PESTICIDES

The organochlorine pesticides, including DDT and its metabolites and HCH were amongst the first compounds to be investigated for their endocrine disrupting properties, and other classes are known or suspected to have endocrine disrupting properties too, but here three classes of compounds which have been the subject of increasing attention over the last 10 years are discussed.

4.8.5.1 Dicarboxamides

Evidence of exposure-disease associations

No direct evidence of exposure-disease associations exists yet for either humans or wildlife.

Evidence of endocrine disruption mechanisms

Results of animal studies indicate that vinclozolin could contribute to male reproductive disorders. In vivo, prenatal vinclozolin exposure causes irreversible adverse effects on male sexual differentiation

Page 62 of 135 in the rat. When pups are exposed in utero during sexual differentiation, androgen dependent processes are inhibited causing AGD decreases and genital deformities. In adulthood the exposed males have testicular abnormalities and poorer spermatogenesis, prostate inflammation and disease, and breast tumours, along with kidney disease and immune abnormalities. The principal dicarboxamide pesticides to receive attention are the fungicides vinclozolin, iprodione and procymidone. The antiandrogenic properties of vinclozolin in particular have been a cause for concern, since there are indications that its prenatal effects on the male reproductive tract may persist for two or more generations (Annex 1, 4.1.4.). Most of the effects ascribed to vinclozolin can be attributed to its properties as an androgen receptor antagonist (Annex 1, 4.1.4.) but in addition to these properties, it causes genome-wide gene methylation changes which may allow these effects to be passed on transgenerationally, although the exact mechanism of action at the genetic level has yet to be determined (Annex 1, 3.4.3.1.). Vinclozolin has also been shown to alter progesterone receptor expression in vivo, having a virilising effect on female mice. Iprodione and procymidone are also AR antagonists, causing male reproductive abnormalities similar to vinclozolin, although it is not yet known whether these are passed to subsequent generations (Annex 1, 4.1.4.).

In wildlife species, vinclozolin can impair reproductive function in male prosobrach molluscs at environmentally relevant concentrations (Annex 1, 7.1.1.4.). It can also inhibit maturation and reduce egg production in fish, females being more sensitive than males possibly because they have far lower endogenous androgen titres and because androgens play an essential role in folliculogenesis (Annex 1, 7.2.1.3.4.). Masculine behaviour is also inhibited in birds exposed in ovo and adult males show reduced blood testosterone levels and have smaller testes (Annex 1, 7.5.3.3), but experiments exposing reptiles in ovo have failed to find any difference in plasma sex steroid levels or sex ratios (Annex 1, 7.4.1.2.).

4.8.5.2 Azole fungicides

Evidence of exposure-disease associations

Azole fungicides can be broadly broken into two categories, triazoles and imidazoles. Prochloraz is the most thoroughly researched imidazole with ED properties, whilst the triazole category includes bitertanol, cyproconazole, febuconazole, epoxiconazole, hexaconazole, metconazole and myclobutanil. Neither they nor the dicarboxymides are persistent, so measuring or estimating exposure at critical times is difficult, especially in humans. The activities of these chemicals in humans have received little epidemiological attention. Studies of agricultural workers have detected statistically significant increases in reproductive abnormalities in the children of mothers exposed to pesticides during pregnancy. For example, one very recent study from Denmark found an increase in the incidence of cryptorchidism in the sons of a cohort of mothers employed in horticulture and exposed to pesticides relative to cohorts of unexposed mothers employed both in horticulture and non-agricultural occupations¹ Unfortunately, these studies do not identify the individual compounds or compound classes that the women were exposed to, so causal links between individual pesticides or pesticide classes cannot be inferred from these data. Little is known about how dicarboximides may affect wildlife populations.

1 Gabel P, Jensen MA, Andersen HR, Baelum J, Thulstrup AM, Bonde JP, Toft G. 2011 The risk of cryptorchidism among sons of women working in horticulture in Denmark: a cohort study. Environmental Health 10:100-

Page 63 of 135 Evidence of endocrine disruption mechanisms

Exposure to prochloraz during organogenesis interferes irreversibly with the formation of the male reproductive tract in a similar manner to other antiandrogens, and there is evidence that other azoles act similarly (Annex 1, 4.1.4.). In addition to being an AR antagonist, prochloraz and the triazoles also interfere with steroid synthesis. In the case of prochloraz, it involves the inhibition the conversion of progesterone to testosterone, the stimulation of aldosterone synthesis at low doses and inhibition of it at high ones. It is also an aromatase inhibitor, allowing it to reduce estradiol levels as well. Other azoles show similar properties, but their individual abilities to inhibit the conversion of progesterone and the action of aromatase vary (4.1.2.). Non-steroidal activity has also been confirmed. Azoles can also enhance the hepatic metabolism and excretion of the thyroid hormones (Annex 1, 4.1.4). Quite a few of the azole fungicides have been used in mixture experiments, and appear to have additive effects in line with dose addition predictions (Annex 1, 3.3.1.2.). Although they are known to affect steroidogenesis in amphibians in laboratory studies (Annex 1, 7.3.1.3.), it is unknown whether they affect them in the field. Fish may be at risk from these compounds, since laboratory studies report reduced fecundity and gonadal abnormalities in individuals exposed during adulthood, and in fish exposed during development increases in the male:female ratio, an increased incidence of intersex and increased vitellogenesis in males were recorded ^1,2. Data for other wildlife groups are lacking.

4.8.5.3 Triazines

Evidence of exposure-disease associations

Atrazine and simazine are the two most widely used herbicidal triazines, and of these atrazine has received the greatest attention for its ED properties, principally because feminised secondary sexual characteristics, intersex and gonadal dysgenesis have been observed in wild frogs collected from contaminated sites (Annex 1, 7.1.1.4.).

Evidence of endocrine disruption mechanisms

Exposure is associated with lower testosterone levels in amphibia, and in female rats, peripubertal exposure to atrazine reduces luteinising hormone (LH) and prolactin levels, delaying pubertal onset.

Its exact mechanism of action, however, is not known. It is not known to interact with any hormone receptor directly, but interferes with steroidogenic enzymes (Annex 1, 7.3.1.3.). Surprisingly, it does not appear to affect aromatase. Similar sexual abnormalities to those seen in male frogs in the wild have been induced experimentally, with some studies using very low doses of atrazine (Annex 1, 7.2.1.3.4.). The effects observed, however, differ between species and strains. Crustacea may have some reproductive vulnerability to triazines (Annex 1, 7.3.1.3.). When exposed to atrazine under experimental conditions, egg bearing water fleas produced a disproportionate number of male offspring. Some studies using fish have found elevated vitellogenin production in exposed males,

1 Ankley GA, Jensen KM, Durhan EJ, Makynen EA, Butterworth BC, Kahl MD, Villeneuve DL, Linnum A, Gray LE, Cardon M, Wilson VS. 2005 Effects of Two Fungicides with Multiple Modes of Action on Reproductive

Endocrine Function in the Fathead Minnow (Pimephales promelas). Toxicological Sciences 86(2):300-308

2 Kinnberg K, Holbech H, Petersen GI, Bjerregaard P. 2006 Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio). Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology 145(2): 165-170

Page 64 of 135 and reduced egg production and numbers of spawning events in females (Annex 1, 7.2.1.3.4.). In these studies, testicular oocytes, oocyte atresia and other histological changes were observed in the gonads of treated fish, although not in a dose dependent manner. Little is known about how these compounds affect the endocrine systems of wild reptiles, birds and mammals. Preliminary studies of effects on the sex ratios of alligator eggs found no effects at male or female producing temperatures, but the temperatures producing mixed sex clutches, which are the most sensitive, were not tested (Annex 1, 7.4.1.3.). Male courtship behaviour and the formation of the cloacal gland can be suppressed in birds by the administration of atrazine in ovo (Annex 1, 7.5.3.3.). Effects in mammals remain largely undetermined.