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ⅥEndocrine Disruptors

Potential Health Effects of Alkylphenols in Japan

JMAJ 46(3): 108–114, 2003

Fujio KAYAMA*, Hyogo HORIGUCHI* and Akihiko HAMAMATSU**

*Division of Environmental Immunology and Toxicology, Department of Health Science, Jichi Medical School **Tokyo Medical Examiner’s Office

Abstract:More than twenty thousand tons of alkylphenols and alkylphenol ethoxylates are used annually in Japan in synthetic rubber industry, plastic, fabric, and metal processing industries as , cleaners, stabilizers for ethyl- cellulose, plasticizes, and resins. Some forms of alkylphenols are reported to induce endocrine disruption especially in fish species. Data monitoring of inland aquatic environments conducted by the Ministry of Environment in 2000 reveal that the concentrations of nonylphenol in 71 monitoring sites (4.5% of all monitoring sites) exceeded the Predicted No Effect Concentration (PNEC) for fish. Even if there is no clear evidence of effects on human health by alkylphenols, the study revealed that the current environmental concentrations in these sites influence reproduction in fish species. As precautionary measures, it is recommended to curb emissions of alkylphenols and its ethoxylates into the aquatic environment. Key words:Alkylphenol; Alkylphenol ethoxylate; Fish; Reproductive toxicity; Endocrine disruptors

Introduction plastic tubes used in experiments. In 1991, Ana Soto et al. from the Tufts Medi- Examining plasticware from various manu- cal School, Massachusetts, USA, observed an facturers led Soto et al. to the discovery that a abnormal proliferation of MCF-7 breast tumor chemical compound extractable from plastic- cells, grown without any stimulating agents in ware of a certain manufacturer is the source of the culture medium. After a thorough investi- contamination. Since the manufacturer refused gation of the possible causes, they ascertained to disclose the chemical, adducing “trade that the abnormal cell response was due to the secret” reasons, identification of the compound

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 127, No. 2, 2002, pages 216–220). The Japanese text is a transcript of a lecture originally aired on October 18, 2001, by the Nihon Shortwave Broadcasting Co., Ltd., in its regular program “Special Course in Medicine”.

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was not initially possible. Analysis of the OH chemical, performed in cooperation with spe- cialists in the field, revealed that the compo- nent in the plastic was nonylphenol. Soto’s group discovered that this synthetic chemical HO HO had -like activity and was responsible Nonylphenol ͱ- for proliferation of the breast tumor cells. (one isomer with branched side chain) Later, other estrogen-mimicking chemicals, Fig. 1 Formula of alkylphenol including a metal can coating compound, A, were also reported. Sumpter et al. observed a high incidence of intersexuality (presence of both male and Alkylphenol production and consumption vol- female gonadal features in the same animal) in umes decrease accordingly in the above men- roaches, a freshwater fish living in the River tioned order, the product with the smallest vol- Lea in the South of Britain. To find the cause of ume being dodecylphenol. Moreover, there are this abnormal condition, they cultivated a num- many alkylphenols with various chemical struc- ber of rainbow trout in cages downstream from tures characterized by branched multi-carbon a number of sewage treatment plants, mea- side chains. sured blood concentrations of (an Alkylphenols are mostly used as alkylphenol egg-yolk protein) in male fish and concentra- ethoxylates, that is, alkylphenols binding an tions of nonylphenol in the river water. ethoxy chain through their hydroxyl groups. The results of their studies indicated that The wide range of alkylphenol ethoxylate containing alkylphenol ethoxylates applications includes surfactants, ethylcellulose used in wool processing factories, particularly stabilizers, hydrophobic phenol resins, which those producing nonylphenol-group metabo- are utilized in detergents, oil varnishes, syn- lites, are potentially the main cause of the inter- thetic rubber vulcanization accelerators, anti- sex gonadal features observed in rainbow oxidants of petroleum products and trout. They demonstrated a positive correlation additives in agriculture. between blood vitellogenin concentrations in male fish and nonylphenol concentration in Usage and Consumption Volume of water and reported that activated sewage Alkylphenol Ethoxylates sludge can degrade alkylphenol ethoxylates, resulting in the release of estrogenic alkyl- The annual alkylphenol ethoxylate produc- phenol metabolites into the water. tion in Japan reached 46,850t in 1998. As shown in Table 1, the largest discharge of What are Alkylphenols? alkylphenol metabolites is considered to be from fabrics, metal processing, and cleaning The chemical structure of alkylphenols is industries, among others; however, accurate based on the phenol ring with multi-carbon data on the volume discharged into the envi- moieties: nonylphenol is a 9-carbon side chain ronment in Japan is unavailable. alkylphenol; octylphenol has an 8-carbon alkyl It has been estimated that distribution of chain; butylphenol has a 4-carbon side chain, alkylphenol compounds after release into the and dodecylphenol is a 12-carbon side chain environment is 58–73% for water and 27–41% alkylphenol (Fig. 1). Nonylphenol and octyl- for estuarine sediments. Furthermore, alkyl- phenol are the most widely used alkylphenols phenol compound levels in organs of animals and have the broadest range of application. high in the chain hierarchy are not neces-

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Table 1 Domestic Consumption of Alkylphenol Ethoxylates (as for 1998) of Fish Species

Synthetic rubber and plastic industry 4,200(t) A committee at the Ministry of Environment has temporarily established 4 testing methods Fabric industry 4,000 to examine the impact of endocrine disruptors Metal processing industry 3,300 on fish species. They are, as follows: Business-use detergents 2,300 1. FLF/d-rR medaka test, 2. Reproduction test, Cleaning industry 1,400 3. Vitellogenin assay, and Dyes, pigments, paints, inks 1,100 4. Medaka partial life cycle test. industry 900 The items 1., 3., and 4. are described below in more detail. Agriculture 800 Regarding the FLF/d-rR medaka test and Paper and pulp industry 700 binding activity, nonyl- Petroleum and fuel industry 600 phenol binds the estrogen receptor 10-times ␤ Civil engineering and construction industry 600 weaker than 17- -estradiol (E2), in concentra- ,M. Further 8מto 10 9מtions ranging from 10 Pharmaceuticals and cosmetics industry 500 based on a reporter gene assay study with Leather auxiliaries 100 estrogen receptor-introduced cells, it was dem- Others 3,400 onstrated that nonylphenol elicits transcrip- tion-stimulating activity hundreds times lower Total consumption 23,900 than E2. On the other hand, in regard to the vitello- genin assay and alkylphenol concentrations affecting fish species, it was reported that an sarily high and do not tend to get concentrated alkylphenol concentration capable of inducing as a result of biological condensation, as dem- abnormal microscopic changes in the testicular onstrated in the results of a study carried out by tissue of fathead minnow (a North-American Tsuda et al. on fish species in Lake Biwa, Japan. cyprinid freshwater fish) was 1.6␮g/l; in juve- nile trout, the effective concentration that pro- Alkylphenols in the Environment duced elevated liver vitellogenin levels was 10␮g/l and in mature male trout, the concen- In sewage treatment plants, alkylphenol tration sufficient to induce elevated serum ethoxylates are degraded aerobically with vitellogenin levels was 20␮g/l. sequential cleavage of ethyl moieties and Concerning medaka partial life cycle test, release of alkylphenols. According to the fact- secondary sex characteristics changes (male finding survey performed by the Japan Envi- fish feminization) were observed in male fish at ronment Agency and Ministry of Construction alkylphenol water concentration of 23.5␮g/l, in 1998/99, the alkylphenol concentrations in with ovotestis and vitellogenesis at an alkyl- water samples ranged from ND (lower than phenol water concentration of 11.6␮g/l. or equal to the lowest detectable limit) to The medaka full life cycle test was per- 21␮g/l, with an average value of 0.22␮g/l, and formed in order to make a detailed examina- 0.59␮g/l (95 percentile) was tentatively set as tion of the alkylphenol effects on subsequent the estimated concentration for the environ- fish generations, revealed abnormal sex differ- ment, the highest alkylphenol compound con- entiation and lower fertilization rates among centration for an average aquatic locality. parental male fish at alkylphenol water concen-

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tration of 17.7␮g/l. In the second generation, foreign countries to reduce alkylphenol etho- the ovotestis condition was found to be present xylate applications, which target a number of at the concentration of 8.2␮g/l, a level not industries, include various initiatives such as observed in the first generation males. legislative regulations and usage limitations. In Japan, such actions include the ban on Ministry of Environment Survey alkylphenol ethoxylate applications in domes- —A Summary tic-use detergents and efforts to find alkyl- phenol substitutes for business- and industrial- The results of the survey on alkylphenols use detergents. Hence, in implementing the performed by the Ministry of Environment Pollutant Release and Transfer Register sys- revealed that fish develop an ovotestis condi- tem, it would be necessary to promote the tion after exposure to low concentrations of automatic administration policy and coopera- octylphenol; which brings the endocrine dis- tion between industry, the government, and rupting activity of this alkylphenol compound academia. into question. On the other hand, nonylphenol was considered a strong in Toxicity in Mammals fish, backed by results of in vitro studies on its nonylphenol ability to bind estrogen receptor Although even very low alkylphenol concen- and transcription-stimulating activity. trations produce harmful effects in fish species, Based on the medaka partial life cycle test alkylphenol toxicity in mammals has been and alkylphenol concentration in water, the demonstrated by acute oral toxicity test results

Lowest Observed Effect Concentration in rats, the lethal dose being 50% (LD50) of (LOEC) that affected fish sexual behavior was 1,200–2,400 mg/kg. Moreover, a repeated dos- demonstrated to be 11.6␮g/l, and the No ing study in rats revealed that pathological Observed Effect Concentration (NOEC) to be changes initially appear in the liver and 6.08␮g/l. The Predicted No Effect Concentra- kidneys. The no observable adverse effect tion (PNEC), the estimated concentration of (NOAEL) dose was found to be 100 mg/kg/ alkylphenols in water that does not affect wild- day for non-branched nonylphenol and 50mg/ life species, was calculated as 1/10 of NOEC kg/day for branched nonylphenol. value, that is 0.608␮g/l. Regarding the toxicity of alkylphenols on the Thus, with the PNEC being 0.608␮g/l, water reproductive system, on the second and third concentrations ranging from ND to 21␮g/l generation tests, endocrine disrupting activities in the domestic aquatic environment (data such as weight increase of the , weight obtained from the environment fact-finding decrease of the ovaries, decrease of sperm den- survey), and alkylphenol concentrations sity in epididymides, elongation of the matura- detected in 71 sampling locations (4.5%) tion period and other abnormalities were dem- exceeded the PNEC value. It was therefore onstrated with alkylphenols in doses of several considered that nonylphenol, in concentrations 10 mg/kg/day. It has been considered that detectable in our country’s aquatic environ- mammals are less sensitive to nonylphenol ment, impacts on the endocrine system func- than fish. Similarly, in vitro studies have also tions in fish species and possibly affects their demonstrated that both binding to the human reproductive system, as well. estrogen receptor and transcription-stimulat- ing activity in mammals are extremely weak in Risk Reduction Measures comparison with binding to the medaka estro- gen receptor and transcription-stimulating The advanced measures undertaken in many activity in fish.

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Estrogenic Activity of Alkylphenols from the oscillating dose-response curve. Using the NOAEL value, a safety factor, NOAEL -was established with the specific differ ,0.1ן -The effects of endocrine disruption by alkyl may be summarized as follows: 1. ence between laboratory animals and humans Added to the culture medium, alkylphenols defined as 0.1, and then taking into account the stimulate in vitro proliferation of the MCF-7 diverse patterns of human sensitivity to chemi- breast tumor cells. 2. They bind to the estrogen cals further multiplied by 0.1. The allowance receptor and elicit transcription-stimulating dose was determined from the estimation that 100-fold as theןactivity. 3. At the water concentration of 10ppb, the safe dose equals NOAEL alkylphenols induce an increase of serum vitel- safety factor value. logenin levels in male rainbow trout. 4. In If endocrine disruptors produce low dose mammalian species, they cause mammary effects, then this necessitates the redesigning of gland cell proliferation and elongation of the safety assessments for a whole range of chemi- sexual cycle, applied in 0.01 mg/kg dose in rat cals, including subjects treated with even lower chronic oral toxicity test. 5. Moreover, male concentrations of a chemical or pharmaceutical rats fed with 1 mg/l alkylphenol in drinking agent into experimental groups, redoubling the water in the prenatal period and from the 22nd cost burden as a result. The “low dose effect” day after birth, have lower-weight testis and hypothesis has already been openly and aggres- lower sperm counts. sively challenged by the chemical industry. Thus, compared with fish species, mammals A similar phenomenon to low dose effects tend to be less sensitive to alkylphenolic has been known to exist in biology; however, compounds. One source of concern, however, since the two arguments are at loggerheads, is the low dose effect problem of endocrine a conclusion has yet to be reached. However, disruptors. the concept of reassessing safety doses for all chemical compounds taking the “low dose Low Dose Effect of Endocrine effect” into consideration does not appear to Disruptors be the opinion of the majority.

Fred von Saal et al. from the University of Studies on Changes in Louisiana reported that treatment of pregnant Male Reproductive Functions rats with very low quantities of resulted in delivery of male offspring with In 1987, Carlsen and Skakkebaek et al. issued lower prostate weights. The dose-response a report based on a meta-analysis of papers curve drawn in their experiment was not published in various countries and related to sigmoid-shaped, as is generally observed, but semen qualities. Their findings indicated a sig- had an inverted U-shape. Furthermore, von nificant decrease in sperm counts—by 50% Saal et al. also reported the early sexual matu- over the past 50 years. The researchers pro- rity of female offspring born to rats treated posed that the cause of such a condition was with ethynyl estradiol during . Their the prenatal disruption of reproductive organs findings, known as the “low dose effects,” have development due to exposure to sex - attracted considerable attention. mimicking chemicals. Though their study drew The reason for the special interest aroused some criticism concerning the differences by the “low dose effect” hypothesis is that until between the subjects examined in the included the publication of von Saal’s results, toxic dose papers, the inability to control the quality of evaluation of a given pharmaceutical or chemi- data in some countries and the inappropriate cal agent was based on the NOAEL calculation statistical methods applied by some authors,

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the work of Carlsen and Skakkebaek et al. (g) made a major contribution to the research on 20.5 male reproductive functions, which, since pub- 20.0 lication, has become more vigorous worldwide. 19.5 Subsequently, reports on reproductive system 19.0 abnormalities observed in wildlife species and 18.5 the decline of in men have gar- Weight of testes 18.0 17.5 nered widespread interest, leading to the estab- 45 Ð 49 50 Ð 54 55 Ð 59 60 Ð 64 65 Ð 69 70 Ð74 75 Ð78 lishment of fair-sized-budget research projects Ages in Europe and America since the mid 1990s. Fig. 2 Changes in weights of testes among men Likewise, in 1998, using what little basic data in their 20s are available, we conducted research on the quality of semen among healthy Japanese men, in collaboration with the Department of Urol- regarding risk assessment of the alkylphenol ogy, School of Medicine, Sapporo University. compounds, we concluded that since exposure In fact, the department mentioned above con- to these endocrine disruptors in prenatal and ducted research on healthy men from Sapporo neonatal period causes suppression of testis some 20 years previously, and a comparison development, alkylphenols cannot continue to with the 1998 survey data revealed no marked be considered safe for a mammalian organism. changes in sperm count or sperm . Since A more detailed and prospective survey is semen quality is influenced by periods of absti- necessary. nence, sperm collection methods and daily fluc- tuations among individuals, the importance of Changes in Environmental Policies a standardized system had been raised. An international quality control program led by In our country, the main objective and start- Skakkebaek from Denmark, related to the ing point for many environmental policies is to research on male reproductive function has protect human health from any environmental been highly promoted. pollulion. The Environment Agency has adopted Additionally, we collected fat tissue from specific measures to act sensibly in compensa- human autopsies to measure endocrine dis- tion cases for victims suffering from Minamata ruptor concentrations and to analyze sperm disease, Itai-itai disease, Yokkaichi asthma, and formation; an estimation of exposure to and similar diseases. It is no exaggeration to say impact from given endocrine disruptors was that to date, the environmental standards have simultaneously conducted on the histopatho- predominantly been based on prevention of logical samples. During the study, our attention environmental harm to human health. was drawn to lifelong changes in testes’ weights. On the other hand, the US Environmental We discovered that, unlike general growth accel- Protection Agency (US EPA), which aims to eration changes observed among young men protect the survival of wildlife species, was from 1945 (the time of administrative autopsy established after the findings that exposure to program implementation), testes’ weights did DDT causes abnormal reproduction in birds. not follow the growth acceleration pattern of In fact, approval of a new pesticide in Europe gradual increases in body height and weight, and America requires reproductive toxicity but entered a plateau phase or even decreased testing in birds; this is not a preventive measure among men in their 20s born in and after 1970 towards potentially harmful effects on the (Fig. 2). human reproductive system. From the aforementioned observations and Regarding the effects of nonylphenol, the

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Ministry of Environment has recognized the policy. Protection of wildlife species, which are harmful effects of this alkylphenolic compound more sensitive to environmental harm than on reproduction in wildlife species and has humans, is an epoch-making decision. established an environmental protection

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