Results
Egg diameters were evaluated upon inspection with a microscope at 4x magnification, which was calibrated using a clear ruler to measure to +/-.01 mm. The initial egg diameters from clutch 1 ranged from 1.40-1.65 mm (mean = 1.416mm), and from 1.15-1.65 mm (mean = 1.442 mm) for clutch 2. The treatment levels were dosed with no additional CO2 added to the control group, one CO2 tank continuously diffusing CO2 at a rate of 1-3 bubbles per second for the medium treatment, and two CO2 tanks continuously diffusing CO2 at a rate of 1-3 bubbles per second for the high treatment.
Figure 1. Egg diameters across treatments. Clutch 1 (blue) and clutch 2 (red) egg diameters with respect to treatment groups. Error bars are added based on standard error (σ/√n). CO2 dose was evaluated by monitoring pH levels with mean values of 8.1, 7.8, and 7.3 for control, medium, and high treatments respectively.
Haphazard samples were taken periodically throughout the experiment with a total sampling size of n=25 for each treatment. For clutch 1 statistical tests using Sigmaplot 12.5 Systat Software, Inc., San Jose California USA to check for statistical differences among treatments. A Shapiro-Wilk test was run to check for normality and failed (P<.050); a Friedman analysis of variance test gave a Chi-square= 7.053 with 2 degrees of freedom. (P = 0.029) for clutch 1, and Chi-square= 21.143 with 2 degrees of freedom. (P = <0.001) for clutch 2. Finally a pair-wise Tukey test for multiple comparisons was used for clutches 1 and 2 with a (p=<.05) for control groups vs. the high-CO2 treatment groups.
pH
pH values were measured using a Vernier handheld pH probe which was 3-way calibrated using pH buffer solutions of 4.00, 7.00, and 10.00. There was a dramatic drop in pH when CO2 was added, but this was quickly followed by a rise in pH and another final drop towards the end of the experiment.
Graph 1. pH fluctuations during the length of the experiment with regression; R2 = 0.1653 for the control tank, R2 = 0.4793 for the medium CO2 tank, and R2 = 0.5496 for the high CO2 tank.
Rate of hatching
The rate in hatching was reported in the number of days it took from the onset of eye pigmentation (pharyngeal phase) until the first signs of hatching. The presence of live, free-swimming larvae confirmed hatching. Percent of hatching was estimated based on the total number of hatched larvae after a period of 1-2 days post initial hatching.
Figure 2. The percentage of hatching observed. The initial sample size was n=200 per treatment for clutch 1 and n=300 for each treatment in clutch 2. The percentage of hatching was based on the number of hatched herring present at the end of the study divided by the initial sample size (# of hatched herring/sample size) X 100.
Developmental morphology
Development was observed under 4X magnification and the presence or absence of developmental features was noted among haphazard samples.
Figure 3. Control tank sample. This represents early development where the body is just starting to form around the yolk sac (segmentation phase).
Figure 4. This figure shows later stage development when the eyes are fully pigmented and the embryo is wrapped around the yolk sac twice (pharyngula stage).
Figure 5. Represents a newly hatched herring larva that is approximately 7 mm in length.