Outbreeding: not what you may think

Mean number of children as a function of geographic distance between Danish marriage partners (Labouriau and Amorim 2008).

Most of us know about the genetics costs of inbreeding. If you do a Google search for "inbreeding is bad," you get 35,900 hits.  "Outbreeding is bad" yields only 2.

Yet outbreeding does incur genetic costs. It can reduce fitness either by introducing alleles that are unsuited to the local environment or by disrupting co-adapted gene complexes. When a native trout species was hybridized with non-native trout, fertility fell by half with as little as 20% admixture (Muhlfeld et al. 2009).

Fertility is the canary in the coal mine. A measurable decline is a sign that some genes are malfunctioning, either at the time of fertilization or during embryonic development. A malfunction can occur because the genes from the mother and father are too similar—the risk is higher that both copies of a gene will be defective. It can also occur because one copy is too different—incompatibilities may develop with other genes.

That's what we know from data on fish and other animals. But what about our species? At what degree of relatedness do the costs of human outbreeding start to exceed the benefits? When you marry a Neanderthal? The answer may surprise you. An Icelandic study found that fertility peaks at marriages between third or fourth cousins. Fertility is lower when the prospective parents are more closely related ... or less.

Our results, drawn from all known couples of the Icelandic population born between 1800 and 1965, show a significant positive association between kinship and fertility, with the greatest reproductive success observed for couples related at the level of third and fourth cousins. Owing to the relative socioeconomic homogeneity of Icelanders, and the observation of highly significant differences in the fertility of couples separated by very fine intervals of kinship, we conclude that this association is likely to have a biological basis. (Helgason et al. 2008)

The data come from a time when birth control was not widely practiced. Nonetheless, there may have been something different about Icelanders who married beyond their fourth cousins. Perhaps they were more likely to go to university, meet someone from the other side of the country, and eventually settle down and have children late in life. 

These socioeconomic factors were controlled in a Danish study that measured geographic distance between marriage partners: 

The Danish study was based on the cohort of all women born in Denmark in 1954 who were alive and living in Denmark in 1969, totaling 42,165 women. This cohort was followed up to the end of 1999. The number of children born to each mother between the ages of 15 and 45 years old was determined and is referred to as fertility. The mean marital radius (MR) associated with each mother in the cohort was estimated using the distance between the centroids of the parish where she was born and the parishes where the partners with which she had children were born. (Labouriau and Amorim 2008)

Fertility peaked at around 75 km. This relationship between fertility and marital radius was not explained by education, family income, urbanicity, or mother's age at first birth. The authors concluded that their findings were consistent with those of the Icelandic study, the cause being the same in both cases: fertility rises with decreasing relatedness up to a peak level and then starts to fall. Inbreeding depression then gives way to outbreeding depression.

How exactly does outbreeding reduce fertility? Joffe (2010) points to the steady decline in sperm quality since the early 20th century, suggesting it may be due to an increase in outbreeding. He rejects the usually cited cause: the rising level of estrogenic compounds in the environment, e.g., dioxin, DDT, PCBs, PBBs, phthalates, etc. This proposed cause fails to explain why the sperm quality decline has varied so much spatially, even within the same country. Why, for instance, has it been steep in Paris and nonexistent in Toulouse? Why is it nonexistent in domestic animals that are just as exposed to estrogenic compounds? Finally, the decline seems to have begun before most of these compounds began to be commercially produced. 

Joffe (2010) also suggests that there may be a parallel decline in egg quality. We don't really know because sperm is much easier to collect than eggs for large-scale study.

Do we now have outbreeding depression?

Today, inbreeding depression has largely disappeared throughout the Western world. For a long time the beneficial effects of outbreeding were shown by a steady increase in height and a steady decrease in the age of menarche. Both trends have now ground to a halt:

In Northern Europe, adult height has largely stabilised, and the age of menarche has also settled at around 13 years, while weight continues to increase due to obesity. (Cole 2003)

The steady rise in IQ, known as the Flynn Effect, has sometimes been attributed to outbreeding, although this explanation has been challenged (Flynn 2007, pp. 101-102; Woodley 2011). In any case, the Flynn Effect, too, is slowing throughout the West (Flynn 2007, p. 143). In Scandinavia, mean IQ peaked during the late 1990s and has since declined (Teasdale and Owen 2005).

Has outbreeding become more problematic than inbreeding? That's what the latest findings suggest, yet that doesn't at all seem to be the current wisdom.

References

Cole, T.J. (2003). The secular trend in human physical growth: a biological view. Economics & Human Biology 1(2): 161-168.

https://doi.org/10.1016/S1570-677X(02)00033-3

Flynn, J.R. (2007). What is Intelligence? Beyond the Flynn Effect. Cambridge University Press.

https://books.google.ca/books?id=qvBipuypYUkC&printsec=frontcover&hl=fr&source=gbs_ge_summary_r&cad=0#v=onepage&q&f=false

Helgason, A., S. Pálsson, D.F. Guðbjartsson, þ. Kristjánsson, K. Stefánsson. (2008). An association between the kinship and fertility of human couples. Science 319(5864): 813-816.

http://facelab.org/debruine/Teaching/EvPsych/files/Helgason_2008.pdf

Joffe, M. (2010). What has happened to human fertility? Human Reproduction 25(2): 295-307.

https://doi.org/10.1093/humrep/dep390
https://academic.oup.com/humrep/article/25/2/295/671754

Labouriau, R., and A. Amorim. (2008). Comment on "An Association Between the Kinship and Fertility of Human Couples" Science 322(5908): 1634

https://doi.org/10.1126/science.1161907
http://science.sciencemag.org/content/322/5908/1634.2.full

Muhlfeld, C.C.,  S.T Kalinowski, T.E. McMahon, M.L. Taper, S. Painter, R.F. Leary, F.W. Allendorf. (2009). Hybridization rapidly reduces fitness of a native trout in the wild. Biology Letters, March 18

http://rsbl.royalsocietypublishing.org/content/early/2009/03/13/rsbl.2009.0033.short  

Teasdale, T.W., and D.R. Owen. (2005). A long-term rise and recent decline in intelligence test performance: The Flynn Effect in reverse. Personality and Individual Differences 39(4): 837-843.

https://doi.org/10.1016/j.paid.2005.01.029

Woodley, M.A. (2011). Heterosis doesn't cause the Flynn effect: A critical examination of Mingroni (2007). Psychological Review 118(4): 689-693.

http://dx.doi.org/10.1037/a0024759 

Why are girls and boys maturing earlier?

For girls, the age of puberty has been falling since the 19th century. The same period has seen a similar decline for boys (source)

In the United States and other Western countries, girls have been reaching puberty at earlier and earlier ages. A recent longitudinal study has examined this trend in white Americans born between 1928 and 1992. Its conclusion? Girls are reaching puberty earlier because of an interaction between a lifestyle factor and a pre-existing genetic predisposition:

Our data also show, for the first time, that the effect of menarche SNPs on prepubertal BMI was stronger in children born more recently compared to those born earlier in the century, thereby suggesting that the developmental genetic susceptibility to elevated BMI may have only been ''uncovered'' in the more obesogenic environments of the recent past. (Johnson et al., 2013)

For the study's authors, the lifestyle factor is that girls are eating more, exercising less, and accumulating more body fat. Because fatty tissue is a significant source of estrogen, an increasing percentage of body fat tends to hasten puberty in young girls (Frisch & Revelle, 1970; Frisch & McArthur, 1974; Kaplowitz et al., 2001; Siiteri & MacDonald, 1973). This effect is stronger in girls with a certain genetic background:

It is possible that over the examined time period, individuals with higher genetic burden for accelerated sexual development are for the first time ''allowed'' by liberalization of the environment to alter dietary intake and energy expenditure to support their genetic potential for rapid weight gain and earlier sexual development. (Johnson et al., 2013)

But why are boys too maturing earlier?

In boys, body fat is not linked to early puberty. In fact, there seems to be a negative correlation, perhaps because fatty tissue is a significant source of estrogen (Wang, 2002). Overweight boys often present signs of disrupted male sexual development, e.g., breast budding, higher voice pitch, etc.

Yet boys likewise are reaching puberty at an earlier age. This is the conclusion of a recent American study:

We observed that onset of secondary sexual characteristics in US boys as seen in office practice appears to occur earlier than in previous US studies and the 1969 British study commonly used for pubertal norms. […] White boys in our study entered stage 2 genital growth 1.5 years earlier than the British boys (10.14 vs 11.60 years of age).

[…] These data are consistent with recent trends from other countries, such as Denmark, Sweden, Great Britain, Italy, and China. For example, urban Han Chinese boys achieve a testicular volume of ≥4 mL (13% by age 9) and spermarche earlier than studies conducted several decades ago; Danish boys achieve a testicular volume >3 mL more than 3 months earlier now than 15 years ago. (Herman-Giddens, 2012)

This trend has also been observed in the age when a boy's voice begins to change:

According to records kept by the Leipzig choir, the most common period of voice breaking for male singers in the mid-18th century was between 17.5 and 18.5 years of age (Daw, 1970); in contrast, children enrolled in the Copenhagen Municipal Choir School from 1994-2003 had a median age of voice breaking of 10.4 years (Juul, Magnusdottir, Scheike, Prytz, & Skakkebæk, 2007), which is consistent with the choir's subjective reports of difficulty retaining children as singers past the age of 12 or 13 years. (Mendle & Ferrero, 2012).

This is a challenge for Occam's Razor, and the task is no easier if we look at other possible causes. If the cause isn’t a higher proportion of body fat, could it be a higher level of estrogens and estrogen-like substances in the environment? (see earlier post). Yes, that might hasten puberty in girls and increase accumulation of body fat. But in boys it would delay puberty by offsetting the rising level of male hormones.

In trying to figure out the causal chain of events, we should keep in mind that the relationship between body fat and age of puberty runs in both directions. On the one hand, estrogen from body fat lowers the age of puberty in girls. On the other hand, earlier puberty increases ovarian production of estrogen, which in turn stimulates deposition of body fat, particularly on the hips, buttocks, and breasts (Van Lenthe et al., 1996). So perhaps some unknown factor is causing earlier sexual development in both sexes and thus greater deposition of body fat in girls.

A response to social cues?

This unknown factor might be something in the social environment. As Hawley (2011) argues, humans unconsciously monitor their social environment for reproductive opportunities and accordingly speed up or slow down their pace of sexual development:

[...] human children, especially girls, may be sensitive to their early socioecological conditions in ways that entrain development toward either a faster (earlier pubertal maturation, more sexual partners, less stable relationships) or slower (later pubertal maturation, fewer sexual partners, more stable relationships) life history strategy.

With the transition to post-traditional societies, there has been an increase in the erotic stimuli that preteens encounter in their surroundings:

Common in traditional societies are adult-supervised adolescent initiation ceremonies (Schlegel & Barry, 1980) that are designed to commemorate the transition from childhood to adulthood and inculcate the adolescent with adult values, duties, behaviors, and sex roles associated with the culture (Schlegel, 1973). That is, these adolescents are taught adult sex roles by adults. We now appear to have a complete turnaround. In modern, Western cultures, adolescents derive sexual relationship expectations from television, cable, music, purveyors of racy lingerie (who target teenage girls), and pornography that they can now access on the Internet and thereby carry around on their cell phones. (Hawley, 2011)

Erotic imagery in particular is today available to a degree that was impossible not so long ago. Young boys and girls have virtual access to an endless supply of picture-perfect sexual partners. Whatever the media—films, TV, magazines, the Internet—we're exposed to images that can stimulate sexual desire as efficiently as what normally exists in the real world. More so, in fact. These images are ‘supernormal’ stimuli.

To date, only one study has looked into possible relationships between erotic imagery and pubertal timing:

The aim of this study was to investigate associations between pubertal timing and boys' Internet use, particularly their viewing of pornography. We used a sample comprising of 97 boys in grade 8 (M age, 14.22 years) from two schools in a medium-sized Swedish town. This age should be optimal for differentiating early, on-time, and later-maturing boys. Boys responded to self-report questionnaires on their Internet use and pubertal timing. Early, on-time, and late-maturing boys did not differ in terms of most Internet activities. However, early maturers reported downloading and viewing pornography more often than the other boys did (p<.001). (Skoog et al.,2009)

Admittedly, the arrow of causality might point in the other direction, i.e., early maturing boys have a stronger sex drive and thus a greater interest in porn. This was, in fact, the authors' explanation. We should also remember the well established correlation between early puberty in girls and the absence of a father in the home. It was long thought that father absence triggers early puberty in girls. In fact, a twin study has shown a genetic cause: absent fathers tend to have genes that favor earlier sexual development in their progeny (Mendle et al., 2006).

One might also object that the decline in the age of puberty began long before the Internet. Before the Internet, however, there were porn magazines. And before them, there were pictures garnered from art books, fashion magazines, or the lingerie sections of mail-order catalogues. One could also bring erotic images to mind by reading certain novels. Thus, modern pornography is merely the latest stage of a lengthy co-evolution between, on the one hand, improvements in photography and other imaging technologies and, on the other hand, a weakening of taboos against masturbation. At the beginning of this co-evolution, in the 19th century, masturbation was much less developed among young boys and girls as a sexual lifestyle. Visual aids were scarce and of poor quality, religious injunctions were strong, and adult supervision inside and outside the home was omnipresent.

Conclusion

The age of puberty might be declining because boys and girls are being exposed to ever more and ever better erotic imagery, but this hypothesis needs confirmation by longitudinal studies to determine which is the cause and which is the effect. Another drawback with current research is its focus on the most extreme forms of pornography, such as child porn. Yet the usual stuff is the kind that most people consume ... and in unparalleled quantities. As the authors of a recent Dutch study remarked:

[...] we can only emphasize that Dutch youth are confronted with and expose themselves to an unprecedented amount of R-rated and Xrated material in the media. Research on its consequences for adolescents' sexual socialization is largely missing but, as this study has shown, is urgently needed. (Peter & Valkenberg, 2006)

And erotic imagery isn't confined to X-rated websites or magazines. It is in fact ubiquitous in modern social environments. Girls might accelerate their sexual development by leafing through fashion magazines just as boys might accelerate theirs by viewing porn.

The erotic imagery hypothesis will have to fit the data better than rival hypotheses. Two of these, the body fat and environmental estrogen hypotheses, can explain the decline in the age of puberty for girls but not for boys. Another possible cause is better nutrition. Yet, among white Americans at least, much of this decline has happened since the 1950s—when nutrient levels were already adequate for this population. Finally, there is the possibility that puberty is happening earlier because genes that favor that developmental trajectory are spreading within the population. Modern social environments favor a reproductive strategy of early puberty, low parental investment and, especially, low paternal investment—in short, the ‘cads’ are outbreeding the ‘dads’ (see earlier post).

References

Frisch, R.E., R. Revelle. (1970). Height and weight at menarche and a hypothesis of critical body weights and adolescent events, Science, 169, 397-399.

Frisch, R.E. & J.W. McArthur. (1974). Menstrual cycles: fatness as a determinant of minimum weight necessary for maintenance or onset, Science, 185, 949-951.

Hawley, P.H. (2011). The evolution of adolescence and the adolescence of evolution: The coming of age of humans and the theory about the forces that made them, Journal of Research on Adolescence, 21, 307-316.

http://www.people.ku.edu/~phawley/Publications/Hawley%202011%20JRA%20Evo%20of%20Adol.pdf

Herman-Giddens, M.E., J. Steffes, D. Harris, E. Slora, M. Hussey, S.A. Dowshen, R. Wasserman, J.R. Serwint, L. Smitherman, & E.O. Reiter. (2012). Secondary sexual characteristics in boys: Data from the Pediatric Research in Office Settings Network, Pediatrics, 130, e1058-e1068.

http://pediatrics.aappublications.org/content/130/5/e1058.full.pdf+html

Johnson, W., A.C. Choh, J.E. Curran, S.A. Czerwinski, C. Bellis, T.D. Dyer, J. Blangero, B. Towne, & E.W. Demerath. (2013). Genetic risk for earlier menarche also influences peripubertal body mass index, American Journal of Physical Anthropology, 150, 10-20

Kaplowitz, P.B., E.J. Slora, R.C. Wasserman, S.E. Pedlow & M.E. Herman-Giddens. (2001). Earlier onset of puberty in girls: relation to increased body mass index and race, Pediatrics, 108, 347-353.

Mendle, J. & J. Ferrero. (2012). Detrimental psychological outcomes associated with pubertal timing in adolescent boys, Developmental Review, 32, 49-66.

Mendle, J., E. Turkheimer, B.M. D'Onofrio, S.K. Lynch, R.E. Emery, W.S. Slutske, N.G. Martin. (2006). Family structure and age at menarche: a children-of-twins approach, Developmental Psycholpgy, 42, 533-542.

Peter, J. & P.M. Valkenberg. (2006). Adolescents' exposure to sexually explicit material on the Internet, Communication Research, 33, 178-204.

Siiteri, P.K. & P.C. MacDonald. (1973). Role of extraglandular estrogen in human endocrinology. In S.R. Geiger (ed.), Handbook of Physiology, Washington D.C. American Physiology Society, sect. 7, vol. 2, part 1, pp. 615-629.

Skoog, T., H. Stattin, & M. Kerr. (2009). The role of pubertal timing in what adolescent boys do online, Journal of Research on Adolescence, 19, 1-7.

Van Lenthe, F.J., C.G. Kemper & W. van Mechelen. (1996). Rapid maturation in adolescence results in greater obesity in adulthood: the Amsterdam Growth and Health Study, American Journal of Clinical Nutrition, 64, 18-24.

Wang, Y. (2002).Is obesity associated with early sexual maturation? A comparison of the association in American boys versus girls, Pediatrics, 110, 903-910.

http://www.pediatricsdigest.mobi/content/110/5/903.short

What you don't know can hurt you

In 1972, the U.S. passed the Clean Water Act, despite a presidential veto by Richard Nixon. Did this act also end an era of unusually high estrogen levels in the environment? (cartoon source)

There has been much concern over the presence of “environmental estrogens” in our drinking water and elsewhere in the environment. These are man-made chemicals, like DDT, PCBs, and dioxins, that mimic the effect of natural estrogens. Among other things, they’re blamed for declining sperm counts and rising male infertility.

Yet estrogen also enters our environment from a source that excites much less concern. This is the estrogen that women excrete every day in their urine. Shouldn’t that source also be cause for worry?

At first thought, no. People have been urinating for a very long time. And other animals for even longer. During that time, microorganisms have evolved to break down and feed on whatever is present in urine. Larger organisms have likewise had plenty of time to adapt to this aspect of their environment. Urine is so ubiquitous and unchanging that it could not possibly pose a danger. Or could it?

Actually, two things have changed in recent times. One is that humans have become much more numerous, with the result that much more urine is being discharged into the environment. Another is the way it is discharged.

Before the late 19th century, urine entered the environment via privies, cesspools, and ditch sewers (Rockefeller, 1996). It was thus discharged into a warm stagnant medium rich in organic matter—ideal conditions for rapid breakdown of the estrogen molecule by nitrifying bacteria (Vader et al., 2000). These same conditions, however, increasingly became a threat to public health, particularly in the ever larger and more numerous urban centers.

And so a new disposal system was developed. Human waste was now expedited via sewers to a central facility where the liquid component would be separated and rapidly discharged into the nearest cold body of water—which often doubled as the city’s source of drinking water. It was a perfect system for discharging urinary estrogen into the environment with as little biodegradation as possible … and then bringing it back into the human organism. As for urinary androgen, it was also present in wastewater but at much lower levels because of its lower solubility in water (Tabak et al., 1981).

That system survived until the late 1960s and early 1970s, when concern about pollution brought an upgrading of almost all sewage treatment facilities. If we look at the total number of Americans who produce untreated wastewater, this number peaked at 70 million in 1960 before falling to 2 million after passage of the Clean Water Act in 1972 (Copeland, 1993; US Council on Environment Quality, 1984). Primary treatment removes 35‑55% of estrogen from wastewater, and this proportion rises to 50‑70% for secondary treatment (Tabak et al., 1981). Today, tertiary treatment removes 90% of all natural and synthetic estrogenic compounds (LeQuire, 1999).

But what about the 100-year period when most wastewater went untreated? During that time, the main sources of drinking water must have been highly contaminated with estrogen. What were the effects? The most obvious ones would have been a decline in sperm counts and a rise in male infertility. But were there others? And should we soon see a reversal of these trends?

I tried to publish a paper on this subject, but the reviewers were skeptical. I was indulging in speculation that could never be proved one way or another. There simply are no records of estrogen levels in the environment for the period in question.

So I published my speculations on my blog (here, here, and here) and let the matter lie … while keeping an eye on the literature. Recently, three findings have come to my attention:

Primary treatment less effective than previously thought

A recent paper confirms that tertiary treatment of wastewater removes over 90% of all estrogen. On the other hand, primary treatment is less effective than previously thought, the removal rate being only 10% (Limpiyakorn et al., 2011).

Estrogen content of wastewater higher than previously thought

In the past, estrogen levels were measured only for the three most common kinds of estrogen: estrone (E1), estradiol (E2), and estriol (E3). Other natural estrogens, however, are present in urine. The total estrogen level in wastewater is thus 18-34% higher than previously thought:

[…] the total excretion rates of EEQ [estrogen equivalent] by estrone (E1), 17β-estradiol (E2), and estriol (E3) only accounted for 66–82% of the total excretion rate of EEQ among four different groups, and the other corresponding natural estrogens contributed 18–34%, which meant that some of the other natural estrogens may also exist in wastewater with high estrogenic activities. (Liu et al., 2009)

River and ocean sediments reveal formerly high estrogen levels in the environment

It is possible to look into the past by taking cores of sediments from the bottoms of lakes, rivers, and coastal waters. A study of the River Thames has found that estrogen levels are higher in river sediments deposited before the 1960s:

There is an indication of higher concentrations of E1 and E3 in samples deemed to be deposited before the mid-1960s, prior to the introduction of biological treatment at STWs [sewage treatment works] discharging to the estuary. […] This provides indirect evidence that historical improvements to wastewater treatment have resulted in a decrease in the concentrations of steroids in the effluent, as observed for PCBs and DDT from the same core (Gomes et al., 2011)

A similar finding comes from a study of sediment cores from Japanese coastal areas:

The concentration of natural estrogens such as 17β-estradiol (E2) and estrone (E1) in the sediment of coastal areas in Japan was determined […] Core samples were sliced every 2cm from the surface to 20cm deep for the measurement of estrogen. Although the concentrations of estrogens decreased with the depth of core samples, fairly high levels of estrogens were again noticed at the layer deeper than 16cm. (Matsuoka et al., 2005)

Conclusion

The most promising line of research seems to be the use of sediment cores to estimate past levels of estrogen in the environment. One problem will be calibration of sediment dating. Gomes et al. (2011) have pointed to a possible solution by noting that the mid-1960s correspond to the first sediment layer that contains synthetic estrogen, i.e., from birth control pills. Another problem is that estrogen seems to degrade gradually over time, even in river or ocean sediments.

References

Copeland, C. (1993). Wastewater Treatment: Overview and Background [93-138 ENR] Washington, D.C.: Congressional Research Service.

Frost, P. (2009). The urinary estrogen theory. Part I, Evo and Proud, March 11

http://evoandproud.blogspot.ca/2009/03/urinary-estrogen-theory-part-i.html

Frost, P. (2009). The urinary estrogen theory. Part II, Evo and Proud, March 18

http://evoandproud.blogspot.ca/2009/03/urinary-estrogen-theory-part-ii.html

Frost, P. (2009). The urinary estrogen theory. Part III, Evo and Proud, March 26

http://evoandproud.blogspot.ca/2009/03/urinary-estrogen-theory-part-iii.html

Gomes, R.L., M.D. Scrimshaw, E. Cartmell, & J.N. Lester.  (2011). The fate of steroid estrogens: partitioning during wastewater treatment and onto river sediments, Environmental Monitoring and Assessment, 175, 431–441.

http://www.springerlink.com/content/052274881l4186g5/

LeQuire, E. (1999). Something in the Water. InSites, 7(1),

http://eerc.ra.utk.edu/insites/ins7-1.htm#Something

Limpiyakorn, T., S. Homklin, & S.K. Ong. (2011). Fate of estrogens and estrogenic potentials in sewerage systems, Critical Reviews in Environmental Science and Technology, 41(13), 1231-1270.

Liu, Z., Y. Kanjo, S. Mizutani. (2009). Urinary excretion rates of natural estrogens and androgens from humans, and their occurrence and fate in the environment: A review, Science of the Total Environment, 407, 4975–4985

Matsuoka, S., R. Sakakura, M. Takiishi, Y. Kurokawa, S. Kawai, & N. Miyazaki. (2005). Determination of natural estrogens in the sediment of coastal area in Japan, Coastal Marine Science, 29(2), 141-146.

http://repository.dl.itc.u-tokyo.ac.jp/dspace/handle/2261/5593

Rockefeller, A.A. (1996). Civilization and sludge: Notes on the history of the management of human excreta. Current World Leaders, 39, 99‑113.

Tabak, H.H., R.N. Bloomhuff, & R.L. Bunch. (1981). Steroid hormones as water pollutants II. Studies on the persistence and stability of natural urinary and synthetic ovulation‑inhibiting hormones in untreated and treated wastewaters. Developments in Industrial Microbiology, 22, 497‑519.

U.S. Council on Environment Quality. (1984). Annual Report. Washington D.C.

Vader, J.S., C.G. van Ginkel, F.M.G.M. Sperling, J. de Jong, W. de Boer, J.S. de Graaf, M. van der Most, & P.G.W. Stokman. (2000). Degradation of ethinyl estradiol by nitrifying activated sludge. Chemosphere, 41, 1239‑1243.