An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations.
Sarah Broadley, BSc (Hons), MSc, PGCE, Research Scientist IV, Specialty Pathology Services; Dr. Catherine Ross, DVM, MSc, MRCVS, Pathologist, Department of Pathology Covance Laboratories Ltd., Harrogate, UK
‘An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations’ WHO-ICPS, 2002.
The Endocrine Disruptor Screening Program (EDSP) is intended to determine if a pesticide, chemical or other substance may pose a risk to human health or environment due to disruption of the endocrine system. The short-term reproduction assay (Test guideline 229) is validated using the fathead minnow (Pimephales promelas) and with a subset of endpoints on the Japanese medaka and zebrafish. The assay considers the reproductive fitness as an integrated measure of toxicant effects using a suite of histological and biochemical endpoints which reflect effects associated with disturbance of the hypothalamus-pituitary-gonadal (HPG) endocrine axis.
In 2018, the OECD issued a revised Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals. The Conceptual Framework consists of five levels of tests and is designed to be used as a toolbox.
Level 1 – Sorting and prioritisation based on existing information.
Level 2 – In vitro assays providing mechanistic data.
Level 3 – In vivo assays providing data about selected endocrine mechanisms and effects.
Level 4 – In vivo assays providing data about adverse effects on endocrine mechanisms.
Level 5 – In vivo assays providing comprehensive data on effects from endocrine and other mechanisms.
The Fish Short-Term Reproduction Assay falls into Level 3 along with the Amphibian Metamorphosis Assay, Uterotrophic Assay and Hershberger’s Assay.
Currently, in the EU there are no statutory requirements to use specific tests for endocrine disruption unless requested to do so.
In the U.S. however, testing is mandatory and the U.S. EPA have developed a two-tiered strategy.
Tier 1 Assays – A selection of in vitro and in vivo assays. The Fish Short-Term Reproduction Assay and the Amphibian Metamorphosis Assay fall into this range of tests.
Tier 2 Assays – In vivo apical assays including Mammalian 2-generation toxicity, Amphibian Growth/Reproduction, Avian 2-generation, Fish 2-generation and Mysid 2-generation.
Fish Short-Term Reproduction Assay
Materials and Methods
The test requires three test concentrations and controls.
Two vessels per treatment for zebrafish (each vessel containing 5 males and 5 females).
Four vessels per treatment for fathead minnow (each vessel containing 2 males and 4 females due to the territorial behaviour of male fathead minnow).
Four vessels or replicates per treatment are used for Japanese medaka (each vessel containing 3 males and 3 females).
The following parameters are assessed:
Analytical chemistry – Weekly water sample collections from tanks to analyse test chemical levels.
Vitellogenin (Vg) – ELISA methods are used to determine the amount of this egg yolk pre-cursor protein. Vg is normally expressed only in female fish and dormant in males. However, when male fish are exposed to endocrine disruptors the Vg gene is expressed in a dose dependent manner. Therefore, the Vg gene expression in male fish has been used as a marker of exposure to oestrogenic endocrine disruptors.
Morphological observations – Appearance and observation of secondary sex characteristics including; male colouration and fat pad (presence and weight at termination), nuptial tubercle mapping, daily assessment of survival and behaviour compared to the controls.
Fertilisation success is assessed, embryos are visually inspected, infertile and fertile eggs counted.
Fecundity – Daily egg production is assessed.
Survival, behaviour and appearance – Fish are observed daily for mortality and abnormal behaviour relative to the controls
Observation of secondary sex characteristics – secondary specialised sex characteristics are also observed such as nuptial tubercles in male fathead minnow and papillary processes in Japanese medaka.
Figure 1. Figure to show nuptial tubercles on the male fathead minnow. Using a location template the quantity and size of the tubercles are recorded and ranked as: 0 - absent, 1 - present, 2 - enlarged and 3 - pronounced.
Image reproduced from OECD (2012), Test No. 229: Fish Short Term Reproduction Assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris. https://doi.org/10.1787/9789264185265-en.
Fathead minnows – The gonads are dissected from the fish and processed whole to paraffin wax.
Japanese medaka and zebrafish – The fish carcass is trimmed if required and the torso processed whole to paraffin wax.
Embedding – The gonads/fish are oriented horizontally to their long axes.
Microtomy – Three step sections are taken through the centre of the gonads and stained with Haematoxylin and Eosin.
Males – primary diagnostic criteria include: Increased proportion of spematogonia Presence of testis-ova
Increased testicular degeneration
Interstitial (Leydig) cell hyperplasia/hypertrophy
Males – secondary diagnostic criteria include:
Decreased proportion of spermatogonia
Increased vascular or interstitial proteinaceous fluid Asynchronous gonad development
Altered proportions of spermatocytes or spermatids Gonadal staging
Figure 1. Male fathead minnow gonad, x20 magnification. Bar = 50 µm.
Figure 2. Male fathead minnow gonad, x40 magnification. Bar = 20 µm. Note all stages of sperm development arranged around lobules: (1) 1˚ Spermatogonia; (2) 2˚ Spermatogonia; (3) 1˚ Spermatocytes; (4) 2˚ Spermatocytes; (5) Spermatozoa.
Females – primary diagnostic criteria include:
Increased oocyte atresia
Perifollicular cell hyperplasia/hypertrophy Decreased yolk formation
Change in gonadal staging
Females – secondary diagnostic criteria include:
Egg debris in the oviduct Granulomatous inflammation Decreased post-ovulatory follicles
Figure 3. Female fathead minnow gonad, x4 magnification. Bar = 200 µm. Note stages of oocyte development in both figures: (1) Perinuclear oocytes; (2) Cortical alveolar oocytes; (3) Early vitellogenic oocytes; (4) Late vitellogenic oocytes.
Figure 4. Female fathead minnow gonad, x40 magnification. Bar = 20 µm.
The Fish Short-Term Reproduction Assay is a screening test intended to provide an indication of potential endocrine activity, not to confirm any specific mechanism, mode of action or adverse effect. Therefore, any statistically significant effect in one or more of the endpoints of the assay may be indicative of a potential of the test material to disturb the HPG axis of fishes.
OECD (2018), Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption, OECD Series on Testing and Assessment, OECD Publishing, Paris. https://doi.org/10.1787/9789264304741-en
OECD (2012), Test No. 229: Fish Short Term Reproduction Assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris. https://doi.org/10.1787/9789264185265-en.