Phyto and algal therapy use in aquaculture

Mr. G. Negroni – April 2018

Introduction
With the lack of future development of extractive world fishing, which sees the largest stocks at
the limit of exploitation or overfished, the increase in the world population that wants to feed
on fish products and the health recommendations that rightly insist on the consumption of fish
products, one of the recommended solutions concerns the development of an aquaculture
industry. Aquaculture could, and already partially, provide the supply of noble proteins to meet
the great growth in world fish demand; the aquaculture sector is growing strongly. Despite
these excellent market prospects and world consumption, there are problems especially in
feed, a key component for fish breeding. One of the present nodes is the excessive use of
chemotherapeutics in farms, which we will deal with in this paper; both against diseases and
against other harmful organisms in structures and directly on animals.
Without forgetting the use of chemical disinfectants, they can leave several unwanted residues
and contaminant in the aquaculture products, in the water and on the treated surfaces.
There are numerous products that can solve the pathologies and disinfection problems in use,
but it is important to put the emphasis on "green" products that we can define as
environmentally and consumer friendly. In this paper it will be described the most common
products originating from land (plants) and ocean (algae).

Technical problem
The use of natural substances from land and sea, used directly in human being and for the
breeding of animal and plant species, have been used for a long time. Here we will treat only
the one for breeding aquaculture animal. The above have obtained a fair recognition in recent
times, compared to the use of synthetic chemotherapy’s, disinfectants, adjuvants and chemical
additives. The synthetic chemistry can leave dangerous residues for the health of animals and
humans. Without forgetting, the dangerous residues and contaminant that remains in the
environment and in animal tissues. The latter (so called chemical products) have certainly
launched the so-called "green revolution" and allowed to increase exponentially the food
production to feed the growing world population, but have also shown their limits, especially if
used massively and indiscriminately without respect for good production practices. Often the
productive purposes of the breeding farms and the mere economic objective do not make the
best use of the great technological capacities that currently allow controlling the main
pathologies in the animal farms; the aquaculture does not fail this principle.
Increasingly, the feed formulators present new components with high protein concentration
that allow them to replace others that are more expensive, scarce and unsustainable (e.g. fish
meal). The above for aquaculture farmed species with different nutritional requirements (tuna,
seriola, frogs, flatfish, etc.).
We also have new technologies and the public's request to use "sustainable" systems,
consistent with animal welfare and "biological", validated internationally. All these new
requests can be met by modern aquaculture technologies with the new feed possibilities that
greatly influence the aquaculture cycle and represent the biggest expense for the producers.
Researching, testing, validating and producing new green components and additives is one of
the great challenges of the modern animal feed industry.

1
Aquaculture and aquaculture related, for its younger age, tries to use what has been discovered
by the breeding industry of terrestrial animals. The use of sustainable feed components (plants
and algae) is a young industry with large commercial space to be explored.
The application of plant compounds to foods for aquatic species has a very innovative value
also due to the presence of completely new applications (for example the anti-inflammatory
functions). For aqua feed, added with natural products, we have some applications that
certainly attract great interest such as: hygiene, pathologies control, gut health, mycotoxins
control, phosphate digestion, attractiveness of feeds in the aquatic environment and others.
Increasing health and reproductive system also has a great interest in aquatic animals with the
administration of additives in the feed or components containing these compounds. More than
the additive it is interesting to develop a full “green feed” developed in a sustainable way for a
specific “bio market” consumers.

Because of the different mediums in which aquatic animals are found compared to terrestrial
animals, appropriate considerations must be made before studying and administering natural
(phytogenic) additives and “green feed” in aquaculture farms, such as:
- will the chosen plant substances be stable in aquatic environments, will they spread rapidly in
the aquatic medium from the pellet casing, or will they remain stable for the consumption of
the species in charge?
- The vegetable substances have a window of temperature that inhibits or increases its
effectiveness and efficiency, this temperature is found in the waters of the breeding of that
species?
- Can the density or structure of the plant substance be affected by both the production
technology and the period of stay in the water?
- Are the active plant components stable through the production process (transport, storage,
extrusion / expansion, pelletizing, storage, transport, consumption?
- Does it positively affect the intake of the food?
Following these investigations we can think of using the new green plant/algal components in
aquatic animals.
In any case, the animal feed industry is going towards an enormous reduction in the use of
antibiotic-based grow promoters to prevent the emergence of antibiotic resistance in the
consumers. OMS (Organisation Mondial de la Santèe) has already launched alarms in this sense.
Moreover the feed impact on the world ecosystem need to be considered and the
public will be more and more interested to eat aquaculture products growth with sustainable
and green feed.

Farm disease
The aquaculture sector is not immune to problems as evidenced by the recent epidemics of
EMS (Early Mortality Syndrome) in shrimp farms, parasite in salmon farms (sea lice), viral
diseases of salmon (ISA / Infectious Salmon Anaemia) and other numerous diseases that create
problems in the fish farming industry. New dangerous pathologies are coming periodically
risking paralyzing the new aquaculture industry. The great epidemics are also the consequence
of the intensification of breeding systems, high density, the poor development of the genetics
of fish species, the poor respect for the welfare of fish species bred and the poor compliance
with best practices in the farm. It is well known that large concentrations of animals create the
conditions for the development of epidemics.

2
In fact, at the time of intensification of livestock farms, there is a strong response in terms of
the spread of diseases and pathogens that become an integral part of the farms and a
formidable obstacle to the development of the animal industry; aquaculture is no exception to
the other zoo technical sectors. In fact, the resistance of bacterial agents to antibiotics available
is one of the great problems of aquaculture. Already in the 1990s the problem of antibiotic
resistance and the low number of antibiotics available (ASM 1994) was highlighted as one of
the biggest obstacles to food production worldwide.
Almost every sector of the intensive aquaculture industry employs chemotherapeutics like
antibiotics and other chemical products that must be used with a lot of precautions and well-
defined suspension times. Many active ingredients are prohibited such as fluoroquinolone,
nitro furans, chloramphenicol and the European Community food safety package requires strict
controls by the competent authorities of the member countries, including imports from third
countries. The border inspection posts of the EU, USA and Japan periodically reject imports of
fish and crustaceans with levels of antibiotics and other substances higher than the maximum
permitted by the hygiene package. For the aquaculture products there were some emergencies
from Bangladesh and now the Vietnam and Indian aquaculture products are on the spot for
residues and contaminant controls in the EU border inspection posts.
In aquaculture, in case of pathological events, there are currently no other solutions to
antibiotics and other chemotherapeutic substances, especially for intensive and hyper-intensive
breeding. In fact the ever-increasing spaces necessary for breeding are no longer available due
to strong competition with other activities; especially in coastal areas oblige high farming
concentration of organisms.
In recent decades, the aquaculture industry has been hit by numerous outbreaks of dangerous
diseases that have brought many sectors to their knees, just remember: in shrimp-producing
countries (Thailand, Vietnam, Ecuador), in salmon farming Chile and Norway with losses of
hundreds of millions of euros from companies in the sector and numerous jobs. Without
forgetting the lack of the security that these productions to the consumers.

The inappropriate use of antibiotics in the aquaculture supply chain

Unfortunately, there is often a redundant use of antibiotics and synthetic chemotherapeutic
substances in both human and veterinary medicine and plant defence. Unfortunately even here
aquaculture follows the trend of other sectors. In fact, the massive and periodical use of
antibiotics, especially those with a wide spectrum, goes hand in hand with resistance to the
same by pathogens. The situation is currently well perceived worldwide for both human
medicine and animal breeding.
But the situation, of resistance to antibiotic agents, is not new and should not be surprising
since there are more resistance to antibiotics and chemotherapeutics in general; on the other
hand, the means to fight pathogens are decreasingi Diseases caused by pathogens are a
constant presence in intensive aquaculture, which is increasingly being applied to our days due
to the aforementioned reasons for lack of space, water pollution and the need for large
quantities of fish products that can be marketed in short periods.
A mortality of 8-25% is considered "normal" in the grow out phase of many species of fish and
crustaceans currently bred, while in the larval stages in the hatcheries (sea bass, sea bream) are
estimated mortality of 15-40% with the most modern technical means availableii.
Mortality is often due and a combination of factors among which we like to mention the

i The responsable use of antibiotics in aquaculture, FAO 2005.

ii Negroni personal evaluations

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environmental ones, the pathological ones, and the poor development of the immune system
(innate in many species). In fact this situation is common to the juvenile phases of numerous
marine and freshwater species. The use of antibiotics, despite numerous prohibitions, is one of
the easiest solutions used to solve the aforementioned problems, both as growth promoters
and specifically against pathogens.
The principles of herbs, algae and plants could replace the present chemotherapeutic agents as
agents against pathogens, promoters of growth and use in increasing feed productivity as in
biosafety. The above needs appropriate studies to better define the characteristics and effects
in farms. Until now little has been done, the industry has decided to merge research resources
on active ingredients other than natural ones. It is recalled that the development, testing and
marketing of active medical and veterinary principles require a costly series of tests to
demonstrate the reliability and lack of hazards in industrial and massive use.

Other natural therapies

Naturopathy (called phytogenic, herbal medicine or phytotherapy) is the use of natural plant
and herbal extract has been known for thousands of years, was developed in the absence of
other systems of animal care. In thousands of years, it was developed with several large
number of tests done on the sick repeatedly, is actually recognized universally, even if it does
not have much scientific evidence, but more often practices.
The main places where this ancient knowledge is used to this day are: Asia, Southeast Asia and
other countries of South and Central America. In other countries, the knowledge, although
present are no longer in use, supplanted by modern medicine that uses other principles
although often "copied" by nature (example aspirin with acetylsalicylic acid "copied" from the
plant kingdom and produced for more than 200 years and other substances). Despite this there
are strong currents of opinion that make use of naturopathy and medicinal herbs in all
countries for both men and animals.
There are many known medicinal herbs or the combination of more than one plant principle
that produce beneficial effects on the health and well-being of aquatic animals, with antifungal
and antibacterial properties and support for digestive, hormonal and immune system functions.
The medicinal herb market has a strong growth and many billions of annual turnovers. Usually a
phototherapeutic principle contains various active ingredients, which best contrast the
pathogenic agents with single or synergistic antimicrobial activity.
The strategies for the prophylaxis and control of pathogens, which attack the activities of
aquaculture farms, include the improvement of environmental conditions (mainly the quality of
water), the production and the growing of specific larval phases (of aquaculture farmed
organism) controlled without pathogens (for example larvae SPF in the prawns), the stimulation
of the resistance of the immune system against pathogens with specific immune stimulants
such as glucans and specific vaccinations with inactivated pathogens.
It has been found that many plant/algal-derived components have a non-specific immuno-
stimulating effect in animals; some have been evaluated in trials with farmed fish and
crustaceans. Numerous herbs and plants have been used as home remedies in many cultures
around the world for thousands of years to treat humans, farmed animals and plants. When
modern medicine was not available and when they were available, normally seasonally. In
addition, the numerous natural active ingredients are present in vegetables in different doses
compared to the season and the organs where they are found (leaves, stems, roots, fruits and
flowers).

4
Some of these remedies have strong characteristics against pathological events against diseases
with antibacterial, antiviral and antifungal effects, so they are also dangerous if used
inappropriately (e.g. rotenone which can be highly toxic in massive doses). Other properties are
recognized for principles such as anti-stress, growth promoters, appetizers, tonics and immune
stimulants.
Other favourable features and benefits of the active ingredients contained in plants and algae,
as an alternative to the industrial principles currently used, are: low toxicity with a greater
"environmentally friendly" feature, biodegradability and biocompatibility, low toxicity that
depends on concentrations. Up to now, no immunological plant pathogens have been
discovered, in fact the plants used usually have a combination of several active ingredients
from which pathogens struggle to defend themselves. In fact the natural remedies do not
stimulate the microorganism resistance, on the contrary is one of the problems of chemicals. It
is typical the rotation of disinfectant in the food safety system to avoid the development of
microorganism resistances.
Despite the wide global knowledge about the benefits of medicinal herbs and algae, currently
few natural plant/algal principles are commercially available for industrial aquaculture.

Phitotherapy utilisation in aquaculture

The strong anti-pathogenic action characteristics of some plant and algal species are well
known and documented also in the aquaculture sector. The action of Flavonoids, Phenols,
Polysaccharides, Terpenes, Proteoglycans, and Flavonoids can considerably prevent and treat
disease and all type of pathogen in aquaculture production (See Table 1-4-6-8) as bacteria’s,
virus and parasites.
Vibrio presence in P. Monodon could be reduced by a third when herbs (S. Triblobatum, A.
Paniculata and P. Coryfolia) are given as feed to Artemia iii during the larval phases of P
Monodon as feed.
Some Chinese researchers have reported remarkable antimicrobial activities from extracts from
5 herbaceous species (Stellaria aquatica, Impatiens biflora, Oenothera biennis, Artemisia
vulgaris, Linocera japonica) against 13 species of bacteria and two viral pathogens of fish
speciesiv. Aeromonas salmonicida and Edwardsiella ictaluri were the major bacteria that are
sensitive to herbal extracts; S. Aquatica was the most effective extract in terms of the higher
number of inhibited bacteria and the degree of inhibition. L. Japonica showed the highest
degree of inhibition against IPN (Infectious Pancreatic Necrosis) and IHN (Infectious
Hematopoietic Necrosis) viruses while A. Vulgaris and S. Aquatica inhibited only the IHN virus.
(Table 4)
It is considered worthy of mention of the evidence of the use of cashew extract (Western
cashew), which contains numerous phenolic compounds contained in the fruit of the cashew.
They have a strong inhibitory power for many bacteria; at high doses it creates strong irritation
on the skin. Cashew is produced in many tropical countries in industrial quantities. Following
the extraction of the board we have a residue that looks like a liquid resin (called LCC - Liquid of
Castanha do Caju), is caustic and dark brown with a characteristic smell. The LCC consists
mainly of: anacardic acid, cardol, cardanol and other substances. Anacardic acid is well known
for its antimicrobial principles. In vitro tests were performed with Staphylococcus sp with
strong inhibitory results in the development of the microbe. Industrially it can be used as:
insecticide, fungicide, germicide, anti-oxidant and for many other activities. The use in soap as a

iii Citrasu et al 2002 and 2009

iv Infectious Disease in Aquaculture: Prevention and Control, edited by B Austin

5
natural antibacterial agent is typical. Because of its great availability and low price, it is not a
good candidate for use in the aquaculture sector.
Numerous products from the vegetable kingdom have shown great antiviral effects against
diseases of crustaceans and fish. Numerous species of Indian herbs and plants such as A.
mamelos, C. dactylon, L. camara, M. charantia and P. Amarus have shown strong antiviral
activity towards White Spot Syndrome Virus (WSSV) when extractedv. Numerous studies have
been published demonstrating the antibacterial and antiviral effects of plant extracts with
different species (See Table 2-7) of fish and crustaceans. .
Plant extracts are also known for their antifungal and antiparasitic properties (See Table 3).
The plant extracts induce the lysis of the fungal cell, altering its permeability; they influence the
metabolism of RNA and the protein sitesis bringing it to death (Citarasu 2009), See Table 3.
Another interesting anti-stress factor for fish, particularly for transport, high stress and as a
growth promoter, is extracted from Yucca ( family Agavaceae) , also used in ponds in production.

For a long time, garlic is used by men against skin parasites, the sweat of people who eat a lot
of garlic (or other plants with highly odorous active ingredients such as manioc) acts as a
repellent against parasites. Similarly, a mixture of plant extracts including Benadenia seriolate
included to fish feed, help to decrease the fish skin and gill parasites. P. Kurroa was used in
experiments to eliminate the free radicals and to reduce the stress in P. Monodon (Citrasu et al
2006) as other herbs.
Hatchery specialists use maturation diet in crustacean (P. monodon) use natural commercial
products (Notrabrood ) that are used in hatchery, the product use the hormone production
properties of some plants extract (W. Sonnifera, Macuna pruita, Ferula asafoetida and Piper
longum, Bobu 1999). In particular they are used for the production of off-season gametes or
with species with problems of fertilization and gonadal development, low sperm mobility and
low spermatic volumes vi. Asparagus racemous used in India as Ayurvedic principle (ancient
Indian medicine system) is used as a grow promoter and has similar effects even on larvae of
Labeo rohita vii.

Finally, scientific experiments were carried out with specific advanced acidifiers in a species
that does not produce acids in its digestive system such as L. Vannamei against the presence of
Vibrio spp (a key pathogen for the species). The results were excellent with a positive linear
effect on the survival of the shrimp at 60 days between the control and the various treatments
with the specific acidifier. In the maximum dose (1.2% of the ration) allowed a decrease of
more than 50% of the presence of Vibrio spp (from more than 600 to 300 Vibrio spp CFU / ml)
and a survival of more than 10% in the treated groups. The same group of researchers has
carried out tests with flavonoids on carp to test the possibilities of: intestinal health, palatability
of the ration, improvement of digestive processes, improved absorption of nutrients thanks to
the antimicrobial and antioxidant effect of phytogenic preparations. The results of the carp
grow were more marked after the 6 week of breeding with differences of more than 20% in the
daily increaseviii.

v Herbal medicine in aquaculture, Dr. S. Kolkovsky

vi
Dr. Sagiv Kolkovski, Herbal Medicine in Aquaculture, Nutra Kol Australia, Aqua Feed international Vol. 16 – iusse
6 - 2013
vii
Kavita K. Sharma LL.. Use of herb (Asparagus recemoius Wild) supplanted diet for promoting growth in the fry of ALbeo rohita. The fourth
Indian Fisheries forum, 1996, p. 136.
viii Microbiota from Litopenaeus vannamei: digestive tract microbial community of Pacific white shrimp (Litopenaeus vannamei)

Jaqueline Tuyub Tzuc, Diana Rendíz Escalante, Rafael Rojas Herrera, Gabriela Gaxiola Cortés, andMaria Leticia Arena Ortiz

6
Other important results derive from a research of the aquatic animal health laboratory of the
Institute of Desert Research which carried out experiments with rosemary extracts with respect
to various pathogens including Streptococcus iniae and S. Agalactie with excellent results in
vitro and in vivo with infected Tilapiaix .

The use of antibiotic ( ossitetracicline ) shown bacterial resistances that were not shown in the
rosemary extractx.

Problems for the application of phytotherapy in aquaculture on an industrial scale

Algal and herb extracts are used from long time in the human sanitary practices but only little
things are done of the aquaculture sector.
The standardization of extractive processes from plants is one of the problems that require
clear solutions in the near future. Furthermore, in the USA, Japan, Australia and the European
Union there are restrictive legislation for the approval of the use of medicinal plants or extracts
in aquaculture. They are often considered surgical medical devices and need long and costly
bureaucratic procedures for legal approvals to introduce new active ingredients onto the
market. Its safety and efficacy must be tested before it can be tested on a commercial scale by
means of tests assays on the absorption, metabolism and extraction of the active ingredients
considered. The procedures can also take 5 years from the conception to the commercial
release of the products (POM Prescription Only Medicine). As a result we have high costs that
discourage the industry that considers the market for natural remedies to be restricted, which
is contrary to the author's thinking.
At other times there are no clear regulations and it is not clear how preparation made from
herbs and medicinal plants should be registered. Legislative clarity and streamlining is one of
the bases for the use of plant principles in animal production and particularly in aquaculture.
Clear harmonized international legislation should take into account the benefits of plant
remedies compared to chemotherapeutic agents that are creating numerous problems.
The plant and algal extracts can also be used as growth promoters, anti-stress, vitamin
complexes, and preventive infections. Often plant extracts can produce beneficial effects for
animals grown at high density, which is a typical situation nowadays. On the other hand, the
concentrates of plant and algae extracts can be dangerous if used improperly and highly toxic
as the extracts of rotenone roots, normally used at 50-200 mg / l kill all the aquatic fauna
present and biodegrade quickly (Negroni personal communication). The collecting season could
widely affect the concentration of plant and algal active principles.
The use of garlic and onion extracts, included in the formula for the production of pellets in the
daily ration of farmed shrimp, contributes to the prevention of bacterial infections xi, a scientific
study highlighted the antibacterial activity of guava (Psidium guajava) against the pathogenic
bacteria of crustaceans, was tested the minimum inhibitory concentration against Vibrio and A.
Hydrophila at 1.25 and 0.625 mg / ml respectivelyxii.
Still Direkbusarankom and others found how guava (Psidium guajava) and neem (Azadirachta

ix www.dri.edu/rosemary-carrol
x
Phyto-therapy in aquaculture, Dina Zilberg, The aquatic Animal helath Lab; The French Association Institute for Agriculture and Biotechnology
of Drylands, J. Blaustein Institutes for Desetr Research, Ben Gurion University of the Negev, Midreshet Ben-Gurion, 84990, Isral – Aquaculture
Europe Vol 36 (1) March 2011
xi Application of Medicinal herbs to aquaculture in Asia, Sataporn Direkbusarakom, School of Agriculture Technology, Walailak University,

Thasala, Nakhon Si Thammarat 80160, Tailandia

xii Direkbusarakom S. Aekpanithangpong U., The efficiency of crude extract from the leaf of guava (Psidium guajava L.) on Vibrio spp isolated

from diseases tiger prawn (Penaesu monodon). Proceedings of the seminar on fisheries. Department of Fisheries, 1992, p. 259-62

7
indica) extracts eliminated luminescent bacteria from the Black tiger (P. Monodon) shrimp
more effectively than tetracyclinexiii.

The use of citrus seed extracts is already in common use in many agro-industrial fields including
aquaculture as a sanitiser against Listeria in the processing industry, as a grow promoter of sea
fish fattening phases and as a sanitizer in reproductions. and larval stages of many species
(frogs, tilapia, catfish) as the author has personally experienced (see separate table on ENSA
Annex 1).

A possible new measurement method

As already mentioned, one of the major needs in the field of phytotherapy applied to
aquaculture is certainly to be able to quickly evaluate the efficacy and biocompatibility of any
new substance intended to be tested and administered to aquatic organisms. It is also difficult
to evaluate the viability of these organisms in response to these administrations, if not with
long tests, often very costly.
One of the reliable, rapid and non-invasive methods to carry out these assessments in such a
difficult sector is provided by the experience of some researchers in the field of bio-
electromagnetism. Starting from unconventional knowledge and methods (not scientifically
validated) but of proven practical effectiveness applied for years in various sectors, today it is
possible to perform refined measurements in the aquaculture sector. It is possible to perform
bioenergetics tests of biocompatibility of any substance on living beings, measuring in very
short time their biological response to that substance. An useful aquaculture application is the
measurements that allow carrying out research with the aim of improving the biological quality
of the medium the animals raised and understand which phyto therapeutic principles (or
combination) will be more effective to stimulate the health and vitality of aquatic animal.
The choice and application of natural substances in the aquaculture sector thus becomes a
faster path, thus reaching the selective identification of the most suitable and specific products
application.
The application of the above-mentioned technique has already made it possible to formulate
effective phytogenic products for the care and support for plants, successfully used in organic
farming.
See www.archibio.it

Use of distillation for the production of essential oils

Distillation is an ancient method for concentrating active plant ingredients; distillation
concentrates and condenses some plant components. The product of the distillation is called
"essential oil" and varies considerably in composition and aroma, it is not dissolvable in water
and can form physicochemical complexes with other components of the ration. It easily
dissolves at room temperature in both fresh and salt water.
The lipolytic structures of essential oils have the ability to expand, fluidify, makes unstable and
permeable cell membranes of pathogens that release cellular fluids to the outside. This type of
action is different from that of antibiotics that inhibit cellular synthesis, metabolism or modify
DNA or RNA. Often essential oils inhibit bacterial species hostile to us while not influencing

xiii
Antibacterial activity of guava (Psidium guajava L.) and Neem (Azadirachta indica A. Juss.) extracts against foodborne pathogens and spoilage
bacteria. Mahfuzul Hoque MD1, Bari ML, Inatsu Y, Juneja VK, Kawamoto S.

8
other more friendly.
Essential oils have the ability to damage membranes and coagulate the cytoplasm, in addition
to damage lipids and proteins of microbes. It has been noted that oils containing high
percentages of phenolic components have higher inhibiting power (high toxic power towards
the cell membranes of the most common pathogens) for numerous pathogens.
If appropriately emulsified, the essential oils do not release biofilm on the surface of the water
avoiding dissociation from the matrix. They can inhibit the formation of biofilm on surfaces,
which are always difficult to inhibit (for example oily extracts from oregano, thyme and other
plants containing carvacol a monoterpene phenol).
The strong odours and flavours, sometimes encourage the encapsulation or microencapsulation
of these oils, which could limit their use. Oily extract of oregano, rosemary and coriander have
excellent results against many bacterial strains both in vitro and in vivo against E.coli,
Salmonella typhinarium and S. Aureous.xiv.

Problems created by antibiotics on a global scale and in particular on farms

Not only does the practice show that the use of plant principles, already widely tested, would
bring benefits to all participants in the chain of fish production, but also the WHO
(http://www.who.int/en/ Antimicrobial resistance: a serious threat to public health 30-04-
2014) requires attention on the subject. The WHO (World Health organisation) report reveals
that antimicrobial resistance, including antibiotic resistance, is not a prediction but a serious
danger and is happening now in every region of the globe and is one of the greatest risks in
public health.

Antimicrobial resistance (AMR) is endangering the treatment and prevention of a large number
of pathologies originating from bacteria, parasites, viruses and fungi. Several governments are
worrying. We have the risk that one day, when antibiotics lose their properties against stronger
pathologies, common infections and small wounds can kill. For the WHO it is not a fantasy but a
real possibility of the 21st century. WHO is closely monitoring all developments of this problem
on a global level (Antimicrobial resistance: global report on surveillance 2014 WHO). The
situation in the aquaculture sector sees a similar situation with antibiotic use in intensive
aquaculture although well controlled and with mandatory withdrawal times. The World

xiv
Optimum essential oils for use in aquaculture, Matt Pearce, Meridien Animal Limited, Aqauculture Europe – Vol
36 (2) June 2011

9
Organization for Animal Health (OIE – Organisation International des Epizoozie) is of the same
opinion and has a website for the problem (http://www.oie.int/for-the-media/amr/) that deals
with the Anti-microbial Activity (AMR) and has just concluded an international conferencexv We
just remember about 100 cases of shrimp imports containing medicinal substances in 2011xvi,
signalled by the Southern Shrimp Alliance.

Actions that mitigate the misuse of antibiotics in aquaculture

The great use of antibiotics as growth promoters can create pathogens resistance and can
increase the incidence of diseases and alter the physiology of digestive metabolism, so
nutritionists have reacted trying to create rations without antibiotics, with considerable effort.
In fact, experience shows how it is not easy to support growth promoters with additives.
We also try to improve the environmental parameters that are little considered for the large
concentrations of animals in intensive farming. Even the excesses of protein in the ration, which
are then partly expelled, favour the development of pathogens; this happens particularly in the
aquaculture sector. In fact the aquatic medium receives the undigested proteins. In many
species of aquatic animals that do not metabolize complex glucose well, the proteins of the
ration are increased for a faster growth. Even the high content of phosphorus, which lowers the
quality of water in farms, can be reduced by the presence of phytasis. Finally, new products
with high protein concentrates such as those derived from soy and other plants can allow the
reduction of fish meal. The new trends recommend increasing the health of the digestive tract
of aquatic animals with natural pre and pro biotics. .

Problem created by chemical disinfectant

It is well known that the most common chemical disinfectant leave contaminants in the surface
washing water and on the surface of the cleaned animal s in some countries is admitted to
wash animals with water and a certain dos of disinfectant. Moreover the continuous use of the
same disinfectant creates resistance to some microorganism obliging to rotate periodically the
disinfectant in some food industries.

Healthcare concept of the digestive tract

The combined efforts of an appropriate formulation of rations, breeding management,
biosafety, and the use of additives can optimize the achievement of "gut health" or "gut
welfare", which is defined as "the ability to execute normal physiological functions, to maintain
homeostasis that support the ability of the bred animal to overcome without consequences the
stresses of infectious and non-infectious diseases "(gut health in English). During the life cycle in
the farm, having a good health care in the digestive tract of aquatic animals is important to
extract maximum productivity without the use of antibiotics the herbal principles can replace
antibiotics for the health of the digestive tract of animals raised in aquaculture.
The growth promoters based on antibiotics used until now can be replaced by additives with
bactericidal or bacteriostatic activities, there are many solutions, we mention some, each has
its own characteristics:

xv
OIE Global Conference on the Prudent Use of Antimicrobial Agents for Animals
International Solidarity to Fight against Antimicrobial Resistance, Paris (France) 13 –15 March 2013
xvi
Southern Shrimp Alliance, P.O. Box 1577 Tarpon Springs, FL 34688 955 E. MLK Dr. Suite D Tarpon Springs, FL 34689 -727-934-5090 Fax 727-
934-5362, January 10, 2012

10
- Zinc oxide and copper sulphate have good antibacterial properties but are harmful to the
environment when used in high doses, in some countries use is limited.
- Not all organic acids have a good efficacy as anti-microbial, their efficacy depends on their
hydrophobicity and dissociation capacity.
- Many plant and algal extracts in vitro demonstrate an antibacterial activity, many of which
confirm the ability to cope with in vivo experiments. Plant extracts and organic acids have a
synergistic effect. Within vivo experiments, synergistic effects have been confirmedxvii.

Apart from what was mentioned in the previous lines, some additives to nutrition can have an
antimicrobial action as in the following cases and can indirectly positively influence the health
of the digestive tract with the following:
- - Enzymes, which reduce the level of polysaccharides (anti-nutritional factors).
- - Probiotics and prebiotics, which increase the number of beneficial bacteria.
- - Immune boosters as a boost to immunity that help prevent infections i13.

Plant extracts, from traditional medicine to zoo technical science

It has long been evident that mankind uses medicinal plants for humans and animals (often in
synergy with magical and religious rituals) to treat, prevent malnutrition and improve health.
There is evidence that their use originates from the Neanderthal period, in the nineteenth
century, the use of medicinal plants began to decrease in medical and veterinary practice and
was replaced by remedies of the pharmaceutical industry, especially in the more developed
countries, but over the years '60 re-emerged as a response to the side effects of “synthetic”
medicine.
The current use of botanical principles is currently very similar and different from the historical
use that scientists call "traditional". Before use on an industrial scale, scientists must perform
reproducible phytogenic tests, the results must be scientifically validated. Furthermore, the
treatment products placed on the market must be effective and safe with the production of
standardized botanical extracts, which must have the following characteristics:
- Specific products of well identified and controlled plant species;
- Use of standardized manufacturing processes that respect the active compounds contained in
vegetal species;
- Products marketed that guarantee a minimum indicated concentration of active ingredients
The scientific application of plant extracts in aquaculture production is a technology that is still
progressing, in many cases the results are very satisfactory, in particular in combination with
organic acids.

The possible role of plant and algal extract in aquaculture

There is a current of thought that favours the use of natural products, the goal is the availability
of safe active ingredients available for the aquaculture industry. Herbal and algal preparations
contain numerous active ingredients that can effectively mitigate many of the problems of
world industrial aquaculture.
Plants contain an abundance of chemical compounds that allow them to resist attacks by micro-
organisms and insects. These compounds defined as "secondary" or "phytochemical" can

xvii
Teresa M. Agulles Teixidò and Qi Wenjuan (PlisVet Animal Health), Can plant extracts replace antibiotic growth promoters? Published on:
04/29/2014 (368) (2)

11
potentially provide growth promoting activities in aquaculture species, as mentioned in the
following points:
- Antioxidant properties: Antioxidants help the body to reduce stress caused by free radical
damage. The flavonoids and phenolic acids, widely present in higher plants, are effective
against the destructive effect of ROS (Reactive Oxygen Species, the so-called free radicals).
Some compounds of basil (Ocimum basilicum), cinnamon and other plants possess a strong
antioxidant activity
- Microcide direct effects: some components have the ability to adhere to the cell wall, thus
forming breaks and causing the cellular content to be externally excreted, killing the
microorganism. The canella and marjoram are examples of plants with micro biocidal
characteristics of their active ingredients, such as guava (Psidium guajava), basil, Indian almond
(Terminalia catappa), oregano (Oreganum vulgare with more than 30 antibacterial principles),
and many morexviii (See table 1-5).

The undersigned has been practicing for long periods the natural extracts of citrus seeds in frog
frogs (Rana catesbeiana and Rana esculenta, amphibians known for their sensitivity to synthetic
antimicrobials), trout, marine fishes and crustacean, with excellent bactericidal and fungicidal
effects on reproducers, eggs and larval stages (See Annex II).
- Antiviral activities: numerous active ingredients present in plants have a potent viricidal
activity in crustaceans11 (YHV, WSSV) and fish (See Table 3-4 e 7)
- Antifungal activity: extracts from herbs can cause the lysis of the cell membranes of
myocytes, alter their metabolism and even bring them to death. Notes fungal infections
are controlled by basil (O. Basilicum), tamarind (Tamarix dioica) and Rhazya strictaxix xx.

The protective effects of herbs against subacute aflatoxicosis in Oreochromis niloticus

were solved with extracts of rosemary and parsley (0, 2 and 4 g kg-1 Body Weight
divided into 2 doses at the beginning and at the 6th day of the experiment) were
compared with three different Aflatoxin concentrations (AFB1 0, 9 and 18 mg kg-1 BW
with a single intraperitoneal administration) were tested individually and in different
groups. In vivo. The study concluded that both rosemary and parsley extracts were able
to protect fish from aflatoxynosis even at low administration volumes.lxxi.
- Anti-stress activity: herbal formulations allow greater physical prowess, clearer mental
functions and a non-specific resistance that allows greater adaptability to the
surrounding environment. Animals have less stress because they do not alter the normal
physiological functions. Numerous herbs, Withinia somnifera, Embalica officanalis,
Asparagus recemosus, Ocimum sanctum, Tribulus terrestris and Piper longum are
believed to have adaptogenic, anabolic effects and the possibility of increasing the vital
energy of the animalsi16. Picrorhiza kurroa fuusata is used as an anti-stress factor in
shrimp farmingxxii like the bioflavonoids extracted from Toona sinensis which acted as

xviiiHerbal medicine-an alternative to synthetic Chemicals in aquaculture feeds in Asia, Thongam Ibemcha Chanu (Department of Aquaculture
Central Institute o fFishery Education, Versova – Mumbai 400061 India) et Al. - World Aquaculture – September 2012
xix
Adiguzel, A. Et Al., Antimicrobial effects of Ocimum basilicum (labiatae) extract. Turkey, Journal of Biology 29:155-160 - 2005
xx Khan, S. et Al, 2004,. Antifungal activity pf Tamarix dioica-an in vitro study. Gomal Journal of medical science 2:2
xxi Manal Ibrahim El-Barbary (National Institute of Oceanography and Fisheries, Egypt)and Ahmed Ismail Mehrim (Mansoura University), 2011.

Protective effect of antioxidant medicinal herbs, Rosemary and Parsley, on subacute aflatoxicosis in Oreochromis niloticus, (839) (0)
xxii Citarasu, T. et Al, 1998. Effects of feeding Artemia enriched with stresstol and cold liver on growth and stress resistance in the Indian white

shrimp Penaesu indicus PL. Asian Fishery Science 12:65-75

12
 antioxidant and anti-stress factors in Litopenaeus vannamei infected by Vibrio
alginolyticus xxiii.
- Hormonal activities: Some plants are recognized as natural "viagra" having effects on
the hormonal and reproductive performances, P. monodon with maturation diets
containing extracts of: W. Sonnifera, Macuna pruita, Ferula asafoetida and Piper
longum, have increased fertility and the weight of the gonads, reducing the period
between the mutes compared to the control shrimp groupsxxiv. Similar results were
obtained with semi-herbal commercial diets based on herbs with P. Vannamei6.
- Probiotic effects: Some plants rich in oligosaccharides, such as chicory, are capable of
stimulating the growth of beneficial bacteria such as lactobacilli and ibifidus bacteria
without promoting the growth of pathogenic bacteria. Usually there is a reduction of
digestive diseases in many animal species.
- Prebiotic effects: instead of stimulating the growth of beneficial bacteria, they also
increase the production of short chain fatty acids (Short Chain Volatile Fatty Acids -
SCVFAs) with lactobacilli and Bifidobacterium. SCVFAs inhibit the growth of numerous
proteolytic putrescent bacteria.
Some plant extracts are not able to stimulate the growth of lactobacilli, so they are not
real prebiotics, but instead can promote the growth of SCVFA with lactobacilli present in
the intestine. cinnamon and garlic are some of these promoters of the production of
SCVFA.
- Blocking the adhesion of bacteria on the surface of the digestive tract: the interactions
with the main receptor of lecithin-carbohydrate interactions are the main mechanism of
adhesion of pathogens to the eyelashes of the epithelium of the gastric mucosa. Some
phytochemical compounds, such as those contained in the carob, can block the
adhesion of pathogens in the intestinal mucous layers, thus preventing the development
of infection.
- Immune-stimulatory effects: An immune stimulant like a chemical drug, can activate
actions that strengthen the physiological defence mechanisms or the immune response
of organisms against pathogens. An immune stimulant can be used to additive to foods
providing greater defences, resistance to pathogenic infections during stressful periods
of the cycle of breeding such as: selection, reproduction, vaccination, withdrawal of
gametes. The immune stimulant activities of plants have long been studied in different
animal species. Interesting immune stimulant properties of plants have been recognized
in extracts from. Emblica officinalis, Cynodon dactylon, Adathoda vasica, O. Sanctum, W.
Sonnifera, Myristica fragrans, Aegle marmelos, T. Cardiofila, P. Kurooa and E. Alba
against bacteria (Vibrio harveyi, A. Hydrophila) and viruses (example WSSV in prawns )
pathogen16.
Lymphoid tissues associated with other digestive tract (gut-associated lymphoid tissue)
GALT play a key role in the digestive immunity of animals raised in aquaculture.
Prebiotic compounds, such as chicory oligosaccharides, can stimulate and induce
beneficial effects on gastric health by positively influencing local digestive immunity
through the prompt response of GALT16. Other plant substances such as fungal
polysaccharides or garlic allacin, can be used in general (on the whole animal) as an
immuno stimulant because they activate both innate and adaptive immunity.

xxiiiHsieh, T.J. et Al, 2008. Effects of Rutin from Toona sinensis on the immune and physiological responses of white shrimp (L. vannamei) under
Vibrio alginolyticus challenge. Fih and shellfish immunologi 25.581-588
xxiv Babu, M.M., 1999. Developing bioencapsulated ayurvedic product for maturation and quality larval production in P. vannamei. PhD Thesis to

MS University, Tirunelveli

13
 - Digestive enzymes secretion and appetite stimulation: some plant components, such
as those contained in the canella (with cinnamaldehyde), pepper (with piperine and
other substances) and or cardamom, are able to stimulate the production of enzyme
secretions digestive tract of the pancreas and intestinal mucosa, inducing a better feed
efficiency and an increase in zoo technical perfomances.10 They have a microbicide
property that stimulates digestive secretions, especially amylases

Conclusions
The news for the development of pathogens resistant to the medication spectrum currently
available for the zoothecnical and aquaculture industry come from all areas of animal
husbandry. The author considers them worrying. The author often works in many EU and not
EU countries, where it is produced and imported the animals; he has a very direct "vision" of
the sector and its problems.
Treat the onset of severe outbreaks in animal and fishery products obliges the treatment of
large quantities of animals with broad-spectrum chemotherapeutic agents with the risk of
residues in meat (and water) that can reach the consumer and harm the environment.
Currently a large part of antibiotics substances are banned for the aquaculture sector in the EU,
USA and other countries.
The plants, algae, herbs and the extracts of their various parts are a great resource and a great
alternative to the use of chemotherapeutic agents, disinfectants and other banned substances
that could improve the treatment of humans, animals reared in aquaculture industry and of '
environment where these substances would spread. The legislative sector must seriously
consider with appropriate legislation and implementing regulations to allow the appropriate
use of the principles of herbs, plants and algae in animal production, particularly in aquaculture.
Furthermore, the certification of herbal products to be used in the breeding sector is essential
for breeders who will use them. Especially for those that are dealing with organic production
aquaculture.
The systems of action of certain phytotherapics that need to be better studied are not yet clear,
but it seems that the attack on the cell membranes takes place in a different way according to
the types of target organisms.
It is clear that you do not quickly succeed in eliminating chemotherapy. It is possible with a
number of measures to mitigate the use of chemotherapy, such as: adjustment of the diets, the
appropriate use of good animal husbandry, the appropriate use of the principles of known
medicinal plants, an increase in the quality of the water and consequently the environment in
which the aquatic animals are kept, could reduce the phyto-therpic and other chemicals use.
The above principles can create good health in the digestive zone of our farmed fish and
crustaceans.
Finally we must realize that feeds with few or no chemotherapy may be more expensive than
others. Increasing the productivity of aquaculture farms with extracts of herbs and plants will
contribute also to pay the difference as the development of organic farms that can sell at more
attractive prices.
The actual legislation about the use of plant, algal and herbs extract must be opportunely
revised to help the diffusion of natural products in the control of animal diseases in the animal
industries.
It is clear that the consumer will be more and more interested and sensitive to the
consumption of safe animal products and bred fish products without the use of
chemotherapeutics, especially following the numerous scandals in the food sector.

14
15
16
Origin Tables 1-2-3: Herbal medicine in aquaculture, Dr. Sagiv Kolkovski, R&D Director Nutra-
Kol, Australia, Aquafeed 2013

17
Table 4 Use of medicinal plant in China

Origin: Rajandra KR. Prevention and control of fish disease by herbal medicine. Fish health
Section News 1990; 3: 3-4

Table 5 Vegetal remedies used against parassites in Vietnam

Table 6 Antibacteries remedies in aquaculture in Vietnam

Origin table 5-6: Dung L. Herbs and their application for control fish disease in Vietnam.
Technical paper. National Aquaculture Research Institute, Vietnam 1990, p. 7

18
Table 7: Traditional herbal antiviral acticvity in Thailand for fish and crustacean

Table 8: Traditional herbs with antibacterial activity in Thailand for fish and crustacean

origin table 7-8:

19
Annexe I

NATURAL CITRUS EXTRACT NOTES

G.Negroni / G. Cappi 2018

Natural Citrus Extract (NCE) or Agrumina (AGR) is a natural organic compound, extracted of
citrus fruits, activated by biological process and stabilized by physical and chemical ways, wich
works as BACTERICIDE, FUNGICIDE AND VIRICIDE with ANTIOXIDANTS characteristics.
NCE is a very complex BIO-MASS, that has POLI-SINERGETIC ACTION of its organic components,
working over natural equilibrated system. The main organic natural traces of NCE are:
ASCORBIC ACID (free, ascorbyls and ascorbates), CITRIC ACID, POLYPEPTIDES, AMINOACIDS,
FATTY ACIDS, BIOFLAVONOIDS, TOCOPHEROLS, SUGARS, AMINE GROUPS AND OTHERS
MINORS.
NCE doesn't have KNOCK-DOWN as synthetic pharmacos, NCE works slowly with residual
MICROBICIDE ACTION over MICROORGANISMS, with combined action on citoplasm and
coagulated the protein.
The ANTIOXIDANT ACTION of NCE is unique, making easy its microbicidal action into ACID and
ALKALINE pH.

NCE has ASCORBIC ACID (AA) of higher bio-availability and shows excellent physiological
properties, thus:
* The AA works with high activity into OXIDATION and REDUCTION process, that required
transfer of HIDROGEN.
* The AA works on BIOCHEMICAL SYNTESIS of STEROID HORMONES, blood coagulation and
increase the body strength AGAINST INFECTIONS caused by BACTERIA, FUNGUS, VIRUSES and
STRESS.
* The AA promotes the formation and action of TISSUES (bones, skin, cartillages).

20
* The AA makes the BIOCHEMICAL union of Vitamins: B1, B6, B12, A and E; and the most
important: ACT AS SEQUESTRANT OF FREE RADICALS, ANTI-TOXIC and HEPATO-PROTECTOR.

SPECIAL PROPERTIES OF NCE:

* ECOLOGICAL product (natural origin and organic).
* BIODEGRADABLE and NON-CONTAMINANT of environment.
* NON-TOXIC, NON-CORROSIVE (doesn't attack metallic materials), NON-VOLATILE (doesn't
evaporate).
* NON-IRRITANT or OFFENSIVE for SKIN or MUCOUS membrane of humans or animals.
* LARGE SPECTRUM GERMICIDE, with effective action against Gram Positive and Gram Negative
Bacteria, Fungi and certain types of Virus.
* It is NOT ANTIBIOTIC, but acts at similar way. It is compatible with antibiotics, sulfa,
nitrofuranes, and so on.
* LONGER RESIDUAL ACTION, even in the presence of organic matter.
* High level of ANTIOXIDANT ACTION.
* OPTIMUM STABILITY in pH of 2 to 9 and temperature up to 130° C; it is stable to changes of
light. The change of colour of the product doesn't affect its efficacy.
* AGRADABLE SMELL. It is an excellent deodorizant, eliminating fetidness.
* Preservative of FOOD and FEED (fungistatic - bacteriostatic).
* Doesn't alter the taste or smell of FOOD and FEED.
* Doesn't alter the vitality of spermatozoids, embryos and incubator eggs.

21
 TECHNICAL PRODUCT DESCRIPTION
I. COMPOSITION
- Bio-activated citrus seed extract 50,00%b/w
- Glycerine (vegetal USP) 50,00%

II. ORIGIN
- Orange (Citrus Sinensis)
- Bergamot (Citrus Aurantium)
- Mandarin (Citrus Reticulata)
- Grapefruit (Citrus Paradisi)
The citrus seed extracts are activated by biological method and stabilized by physical (U.V.)
and chemical (vegetal glycerine) methods.
The bio-activated citrus seed extracts contain traces of: Ascorbic Acids (free, ascorbyls and
ascorbates), Citric Acid, Polypeptides, Aminoacids, Fatty Acids, Bioflavonoids, Tocopherols,
Sugars, Amine Groups and other minors.

III. PHYSICAL-CHEMICAL PROPERTIES

- Chemical formula: Doesn't have, because it is a very complex natural organic
compound.
- Appearance and aspect: Cristaline Liquid viscous.
- Organoleptic characteristics:
Taste: Bitter (as citrus seeds) and astringent.
Odour: Agradable very light citric.
Colour: Lemmon. The change to darkness doesn't affect its efficacy.
- Density (g/ml at 25°C): 1,110 to 1,220.
- Viscosity (centist. at 25°C): 125 to 140.
- pH (10% dilution at 25°C): 2,0 to 3,5.
- Solubility: 100% in water, alcohol, liquid organic acids, glycerine and
propilen glycol.
- Corrosivity: 2000 ppm (water dilution), its corrosivity is similar as potable
water.
- Use stability: Better for pH: 2,0 to 9,0.
- Use efficacy: Hard waters (Ca, Mg) can reduce its efficacy. This problem
has easy
solution, using lactic acid and citric acid (organic natural acids)
acting as
chelactants to Ca and Mg, mainly in by-products formulation.

IV. STANDARD OF QUALITY CONTROL

- Water solubility: 100%
- Organoleptic characteristics:
Taste: Bitter and astringent (as citrus seeds).
Odour: Agradable, very light citric.
Colour: Lemmon to darkness.
- Density (g/ml at 25°C): 1,110 to 1,220.
- pH (10% water sol. at 25°C): 2,0 to 3,5.
- Ascorbic Acid (AOAC Meth.): 2,00% (minimum) - (limits: 2,0 and 4,5%).

22
 - Organic Acid (TOTAL): 1400 mg (minimum) - (limits: 1400 and 1780 mg).
Method: Quantity of product (mg) neutralized by 1 ml of NaOH-1N.
- Nitrogen (KJELDAHL Meth.): 0,52% (minimum) - (limits: 0,52 and 0,72%).

V. STABILITY AND EXPIRATION DATE

According to international legislation the expiration date is 3 (three) years after
manufacturing date, but Natural Citrus Extract into right closed drums, reaches 5 (five) years
or more, with stability over 95%.
The ageing transforms darkness (suicide of antioxidant radicals), but its biocide efficacy and
cristalinity remain right.
At normal temperatures (4° C to 40° C) this technical product is very stable, but over 120° C its
stability go down.
Incompatibility with anionic products.
Very stable at light.

VI. BIOCHEMICAL PROPERTIES

Natural Citrus Extract
- is a BIOMASS that has MICROBIOSTATIC characteristics (BACTERIA and FUNGUS), acting with
excellent ANTIOXIDANT property.
- works better against MESOPHILES (microorganism to ideal temperature between 25° and 40°
C, but growths between 10° C and 45° C).
- has EXCELLENT ACTION against GRAM-POSITIVE BACTERIA. Ex: Staphylococcus,
Streptococcus, Bacillus, and so on.
- has GOOD ACTION against GRAM-NEGATIVE BACTERIA. Ex: Salmonella, E. Coli, Proteus,
Shigella, and so on.
- has EXCELLENT ACTION against DERMATOPHYTES FUNGUS. Ex: Candida albicans, and so on.
- has GOOD ACTION against FUNGUS that produce MICOTOXINS. Ex: Aspergillus, and so on.
- its ANTIOXIDANT action is unique, making easy its MICROBIOSTATIC action in large ACID and
ALKALINE pH, helping the field work of Natural Citrus Extract.
- its PHYSIOLOGICAL action into DIGESTIVE TRACT of animals is very complex, and NON
STERILIZANT.
- its MICROBIOSTATIC and MICROBICIDE action is generated by polisinergism of its
biochemical compounds: Ascorbic Acid, Citric Acid, Bioflavonoids, Peptides and others.
- works at different way of traditional chemotherapics, because its initial KNOCK-DOWN is
slow, but its RESIDUAL MICROBIOSTATIC /MICROBICIDE action works during long time, when
the traditional chemotherapics don't work any more.
- acts by COMBINATED ACTION UPON CITOPLASM and COAGULATED THE CELULAR PROTEIN,
stopping the celular vital cicle.

VII. MINIMUM INHIBITORY CONCENTRATION (PPM - IN VITRO)

Staphylococcus aureus ATCC 6538 Proteus vulgaris ATCC 8427
5 3
Streptococcus faecalis Proteus mirabilis ATCC # 32 3
14 Pseudomonas aeruginosa ATCC1014520 4
Klebsiella pneumoniae CCH Salmonella schottmuelleri 4
10 Salmonella anatum 5

23
Salmonella pullorum 5
Salmonella cholerae suis 9
Salmonella typhi 3
Salmonella partiphy 3
Shigella dysenteriae 7
Escherichia coli cepa B ATCC 11303 7
Escherichia coli ATCC 9337 5
Erwinia carotovora ATCC 8061 4
Bacillus subtilis ATCC 6633 5
Candida albicans 10
Aspergillus niger ATCC QM-458 90
Aspergillus oryzae ATCC 4814 100
Aspergillus oryzae ATCC 9102 100
Aspergillus tamarr ATCC 13450 50
Penicillum funiculosum ATCC 10445
20

24
 SAFETY DATA SHEET
I. HAZARDOUS INGREDIENTS:
The Natural Citrus Extract was tested according to the techniques specified by "Regulation for The
Enforcement of the U.S. Federal Hazardous Substances Act - Code of Federal Regulations, Title 16,
Chapter II, 1976"; then the citrus seed extract is considered AS NON TOXIC by ORAL INGESTION,
PRIMARY SKIN IRRITATION and NON CORROSIVE by use dilutions.
The organic compounds of Natural Citrus Extract are considered AS G.R.A.S. (Generally Recognized
AS SAFE), according to the "U.S. CODE OF FEDERAL REGULATIONS of FOOD AND DRUG
ADMINISTRATION - F.D.A.".
Natural Citrus Extract was also tested according to the uptoday techniques about MOLECULAR
TOXICOLOGY: Pro-Tox (C), Bacterial Stress Gene Assay = CAT-TOX (L), Human Liver Cell Stress Gene
Assay = AMES II (Complete), Salmonella Mutagenesis Assay; then Natural Citrus Extract doesn't
affect the DNA of cells, it isn't MUTAGENIC and it isn't CARCINOGENIC.

Tests on Natural Citrus Extract:

a) "ACUTE ORAL TOXICITY":
- LD50: 2,2 ml/Kg (2,68 g/Kg) with 95% of confidence.
- Method: OECD Guidelines for testing of chemicals. December 1979.
- Animals: Rats EPM-1 Wistar (youngs).
b) "SKIN IRRITATION".
- NON IRRITANT.
Method: F.Lautier, D.Razafitsalama, J.Lavillaurex, Le Cobaye, Animal de Choix Pour Teste
D'Irritabilite
Congress International organise à L'Ecole Veterinaire de Lyon, Fondation Marcel Mérieux 1978 -
France.
- Animals: Guinea Pigs (adults), 400-700 g.
II. FIRE AND EXPLOSION HAZARD DATA:
- Flash point (°C):..........................140 to 152.
- Fire:..............................................Non flammable.
- Explosion:....................................Non explosive.
- Special fire fighing procedures:. Doesn't have.
- Unusual fire explosion hazard:.. Doesn't have.

III. REACTIVITY DATA:

- Stability:................................................. Excellent (4 to 40°C).
- Incompatibility:...................................... Anionic products.
- Caducity:........................ .......................3 (three) years into closed drums.
- Hazardous decomposition products: ..Avoid breathing fumes or
vapor full strenght if is heated.
- Hazardous polimerization:........ It will not occur.

IV. TOXICITY AND HEALTH HAZARD DATA:

- Oral LD 50:...................... 2,2 ml/Kg (2,68 g/Kg).
- Non carcinogenic.
- Non mutagenic.
- Non teratogenic.

25
 - BIODEGRADABLE.

* EFFECTS OF OVEREXPOSURE:
- Maybe harmful if swallowed in full strenght.
- Full strength can produce eye irritation.

* EMERGENCY AND FIRST AID PROCEDURES:

- If ingestion (full strenght):
Don't induce vomiting.
Give milk and white of egg beaten on water, and call a
physician.
Avoid alcohol.
- If contact with eyes (full strenght):
Flush immediately with plenty of water and call a
physician.

V. SPILL OR LEAK PROCEDURES:

- Don't spill Natural Citrus Extract into water stream in full strenght, because coagulates (lower
pH) the
protein of eggs and larvas of fishes.
- Keep drums closed when not in use.
- In case of leaks, then washing with water in abundance.

VI. SPECIAL PROTECTION INFORMATIONS:

- Ventilation:...................................Normal.
- Breathing (normal temperature):Doesn't need special precautions.
- Eyes:.......................................... Security eye-glasses.
- Hands:........................................ Latex gloves.
- Body:.......................................... Doesn't need special precautions.

VII.HANDLING AND STORING PRECAUTIONS:

According to the normal systems to handled and stored products as GLYCERINE and PROPYLENE-
GLYCOL.
NCE recommended dosages for fishery and aquaculture industry
Item USE PPM % Ml\Lt Water
for liquid
preparation
1. Sanitizer by fogging application into rooms 1,200 0,12 6 ml\5 Lt
2. General disinfections of contact surface 800 0,08 4 ml \ 5 Lt
(equipment and tank) and surface (wall,
floor, ceiling, ect)
Special disinfections of air conditioning 1,600 0,16 8 ml \ 5
3. system
26
 4. Disinfections of vehicles for live and dead 800 0,08 4 Ml \ 5 Lt
fish transport
5. General disinfections of fish cold storage 800 0,08 4 Ml \ 5 Lt
plants
6. Special disinfections of fish and seafood 1200 0,12 6 Ml \ 5 Lt
industries
7. External decontamination of fishes, 600 0,06 3 Ml \ 5 Lt
crustaceans and gametes
8. Water for ice, preserving ice 600 0,06 3 Ml \ 5 Lt
9. ENSA concentration for injection pump 1000 5 Ml \ 100 Ml
(max 2% pumping) *
10. ENSA concentration for needle machine in 1200 0,12 6 Ml \ 5 Lt
brine (change the brine solution every
hour) **
11 Probiotics use in aquaculture feed 400 0.04 2 ml / 5 Lt
12 External decontamination for gametes in 400 0.04 2 ml 5 Lt
hatchery

* verification test before full scale use of these concentration

** it is possible to use a lower concentrated brine solution, ENSA lose effectiveness in high salt
solution

Annexe II

May 9, 2014, Shrimp News International

United States

Louisiana—Antibiotics in Imported Shrimp

Veterinary drugs are commonly used to prevent and treat disease outbreaks on shrimp farms. Several of these drugs are banned fo
use in shrimp farming in the United States. They can be toxic to humans, with side effects that can be fatal. There is also an increased
isk of developing antibiotic resistant strains of human pathogens, including Bacillus and Vibrio species. The Food and Drug
Administration is responsible for the safety of all fish and fishery products entering the United States, but funding for testing i
imited. Examples of drugs with high enforcement priority include chloramphenicol, nitrofurans, fluoroquinolones and quinolones
malachite green and steroid hormones. State testing has repeatedly resulted in the detection of banned drugs. The objective of thi
tudy was to quantify veterinary drug residues in commercially available frozen shrimp.

Imported, farm-raised shrimp samples were purchased from local supermarkets and include shrimp from seven brands and si
27
different countries. A preliminary screening was done using rapid ELISA kits to test for chloramphenicol, malachite green, nitrofurans
and fluoroquinolones. Samples tested positive for malachite green and fluoroquinolones; all samples tested negative fo
hloramphenicol and nitrofurans. ELISA results were confirmed using liquid chromatography with tandem mass spectrometry. Drug
esidues in shrimp samples were confirmed for chloramphenicol at concentrations ranging from 0.30 to 0.49 parts per billion, and
enrofloxacin from 1.22 to 5.95 ppb. Results suggest that current testing by the FDA may not be adequately addressing imported
eafood safety. Concurrently analyzed wild-caught shrimp from the USA tested negative for all veterinary drugs considered.

Twenty-seven frozen samples of imported, farm-raised shrimp were obtained from five retail grocery stores in Baton Rouge, Louisiana
Samples included shrimp from Thailand, India, Indonesia, Vietnam, China, and Ecuador. Fourteen frozen samples of domestic, wild
aught shrimp were obtained from six retail grocery stores in Baton Rouge, Louisiana. Frozen samples were transported to the
Department of Food Science at Louisiana State University Agricultural and Mechanical College in Baton Rouge, Louisiana and were
tored at 80°C until further processing.

Detection and Confirmation of Drug Residues ELISA kits were used to rapidly screen shrimp samples to determine which drug residues
were present. Chloramphenicol, fluoroquinolones, malachite green and nitrofurans were the drugs chosen for screening because of
heir ability to cause severe adverse effects in humans and their high enforcement priority status.

The results of this study confirm the presence of illegal veterinary drug residues in shrimp sold at the retail level in the United
States. Ninety-two percent of imported, farm-raised shrimp samples tested positive for at least one drug that is banned for use in
ood-producing animals in the United States. Two of the four drugs considered in this study were detected using ELISA:
luoroquinolones and malachite green. The fluoroquinolone enrofloxacin was confirmed in two out of five samples using LC-
MS/MS. Malachite green could not be detected using confirmatory methods. Chloramphenicol was not detected using ELISA, but was
detected in three out of five samples using LCMS/ MS.

Sources: Detection and Confirmation of Veterinary Drug Residues In Commercially Available Frozen Shrimp. A Thesis Submitted to the
Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in Partial Fulfillment of the Requirements
or the Degree of Master of Science. Jessica Danielle Johnson. May 2014. 2. Aquafeed.com Confirmation of Veterinary Drug Residues
n Commercially Available Frozen Shrimp. Issue 17, Volume 14, May 8, 2014. And Shrimp News International 09 / 05 / 2014

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