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Environmental impacts 1. Effect of net pen structures 2. Organic enrichment 3. Chemicals - solid wastes - nitrogen and phosphorus (nutrient enrichment) - biological oxygen demand (BOD) -chemicals used in medication a) antibiotics b) anti-fungal c) pesticides - antifoulants 4. Disease and parasites 5. Interactions with wild fish 6. Interactions with other wildlife

Impact of chemicals Chemicals used in medication a) Trends in use of different drugs b) Pathways into the environment c) Concerns about impacts 1. Environmental 2. Human

Druguseadvice USA example

Trends in use Chemicals used in medication a) Antibiotics. A broad range of antibiotics (e.g. oxytetracycline, erythromycin, amoxycillin, chloramphenicol) are presently used in aquaculture to treat a variety of bacterial infections Antibiotics are also commonly employed in most forms of aquaculture especially shrimp culture, and in the ornamental fish trade. Antibiotics are used intermittently for short periods (5-14 days), and are administered by bath, injection or oral delivery.

Drug class Aminoglycosides Amphenicols Beta-lactams Beta-lactams Fluoroquinolones Macrolides The different classes of antibiotics used in aquaculture their importance for human medicine and examples of (multi)resistant pathogenic bacteria isolated from aquaculture settings. Importance for human Multiple medecine Example Resistant bacteria Critically important Streptomycin Edwardsiella ictulari Yes Enterobacter spp. and resistance? Isolated from Diseasedstripedcatfish(Pangasianodonhypophthalmus) Vietnam Important Florfenicol Pseudomonas spp. Yes Freshwater salmon farms Chile Vibrio spp. Aeromonas Critically spp. and Edwardsiella important Amoxicillin tarda Yes Different aquaculture settings Australia Critically important Ampicillin Vibrio harveyi Yes Shrimp farms and coastal waters Indonesia Critically Tenacibaculum important Enrofloxacin maritimum Yes Critically important Erythromycin Salmonella spp. Yes Marketed fish China Diseased turbot(scophthalmus maximus) and sole (Solea senegalensis) Spain and Portugal Nitrofurans Critically important Furazolidone Vibrio anguillarum Yes Diseased sea bass and sea bream Greece Nitrofurans Important Nitrofurantoin Vibrio harveyi Yes Diseased penaeid shrimp Taiwan Aeromonasspp. Pseudomonasspp. and Vibrio spp. Quinolones Critically important Oxolinic acid Yes Pond water pond sediment and tiger shrimp (Penaeus monodon) Philippines Diseasedkatla(Catlacatla) mrigel(cirrhinusmrigala) and Sulphonamides Important Sulphadiazine Aeromonas spp. Yes punti(puntius spp.) India Highly Tetracyclines important Tetracycline Aeromonas hydrophila Yes Water from mullet and tilapia farms Egypt Highly Tetracyclines important Oxytetracycline Aeromonas salmonicida Yes Atlantic salmon(salmo salar) culture facilities Canada Defoirdt et al. 2011

Administration of antibiotics 1) Injection - most direct and effective method - labour intensive - stressful to fish 2) Oral -mixed in with feed - most cost effective and commonly used method - dependent upon the fish still eating 3) Bath treatment -much more of the drug is needed to have the required effect -excessive amounts of antibiotics in the water can increase the likelihood of water-borne bacteria developing resistance to the drug.

Use of antibiotics in the Norwegian salmon farming industry between 1974 and 2002 50 use of antibiotics, mt/year Antibiotics Salmon production 600,000 annual production of farmed salmon mt/year 0 1987 2002 The dramatic decline in antibiotic use in the Norwegian salmon industry has been due to: improved farm management development and implementation of new salmon vaccines

antibiotics Antibiotic use in Norwegian, 1987 and 2002. A comparison between aquaculture, human health and farm animals Antibiotics used in 1987 Antibiotics used in 2002 Farmed fish 48,570 kg 1,051 kg Humans 24,185 kg 35,000 kg Terrestrial animals 11,075 kg 5,712 kg Between 1987 and 2002 the use of antibiotics has declined in the Norwegian salmon industry while antibiotic use has increased in human health

Use of antibiotics in aquaculture - Canada Health Canada allows for the use of 4 antibiotics in Canadian aquaculture. On average, 2.5% of all feed is medicated. Most antibiotics are used for fish < 2 kg in size (non-food fish size)

Use of antibiotics in aquaculture - USA The FDA (Food and Drug Administration) approves the use of only two antibiotics for use in aquaculture. Oxytetracycline - catfish, salmonids and lobster Ormetoprim: sulfadimethoxine - catfish, salmon In line with Norway, usage of antibiotics is starting to decline in recent years having peaked during the 1990s. 2001-32,540 kg 2002-24,475 kg Antibiotics recorded in the outflow from raceways used for salmon/trout production Oxytetracycline Ormetoprim 0-2.3 µg/l 0-15 µg/l

Druguseadvice USA example

Use of antibiotics in US aquaculture total 92,900 kg 197,000 kg

Antibiotics used for aquaculture purposes in certain countries

Antibiotics used in Japan for aquaculture of yellowtail, rainbow trout and kuruma prawn

Antibiotics used for aquaculture in the People`s Republic of China.

Use of antibiotics in shrimp farming Major issues Presently very little legislation. Although antibiotic use is very widespread in shrimp farming, information on quantities lacking. Reports of antibiotic abuse: 1) The antibiotics oxytetracyclin and oxolinic acid were detected above permissible levels in almost 10% of Penaeus monodon sampled from Thai domestic markets in 1990-91. 2) Between 1992-94, Japanese quarantine stations found anti-microbial residues in 30 shipments of cultured shrimp from Thailand. 3) Recently, the Swedish National Food Administration found residues of the highly toxic antibiotic chloramphenicol in shrimps imported from Vietnam.

Ornamental fish trade - antibiotic use Increasingly valuable fish trade Fish subjected to extreme stress (capture and transport), often leading to immuno-suppression, and in turn the increased likelihood of disease. Unregulated, and widespread abuse of antibiotics. Dangers associated with antibiotic abuse: 1. Too high a dose -may be toxic to fish 2. Antibiotic resistance can occur with improper antibiotic use. shotgunning method This involves administering one antibiotic after another. This method increases the risk of producing populations of bacteria that are resistant to antibiotics. Can lead to superinfection, where bacteria cannot be controlled by antibiotics.

Impact of chemicals Chemicals used in medication a) Trends in use of different drugs b) Pathways into the environment c) Concerns about impacts 1. Environmental 2. Human

Fate of Antibiotics after application A large proportion of the used antibiotic is not absorbed by the fish, and is subsequently excreted in an active form via the faeces. Depending on the antibiotic, between 60-85% of the drug can be excreted via the faeces unchanged. Rico et al 2013 EnvTox& Chem

Fate of Antibiotics after application Rico et al 2013 EnvTox& Chem

Evidence of antibiotic residue Violations on inspection of imports Violationsoninspectionof domestic products Love et al. 2011

Evidence of antibiotic(and other) residues Concentrationsfoundin imported aquaculture products USA and EU imports compared Love et al. 2011

Impact of chemicals Chemicals used in medication a) Trends in use of different drugs b) Pathways into the environment c) Concerns about impacts 1. Environmental 2. Human

The major issues are: Leakage into the environment = disruption of microbial communities Residuesin fish = enterintothehuman foodchain Antibiotic resistance

Antibiotics entering the environment Overall, large amounts of antibiotics can accumulate in the sediments, and in turn find their way into the food web (invertebrates and fish). Antibiotics are generally quite persistent-can persist up to 1-2 years in sediments. For example, the most commonly used antibiotics, oxytetracyclineand oxolinicacid, can persist in sediments for 10 and 6 months respectively. As a consequence, antibiotics can accumulate in fish and shellfish at levels considered unacceptable for human consumption. The antibiotics typically used are also important in treating human disease and infection. One of the main dangers of widespread antibiotic use is the potential for the development of drug resistance among target pathogens. Drug resistance has been reported in natural sediment bacteria from antibiotics that have accumulated below net pens, i.e. strains of A.salmonicida, the bacteria responsible for furunculosis.

Drug resistance evidence: Studies have demonstrated an increase in resistant bacteria in the intestines of fish receiving antibiotic drugs. Recent studies indicate the level of resistant bacteria in the gut of wild fish is affected during antibiotic treatment of farmed fish. A total of 74-100% of wild fish caught in close proximity of treated ponds contained quinolone residues -a group of antibiotics important in human health.

A number of European studies have highlighted the dangers associated with antibiotic use in aquaculture. Rhodes et al (2000) documented the movement of resistant pieces of DNA from fish hatcheries into E. Coliand Aeromonas species isolated from patients in hospitals. They concluded that collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment. Schmidt et al. (2000) found that many bacteria sampled in and around Danish trout farms were resistant to most antibiotic agents presently available for use in Danish aquaculture.

Use of antibiotics in aquaculture In a further case, in Ecuador, which exports large quantities of pond-raised shrimp to the US, a cholera outbreak was suspected to be linked to inappropriate use of antibiotics in industrial shrimp farming practices. Conclusion What becomes clear in each of these cases is that a number of highly complex scenarios emerge that can lead to bacterial resistance transfers from aquaculture practices to humans. BIO 208-2016

Antibiotics Monitoring use and Enforcement In light of the environmental and health dangers associated with the unrestricted use of antibiotics in aquaculture, there is an urgent need to introduce new legislation aimed at controlling antibiotic use. Suggested measures include: 1. On-farm visits to review usage before receipt of the drug. 2. Receipt of supplier s lot-by-lot certification of proper drug usage, with appropriate verification. 3. Review of drug usage records at receipt of the product. 4. Drug residue testing. 5. Receipt of evidence that the producer operates under a third party-audited quality assurance programme (eg. Government Agency veterinarian) for aquaculture drug use.

b) Pesticides Chemicals used in medication In the salmon industry, pesticides are used primarily to control sea lice infestations. A salmon smolt heavily infested with sea lice

Trends in use Pesticides In the salmon industry, pesticides are used primarily to control sea lice infestations. Roth (2000) reports that eleven compounds representing five pesticide types are currently being used for sea lice control. - two organophosphates - three pyrethrin/pyrethroid compounds - three avermectins - two benzoylphenyl ureas - one oxidizing agent (hydrogen peroxide) With theexception ofhydrogen peroxide, all these pesticides were originally developed for terrestrial agriculture. All are potent neurotoxins and are listed by regulatory agencies as being toxic or extremely toxic to aquatic invertebrates and/or fish. Administered either by bath treatments or given orally with the diet.

Diseases and control measures: Ectoparasites Disease Agent Type Syndrome Measures Saprolegniasis Saprolegnia Fungus White or grey patches of filamentous threads on surface; cotton-like appearance radiating in circular, crescent-shaped or whorled pattern; usually begins on head or fins Bronopol/formalin bath Sea lice Lepeophtheirus salmonis; Caligus elongatus Ectoparasites Reduced growth; loss of scales; haemorrhaging of eyes and fins Pesticides: bath (e.g. Cypermethrin, Hydrogen peroxide); in feed (e.g. Emamectin, teflubenzuron) Cleaner fish Gill amoeba Paramoeba pemaquidensis Ectoparasite Gill infestation Freshwater baths Tapeworms Eubothrium spp.; Diphillobothrium spp. Endoparasites Reduced growth; reduced condition factor; aesthetically unacceptable to consumers Fenbendazole/praziquante l in feed for Eubothrium; avoidance of early hosts Freshwater protozoa Ichthyobodo; Trichodina; Ichthyophthirius Ectoparasites Irritation response; heavy and laboured operculum movements; flashing and rubbing; skin cloudiness caused by excess mucus; focal redness; lethargy Formalin baths

b) Pesticides Most commonly used pesticides for sea lice control include: 1. Azamethiphos(Salmosan) This organophosphate is given by in-feed treatments. Use of this pesticde is predicted to decline. 2. Cypermethrin(Excis) This pyrethroid compound is given by bath treatment, and widely used in both Scotland and Norway. A recent study concluded that a single cage application of cypermethrin has the potential to create a plume of up to 1 km 2 that may retain its toxicology for several hours. 3. Emamectin benzoate(slice) and Ivermectin These pesticides are administered by bath treatment. The use of Slicein sea lice control is increasing in both Scotland and Norway. Characterised by low solubility and readily binds to suspended particulate material. These pesticides are very persistent, and have a biological half-life of between 90-240 days.

Impacts of Pesticides on non-target species Information on the biological impact of pesticides on aquatic organisms, in terms of mode of action and toxocity, shows areas of concern. Pesticides used for sea lice control tend to be persistent, readily binds to particulate material (uneaten feed and faecal material) -readily concentrates in sediments under sea cages. Ivermectin levels as high as 6.8 mg/kg sediment have been recorded in sediments under salmon cages. Studies of the lethal and sublethal effects of these potent neurotoxins show: 1) crustaceans and polychaetes are most sensitive to these pesticides. 2) early life-history stages are more sensitive than later life stages. Further, the inert component of the treatment (acting as a solvent or carrier for the active ingredient) can also be toxic, and in fact in some cases are a greater concern than the pesticide itself. For example, the phthalate acid ester DBP is a potent endocrine disrupter, and 8 tonnes are released annually into the marine environment through aquaculture.

b) Pesticides Known lethal effects of pesticides on marine organisms a) Ivermectin at concentrations of 8-80 mg/m 2 sediment was found to be lethal to 8 species of polychaete. The lethal effect on polychaetes is disturbing as these marine worms are a crucial component in many marine food chains. Polychaetes are particularly important to sea cage facilities where they constantly turn over the marine sediment under the cages, allowing oxygenated water to reach aerobic decomposing bacteria. b) Cypermethrin is lethal to lobster larvae at concentrations of 0.06-0.16 mg/l seawater. c) Azamethiphos at concentrations of 50 mg/l seawater was lethal to both adult shrimp and lobster. d) Sub-lethal concentrations of Azamethiphos at 5 and 10 mg/l seawater impaired spawning in female lobsters.

b) Pesticides Most commonly used pesticides for sea lice control include: 1. Azamethiphos(Salmosan) This organophosphate is given by in-feed treatments. Use of this pesticde is predicted to decline. 2. Cypermethrin(Excis) This pyrethroid compound is given by bath treatment, and widely used in both Scotland and Norway. A recent study concluded that a single cage application of cypermethrin has the potential to create a plume of up to 1 km 2 that may retain its toxicology for several hours. 3. Emamectin benzoate(slice) and Ivermectin These pesticides are administered by bath treatment. The use of Slicein sea lice control is increasing in both Scotland and Norway. Characterised by low solubility and readily binds to suspended particulate material. These pesticides are very persistent, and have a biological half-life of between 90-240 days.

c) Antifoulants The growth of seaweed, barnacles, mussels, tunicates on marine cages is a significant management problem. - reduces water exchange - increases cage resistance in the water In the 1980s, anti-fouling paints were based on tributyltin (TBT). However, TBT was found to act as a potent endocrine disrupter, causing imposex in marine gastropods

Imposex in molluscs Imposex in marine gastropods imposex is the masculinization of marine gastropods. imposex females develop non-functional male genitalia. in severe cases females become sterile. imposex has been clearly linked to exposure to tributyltin (TBT) It is believed that TBT exerts its effect by inhibiting the action of the enzyme complex aromatase SEM images showing the effects of TBT in female mudsnails (Hydrobia ulvae), producing abnormal penis (pp), sperm canal (Vd) and blocking the oviduct (OvL).

c) Antifoulants The growth of seaweed, barnacles, mussels, tunicates on marine cages is a significant management problem. - reduces water exchange - increases cage resistance in the water In the 1980s, anti-fouling paints were based on tributyltin (TBT). However, TBT was found to act as a potent endocrine disrupter, causing imposex in marine gastropods Copper-based paints have now replaced TBT. These effective paints contain copper (as copper, copper oxide or copper sulphate) and generally smaller quantities of zinc. 1 kg copper/cage Copper can leach out of the paints into the water, and subsequently accumulate in the underlying sediments. In two separate studies in Canada and Scotland, it was found that levels of both copper and zinc in sediments under salmon farms were above Government recommended safe levels. Although the long-term ecological impact of ofhigh metal concentrations in marine sediments are not known, it is known that high levels of copper and other heavy metals in seawater are toxic to marine organisms.

Impact of Antifoulants Known lethal and sub-lethal effects of heavy metals on aquatic organisms: 0.15 ppm copper induced hepatopancreas damage in a penaeid shrimp. Copper prevented seaweed germination at concentrations of 0.320 to 0.470 ppm. Sea urchin embyos were killed at 1.4 to 11.4 ppb copper (depending on exposure time) and 0.327 ppm of zinc. It has been found that copper and zinc can synergistically in eliciting toxic effects. Copper and zinc can also have many sub-lethal effects. For example: Concentrations of 0.212 ppm copper and 0.525 ppm zinc can reduce feeding and oxygen consumption in some crustaceans. Heavy metals can inhibit chemoreception Copper and zinc can disrupt the nervous system in shrimps, disrupting prey capture.

Other aquaculture sources of Heavy Metals Heavy metals, notably copper and zinc, are also present in fish feed. Often added as supplements to the feed. For example, zinc sulphateis added to salmon feeds as a way to help fish avoiding contracting cataracts. Copper, mg/kg Zinc, mg/kg Dietary requirements 5-10 37-67 Concentration in feed 3.5-25 68-240 Metal concentrations in some feeds are unnecessarily high as they exceed salmon dietary requirements.

Additional Reading Rico et al. 2013 Rico A, GengY, FocksA, Van den Brink PJ. Modelingenvironmental and human healthrisks of veterinarymedicinalproductsappliedin pond aquaculture. Environ Toxicol Chem. 2013-32(5):1196-207. doi: 10.1002/etc.2153 Love et al. 2011 Love DC, Rodman S, NeffRA, NachmanKE. Veterinary drug residues in seafood inspectedby theeuropean Union, United States, Canada, and Japan from 2000 to 2009. Environ Sci Technol. 2011. 45(17):7232-40. doi: 10.1021/es201608q Sapkota et al. 2008 Amir Sapkota, Amy R. Sapkota, Margaret Kucharski, Janelle Burke, Shawn McKenzie, Polly Walker, Robert Lawrence, Aquaculturepracticesand potentialhuman healthrisks: Currentknowledgeand futurepriorities, Environment International, 34 (8): 1215-1226, http://dx.doi.org/10.1016/j.envint.2008.04. Defoirdt et al. 2011 Defoirdt T, Sorgeloos P, Bossier P. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr Opin Microbiol. 2011 Jun;14(3):251-8. doi: 10.1016/j.mib.2011.03.004. Epub2011 Apr12. Review. PubMedPMID: 214 Burridge et al. 2011 Les Burridge, Judith S. Weis, Felipe Cabello, Jaime Pizarro, Katherine Bostick, Chemical usein salmonaquaculture: A reviewof currentpracticesand possibleenvironmentaleffects, Aquaculture, 306: 7-23 http://dx.doi.org/10.1016/j.aquaculture.2010.05.020.