The two main questions of the present study were: Is an ecologically relevant exposure to EE2 toxic to embryos and larvae of a population of river-spawning grayling, and is there additive genetic variance for the tolerance to EE2 in this population, i.e. does the population currently have a potential to rapidly adapt to this type of pollution? The first question is relevant even if the toxicity of EE2 has been demonstrated in many other fish taxa [e.g. 30, 31-33], because (i) the study population is declining for unknown reasons and a lack of an evolutionary response to toxicity could be contributing to the problem, and (ii) the chemical pollution of freshwater habitats that has happened since the market launch of the contraceptive pill, i.e. during more than 5 decades, could have led to adaptation and hence to reduced toxicity in some fish. The answer to the latter question may help us to better understand if pollution by EE2 has induced rapid evolution because, in our study population, the period of exposure is likely to span around 10 to 15 generations, i.e. there could have been enough time for evolution to deplete any genetic variance for tolerance to EE2 that the population could have had at the beginning of the exposure. Moreover, these questions are of ecotoxicological relevance [10, 34, 35] because standard ecotoxicological testing often ignores potential taxon-specific toxicities .
Regarding our first main question: We found a statistically non-significant increase in mortality of 1.5 pp for embryos and 0.4 pp for larvae. These effect sizes seem comparable to the observed increase in embryo mortality of 0.9 pp in brown trout that was only significantly different from zero because of an extra-ordinary large sample size (N = 7,302 singly raised embryos) . In whitefish, the EE2-induced increase in embryos mortality was significant and around 3 percent points (pp) in C. palaea  and around 13 pp in C. albellus .
With the observed low mortality, the question of whether there is EE2-induced sex-specific mortality in grayling cannot be solved yet. The study population suffers from a skewed sex ratio (more males than females ) that seems not due to EE2-induced sex reversal [28, 31] but rather caused by sex-specific mortality . It is still possible that there are sex-specific susceptibilities to combined effects of EE2 and other environmental stressors. Other types of environmental stressors such as microbes , temperature variations , or other micropollutants [40, 46] could interact with the effects of EE2 and thereby amplify its toxicity [47, 48]. Therefore, single-factor laboratory studies like ours are likely to underestimate the ecotoxicological relevance of EE2 in the wild.
While EE2- and sham-exposed grayling embryos hatched at similar size, exposure to EE2 reduced larval growth and consumption of yolk sac after hatching by about 4% each during the first 8 days after hatching. We therefore conclude that EE2 is toxic to grayling at early developmental stages. Such a reduction in growth was predicted from recent analyses of physiological reactions to EE2 in Atlantic salmon [37, 38] but was not observed in brown trout . One possible explanation for this apparent discrepancy between brown trout and grayling larvae is that hatching was not induced in the study on brown trout  but induced by an increase in temperature in the present study on grayling. Under the given conditions, EE2-exposed brown trout embryos hatched later and at smaller size than sham-exposed ones, while, in the present study on grayling, no treatment-related difference in the timing of hatching nor on hatchling size could be observed. If growth rate after hatching is dependent on larval size and developmental stage, such differences in the experimental protocols could be responsible for the apparent differences in treatment effects on growth rates. However, in both cases, the combined effects of EE2 on embryo and larval development would be expected to delay the emergence from gravel at the end of the yolk sac stage and could even lead to smaller body sizes at emergence. Time to emergence, and body size at emergence, is likely to be linked to fitness in salmonids because larvae that emerge earlier and larger than others may face less competition for resources (e.g. feeding territory) and are more prone to outcompete their late emerging counterparts [50, 51].
Regarding our second main question: Because grayling males do not provide any parental care, significant sire effects on offspring traits reveal additive genetic variance in full-factorial breeding experiments . The dam effect then represents a combination of additive genetic variance and maternal environmental effects . In salmonids, maternal environmental effects comprises characteristics such as egg size  and compounds that females allocate to their eggs [e.g. 47, 48-51]. We found strong direct maternal effects on every offspring trait that we measured, and a dam x EE2 interaction on the timing of hatching. We conclude that maternal sib groups reacted differently to exposure to EE2. However, these maternal effects seem to be mainly due to maternal environmental effects , because we found no significant additive genetic variance for tolerance to EE2 pollution in any of the analysed traits.
No significant additive genetic variance could potentially be due to a type II error (false negative). However, such an error is unlikely here because (i) our analysis is based on a large sample size (1,555 singly-reared embryos) and 40 sib groups, (ii) our sample revealed overall additive genetic variance (i.e. significant sire effects) on embryo mortality and the timing of hatching, (iii) a parallel study (Marques da Cunha, Mobley, Maitre, de Guttry, Wedekind, in preparation) on other samples of the same 40 families revealed genetic variation in the tolerance to infection by a bacterium, and (iv) singly-reared salmonid embryos are sensitive indicators of environmental stress, and studies based on comparable breeding designs have demonstrated additive genetic variance for the tolerance to other types of stressors, including other types of pollutants [52, 53], pathogens  or even water-borne cues linked to infection .
The finding of no significant additive genetic variance for tolerance to EE2 pollution in grayling is in sharp contrast to the findings of Brazzola et al.  on lake-spawning whitefish. However, our findings correspond well with the ones of Marques da Cunha et al.  who used a similar experimental protocol to test for this type of genetic variation in 7 genetically distinct populations of river-spawning brown trout and found none (in a total sample size of 7,302 singly embryos, i.e. a type II error was also unlikely in their case). Taken together, these observations support the view that the appearance of the novel stressor EE2 has induced evolution and thereby used up the corresponding additive genetic variance in river-spawning salmonid that are exposed to the pollutant, while lake-spawning salmonids who are less exposed still have a strong potential to evolve rapidly to EE2. However, alternative explanations are possible. Future studies could therefore compare exposed and non-exposed populations of the same species (if at all possible, given human population density and the finding that very low doses of EE2 can induce selection), add analogous tests on further river-or lake-spawning salmonids, or test for signatures of selection in the EE2 response pathways [56, 57].
As far as we know, there exist no measurements of estrogenic pollution around the spawning ground of our study population. However, this spawning ground is located in the river Aare within a city of more than 40,000 inhabitants, a large sewage treatment plant about 4 km downstream, and several nearby villages (with several thousand inhabitants each) upstream. The sewage treatment process typically removes only about two thirds of the EE2 , and exposure to EE2 is therefore likely in rivers of the Swiss Plateau [8, 64]. Marques da Cunha et al.  sampled brown trout from 7 different streams (the river Aare and 6 tributaries) to test whether variation in estrogenic pollution creates population differences in toxicity of EE2. They found population differences in various embryo and larval traits, but none in the reaction to EE2. They argued that very low concentrations on EE2 and exposure during only short periods can cause selection and hence induce rapid evolution. The hypothesis is supported by the observation that the 2 pg EE2 in the aqueous exposure seemed to be continuously taken up by the embryo (about 80% within 4 weeks) while the concentration remained constant in empty plates . This suggests that salmonid eggs take up EE2 at concentrations that are far lower than the 1 ng/L that are sometimes even found in groundwater . On the other side of the scale: when Brazzola et al.  exposed whitefish embryos to 1 ng/L, 10 ng/L, or 100 ng/L EE2, increasing concentration seemed only weakly linked to increased toxicity. Similar observations were made by Duffy et al.  who exposed Atlantic salmon to 1.2 ng/L, 11.9 ng/L, and 118.6 ng/L EE2, respectively. We therefore argue that our one-dose aqueous exposure to 2 pg EE2 was ecologically relevant for grayling embryos and likely to reveal additive genetic variance for tolerance, should it exist.
Our study adds the grayling to the list of salmonids whose embryos and larvae could be experimentally exposed to ecologically relevant concentrations of around 1 ng/L EE2. With the present study, at least one species of each subfamily of the Salmonidae (Coregoninae, Salmoninae, and Thymallinae) has now even been tested using the same method of applying a one-dose exposure of 2 pg to embryos developing in 2 mL wells [11, 22]. Together, these studies reveal strong species-specific reactions to EE2 within the salmonids, and various amounts of additive genetic variance in the tolerance to this synthetic stressor.