The APE Research Council is composed of manufacturers, processors, users and raw material suppliers. Its mission is to develop an accurate and scientifically sound understanding of the human health and environmental profile of alkylphenols and alkylphenol ethoxylates.

For additional information, contact Robert J. Fensterheim, Executive Director at (202) 419-1506 or info@aperc.org.

Testing and Testing Methodology

Identification and Quantitation of Alkylphenols in Fish Tissue

Since sediments in polluted waters can contain nonylphenol (NP), there is the potential for fish to be exposed and ingest NP. A few reports of NP in fish tissue have appeared, but the sites and sample handling procedures have not been well documented. As a result, the APE Research Council provided a research grant to Michigan State University (MSU) to: 1) develop an improved analytical method for measuring NP in tissue; 2) determine the ranges of NP and lower-oligomer NP-ethoxylates in whole fishes of the north-central United States; and, 3) to determine concentrations of NP in fish inhabiting rivers receiving effluents that are likely to contain NP-ethoxylates or their degradation products. The method validation for extractive steam distillation and normal phase high-pressure liquid chromatography was completed in March 1999, and detection limits of 2 ppb have been demonstrated in fish tissue. The protocol for fish collection and handling is now under development. It is planned that twenty fish will be collected at each of ten sites over a period of six months, for a total of 200 extractions and analyses.

An International Round Robin Analysis to Determine the Effect of Analytical Methodology on Reported Nonylphenol/ Nonylphenol Ethoxylate Levels in the Environment

This project was an interlaboratory study to examine whether analytical methodology is a factor for differences in nonylphenol/nonylphenol ethoxylates measured in environmental samples. There was significant variability in the reported results from the eight participating laboratories' analyses of standard samples which emphasizes the critical need for full documentation and high quality assurance for all environmental monitoring data.

Aquatic Toxicology Studies

Nonylphenol-1.5EO - Chronic Toxicity to Mysids (Mysidopsis bahia) Under Flow-Through Conditions

The objective of this study was to determine the chronic effects of Nonylphenol-1.5EO (NPE1.5) on the survival, reproduction and growth (average total body length and dry weight) of the mysid, Mysidopsis bahia. The study was performed under flow-through conditions which provided continuous aqueous exposure for a period of 28 days (approximately one generation). Based on statistical analysis of mysid reproduction, the lowest-observed-effect concentration (LOEC) was 16mg/L. The no-observed-effect concentration (NOEC) for NPE1.5 was 7.7 mg/L. The final report was issued on March 30, 1999.

Aquatic Toxicity of Nonylphenol

A comprehensive program of testing for toxicity of nonylphenol (NP) and/or nonylphenol ethoxylate (NPE) to aquatic life was undertaken. The toxicity testing included acute and chronic exposures, and a wide range of organisms in terms of taxonomy and ecology. Saltwater and freshwater fish, algae, and invertebrate animals were represented. Water column exposures were done via flow-through systems, and dosed sediment exposures were done for bottom-dwelling species. All tests were conducted using EPA-approved protocols under Good Laboratory Practice procedures, by laboratories subject to independent audits. The following studies were conducted:

Freshwater Species

1. Chronic Toxicity of NP to Ceriodaphnia dubia (water flea)

2. Chronic Toxicity of NPE to Ceriodaphnia dubia

3. Chronic Toxicity of NP to Chironomus tentans (midge)

4. Acute Toxicity of NP to Chironomus tentans

5. Acute Toxicity of NP to Hyalella azteca (amphipod)

6. Acute Toxicity of NP to Selenastrum capricornutum (alga)

7. Early Life Stage 3C-Day of NP to Pimephales promelas (fathead minnow)

Saltwater Species

1. Chronic Toxicity of NP to Mysidopsis bahia (shrimp)

2. Acute Toxicity of NP to Mysidopsis bahia

3. Acute Toxicity of NP to Cyprinodon variegatus (sheepshead minnow)

4. Acute Toxicity of NP to Skeletonema costatum (alga)

Sediment Species

1. Chronic Toxicity of NP to Chironomus tentans (tube-dwelling midge)

2. Chronic Toxicity of NP to Rana catesbiana (frog tadpole)

Staples, C.A., et al. (1998) "An evaluation of the aquatic toxicity and bioaccumulaion of C8- and C9-alkylphenol ethoxylates," Environmental Toxicology and Chemistry 17, 2470-2480.

Aquatic Toxicity of Some Nonylphenol Ethoxylate (NPE) and Octylphenol Ethoxylate (OPE) Metabolites

This study performed chronic toxicity tests on the degradation intermediates of the most common APE surfactants: nonylphenoxyacedic acid (NPEC) and octylphenoxyacetic acid (OPEC). The following tests were conducted:

Nonylphenoxyacetic Acid (NPEC) Studies

1. Acute Toxicity of NPEC to Mysidopsis bahia

2. Acute Toxicity of NPEC to Daphnia magna

3. Chronic Toxicity of NPEC to Ceriodaphnia dubia

Octylphenoxyacetic Acid (OPEC) Studies

1. Acute Toxicity of OPEC to Mysidopsis bahia

2. Acute Toxicity of OPEC to Daphnia magna

3. Chronic Toxicity of OPEC to Ceriodaphnia dubia

Bioconcentration of Nonylphenol

A bioconcentration test was conducted of nonylphenol (NP) in the fathead minnow, Pimephales promelas. Fish were exposed to either 4.9 or 22.7 mg/L of NP under flow through conditions for 20 days and then to dilution water without test substance for 7 days. Exposure of fathead minnows to 4.9 mg/L in water for 20 days resulted in a bioconcentration factor of 271, based on an uptake rate constant of 133 ml/g wet-d and a depuration rate constant of 0.49/d. Exposure of fathead minnows to 22.7 mg/L in water for 20 days resulted in a bioconcentration factor of 344, with an uptake rate constant of 193 ml/g wet-d and a depuration rate constant of 0.56/d.

Staples, C.A., et al. (1998) "An evaluation of the aquatic toxicity and bioaccumulaion of C8- and C9-alkylphenol ethoxylates," Environmental Toxicology and Chemistry 17, 2470-2480.

Avian Toxicity Studies

Avian Dietary Toxicity with Nonylphenol Ethoxylate 9 (NPE9) in the Northern Bobwhite Quail

The objective of this study was to determine the acute dietary LC50, lowest-lethal concentration (LLC) and no-observable effect concentration (NOEC) for mortality of nonylphenol ethoxylate 9 (NPE9) administered via the diet to Northern Bobwhite Quail. Methods used were provided by the U.S. EPA for data requirement 40 CFR 158, Series 71-2, Subdivision E; U.S. EPA Ecological Effects Test Guidelines, OPPTS 850-2200, Avian Dietary Toxicity Test, January, 1995; and, OECD Guideline for Testing of Chemicals, "Avian Dietary Toxicity Test," 205, April 4, 1984. It appears there were no effects up to 5,000 ppm, which was the highest dose tested. The draft report is currently being prepared.

Environmental Exposure Studies

Monitoring the Fate of Nonylphenol Ethoxylates Released from Septic Systems

A significant percentage of North American homes employ on-site septic systems for treatment and disposal of wastewater. The surfactants in household laundry detergents must therefore degrade in these systems. The objective of this study is to generate additional information on the fate of nonylphenol ethoxylates (NPE) as they move through septic tanks and leach fields into the underlying soil and potentially into groundwater. The study objective is being achieved by introducing and tracking a known amount of NPE9 through two septic systems; Site A (low permeability, poorly drained) and Site B (porous, rapidly drained). Terran Corporation continues to collect samples at both sites for the septic project. The dose of NPE at site A was tripled in November 1998, but only traces of NPE have been detected in the leach field. Traces have not been detected past a depth of four feet. The salt tracer was detected in groundwater after eight days at site B, but NPE has not yet appeared in samples from this site after six months. Sampling is expected to continue at least through 1999.

U.S.A. 30 Rivers Study

A comprehensive monitoring study, designed in conjunction with the EPA, measured the levels of nonylphenol (NP) and nonylphenol ethoxylate (NPE) in 30 U.S. rivers. The sites, all receiving municipal or industrial wastewater, were selected at random by a statistical procedure from the EPA's river reach database. Water column and bottom sediment samples were collected along a perpendicular transect at each site. All samples were assayed for NP and NPE1, and the higher ethoxylates (NPE2 to NPE17) were determined in the water samples. Analysis was by high-performance liquid chromatography (HPLC) with fluorescence detection of microgram quantities of NPE obtained by extractive steam distillation (NP and NPE1) or a dual - column extraction procedure (NPE2 to NPE17). Sample collection and analytical procedures were validated according to rigorous EPA guidelines, and quality assurance standards were met throughout the study. NP and NPE concentrations in river water were below detection limits (0.1 ppb for NP, NPE1 and NPE2: 1.6 ppb for NPE3-17) in 60 to 75% of the samples. The highest levels found were about 1 ppb for NP, NPE1, and NPE2, 15 ppb for NPE3-17. A majority of sediment samples contained detectable amounts of NP and NPE1, ranging up to 3000 ppb for NP and 170 ppb for NPE1. Sediment interstitial water concentrations of NP were estimated to be similar to concentrations in the water column.

Naylor, C.G., Mieure, J.P., Adams, W.J., Weeks, J.A., Castaldi, F.J., Ogle, L.D. and Romano, R.R. (1992) "Alkylphenol ethoxylates in the environment," Journal of the American Oil Chemists Society 69, 7, 605-703.

Nonylphenol Ethoxylates in an Industrial River

Information essential for judging the environmental risk of nonylphenol ethoxylates (NPE) in an important industrial river in Wisconsin was obtained by a unique cooperative agreement among the Wisconsin Department of Natural Resources, the local industry, local wastewater authorities and NPE manufacturers. Three rounds of monitoring the lower Fox River in Wisconsin for residues of NPE during a period of two years showed the concentrations to be well within the statistical model of NPE exposure in American rivers. The Fox River receives discharges from over 20 paper mills and publicly owned treatment works (POTWs) which treat NPE -containing wastewater from mills and industrial laundries. The influx of NPE to the river from paper mills and cities was measured in winter and summer. Levels in the Fox River showed low concentrations of nonylphenol (NP) and 1 mole and 2 mole ethoxylates in sediment and generally non-detectable concentrations in water. The concentrations of NP and NPE in the Fox River were well below the most sensitive thresholds of adverse effects on aquatic organisms. Use of NPE by industry, commercial cleaning businesses and households does not appear to pose a significant risk to the Fox River environment. Because NPE is so widely used, measurement of NP and NPE provides a rapid and sensitive means for evaluating the general quality of water after its use by cities and industry. The results were comparable to those obtained in the 30 Rivers Study where there were industrial plants and secondary treatment facilities.

Naylor, C.G., Williams, J.B., Varineau, P.T., Webb, D.A. and Sanders, D.F. (1996) "Nonylphenol ethoxylates in an industrial river," Presented at the 4th CESIO World Surfactants Congress, Proceedings, Vol. 4, 378-391.

Nonylphenol Ether Carboxylates in an Industrial River

Monitoring of nonylphenol ethoxylates (NPE) in the Fox River of Wisconsin was enhanced by including their carboxylate biodegradation intermediate metabolites. The carboxylates (NPECs) were measured in all significant wastewater effluent streams entering the river and in the river itself. NPECs were present in the river at its mouth in low concentrations, but at levels higher than NPE. NPE2C (nonylphenoxyethoxy acetic acid) was the most prevalent carboxylate species. NPEC/NPE ratios in the effluents were highly variable.

Naylor, C.G., Varineau, P.T. and Field, J. "Nonylphenol ether carboxylates in an industrial river." Presented at the 17th Annual Meeting of the Society of Environmental Toxicology and Chemistry, November, 1996 and the Annual Meeting of the American Oil Chemists Society, May, 1997.

Environmental Fate of Nonylphenol Ethoxylates

Nonylphenol ethoxylates (NPE) are surfactants used in textile manufacture, as well as in industrial and household cleaning products. NPE exhibit toxicity patterns very similar to other surfactants. They are highly treatable in aerobic biological treatment plants and studies in U.S. wastewater plants have shown up to 99.8% removal. Nationwide river monitoring has provided a statistically valid model of the distribution of p-nonylphenol (NP) and nonylphenol ethoxylates in U.S. river waters. The treatability studies demonstrate their high degree of degradability under real-world conditions. The large body of data on toxicity, combined with environmental exposure data from this study, allow confident predictions of the risks NP and NPE pose to the aquatic environment.

Naylor, C.G., Presented at the 17th Meeting of the Society of Environmental Toxicology and Chemistry, November, 1996.

Nonylphenol Ethoxylates: Chemistry Applications and Routes into the Aquatic Environment

The uses for nonylphenol ethoxylate (NPE) surfactants are wide and diverse. In most applications, the surfactants are used briefly then disposed into wastewater streams. Wastewater treatment is essential for minimizing the environmental impact of residual NPE. Studies of NPE treatability and environmental exposure conducted by the Alkylphenols and Ethoxylates Panel were reviewed.

Naylor, C.G., Presented at the 17th Annual Meeting of the Society of Environmental Toxicology and Chemistry, November, 1996.

Environmental Chemistry Studies

Nonylphenol and Octylphenol and their Ethoxylates - Determination of the Biodegradability by the CO2 Evolution Modified Sturm Test (OECD 301B)

This study was performed to determine the potential for biodegradation of nonylphenol (NP) and octylphenol (OP) and their ethoxylates in water by the carbon dioxide evolution method following OECD Test Guideline 301B. The amount of carbon dioxide released upon biodegradation of the test substance and two reference substances, sodium benzoate and di-isotridecyl adipate, was measured. Nonylphenol ethoxylate 9 (NPE9), octylphenol ethoxylate 9 (OPE9), OP, octylphenol ethoxylate 1.5 (OPE1.5), and the di-isotridecyl adipate were extensively mineralized (> 60% CO2) during the 35-day study; NP reached 48% and NPE1.5 57% mineralization. The final amended report was received from Springborn Laboratories on February 10, 1999. The Council is in the process of incorporating the results from the 301B and ISO projects into a manuscript for publication.

Biodegradation of Detergent Products Using the ISO Headspace CO2 Biodegradation Test (Methodology - ISO/DIS - 14593)

Surfactants Marlophen® NP9, Suframin® Acid TPB (branched dodecylbenzene sulfonate), Amonyl® 675-SB (a sulfobetaine amphoteric surfactant) and a reference substance benzoic acid were tested according to the ISO Headspace CO2 Biodegradation Test. Both NP9 and 675-SB were extensively biodegraded (69-71% CO2 production), while TPB was not (11-15%). The final summary report for this project was issued on November 9, 1998.

Fate of Nonylphenol in Composted Sludge

Residual surfactants and surfactant metabolites can be detected in the waste sludge from biological wastewater treatment plants. An increasingly popular way to utilize these waste biosolids is composting to produce a salable product. The major nonylphenol ethoxylate (NPE) residue in sludge is nonylphenol (NP). This project involves analyzing composted sewage sludge from Austin, Texas (Dillo Dirt) for nonylphenol. Temperature is monitored daily at three points as a measure of biological activity until the composting process is complete. Analysis of samples after 125 days showed over 90% reduction of NP in the sludge through composting. Samples continue to be collected and analyzed.

Synthesis of [U-ring ¹⁴C] NPE4 and NPE9 Ethoxylates

The purpose of this project was to synthesize [¹⁴C-U-Ring] p-nonylphenol with a chemical composition in close agreement to a typical manufactured batch of the material. The material synthesized is now being used for dermal penetration studies at North Carolina State University. The final report for this project was issued by BioDynamics Radiochemicals on November 24, 1998.

The Biodegradation of ¹⁴C Ring-labeled Nonylphenol Ethoxylate in a Semi-Continuous Activated Sludge and River Water Systems

The aerobic biodegradation of a ¹⁴C ring-labeled nonylphenol 9-mole ethoxylate (¹⁴C-NPE9) was examined in laboratory semi-continuous activated sludge (SCAS) and river water environments. In the SCAS experiments primary effluent from a local wastewater treatment plant was dosed with ¹⁴C NPE9 and fed into SCAS systems, and the levels of ¹⁴C in the clarified effluent, settled sludge solids, and CO2 were monitored. A significant portion of the ¹⁴C consisted of soluble metabolites that had degraded beyond the phenol ring. Dosing of the SCAS system ended after 29 days, and dissipation of the residual radioactivity was followed for another 19 days. CO2 evolution and decline of radioactivity in the sludge solids both followed first order rate kinetics, with half-lives of 2.8 days and 5.8 days, respectively. A portion of the residual sludge activity was incorporated into the biomass.

In the river die-away experiment, the extent of the ¹⁴CO2 evolution from river water dosed with ¹⁴C NPE was monitored for 128 days. After an induction period of 21 days, ¹⁴CO2 evolution followed first order kinetics; the half-life was 22 days.

This is the first unequivocal demonstration that the phenolic ring of NPE is mineralized under activated sludge and die-away conditions.

Naylor, C.G., Williams, J.B., Varineau, P.T., Yunick, R.P., Serak, K., Cady, C. and Severn, D.J. (1998) "Biodegradation of the 14C ring-labeled nonylphenol ethoxylate in activated sludge and in river water," Presented at the 19th Annual Society of Environmental Toxicology and Chemistry.

Treatability Studies of Nonylphenol Ethoxylates

These studies determined the extent of nonylphenol ethoxylates (NPE) degradation and removal from a variety of treatment plants, both publicly owned and industrial. The plants treated wastewaters from textile mills, paper and pulp mills, and domestic sources. Removal rates averaged over 95% with no accumulation of nonylphenol or lower mol NPE in the effluents or sludge. Researchers also found that oligomer distribution of NPE in influents and effluents can be a sensitive measure of degradation conditions. This work was presented in part at the 1993 American Chemical Society Southwest Regional Meeting and published:

Naylor, CG. Soap/Cosmetics/Chemical Specialties, August 1992.

Naylor, CG. Textile Chemist and Colorist, April 1993.

Nonylphenol Polyethoxy Carboxylate Metabolites of Nonionic Surfactants in U.S.A. Paper Mill Effluents, Municipal Sewage Treatment Plant Effluents, and River Waters

An analytical method was developed for the quantitative determination of nonylphenol polyethoxy carboxylate (NPEC) metabolites of nonylphenol ethoxylates surfactants in paper mill effluents, municipal sewage treatment plant effluents, and river waters. Strong anion-exchange solid-phase extraction disks were used for NPEC isolation from aqueous samples and NPEC elution from the disk was simultaneously combined with derivatization. Extracts were analyzed by gas chromatography and positive-mode chemical-ionization mass spectrometry. The detection limit of the method ranged from 0.2 mg/L for NPE1 EC to 2 mg/L for NP4EC. The total concentration of NPEC in a group of paper mill and municipal sewage effluents ranged from below detection to 1300 mg/L. The total concentration of NPEC in several river waters ranged from below detection to 13.8 mg/L.

Field, J.A. and Reed, R.L. (1996) "Nonylphenol Polyethoxy Carboxylate Metabolites of Nonionic Surfactants in US Paper Mill Effluents, Municipal Sewage Treatment Plant Effluents, and River Waters," Environmental Science & Technology, Vol. 30, No 12, 3544-3550.

Animal Toxicology Studies

Effects of Nonylphenol on Mammary Gland Development and Mammary Gland Cell Proliferation in the Rat

These studies were designed to replicate and expand on the results of published studies on the effects of p-nonylphenol (NP) on mammary gland development (Colerangle, J.B., and Roy, D. (1996) "Exposure of environmental estrogenic compound nonylphenol to Noble rats alters cell-cycle kinetics in the mammary gland," Endocrine, 4, 115-122). The studies were completed in 1999 and evaluated the effect of route of exposure and rat strain on the ability for relatively low doses of NP to induce mammary gland development and cell proliferation in rats. Three strains of rats and two routes of exposure were tested. Comparison of the response of the mammary gland to more traditional uterotrophic responses were also made.

Odum, J., Payrah, I.T.G., Foster, J.R., Van Miller, J.P., Joiner, R.L., Ashby, J. (1999) "Comparative activities of p-nonylphenol and diethylstilbestrol in Noble rat mammary gland and uterotrophic assays." Reg. Toxicol. Pharmacol. 29, 184-195.

Odum, J., Payrah, I.T.G., Soames, A.R., Foster, J.R., Van Miller, J.P., Joiner, R.L., Ashby, J. (1999) "Similar sensitivity of the rat mammary gland and uterus to p-nonylphenol (NP) following oral administration or via subcutaneous mini-pumps, and comparison with diethylstilbestrol (DES)" - IN PRESS.

Skin Penetration of NP and NPE

To aid in evaluation of exposure to nonylphenol ethoxylate (NPE) and nonylphenol (NP), studies evaluating the in vitro skin penetration of radiolabeled NPE4, NPE9, and NP have been initiated. Phase I studies evaluated the penetration through full-thickness skin from humans, rats and pigs. Subsequent studies will be conducted using porcine skin flaps. The studies are being conducted at North Carolina State University. A manuscript for the initial studies is in preparation.

Nonylphenol Effects on Aromatase Activity

Results presented recently at a conference (Geisy, 1999) indicated that nonylphenol (NP) produced effects on fathead minnows at concentrations from 0.05 to 3.4mg NP/L for 42 days, considerably below all previous effect doses. Effects described were for egg production, plasma vitellogenin and plasma E2. Exposure to NP caused a 9-fold increase in the plasma concentration of E2 suggesting that the effects of NP are due to indirect effects on E2 rather than NP itself.

To determine if NP can affect aromatase activity in mammals, a study is planned to evaluate whether the uterotrophic response of NP can be attributed to aromatase induction as opposed to direct interaction of NP with the estrogen receptor.

Pharmacokinetecs of p-Nonylphenol in the Rat

Studies were conducted to determine the kinetics of tissue distribution of p-nonylphenol in male and female rats.

Fennell, T. and MacNeela, J. (1997) "Disposition and Metabolism of p-nonylphenol in Male and Female Rats," Fundamental and Applied Toxicology, 36, No. 1 part 2, 142 [Abstract No. 722].

Fennell, T., MacNeela, J. and Manaugh, C. (1997) "Metabolism, Disposition, and Pharmacokinetics of p-nonylphenol in Male and Female Rats," ISSX Proceedings, 12, 125 [Abstract No. 250].

Fennell, T., MacNeela, J. and Manaugh, C. (1998) "Pharmacokinetics of p-nonylphenol in Male and Female Rats." Fundamental and Applied Toxicology, 42, No. 1-S, 213 [Abstract No. 1051].

The APE Research Council is currently conducting additional kinetics studies. Results are expected in early 2001.

Subchronic Toxicity Study of Nonylphenol

A 90-day subchronic toxicity study of p-nonylphenol was administered in the diet to Sprague-Dawley rats at dose levels of 0, 200, 650 and 2000 ppm. In addition to the customary measurement parameters (body weight, feed consumption, ophthalmology, hematology, clinical chemistry, organ weights and histopathology), the study included evaluation specifically for hormonal perturbations. The additional measurements included vaginal smears (for 7 days beginning on treatment day 50) and semen evaluations for sperm count, motility and morphology. The study also included a 4-week recovery segment for the 0 and 2000 ppm groups. A final report is available.

Cunny, H.C., et al. (1997) "Subchronic Toxicity (90-day) Study with Para-nonylphenol in Rats." Regulatory Toxicology and Pharmacology, 26, 172-178.

Uterine Weight Assay of Nonylphenol and Octylphenol Ethoxylate

Two separate uterine weight assays of p-nonylphenol (NP) and octylphenol ehtoxylate-5 (OPE5) in ovariectomized adult female Sprague-Dawley rats have been conducted. Both substances were administered by gavage at doses of 30, 100, and 300 mg/kg daily for three consecutive days. Ethynylestradiol and 17b estradiol served as positive reference standards. A final report for these studies is available.