Optimizing Sex Ratios of Hyalella azteca to Reduce Variability in Reproduction and Improve Reproductive Toxicity Test Methods

Abstract

Standard toxicological test methods with Hyalella azteca incorporate both lethal and sublethal (growth, reproduction) endpoints, though lethal endpoints are often favoured in routine testing. However, sublethal endpoints are important to consider as they are ecologically relevant and are often more sensitive than lethality. It is difficult to achieve robust data for reproduction in H. azteca because a high biological variability associated with reproductive yield, likely due to complex interactions in mate selection which can add to the variability in brood sizes that female produce. In addition, effects on reproduction often co‐occur with effects on growth, making it difficult to differentiate effects of growth from those on reproduction in standard tests initiated with juveniles. This study characterized the reproductive capacity of H. azteca by investigating the role of sex ratios in reproductive yield. Experiments were initiated in the absence of toxicants with sexually mature (6‐8‐week‐old) individuals that were placed in different female to male ratios (1:1, 2:3, 3:2 and 7:3). Reproduction was monitored during weekly static renewals for seven weeks. Results indicated that a higher female:male ratio (7 females to 3 males) improved reproductive success and lowered biological variability in reproduction. In addition, the body lengths and weights of newly mature amphipods were used to create a size distribution to aid in the identification of small female amphipods. Based on the results of this study, a novel reproductive toxicity test method is proposed that begins with sexually mature amphipods in a ratio of seven females to three males to minimize reproductive variability in amphipods and to enable a more effective assessment of contaminants using reproduction as a highly sensitive endpoint.

Novel Reproduction Toxicity Test Method for Hyalella azteca Using Sexually Mature Amphipods Provides More Robust Data Than Standard Methods in Exposures to Imidacloprid

“…The amphipods were fed a ramped diet of ground TetraMin three times a week: 5 mg three times in weeks 1 and 2, 10 mg three times in weeks 3 and 4, and 15 mg three times in weeks 5 and 6. At 6 weeks old, the amphipods were identified and separated by sex for 7 days before use in testing to allow females to release their current broods (Khan et al, 2023). Novel tests were then initiated by adding 20 sexually mature amphipods to each replicate in a ratio of seven females to three males (Khan et al, 2023).…”

“…At 6 weeks old, the amphipods were identified and separated by sex for 7 days before use in testing to allow females to release their current broods (Khan et al, 2023). Novel tests were then initiated by adding 20 sexually mature amphipods to each replicate in a ratio of seven females to three males (Khan et al, 2023). A subset of females and males was weighed (dry weights) at the start of each test.…”

“…Novel (28‐day) tests were initiated with sexually mature amphipods (7 weeks old). Methods were optimized to grow enough sexually mature amphipods for use in tests: 400 juvenile amphipods were placed in a 5‐L high‐density polyethylene bucket containing 4 L of culture water and eight pieces of 7.5‐ × 7.5‐cm cotton gauze, and water was renewed weekly (Khan et al, 2023). The water was not aerated, to minimize disturbance and physical stress on the growing amphipods (Khan et al, 2023).…”

“…To address the challenges of using reproduction as an endpoint in H. azteca toxicity testing, a novel toxicity test method was developed and evaluated to determine the applicability and efficacy of assessing reproduction effects from water-soluble contaminants in environmental monitoring (Khan et al, 2023). This 28-day method was initiated with sexually mature amphipods to address the confounding effects of growth on reproduction and ensure that reproductive success during the test was not limited by reduced and/or delayed growth.…”

“…This 28‐day method was initiated with sexually mature amphipods to address the confounding effects of growth on reproduction and ensure that reproductive success during the test was not limited by reduced and/or delayed growth. In addition, the roles of sex ratios were investigated, and it was determined that a sex ratio with excess females (seven females:three males) resulted in reduced variability in reproduction, likely due to increased female availability and less male competition for mates in the first 28 days of experiments (Khan et al, 2023). Finally, these methods were developed as water‐only exposures to simplify the recovery of juveniles and expand the focus of toxicity testing to contaminants found in surface waters.…”