FISHERY BULLETIN: VOL. 73, NO. 1 



bath that recycled through both charcoal and a 

 gravel bacterial filter at a rate of 3 gallons/min. 

 Ammonia levels were measured periodically 

 and never attained 0.1 ppm. All ammonia 

 analyses w^ere made by separating ammonia by 

 diffusion (Conway and Cooke 1939) and followed 

 by nesslerization of the separated ammonia. For 

 the ammonia bioassays, the small trays were 

 transferred directly to the experimental medium. 

 By conducting the bioassays in the same trays 

 in which the eggs or larvae were incubated, 

 we did not have to pipette them to other con- 

 tainers — a process that might have injured them. 



Two series of duplicated ammonia toxicity bio- 

 assays were conducted according to standard pro- 

 cedures outlined by DoudorofF et al. (1951) and 

 results were expressed as 24-h median tolerance 

 limits (24-h TLm)^. The bioassays were conducted 

 every 4 to 7 days from fertilization to the com- 

 pletion of yolk sac absorption. Toxicity of am- 

 monia to adult rainbow trout (length 7-9 inches) 

 was also measured with static bioassays (12 fish 

 per concentration tested, 1 fish per 10-liter aquar- 

 ium) at the same water temperature and pH 

 used with the eggs and larvae. 



All bioassays were conducted in aged tap water 

 (total hardness 5.94 ppm as calcium carbonate 

 at pH 7.8) adjusted to pH 8.3 with tris buffer 

 (final concentration 0.05 M). Ammonia, in the 

 form of ammonia sulfate, was added to arrive 

 at the various test concentrations. The resulting 

 conditions made the ammonia toxicity assays 

 more severe than would normally be encoun- 

 tered because the toxicity of ammonia increases 

 as pH increases due to the conversion of ionized 

 NH^ into the un-ionized NHg form. At 10°C and 

 pH 8.3, 3.58% of the ammonia in water is un- 

 ionized, considerably more than the 0.19% un- 

 ionized ammonia at pH 7 (Trussell 1972). 



Since the un-ionized form of ammonia has been 

 identified as the toxic form, we report our results 

 in units of un-ionized ammonia rather than total 

 ammonia. 



Several water quality parameters were mea- 

 sured at the beginning and end of the bioassays, 

 since changes could affect the results. Ammonia 

 levels never dropped below 93% of the initial 

 bioassay concentrations during the course of the 

 24-h experiments. Very low levels of un-ionized 

 ammonia (0.011 ppm) were detected in the con- 



trol exposures after 24 h. The tris buffer prevented 

 any changes in pH from occurring during the 

 24-h tests. Dissolved oxygen remained above 

 91% saturation in the shallow egg-alevein bio- 

 assay containers and above 88% saturation in the 

 adult bioassays (measured with YSI oxygen 

 probe).'* Carbon dioxide was not measured in any 

 of the bioassays. 



We tested the influence of ammonia on egg 

 fertilization and viability during the water- 

 hardening stage by exposing some eggs to am- 

 monia at Bowden Hatchery on the day our experi- 

 mental eggs were collected. Approximately 200 

 to 300 eggs from one female were stripped into 

 each of several pans containing tris buffered 

 water (pH 8.3, temperature 8°-10°C), some with 

 added ammonia at concentrations up to 1.79 

 mg/liter of un-ionized ammonia. Milt from at 

 least two young males was stripped into each 

 pan of water and eggs 15 to 30 s later. Buss and 

 Corl (1966) determined that fertilization must 

 be completed within the first 1 or 2 min because 

 the sperm are viable in water for only a few 

 seconds. By replacing the ammonia solutions with 

 fresh water in one-half of the pans after 2 or 3 min 

 of ammonia exposure and in the remaining pans 

 after 1 h, we hoped to separate the effects of 

 ammonia on fertilization per se from the effects 

 on the viability of the fertilized eggs during the 

 water-hardening stage of the first hour. The 

 effects were measured by determining the per- 

 centage of eggs that hatched. 



RESULTS AND DISCUSSION 



Neither fertilized eggs, embryos, nor alevins 

 (embryo after hatching) were susceptible to a 24-h 

 exposure of un-ionized ammonia (3.58 mg/liter) 

 until about the 50th day of development (Figure 

 1). At that time, susceptibility increased dramati- 

 cally and continued to increase until most of the 

 yolk was absorbed (when alevins became fry). 

 The median tolerance limits (24-h TLm) for 85- 

 day-old fry were 0.068 mg/liter, slightly less 

 than the 0.097 mg/liter value we observed for 

 adult trout; in the bioassays for both the fry 

 and the adults, temperature was 10°C and pH 

 was 8.3. 



Buss and Corl (1966) found that the viability 

 of eggs of brook trout, Salvelinus fontinalis, and 



'24-h TLm = the concentration resulting in 50% survival 

 after 24-h exposures. 



■•YSI = Yellow Springs l.astrument Company, Inc., Yellow 

 Springs, Ohio. Reference to trade name does not imply endorse- 

 ment by the National Marine Fisheries Service, NOAA. 



208 



