively.
Treatment 10 hours 22 hours

Control (no treatment) -2 % 1%
S 600 mg/L -23 % -2 %
S 600 mg/L + P 200 mg/L -20 % -18 %
S 600 mg/L + P 400 mg/L -20 % -14 %
P 200 mg/L -25 % -71 %
P 400 mg/L -23 % -96 %
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The survival rates are also shown in table 10 in the Appendix. Population density
changes over time are illustrated in Table 5.
3.3.2 Effect of disinfection on total microbial counts from brine shrimp cultures
The microbial load of individual brine shrimp was calculated based on the assumption
of 30,000 brine shrimp/ml sample or 30 brine shrimp/µl sample (1000-fold dilution).
The microbial loads in CFU/brine shrimp were presented on a linear scale to quantify
the relative treatment effects (figure 13).
9,000
10 hours
8,000
22 hours
7,000
6,000
CFU/artemia
5,000
4,000
3,000
2,000
1,000
0
control S S+P200 S+P400 P200 P400
Treatment groups
Figure 13: Total microbial counts (CFU per brine shrimp) in the brine shrimp
disinfected with Sanocare ACE (S) and Pyceze (P). P200 and P400 mean 200 ppm
and 400 ppm Pyceze, respectively (100 ppm and 200 ppm active bronopol,
respectively). The error bars represent the standard error of the mean.
Figure 13 shows that both the antimicrobial agents tested were highly effective and
also positively synergistic in their effect. The effectiveness was also quite duration
dependent and the 10 and 22 hour treatments produced quite different results. After 10
hours all treatments were highly effective but the S+P400 combination was extremely
effective with only 383 CFU/ brine shrimp (-95 % effect). The P200 and P400
treatments were also very effective with CFU’s of 1000-1200/ brine shrimp.
After the 22 hour treatment, the situation was, however, quite different. The CFU’s
were now higher for the previously lowest treatments (S+P400, P200, P400), most
likely due to induced mortality from the treatments. The S treatment was now very
effective (1150 CFU/ brine shrimp) but the S+P200 treatment was extremely effective
with only 433 CFU/ brine shrimp. There was therefore a clear synergistic effect of
both the chemicals used and it can be concluded that the S+P200 for 22 hour
treatment was the best protocol tested. This protocol reduced the microbial load on the
brine shrimp by 95 % as compared to initial controls, without only moderate effects
on brine shrimp viability.
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No significant differences were observed after 10 hours of treatment (p > 0.05, one-
way-ANOVA). A one-way-ANOVA did not show significant differences between
treatments after 22 hours (p = 0.138). However, Fisher LSD showed significant
differences between S+P200 and P400 (p ˂ 0.05) and also between S+P200 and
S+P400 (p ˂ 0.05).
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4 DISCUSSION
Despite the improved technology in the batch culture of rotifers (Dhert et al. 2001 and
Yoshimura et al. 2003), poor performance is still a common problem in aquaculture
hatcheries. Many factors have been linked to rotifer growth performance, such as
nutrition and different microorganisms. The bacterial communities associated with
rotifers might be one of the most important factors affecting the culture of rotifers.
In the feeding trial, rotifers fed with Rocult showed the best daily production, best
fertility and best growth rates as compared to rotifers fed with yeast and algae paste.
Therefore Rocult was considered to be the best feed diet for rotifers. The Rocult diet
may have performed best because it is made up of live yeast that is rich in proteins
and green algae that provide necessary vitamins to rotifers. Yeast, however,
performed almost as well as Rocult and much better than the algae paste. The density
decline on the second day with yeast may have been caused by overfeeding. The poor
performance of rotifers in tanks fed with algae paste may also be the result of the
amount of feed provided. Higher food quantities may result in higher mortalities of
rotifers while insufficient food may result in poor growth rates as a result of
competition between live feed and protozoans. The population densities of rotifers are
known to be affected by the amount of food given to the animals on each day
(Theilacker and McMaster 1971). It is also possible that the poor performance from
the algae paste fed rotifers may have been caused by an overdosing of ClorAm-X
added while mixing the feeds. There was also a massive decline in oxygen levels on
the last day, which was found to be the most contributing factor to high mortalities in
the algae paste tanks.
Rotifers are mostly cultured in batch culture systems that generate highly variable
conditions, which can influence growth performance and the composition of microbial
communities (Rombaut et al. 2001). High rotifer densities demand high feeding
levels and produce high concentrations of waste products and thus creating high loads
of organic material that is utilized as a feed source by bacteria. These intensive rearing
conditions allow heterotrophic bacteria to grow fast in culture tanks (Skjermo et al.
1997, Verschuere et al. 1997, Skjermo and Vadstein 1999). Bacteria are always
associated with mass production of rotifers and may cause unexpected mortalities or
suppressed growth of rotifers. Moreover, since they are used as the first food of
larvae, rotifers are often suspected as vectors of potential harmful bacteria to the
cultured animals (Dhert et al. 2001).
The density of egg carrying rotifers in the feeding trial was less than 30 % throughout
the experiment, which is a desirable fertility minimum during the culture of rotifers.
The percentage of rotifers with eggs is a useful indicator of the health of rotifer
population. A rotifer population with ≥30 % egg percentage is consistent with a
healthy culture, while a drop in this percentage to 15-20 % is still acceptable, but the
growth of the culture will be reduced. A decrease to <15 % often indicates that
conditions in the culture tank are sub-optimal, likely due to feeding rate or low water
quality (Delbos et al. 2001). Low egg percentages were indeed observed and
associated with the high mortalities in tanks fed with algae paste.
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The disinfection of rotifers with Pyceze (50 % bronopol) proved very effective in
lowering microbial counts but the 6 hour treatment caused high mortalities of the
rotifers. A disinfection protocol including 0.1 g/L Pyceze (50 ppm bronopol) for 2
hours was highly effective without killing the rotifers and was considered to be the
best treatment protocol for rotifers in the study. A lower bronopol concentration was
not tested in this study but it might have been more effective in the long term
treatment. The producer, Novartis Animal Health, has also tested the effect of
bronopol for rotifer disinfection and found out that the disinfection efficiency
increases with the increase in chemical dosages, as well as the rotifer mortalities
(Novartis Animal Health 2014).
The brine shrimp disinfection trials showed that the initial densities were mostly
maintained in the control and Sanocare ACE groups. Pyceze, however, cannot be used
as a sole treatment agent for brine shrimps as proven by the study. It has been
observed that bronopol has adverse and potentially lethal effects on brine shrimps,
depending on duration and concentration of treatment. Sanocare ACE is needed for
water quality and provides good protection from the harmful effects of bronopol.
Sanocare ACE also had a significant antimicrobial effect but this effect was greatly
enhanced when combined with bronopol. A combination of Sanocare ACE (0.6 g/L)
and Pyceze (0.2 g/L, 100 ppm bronopol) for 22 hours was very effective in reducing
microbial counts, with a moderate negative density effect (-15 to -20 %) and was
therefore regarded the best treatment for brine shrimp disinfection. National Lobster
Hatchery (2014) had similar results as in the present study, with 14-22 % mortalities
and 150 mg/L as the most effective treatment (87 % reduction in CFU as compared
with 95 % in the present study).
All the specific objectives defined for this study were realised with potentially
interesting results. The study has successfully tested disinfection protocols that were
highly effective with regards to survival and feed quality of rotifers and brine shrimp.
It is both easy and essential to disinfect live feed to eliminate any pathogens which
could have undesirable effects on the culture of marine fish larvae. The results from
this study may prove very useful in improving marine larvae culture in South Africa
as it has been suspected that the mortalities observed during the larval state of dusky
kob (Argyrosomus japonicus) might be the result of high microbial loads in live feed
cultures. The results from this study may be regarded to improve the management
practices of aquaculture in South Africa in accordance to the objectives specified in
the National Aquaculture Strategic Framework for 2013.
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ACKNOWLEDGEMENTS
The research funding and formatting were provided by United Nations University
(UNU-FTP), Reykjavík, Iceland.
A special thanks to my supervisor Agnar Steinarsson and his colleagues for assistance
during the experiment. The experiments were done at Marine Research Institute
(MRI) facility, Grindavík, Iceland.
The microbial analyses were done at Matís: Food and Biotech Research and
Development, Iceland.
The research idea and editing were provided by Mr. Esau Mbokane and Dr. Chris
Fouche, Department of Agriculture Forestry and Fisheries (DAFF), Cape Town,
South Africa.
Thanks to the aquaculture directors (Mr. Belemane Semoli, Mr. Asanda Njobeni, and
Ms. Khumo Morake) for the nomination to attend the UNU-FTP course, DAFF, Cape
Town, South Africa.
Mr. Tumi Tómasson, Mr. Þór Ásgeirsson, Ms. Sigríður Kr. Ingvarsdóttir and Mrs.
Mary Frances Davidson for their support and encouragement throughout the
programme, Reykjavík, Iceland.
Mr Stanley Onyango, Mr Yosvani Crus and Mrs Dang Thi Thu Huong for their
assistance with statistical analysis, UNU-FTP 2013 fellows
Thanks to my family for their understanding and support
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Brine Shrimp Direct (2014), January, 19. Retrieved from Selco:

http://www.brineshrimpdirect.com/SELCO-c47.html.
Bryce, D.M; Croshaw, B; Hall, J.E; Holland, V.R; and Lessel, B. (1978). The activity
and safety of the antimicrobial agent Bronopol (2-brono-2nitropropan-1, 3-dio).
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34
Day Silo Number Temperature (° C) Oxygen (ppm) Salinity (ppt) Wv (L) D (NEC) D(EC) Total D/ml Total D (M) Egg % AVR D(ml) IN D (ml) AVR D (M) SGR (rotifers/day) Mean Egg %
1 1A 26.8 20.2 32.5 25 952 192 1144 28600000 17
1 1B 26.8 17.7 32.6 25 860 232 1092 27300000 21
1 1C 26.8 21.0 32.6 25 772 164 1006 25150000 16 1081 6,99 27016667 0 18
Mzimba
35
Table 6: Water quality parameters and rotifer population densities during the rotifer
Table 7: Water quality parameters recorded during the rotifer disinfection experiment.
1 2A 26.7 19.6 32.6 25 1056 254 1092 27300000 23
1 2B 26.6 24.9 32.6 25 1204 284 1134 28350000 25
1 2C 26.4 23.6 32.6 25 1112 204 1028 25700000 20 1085 6,99 27116667 0 23
1 3A 26.3 18.5 32.4 25 684 120 1092 27300000 11
1 3B 26.3 17.0 32.5 25 916 152 1068 26700000 14
1 3C 26.3 19.2 32.5 25 816 152 1281 32025000 12 1147 7,04 28675000 0 12
2 1A 26.7 20.5 31.1 25 1106 235 1341 33524000 18
2 1B 26.8 19.8 32.0 25 1134 244 1378 34452000 18
2 1C 26.6 14.5 31.9 25 1206 274 1480 37004000 19 1400 7,24 34993333 13 18
2 2A 26.4 16.8 32.0 25 1382 231 1513 37816000 15
2 2B 26.7 16.8 31.8 25 1074 225 1299 32480000 17
2 2C 26.2 16.7 31.9 25 1454 249 1703 42572000 15 1505 7,32 37622667 16 16
2 3A 25.5 20.7 32.3 25 994 166 1160 29000000 14
2 3B 25.4 17.3 32.0 25 848 164 1012 25288000 16
2 3C 25.2 19.9 32.2 25 1334 269 1603 40078000 17 1258 7,14 31455333 5 16
3 1A 27.7 19.6 30.9 25 982 174 1146 28652000 15
3 1B 28.0 23.0 30.9 25 1220 341 1661 41528000 21
3 1C 27.6 18.3 30.8 25 1122 168 1290 32248000 13 1366 7,22 34142667 12 16
3 2A 27.2 11.6 31.3 25 836 92 928 23200000 10
3 2B 27.2 19.0 31.6 25 1008 189 1197 29928000 16
3 2C 26.9 16.0 30.9 25 1154 247 1401 35032000 18 1175 7,07 29386667 4 14
3 3A 26.5 10.2 31.3 25 776 41 817 20416000 5
APPENDIX: TABLES AND FIGURES
3 3B 26.5 11.6 31.2 25 1024 159 1183 29580000 13

3 3C 26.4 14.5 31.2 25 1170 111 1281 32016000 9 1093 7,00 27337333 -2 9
4 1A 27.2 16.9 30.7 25 1364 316 1680 41992000 19

4 1B 27.4 22.4 30.7 25 914 153 1067 26680000 14
4 1C 27.2 19.2 30.8 25 1346 352 1698 42456000 21 1482 7,30 37042667 16 18
4 2A 26.9 15.0 30.9 25 756 149 905 22620000 16
4 2B 27.0 15.8 30.9 25 1058 236 1292 32306000 18
4 2C 25.8 18.3 30.5 25 1520 280 1800 45008000 16 1332 7,19 33311333 10 17
4 3A 26.2 15.7 31.2 25 326 91 617 15428000 15
4 3B 26.3 15.1 29.2 25 780 64 844 21112000 8
4 3C 26.2 15.2 30.7 25 872 135 1007 25172000 13 823 6,71 20570667 -17 12
5 1A 27.7 16.4 30.1 25 1584 374 1958 48952000 19
5 1B 28.1 21.6 30.0 25 1178 335 1513 37816000 22
feeding..trial.
5 1C 27.6 15.3 29.9 25 1462 515 1977 49416000 26 1816 7,50 45394667 26 22
5 2A 27.1 16.8 30.4 25 1184 343 1527 38164000 22
5 2B 27.3 17.7 30.2 25 1324 230 1554 38860000 15
5 2C 27.1 11.3 29.5 25 1444 231 1675 41876000 14 1585 7,37 39633333 19 17
5 3A 27.3 0.01* 29.8 25 47* 0* 51 1276000 0
5 3B 26.4 0.01* 29.6 25 350* 35* 385 9628000 11
5 3C 26.5 0.86* 29.8 25 1028 192 1220 30508000 16 552 6,31 13804000 -37 9
Mzimba
Tank No Temperature Oxygen (ppm) Salinity (ppt) V (L)

1A: 2hours 26.3 20.3 32.0 6
1B: 2 hours 26.0 23.9 32.1 6
1A: 6 hours 26.2 20.3 32.1 6
1B: 6 hours 26.3 23.8 32.1 6
2A: 2 hours 26.1 25.2 32.0 6
2B: 2 hours 25.9 22.3 32.0 6
2A: 6 hours 26.2 25.3 32.0 6
2B: 6 hours 25.8 22.1 32.1 6
3A: 2 hours 26.2 26.5 32.0 6
3B: 2 hours 26.1 25.8 32.0 6
3A: 6 hours 26.0 26.3 32.0 6
3B: 6 hours 26.2 25.7 32.0 6
Key: V = Water volume
Table 8: Population densities after two- and six hour treatment of rotifers during the
disinfection trial.
Duration
(hours) ID FD ID FD ID FD SE SE
2 2000 1940 2000 1940
2 1510 1504 2000 1992 2000 1966 0 26
6 2184 2040 2000 1868
6 1976 1830 2000 1852 2000 1860 0 8
2 1891 1888 2000 1997
2 2108 2104 2000 1996 2000 1997 0 0
6 1680 796 2000 948
6 1968 1012 2000 1028 2000 988 0 40
2 1975 1972 2000 1997
2 1730 1724 2000 1993 2000 1995 0 2
6 1752 720 2000 822
6 1472 620 2000 842 2000 832 0 10
1853,8 1512,5 2000 1606,3
Key:
ID = Initial density (normalised).
FD = Final density (normalised).
SE = Standard error.
36
Mzimba
Table 9: Water quality parameters recorded during the disinfection trial of brine
shrimps.
Tank no Temperature Oxygen (ppm) Salinity (ppt) V (L)
1A: 10 hours 26.2 21.2 31.8 6
1B: 10 hours 26.0 26.6 31.9 6
1A: 22 hours 26.1 21.1 31.7 6
1B: 22 hours 26.1 26.4 31.8 6
2A: 10 hours 26.22 18.8 31.9 6
2B: 10 hours 25.9 24.7 31.9 6
2A: 22 hours 26.9 18.4 31.6 6
2B: 22 hours 25.8 25.3 31.8 6
3A: 10 hours 26.2 23.3 31.9 6
3B: 10 hours 26.0 23.6 31.9 6
3A: 22 hours 26.2 23.1 31.8 6
3B: 22 hours 26.1 23.0 31.7 6
4A: 10 hours 26.2 22.6 31.9 6
4B: 10 hours 26.0 21.5 31.9 6
4A: 22 hours 26.1 22.6 31.9 6
4B: 22 hours 26.1 22.5 31.8 6
5A: 10 hours 26.2 19.9 31.9 6
5B: 10 hours 26.0 21.1 31.9 6
5A: 22 hours 26.2 21.0 31.9 6
5B: 22 hours 26.0 19.7 31.9 6
6A: 10 hours 26.2 19.7 31.9 6
6B: 10 hours 26.0 18.8 31.9 6
6A: 22 hours 26.1 19.7 31.9 6
6B: 22 hours 26.0 18.5 31.9 6
37
Mzimba
Table 10: Population densities counted before and after disinfection of brine shrimps
during the disinfection trial. The chemicals uded were Sanocare ACE (S) and Pyceze
(P). P200 and P400 mean 200 ppm and 400 ppm Pyceze, respectively.
Duration
Treatment Tank Repl. (hours) ID FD ID FD SE SE
0 1 A 10 300 308
0 1 B 10 300 278 300 293 0 15
0 1 A 22 300 296
0 1 B 22 300 308 300 302 0 6
S 2 A 10 300 226
S 2 B 10 300 239 300 233 0 7
S 2 A 22 300 290
S 2 B 22 300 298 300 294 0 4
S+P200 3 A 10 300 211
S+P200 3 B 10 300 272 300 242 0 31
S+P200 3 A 22 300 246
S+P200 3 B 22 300 246 300 246 0 0
S+P400 4 A 10 300 240
S+P400 4 B 10 300 238 300 239 0 1
S+P400 4 A 22 300 280
S+P400 4 B 22 300 238 300 259 0 21
P200 5 A 10 300 252
P200 5 B 10 300 200 300 226 0 26
P200 5 A 22 300 100
P200 5 B 22 300 74 300 87 0 13
P400 6 A 10 300 242
P400 6 B 10 300 218 300 230 0 12
P400 6 A 22 300 10
P400 6 B 22 300 12 300 11 0 1
Key:
ID = Initial density.
FD = Final density.
SE = Standard error.
38
Mzimba
Table 11: Total CFU counted after disinfection of rotifers with Pyceze (50%
bronopol) for 2 and 6 hous.
3
Tank No Time (Hours) Treatment (Pyceze) CFU count 10 CFU Rotifers/µl Rotifers/µlCFU/rotifer CFU/rotifer SD SE
1A 2 0 364 364.000 300 300 1.213 1.335 172 122
1B 2 0 437 437.000 300 1.457
2A 2 50 271 271.000 300 300 903 890 19 13
2B 2 50 263 263.000 300 877
3A 2 100 245 245.000 300 300 817 713 146 103
3B 2 100 183 183.000 300 610
1A 6 0 344 344.000 300 300 1.147 998 210 148
1B 6 0 255 255.000 300 850
2A 6 50 217 217.000 300 300 723 657 94 67
2B 6 50 177 177.000 300 590
3A 6 100 150 150.000 300 300 500 518 26 18
3B 6 100 161 161.000 300 537
Key: CFU=Colony forming units.
39
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Table 12: Total CFU counted after 10 and 22 hours of disinfection of brine shrimp
with Pyceze (50% bronopol) and Sanocare ACE.
3
Tank No Time Treatment CFU counts 10 CFU Artemia/µl Artemia/µl CFU/artemia CFU/artemia SD SE
1A 10 0 232 232.000 30 30 7.733 7.433 424 300
1B 10 0 214 214.000 30 7.133
2A 10 600 S 166 166.000 30 30 5.533 3.467 2.923 2.067
2B 10 600 S 42 42.000 30 1.400
3A 10 200 P+600 S 143 143.000 30 30 4.767 2.483 3.229 2.283
3B 10 200 P+600 S 6 6.000 30 200
4A 10 400 P+600 S 9 9.000 30 30 300 383 118 83
4B 10 400 P+600 S 14 14.000 30 467
5A 10 200 P 36 36.000 30 30 1.200 1.017 259 183
5B 10 200 P 25 25.000 30 833
6A 10 400 P 53 53.000 30 30 1.767 1.167 849 600
6B 10 400 P 17 17.000 30 567
1A 22 0 71 71.000 30 30 2.367 2.217 212 150
1B 22 0 62 62.000 30 2.067
2A 22 600 S 27 27.000 30 30 900 1.150 354 250
2B 22 600 S 42 42.000 30 1.400
3A 22 200 P+600 S 14 14.000 30 30 467 433 47 33
3B 22 200 P+600 S 12 12.000 30 400
4A 22 400 P+600 S 105 105.000 30 30 3.500 3.250 354 250
4B 22 400 P+600 S 90 90.000 30 3.000
5A 22 200 P 29 29.000 30 30 967 1.767 1.131 800
5B 22 200 P 77 77.000 30 2.567
6A 22 400 P 113 113.000 30 30 3.767 3.583 259 183
6B 22 400 P 102 102.000 30 3.400
Key:
B = Bronopol
S = Sanocare ACE
Figure 14: An s-shaped tray used for rotifer- and brine shrimp density determination.
40
Mzimba
Figure 15: Sponge filters used for trapping suspended particles in the rotifer feeding
trial.
Figure 16: Appearance of rotifers under a 40X magnification using a light microscope
during population density counting.
Figure 17: Appearance of brine shrimp under a 40 X magnification in a light

microscope used during population density counting.
41
Mzimba
Figure 18: Colonies observed from plates during microbial analyses of rotifers.
Figure 19: Colonies and fungal growth observed from plates during the microbial
analysis of brine shrimp.
42

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unuftp.

is Final Project 2013

THE EFFECT OF DISINFECTION ON SURVIVAL AND FEED

Lucia Matlale Mzimba

This paper should be cited as:

LIST OF TABLES ...................................................................................................... 3

Figure 17: Appearance of brine shrimp under a 40 X magnification in a light

1.1 Background of marine larval culture in South Africa

South Africa is known to be one of the leading countries in abalone production.

The Department of Agriculture, Forestry and Fisheries: Directorate: Aquaculture

1.2 Rotifer culture

1.2.1 The culture conditions for rotifers

1.2.2 Feeding and enrichment of rotifers

1.2.3 Batch culture of rotifers

1.2.4 Rotifer culture contamination

1.3 Brine shrimp culture

1.3.1 The culture methods for brine shrimp

1.3.2 The culture conditions for brine shrimp

1.3.3 Feeding or enrichment of brine shrimp

1.3.4 Brine shrimp culture contaminations

1.4 Use of antimicrobial agents in live feed culture

1.5 The objectives of the study

2 MATERIALS AND METHODS

2.1 Location and dates of the experiments

2.2 Density determination

2.3 Rotifer trials

2.3.1 Feeding trial

The feeds were used in different quantities as shown in table 1 below.

2.3.2 Rotifer disinfection trial

2.4 Brine shrimp trials

2.4.1 Brine shrimp disinfection trial

2.5 Microbial analysis

2.6 Water quality parameters

2.7 Statistical analyses of data

3.1 Feeding of rotifers

3.1.1 Effect of different feeds on population density of rotifers

Figure 5: Rotifer population densities counted on a daily basis based on different

3.1.2 Rotifer production and harvesting

3.1.3 Egg percentage

3.1.4 Specific population growth rates

-10.0% Feed diets

3.1.5 Effect of water quality parameters

3.2 Disinfection of rotifers

3.2.1 Effects of disinfection on rotifer densities

3.2.2 Effect of disinfection on total microbial counts from rotifer cultures

A one-way-ANOVA showed a significant effect of treatment duration and treatment

3.3 Culture of brine shrimp

3.3.1 Effect of disinfection on brine shrimp densities

Treatment 10 hours 22 hours

Brine Shrimp Direct (2014), January, 19. Retrieved from Selco:

Collette. P. D, Vine. N. G, and Kaiser. H. (2008). Determination of the optimal water

De Donno, A; Lugoli, F; Bagordo,F; Vilella, S; Campa, A; Grassi,T; Guido,M.

Department of Agriculture, Forestry and Fisheries. (2012). Aquaculture Year Book

Department of Agriculture, Forestry and Fisheries. (2013). National Aquaculture

Dhert, P., Rombaut, G., Suantika, G. and Sorgeloos, P. (2001). Advancement of

Hecht, T. (2000). Considerations on African aquaculture. World Aquaculture, 12-15.

Hirayama, K. (1987). A consideration of why mass culture of the rotifer Brachionus

Inve Aquaculture. (2014, February 1). Retrieved from http://www.inve.com/INVE-