The Uptake of Ethinyl-Estradiol and Cortisol From Water by Mussels (Mytilus spp.)

Abstract

Previous toxicokinetic studies have shown that mussels (Mytilus spp.) can readily absorb the three main mammalian sex steroids, estradiol (E2), testosterone (T) and progesterone (P) from water. They also have a strong ability to store E2 and the 5α-reduced metabolites of T and P in the form of fatty acid esters. These esters were shown to have half-lives that were measured in weeks (i.e. they were not subject to fast depuration). The present study looked at the toxicokinetic profile of two other common steroids that are found in water, the potent synthetic oestrogen, (ethinyl-estradiol) (EE2; one of the two components of ‘the pill’), and cortisol, a natural stress steroid in vertebrates. In the first three hours of uptake, tritiated EE2 was found to be taken up at a similar rate to tritiated E2. However, the levels in the water plateaued sooner than E2. The ability of the animals to both esterify and sulphate EE2 was found to be much lower than E2, but nevertheless did still take place. After 24 h of exposure, the majority of radiolabelled EE2 in the animals was present in the form of free steroid, contrary to E2, which was esterified. This metabolism was reflected in a much lower half-life (of only 15 h for EE2 in the mussels as opposed to 8 days for E2 and >10 days for T and P). Intriguingly, hardly any cortisol (in fact none at all in one of the experiments) was absorbed by the mussels. The implications of this finding in both toxicokinetic profiling and evolutionary significance (why cortisol might have evolved as a stress steroid in bony fishes) are discussed.

Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks

“…Extraction and phase separation of the metabolites in the snail tissues showed the presence of a high proportion of radioactivity (40, 50 and 85%) for P, E 2 , and T, respectively, in the lipid-rich heptane fraction where esterified steroids would be expected to accumulate ( Figure 2 ). The proportion of EE 2 that had been potentially esterified was, as expected ( 18 ), much lower (8%). Most of the rest of the radioactivity was recovered in the 80% ethanol fraction which contains free steroids.…”

“…We confirmed that snails do indeed, like many other mollusks, absorb vertebrate steroids. Compared to M. edulis ( 15 – 18 ), the rate was approximately ten times slower on a per animal basis. On a gram for gram basis, however, the difference was c .…”

“…We have confirmed not only that L. stagnalis snails can absorb the natural steroids E 2 , T and P from the water, but also that they are able to esterify them. Just as in mussels ( 18 ), EE 2 appeared to be the least easily esterified steroid. Since P has no hydroxyl groups by which it can be esterified, the presence of radioactivity in the ester fraction implied that this steroid had to have been metabolized following uptake.…”

“…The experiment was designed to investigate uptake, metabolism, and depuration of radioactive-labeled steroids by L. stagnalis . The methodology was based on previously published protocols ( 15 – 18 ) with slight modifications (e.g., freshwater instead of seawater, shorter incubation and depuration period) - the details are provided in Table 1 . All procedures involved placing the animals in containers with freshwater (pre-aerated water), adding labelled steroids to the water using ethanol as a carrier (<0.01% final solvent concentration), and then taking 1 mL water samples (immediately mixed with 7 mL scintillation fluid) every two hours to measure the amount of radioactivity remaining in the water.…”

From Dark to Light – An Overview of Over 70 Years of Endocrine Disruption Research on Marine Mollusks

“…Since no live animal studies have been carried out under immaculate steroid-free conditions (humans are a substantial potential source of contamination in laboratories) (11), it is impossible to know for sure whether a steroid level has changed because that is what it should have done if it really was a hormone, or because there was increased contamination, or a reduction in esterification, or an increase in de-esterification [or at least fourteen other potential mechanisms that could produce the same results (12)]. In fact, uptake and esterification of steroids by marine mollusks were firmly demonstrated in 2001 (57) and have since been confirmed many times by investigating different marine molluscan species (26,31,(58)(59)(60)(61)(62). Yet publications that make no mention of these mechanisms continue to appear in the literature.…”