Out-of-season production of 17,20ß-dihydroxypregn-4-en-3-one in the roach Rutilus rutilus.

In this study, although the highest production of two physiologically significant progestins in teleosts [17,20ß-dihydroxypregn-4-en-3-one (17,20ß-P) and 17,20ß,21-trihydroxypregn-4-en-3-one (17,20ß,21-P)] was observed in the period just prior to spawning in both male and female roach Rutilus rutilus, there was also a substantial production (mean levels of 5-10 ng ml(-1) in blood; and a rate of release of 5-20 ng fish(-1) h(-1) into the water) in males and females in the late summer and early autumn (at least 7 months prior to spawning). During this period, the ovaries were increasing rapidly in size and histological sections were dominated by oocytes in the secondary growth phase [i.e. incorporation of vitellogenin (VTG)]. At the same time, the testes were also increasing rapidly in size and histological sections were dominated by cysts containing mainly spermatogonia type B. Measurements were also made of 11-ketotestosterone (11-KT) in males and 17ß-oestradiol and VTG in females. The 3 months with the highest production of 11-KT coincided with the period that spermatozoa were present in the testes. In females, the first sign of a rise in 17ß-oestradiol concentrations coincided with the time of the first appearance of yolk globules in the oocytes (in August). The role of the progestins during the late summer and autumn has not been established.

Preliminary data on the influence of rearing temperature on the growth and reproductive status of fathead minnows Pimephales promelas.

An investigation into the influence of temperature on the growth and reproductive status of the fathead minnow Pimephales promelas revealed that, while there was no clear effect of treatment on sex differentiation, ovarian tissue from female fish reared under the highest temperature regime contained large amounts of undefined tissue containing no germ cells. Furthermore, both male and female fish exhibited differences in length mass, condition and somatic indices, and in the expression of secondary sexual characteristics. The patterns observed are discussed in the context of climate change.

Steroid hormones in the aquatic environment.

Steroid hormones are extremely important natural hormones in all vertebrates. They control a wide range of physiological processes, including osmoregulation, sexual maturity, reproduction and stress responses. In addition, many synthetic steroid hormones are in widespread and general use, both as human and veterinary pharmaceuticals. Recent advances in environmental analytical chemistry have enabled concentrations of steroid hormones in rivers to be determined. Many different steroid hormones, both natural and synthetic, including transformation products, have been identified and quantified, demonstrating that they are widespread aquatic contaminants. Laboratory ecotoxicology experiments, mainly conducted with fish, but also amphibians, have shown that some steroid hormones, both natural and synthetic, can adversely affect reproduction when present in the water at extremely low concentrations: even sub-ng/L. Recent research has demonstrated that mixtures of different steroid hormones can inhibit reproduction even when each individual hormone is present at a concentration below which it would not invoke a measurable effect on its own. Limited field studies have supported the conclusions of the laboratory studies that steroid hormones may be environmental pollutants of significant concern. Further research is required to identify the main sources of steroid hormones entering the aquatic environment, better describe the complex mixtures of steroid hormones now known to be ubiquitously present, and determine the impacts of environmentally-realistic mixtures of steroid hormones on aquatic vertebrates, especially fish. Only once that research is completed can a robust aquatic risk assessment of steroid hormones be concluded.

The role of the maturation-inducing steroid, 17,20beta-dihydroxypregn-4-en-3-one, in male fishes: a review.

The major progestin in teleosts is not progesterone, as in tetrapods, but 17,20beta-dihydroxypregn-4-en-3-one (17,20beta-P) or, in certain species, 17,20beta,21-trihydroxy-pregn-4-en-3-one (17,20beta,21-P). Several functions for 17,20beta-P and 17,20beta,21-P have been proposed (and in some cases proved). These include induction of oocyte final maturation and spermiation (milt production), enhancement of sperm motility (by alteration of the pH and fluidity of the seminal fluid) and acting as a pheromone in male cyprinids. Another important function, initiation of meiosis (the first step in both spermatogenesis and oogenesis), has only very recently been proposed. This is a process that takes place at puberty in all fishes and once a year in repeat spawners. The present review critically examines the evidence to support the proposed functions of 17,20beta-P in males, including listing of the evidence for the presence of 17,20beta-P in the blood plasma of male fishes and discussion of why, in many species, it appears to be absent (or present at low and, in some cases, unvarying concentrations); consideration of the evidence, obtained mainly from in vitro studies, for this steroid being predominantly produced by the testis, for its production being under the control of luteinizing hormone (gonadotrophin II) and, at least in salmonids, for two cell types (Leydig cells and sperm cells) being involved in its synthesis; discussion of the factors involved in the regulation of the switch from androgen to 17,20beta-P production that seems to occur in many species just at the time of spermiation; discussion of the effects of in vivo injection and application of 17,20beta-P (and closely related compounds) in males; a listing of previously published evidence that supports the proposed new function of 17,20beta-P as an initiator of meiosis; finally, discussion of the evidence for environmental endocrine disruption by progestins in fishes.

How we can make ecotoxicology more valuable to environmental protection.

There is increasing awareness that the value of peer-reviewed scientific literature is not consistent, resulting in a growing desire to improve the practice and reporting of studies. This is especially important in the field of ecotoxicology, where regulatory decisions can be partly based on data from the peer-reviewed literature, with wide-reaching implications for environmental protection. Our objective is to improve the reporting of ecotoxicology studies so that they can be appropriately utilized in a fair and transparent fashion, based on their reliability and relevance. We propose a series of nine reporting requirements, followed by a set of recommendations for adoption by the ecotoxicology community. These reporting requirements will provide clarity on the the test chemical, experimental design and conditions, chemical identification, test organisms, exposure confirmation, measurable endpoints, how data are presented, data availability and statistical analysis. Providing these specific details will allow for a fuller assessment of the reliability and relevance of the studies, including limitations. Recommendations for the implementation of these reporting requirements are provided herein for practitioners, journals, reviewers, regulators, stakeholders, funders, and professional societies. If applied, our recommendations will improve the quality of ecotoxicology studies and their value to environmental protection.

Balancing the health benefits and environmental risks of pharmaceuticals: Diclofenac as an example.

Pharmaceuticals are designed to improve human and animal health, but even the most beneficial pharmaceuticals might raise some questions concerning the consequences of exposure to non-target organisms. To illustrate this situation and using diclofenac as a case-study, we analyze global consumption and occurrence data to identify hot spots of consumption without occurrence data, review the scientific literature on the harmful environmental effects to determine whether the observed concentrations in freshwater are of environmental concern, summarize the current pharmaceutical and environmental policies to highlight policy gaps, and suggest a series of research and policy recommendations, which can be summarized as follows: we need to improve the current knowledge on occurrence in freshwaters to properly implement environmental policies (i), diclofenac might pose a risk to non-target organisms in freshwater (ii); the harmful effects that some pharmaceuticals may have on the environment are not always addressed by environmental policies (iii).

Environmentally persistent alkylphenolic compounds are estrogenic.

We show that a number of alkylphenolic compounds, used in a variety of commercial products and found in river water, are estrogenic in fish, birds, and mammals. 4-Octylphenol (OP), 4-nonylphenol, 4-nonylphenoxycarboxylic acid, and 4-nonylphenoldiethoxylate were each capable of stimulating vitellogenin gene expression in trout hepatocytes, gene transcription in transfected cells, and the growth of breast cancer cell lines. The most potent of the chemicals is OP, which was able to stimulate these biological responses to a similar extent as 17 beta-estradiol itself, albeit at a 1000-fold greater concentration. The action of alkylphenols is mediated by the estrogen receptor, as their effects depended on its presence and was blocked by estrogen antagonists. OP, 4-nonylphenol, and 4-nonylphenoxycarboxylic acid appear to possess intrinsic estrogenic activity, because they compete for binding to the estrogen receptor. Moreover, it is likely that they interact with a similar region of the hormone-binding domain as 17 beta-estradiol, because the mutant receptor G-525R, which is defective in estrogen binding, is also insensitive to OP. Like 17 beta-estradiol, OP is capable of stimulating the activity of both transcriptional activation functions, TAF-1 and TAF-2, in the receptor, as judged by analyzing the activity of the wild-type and mutant receptors in transiently transfected cells. The significance of our results will depend to a large extent on the degree of exposure of wildlife and humans to these estrogenic alkylphenolic compounds.

Molecular characterisation of ovarian cathepsin D in the rainbow trout, Oncorhynchus mykiss.

In fish, cathepsin D, an aspartyl protease, is believed to mediate the processing of yolk proteins in the oocyte. Cathepsin D, therefore, is vital for the production of a viable egg. This study set out to isolate and sequence the cDNA encoding cathepsin D, and to determine the developmental expression of the message in the ovary and subsequently during embryogenesis in the rainbow trout, Oncorhynchus mykiss. The full-length trout cathepsin D cDNA is 1847 base pairs (bp) long, encoding a protein of 400 amino acids (aa). The sequence consists of a putative signal peptide of 18 aa, a prosequence extending 46 aa and a mature peptide of 336 aa. The deduced sequence of rainbow trout ovarian cathepsin D shows significant homology with cathepsin D in mammals (human; 81% aa similarity), in the chicken (80% aa similarity) and in Xenopus (74% aa similarity). Our data support the contention that the primary structure of cathepsin D is highly conserved across the vertebrate phyla, from mammals to fish. Unlike cathepsin Ds in other species, however, rainbow trout cathepsin D appears to have only one putative N-glycosylation site, rather than two. The mRNA for 'ovarian' cathepsin D was expressed in both ovarian and non-ovarian tissues (liver, muscle, spleen and testis). During the development of the ovary, the highest expression levels of cathepsin D mRNA were seen at around the onset of vitellogenesis, a time when the oocytes are starting to sequester large quantities of yolk proteins. Northern hybridisation did not detect cathepsin D mRNA in either unfertilised eggs, or in fertilised eggs until after gastrulation, indicating that there is little, if any, de novo synthesis of this message at these stages of development. However, the mRNA for cathepsin D was detectable at the eyed embryo stage, and the expression of the gene increased towards the end of embryonic development.

Isolation and characterisation of mRNA encoding the salmon- and chicken-II type gonadotrophin-releasing hormones in the teleost fish Rutilus rutilus (Cyprinidae).

The complementary DNAs (cDNA) encoding the [Trp7,Leu8]-gonadotrophin-releasing hormone (salmon-type GnRH; sGnRH:GeneBank accession no. u60667) and the [His5,Trp7,Tyr8]-GnRH (chicken-II-type GnRH; cGnRH-II: GeneBank accession no. u60668) precursor in the roach (Rutilus rutilus) were isolated and sequenced following reverse transcription and rapid amplification of cDNA ends (RACE). The sGnRH and cGnRH-II precursor cDNAs consisted of 439 and 628 bp, and included open reading frames of 282 and 255 bp respectively. The structures of the encoded peptides were the same as GnRHs previously identified in other vertebrates. The sGnRH and cGnRH-II precursor cDNAs, including the non-coding regions, had 88.6 and 79.9% identity respectively, to those identified in goldfish (Carassius auratus). However, significant similarity was not observed between the non-coding regions of the GnRH cDNAs of Cyprinidae and other fish. The presumed third exon, encoding partial sGnRH associated peptide (GAP) of roach, demonstrated significant nucleotide and amino acid similarity with the appropriate regions in the goldfish, but not with other species, and this may indicate functional differences of GAP between different families of fish. cGnRH-II precursor cDNAs from roach had relatively high nucleotide similarity across this GnRH variant. Cladistic analysis classified the sGnRH and cGnRH-II precursor cDNAs into three and two groups respectively. However, the divergence between nucleotide sequences within the sGnRH variant was greater than those encoding the cGnRH-II precursors. Consistent with the consensus developed from previous studies, Northern blot analysis demonstrated that expression of sGnRH and cGnRH-II was restricted to the olfactory bulbs and midbrain of roach respectively. This work forms the basis for further study on the mechanisms by which the tapeworm, Ligula intestinalis, interacts with the pituitary-gonadal axis of its fish host.

Effects of background adaptation on the pituitary and plasma concentrations of some pro-opiomelanocortin-related peptides in the rainbow trout (Salmo gairdneri).

Radioimmunoassays for alpha-MSH, beta-MSH, ACTH and endorphin were used to measure pituitary concentrations of these peptides in rainbow trout during adaptation to black and white backgrounds. There was no difference in the pituitary content of any of these peptides between long-term black- and white-adapted trout. Plasma levels of alpha-MSH immunoreactivity were significantly higher in black-adapted trout than in white-adapted trout. Time-course studies revealed that although the body colour of trout showed an initial rapid adaptation to background colour, this was not paralleled by a corresponding change in plasma alpha-MSH levels. These only showed significant changes after 7 or more days of background adaptation, when melanophore recruitment or degradation occurred on black or white backgrounds respectively. Intravenous administration of mammalian alpha-MSH, salmon beta-MSH I or antibodies to these peptides did not affect short-term background adaptation. However, long-term administration of mammalian alpha-MSH via osmotic minipump maintained melanophore numbers in grey-adapted trout transferred to a white background, although this observation was based on only two fish. It is concluded that peptides derived from pro-opiomelanocortin do not appear to be involved in controlling physiological colour change but may be involved in regulating morphological colour change of the rainbow trout.

Several environmental oestrogens are also anti-androgens.

There is presently considerable interest in endocrine disruption which is a new area of endocrinology concerned with chemicals that mimic hormones, in particular sex steroids. It has been hypothesised that exposure to such chemicals may be responsible for adverse effects in both humans and wildlife. Until now, chemicals that mimic oestrogens (so-called xenoestrogens) have been the main focus of endocrine disruption research. However, recent evidence suggests that many abnormalities in the male reproductive system may be mediated via the androgen receptor. By blocking androgen action, exposure to an anti-androgen may cause changes similar to those associated with oestrogen exposure. We have used in vitro yeast-based assays to detect oestrogenic, anti-oestrogenic, androgenic and anti-androgenic activities in a variety of chemicals of current interest. We show that many of the so-called 'environmental oestrogens' also possess anti-androgenic activity. The previously reported anti-androgenic activities of vinclozolin and p,p'-1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) were confirmed. We also found that o,p'-1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), bisphenol A and butyl benzyl phthalate were anti-androgenic. However, not all xenoestrogens are also anti-androgenic, because nonylphenol was found to be a weak androgen agonist. Our results demonstrate that hormone-mimicking chemicals can have multiple hormonal activities, which may make it difficult to interpret their mechanisms of action in vivo. Although not a specific objective of this study, our results also demonstrate that yeast-based assays are powerful tools with which to investigate both agonist and antagonistic hormonal activities of chemicals.

Plasma somatolactin concentrations in salmonid fish are elevated by stress.

The preliminary finding that plasma levels of somatolactin (SL) were markedly elevated following stress caused by confinement in chinook salmon (Oncorhynchus tshawytscha) prompted a more detailed study of SL dynamics during stress. SL levels have been determined in the plasma of rainbow trout (Oncorhynchus mykiss) during exposure to acute (0-30 min) and short (0-24 h) periods of stress resulting from handling and confinement. The results show that SL levels increase rapidly within minutes following the onset of stress, reach a peak between 1 and 2 h, decline over the next 3 h, and then show an additional increase again by 24 h. During acute stress caused by confinement, the increase in plasma SL levels occurred within 2 min, thus showing a more rapid response than cortisol. This suggests that the response is mediated directly by the hypothalamus and is not a result of a feedback mechanism. The results also demonstrate that SL secretion in response to stress is at least partially under genetic control. In the short-term stress experiment, progeny of fish selected as high responders or low responders to stress, based on the magnitude of the plasma cortisol levels induced by stress, were used, and these fish showed similarly accentuated or attenuated release of SL following stress. These results clearly demonstrate that non-specific environmental stress causes rapid activation of SL-secreting cells in the pars intermedia, suggesting that this hormone has an important role in the adaptive response of fish to stress.

Pronounced seasonal rhythms in plasma somatolactin levels in rainbow trout.

The aim of this study was to establish whether there are seasonal or age-related changes in circulating levels of somatolactin (SL) in rainbow trout (Onchorhynchus mykiss). SL levels were determined in blood sampled at monthly intervals over a 2-year period from a population of rainbow trout maintained under a natural daylength and temperature regime (North-West England, latitude 54 degrees 20' N). SL levels displayed a distinct circannual cycle, with peak levels in summer (17-20 micrograms/l) and lowest levels occurring in winter (0.2-2 micrograms/l). This variation in SL levels was closely correlated with water temperature (P < 0.001) but was out of phase with changes in daylength. Plasma SL levels were significantly higher in both mature male and female fish compared with immature fish. Plasma prolactin levels were determined to provide information on a hormone structurally related to SL, and also because mammalian prolactin is known to show distinct seasonal cycles. However, trout prolactin, in contrast to SL, did not show a pronounced seasonal pattern, although prolactin levels were significantly, but inversely, correlated with water temperature (P < 0.001).

Peptides derived from pro-opiocortin in the pituitary gland of the dogfish, Squalus acanthias.

Combined rostral, median and neurointermediate lobes from 650 pituitary glands of the dogfish Squalus acanthias were extracted in 0.1 M-HCl and subjected to filtration on Sephadex G-50. Fractions were monitored for ACTH, alpha-MSH, gamma-MSH and endorphin by heterologous radioimmunoassay, corticotrophin-like intermediate lobe peptide (CLIP) and gamma-MSH by homologous radioimmunoassay, and methionine enkephalin after enzymatic digestion. The majority (about 99%) of the immunoreactivity detected was present as small peptides, with alpha-MSH, CLIP and gamma-MSH predominant. The single peaks of ACTH, alpha-MSH and CLIP contrasted with many distinct peaks of endorphin-like immunoreactivity. The gamma-MSH radioimmunoassays monitored different peptides; the heterologous assay revealed a single major peak, while the homologous assay detected a number of distinct small peptides. The results suggested that there may be more than three distinct forms of MSH in the dogfish pituitary gland. Small amounts of much larger proteins were detected by a number of the radioimmunoassays, suggesting that peptides related to ACTH, gamma-MSH and lipotrophin are derived from common pro-opiocortin-type precursor molecules in this phylogenetically ancient cartilaginous fish.

Biosynthesis of pro-opiomelanocortin-related peptides in the neurointermediate lobe of the pituitary gland of the rainbow trout (Salmo gairdneri).

This study concerns the biosynthesis of a number of peptides in the neurointermediate lobe of the rainbow trout, Salmo gairdneri. Among the newly synthesized peptides positively identified were four forms of alpha-MSH, a beta-MSH and two forms of endorphin. The identification of two of the products as sequentially distinct endorphins suggests that the trout neurointermediate lobe synthesizes two pro-opiomelanocortin (POMC) molecules. This may account for the large number of peptides synthesized and secreted by the trout neurointermediate lobe. The identification of some of the products is discussed. Pulse-chase analysis revealed no positive identification of a precursor molecule, which may indicate that trout POMC is very rapidly processed into smaller products. All of the newly synthesized peptides were secreted concomitantly. There was little if any evidence of post-translational modification of any of the peptides during their secretion.

Obstacles to the prediction of estrogenicity from chemical structure: assay-mediated metabolic transformation and the apparent promiscuous nature of the estrogen receptor.

Information on structure-activity relationships (SAR) and pathways of metabolic activation would facilitate the preliminary screening of chemicals for estrogenic potential. Published crystallographic studies of the estrogen receptor (ER) imply an essential role of the two hydroxyl groups on estradiol (17beta-E(2)) for its binding to ER. The influence of these hydroxyl groups on ER binding and estrogenicity was evaluated by the study of 17beta-E(2) with one or both of these hydroxyl groups removed (17beta-desoxyestradiol and 3, 17beta-bisdesoxyestradiol, respectively). 6-Hydroxytetralin (17beta-E(2) with its C- and D-rings removed) and other synthetic estrogens were also studied. The estrogenicity assays comprised a yeast ER-mediated transcription assay, mammalian cell transcription assays incorporating either ER alpha or ER beta, and the immature rat uterotrophic assay. With the exception of 6-hydroxytetralin in the uterotrophic assay, all the chemicals were active in all the assays. Hydroxylation of the two desoxy compounds to estradiol was shown to occur in immature female rats, but metabolism was not implicated in the responses observed in the ER-binding and yeast systems. It is concluded that the 3-hydroxyl and 17beta-hydroxyl groups of 17beta-E(2) are not absolute requirements for estrogenicity. It would therefore be of value to the derivation of SAR for estrogenicity were the crystal structure of the bisdesoxy-E(2)/ER complex to be evaluated.

Effects of estrogenic substances in the aquatic environment.

This review describes the research that has been carried out into estrogenic effects occurring in aquatic environments, both freshwater and marine, and the substances found to be responsible. In summary, estrogenic (and probably some anti-androgenic) activity has mainly been detected in a variety of treated sewage and other effluents, but also as a result of certain chemical spills and deliberate applications. This activity has resulted in a number of effects in vertebrate wildlife that can best be described as feminization, although the severity of these effects ranges from biomarkers of exposure such as vitellogenin induction in males through to morphological changes in sex organs and complete sex reversal. The implications of these changes for the future of aquatic wildlife populations have not yet been thoroughly explored. It is unlikely that all the causative substances have yet been discovered, but those which have been positively identified include natural and synthetic estrogenic hormones, natural plant sterols, synthetic alkylphenols, and certain organochlorine substances. The review concludes that there is now a need to investigate the consequences for wildlife populations of exposure to these materials, by means of a variety of field experiments and investigations.

Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment.

A rapidly increasing number of chemicals, or their degradation products, are being recognized as possessing estrogenic activity, albeit usually weak. We have found that effluent from sewage treatment works contains a chemical, or mixture of chemicals, that induces vitellogenin synthesis in male fish maintained in the effluent, thus indicating that the effluent is estrogenic. The effect was extremely pronounced and occurred at all sewage treatment works tested. The nature of the chemical or chemicals causing the effect is presently not known. However, we have tested a number of chemicals known to be estrogenic to mammals and have shown that they are also estrogenic to fish; that is, no species specificity was apparent. Many of these weakly estrogenic chemicals are known to be present in effluents. Further, a mixture of different estrogenic chemicals was considerably more potent than each of the chemicals when tested individually, suggesting that enhanced effects could occur when fish are exposed simultaneously to various estrogenic chemicals (as is likely to occur in rivers receiving effluent). Subsequent work should determine whether exposure to these chemicals at the concentrations present in the environment leads to any deleterious physiological effects.

Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay.

We used a recombinant yeast estrogen assay to assess the activity of 73 phenolic additives that are used as sunscreens, preservatives, disinfectants, antioxidants, flavorings, or for perfumery. Thirty-two of these compounds displayed activity: 22 with potencies relative to 17beta-estradiol, ranging from 1/3,000 to < 1/3,000,000, and 10 compounds with an impaired response that could not be directly compared with 17beta-estradiol. Forty-one compounds were inactive. The major criteria for activity appear to be the presence of an unhindered phenolic OH group in a para position and a molecular weight of 140-250 Da.

The estrogenic activity of phthalate esters in vitro.

A large number of phthalate esters were screened for estrogenic activity using a recombinant yeast screen. a selection of these was also tested for mitogenic effect on estrogen-responsive human breast cancer cells. A small number of the commercially available phthalates tested showed extremely weak estrogenic activity. The relative potencies of these descended in the order butyl benzyl phthalate (BBP) > dibutyl phthalate (DBP) > diisobutyl phthalate (DIBP) > diethyl phthalate (DEP) > diisiononyl phthalate (DINP). Potencies ranged from approximately 1 x 10(6) to 5 x 10(7) times less than 17beta-estradiol. The phthalates that were estrogenic in the yeast screen were also mitogenic on the human breast cancer cells. Di(2-ethylhexyl) phthalate (DEHP) showed no estrogenic activity in these in vitro assays. A number of metabolites were tested, including mono-butyl phthalate, mono-benzyl phthalate, mono-ethylhexyl phthalate, mon-n-octyl phthalate; all were wound to be inactive. One of the phthalates, ditridecyl phthalate (DTDP), produced inconsistent results; one sample was weakly estrogenic, whereas another, obtained from a different source, was inactive. analysis by gel chromatography-mass spectometry showed that the preparation exhibiting estrogenic activity contained 0.5% of the ortho-isomer of bisphenol A. It is likely that the presence of this antioxidant in the phthalate standard was responsible for the generation of a dose-response curve--which was not observed with an alternative sample that had not been supplemented with o,p'-bisphenol A--in the yeast screen; hence, DTDP is probably not weakly estrogenic. The activities of simple mixtures of BBP, DBP, and 17beta-estradiol were assessed in the yeast screen. No synergism was observed, although the activities of the mixtures were approximately additive. In summary, a small number of phthalates are weakly estrogenic in vitro. No data has yet been published on whether these are also estrogenic in vitro. No data has yet been published on whether these are also estrogenic in vivo; this will require tests using different classes of vertebrates and different routes of exposure.