1. UoP 677465
Abstract The study investigated the potential of novel essential oils to be used as treatments in reducing the bacterial load of Artemia
fransicana when used as a live food in aquaculture, compared to existing treatments. The essential oil Origanum vulgare completely
inhibited hatching efficiency. All treatments significantly reduced presumed Vibrio spp. counts (P=0.001). None of the essential oils
tested significantly reduced general bacteria loading. Rosmarinus offcinalis was the only essential oil tested that reduced presumed Vibrio
spp. without inhibiting hatching efficiency.
Introduction Artemia fransicana are a valuable live food in aquaculture;
nutritionally, and conveniently as they can be hatched from cysts as required
[1,2,3,4]. Artemia fransicana cysts harbour bacteria, this combined with the release
of glycerol during hatching can lead to bacterial blooms that may be exposed
to the organisms being fed [5,6,7,8,9]. Existing methods of bacterial load control
are available including; antibiotics [10], probiotics [11], de-capsulation [10],
chemical [12], water exchange [13], bio-flocs [14], and commercial purpose specific
disinfectants [15]. Existing methods can be time consuming, or do not
significantly enough reduce the bacterial loading, some also using chemicals
that may in the future face bans [16,17,18]. Concerns with current methods
leaved potential for active ingredient rich essential oils to be explored, with
research available in-vitro on essential oils antibacterial capabilities [19,20,21], but
limited existing published work available for their use in aquaculture [22].
Methodology The concentrations of essential oils tested were based on in-
vitro studies; Oreganum vulgare 100/50mg/l [22,23,24], Cinnamomum verum 50mg/l
[26,27,28], and Rosmarinus offcinalis 10mg/l [20,29,30]. Methanol control at 10mg/l
(same dose as solvent for each essential oil), and existing treatments dosed
following manufactures specifications. Culture equipment was sterilised and
kept sterile using procedures including; shock chlorination, hydrochloric acid,
air filters, de-humidifiers, and fume cupboards. Artemia fransicana cysts were
incubated for 24 hours, and hatched in triplicated batches at; 1g/l, 28°C, and
~2000 lux. Bacteria loads were assessed by counting CFU (Colony Forming
Units) on TSA (Trypticase Soy Agar), presumed vibrio strains were assessed
using CFU on TCBS (Thiosulfate-Citrate-Bile Salts-Sucrose). Photo-microscopy
used FoldScope to observe any biological impacts of treatments [31]. Results
compared for significant differences between treatments using Minitab 17.0,
tests conducted included; nested anovas, one-way anovas, Box-Cox
transformations, and Tukey post-hocs.
for average values (±SE) of presumptive vibrio counts and unspecified
bacterial counts. Presumed vibrio counts showed Oreganum vulgare (50mg/l &
100mg/l), Cinnamomum verum, Rosmarinus olfinalis, Sanolife ACE, and Sanolife
Mic-F had significantly lower counts than the control, Sanolife Ace showed
significantly lower counts than all other treatments for TSA agar.
PresumedVibrio Count
(10-3 CFU/mL)
Unspecific Bacteria
Count (10-5 CFU/mL)
Oregano vulgare (100mg/l) 0±0d* 31.57±14.01b
Cinnamomum verum (50mg/l) 107.47±64.45bc 273.33±194.27b
Rosmarinus offcinalis (10mg/l) 126.88±65.64bc 209.67±131.51ab
Control (NoTreatment) 753.33±64.90a 228.67±115.74ab
Sanolife ACE 0±0d* 0.00087±0.00045c
Sanolife Mic-F 238.67±110.76bc 394±326a
Methanol (10mg/l) 586.67±202.76ab 208.33±70.28ab
Origanum vulgare (50mg/l) 1.44±1.38c 181.97±144.80b
a Different superscript letters indicate significant difference between treatments.Asterisks
indicates significant difference determined by all 0 value results.
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HE count/mL
Oreganum vulgare (100mg/l) 0±0d*
Cinnamomum verum (50mg/l) 18±3.27c
Rosmarinus offcinalis (10mg/l) 94±8.38ab
Control 131.33±16.64a
Sanolife ACE 98±6.41ab
Sanolife Mic-F 62.67±6.58b
Methanol 109.33±7.90a
Origanum vulgare (50mg/l) 0±0d*
a Different superscript letters indicate significant difference between treatments (P= 0.000).
Asterisks indicates significant difference determined by all 0 repeated results.
Figure 1 Cultures Subjected to DifferentTreatments,Taken Using FoldScope [Primary source].
Results Developmental differences can be seen in figure 1; ‘A’ Oreganum
vulgare showing rupture cysts with inert nauplii, ‘B’ non motile nauplii from
Cinnamum verum treatment , and ‘C’ all other cultures motile nauplii.
Table 2 AverageValues (±SE) of PresumptiveVibrio Counts and Unspecified Bacterial
Counts from Artemia fransicana CultureWater with DifferentTreatments.
Conclusions All three essential oils tested showed significant
reductions on the levels of presumed vibrio bacterium within the
Artemia fransicana culture medium. Oreganum vulgare and
Cinnamomum verum both significantly inhibited the hatching
efficiency of the Artemia fransicana cysts. The unexpected toxicity
rules out these essential oils for use for Artemia fransicana when
used at the concentrations they were. It’s likely that the following
previous studies concentrations for toxicity levels were inaccurate
because of the nature of distilling essential oils, and the wide
variation of levels of active ingredients between batches. In future
studies it should first be calculated what the maximum
concentration of the essential oil is before its shows signs of
significant toxicity. Rosmarinus olfinalis didn’t inhibit hatching
efficiency, but reduced vibrio levels compared to the control. Results
for Rosmarinus olfinalis highlighted its potential, and it should be
further tested to identify; maximum non-lethal concentrations, the
effect on Artemia fransicana nutritional content, and the potential
risk of bioaccumulation of active ingredients from the essential oil.
Table 1 Average Hatching Efficiency (±SE) 0.001g cysts/mL withTested Treatments.
Table 1 shows average hatching efficiency (HE) of 0.001g cysts/mL for each
treatment (±SE). Both Oreganum vulgare treatments completely inhibited
hatching, with no other showing significant difference except Cinnamomum
verum with significantly lower hatch hatching efficiency.Table 2 showed results