Lisa N. Taylor

Efficient induction of spawning of Northern leopard frogs (Lithobates pipiens)during and outside the natural breeding season

BackgroundAmphibian declines are now recognized globally. It is also well known that many anurans do not reproduce easily in captivity, especially when held over long periods, or if they require hibernation before breeding. A simple method to induce spawning and subsequent development of large numbers of healthy tadpoles is therefore required to meet research and conservation goals.MethodsThe method is based on simultaneous injection of both female and male leopard frogs, Lithobates pipiens (formerly called Rana pipiens) with a cocktail of a gonadotropin-releasing hormone agonist (GnRH-A) and a dopamine antagonist. We call this the AMPHIPLEX method, which is derived from the combination of the words amphibian and amplexus. Following injection, the animals are thereby induced, and perform amplexus and natural fertilization under captive conditions.ResultsWe tested combinations of a GnRH agonist with 2 different dopamine antagonists in L. pipiens in the breeding season. The combination of des-Gly10, D-Ala6, Pro-NHEt9-GnRH (0.4 micrograms/g body weight; GnRH-A) with metoclopramide hydrochloride (10 micrograms/g body weight; MET) or domperidone (DOM) were equally effective, producing 89% and 88% successful spawning, respectively. This yielded more than 44,000 eggs for the 16/18 females that ovulated in the GnRH-A+MET group, and more than 39,000 eggs for the 15/17 females that ovulated in the GnRH-A+DOM group. We further tested the GnRH-A+MET in frogs collected in the wild in late autumn and hibernated for a short period under laboratory conditions, and report a low spawning success (43%). However, GnRH-A priming 24 hours prior to injections of the GnRH-A+MET cocktail in animals hibernated for 5–6 weeks produced out-of-season spawning (89%) and fertilization (85%) comparable to those we observed for in-season spawning. Assessment of age and weight at metamorphosis indicated that L. pipiens tadpoles resulting from out-of-season spawning grew normally and metamorphosed successfully.ConclusionWe provide evidence for successful captive breeding of the leopard frog, L. pipiens. This simple protocol can be used to obtain large numbers of eggs in a predictable, timed manner.

Using an interlaboratory study to revise methods for conducting 10-d to 42-d water or sediment toxicity tests with Hyalella azteca.

Studies have been conducted to refine US Environmental Protection Agency, ASTM International, and Environment Canada standard methods for conducting 42-d reproduction tests with Hyalella azteca in water or in sediment. Modifications to the H. azteca method include better-defined ionic composition requirements for exposure water (i.e., >15 mg/L of chloride and >0.02 mg/L of bromide) and improved survival, growth, and reproduction with alternate diets provided as increased rations over time in water-only or whole-sediment toxicity tests. A total of 24 laboratories volunteered to participate in the present interlaboratory study evaluating the performance of H. azteca in 42-d studies in control sand or control sediment using the refined methods. Improved growth and reproduction of H. azteca was observed with 2 alternate diets of 1) ramped diatoms (Thalassiosira weissflogii) + ramped Tetramin or 2) yeast-cerophyll-trout chow (YCT) + ramped Tetramin, especially when compared with results from the traditional diet of 1.8 mg YCT/d. Laboratories were able to meet proposed test acceptability criteria and in most cases had lower variation in growth or reproduction compared with previous interlaboratory studies using the traditional YCT diet. Laboratory success in conducting 42-d H. azteca exposures benefited from adherence to several key requirements of the detailed testing, culturing, and handling methods. Results from the present interlaboratory study are being used to help revise standard methods for conducting 10-d to 42-d water or sediment toxicity exposures with H. azteca. Environ Toxicol Chem 2016;35:2439-2447.

An in vitro approach for modelling branchial copper binding in rainbow trout

The main objective of this study was to characterize the individual effects of water chemistry (Ca(2+), Na(+), dissolved organic matter (DOM), pH, alkalinity) on the rapid binding of copper to the gill surface of rainbow trout using an in vitro gill binding assay. In this assay, individual gill arches were exposed for 5 min to (64)Cu labelled copper solutions ranging from 0.02 to 0.16 microM in water chemistries reflecting the full range of fresh water values for the Great Lakes. The gills displayed saturable Cu binding within this Cu range but gill-Cu binding was completely unaffected over the full range of calcium, sodium and alkalinity concentrations used. Only low pH (pH 4.0) and commercial DOM (Aldrich humic acid at > or =3 mgC/l) altered copper binding to rainbow trout gills in vitro. These findings were consistent with the results of geochemical modelling of our water chemistry (using MINEQL+, Version 4.5) which showed that H(+) and DOM affected the free cupric ion concentration. However, DOM (up to 80 mgC/l) was only able to reduce Cu on the gills by 50%. We hypothesize that in the range of 0.02-0.16 microM Cu there are two high affinity Cu binding sites on the gills, one having a substantially higher affinity for copper than DOM. The absence of a calcium effect on gill copper binding was in accord with in vivo evidence that calcium primarily acts to alter the physiology of the gill binding sites through acclimatory processes, rather than through competitive interactions. It was a surprise that water chemistry parameters influence rapid gill-metal binding in a manner different to their influence on acute toxicity and different from the effects on long-term binding reported in other studies. Currently, the biotic ligand model uses the rapid increase of gill copper (believed to reflect binding to the physiologically active receptor sites) to model gill binding characteristics. The distinction between rapid surface binding and metal uptake obviously plays an important role in determining the toxic effects of copper, especially when regulators need to predict the modifying effects of water chemistry.

Validation of a new standardized test method for the freshwater amphipod Hyalella azteca: Determining the chronic effects of silver in sediment

Environment Canada has developed a new 42-d sediment toxicity test method that includes a reproduction test endpoint with the freshwater amphipod Hyalella azteca. Because of concerns that existing standard methodologies, whereby adults are transferred to a water-only exposure before release of their first brood at day 28, will lead to internal contaminant depuration and loss of sensitivity, the Environment Canada methodology conducts the entire exposure in sediment. To demonstrate applicability of the method for assessing the toxicity of chemical-spiked sediment, H. azteca were exposed for 42 d to sediment amended with silver nitrate (AgNO3 ). Mortality was significantly higher at the highest sediment concentration of Ag (2088 mg/kg dry wt); however, there was no significant reduction in biomass or reproduction as a result of Ag exposure despite significant bioaccumulation. Based on Ag measurements and speciation modeling, the principle route of Ag exposure was likely through the ingestion of complexed colloidal or particulate Ag. The techniques used to recover young amphipods from sediment were critical, and although this effort can be labor intensive (20-45 min/replicate), the technicians demonstrated 91% recovery in blind trials. For the first time, Environment Canada will require laboratories to report their recovery proficiency for the 42-d test-without this information, data will not be accepted. Overall, the reproduction test will be more applicable when only a few chemical concentrations need to be evaluated in laboratory-amended sediments or for field-collected contaminated site assessments (i.e., contaminated site vs reference site comparisons). Environ Toxicol Chem 2016;35:2430-2438.

Sublethal Toxicity Testing of Canadian Metal Mining Effluents: National Trends and Site-Specific Uses

ABSTRACT As part of the Canadian Environmental Effects Monitoring program under the National Metal Mining Effluent Regulation, there is a requirement to conduct sublethal toxicity tests twice per year for the first three years. These first three years (2003 to 2005) were considered a period of initial monitoring and resulted in test endpoints for each of the required standardized methods on a fish, an aquatic plant, an invertebrate, and an algal species. On a national level (based on 1648 valid results), the test from most to least sensitive was: the inhibition of reproduction with Ceriodaphnia dubia, the growth inhibition (frond number) with Lemna minor, the inhibition of cell yield with Pseudokirchneriella subcapitata, the growth inhibition (dry weight) with Lemna minor, the growth inhibition of fathead minnows, and the effect on embryo viability with rainbow trout. This sensitivity ranking changed when tests were further evaluated on a geographical region and mine-type basis (e.g., base metal, precious metal, uranium, iron ore). Site-specific examples show how sublethal toxicity data are being used to track changes in effluent quality, choosing a final discharge point, monitoring multiple discharges to the same watercourse, and to identify study design weaknesses by comparing laboratory results to field survey conclusions.

Inter‐laboratory validation of organism recovery for use in 42 day sediment toxicity tests with Hyalella azteca

Environment and Climate Change Canada has developed a 42-d sediment toxicity test that includes a reproduction endpoint with the freshwater amphipod Hyalella azteca. The new methodology conducts the entire exposure in sediment, in contrast to existing standardized methods whereby adults are transferred to a water-only exposure before release of their first brood at day 28. This midtest transfer to clean water was because of the results of a juvenile H. azteca recovery trial conducted in the 1990s concluding that reproductive endpoints could be biased because of low recovery of young amphipods from sediment. Using a new procedure and reduced volume of sediment, an interlaboratory recovery trial was conducted using 2-d to 5-d old H. azteca added to control sediment. A total of 29 technicians from 8 laboratories participated in the present study. The average recovery for all laboratories and all technicians was 76% (coefficient of variation [CV] = 30%). Based on an initial target recovery of at least 80%, 19 of 29 (66%) technicians met this criterion, with an average recovery for this group of 88% (CV = 8.3%). Factors that reduced recovery success included: not using a light table, technicians with minimal sediment testing experience, and the use of imported young amphipods with limited acclimation. Excluding those results, the overall average recovery, which included 17 participating technicians, increased from 76% to 88% and lowered the CV from 30% to 8.6%. Based on these results, Environment and Climate Change Canada will recommend ≥85% average recovery of young in control sediment and require ≥80% as a technician performance criterion in its new test design for the reproduction methodology. Environ Toxicol Chem 2017;36:1085-1089.