1. Azasure® vet in freshwater
Method for use in well boats or similar treatment units
Juni 2016.

2. Norwegian Food Safety Authority (NFSA) about responsible use of pharmaceuticals in sea lice treatment
Food safety
Environmental impact
Safety for operators
Efficacy (not mentioned by NFSA)
Fish welfare
Resistance development against the pharmaceutical product

3. Food safety
Azasure can be use with standard doses in combination with fresh water or brackish water
Recommended withdrawal time is 10 day degrees, similar to salt water treatment.

4. Environmental impact
When treatment water is released, the environmental impact would be approximately similar as with use of Azasure in seawater
Degradation starts when Azasure comes into contact with seawater. pH>7 increase the speed of degradation

5. Safety for operators
Use safety procedures as described in the SPC for Azasure

6. Efficacy observed in bioassays

7. Azasure and freshwater. Bioassays
Treatment
# lice
Concentration
Exposure time
Neg. Control SW 25
100 % SW
24 hours in SW
Control FW 23
100 % Fw for 1 hour
1 hour in FW → 23h SW
High Aza SW 22
2ppb Aza for 1 hour
1 hour in Aza → 23h SW
Low Aza SW 19
0,4ppb Aza for 1 hour
100 % FW + High Aza
100% FW + 2ppb Aza for 1 hour
1 hour in FW+Aza → 23h SW
100 % FW + Low Aza 20
100% FW + 0,4ppb Aza for 1 hour
75 % FW + High Aza
75% FW + 2ppb Aza for 1 hour
75 % FW + Low Aza
75% FW + 0,4ppb Aza for 1 hour
50 % FW + High Aza
50% FW + 2ppb Aza for 1 hour
50 % FW + Low Aza 24
50% FW + 0,4ppb Aza for 1 hour
25 % FW + High Aza
25% FW + 2ppb Aza for 1 hour
25 % FW + Low Aza 21
25% FW + 0,4ppb Aza for 1 hour

8. Azasure in Freshwater. Bioassay

9. Efficacy and safety observed in small scale treatments

10. Water treatment
Fresh water and brackish water were buffered with NaOH to approximately pH 8, before treatment.
pH in original fresh water was approximately 5,8
The freshwater was rich in Aluminium
The fish produces CO2 under treatment, and this is expected to give a drop in pH in freshwater, in accordance with the bicarbonate equilibrium.
By using a buffer, pH can be kept at a level that does not cause problems for the fish, from Al – ions.
pH reaches 6,1 after 60 minutes treatment time.

11. n= 10 in each treatment group

12. Evaluation of efficay and safety in small scale treatment.
Sea lice was counted before treatment, immediately after treatment and 48 hours after treatment. The treatment results are partly caused by physical impact on the sea lice from handling, and use of anaesthesia on the fish.
The results show that use of freshwater 0 ‰ gives the best results on reduction in total number of sea lice and mature females.
Use of brackish water with salinity of 16 and 25 ‰, cause good treatment results in reduction of total number of sea lice. The effect on mature females are less effective compare to freshwater only.
There was no mortality under the treatment
The fish behaved normal during treatment
Evaluation of efficay and safety in small scale treatment.

13. Efficacy and safety observed under large scale treatment

16. Behavior under treatment. Mortality under/after treatment
Fresh Water is buffered with NaOH to approximately pH 8, before adding fish to the well room.
When using NaOH, pH dropped in freshwater to approximately 5,8 under treatment. The fish responded with uneasy behavior
CO2 value in treatment water was 7-10 mg/l
Presumable there are Al-ions in the freshwater at pH 5,8 which causes gill irritation and the uneasy behavior
When useing NaHCO3 behavior was normal. pH was measured to be 6,7, a level which not cause Al-ions to be toxic CO2 was measured at 5-10 mg/l
Mortality was low during and after the treatment. It was observed that cages with the highest mortality was most likely caused by handling failures
Behavior under treatment. Mortality under/after treatment

18. Observations
Well boats performing such treatment, need sea lice filters and aeration equipment available and in functioning.
Release of treatment water should take place at a proper distance from cages with fish, to prevent reinfection of sea lice.
NaHCO3 is the preferred buffer. Buffer freshwater to pH 8. During 180 minutes treatment time, pH will drop to 6,7 – 6,9.
Survey amount of CO2 in the treatment water with LaMotte test kit or other suitable equipment. Adjust the biomass in the well boat in accordance with CO2 levels. Treatment water should not contain CO2 > 15 mg/ml.
Treat the fish with freshwater for 180 minutes. The last 60 minutes with Azasure 0,2 mg/m3 (standard dose) according to the SPC.
In this case, we used 50 tons of biomass in 500 m3 well volume.

19. Resistence Genotype % of sea lice population before treatment
% of sea lice population after treatment
SS (sensitive) 3
RS (partly sensitive) 50 40
RR (resistant) 47 60
Samples analysed at Patogen Analyse AS, June 2016
There are no change in the selection profile compare with traditional seawater treatment.