Challenges in current adult fish laboratory reproductive tests: Suggestions for refinement using a mummichog (Fundulus heteroclitus) case study

Abstract

Concerns about screening endocrine-active contaminants have led to the development of a number of short-term fish reproductive tests. A review conducted of 62 published adult fish reproductive papers using various fish species found low samples sizes (mean of 5.7 replicates with a median of 5 replicates) and high variance (an average coefficient of variance of 43.8%). The high variances and low sample sizes allow only relatively large differences to be detected with the current protocols; the average significant difference detected was a 68.7% reduction in egg production, while only differences above 50% were detected with confidence. This result indicates low power to detect more subtle differences and a high probability of type II errors in interpretation. The present study identifies several ways to increase the power of the adult fish reproductive test in the mummichog (Fundulus heteroclitus). By identifying the peak timing of egg production (before and after the new moon), extending the duration of the experiment (increased from 7 to 14 d), and determining that a sample size of eight replicate tanks per treatment accurately predicts variance in the sample population (based on pre-exposure variation calculations of replicate tanks), the power of the test has been significantly increased. The present study demonstrates that weaknesses in the current adult fish reproductive tests can easily be addressed by focusing on improved understanding of the reproductive behavior of the test species and developing study designs that include calculating desired variability levels and increasing replicates.

Transgenerational effects of polycyclic aromatic hydrocarbon exposure on sheepshead minnows (Cyprinodon variegatus)

“…High variability between tanks lowered statistical power and limited interpretation of F 1 egg production data. Although there is no consensus on critical effect size for fish reproductive tests, a commonly used size is 25%, indicating that a 25% change in egg production would cause adverse effects on the fish population (Bosker et al ; Munkittrick et al ). This suggests that the changes observed in egg production in the present study, although not statistically significant, could be biologically significant and lead to population‐level consequences in sheepshead minnow.…”

“…After acclimation to the environmental conditions, a 10‐d pre‐exposure was performed to establish baseline egg production and determine exposure groups (Bosker et al ). During this pre‐exposure period, one breeding net made of a PVC ring with fine nylon mesh (335 μm) was placed into each tank to provide a spawning substrate for female fish.…”

“…Six tanks were assigned to each of 3 exposure groups based on average daily egg production over the 10 d (control 25.3 ± 3.4, low HEWAF 26.1 ± 2.9, high HEWAF 27.0 ± 3.2). Tank assignments were performed following guidelines set forth in Bosker et al () to ensure that any changes in egg production between groups during the exposure phase were attributable to the HEWAF, not to natural variation in egg production between fish.…”

Mummichog (Fundulus heteroclitus) continue to successfully produce eggs after exposure to high levels of 17α‐ethinylestradiol

“…This approach is directly comparable to other short‐term reproductive tests (e.g., fathead minnow and zebrafish ( Danio rerio ) ), in which eggs are collected in a (pre‐) exposure phase. This elongated period of egg collection reduces the variance in cumulative egg production, thereby reducing the chance of making a type I error (i.e., saying there is an impact when, in fact, there is none) . The present study was conducted to determine whether reproductive success measured as fecundity was affected when mummichog were exposed to 100 ng EE2/L.…”

Effects of 17α-ethynylestradiol on early-life development, sex differentiation and vitellogenin induction in mummichog (Fundulus heteroclitus)

“…Partial lifecycle and short-term bioassays have been developed for estuarine and marine species; examples include sheepshead minnow, Cyprinodon variegates (Folmar et al, 2000;Zillioux et al, 2001;Hemmer et al, 2008) and mummichog, Fundulus heteroclitus (MacLatchy et al, 2003;Boudreau et al, 2004;Peters et al, 2007;Bosker et al, 2009). Effects of EDSs on species living in salt water may be quite different than 0141-1136/$ -see front matter Ó 2009 Elsevier Ltd. All rights reserved.…”