Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School

Natural Resource Management

First Advisor

Michael Brown


aquaculture, fugacity, genistein, phytoestrogen, rainbow trout


Fish production from aquaculture has continued to grow over the past several decades, with finfishes being fed formulated, fish meal-based diets supporting most of that industry’s growth. However, increased demand and costs, coupled with static volume and erratic supplies of fish meal, has resulted in the use of alternative plant-derived protein sources. A common oilseed protein source is soybean meal (SBM), because of its protein content and amino acid profile; however, it also contains multiple anti-nutritional factors, including phytoestrogens. Phytoestrogens are naturally produced, estrogenmimicking compounds, with isoflavones receiving the most investigation as higher levels of these compounds occur within SBM. Isoflavone content of SBM can vary widely based on numerous factors from varieties and growing conditions to processing effects. A wide array of in vivo and in vitro studies on isoflavones have been performed on numerous fish species, with results ranging from competition for estrogen receptor sites to intersex characteristics in gonads. Likewise, a variety of different factors (i.e., phytoestrogen compounds, inclusion levels, species, life stage, etc.) and various combinations appeared to influence the potential effects (if any) of phytoestrogens upon finfishes with no distinct relationships occurring among species or with specific phytoestrogens or inclusion levels were discovered in the literature. To study the effects of the isoflavone genistein on growth, condition, and estrogenic responses of first-feeding Rainbow Trout (Oncorhynchus mykiss; initial weight = 109 ± 22 mg [mean ± SD]), a 112-day feeding trial was performed by impartially and randomly stocking larvae into 24, 110-L glass aquaria providing six replicate aquaria per treatment. The larvae were fed one of four experimental diets containing the following SBM:genistein (g/100 g) ratios: 0:0 (Ref), 20:0 (NoGen), 20:0.124 (LoGen), and 20:0.249 (HiGen). Individual mean total length and weight was measured at 2-week intervals for the duration of the feeding trial. Plasma vitellogenin (VTG) was measured from blood samples collected nine days after feeding trial termination. Growth and condition parameters were significantly higher for fish fed the Ref diet, while growth was significantly lower for HiGen when comparing SBM-based diets only. Hepatosomatic index values and mortality rates were not significantly different. Plasma VTG levels formed a U-shaped response curve with fish fed NoGen and HiGen diets having statistically lower and higher plasma levels, respectively, while those fed Ref and LoGen diets were similar. These results indicated that SBM inclusion and increasing levels of dietary genistein did negatively impact growth and condition of firstfeeding Rainbow Trout. Also, the U-shaped VTG response curve lends further evidence to the dualistic role of genistein as an estrogen agonist and antagonist. While numerous studies have looked at the various biological and physiological effects of dietary and water-borne exposure to genistein and other phytoestrogens, not much emphasis has been placed on the incorporation and/or pathways of such chemicals within and out of aquaculture systems. Likewise, the use of fugacity models to depict chemical partitioning within such systems remains limited. As such, genistein concentrations were measured in the culture water and fish compartments of 18, 110-L microcosms containing larval Rainbow Trout fed the same experimental diets as mentioned above, except for the LoGen treatment that was removed from analyses due to funding constraints. Genistein concentrations were measured in whole-body fish tissue samples, fish livers, fecal material, and culture water samples at mid- (56 or 63 days of experiment [DOE]) and end-points (112 or 119 DOE). Significant differences occurred between the mid- and end-point measurements for all four parameters along with statistically significant differences among all treatments for whole-body, fecal, and livers samples. For water samples, the Ref treatment was statistically lower than both the NoGen and HiGen. Level I and II fugacity models consisting of three environmental compartments (water, fish, and air) were developed using parameters derived from the literature. Estimated compartment concentrations were determined at both the mid- and end-points using genistein amounts from the last 2 weeks of feeding (L2W) or total amounts fed (TGA) to each study point. The model estimates were then compared against the empirically derived data using percent bias (PBIAS) to determine goodness-of-fit of the models resulting in a wide range of PBIAS values. For Level I models, PBIAS ranged from (-2,370 to 96%) with several models falling in the acceptable PBIAS range (-70 to 70%). For Level II models, all models underestimated and were >94% PBIAS except for water at the mid-point using TGA data. Genistein concentrations in the samples indicated that over time, the fish appeared to excrete larger amounts of the genistein, thereby, increasing concentrations within the culture water, most likely due to ontogenetic changes. The concentrations within the culture water were similar to genistein levels seen from other effluent sources and at levels deemed biologically relevant; therefore, dietary genistein from aquaculture diets could be considered a contaminant of emerging concern. Results from the fugacity models indicated that, while relatively simplistic in nature and capable of determining compartment partitioning, several necessary assumptions needed for Level I and II fugacity models were unmet resulting in poor compartment concentration estimates. However, PBIAS results from several models did show acceptable estimates meriting further investigation into the use of such models due to the relative simplicity and ease of use.


South Dakota State University



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