Abstract

Microbial Inhibition in Response to Treatments of Hydrogen Peroxide and Formalin on Landlocked Fall Chinook Salmon Eyed Eggs as Determined by Scanning Electron Microscopy
by Hans Stephenson, Mike Barnes, and Mark Gabel

Scanning electron microscopy was used to compare microbial growth in landlocked fall Chinook salmon eggs receiving daily 15 min treatments of 1,667mg/L. formalin, 700 mg/L. hydrogen peroxide treatments, or no chemical treatments during incubation in vertical-flow incubators from egg eye-up to hatch. In eggs that were not chemically treated, bacterial numbers significantly increased from 2,413 bacteria/mm² on the external egg membrane surface at the start of the experiment to 69,598 bacteria/mm² 12 days later. Eggs receiving no chemical treatment had bacterial numbers at the end of the experiment that were significantly greater than those on eggs receiving either of the chemical treatments. The number of bacteria attached to the external egg membranes did not significantly differ between eggs treated with either formalin or hydrogen peroxide throughout the study. Fungal growth was negligible and only observed in control eggs. The external membranes of control eggs were visibly degraded over the 12-day period. Egg survival was significantly lower in control eggs compared to eggs receiving either the chemical treatments, and was significantly correlated to bacterial numbers.

Materials & Methods

Incubator trays were loaded with approximately 1522 eggs per tray. Water (11 degrees C) flowing over the trays at 8 L per minute was untreated (control), treated with 1667 mg/L of formalin, or treated with 700 mg/L of hydrogen peroxide (H₂0₂). All trays were “hand-picked” to eliminate fungal mortality in the control trays. Treatments were continued for twenty days.

The eggs were taken from the hatchery on selected days and transported in a buffer solution. The specimens then underwent inspection using the scanning electron microscope. During low vacuum observations, no additional preparation was necessary. However, for high vacuum observations eggs were dissected in buffer, treated with gluteraldhyde, osmium tetroxide, dehydrated, critical point dried, and coated with gold or gold/palladium. Five randomly selected sampling sites were viewed from three to five eggs for each sampling date, treatment, and incubator tray location. Each sample site was 32.5 x 42.5mm. Eggs were observed at 300x. Each micrograph was reviewed in order to record microbe levels and determine membrane condition.

Data were analyzed using analysis of variance with the SPSSS (9.0) statistical analysis program (SPSS 1999). Pairwise mean comparisons were performed using Fisher’s Protected Least Significance Difference, with significance predetermined at P<0.05 (Ott 1984). All embryo survival percentage data were arcsine transformed prior to analysis to stabilize the variances (Ott 1984).

 Results

Survival was 91.0%, 90.5%, and 86.7% for hydrogen peroxide, formalin, and the control treatments respectively. We found that there was a significant difference to percent of eggs hatched between the formalin treated eggs and the control treatment. There was also a significant difference between the H₂0₂ treated eggs and the control treatment. There was not a significant difference in the percent of the eggs hatched between formal in and H₂0₂ treatments.

Mean bacterial counts from formalin treated eggs on the first day of the study were 2,075/ mm² and were not significantly different by day 12 at 3,764/ mm² . Eggs treated with hydrogen peroxide averaged 5,406 bacteria/ mm² on day 1, and 6,033/ mm² on day 7. By day 12, bacterial numbers in the hydrogen peroxide treated eggs were about 2.7 times greater than the first day of the study at 14,431/ mm² although these means were not statistically significantly different. In the eggs receiving no chemical treatments, bacterial levels rose significantly from day 1(2,413/ mm²) to day 12(69,598/ mm²). Bacterial levels did not significantly differ by tray location within treatments.

 Discussion

All of the treatments used in our study appeared to restrict fungal growth on the egg surfaces. However, only the two chemical treatments adequately controlled bacterial populations attached to the external egg membranes. Given the significant correlation between attached bacterial numbers and embryo survival, we hypothesize that the increase in embryo survival from egg eye-up to fry hatch associated with either daily formalin or hydrogen peroxide treatments compared to handpicking alone is due to the inhibition of bacterial growth on the external egg membrane. The increased hatching success associated with chemical treatments may also be related to the bacteria observed on the surface of the eggs as reported by Barker et al. (1989).

We feel it is important for hatcheries to monitor their attached bacterial populations more closely. Because we feel that these attached microbes are linked to mortality, estimating the severity and spread of microbial populations would provide the basis for a prescribed anti-microbial treatment regime using formula or hydrogen peroxide.

We are currently investigating the use of simpler methodology involving low vacuum electron microscopy to increase the expediency and efficiency of monitoring microbe populations attached to the surface of salmonid eggs. Chemical could be increased as microbial populations increase and decreased as populations decrease, thereby reducing chemical costs and decreasing the discharge of chemicals in the hatchery effluent and environment.

 Pictures

Bacterial numbers per square millimeter of external egg membrane from eyed landlocked fall Chinook salmon eggs treated with either hydrogen peroxide, formalin, or no chemical treatment during incubation. Bars indicate standard error.

We would like to thank the BRIN Undergraduate Fellows Program, BHSU Nelson Grant, the hatchery spawning crews at Whitlock’s Spawning Stations, and the hatchery staff at McNenny State Fish Hatchery.