# Ecosystem Health

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Environment Environnement Canada Canada 

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# Canadian Environmental Quality Guidelines for Nonylphenol and its Ethoxylates (Water, Sediment, and Soil)

Report No. 1-3

### Prepared and published by National Guidelines and Standards Office Environmental Quality Branch Environment Canada Ottawa

August, 2002

ISSN 1497-2689 ISBN 0-662-32230-4 Cat. no. En1-34/4-2002E

Scientific Supporting Document (Water, Sediment, and Soil)



# Canadian Environmental Quality Guidelines for Nonylphenol and its

Ethoxylates

Report No. 1-3

© Her majesty the Queen in Right of Canada, represented by the Minister of the Environment, 2002. All rights reserved. Reproduction authorized if source is acknowledged. The reproduction must be presented within its proper context and must not be used for profit.

### ii

NOTE TO READERS

The Ecosystem Health: Science-based Solutions series is dedicated to the dissemination of scientific knowledge, information and tools for monitoring, assessing, and reporting on ecosystem health to support Canadians in making sound decisions. Documents published in this series include the scientific basis, methods, approaches and frameworks for environmental quality guidelines and their implementation; monitoring, assessing, and rehabilitating environmental quality in Canada; and, indicator development, environmental reporting and data management. Issues in this series are published ad libitum

. This particular issue provides the scientific supporting information and rationale for the development of Canadian Environmental Quality Guidelines for nonylphenol and its ethoxylates. The information in this document is current as of April 2001. Minor revisions and editorial changes have been made for publication in 2002. For additional information regarding these guidelines, please contact:

Environment Canada Phone: 819-953-1550 Environmental Quality Branch Fax: 819-953-0461 National Guidelines and Standards Office ceqg-rcqe@ec.gc.ca 351 Saint-Joseph Boulevard http://www.ec.gc.ca/ceqg-rcqe Hull, Quebec K1A 0H3

Canadian Water and Sediment Quality Guidelines are approved by the Water Quality Task Group of the Canadian Council of Ministers of the Environment (CCME), and Canadian Soil Quality Guidelines are approved by the CCME Soil Quality Guidelines Task Group. Environment Canada acts as the federal representative and serves as the technical secretariat to both of these Task Groups. These guidelines are included as a 2002 update to the Canadian Environmental Quality Guidelines, which was published by the CCME in October of 1999. For CCME publications, please contact:

CCME Documents Phone: 1-800-805-3025 c/o Manitoba Statutory Publications Fax: 204-945-7172 200 Vaughan Street spccme@gov.mb.ca W innipeg, MB R3C 1T5 http://www.ccme.ca

This scientific supporting document is available in English only. Factsheets are also available in English under the title Canadian Environmental Quality Guidelines (CCME 1999). Ce document scientifique du soutien n’est disponible qu’en anglais avec un résumé en français. Des feuillets d’information sont aussi disponibles en français sous le titre Recommandations canadiennes pour la qualité de l’environnement (CCME 1999).

Reference listing:

Environment Canada. 2002. Canadian Environmental Quality Guidelines for Nonylphenol and its Ethoxylates (Water, Sediment, and Soil). Scientific Supporting Document. Ecosystem Health: Science-based Solutions Report No. 1-3. National Guidelines and Standards Office, Environmental Quality Branch, Environment Canada. Ottawa.

### iii

TABLE OF CONTENTS

NOTE TO READERS

................................ ................................ ................................ ........................ ii

TABLE OF CONTENTS

................................ ................................ ................................ ................... iii

LIST OF TABLES

................................ ................................ ................................ ............................. v

LIST OF FIGURES

................................ ................................ ................................ ........................... v

LIST OF APPENDICES

................................ ................................ ................................ .................... vi

EXECUTIVE SUMMARY

................................ ................................ ................................ ................. vii
RÉSUMÉ

................................ ................................ ................................ ................................ .......... x

ACKNOWLEDGEMENTS

................................ ................................ ................................ .............. xiii

GLOSSARY OF ACRONYMS

................................ ................................ ................................ ........ xiv

CHAPTER 1. INTRODUCTION

................................ ................................ ................................ ...... 1 Physical and Chemical Properties

................................ ................................ ......................... 2 Production and Uses

................................ ................................ ................................ .............. 6 Sources to the Environment

................................ ................................ ................................ ... 8 Water

................................ ................................ ................................ ........................... 8 Sediment

................................ ................................ ................................ ...................... 8 Soil

................................ ................................ ................................ ............................... 8 Air

................................ ................................ ................................ ................................ . 9 Analytical Methods

................................ ................................ ................................ ................. 9

CHAPTER 2. CONCENTRATIONS IN CANADA

................................ ................................ .......... 11 Surface Waters

................................ ................................ ................................ .................... 11 Sediment

................................ ................................ ................................ .............................. 12 Soil

................................ ................................ ................................ ................................ ....... 12 Sewage and Sludge

................................ ................................ ................................ ............. 12 Industrial Effluents and Sludge

................................ ................................ ............................ 13

CHAPTER 3. ENVIRONMENTAL FATE AND BEHAVIOUR

................................ ........................ 14 Physical and Chemical Processes

................................ ................................ ....................... 14 Water

................................ ................................ ................................ ......................... 14 Sediment

................................ ................................ ................................ .................... 15 Soil

................................ ................................ ................................ ............................. 16 Biodegradation

................................ ................................ ................................ ..................... 17 Primary Biodegradation

................................ ................................ ............................. 17 Ultimate Biodegradation

................................ ................................ ............................ 20 Uptake/Elimination Pathways, and Metabolism

................................ ................................ ... 21 Bioaccumulation

................................ ................................ ................................ ................... 23 Toxicity

................................ ................................ ................................ ................................ . 29 Mode of Action

................................ ................................ ................................ ........... 29 Narcosis

................................ ................................ ................................ .......... 29 Endocrine Disruption

................................ ................................ ....................... 30 Physical Surfactancy

................................ ................................ ....................... 32 Toxic Effects

................................ ................................ ................................ .............. 33 Mixtures and the TEQ Approach

................................ ................................ .......................... 34

CHAPTER 4. GUIDELINES FROM OTHER JURISDICTIONS

................................ .................... 37

### iv Water

................................ ................................ ................................ ................................ ... 37 Sediment

................................ ................................ ................................ .............................. 37 Soil

................................ ................................ ................................ ................................ ....... 38

CHAPTER 5. DEVELOPMENT OF CANADIAN WATER QUALITY GUIDELINES

...................... 41 Freshwater

................................ ................................ ................................ ........................... 41 Toxicity Data

................................ ................................ ................................ .............. 41 Fish

................................ ................................ ................................ ................. 41 Invertebrates

................................ ................................ ................................ ... 41 Plants/Algae

................................ ................................ ................................ .... 41 Guideline Derivation

................................ ................................ ................................ ... 42 Marine/Estuarine Water

................................ ................................ ................................ ....... 46 Toxicity Data

................................ ................................ ................................ .............. 46 Fish

................................ ................................ ................................ ................. 46 Invertebrates

................................ ................................ ................................ ... 46 Algae

................................ ................................ ................................ ............... 46 Guideline Derivation

................................ ................................ ................................ ... 47 Recommended Canadian Water Quality Guidelines

................................ ........................... 51 Guideline for the Protection of Freshwater Life

................................ ......................... 51 Guideline for the Protection of Marine Life

................................ ................................ 51

CHAPTER 6. DEVELOPMENT OF CANADIAN SEDIMENT QUALITY GUIDELINES

................ 52 Toxicity Data

................................ ................................ ................................ ........................ 52 Invertebrates

................................ ................................ ................................ .............. 52 Amphibians

................................ ................................ ................................ ................ 52 Protocol Approaches

................................ ................................ ................................ ............ 52 Equilibrium Partitioning Approach

................................ ................................ ........................ 53 Guideline Derivation

................................ ................................ ................................ ... 54 Recommended Canadian Sediment Quality Guidelines

................................ ...................... 55 Freshwater Sediments

................................ ................................ ............................... 55 Marine Sediments

................................ ................................ ................................ ...... 55

CHAPTER 7. DEVELOPMENT OF CANADIAN SOIL QUALITY GUIDELINES

........................... 58 Toxicity Data

................................ ................................ ................................ ........................ 58 Microorganisms

................................ ................................ ................................ ......... 58 Plants

................................ ................................ ................................ ......................... 59 Invertebrates

................................ ................................ ................................ .............. 59 Mammals

................................ ................................ ................................ ................... 59 Agricultural and Residential/Parkland Land Uses

................................ ................................ 62 Soil Contact

................................ ................................ ................................ ................ 62 Nutrient and Energy Cycling Check

................................ ................................ 64 Soil and Food Ingestion

................................ ................................ ............................. 64 Commercial and Industrial Land Uses

................................ ................................ ................. 65 Soil Contact

................................ ................................ ................................ ................ 65 Nutrient and Energy Cycling Check

................................ ................................ 67 Groundwater Check

................................ ................................ ................................ ............. 67 Off-site Migration Check

................................ ................................ ................................ ...... 68 Environmental Soil Quality Guideline Derivation (SQG E 

### )

................................ ..................... 68 Recommended Canadian Soil Quality Guidelines

................................ ............................... 69

CHAPTER 8. AREAS FOR FUTURE RESEARCH

................................ ................................ ....... 71 Concentrations in Canada

................................ ................................ ................................ .... 71 Fate and Behaviour

................................ ................................ ................................ .............. 71 Toxicity Testing

................................ ................................ ................................ .................... 71 Guideline Derivation and Implementation

................................ ................................ ............ 71

CHAPTER 9. GUIDANCE ON IMPLEMENTATION

................................ ................................ ...... 73

### v General Guidance for all Guidelines

................................ ................................ .................... 73 Application of the TEQ Approach to EQGs

................................ ................................ .......... 73 Water Quality Guidelines

................................ ................................ ................................ ..... 74 Sediment Quality Guidelines

................................ ................................ ................................ 74 Soil Quality Guidelines

................................ ................................ ................................ ......... 75

REFERENCES

................................ ................................ ................................ ............................... 76

ANNEX I. ECOLOGICAL RELEVANCE OF ENDOCRINE DISRUPTION

................................ .. 102

ANNEX 2. DATASET REQUIREMENTS FOR DERIVING WATER QUALITY GUIDELINES

.... 104

ANNEX 3. SUMMARY OF SEDIMENT QUALITY GUIDELINE PROTOCOL

............................. 106

LIST OF TABLES Table 1. Summary of Canadian Environmental Quality Guidelines for nonylphenol and its

ethoxylates.

................................ ................................ ................................ .................... ix Tableau 1. Résumé des recommandations canadiennes pour la qualité de l’environnement,

concernant le nonylphénol et ses dérivés éthoxylés

................................ ..................... xii Table 2. Physical and chemical properties of nonylphenol and select nonylphenol ethoxylates.

.. 5 Table 3. Bioconcentration and bioaccumulation factors for nonylphenol and nonylphenol

ethoxylates in aquatic organisms.

................................ ................................ ................ 26 Table 4. Summary of relative toxicity of nonylphenolic and related compounds.

........................ 36 Table 5. Freshwater quality guidelines for nonylphenol from other jurisdictions.

........................ 38 Table 6. Freshwater quality guidelines for nonylphenol ethoxylates and ethoxycarboxylates

from other jurisdictions.

................................ ................................ ................................ 39 Table 7. Marine water quality guidelines for nonylphenol from other jurisdictions.

..................... 40 Table 8. Soil and sludge guidelines from other jurisdictions.

................................ ...................... 40 Table 9. Data used in derivation of guideline for toxicity of nonylphenol to freshwater aquatic

life.

................................ ................................ ................................ ................................ 43 Table 10. Data used in derivation of guideline for toxicity of nonylphenol to marine aquatic

life.

................................ ................................ ................................ ................................ 48 Table 11. Primary data available for toxicity of nonylphenol though direct soil contact.

............... 61 Table 12. Summary of soil quality guideline derivation for nonylphenol.

................................ ..... 70 Table 13. Summary of the available biological effects and related physicochemical data for

sediment-associated nonylphenol (mg·kg -1 

### dry weight) in freshwater ecosystems

.... 112 Table 14. Summary of the available biological effects and related physicochemical data for

sediment-associated nonylphenol (mg·kg -1 

### dry weight) in marine ecosystems

......... 114

LIST OF FIGURES Figure 1. The structure of nonylphenol and its derivatives.

................................ ............................ 3 Figure 2. Pathways for biodegradation of nonylphenol polyethoxylates.

................................ ...... 19 Figure 3. Select freshwater toxicity data for nonylphenol.

................................ ............................ 45 Figure 4. Select marine toxicity data for nonylphenol.

................................ ................................ .. 50 Figure 5. Distribution of nonylphenol concentrations in freshwater sediments that are associated with adverse biological effects ( l ) and no adverse effects ( m ).

................. 57 Figure 6. Distribution of nonylphenol concentrations in marine and estuarine sediments that are associated with adverse biological effects ( l ) and no adverse effects ( m ).

........... 57 Figure 7. Overview of steps leading to the development of final Environmental Soil Quality

Guidelines for nonylphenol.

................................ ................................ .......................... 60 Figure 8. Distribution of soil contact toxicity data used in the guideline derivation for agricultural and residential/parkland land uses.

................................ ............................ 63 Figure 9. Distribution of soil contact effects toxicity data used in guideline derivation for

commerical and industrial land uses.

................................ ................................ ........... 66

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LIST OF APPENDICES

Appendix I. Toxicity of nonylphenol and its ethoxylates to freshwater life through water

column exposure

a. Toxicity of nonylphenol to freshwater algae and macrophytes through water column exposure b. Toxicity of nonylphenol to freshwater invertebrates through water column exposure

c. Toxicity of nonylphenol to freshwater fish through water column exposure d. Toxicity of nonylphenol ethoxylates to freshwater algae and macrophytes through water column exposure

e. Toxicity of nonylphenol ethoxylates to freshwater invertebrates through water column exposure

f. Toxicity of nonylphenol ethoxylates to freshwater fish through water column exposure

Appendix II. Toxicity of nonylphenol and its ethoxylates to marine aquatic life through water

column exposure a. Toxicity of nonylphenol to marine algae through water column exposure b. Toxicity of nonylphenol to marine invertebrates through water column exposure c. Toxicity of nonylphenol to marine fish through water column exposure d. Toxicity of nonylphenol ethoxylates to marine algae and plants through water column exposure e. Toxicity of nonylphenol ethoxylates to marine invertebrates through water column exposure f. Toxicity of nonylphenol ethoxylates to marine fish through water column exposure

Appendix III. Toxicity of nonylphenol to sediment-dwelling life

a. Toxicity of nonylphenol to freshwater life through spiked-sediment exposure b. Toxicity of nonylphenol to marine life through spiked-sediment exposure

Appendix IV. Toxicity of nonylphenol and its ethoxylates to terrestrial life

a. Toxicity of nonylphenol to soil microorganisms b. Toxicity of nonylphenol to soil-dwel ling invertebrates c. Toxicity of nonylphenol to terrestrial plants d. Toxicity of nonylphenol to terrestrial vertebrates e. Toxicity of nonylphenol ethoxylates to terrestrial organisms

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EXECUTIVE SUMMARY

This scientific supporting document provides the rationale for the development of Canadian Environmental Quality Guidelines for nonylphenol (NP) and nonylphenol ethoxylates (NPEs). It contains a review of technical background information on the chemical and physical properties of NP and NPEs, a review of sources and emissions in Canada, the distribution and behaviour of NP and NPEs in the environment, and their toxicological effects on a variety of aquatic and terrestrial species. This information is used to derive water quality, sediment quality, and soil quality guidelines for NP and NPEs to protect ecological receptors in each of these environmental compartments. All of the Canadian Environmental Quality Guidelines in this document are based on the best available toxicity data at the time of writing. The most recent literature search was conducted in August 2000, but some information obtained since that date has also been included in response to reviewers’ comments.

NPEs are a group of nonionic organic chemicals that are used as surfactants, detergents, emulsifiers, wetting agents, and dispersants in a wide variety of industrial processes and commercial products. They are primarily released to the environment through sewage effluents or industrial wastewaters. In sewage treatment systems, and upon entering the environment, NPEs readily undergo an initial primary degradation which largely involves the loss of ethoxylate groups. This results in the formation of more refractory degradation products such as nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), nonylphenoxy acetic acid (NP1EC), and nonylphenoxy ethoxy acetic acid (NP2EC).

The major pathway for nonylphenolic substances to enter the environment is through the discharge of municipal and industrial wastewaters into surface waters. Because they are used as formulants for some pesticides, nonylphenolic substances may also be deposited on surface waters through aerial spraying of pesticides, or as runoff from agricultural land or forests which have been sprayed. Once in the aquatic environment, the more hydrophobic chemicals, such as NP, will tend to adsorb to particulates and accumulate in bottom sediments. In wastewater treatment plants, NP will also partition to the sludge, and may then become a contaminant of terrestrial systems when the sludge is landfilled or applied as a soil amendment to agricultural soils. Leaching of nonylphenolic substances into groundwater has also been documented.

It has been proposed that nonylphenol and its ethoxylates be designated as “CEPA toxic” under the Canadian Environmental Protection Act (CEPA 1999). Large quantities of NPEs are used in Canada each year. Therefore, their degradation products (i.e., NP and short-chain NPEs), which are more persistent, toxic, and estrogenic, may be released at concentrations that could affect aquatic and terrestrial life in the receiving environments. In light of these possible impacts, the development of Canadian Environmental Quality Guidelines (CEQGs) has been deemed necessary. CEQGs are nationally-endorsed, science-based goals that are recommended as levels that should result in negligible risk to biota and ecosystem health. These guidelines are developed according to the procedures described in nationally approved protocols for the derivation of Canadian Environmental Quality Guidelines (CCME 1991; CCME 1995; CCME 1996).

A summary of the Canadian Environmental Quality Guidelines developed in this document is provided in Table 1. Guidelines in this document were developed based on data for nonylphenol, but, as described in Chapter 3, it is recommended that a toxic equivalency (TEQ) approach be used to apply these guidelines to mixtures of nonylphenolic substances. When comparing an environmental sample to the guidelines, concentrations of each nonylphenolic substance in the sample should be multiplied by their respective toxic equivalency factors (TEFs) and then summed to yield a total concentration in NP TEQ units. Based on the toxicity of nonylphenol ethoxylates and nonylphenoxy carboxylates relative to nonylphenol, an initial set of TEFs has been recommended. Future work to develop and validate ethoxamer-specific TEF values is needed, but, in the interim, it is recommended that the TEFs presented here be applied.

### viii

Tissue residue guidelines in aquatic biota for the protection of wildlife consumers of aquatic life have not been derived due to the lack of available toxicity data for mammalian and avian species.

Sufficient toxicity data were available for the derivation of a full freshwater water quality guideline, but not for a marine water quality guideline. The freshwater guideline was based on the most sensitive chronic endpoint from a primary study, which was a 91-day lowest observed effect concentration (LOEC) for growth reduction in rainbow trout at a nonylphenol concentration of 10.3μg·L-1 

### (Brooke 1993a). This concentration was multiplied by a safety factor of 0.1 to obtain a water quality guideline for nonylphenol and its ethoxylates of 1.0 μg·L-1 

### , expressed in NP TEQ units, for the protection of freshwater aquatic life. The toxicity data available for marine aquatic species were sufficient to meet the minimum data requirements for an interim water quality guideline. The critical study was a 28-day test with Americamysis bahia which had a LOEC for growth reduction of 6.7 μg·L-1 

### (Ward and Boeri 1991a). Multiplying this by a safety factor of 0.1, an interim water quality guideline of 0.7 μg·L-1 

### , expressed in NP TEQ units, is recommended for the protection of marine (including estuarine) aquatic life. These guidelines, which refer to concentrations in unfiltered samples, are intended to protect all forms of aquatic life and all aspects of aquatic life cycles during an indefinite period of exposure to the water column (CCME 1991). Current limits of detection for nonylphenolic substances in water range from 0.01 μg·L-1 

### (for NP) to 1.6 μg·L-1 

### (for higher molecular weight NPEs).

For the development of Canadian sediment quality guidelines, there were insufficient data to meet the minimum requirements for any of the three approaches described in the protocol (i.e., National Status and Trends Program Approach (NSTP), Spiked-Sediment Toxicity Test Approach (SSTT), adoption of guidelines from other jurisdictions). Instead, an equilibrium partitioning approach was used to derive provisional interim sediment quality guidelines (ISQGs) from the Canadian water quality guidelines. The revision and re-assessment of these provisional ISQGs will be possible when sufficient data become available to support the use of the modified NSTP or SSTT approaches as described in the formal protocol (CCME 1995). Based on equilibrium partitioning calculations, a provisional interim sediment quality guideline of 1.4 mg·kg -1 

### dry weight, expressed in NP TEQ units, is recommended for the protection of freshwater aquatic life. For the protection of marine (including estuarine) life, a provisional interim sediment quality guideline of 1.0 mg·kg -1 

### dry weight, expressed in NP TEQ units, is recommended. These guidelines are normalized to 1% total organic carbon, but should be adjusted based on local levels of organic carbon in the sediments. These guidelines are intended to be protective of all forms of aquatic life and all aspects of aquatic life cycles during an indefinite period of exposure to bed sediments (CCME 1995). These guidelines refer to total concentrations of nonylphenolic substances in surficial sediments (i.e., top 5 centimetres), as quantified by extraction using standard analytical methods (e.g., soxhlet extraction, steam distillation, supercritical fluid extraction) followed by detection with high pressure liquid chromatography or gas chromatography/mass spectrometry. Current limits of detection for nonylphenolic substances in sediment range from 0.015 to 0.1 mg·kg -1 

### dry weight.

There were sufficient soil toxicity data to meet the minimum requirements for a weight of evidence method for soil contact guideline derivation. However, adequate data were not available for the derivation of a food ingestion guideline value or a nutrient and energy cycling check. For agricultural and residential/parkland land uses, the soil contact guideline was calculated as the 25th

### percentile of the effects and no effects data distribution, divided by an uncertainty factor of 5. For commercial and industrial land uses, the soil contact guidelines were calculated as the 25 th 

### percentile of the effects data distribution only. Therefore, soil quality guidelines for nonylphenol and its ethoxylates for agricultural and residential/parkland land use are both recommended at 5.7mg·kg -1 

### , expressed in NP TEQ units. For both commercial and industrial land uses, soil quality guidelines of 14 mg·kg -1 

### , expressed in NP TEQ units, are recommended. These guidelines are optimized for soils within the pH range of 5.8 to 7.5 because the toxicological studies upon which they are based were conducted within this range. The current limit of detection for nonylphenol in soil is 0.05 mg·kg -1 

### . A groundwater check of 21 mg·kg -1 

### , expressed in

### ix

NP TEQ units, was calculated as the concentration of NP and NPEs in soil that should not be exceeded in order to protect freshwater aquatic life from possible groundwater contamination.

Table 1. Summary of Canadian Environmental Quality Guidelines for nonylphenol and its ethoxylates.

Environmental Media Media Type Guideline Type Guideline Value*
Water Freshwater Full 1.0 μg·L-1 

### Marine Interim 0.7 μg·L-1 

### Sediment † 

### Freshwater Provisional Interim 1.4 mg·kg -1 

### Marine Provisional Interim 1.0 mg·kg -1 

### Soil Agricultural SQG E ‡ 

### 5.7 mg·kg -1 

### Residential/Parkland SQG E 

### 5.7 mg·kg -1 

### Commercial SQG E 

### 14 mg·kg -1 

### Industrial SQG E 

### 14 mg·kg -1 

### Groundwater Check value 21 mg·kg -1



### * all guidelines are expressed in nonylphenol toxic equivalent units †

### these guideline values are for sediments containing 1% total organic carbon ‡

### environmental soil quality guideline; a full soil quality guideline would also consider protection for human health (CCME 1996)

### x

RÉSUMÉ

Le présent document d’évaluation scientifique présente les principes qui sont à la base de l’élaboration des Recommandations canadiennes pour la qualité de l'environnement pour le nonylphénol (NP) et les dérivés éthoxylés du nonylphénol (NPE). Il contient une revue de l’information technique de base sur les propriétés chimiques et physiques du NP et des NPE, les sources et les émissions au Canada, la distribution et le comportement des NP et des NPE dans l’environnement et enfin leurs effets toxicologiques sur diverses espèces aquatiques et terrestres. Cette information sert à l’élaboration de recommandations pour la qualité de l’eau, des sédiments et des sols concernant le NP et les NPE, de façon à protéger les récepteurs écologiques dans chacun de ces compartiments de l’environnement. Toutes les Recommandations canadiennes pour la qualité de l'environnement présentées dans le présent document sont basées sur les meilleures données toxicologiques disponibles au moment de sa rédaction. Les recherches de littérature les plus récentes ont été effectuées en août 2000, mais certains renseignements obtenus depuis cette date ont également été inclus suite aux commentaires des examinateurs.

Les NPE sont un groupe de substances chimiques, organiques et non ioniques, utilisées comme tensioactifs, détergents, émulsifiants, agents de mouillage et dispersants dans une vaste gamme de procédés industriels et de produits commerciaux. Ils sont principalement rejetés dans l’environnement via les effluents d’eaux usées provenant des égouts ou les eaux usées industrielles. Les NPE sont soumis à une dégradation primaire initiale impliquant principalement une perte de groupes éthoxy dans les installations de traitements d’eaux usées et à leur entrée dans l’environnement. Des produits de dégradation plus réfractaires, comme le nonylphénol (NP), le nonylphénol monoéthoxylé (NP1EO), le nonylphénol diéthoxylé (NP2EO), l’acide nonylphénoxyacétique (NP1EC) et l’acide nonylphénoxyéthoxyacétique (NP2EC) sont alors formés.

Les substances nonylphénoliques pénètrent l’environnement majoritairement par la voie des rejets d’eaux usées des municipalités et des usines à travers les eaux de surface. Étant donné qu’elles sont employées comme produits de formulation dans certains pesticides, les substances nonylphénoliques peuvent également être déposées sur les eaux de surface suivant l’épandage aérien de pesticides, ou encore, être lessivées à partir de terres agricoles ou forestières traitées par pulvérisation. Dans le milieu aquatique, les substances chimiques les plus hydrophobes, comme le NP, auront tendance à être adsorbées sur les particules et à s’accumuler dans les sédiments de fond. Dans les usines de traitement des eaux usées, une partie du NP sera également distribuée dans les boues et pourra alors devenir un contaminant des systèmes terrestres, lorsque ces boues sont utilisées comme remblai ou appliquées comme amendement du sol sur les terres agricoles. La documentation mentionne le lessivage des substances nonylphénoliques dans les eaux souterraines.

Il a été proposé que le nonylphénol et ses dérivés éthoxylés soient désignés « toxiques » sous la Loi canadienne sur la protection de l'environnement (LCPE 1999). De grandes quantités de NPE sont utilisées chaque année, au Canada. Par conséquent, leurs produits de dégradation (c. -à-d. le NP et les NPE à courte chaîne), qui sont persistents, toxiques et qui possèdent un effet oestrogène plus marqués, peuvent être rejetés à des concentrations qui pourraient altérer la vie aquatique et terrestre dans les milieux récepteurs. À la lumière de ces impacts possibles, l’élaboration des Recommandations canadiennes pour la qualité de l'environnement (RCQE) s’est avérée nécessaire. Les RCQE, approuvées à l’échelle nationale et fondées sur des données scientifiques solides, sont des objectifs recommandés sous forme de concentrations qui ne devraient entraîner qu’un risque négligeable pour la santé du biote et de l’écosystème. Ces recommandations ont été élaborées selon les méthodes décrites dans les protocoles approuvés à l’échelle nationale visant l’établissement des Recommandations canadiennes pour la qualité de l’environnement (CCME, 1991; CCME, 1995; CCME, 1996).

### xi

Les Recommandations canadiennes pour la qualité de l'environnement élaborées dans le présent document sont résumées dans le tableau 1. Les recommandations figurant dans ce document sont fondées sur les données relatives au nonylphénol, mais, comme décrit au chapitre3, il est conseillé d’utiliser une approche d’équivalent toxique (QÉT) lors de l’application de ces recommandations à des mélanges de substances nonylphénoliques. En comparant un échantillon de l’environnement aux recommandations, les concentrations de chaque substance nonylphénolique présente dans l’échantillon devraient être multipliées par leur facteur d’équivalence de toxicité (FÉT) respectif, et additionnées pour donner la concentration totale en unités de QÉT de NP. Un ensemble initial de FÉT, fondé sur la toxicité des dérivés éthoxylés et carboxylés du nonylphénol, a été recommandé. D’autres travaux seront nécessaires pour obtenir et valider les valeurs FÉT pour des congénères éthoxylés individuels, mais, d’ici là, il est recommandé d’appliquer les FÉT présentés ici.

Les recommandations pour les résidus dans les tissus du biote aquatique, visant à protéger les espèces fauniques consommant les organismes aquatiques, n’ont pas été déterminées en raison du manque de données de toxicité disponibles pour les mammifères et les oiseaux.

Suffisamment de données de toxicité étaient disponibles pour l’élaboration d’une recommandation complète concernant la qualité de l’eau douce, mais non pour une recommandation concernant la qualité de l’eau marine. La recommandation pour l’eau douce a été basée sur l’effet toxicologique chronique le plus sensible provenant d’une étude primaire de 91 jours, dont la concentration minimale avec effet observé (CMEO) de 10,3 μg·L-1 

### (Brooke 1993a) de nonylphénol a entraîné une réduction de la croissance chez la truite arc-en-ciel. Afin de protéger la vie aquatique en eau douce, cette concentration a été multipliée par un facteur de sécurité de 0,1 pour obtenir une recommandation de 1,0 μg·L-1 

### , exprimée en unités de QÉT de NP, pour la qualité de l’eau vis à vis le nonylphénol et ses dérivés éthoxylés. Les données de toxicité disponibles pour les espèces aquatiques marines étaient suffisantes pour répondre aux exigences de données minimales pour élaborer une recommandation provisoire pour la qualité de l’eau. L’étude critique était un essai de 28 jours avec Americamysis bahia démontrant une réduction de croissance associée à une CMEO de 6,7 μg·L-1 

### (Ward et Boeri, 1991a). En multipliant cette valeur par un facteur de sécurité de 0,1, une recommandation provisoire pour la qualité de l’eau de 0,7 μg·L-1 

### est obtenue, en unités de QÉT de NP, aux fins de la protection de la vie aquatique marine (incluant la vie estuarienne). Ces recommandations, qui correspondent à des concentrations dans des échantillons non filtrés, visent à protéger toutes les formes de vie aquatique et tous les aspects de leurs cycles de vie pendant une période indéfinie d’exposition à la colonne d’eau (CCME, 1991). Les limites de détection actuelles des substances nonylphénoliques dans l’eau se situent dans une plage de 0,01 μg·L-1 

### (pour le NP) à 1,6 μg·L-1 

### (pour les NPE de masse moléculaire plus élevée).

Aux fins de l’élaboration des recommandations canadiennes pour la qualité des sédiments, les données disponibles étaient insuffisantes pour satisfaire aux exigences minimales de n’importe laquelle des trois démarches décrites dans le protocole (« National Status and Trends Program » (NSTP), tests de toxicité des sédiments avec dopage (TTSD) et adoption de recommandations provenant d’autres jurisdictions). Une approche de partage à l’équilibre a plutôt été adoptée pour élaborer une version intérimaire des recommandations provisoires pour la qualité des sédiments (RPQS) à partir des recommandations canadiennes pour la qualité de l’eau. La révision et la réévaluation de ces RPQS provisoires seront possibles lorsque les données seront suffisantes pour justifier l’emploi des variantes des méthodes NSTP et TTSD, telles que décrites dans le protocole officiel (CCME 1995). Basée sur des calculs de partage à l’équilibre, une version intérimaire de la RPQS de 1,4 mg·kg -1 

### de poids sec, exprimée en unités de QÉT de NP, fut déterminée pour la protection de la vie aquatique en eau douce. Pour la protection de la vie marine (y compris la vie estuarienne), une version intérimaire de la RPQS de 1,0 mg·kg -1 

### de poids sec, exprimée en unités de QÉT de NP est proposée. Ces recommandations sont normalisées à 1 %de carbone organique total, mais devraient être ajustées en fonction des concentrations locales de carbone organique dans les sédiments. Les recommandations visent à protéger toutes les formes de vie aquatique et tous les aspects de leurs cycles de vie pendant une période

### xii

indéfinie d’exposition aux sédiments du lit (CCME, 1995). Ces recommandations correspondentaux concentrations totales de substances nonylphénoliques dans les sédiments de surface (c.-àd. dans la couche supérieure de 5 cm), mesurées par extraction à l’aide de méthodes analytiques standardisées (p. ex. extraction au soxhlet, distillation à la vapeur, extraction par fluide supercritique), suivie de détection par chromatographie liquide à haute pression ou chromatographie gazeuse/spectrométrie de masse. Les limites de détection actuelles des substances nonylphénoliques dans les sédiments se situent dans une plage de 0,015 à 0,1 mg·kg -1 

### de poids sec.

Suffisamment de données de toxicité étaient disponibles en ce qui a trait au sol pour satisfaire aux exigences minimales d’une méthode fondée sur le poids de la preuve permettant d’élaborer une recommandation pour le contact avec le sol. Par contre, un manque de données ne permettait pas l’obtention d’une valeur recommandée concernant l’ingestion par les aliments ni une vérification du cycle nutritif et énergétique. Dans le cas des utilisations sur les terres à vocation agricole, résidentielle, ou encore destinées aux parcs, la recommandation pour le contact avec le sol a été calculée au 25 e

### percentile de la distribution des données avec et sans effets, valeur par la suite divisée par un facteur d’incertitude de 5. Pour les utilisations sur les terres à vocation commerciale ou industrielle, les recommandations pour le contact avec le sol ont été calculées au 25 e

### percentile de la distribution des données avec effets seulement. Par conséquent, les recommandations pour la qualité du sol dans le cas du nonylphénol et de ses dérivés éthoxylés utilisés sur les terres à vocation agricole, résidentielle, ou encore destinées aux parcs sont toutes deux de 5,7mg·kg -1 

### , en unités de QÉT de NP. Pour les utilisations sur les terres à vocation tant commerciale qu’industrielle, les recommandations pour la qualité du sol sont de 14,2 mg·kg -1 

### , en unités de QÉT de NP. Ces recommandations sont optimalisées pour les sols dont le pH se situe entre 5,8 à 7,5, du fait que les études toxicologiques sur lesquelles elles sont fondées ont été effectuées dans cette étendue. La limite de détection actuelle pour le nonylphénol dans le sol est 0,05 mg·kg -1 

### . Une valeur de vérification d’eau souterraine de 21,2 mg·kg -1 

### , en unités de QÉT de NP, a été calculée comme concentration de NP et de NPE du sol à ne pas dépasser afin de protéger la vie aquatique en eau douce contre une contamination possible par l’eau souterraine.

Tableau 1. Résumé des recommandations canadiennes pour la qualité de l’environnement, concernant le nonylphénol et ses dérivés éthoxylés

Milieu environnemental Type de milieu Type de recommandation Valeur recommandée*
Eau Eau douce Complète 1,0 μg·L-1 

### Eau marine Provisoire 0,7 μg·L-1 

### Sédiments † 

### Eau douce Provisoire (intérimaire) 1,4 mg·kg -1 

### Eau marine Provisoire (intérimaire) 1,0 mg·kg -1



### Sol Agricole RQS E ‡ 

### 5,7 mg·kg -1 

### Résidentiel/parcs RQS E 

### 5,7 mg·kg -1 

### Commercial RQS E 

### 14 mg·kg -1 

### Industriel RQS E 

### 14 mg·kg -1 

### Eau souterraine Valeur pour vérification 21 mg·kg -1



### * toutes les recommandations sont exprimées en unités d’équivalent toxique de NP †

### ces recommandations sont valables pour des sédiments renfermant 1% de carbone organique total ‡

### recommandation pour la qualité environnementale du sol; une recommandation complète pour la qualité du sol tiendrait également compte de la protection de la santé humaine (CCME, 1996)

### xiii

ACKNOWLEDGEMENTS

This scientific supporting document for the development of Canadian Environmental Quality Guidelines for nonylphenol and its ethoxylates was prepared by the National Guidelines and Standards Office, Environmental Quality Branch of Environment Canada. Authors of this document include Kelly Potter, Kathie Adare and Sherri Smith (National Guidelines and Standards Office, Environment Canada). Special thanks are extended to Philippa Cureton, Nicole Davidson,and Thea Rawn (Commercial Chemicals Evaluation Branch, Environment Canada), and to MarcAndré Roy (Ministère de l’Environnment du Québec) for sharing information and references.

This document incorporates review comments received from various scientists representing federal and provincial government organizations, academic institutions, and the private sector. Thanks are extended to all those who provided input, including: Pierre-Yves Caux, Janet McCann, Elizabeth Roberts, Susan Roe, Doug Spry, and Lise Trudel (National Guidelines and Standards Office, Environment Canada); Bijan Aidun, Isabelle Guay, and Darrell Taylor (CCME Water Quality Guidelines Task Group); Ruth Hall, Marius Marsh, Ted Nason, Edwin Yee (CCME SoilQuality Guidelines Task Group); Nicole Davidson, Lucie Desforges, Dominique Dore, MarieLouise Geadah, Colin Gray, Catherine Jefferson, Céline Labossière, Bill Lee, Paul Mudroch, Thao Pham, Linda Porebski, Louise Power, Thea Rawn, Chantal Roberge, Danielle Rodrique, Mark Servos, Cecelia Wong (Environment Canada); Wayne Fairchild (Fisheries and Oceans Canada); Donald Rodier (U.S. Environmental Protection Agency), Nile Kemble (U.S. Geological Survey); Sylvia Talmage (Oak Ridge National Laboratory); Vince Kramer, Barbara Losey, Richard Mast, Ellen Mihaich, Carter Naylor, Charles Staples (Alkylphenols and Ethoxylates Research Council).

Funding for the development of Canadian Environmental Quality Guidelines for Nonylphenol and its Ethoxylates was provided by Environment Canada (National Guidelines and Standards Office, Environmental Quality Branch).

### xiv

GLOSSARY OF ACRONYMS
AP alkylphenol APE alkylphenol ethoxylate (as a general class) AP nEO alkylphenol ethoxylate, where n = specific number of EO groups BEDS Biological Effects Database for Sediments BOD, COD biological oxygen demand, chemical oxygen demand CAS Chemical Abstracts Service CCME Canadian Council of Ministers of the Environment CEC cation exchange capacity CEPA Canadi an Environmental Protection Act DTED daily threshold effects dose DT50 

### , DT 95 

### mean time to 50% dissipation, mean time to 95% dissipation dw dry weight ECL, ERL effects concentration low, effects range low EC 50 

### , ED 50 

### median effective concentration, median effective dose EDC endocrine disrupting chemical EO ethoxylate group EQGs Environmental Quality Guidelines GC/MS gas chromatography/mass spectrometry HLB hydrophile-lipophile balance HPLC high pressure liquid chromatography ISQG interim sediment quality guideline LC 50 

### median lethal concentration LOEC, LOEL lowest observed effect concentration, lowest observed effect level LOAEL lowest observed adverse effect level NE no effect NOEC, NOEL no observed effect concentration, no observed effect level NP nonylphenol NPEC nonylphenol ethoxycarboxylate or nonylphenoxy acetic acid (as a general class) NP nEC nonylphenol ethoxycarboxylate, where n = specific number of EO groups NPE nonylphenol ethoxylate (as a general class) NP nEO nonylphenol ethoxylate, where n = specific number of EO groups NPER no potential effects range NR not reported NSTP National Status and Trends Program OECD Organisation for Economic Co-operation and Development OP octylphenol OP nEO octylphenol ethoxylate, where n = specific number of EO groups PEL probable effect level PSL Priority Substance List QSAR quantitative structure-activity relationship SQG E 

### , SQG HH 

### environmental soil quality guideline, human health soil quality guideline SQG I 

### , SQG SC 

### soil quality guideline for ingestion, soil quality guideline for soil contact SSTT Spiked Sediment Toxicity Test WWTP wastewater treatment plant TEC, TEL threshold effect concentration, threshold effect level TEF, TEQ toxic equivalency factor, toxic equivalency TOC total organic carbon UF uncertainty factor USEPA United States Environmental Protection Agency WQG water quality guideline

ww wet weight

### 1

CHAPTER 1. INTRODUCTION

Nonylphenol (NP) is a synthetic substance (typically an isomeric mixture) that belongs to the class of organic chemicals known as alkylphenols (APs), and consists of a nine-carbon alkyl chain attached to a phenol ring (Fig. 1). APs, such as NP, may be used to produce various derivatives, including a class of nonionic surfactants 1 

### called alkylphenol ethoxylates (APEs). APEs are composed of a phenol group attached to both an alkyl chain [i.e., -(CH 2

### ) n 

### H] and an ethoxylate chain [i.e., -O(CH 2

### CH 2

### O) n 

### H]. In the case of nonylphenol ethoxylates (NPEs), the alkyl chain contains nine carbon atoms, and the ethoxylate chain may range in length from 1 to 100 ethoxylate groups.

As nonionic surfactants, NPEs are commonly used in a wide variety of industrial processes, as well as in cleaning products for residential and commercial use. Upon their release to the environment, the major degradation product of NPEs is NP. Therefore, NP is both the parent compound of NPEs during production, and a breakdown product of NPEs upon degradation. There is concern about the occurrence of NP and NPEs in the Canadian environment for a number of reasons. NPEs are released through sewage and industrial effluents in large quantities, and although they undergo a quick initial degradation, their degradation products, including nonylphenol, are much more persistent. NP and NPEs have shown evidence of toxicity to both aquatic and terrestrial organisms, including effects on endocrine systems.

Nonylphenol and its ethoxylates are among the substances on the second Priority Substances List (PSL2) that were required to undergo an environmental risk assessment under the Canadian Environmental Protection Act (CEPA, Bill C-32, June 1, 1999). The draft PSL2 assessment of nonylphenol and its ethoxylates has found that nonylphenol is entering the Canadian environment in quantities or concentrations, or under conditions, that are having, or may have, a harmful effect on the environment or its biological diversity (Government of Canada 2000a). Therefore, it has been proposed that nonylphenol and its ethoxylates be designated as “toxic” according to Section 64 of CEPA. An estimated no-effects value (ENEV) of 1.0 μg·L-1 

### was calculated from available water toxicity data for the protection of aquatic biota (Servos et al. 2000). Risk management strategies, that may be developed as a follow-up to the CEPA assessment and the likely designation of NPEs and NP as toxic, will require the use of targets or benchmarks for ensuring sustainability of ecosystem health. Therefore, in complement to the CEPA assessment, this report has been undertaken to develop Canadian Environmental Quality Guidelines (EQGs) to provide such benchmarks for NP and NPEs.

Under the auspices of the Canadian Council of Ministers of the Environment (CCME), Canada has been developing environmental quality guidelines for water, sediment, soil, and tissue qualitysince the 1980s (CCREM 1987; CCME 1999). Canadian EQGs are nationally endorsed, sciencebased goals that are recommended as levels that should result in negligible risk to biota and ecosystem health. These guidelines serve as benchmarks or indicators of environmental quality that can be used nationally for the protection, evaluation, and enhancement of the Canadianenvironment and its beneficial uses. Often the Canadian EQGs form the scientific basis for sitespecific criteria, guidelines, objectives, or standards set by the various Canadian provincial and territorial jurisdictions.

This document describes the development of Canadian EQGs for nonylphenol and its ethoxylates for the protection of water, sediment and soil quality. These guidelines are numerical concentrations that are recommended to support and maintain aquatic (both freshwater and marine), sediment-dwelling (both freshwater and marine), and terrestrial life, and are developed

1 

### Nonionic surfactants (as opposed to cationic, anionic, or amphoteric surfactants) contain no ionic constituents and produce electrically neutral colloidal particles in solution (Davidsohn and Milwidsky 1967).

### 2

from the available scientific information on the biological effects of nonylphenolic compounds in the environment. Water, sediment and soil have been identified as the key compartments of the environment for which Canadian EQGs should be developed as a result of the potential for impacts on the beneficial uses of the environment from NP and NPEs. Tissue residue guidelines in aquatic biota for the protection of wildlife consumers of aquatic life have not been derived due to the lack of available toxicity data for mammalian and avian species. Nationally approved protocols have been followed in developing Canadian EQGs for the protection of water, sediment and soil quality (CCME 1991; CCME 1995; CCME 1996).

All of the guidelines that have been developed in this document are based on toxicity data for nonylphenol; however, they may be applied to mixtures of nonylphenol, nonylphenol ethoxylates and nonylphenol ethoxycarboxylates (NPECs) with the use of toxic equivalents. In Chapters 3 and 9, guidance is provided on how to account for NPEs and NPECs when implementing the guidelines.

In addition to describing the development of environmental quality guidelines, this document also reviews other available scientific information on nonylphenol and its ethoxylates. This discussion includes a review of the physical and chemical properties of NP and NPEs, sources of these substances to the environment, analytical methods used to detect NP and NPEs, levels of these substances found in the various compartments of the Canadian environment, and their environmental fate and behaviour. Although the EQGs are solely developed using toxicological data, this additional information is discussed in order to provide important considerations for the implementation of the guidelines. More detailed information on some of these aspects has also been summarized and reviewed to support the CEPA PSL2 assessment of NP and NPEs and can be found elsewhere (see Servos et al. 2000).

Physical and Chemical Properties

Nonylphenol is a generic term used to refer to a group of isomeric compounds, each consisting of a nine-carbon alkyl chain (-C 9 

### H 19

### ) attached to a phenol group, with the molecular formula C 15 

### H 24

### O. The various isomers can differ both in the degree of alkyl chain branching, and in the position on the phenol ring at which the alkyl chain is attached. Most nonylphenol in production is of the form 4-nonylphenol (i.e., attachment at the para-position) with varied alkyl chain branching. Very little straight chain nonylphenol ( n-nonylphenol) is produced. Commercially produced 4-nonylphenol may also contain small amounts of 2 -nonylphenol (i.e., attachment of the nonyl chain to the ortho-position of the phenol ring), or 2,4-dinonylphenol (i.e., two nonyl chains attached to a phenol ring) as impurities (European Commission 1999). The Chemical Abstract Service (CAS) registry number for a mixture of various branched isomers of nonylphenol is 84852 -15 -3 (which may be followed by an asterisk) and is given the IUPAC name of “4-nonylphenol (branched)”. Some of the individual branched isomers also have their own specific CAS numbers. Straight chain nonylphenol goes by the IUPAC name of “nonylphenol” and has the CAS number 25154-52 -3. NP may also be referred to as “isononylphenol” (CAS n umber 11066 -49 -2) or “phenol, nonyl-, branched” (CAS number 90481 -04-2) (European Commission 1999). In this document, the term nonylphenol is used to refer to all isomers and is best represented by CAS number 84852-15-3. Data specific to particular isomers generally have not been differentiated.

The major derivatives of nonylphenol are nonylphenol ethoxylates (NPEs) and nonylphenol ethoxycarboxylates (or nonylphenoxy acetic acids, NPECs) (Figure 1). Nonylphenol ethoxylates have the general formula C 9 

### H 19

### -C 6 

### H 4

### O(CH 2

### CH 2

### O) n 

### H, with n ranging from 1 to 100, but with the vast majority of commercially produced NPEs containing between 6 and 12 ethoxylate (EO) groups. Specific NPE oligomers may be reported as NP nEO, where n refers to the mean number of EO groups in the ethoxylate chain. For example, an oligomer with 9 EO groups is referred to as NP9EO. The term NP9EO may also refer to a mixture of various oligomers for which the mean number of EO groups per molecule is 9 (i.e., the mixture may also contain NP8EO, NP10EO,

### 3

etc.). A mixture containing equal proportions of both NP1EO and NP2EO, for example, would be given the abbreviation of NP1.5EO.

Nonylphenol ethoxycarboxylates have the general formula C 9 

### H 19

### -C 6 

### H 4

### O(CH 2

### CH 2

### O) n

### CH 2

### COOH. As with NPEs, nonylphenol ethoxycarboxylates with specific ethoxylate chain lengths may be reported as NP nEC, where n refers to the number of EO groups in the ethoxylate chain.

The basic structures of the three main types of nonylphenolic compounds are shown in Figure 1.



## C 9 

## H 19 

## OH

C 9 

## H 19 

## O(CH 2

## CH 2

## O) n 

## H

C 9 

## H 19 

## O(CH 2

## CH 2

## O) n 

## CH 2

## COOH

Nonylphenol Nonylphenol ethoxylate Nonylphenol ethoxycarboxylate (NP) (NP nEO) (NP nEC)



### Figure 1. The structure of nonylphenol and its derivatives.

Nonylphenol is a colourless to pale yellow, or straw-coloured, viscous liquid with a slight characteristic phenolic odour (Reed 1978; CCOHS 1995; Budavari et al. 1996). The physical and chemical properties of nonylphenol are listed in Table 2. NP has a molecular weight of 220.3 (Reed 1978). The vapour pressure is 4.55 (± 3.5) x 10 -3

### Pa at 25°C, and Henry’s Law Constant is 11.02Pam 3 

### ·mol -1 

### (Romano 1991 as cited in Maguire 1999; European Commission 1999). This low Henry’s Law Constant suggests that volatilization of nonylphenol from water would not be significant (Maguire 1999). The pKa

### (negative logarithm of the acid dissociation constant) of NP is 10.7, indicating that in most natural waters virtually all NP is present in the undissociated form (Romano 1991 as cited in Maguire 1999).

Nonylphenol has a low solubility in water or dilute aqueous NaOH, but is readily soluble in acetone, benzene, chlorinated solvents, aniline, heptane, aliphatic alcohols, and ethylene glycol (Reed 1978; Budavari et al. 1996). Aqueous solubility of nonylphenol (at 20.5oC) is 5.43 mg·L-1



### , with only slight variation of this solubility over the temperature range of 2 to 25oC (Ahel and Giger 1993a). Water solubility is increased by alkyl branching (Talmage 1994). There is also a linear increase in solubility with increasing number of ethoxylate groups. Solubilities (at 20.5oC) for NP1EO, NP2EO, NP3EO, NP4EO, and NP5EO are 3.02, 3.38, 5.88, 7.65, and 9.48 mg·L-1



### , respectively (Ahel and Giger 1993a). NP nEOs are water soluble for values of n Ú7 (Talmage 1994). Similar trends were observed with octylphenol (OP) and its ethoxylates (OP nEO). Octylphenol ethoxylates have the same chemical structure as NPEs, except that their alkyl chain consists of eight carbons, rather than nine. 1 

### The solubility of the OP nEO oligomers were greater than those of the corresponding NP nEO oligomers. Therefore, the lengths of both the ethoxylate and alkyl chains of APEs affect their solubility.

1 

### OP and NP also differ with respect to the branching of their alkyl chains. While NP is always present as a mixture of isomers with varied branching of the nine-carbon alkyl chain, OP found in the environment is almost entirely a single isomer with a 1,1,3,3-tetramethylbutyl eight-carbon chain (Swisher 1987; Bennie et al. 1997, 1998).

### 4

Nonylphenol ethoxylates have an amphipathic nature, with the alkylphenyl moiety being hydrophobic (i.e., lipophilic) in nature, and the polyethoxylate chain being hydrophilic. This characteristic enables NPEs to concentrate at interfaces between non-mixable phases or liquids (e.g., air and water, oil and water, oil and minerals, biological membranes). Most commercial and industrial applications of nonylphenol ethoxylates utilize this characteristic surfactant property. The sorption and solubilization abilities of a surfactant are related to their hydrophile-lipophile balance (HLB). The higher the molecular weight of the lipophilic component, the more moles of hydrophilic ethoxylate units are needed to make the surfactant water-soluble (Davidsohn and Milwidsky 1967). By calculating the percentage of hydrophilic ethoxylate units compared to the total mass of the surfactant, HLBs for NP5EO, NP8EO, NP9EO, NP14EO, NP17EO, and NP40EO have been estimated at 10, 12.3, 12.8, 14.9, 15.8, and 17.8, respectively (Beigel et al. 1998). When present in solution at high enough concentrations, surfactants will form colloidalsized clusters, called micelles, in which the hydrophobic tails of the molecules are grouped together in the centre, with the hydrophilic heads pointing out towards the solvent (Haigh 1996). The detergency and solubilisation properties of nonylphenol polyethoxylates are due to this formation of micelles. The concentration at which thermodynamics favour micelle formation is referred to as the critical micelle concentration. Critical micelle concentration values for NP5EO, NP8EO, NP9EO, NP14EO, NP17EO, and NP40EO have been estimated at 13, 32, 42, 66, 112, and 324 mg·L-1 

### , respectively (Beigel et al. 1998). Therefore, the more hydrophobic compounds (i.e., those with fewer ethoxylate groups and a lower HLB) are more efficient at micelle formation. Critical micelle concentration values for nonylphenol ethoxylates increase with increasing HLB (Beigel et al. 1998).

### 5

Table 2. Physical and chemical properties of nonylphenol and select nonylphenol ethoxylates.
Property Nonylphenol NP1EO NP4EO NP9EO NP40EO CAS registry number 84852-15-3

-- -- -- -- chemical formula C 15 

### H 24

### O C 17 

### H 28

### O 2 

### C 23 

### H 40

### O 5 

### C 33 

### H 60

### O 10 

### C 95 

### H 184

### O 41 

### molecular weight (g·mol -1 

### ) 220.3 281.4 396.6 616.8 1982.5 freezing point 1 

### (°C) <20

-- -- -- -- melting point 1 

### (°C) 81-83

-- -- -- -- boiling point2

### (°C) 295-320, 310, 304, 293-297 -- -- -- --

flash point3

### (°C) 146; 300 (open cup), 285 (closed cup) -- -- -- --
density 1 

### (g·mL-1 

### at 43°C) 0.933

-- -- -- -- specific gravity4

### (at 20°C referred to water at 4°C) 0.95

-- -- -- -- aqueous solubility5

### (mg·L -1 

### at 20.5°C) 5.43 3.02 7.65 water soluble water soluble pKa 6 

### 10.7

-- -- -- -- log Kow7 

### 4.1, 4.2, 4.48, 4.7 4.17, 4.4 4.24, 4.3 4.1

-- vapour pressure 6

### (Pa at 25°C) 4.55 x 10 -3



### -- -- -- -- Henry’s law constant8

### (Pa m 3 

### ·mol -1 

### ) 11.02

-- -- -- -- hydrophile-lipophile balance 9



### -- -- -- 12.8 17.8

1 

### Lorenc et al. 1992
2

### as determined by Reed 1978, Lorenc et al. 1992, CCOHS 1995, and Budavari et al. 1996, respectively
3

### as cited in Lorenc et al. 1992 and Chemical Hazard Response Information System 1995, respectively
4

### Reed 1978
5 

### Ahel and Giger 1993a; Hall et al. 1989
6 

### Romano 1991 as cited in Maguire 1999
7

### as cited by McLeese et al. 1981, Barber et al. 1988, Freij 1991, W ild and Jones 1992, and Ahel and Giger 1993b (both experimentally determined values and calculated estimations) 8

### European Commission 1999
9

### as cited in Beigel et al. 1998

### 6

Production and Uses The industrial manufacture of nonylphenol involves alkylating phenol with a mixture of isomeric nonenes (propylene trimers) in the presence of an acid catalyst. The resulting nonylphenols (with various isomers of the branched nonyl chain) are recovered by fractional distillation under reduced pressure (Reed 1978). As of 1997, three Canadian companies were producing nonylphenol: Huntsman Canada (Guelph, Ontario), Rhone-Poulenc (Mississauga, Ontario), and Stepan Canada (Longford Mills, Ontario). Total Canadian domestic production of nonylphenol in 1996 (the most recent year for which data are available) was 7700 tonnes, with 5000 tonnes exported (Camford Information Services Inc. 1997). In 1996, a total of 1200 tonnes of NP were imported into Canada, all from the United States. Therefore, the total Canadian domestic demand for nonylphenol in 1996 was 3900 tonnes (Camford Information Services Inc. 1997).

The domestic demand for nonylphenol can be subdivided into its various use applications. In 1989, 1600 tonnes of NP were used to produce ethoxylated pulp mill specialties, 1000 tonnes were needed for ethoxylated textile specialties, 500 tonnes were used to produce miscellaneous ethoxylates, another 500 tonnes were used to produce trinonylphenol phosphite, and 900 tonnes went towards other miscellaneous products (Camford Information Services Inc. 1990). These other miscellaneous products include the use of nonylphenol as an additive in lube oils, and a formulant in pesticides (Camford Information Services Inc. 1990). The most notable Canadian historical example of NP use in pesticides was as a solvent in the Matacil 1.8D formulation of aminocarb. This insecticide, which contained NP at almost three times the weight of the aminocarb, was applied aerially to extensive forested areas of Quebec, New Brunswick, and Newfoundland between 1975 and 1985 to control spruce budworm (Fairchild et al. 1999). Tris(4- nonylphenyl) phosphite is an antioxidant used in the stabilization of rubber, vinyl polymers, polyolefins and styrenics (Lorenc et al. 1992). Nonylphenol may also be used as a catalyst in the curing of epoxy resins, or it may be converted to barium and calcium phenolates which are used as heat stabilizers in poly(vinyl chloride) (Lorenc et al. 1992). As evidenced in the above statistics though, the main use (approximately 70%) of nonylphenol is in the production of nonylphenol ethoxylates.

Nonylphenol ethoxylates are formed through the process of etherification, whereby a basic catalyst is used to condense nonylphenol with ethylene oxide (Reed 1978):

C 9 H 19 OH

C 9 H 19 O(CH 2 CH 2 O) n H



## + nCH 2CH 2

O catalyst



### Nonylphenol ethoxylates belong to a class of compounds called alkylphenol ethoxylates (APEs). Of the nonionic surfactants, APEs are the second largest group, after alcohol ethoxylates, in commercial production. Since their introduction in the 1940s, APEs have been widely used in cleaning products and industrial processes (Talmage 1994; White et al. 1994). More than 300000tonnes of APEs are produced annually world-wide (Freij 1991; Metcalfe et al. 1996). Nonylphenol ethoxylates account for about 82% of the APE world market, followed by octylphenol ethoxylates (15%) and small amounts of dodecylphenol and dinonylphenol ethoxylates (1-2%; Naylor 1992). In 1990, total use of NPEs in Canada was estimated at 7000 tonnes per year, of which 4100 tonnes of NPEs were produced domestically, while the remainder was imported in cleaning product formulations (Metcalfe et al. 1996). In a 1997 survey of 4500 companies operating in Canada, 189 companies were involved in the production, import, export, shipping, acquisition, use or release of at least 1 tonne per year of NP and/or NPEs (Servos et al. 2000).

Nonylphenol ethoxylates have a broad range of uses due to their effectiveness as detergents, emulsifiers, wetting agents, and dispersing agents. There are eleven major Canadian industrial sectors that use NPEs. These include the pulp and paper industry, textile manufacturing, plastic

### 7

and elastomer manufacturing, oil extraction and petroleum production, pesticide formulations, domestic/industrial/institutional cleaners, metal processing, paint and protective coatings, leather manufacturing, building and construction, and the food and beverage sectors. Smaller amounts of nonylphenol ethoxylates are also used in spermicides (Metcalfe et al. 1996).

The commercial and industrial applications of NPEs depend partly upon the length of their ethoxylate chains. Nonylphenol ethoxylates with 4 or 5 ethylene oxide residues per molecule of nonylphenol are typically used as oil-soluble detergents and emulsifiers, and if sulphonated or phosphorylated, can be used as anionic detergents, lubricants and antistatic agents (Reed 1978). NP nEOs with 8 to 12 EO groups are the most commonly used compounds; they are utilized in high performance detergents (such as for textile scouring) and in industrial cleaning agents forhard surfaces. Higher ethoxylates (13 to 15 EO groups per molecule), when combined with an oilsoluble anionic surfactant, make excellent emulsifiers for a wide range of solvents and agricultural pesticides (Reed 1978). NP nEOs with up to 80 ethoxylate groups are effective as dispersants (Freij 1991).

Directories are available that contain extensive listings of the trade names, manufacturers and distributors of the various NPE products available (Ash and Ash 1997; Anonymous 1999). Examples of some commercial preparations of NPEs include the following products: the Alkasurf CO series manufactured by Rhodia, Inc.; Caloxylate N-9 (NP9EO) manufactured by Pilot Chemical Co.; the Carsonon N series manufactured by Lonza, Inc.; the Cedepal CO series manufactured by Stepan Canada Inc.; the Chemax NP series manufactured by Chemax Inc.; the Delonic NPE series manufactured by DeForest Enterprises, Inc.; the Iconol NP series manufactured by BASF Corp. Performance Chemicals; the Igepal CO series manufactured by GAF Corp. (Rhodia, Inc.); Imbentin-N/7A (NP5EO) and Imbentin-N/200 (NP20EO) manufactured by W . Kolb, AG; Lissapol TN 450 and Lissapol TN XP (both NP8-9EO) manufactured by ICI Chemicals; the Marlophen NP series manufactured by CONDEA Chemie GmbH; the Norfox NP series manufactured by Norman, Fox & Co.; the Polystep F series by Stepan Company; the Surfonic N series manufactured by the Huntsman Corporation; the Tergitol NP series manufactured by Union Carbide Corporation; the TERIC N series manufactured by Huntsman Corp. Australia; and TRITON N by Union Carbide Corporation (Ash and Ash 1997; Anonymous 1999; Lee 1999).

Due to concerns about the toxicity and persistence of NPE degradation products, regulatory and voluntary initiatives have been taken to eliminate many of the uses of NPEs in Europe. In 1986, Switzerland banned the use of NPEs in laundry detergents for domestic use (WW F Canada 1997). In an ordinance enacted in 1991, Swedish parliament decided to phase out 90% of NP and NPE use by the year 2000 (WW F Canada 1997). The Cosmetic, Toiletries, Soap and Detergent Industries Association (SPT) of Denmark have already phased out alkylphenols completely under a voluntary agreement with the Danish EPA (Metcalfe et al. 1996). Germany, Norway, the Netherlands, UK, and France are currently discussing a potential ban and phase out of NPEs in commercial products such as domestic and industrial detergents. In the UK, domestic use of NPEs in detergents has been phased-out since 1976, and some agrochemical companies have voluntarily phased-out NPEs in their pesticides since 1992. In 1995, UK wool scourers volunteered to eliminate discharges (though not use) of APEs by the end of 1996, and several have switched to the use of alcohol ethoxylates (WW F Canada 1997). As of 1990, German wool manufacturers had voluntarily phased out the use of NPEs in wool scouring, and in 1997 a voluntary agreement was drawn up to phase out the use of NPEs in Germany as flocculants in wastewater purification (WW F Canada 1997).

This widespread European phase-out of NPEs is the result of a 1992 recommendation from the Paris Commission. Under Paris Commission Recommendation 92/8, all of the North Sea States and the European Community agreed to phase NPE out of domestic cleaning products by 1995 and out of industrial cleaning products by 2000 (WW F Canada 1997). The Working Group on Diffuse Sources of the Oslo and Paris Conventions for the Prevention of Marine Pollution is currently performing a full risk assessment on nonylphenol (Maguire 1999).

### 8

In the United States, nonylphenol was first placed on the Environmental Protection Agency’s Inerts List 1 in 1987, identifying it as a pesticide formulant of toxicological concern because of human health and/or ecological considerations (PMRA 2000). Pesticide products in the U.S. which contain nonylphenol must state so on the label. In Canada, the Pest Management Regulatory Agency is proposing regulations to phase out the use of nonylphenol in pesticide formulations by December 31, 2002 (PMRA 2000).

Sources to the Environment

Water Almost all releases of nonylphenolic compounds are initially routed to the aquatic environment in the form of industrial or municipal wastewater effluents. The bulk of industrial releases to the Canadian environment originate from formulators/distributors of surfactants, and industrial users of cleaning products, degreasers and detergents (Servos et al. 2000). Other significant industrial sources include pulp mills and textile mills, as well as the leather manufacturing, oil extraction, and petroleum production sectors (Metcalfe et al. 1996). Household, industrial, and institutional cleaning products are the major source of the nonylphenolic content in effluents from municipal wastewater treatment plants (WWTPs). Although most nonylphenol and nonylphenol ethoxylates that enter the aquatic environment are introduced via effluent discharge, there is also the potential for these substances to be released to the environment during production, transportation, processing, and during the use of the products (Metcalfe et al. 1996).

Due to their use in some pesticides as adjuvants and dispersants, nonylphenol and nonylphenol ethoxylates may potentially enter the aquatic environment as residues deposited on surface waters from aerial spraying (Ernst et al. 1980; Sundaram et al. 1980; Holmes and Kingsbury 1980; Paveglio et al. 1996) or as runoff from fields. Application of pesticides to forests can introduce NP and NPEs to forest streams and ponds. Application to agricultural fields and orchards is a source of NP and NPEs to drainage ditches and farm ponds, and application to emergent vegetation provides a pathway for entry of NP and NPEs to wetlands and intertidal zones.

Sediment Once introduced to the aquatic environment, NP and NPEs may adsorb to particulates and be deposited to bottom sediments. Their amphipathic nature enables adsorption to organic matter through hydrophobic reactions as well as adsorption to the mineral components of sediment through hydrophilic reactions (John et al. 2000). Movement of NP and NPEs to the sediments may involve direct adsorption to sediment particles at the surface of the sediment-water interface, or adsorption to suspended sediment particles in the water column that may eventually settle out. Once present in the bottom sediments, there is also the possibility for re-entry of NPEs to the water column through resuspension of sediment, particularly during storms or through dredging.

Soil
The sources of NP and NPEs to the terrestrial environment are much more limited than for the aquatic environment. Some NP or NPEs may enter the soil through the spraying of pesticides on agricultural fields, orchards, golf courses and forests (Sundaram et al. 1980). Another pathway is through the application of sewage sludge or sludge from recycled paper plants to agricultural soils (Diercxsens and Tarradellas 1987; Marcomini et al. 1989a,b; Kirchmann and Tengsved 1991; Hawrelak et al. 1999). Disposal of sewage sludges in landfills may also introduce nonylphenolic compounds into soil (Marcomini et al. 1989a,b; Marcomini et al. 1991).

Despite the strong tendency for adsorption to soil and sediment particles, alkylphenolic compounds (such as NP and NPEs) present in surface waters or landfills may leach into groundwaters (Reinhard et al. 1984; Barber et al. 1988; Zoller et al. 1990; Ahel 1991; Ahel et al. 1996).

### 9

Air Some authors have stated that due to the low volatility of nonylphenolic substances, they are generally not contaminants of the air (e.g., Metcalfe et al. 1996). However, recent studies indicate that in some locations there may be considerable water-to-air volatilization, resulting in high atmospheric concentrations of nonylphenolic substances (Dachs et al. 1999).

The fate processes that control the movement of NP and NPEs through various environmental compartments are discussed further in Chapter 3.

Analytical Methods Over the past few years there have been major improvements in the methods used for sample extraction and detection of nonylphenol and nonylphenol ethoxylates in environmental samples, both in terms of ease of analysis, and in sensitivity and precision (Kubeck and Naylor 1990). Comprehensive discussions of both historical and more recent analytical methods used for determination of nonylphenolic compounds are provided by Talmage (1994) and Lee (1999). In this document, however, brief descriptions of only the most up-to-date and currently accepted methods are presented. For more detailed information on any of the particular methods that are mentioned below, readers are advised to consult the references given.

Determining concentrations of nonylphenolic compounds in environmental samples involves two main procedures: extraction/clean-up, and detection. The optimal methods to use for extraction and clean-up depend upon the type of environmental sample being analyzed, and the particular oligomer range of interest.

Extraction of nonylphenol and the lower molecular weight nonylphenol ethoxylates (e.g., NP1EO, NP2EO) from aqueous samples can be accomplished through liquid-liquid extraction techniques (Bennie et al. 1997, 1998), or through a combination of steam distillation and solvent extraction by heating the sample to reflux with iso-octane (Kubeck and Naylor 1990) or cyclohexane (Ahel and Giger 1985). W ith steam distillation methods, no further clean-up of the sample is required before detection analysis. Extraction of higher molecular weight NP nEOs requires other methods due to their lower volatility and their higher polarity and solubility. Successful extraction and preconcentration of NPEs, as well as NP and NPECs, from water and effluent samples has been achieved through solid phase extraction (Marcomini et al. 1987, 1990; Kubeck and Naylor 1990; Blackburn and Waldock 1995; Field and Reed 1996; Lee et al. 1998; Lee and Peart 1998; Sekela et al. 1999). Solid phase extraction involves passing the sample through a cartridge or disk containing an adsorbent such as octadecylsilane, graphitized black carbon, or a strong anion exchange resin (Lee et al. 1998). For example, Kubeck and Naylor (1990) used a dual columnapparatus in which the aqueous sample was deionized by passing through a mixed-bed ionexchange resin, then passed through octadecylsilica, which adsorbed the organic material. The organics were flushed from the octadecylsilica with warm methanol, the methanol was removed under a stream of nitrogen, and the remaining residue was redissolved in a dichloromethane/hexane mixture for detection analysis (Kubeck and Naylor 1990).

For extraction of NP, NP1EO and NP2E0 from solid samples (i.e., soil, sediment, sludge, or tissue), Soxhlet extraction (usually with dichloromethane) or steam distillation may be used (Marcomini et al. 1989a, 1989b, 1991; Bennie et al. 1997, 1998). Marcomini et al. (1990) also used a combination of Soxhlet extraction and elution through an aminosilica minicolumn cartridge to extract NP and NPEs from marine sediment and macroalgae. A more recent method using supercritical fluid extraction is a more efficient alternative that may be applied to a wider range of nonylphenolic compounds (Lee and Peart 1995; Lee et al. 1997).

For sensitive detection and quantification, it is now common practice to use high pressure liquid chromatography (HPLC) with fluorescence detection. The phenolic ring in nonylphenolic compounds absorbs ultraviolet light, giving these substances a distinctive fluorescent profile that

### 10

can be readily detected (Naylor 1992). W ith normal-phase HPLC, various nonylphenol ethoxylates can be separated according to their EO chain length on a silica gel matrix using an organic solvent gradient (Talmage 1994). The separated components are then detected byfluorescence and quantitated by comparison with external standards (Talmage 1994). Reversedphase HPLC may be used to separate different alkylphenol ethoxylates by their alkyl chain (i.e., into nonylphenol, octylphenol, and dodecylphenyl ethoxylates, etc.) (Talmage 1994). Gas chromatography/mass spectrometry (GC/MS) is also widely used for analysis. Extracts containing NP may be analyzed by GC/MS with either electron ionization or chemical ionization (Wahlberg et al. 1990; Clark et al. 1992; Blackburn and Waldock 1995; Bennie et al. 1997; Bennett and Metcalfe 1998). Determination of NPECs is also possible though GC/MS, although they must be derivatized to their volatile esters prior to analysis (Field and Reed 1996). It appears that of the NPEs, only the lower molecular weight compounds can be adequately determined using GC/MS. Wahlberg et al. (1990) were able to quantify NP1EO, NP2EO and NP3EO by first derivatizing them with pentafluorobenzoyl chloride. Clark et al. (1992) found that NPEs with low to moderate degrees of ethoxylation could be determined by GC/MS if present at high concentrations. Clark et al. (1992) also found that NP3-8EO could be detected at the parts per trillion level by using particle-beam liquid chromatography/mass spectrometry.

Although analytical techniques are constantly improving, the analysis of environmental samples for nonylphenolic substances still remains a complicated procedure. Often environmental samples will contain other alkylphenolic surfactants which must be separated from NP and NPEs. Samples may also contain hundreds of different isomers and ethoxamers of NPEs, requiring the use of several different analytical techniques for the detection of all.

Using various combinations of the analytical methods described above, sensitive analyses may be conducted with low limits of detection. The detection limits discussed below were determined through the analysis of environmental samples, and therefore represent concentrations that are environmentally relevant and can be realistically determined from field samples. Reported limits of detection and method detection limits for NP, NP1EO, and NP2EO in water, sewage or effluents are as low as 0.01, 0.02 and 0.02 μg·L-1 

### , respectively (Bennie et al. 1997, 1998; Rudel et al. 1998). For larger molecular weight NPEs (i.e., NP nEOs where n = 3 to 17) method detection limits are higher, at about 1.6 μg·L-1 

### (Radian Corporation 1990). Detection limits for individual NPECs in water have been reported between 0.04 and 0.4 μg·L-1 

### (Field and Reed 1996). For NP, NP1EO, and NP2EO in sediments, current limits of detection are reported at concentrations as low as 0.004 mg·kg -1 

### (Marcomini et al. 2000). Detection limits for NP in soil have been reported at concentrations as low as 0.05 mg·kg -1 

### (Jobst 1987). For tissue samples, detection limits for NP and each of NP1EO to NP3EO have been reported at 0.001 mg·kg -1 

### and 0.006 mg·kg -1



### , respectively (Wahlberg et al. 1990). W ith refinements to the standard analytical methods, it may be possible to obtain detection limits lower than those reported here, and in some cases (e.g., Bennie 1998; Sekela et al. 1999) environmental measurements have been noted at concentrations below these detection limits.

### 11

CHAPTER 2. CONCENTRATIONS IN CANADA

Nonylphenol and nonylphenol ethoxylates are synthetic compounds that do not occur naturally. Therefore, environmental concentrations can be attributed solely to human activities. A considerable amount of data has been collected on environmental concentrations of nonylphenolic compounds in North America and Europe for fresh water, marine or estuarine water, groundwater, freshwater sediment, and marine or estuarine sediment. Many measurements of these compounds have also been made in sewage effluents, sewage sludge, and industrial effluents. Considerably less data have been collected on soil concentrations of nonylphenolic compounds, and the data that do exist focus on soils that have been amended with sewage sludge.

A comprehensive review and discussion of environmental concentrations of nonylphenol and nonylphenol ethoxylates, both within Canada and outside of Canada, are provided in the CEPA PSL2 assessment of nonylphenol and its ethoxylates (Servos et al. 2000). Therefore, the summary of Canadian environmental concentrations included in this document largely draws upon the above-mentioned assessment. Data were available for NP and NPE concentrations in Canadian fresh surface waters, freshwater and marine sediments, sludge-amended soil, raw sewage, municipal wastewater treatment plant (WWTP) effluent and sludge, and industrial effluents. No data on concentrations in Canadian marine/estuarine waters or groundwater were available.

Surface Waters Canadian freshwater concentrations of NP and NPEs have been reported for various sites in southern Ontario (largely from rivers, lakes and harbours throughout the Great Lakes - St. Lawrence River system), for freshwater rivers in Quebec, Prince Edward Island, and Nova Scotia, and for sites in the Fraser River of British Columbia. Most reported concentrations of NP fall within the range of <0.01 to 1.7 μg·L-1 

### (Bennie et al. 1997; Sekela et al. 1999; Sabik et al. 2000; Rutherford 2001). Agricultural streams monitored in Prince Edward Island in 1999 contained concentrations of NP as high as 0.25 μg·L-1 

### (Ernst 2000). The highest measured concentrations occurred in waters near heavily industrialized areas. For example, nonylphenol concentrations directly adjacent to an outfall of a secondary municipal wastewater treatment plant treating textile mill effluent on the Eaton River in Quebec were measured at 94.2μg·L-1 

### (Rutherford 2001). Carey et al. (1981, as cited in Servos et al. 2000) reported NP concentrations as high as 2600μg·L-1 

### in the Canagagigue Creek near Elmira, Ontario; however, these measurements were made following an industrial spill into the small creek, and are not considered representative of typical levels (Servos et al. 2000). NPEs tend to be more ubiquitous than NP, and also occur at higher concentrations. Concentrations of NP1EO and NP2EO have been measured in Canadian fresh waters at <0.02 to 7.8 μg·L-1 

### , and <0.02 to 16.9 μg·L-1 

### , respectively (Bennie et al. 1997; Rutherford 2001). Unpublished results from an ongoing survey of 8 rivers in Quebec indicate that the sum of NPE (with 1 to 17 EO groups) ranges between <0.05 and 14.6 μg·L-1 

### (Berryman 2000). For the sum of NP1EC and NP2EC, concentrations in Quebec rivers range between <0.05 and 23μg·L-1 

### (Berryman 2000). Levels of NP1EC in water from the St. Lawrence River near Montreal have been reported at 0.014 to 0.51 μg·L-1 

### , and levels of NP nEOs (with n ranging from 7 to 16) from the same area have been reported to range from below detection limit to 10.3 μg·L-1 

### (Sabik et al. 2000). Extremely high levels of NP and NPEs have been measured in the Annapolis River of Nova Scotia downstream from a textile mill; at 30 metres below the outfall, concentrations of NP, NP1EO, NP2EO, and the sum of NPEs (with 3 to 17 EO groups) were 644, 180, 44.4, and 186 μg·L-1 

### , respectively (Rutherford 2001). These high levels were attributed to a poorly operating primary treatment system at the mill (Rutherford 2001).

There is currently a lack of data on all nonylphenolic substances for surface fresh waters in Alberta, Manitoba, New Brunswick, Newfoundland, Saskatchewan, and the three territories.

### 12

Sediment Concentration data for nonylphenolics in Canadian freshwater sediments only exist for the Great Lakes - St. Lawrence system, and freshwater locations in British Columbia. Reported freshwater bed sediment concentrations of NP range from below detection limits to 110 mg·kg -1 

### on a dry weight basis (Lee and Peart 1995; Bennie et al. 1997; Brewer et al. 1999; Bennett and Metcalfe 1998, 2000; Sabik et al. 2000). Sekela et al. (1999) reported NP concentrations in suspended solids of the Fraser River, B.C., at 0.021 to 0.087 mg·kg -1 

### dry weight. Canadian freshwater bed sediment concentrations of NP1EO and NP2EO fall within the ranges of <0.015 to 75 mg·kg -1 

### and <0.015 to 18.0 mg·kg -1 

### dry weight, respectively (Bennie et al. 1997; Bennett and Metcalfe 2000; Sabik et al. 2000).

Data on the concentrations of nonylphenolics in Canadian marine sediments are only available for the Strait of Georgia, British Columbia. Concentrations of NP in surficial marine sediments ranged from 0.122 to 0.541 mg·kg -1 

### dry weight (mean = 0.273 mg·kg -1 

### , n = 21) (Shang et al. 1999; MacDonald and Ikonomou 2001). Surficial marine sediment concentrations of NP1EO and NP2EO were measured at 0.065 to 0.558 mg·kg -1 

### dry weight (mean = 0.279 mg·kg -1 

### , n = 21) and 0.034 to 0.198 mg·kg -1 

### dry weight (mean = 0.069 mg·kg -1 

### , n = 21), respectively. Total concentrations of NP nEO (3 ≤ n ≤19) were measured at 0.29 to 1.42 mg·kg -1 

### dry weight (mean = 0.78 mg·kg -1 

### , n = 21). These same researchers also collected marine sediment cores and examined the concentrations of NP and NPEs at various sediment depths. They found that both the total amount of NP nEO, and the profile of relative concentrations for individual NP nEO ethoxamers did not change significantly with depth (Shang et al. 1999).

Most of the sediments that have been analyzed for NP and NPEs were collected from locations

near WWTP outfalls or industrialized areas. Therefore, the data reported here likely represent the upper range of concentrations present in Canadian sediments.

Soil Only one study has looked at Canadian soil concentrations of nonylphenolic substances, and that study only examined soil from a single site that had been amended with sludge. Water Technology International Corporation (1998a as cited in Servos et al. 2000) measured 2.72mg·kg -1 

### of NP in sludge-amended soil, <0.538 mg·kg -1 

### each of NP1EC and NP2EC, and only trace amounts of NP1EO, NP2EO, and NP nEO.

Sewage and Sludge Several researchers have reported concentrations of nonylphenolic substances in the raw sewage, primary effluent, final effluent, and sludge of various municipal WWTPs across Canada.

Reported concentrations of NP, NP1EO, NP2EO, NP nEO, NP1EC, and NP2EC in raw sewage fall within the ranges of 0.69 to 155 μg·L-1 

### , 2.8 to 43 μg·L-1 

### , 0.26 to 38 μg·L-1 

### , 98.5 to 1300 μg·L-1



### , 0.9 to 71.3 μg·L-1 

### , and 1.4 to 35 μg·L-1 

### , respectively (Bennie et al. 1998; Lee and Peart 1998; Lee et al. 1998; Water Technology International Corporation 1998b as cited in Servos et al. 2000).

Most studies have found that concentrations of NP and NPEs are lower in the final effluent from municipal WWTPs relative to the incoming raw sewage, with the greatest decreases occurring for the larger NPEs. Reported concentrations of NP, NP1EO, NP2EO, and NP nEO in final effluents from Canadian municipal WWTPs are <0.02 to 15.1 μg·L -1 

### , 0.072 to 26 μg·L-1 

### , 0.099 to 21 μg·L-1



### , and from below detection to 13.3 μg·L-1 

### , respectively (Lee and Peart 1995; Bennie et al. 1998; Lee and Peart 1998; Sekela et al. 1999; Water Technology International Corporation 1998b as cited in Servos et al. 2000). Rogers et al. (1986) also identified NP, NP1EO and NP2EO in municipal wastewater effluents from Vancouver, B.C.; however, they did not report concentrations.

Sewage treatment appears to have the opposite effect on NPECs, with higher concentrations occurring in the final effluent relative to the raw sewage. Concentrations of NP1EC and NP2EC in

### 13

final effluents from Canadian municipal WWTPs have been reported at 1.9 to 703 μg·L-1 

### and 11 to 565 μg·L-1 

### , respectively (Lee and Peart 1998; Lee et al. 1998; Water Technology International Corporation 1998b as cited in Servos et al. 2000). Similar observations of elevated levels of NPECs following secondary treatment have also been observed in several Swiss municipal WWTPs (Ahel et al. 1987). NP1EC and NP2EC are biodegradation products of NPEs, which explains their occurrence at higher concentrations in sewage following treatment (Ahel et al. 1987).

Substances that are more hydrophobic, such as NP and the lower NPEs, have a greater tendency to adsorb to particles and thus accumulate in the sludge during treatment. Concentrations of NP, NP1EO, and NP2EO in the sludge of Canadian municipal WWTPs have been reported at 8.4 to 850 mg·kg -1 

### , 3.9 to 437 mg·kg -1 

### , and 1.5 to 297 mg·kg -1 

### , respectively (Lee and Peart 1995; Lee et al. 1997; Bennie et al. 1998; Water Technology International Corporation 1998b as cited in Servos et al. 2000). More hydrophilic substances, such as the NPECs and the higher NPEs, occur at lower concentrations in sludge. Reported sludge concentrations for NP nEO, NP1EC and NP2EC are 9 to 169 mg·kg -1 

### , <0.5 to 25 mg·kg -1 

### , and <0.5 to 38 mg·kg -1 

### , respectively (Lee et al. 1997; Water Technology International Corporation 1998a,b as cited in Servos et al. 2000).

The concentrations and relative proportions of various NPEs in final effluents and sludges vary considerably among municipal WWTPs and reflect differences in the type of effluent and sludge treatment used. The operating efficiencies of individual WWTPs may also affect the levels of NPEs in final effluent and sludge (Ahel et al. 1994a). Differences in NP and NPE concentrations among the raw sewages entering municipal WWTPs are likely a function of several factors including the size of the population served by the WWTP, and the number and types of commercial or industrial clients contributing to the WWTP.

Industrial Effluents and Sludge Little data exist for concentrations of nonylphenolic compounds in Canadian industrial effluents and sludge, but most of the data that do exist are from pulp and paper mills or textile mills.

Pulp mill effluents have been found to contain NP, NP1EO and NP2EO in concentrations of <0.02 to 26.2 μg·L-1 

### , <0.015 to 25.9 μg·L-1 

### , and <0.015 to 35.9 μg·L-1 

### , respectively (Bennie 1998 as cited in Servos et al. 2000). Unbleached whitewater from a Canadian pulp mill was also reported to contain NP nEO at a concentration of 310 μg·L-1 

### (Sithole and Allen 1989). Lee and Peart (1999) measured concentrations of nonylphenolics in both the effluent and sludge of 19 Canadian (and 1 U.S.) pulp and paper mills that use a variety of process types and waste treatment methods. Secondary treated effluent contained NP, total NPEs, NP1EC, and NP2EC of <0.1 to 4.32 μg·L-1



### , <2 to 71.3 μg·L-1 

### , <1.0 to 10.13 μg·L-1 

### , and <1.0 to 32.32 μg·L-1 

### , respectively (Lee and Peart 1999). Effluents commonly contained NPE oligomers with 10 or fewer ethoxy groups, but ethoxylates with greater than 10 ethoxy groups were seldom detected. Sludge samples from the mills contained NP, total NPEs, NP1EC, and NP2EC at concentrations of <0.05 to 120.9 mg·kg -1



### , <1 to 90.8 mg·kg -1 

### , <1 to 5.78 mg·kg -1 

### , and <1 to 18.5 mg·kg -1 

### dry weight, respectively (Lee and Peart 1999). Generally, the NPE oligomers that occurred at the highest concentrations in the sludge were NP8EO and NP9EO. Sludge from a Canadian recycled paper plant that was used as an agricultural soil amendment was found to contain NP, NP1EO and NP2EO at concentrations as high as 4.61, 1.21, and 0.39 mg·kg -1 

### dry weight, respectively (Hawrelak et al. 1999).

Textile mill effluents appear to contain considerably greater levels of nonylphenolic substances than pulp and paper mills, with concentrations of NP, NP1EO, NP2EO, and NP nEO ( n=3 to 17) reported at 1.1 to 13.3 μg·L-1 

### , 0.74 to 260 μg·L-1 

### , 0.64 to 590 μg·L-1 

### , and 50.2 to 8600 μg·L-1



### , respectively (Bennie 1998 as cited in Servos et al. 2000).

Analysis of two oil refinery effluent samples did not reveal any detectable concentration of NP (Bennie 1998 as cited in Servos et al. 2000).

### 14

CHAPTER 3. ENVIRONMENTAL FATE AND BEHAVIOUR

The occurrence of nonylphenol and its ethoxylates in aquatic or terrestrial ecosystems poses a potential risk to exposed organisms. The bioavailability and toxicity of nonylphenolic compounds are dependent on a number of physical and chemical processes that affect the fate and behaviour of these compounds in the environment. Therefore, understanding the role of these factors is essential for interpreting toxicity data, developing and using environmental quality guidelines, and implementing site-specific objectives. This chapter provides a discussion of the factors that govern the fate and behaviour of nonylphenolic compounds in the environment. The major processes affecting the persistence, distribution and bioavailability of nonylphenolic compounds in the environment include biodegradation, adsorption to suspended solids with subsequent sedimentation, soil particle adsorption, infiltration to groundwater, volatilization, and photolysis. Aerobic and anaerobic biodegradation of nonylphenolic substances by microorganisms is the most important factor affecting their fate. Various other biotic interactions are also involved in the fate and behaviour of nonylphenolic compounds. Uptake and bioaccumulation of nonylphenolic compounds by higher organisms may occur, as well as biotransformation, metabolism, and excretion. Nonylphenolic compounds, if available to biota at sufficiently high concentrations, have the potential to cause toxicity and/or endocrine disruption.

Physical and Chemical Processes The most important physicochemical process affecting nonylphenolic substances is particle adsorption. Abiotic degradation is not thought to contribute significantly to the dissipation of nonylphenolic substances. Laboratory studies have shown that under aseptic conditions nonylphenol is very stable (Sundaram and Szeto 1981; Corti et al. 1995). Therefore, the effects due to physicochemical transformations such as hydrolysis and photolysis are thought to be negligible (Trocmé et al. 1988; European Commission 1999). Volatilization from either water or soil is also considered an insignificant fate process for nonylphenolic substances (European Commission 1999). Nonetheless, these processes may occur to a minor degree, and therefore are discussed below.

Water Most of the nonylphenol and nonylphenol ethoxylates entering the environment are initially released to surface waters. Parent nonylphenol polyethoxylates do not appear to be persistent in natural waters, while some breakdown products (e.g., NP, NP1EO, and NP2EO) are moderately persistent (Maguire 1999). The mean time to 50% dissipation (DT50

### ) of 4-NP from the water column was estimated in a mesocosm study to be 0.74 days, and mean time to 95% dissipation (DT 95

### ) was 13.8 days (Heinis et al. 1999). In another study where nonylphenol was aerially applied to a forested plot containing a stream, nonylphenol residues were found to persist in the stream water for no more than five days, and a half-life of 2.5 hours was estimated (Sundaram et al. 1980). While in the aquatic environment, potential abiotic fate processes for nonylphenolic compounds include photolysis, volatilization and adsorption to suspended solids.

Photolysis, the decomposition of chemicals by the action of radiant energy, is one process by which nonylphenol and its ethoxylates may be degraded. This process is most effective in the surface layers of the water column where compounds are exposed to sunlight at higher intensities. The first order rate constant for sunlight photolysis of nonylphenol in fresh water has been estimated to range from 0.05 to 0.09 m 2 

### kW -1 

### h -1 

### (Ahel et al. 1994d). In other words, if exposed to continuous noon, summer sunlight, NP in the surface layer of natural waters would have a half-life of 10-15 hours (Ahel et al. 1994d). In deeper water layers this half-life would be considerably longer (e.g., at a depth of 20-25 cm the photolysis rate would be 1.5 times slower than at the surface). The photochemical oxidation of nonylphenol ethoxylates is significantly slower than that of NP (Ahel et al. 1994d). Often the photolytic process is aided by the presence of metals or metallic oxides that can act as photocatalysts. Degradation of alkylphenol ethoxylates is known to be induced by the catalysts Fe (III) and titania (Ti O 2

### ) in the presence of

### 15

sunlight. The degradation process involves a hydroxyl radical attack on the ethoxylate chain, and, at a much slower rate, on the benzene ring (Pelizzetti et al. 1989; Brand et al. 1998). Further degradation involves oxidation to lower molecular weight products such as acidic compounds, polyethylene glycols, ethylene glycols, and ultimately to carbon dioxide (CO 2

### ).

Variable results have been found for the effect of temperature on the rate of nonylphenol degradation in water. Ahel et al. (1994b) found that concentrations of nonylphenolic compounds in fresh water tend to decrease with higher temperatures. Schöberl and Mann (1976) found little effect of temperature on degradation of NP9EO in fresh water, with 33-36% decomposition occurring at 20-23oC, and 37% at 3-4oC. In contrast, they found a substantial temperature effect in seawater, with 95% degradation occurring at 20-23oC, and only 15% at 3-4oC. Manzano et al. (1999) observed significant increases in the rates of both primary biodegradation and mineralization with increased temperature. They also observed shorter acclimation periods at higher temperatures. Tanghe et al. (1998) found that degradation of nonylphenol in lab-scale activated sludge units was almost complete at 28oC, but slowed down to 13-86% elimination capacity at temperatures of 10-15oC, a finding that is significant considering Canadian conditions. Marcomini et al. (2000) observed considerable seasonality in the concentrations of nonylphenol ethoxylates present in the lagoon of Venice, Italy, and attributed this to the dependence of their biodegradation on temperature.

One potential mechanism by which organic compounds may leave surface waters is through volatilization. In a recent study, nonylphenol was measured in the atmosphere over the Hudson River Estuary at concentrations ranging from 0.1 to 68.6 ng·m -3

### (Dachs et al. 1999). The authors concluded from these high concentrations that a considerable amount of water-to-air volatilization must have occurred. These results are surprising because most of the previous literature has claimed that volatilization of nonylphenol is not likely to occur under most conditions. The Henry’s Law Constant for nonylphenol, which describes the likelihood for a chemical to move from surface water to the air, is 11.02 Pa m 3 

### ·mol -1 

### (Reed 1978; European Commission 1999). This low Henry’s Law Constant indicates that volatilization is not likely to be a significant mechanism for removal of nonylphenol from water systems and that it is unlikely to be transported very far in the atmosphere (Maguire 1999; European Commission 1999).

Movement of dissolved compounds from the aqueous phase of the water column to the particulate phase may occur through adsorption. Studies have shown that nonylphenol in water is readily adsorbed onto suspended solids (Kvestak et al. 1994; Sekela et al. 1999). Using field data from the Fraser River in British Columbia, the tendency for nonylphenol to partition from the aqueous phase to the suspended solid phase has been evaluated based on organic carbon (OC) content. The sorption partition coefficient (log KOC

### ) for nonylphenol was estimated to be in the range of 4.7 to 5.6 (Sekela et al. 1999). Partitioning of nonylphenol polyethoxylates between the dissolved and particulate phases can vary slightly among various oligomers; NP6EO and NP7EO have shown higher relative tendencies to partition into the particulate phase than any other NPEs (Kvestak et al. 1994).

Sediment NP and NPEs adsorbed to suspended particles may settle out of the water column into the sediments. Nonylphenolic compounds dissolved in the water may also directly adsorb to particles on the sediment surface. John et al. (2000) suggested that the adsorption of nonylphenol polyethoxylates to sediment involves a combination of two main processes: hydrophobic interactions with organic matter in the sediment, and hydrophilic interactions with the mineral components. These two processes have opposing effects in terms of the ethoxylate chain length. The hydrophobic interactions increase with decreasing ethoxylate chain length since the lower ethoxamers have higher octanol-water partition coefficients (see Table 2) and are more hydrophobic. The hydrophilic interaction involves hydrogen-binding due to the hydration of metal oxides in the sediment to the ether oxygens of the ethoxylate chain. Therefore, the amount of adsorption due to hydrophilic interactions increases with increasing ethoxylate chain length. Partitioning of nonylphenol ethoxylates between the water and sediment phases, and

### 16

achievement of adsorption equilibrium occurred rapidly under artificial conditions (John et al. 2000). In a littoral enclosure study, some of the nonylphenol applied through subsurface injection into the water column was found to adsorb to the sediment within two days of the initial application, as well as adsorbing to the plastic walls of the mesocosm enclosures (Heinis et al. 1999). Nonylphenol also appears to be persistent in sediments; in the same freshwater mesocosm study, the DT50

### of nonylphenol from sediments was estimated at 66.0 days, and the DT 95

### was estimated at 401 days (Heinis et al. 1999). This indicates that there is the potential for carry-over from one year to the next, such that a site which receives regular inputs will have increasing sediment concentrations of NP over time. More studies are needed though to confirm the fate of nonylphenol in freshwater sediments under natural conditions.

Persistence has also been observed in marine sediments. NPEs (with 1 to 14 ethoxylate units) applied to estuarine intertidal plots as part of a herbicide formulation showed decreasing concentrations in the sediments during the first 14 days post-treatment, but then stabilized and remained constant even after 119 days (Paveglio et al. 1996). The authors suggest that this stabilization may have been due to partitioning of the NPEs to sediment with reduced availability for biodegradation; also, lower temperatures at the later dates may have resulted in reduced microbial degradation.

Soil The abiotic fate processes affecting nonylphenol and its ethoxylates in soils, in decreasing order of importance, include particle adsorption, infiltration to groundwater, and volatilization.

The major route for nonylphenolic compounds to enter terrestrial ecosystems is through the application of sewage sludge or pulp and paper mill sludge to agricultural soils, or through disposal of sludge in landfills. There appear to be conflicting reports on the persistence of nonylphenolic compounds in soil. Some researchers report that under aerobic conditions, nonylphenolic compounds are not persistent and degradation occurs readily (W ild and Jones 1992; Maguire 1999). For example, concentrations of nonylphenol introduced into agricultural soils through sludge application decreased by 90% in ten days (Kirchmann et al. 1991) and by 92% in three months (Diercxsens and Tarradellas 1987). In contrast, there is also evidence of persistence with complete degradation taking considerably longer. In a Swiss study where digested sewage sludge was applied to an experimental field plot, soil concentrations of NP, NP1EO and NP2EO decreased rapidly during the first 10 days after application, with half-lives of 7 to 8 days for each compound (Marcomini et al. 1988). This was followed by a transition period of about 90 days, during which the compounds disappeared at a much slower rate; the half-lives for NP, NP1EO and NP2EO were 90, 150, and 110 days, respectively. The authors continued to measure the concentrations of the nonylphenolic compounds until 330 days after the sewage application, and for the rest of that period, the concentrations remained relatively constant, with estimated half-lives of >360 days. Thus, a small fraction of the initial NP, NP1EO and NP2EO was highly persistent, possibly because its strong sorption to the soil made it unavailable for biodegradation. It is unknown whether this constant residual concentration of NP simply represents the threshold for biological transformation, and therefore will remain the same, or whether the soil is at risk of increasing accumulation of NP with further sludge applications (Marcomini et al. 1988, 1989b). Topp and Starratt (2000) also found that NP underwent a biphasic mineralization in agricultural soil. During a rapid initial phase, that lasted 10 days, about 30% of the nonylphenol (radioactively-labelled at one of the phenolic ring carbons) was converted to carbon dioxide; this was followed by a much slower second phase. Under anaerobic conditions, such as in landfills, it appears that nonylphenolic compounds are persistent in soil (Maguire 1999). For example, in a survey of several German sludge-only landfills, concentrations of NP and NP1EO of 4 and 19 mg·kg -1 

### dry weight, respectively, were detected in samples of sludge that had been landfilled 30 years previously (Marcomini et al. 1989b).

Based on physical/chemical properties, W ild and Jones (1992) speculated that nonylphenol in sludge applied to agricultural soils would likely have high adsorption to soil particles. Various NPEs have also exhibited high adsorption on soil (Beigel et al. 1998). The relative degree of

### 17

adsorption among NPE oligomers appears to increase with decreasing number of ethoxylate units, to a maximum soil affinity at about NP9EO, after which the tendency for adsorption decreases with decreasing number of ethoxylate units (Beigel et al. 1998). It is suggested that adsorption increases due to increasing hydrophobicity, but with the shorter ethoxylate chains, that have a lower critical micelle concentration, there may be a preference for surfactant-surfactant interactions and micelle formation, rather than surfactant-soil surface interactions (Beigel et al. 1998).

Although the high adsorption of nonylphenolic compounds to soil particles suggests that there would be a low probability of leaching (W ild and Jones 1992), numerous studies have shown evidence of infiltration of nonylphenolic compounds into groundwater from municipal landfills (Ahel 1991), from contaminated rivers and wastewater canals (Zoller et al. 1990; Ahel 1991; Ahel et al. 1996), and due to sewage disposal through rapid infiltration (Barber et al. 1988; Rudel et al. 1998). Due to their higher water solubility and mobility, nonylphenol ethoxycarboxylates (i.e., NP1EC and NP2EC) are thought to pose a greater risk as groundwater contaminants than other nonylphenolic compounds (Ahel et al. 1996). Although NP, NP1EO and NP2EO are more effectively retained in the soil, significant concentrations of these substances have been measured in groundwater at distances greater than 100 metres from the original source (Ahel 1991; Ahel et al. 1996).

As in water, nonylphenol is not likely to volatilize from soils in significant quantities either. For example, of the initial 1000 mg·kg -1 

### nonylphenol that was spiked into a compost-sandstone mixture, only 0.22% was lost through volatilization over a period of 40 days (Trocmé et al. 1988).

There is currently no information available in the literature on photolysis of nonylphenolic substances on soils.

Biodegradation The most important mechanism determining the fate of nonylphenolic compounds in water, sediment, or soil, is likely biological degradation and transformation (Ahel et al. 1994b). NPEs undergo a two-stage biodegradation process (refer to Figure 2). Primary biodegradationis the process by which the molecules of NPE are oxidized or altered sufficiently to remove their characteristic detergent properties (i.e., surfactancy, formation of foam). Often this results in the formation of intermediate biodegradation products such as mono- (NP1EO) and di-ethoxylates (NP2EO), nonylphenoxy acetic acid (NP1EC), nonylphenoxy ethoxy acetic acid (NP2EC), and NP. Ultimate biodegradation(or total biodegradation) refers to the complete conversion of the molecules to CO 2 

### , H 2

### O, and inorganic salts (Swisher 1987). In sewage treatment processes, efficient elimination of nonylphenol ethoxylates through primary biodegradation may occur, however, ultimate biodegradation is limited due to the formation of products that are biorefractory, i.e., that resist further biodegradation (Ahel et al. 1994a; Maguire 1999).

Primary Biodegradation Nonylphenol ethoxylates undergo a relatively slow degradation process compared with some other types of surfactants. Steric effects due to the aryl group prevent the occurrence of central ether fission, and a branched alkyl chain reduces the likelihood of ω -/ß-oxidation 1 

### (White 1993). The major route of biodegradation is most likely to be ethoxylate shortening through the stepwise removal of ethoxy groups (White 1993; Jones and Westmoreland 1998; Maguire 1999). Therefore, the intermediate degradation products of primary biodegradation are lower NPE ethoxamers, or NPECs (depending on environmental conditions), and nonylphenol. Under anaerobic conditions (as in the anaerobic digestion of sludge in sewage treatment plants) the

1 

### ω -/ß-oxidation is a two-step process which results in the shortening of an alkyl chain. Through

ω -oxidation, the terminal unsubstituted carbon of the alkyl chain is oxidized to form a carboxylic acid (White 1993). W ith ß-oxidation, the carboxylic acid is further oxidized at the ß-carbon atom, resulting in the removal of a 2-carbon fragment (Manahan 1994).

### 18

intermediate breakdown products include NP1EO and NP2EO; under aerobic conditions (that would be found in most natural river waters and wastewater effluents) major degradation products include NP1EC and NP2EC (Ahel et al. 1994b; Maki et al. 1996). Nonylphenol is a degradation product under both aerobic and anaerobic conditions, although formation of NP appears to be more likely under anaerobic conditions (Giger et al. 1984). Degradation of the hydrophobic moiety (i.e., the alkyl chain and phenol group) of NPEs has also been observed, but, it occurs at only half the degradation rate of the hydrophilic polyethoxylate chain (Jones and Westmoreland 1998).

Biodegradabilities of both NP1.5EO and NP9EO have been assessed using the CO 2

### Evolution Modified Sturm Test, as described in the Organisation for Economic Co-operation and Development (OECD) guideline 301B (Gledhill, 1999). According to the results from these tests, although the substances are both inherently biodegradable (with >50% mineralization occurring over the 35-day test period), neither meets the requirements for classification as “readily biodegradable” (i.e., ≥60% CO 2

### produced within 28 days, with this value being achieved within a 10-day window). Among the higher molecular weight NPEs (i.e., with 8 to 30 EO groups) individual ethoxamers do not appear to differ significantly in their biodegradabilities (Rudling andSolyom 1974). Sulphated nonylphenol ethoxylates are less readily biodegraded than nonsulphated NPEs (Pitter and Fuka 1979).

Studies examining biodegradation efficiencies in wastewater treatment plants (WWTPs) using activated sludge have reported greater than 90% primary degradation of NPEs (Rudling and Solyom 1974; Lee and Peart 1998). Lee and Peart (1998) examined biodegradation in a southern Ontario municipal WWTP using secondary treatment with activated sludge. Through aerobic biodegradation, concentrations of NPEs and NP entering the WWTP in raw sewage were reduced by more than 90 and 82%, respectively, in the final effluent. The degradation of NPEs resulted in the formation of carboxylates, as evidenced by 418 and 468% increases in concentrations of NP1EC and NP2EC, respectively, from the raw influent to the effluent. The median total alkylphenolic concentrations were reduced by 53%. The authors speculated that much of the “eliminated” nonylphenolics may have been bound to the sludge, that was used for landfill applications, thus making the total environmental loading even higher. It has been estimated that at least 63% of the total molar mass of all nonylphenolic compounds entering WWTPs is released to the environment; 25% as NP, 19% as NPECs, 11% as lipophilic nonylphenol ethoxylates (NP1EO + NP2EO), and 8% as higher NPEs (Ahel et al. 1994a). The majority of these compounds are released through the secondary effluents, except for NP which is primarily disposed via the digested sewage sludge (Ahel et al. 1994a; Brunner et al. 1988).

Degradation of NPEs has also been observed during the composting of sludges from wool scour effluents (Jones and Westmoreland 1998). Over a 14 week period, NPE concentrations were observed to decrease by more than 96%.

Laboratory studies have been conducted to simulate biodegradation processes that would occur in natural waters. For example, Potter et al. (1999) examined the static die-away of NP18EO added to estuarine water collected from Tampa Bay, Florida. They observed a lag-period of 0 to 12 days, followed by a rapid primary degradation, with primary degradation complete within 4 to 24 days. The first major degradation product to form was NP2EO, which then decayed to NP2EC, a more persistent degradation product. No formation of NP was observed, perhaps because the test conditions were aerobic. Ahel et al. (1994c) also conducted die-away tests by adding NP1EO and NP2EO to river water containing pre-exposed bacterial cultures. They found that biotransformation rates were 1.5 times faster when the water was stirred, than when it remained static. Biotransformation rates were also faster at higher temperatures, with 50-60%

### 19



## C 9 

## H 19

## O -

# ( CH 2

## - CH 2

## - O

# )
n 

## - CH 2

## - CH 2

## - OH Nonylphenol polyethoxylate (NPnEO; n = 0 to 99) aerobic anaerobic (e.g., water, effluent) (e.g., sludge, soil) Gram negative bacteria?



## C 9 

## H 19

## O - 

# ( CH 2

## - CH 2

## - O

# ) 2

## - H Nonylphenol diethoxylate (NP2EO)

+

C 9 

## H 19

## O - CH 2

## - CH 2

## - OH Nonylphenol monoethoxylate (NP1EO)

C 9 

## H 19

## O - CH 2

## - CH 2

## - O- CH 2

## - COOH Nonylphenoxy ethoxy acetic acid (NP2EC)

+

C 9 

## H 19

## O - CH 2

## - COOH Nonylphenoxy acetic acid (NP1EC)

Yeast?



## C 9 

## H 19

## OH Nonylphenol Stage 1 PRIMARY (NP)

Carbon dioxide, water, and Stage 2 CO 2

## + H 2

## O + inorganic salts ULTIMATE inorganic salts

Figure 2. Pathways for biodegradation of nonylphenol polyethoxylates.

### 20

transformation after 12 days at 20oC compared with 10-20% at 4oC. A control sterile sample showed no evidence of analyte transformation throughout the entire study (Ahel et al. 1994c).

In a few studies, specific microorganisms capable of biodegrading nonylphenolic compounds have been identified. A strain of the bacteria Pseudomonas sp. isolated from activated sludge of a municipal WWTP was observed to degrade the EO chain of nonylphenol polyethoxylates to form NP2EO and NP2EC (Maki et al. 1994). Frassinetti et al. (1996) also isolated two strains of Pseudomonas

sp. and a strain of Xanthomonas sp. capable of degrading NP9EO, however none of these were able to assimilate NP. Biotransformation of nonylphenol has been observed with a strain of the yeast Candida maltosa, isolated from the sludge of a plant treating textile industry wastewater (Corti et al. 1995). The major degradation product was 4-acetylphenol, and the authors suggest ß-oxidation, followed by decarboxylation, as a possible biodegradation process. It should be noted, however, that the study by Corti et al. (1995) used straight-chain NP; with branched-chain isomers of NP, it is likely that ß-oxidation would occur much less readily (see White 1993).

Microorganisms are more efficient at the biodegradation of nonylphenolic compounds if they have been previously exposed and given time for adaptation; however, pre-exposure is not necessary for biodegradation to occur. For example, biotransformation of NP1EO and NP2EO in synthetic sewage using aerobic bacterial cultures was examined by Ahel et al. (1994c). The bacterial cultures were isolated from three different locations: the wastewater of a detergent manufacturing plant, from the water of a polluted river, and from a pristine forest soil in Croatia. Biotransformation occurred quite rapidly with both the river water and wastewater cultures, while biotransformation with the forest soil culture was less efficient.

Ultimate Biodegradation Nonylphenol, NP1EO, NP2EO, NP1EC, and NP2EC are very refractory compounds that are much less readily degraded than their parent compounds, the nonylphenol polyethoxylates. Partial biodegradation and biotransformation of these compounds will occur, but as evidenced below, there are fewer reports of complete degradation (i.e., to CO 2 

### , H 2

### O, and inorganic salts) than there are of primary degradation of NPEs.

Ekelund et al. (1993) examined the biodegradation of 4-nonylphenol in seawater, both with and without sediment present, through the use of radioactive labelling. Initial degradation in the seawater alone was very slow, with about 0.06% of the NP degraded per day, but after four weeks this rate had increased to 1% per day. The authors suggested that this increase was due to adaptation of the microorganisms to the substrate. In the presence of sediment, the degradation rate was much quicker, at 1.2% per day from the onset, and remained relatively constant. The faster rate with sediment can be accounted for by the larger number of microorganisms that would be available, thus increasing the probability of bacteria able to degrade the NP being present. This rate did not increase with adaptation (as it did in the water only treatment) because adsorption to sediment particles likely reduced NP bioavailability. In the presence of sediment, but under anoxic conditions, the biodegradation rate was reduced by 50% (Ekelund et al. 1993).

Biodegradation of NP, NP1EO, and NP2EO in soil-sludge mixtures and sludge slurries were examined by Madsen et al. (1998). Three different sludges collected from Danish WWTPs, either activated or anaerobically digested, were tested. After 28 days, no change in concentration was observed for NP, NP1EO or NP2EO; however, the analysis did not examine longer-chained nonylphenol ethoxylates, nor nonylphenoxy carboxylates. As such, it is possible that somedegradation of NP and NP1-2EO did occur, but was masked by the transformation of longerchained NPEs to short-chain NPEs and NP (Madsen et al. 1998). Two of the sludges were also examined for biodegradation by preparing slurries of the sludges and incubating them in the dark in closed beakers connected to an oxygen supply, and equipped with stirrers, at 15°C for 14 days. The pre-conditioned sludge bacteria in the slurry, possibly aided by desorption of sludge-bound contaminants through stirring, resulted in decreases of 70-80% of the initial levels of NP, NP1EO

### 21

and NP2EO for both sludges. How much of the decrease in NP and NPEs was due to mineralization is unknown since there also may have been formation of other degradation products such as NPECs.

Ultimate biodegradation of NP9EO has been observed in soil under aerobic conditions. During a nine week experimental period, 57% of the initially applied NP9EO was mineralized, including breakdown of the benzene ring, as confirmed through HPLC with UV detection (Hughes et al. 1996). Mineralization of nonylphenol has been observed in a variety of soil types, including agricultural soils of various textures, non-cultivated temperate soils, and soils from the Canadian tundra (Topp and Starratt 2000). The fact that biodegradation occurred in the arctic soils indicated that the microorganisms already possessed the biochemistry for nonylphenol metabolism without prior exposure and adaptation. The authors suggested that exposure of microorganisms to phenolic substances released by the decomposition of plant matter may have enabled them to degrade nonylphenol (Topp and Starratt 2000).

Mineralization of nonylphenol by soil bacteria can be inhibited by either an overabundance or a lack of water content (Topp and Starratt 2000). Temperature has also been observed to affect rates of nonylphenol mineralization in soil. Although biodegradation will occur over the temperature range of 4 to 30°C, at lower temperatures the rate decreases, and there is a greaterlag period (Topp and Starratt 2000). Overall, the authors conclude that nonylphenol in landapplied sewage sludge would readily undergo biodegradation under the soil conditions of a normal Canadian growing season.

W ild and Jones (1992) suggest a half-life of <10 days for nonylphenol in sewage sludge. Data from Ekelund et al. (1993) indicate a half-life for NP in seawater of 56 days, while Sundaram and Szeto (1981) measured a freshwater half-life of about 16 days. Marcomini et al. (1988), studying degradation of NP in soil, measured a half-life of 8 days during the first 10 days after application, followed by a 90 day period in which the degradation rate slowed to give a half-life of 90 days. An analysis of data from various sources indicated that half-lives measured from the end of the lag phase to the beginning of the plateau for the NP degradation curves results in half-lives ranging from 6 to 26 days for water, soil, and water-sediment slurries (Naylor 2000).

Uptake/Elimination Pathways, and Metabolism The degree to which a substance bioaccumulates in an organism is dependent on the relative rates of uptake and elimination. These processes can also have a bearing on the type, and magnitude, of toxic effect that a substance will have. Metabolism, or biotransformation, of a substance within an organism will produce metabolites that may be more or less toxic than the parent substance. Therefore, an understanding of the processes affecting a substance within an exposed organism will permit a greater understanding of the toxicological risks of that substance to the organism. A discussion of the physiological and metabolical pathways for NP and NPEs in fish, invertebrates, plants, and wildlife is provided below.

Numerous researchers have examined uptake and elimination pathways for nonylphenolic compounds in fish. Uptake occurs primarily through the gills with transport to the various tissues/organs via the blood (Granmo and Kollberg 1976; Arukwe et al. 2000b). For example, Granmo and Kollberg (1976) found that after only five minutes of exposure to NP10EO, high levels of the contaminant were observed in the gill tissue and blood of cod ( Gadus morhua). Smaller amounts of NP10EO were also taken up through intestinal resorption. Uptake of nonylphenolic compounds is fairly rapid, with tissue concentrations reaching a steady state during the first 3 to 10 days of exposure (Granmo and Kollberg 1976; Ward and Boeri 1991c). W ithin a fish, nonylphenol can concentrate in the viscera at levels 1.6 to 7.1 times higher than in the carcass (Ward and Boeri 1991c). Once a stable equilibrium has been reached, the highest concentrations of nonylphenolics in fish can be found in the gall bladder, bile, pyloric caecae, intestine, and faeces (Granmo and Kollberg 1976; Lewis and Lech 1996; Coldham et al. 1998; Arukwe et al. 2000b). Intermediate levels of nonylphenolic compounds are found in the liver, kidney and fat tissue, and comparatively low concentrations can be found in the gills, heart,

### 22

gonad, muscle, spleen, skin, blood, eye and brain (Granmo and Kollberg 1976; Lewis and Lech 1996; Coldham et al. 1998; Arukwe et al. 2000b).

When fish exposed to nonylphenolic compounds are transferred to clean water, elimination of the contaminants will occur for all tissues, although there appears to be a longer time lag for the gall bladder, where concentrations of nonylphenolic substances have been observed to remain stable for extended periods (Granmo and Kollberg 1976; Coldham et al. 1998). For most of the tissues, Coldham et al. (1998) found that the elimination process was biphasic and fit a two compartment model. The highest rate of elimination occurs in the blood and gills (Granmo and Kollberg 1976), and the lowest rates of elimination occur in the liver, muscle, and skin tissues, as evidenced by the half-lives (Coldham et al. 1998). In rainbow trout ( Oncorhynchus mykiss), the estimated halflife of NP in fat was 20 hours, in muscle it was 19 hours, and in the liver the half-life was six hours (Lewis and Lech 1996). Arukwe et al. (2000b) found that the half-lives for nonylphenol residues in the carcass, viscera and muscle of Atlantic salmon ( Salmo salar ) were all between 24 and 48 hours. For all tissues, rates of elimination are slower than rates of uptake (Coldham et al. 1998). The whole-body excretion half-life for juvenile Atlantic salmon exposed to technical grade p-nonylphenol was estimated at four days (McLeese et al. 1981). Some excretion is thought to occur via the kidney, with high levels of nonylphenolics being observed in the urine (Granmo and Kollberg 1976). Thibaut et al. (1999) gave rainbow trout a single dose of radioactively labelled nonylphenol and observed urinary elimination of 1.1% of the radioactivity after 24 hours, and 3.0% elimination through urine after 48 hours. In contrast, Coldham et al. (1998) suggest that the main pathway for elimination of nonylphenol follows a biliary/faecal route of excretion. Evidence for this pathway comes from the high equilibrium concentrations of nonylphenolics that have been observed in tissues associated with the biliary/faecal system (Granmo and Kollberg 1976; Lewisand Lech 1996; Coldham et al. 1998; Arukwe et al. 2000b). Ekelund et al. (1990) exposed threespined sticklebacks ( Gasterosteus aculeatus) to 14

### C-labelled NP in a flow-through system with seawater for 16 days, followed by an elimination period of 32 days. They found that the sticklebacks reached steady states of bioaccumulation after about ten days and elimination of NP was quite rapid and almost complete after a few days.

W ithin the fish tissue, nonylphenol may be biotransformed into various metabolic products. Researchers have suggested that metabolic degradation of nonylphenol in fish occurs through oxidation and glucuronic acid formation in bile (Lewis and Lech 1996; Meldahl et al. 1996; Arukwe et al. 2000b). Through in vitro tests with rainbow trout hepatic microsomes, Meldahl et al. (1996) also observed metabolism of nonylphenol by hepatic cytochrome P450. Some possible metabolites of nonylphenol have been tentatively identified in rainbow trout as 9 -(4-hydroxylphenyl)-nonanoic acid, 4-hydroxybenzoic acid, 3-(4-hydroxyphenyl) propionic acid, and 3-(4-hydroxylphenyl)-2-propenoic acid (Thibaut et al. 1999). The process of ω -oxidation followed by ß-oxidation has been proposed as the most likely biotransformation pathway (Thibaut et al. 1999).

There are few studies that have investigated uptake and elimination rates in invertebrates. Ekelund et al. (1990) exposed common blue mussels ( Mytilus edulis) and common shrimp ( Crangon crangon) to 14

### C-labelled NP in a flow-through system with seawater for 16 days, followed by an elimination period of 32 days. The shrimp reached steady states of bioaccumulation after approximately ten days, however, with the mussels, steady state was not reached by the end of the 16 day exposure period. Elimination of NP from the shrimp took about four days, but was much slower in the mussels which still had significant concentrations present after 30 days in clean water. McLeese et al. (1980b) also found that common blue mussels have low uptake rates and have high excretion rates of nonylphenol.

There has been considerably greater investigation into the uptake/elimination and metabolism of nonylphenol in plants than in invertebrates. Much of the literature has focussed on agricultural plant species exposed to nonylphenolic compounds through contaminated soil. Based on the physical and chemical properties of nonylphenol, it has been hypothesized that high retention of nonylphenol at the root surface of plants would be likely, with only moderate uptake and

### 23

translocation occurring within the plants (W ild and Jones 1992). Kirchmann and Tengsved (1991) examined the grains of wheat plants grown in soil contaminated with nonylphenol due to sewage sludge application. They found no significant increase in nonylphenol concentrations in the grains, though were uncertain whether this indicated a lack of root uptake and transport to the grains, or rapid decomposition of the nonylphenol in the soil. Harms (1992) also did not observe any evidence of nonylphenol in the shoots of tomato plants whose roots were exposed to nonylphenol. In contrast, translocation of nonylphenol or its metabolites from roots to shoots within wheat, tomato and garden orache plants has been observed by other researchers (Bokern et al. 1998).

Bokern et al. (1996) examined the metabolism of radioactively labelled 4- n-nonylphenol in wheat cell suspension cultures. Metabolic products observed were 4-(hydroxy)- and 4-(dihydroxy)nonylphenols that were glucosylated at the phenolic OH-group and further glucosylated, glucuronidated, and acylated with acetic acid or malonic acid. Metabolism of nonylphenol was also examined in cell suspension cultures of Daucus carota (carrot) and Atriplex hortensis (garden orache) (Bokern and Harms 1997). Radiolabelled nonylphenol was taken up quickly in both cultures with 93-97% of the radioactivity disappearing from the culture medium after six hours, and concentrations reaching a steady state after 48 hours. Metabolism of nonylphenol within the cells occurred quickly. In the carrot cell cultures, 80% of the parent compound had been metabolized within 24 hours, and in the A. hortensis cells about 50% of the nonylphenol was metabolized after 30 hours (Bokern and Harms 1997). The authors also looked at the uptake and metabolism of nonylphenol in 14 different cell cultures (predominantly agricultural crop species). All of the species took up NP and metabolized it. Metabolites were ß -glucosides of differently hydroxylated nonylphenol, some of which were further acylated with, for example, glucuronic acid or malonic acid. Cell cultures of plants from similar taxonomic groups tended to produce similar metabolites (Bokern and Harms 1997). Distribution of radioactivity among the various cell wall fractions appeared to be species specific. In the majority of species, high concentrations were associated with the lignin, though in some species it was associated with the protein or hemicellulose. Although most plant species seem to readily take up nonylphenol (at least when exposed in vitro ) and metabolize it, no mineralization of nonylphenol to CO 2

### appears to occur in plants (Bokern et al. 1998).

Considerably less information exists on the uptake of nonylphenolic compounds by aquatic plants. In a littoral mesocosm study, nonylphenol applied to the water column was found to partition to macrophytes, with maximum concentrations of 4-NP observed in macrophytes within 1 to 2 days of application. The authors estimated the mean DT50

### and DT 95

### for nonylphenol persistence in/on macrophytes at ten and 189 days, respectively, indicating persistence of nonylphenol in macrophytes (Heinis et al. 1999).

There is a paucity of data on uptake of nonylphenolic compounds by terrestrial animals. Based on the physical and chemical properties of nonylphenol, W ild and Jones (1992) have speculated that there is a moderate chance of transfer of nonylphenol to animal tissues through foliage ingestion, with less chance of transfer occurring through soil ingestion.

Bioaccumulation Bioaccumulation is defined as the net accumulation of a substance by an organism as a result of uptake from all routes of exposure (e.g., water intake, food ingestion, dermal absorption, etc.). Bioconcentration also refers to net accumulation of a substance, but with respect to aquatic organisms, and involving only direct uptake from an aqueous solution. Thus, bioaccumulation includes the process of bioconcentration, but not vice versa. Nonylphenolic compounds have been evaluated for their potential to bioaccumulate or bioconcentrate both through direct investigations involving exposure of organisms to these substances under natural or artificial conditions, and through studies of their physical/chemical properties.

Numerous studies have examined bioaccumulation potential through examination of nonylphenol concentrations in aquatic biota relative to water concentrations, and nonylphenolic compounds

### 24

appear to bioaccumulate and bioconcentrate to a low or moderate degree. Several researchers have observed bioaccumulation of NP and NPEs in common blue mussels when placed in cages near industrial wastewater outflows or when exposed to NP (or wastewater containing NP and NPEs) under laboratory conditions (McLeese et al. 1980b; Wahlberg et al. 1990; Granmo et al. 1991a,b). Granmo et al. (1991b) found that the amount of bioaccumulation of NP, NP1EO, NP2EO, and NP3EO depended on the length of the ethoxylate chain with greater bioaccumulation of compounds with shorter ethoxylate chains (and therefore greater hydrophobicity). Ethoxylate chain length appears to affect bioconcentration in aquatic plants and algae as well. In their field study of the Glatt River, Switzerland, Ahel et al. (1993) found that although nonylphenol was present in the river water at a lower concentration (3.9 μg·L-1 

### ) than NP1EO or NP2EO (24 and 9.4μg·L-1 

### , respectively), it occurred at higher concentrations (2.5 to 38.0 mg·kg -1 

### dry weight) in the resident algae and macrophytes than the other two compounds (0.6 to 4.7 mg·kg -1 

### dry weight).

For the purposes of comparing bioaccumulation potentials from different studies, or across different substances and different species, bioaccumulation factors (BAFs) and bioconcentration factors (BCFs) are calculated. Generally, BAFs are calculated in field studies and BCFs are determined in laboratory studies. Because BCFs do not include dietary uptake, they tend to underestimate the bioaccumulation potential of a substance. Table 3 lists BAF and BCF values that have been determined for NP and NPEs on a variety of aquatic species. In general, BAF and BCF values are higher for NP than for NPEs (i.e., BAFs and BCFs decrease with increasing ethoxylation). Considerable variation in BAF and BCF values are observed within taxa, and even within the same species, which makes it difficult to discern any significant differences in bioaccumulation between algae, plants, invertebrates, fish, and waterfowl. Under the Toxic Substances Management Policy (Government of Canada 1995) and under Canada’s Persistence and Bioaccumulation Regulations (Government of Canada 2000b), a substance is considered bioaccumulative if its BAF (or BCF) is equal to or greater than 5000. Ahel et al. (1993) reported BAFs greater than 5000 in the algae Cladophora glomerata . Ekelund et al. (1990) calculated BCFs greater than 5000 in a crustacean, a mollusc, and a fish when based on lipid concentrations, but not when based on total wet weight. All other reported BAFs and BCFs fall below the critical value of 5000 (see Table 3), suggesting that nonylphenolic substances are not highly bioaccumulative.

Based on their physical and chemical properties, little bioaccumulation and bioconcentration of nonylphenolic compounds are likely to occur. The potential for a contaminant to bioaccumulate is largely affected by its hydrophobicity, and therefore is often evaluated based on its octanol-water partition coefficient (Kow

### ). Estimates of the log Kow

### for nonylphenol of 4.7 (Barber et al. 1988), 4.48 (Ahel and Giger 1993b), 4.2 (McLeese et al. 1981), and 4.1 have been made (W ild and Jones 1992). Partition coefficients between n-octanol and water have also been determined for several of the smaller NPEs. The log Kow

### s for NP1EO, NP2EO and NP3EO are estimated at 4.17, 4.21, and 4.20, respectively (Ahel and Giger 1993b). According to the Toxic Substances Management Policy (Government of Canada 1995) and Canada’s Persistence and Bioaccumulation Regulations (Government of Canada 2000b), in the absence of BAF or BCF data, a substance may be considered as bioaccumulative if it has a log Kow

### value equal to or greater than 5. As estimates of the log Kow

### for nonylphenolic substances all fall below 5, this reinforces the conclusion based on BAF values that these substances are not bioaccumulative. Partition coefficients (Khw

### ) have also been determined for the partitioning of nonylphenolics between n-hexane and water. Log K hw

### s for NP, NP1EO, and NP2EO were estimated at 3.7, 3.4, and 3.3, respectively (Ahel and Giger 1993b). The compounds generally had a lower solubility in hexane than in octanol. W ith both organic solvents, the difference of a single ethoxylate group had a negligible influence, with NP1EO and NP2EO having almost equal lipophilicity (Ahel and Giger 1993b). Although neither log Kow 

### nor log Khw

### values have not been calculated for the higher NPEs, they are known to be less hydrophobic than nonylphenol and the lower molecular weight NPEs and would therefore have lower potential for bioaccumulation as well.

Substances that bioaccumulate or bioconcentrate may also have the potential to biomagnify. There is no evidence in the current literature to suggest that NP or NPEs biomagnify. For

### 25

example, Ahel et al. (1993) examined concentrations of NP, NP1EO and NP2EO in biota, at several trophic levels, of the Glatt River, Switzerland. For each of these substances, bioaccumulation factors in the algae Cladophora glomerata were significantly higher than the BAFs in three fish species ( Squalius cephalus

, Barbus barbus, and Oncorhynchus mykiss). Concentrations of nonylphenolic compounds detected in the tissues of a mallard duck (Anas boscas) were not significantly different from concentrations observed in fish tissues. Because the concentrations of NP and NPEs did not increase with increasing trophic level of organisms, the authors concluded that the compounds did not biomagnify.

### 26

Table 3. Bioconcentration and bioaccumulation factors for nonylphenol and nonylphenol ethoxylates in aquatic organisms.

Chemical Species Water type BCF BAF Tissue Reference

Plants/Algae NP Cladophora glomerata (algae) fresh NR 10000 * 6600-7700 dw Ahel et al. 1993

Fontinalis antipyretica (macrophyte) fresh NR 1100 dw Ahel et al. 1993

Potamogeton crispus (macrophyte) fresh NR 600 dw Ahel et al. 1993

NP1EO Cladophora glomerata

(algae) fresh NR 200 * 3500-5000 dw Ahel et al. 1993

Fontinalis antipyretica (macrophyte) fresh NR 40 dw Ahel et al. 1993

Potamogeton crispus (macrophyte) fresh NR 50 dw Ahel et al. 1993

NP2EO Cladophora glomerata

(algae) fresh NR 500 * 1000-1800 dw Ahel et al. 1993

Fontinalis antipyretica (macrophyte) fresh NR 60 dw Ahel et al. 1993

Potamogeton crispus (macrophyte) fresh NR 200 dw Ahel et al. 1993

Invertebrates NP Crangon crangon (common shrimp) marine 90,110 NR based on total ww Ekelund et al. 1990

7500,5500 NR lipidbased Ekelund et al. 1990

Mytilus edulis (common blue mussel) marine 1.4-7.9 (K 1 

### /K2

### = 10) NR NR McLeese et al. 1980b

NR 340 NR Granmo et al. 1991b 2740,4120 NR based on total ww Ekelund et al. 1990

169300, 216600 NR lipidbased Ekelund et al. 1990

NP1EO Mytilus edulis

(common blue mussel) marine NR 170 NR Granmo et al. 1991b

NP2EO Mytilus edulis

(common blue mussel) marine NR 100 NR Granmo et al. 1991b

NP3EO Mytilus edulis

(common blue mussel) marine NR 60 NR Granmo et al. 1991b

### 27

Table 3 (continued). Bioconcentration and bioaccumulation factors for nonylphenol and nonylphenol ethoxylates in aquatic organisms.

Chemical Species Water type BCF BAF Tissue Reference

Fish NP Barbus barbus (barb) fresh NR 92 muscle, gut, liver, heart, roe (dw) Ahel et al. 1993

Cyprinus carpio (carp) fresh 2.5-3.3 0.9-2.2 NR NR CITI 1992 as cited in Staples et al. 1998 Gasterosteus aculeatus (3-spined stickleback) marine 1200,1300 NR based on total ww Ekelund et al. 1990

17800, 16700 NR lipidbased Ekelund et al. 1990

Lepomis macrochirus (bluegill sunfish) fresh 220 NR ww Brooke 1993b

NR 87 ww Liber et al. 1999a Oncorhynchus mykiss (rainbow trout) fresh 21-29 NR carcass Lewis and Lech 1996

90-110 NR viscera minus gall bladder/ bile Lewis and Lech 1996

NR 224 muscle, gut (dw) Ahel et al. 1993

Pimephales promelas (fathead minnow) fresh 271, 344 NR whole fish Ward and Boeri 1991c 741 NR ww Brooke 1993b Salmo salar (Atlantic salmon) marine 75 NR NR McLeese et al. 1981

190 NR NR McLeese et al. 1981 235 NR NR McLeese et al. 1981 280 NR NR McLeese et al. 1981 Squalius cephalus** (chub) fresh NR 246 muscle, gut, liver, gills (dw) Ahel et al. 1993

NP1EO Barbus barbus

(barb) fresh NR 31 muscle, gut, liver, gills, roe, heart (dw) Ahel et al. 1993

Oncorhynchus mykiss (rainbow trout) fresh NR 155 muscle, gut (dw) Ahel et al. 1993

Squalius cephalus*

* (chub) fresh NR 263 muscle, gut, liver, gills (dw) Ahel et al. 1993

### 28

Table 3 (continued). Bioconcentration and bioaccumulation factors for nonylphenol and nonylphenol ethoxylates in aquatic organisms.

Chemical Species Water type BCF BAF Tissue Reference

NP2EO Barbus barbus

(barb) fresh NR 88 muscle, gut, liver (dw) Ahel et al. 1993

Oncorhynchus mykiss (rainbow trout) fresh NR 162 muscle, gut (dw) Ahel et al. 1993

Squalius cephalus*

* (chub) fresh NR 73 muscle, gut, liver, gills (dw) Ahel et al. 1993

Wildlife NP Anas boscas (mallard duck) fresh NR 308 muscle (dw) Ahel et al. 1993

NP1EO Anas boscas

(mallard duck) fresh NR 88 muscle (dw) Ahel et al. 1993

NP2EO Anas boscas

(mallard duck) fresh NR 37 muscle (dw) Ahel et al. 1993

NR = not reported ww = wet weight dw = dry weight

* the authors (Ahel et al. 1993) note that due to various sources of uncertainty these calculated BAFs for Cladophora glomerata are only intended to provide an approximation of the order of magnitude and should not be interpreted as exact bioaccumulation factors ** the more commonly accepted name for this species is Leuciscus cephalus

### 29

Toxicity

Mode of Action Although extensive research has been conducted on the toxic effects of nonylphenolic compounds, much less is known about their actual mode of toxicity. It has been suggested that the mode of toxicity for nonylphenol and the lower chain length NPEs and NPECs is narcosis, while toxicity from longer chain length NPEs may be due to physical surfactant effects (Fay et al. 2000; Servos et al. 2000). It is also likely that the mode of toxicity will depend on the dose, with high concentrations above the critical micelle concentration resulting in more effects due to physical surfactancy. Nonylphenolic substances may also act as estrogen agonists to disrupt endocrine functions.

Narcosis Narcosis is a nonspecific, reversible mode of toxic action in which the presence of hydrophobic organic chemicals causes a disruption of cellular activity, possibly through binding to proteins in the cell membrane, or through “swelling” of the lipid portion of cell membranes (Rand 1995). Numerous researchers have observed changes in the cellular membranes of organisms exposed to NP and NPEs, thus giving support to the theory that these substances exert a narcotic-type effect. For example, increased cell membrane permeability occurs in plants exposed to nonylphenol (Silcox and Holloway 1986). Linsel et al. (1990) found that NPEs caused membrane depolarization affecting electron transport across the plasmalemma of the aquatic plant Egeria densa. Octylphenol ethoxylates, that likely have a similar mode of toxicity to their nonylphenol counterparts, are thought to act on aquatic macrophytes at the cellular membrane (Caux and Weinberger 1993). It has been proposed that at low surfactant concentrations (i.e., below the critical micelle concentration) the surfactant molecules mimic membrane phospholipids and intercalate in the phospholipid bilayer of the cell membrane causing compression of the lipid constituents, thereby inducing fatty acid desaturate enzymes to render membranes more fluid (Caux and Weinberger 1993). In unicellular algae, nonylphenol concentrations greater than 1000μg·L-1 

### cause membrane disorganization including crennulation, vesiculation and pinocytotic invaginations of the nuclear membrane, as well as cytoplasm friability and distortion of flagellae indicative of microtubular damage (Weinberger and Rea 1981; Weinberger and Greenhalgh 1984). Tree seedlings grown in aqueous solutions of nonylphenol display excessive swelling of cell mitochondria, loss of chloroplast integrity, and convolution of the plasmalemma at concentrations of 1000 μg·L-1 

### (Weinberger and Greenhalgh 1984). Massive K+

### ion leakage from the roots of tree seedlings also occurs at NP concentrations of 1500 μg·L-1 

### (Weinberger and Greenhalgh 1984).

There are indications that nonylphenolic compounds may alter the permeability of cellular membranes in animals. For example, tissues of saltwater mussels became swollen when exposed to a nonylphenol polyethoxylate. The authors concluded that the swelling was likely caused by some nonspecific damage to the cell membranes that could have affected the osmoregulatory capacity of the tissues (Braaten et al. 1972). At a very high concentration (63,000μg·L-1 

### ) NP10EO also caused an increase in ion permeability in the gill epithelial cells of a rainbow trout perfused head (Pärt et al. 1985). A decreased oxygen diffusion capacity was measured in the gills, possibly because the altered permeability caused swelling of the epithelium (Pärt et al. 1985). There was also an increase in the vascular resistance of the gills that may have been caused by an inhibition of the adrenergic dilatation of the gill vasculature by the surfactant (Pärt et al. 1985).

Nonylphenol also causes vasodilation in the vascular smooth muscle of rats by inhibiting Ca2+ 

### influx through L-type Ca2+

### channels in the smooth muscle cells (Ruehlmann et al. 1998). The sarcoplasmic reticulum (Ca2+

### - Mg 2+

### )-ATPase may be another specific target site of nonylphenol action in animal cells (Michelangeli et al. 1990). At high concentrations (5 to15 μM), nonylphenol inhibits the ATPase activity, while at low concentrations (1 to 3 μM) it increases ATPase activity

### 30

(Michelangeli et al. 1990). Nonylphenol may, therefore, affect other P-type ATPase electroenzymes such as the plasma membrane H +

### -ATPase of plants and fungi (Karley et al. 1997). It appears that NP may partition into the mitochondrial membranes of fungi and induce ionophoretic uncoupling of respiration (Karley et al. 1997). This process would restrict the cytosolic ATP supply, thus indirectly inhibiting the plasma membrane H +

### -ATPase that is essential in maintaining cytosolic ionic homeostasis (Karley et al. 1997). Weinberger and Greenhalgh (1984) found that NP at 6.25 μg·L-1 

### causes disruption in the normal sequence of ATP production in birch and pine seedlings.

QSAR modelling also supports the theory that acute toxicity of APEs follows a narcotic-type mechanism that is likely to be intermediate with respect to the nonpolar and polar narcosis syndromes (Schüürmann 1991). Although information is not available for NP itself, QSAR modelling has shown that other APs, such as 4-propylphenol and p-tert -pentylphenol, also have a mode of action that is between nonpolar (type I) and polar (type II) narcosis (Veith and Broderius 1990).

Endocrine Disruption There is considerable evidence to suggest that nonylphenol is an endocrine disrupting chemical (EDC) with estrogenic properties. The discovery was first made unintentionally when Soto et al. (1991), who were working with human MCF 7

### breast tumour cells, found that the use of a new brand of polystyrene centrifuge tubes resulted in cell proliferation. They were able to extract the substance being released from the polystyrene using methanol, purify it through flash chromatography and reverse-phase HPLC, and identify it by GC-MS as nonylphenol. Further tests showed that the nonylphenol induced progesterone receptors, as well as cell proliferation in the MCF 7

### cells, and it induced mitotic activity in rat endometrium - all indicators of estrogenic activity (Soto et al. 1991).

It has since been determined that NP, NP2EO, and NP1EC mimic the effects of 17ß-estradiol by binding to the estrogen receptor, thereby acting as an estrogen agonist (White et al. 1994). Moreover, these compounds will competitively displace 17ß-estradiol from binding to the estrogen receptor, suggesting that they have intrinsic estrogenic activity (White et al. 1994). Estrogen receptor competitive binding assays have been conducted with rainbow trout hepatocyte cultures to ascertain relative potencies of the various nonylphenolics. Relative potencies of nonylphenol compared to 17ß-estradiol have been measured at 1.0 x 10 -3

### (Tremblay et al. 1995) and 2.5 x 10 -4 

### (Tremblay and Van Der Kraak 1998). Estrogenicity of NP and NP2EO is similar, while NP1EC shows slightly greater estrogenicity (White et al. 1994). White et al. (1994) found that APEs with more than 3 EO groups had little or no estrogenicity. This seems to indicate that the larger NPEs that are used commercially are unlikely to act as estrogen agonists in their parent form.

Numerous studies have since examined the estrogenicity of nonylphenol in fish through vitellogenin production. Vitellogenin is a lipoglycophosphoprotein that is normally produced in the liver of sexually mature female fish under estrogenic control (Jobling and Sumpter 1993; Bjerregaard et al. 1998). Vitellogenin secreted by the liver is transported via the blood to the ovaries where it forms the major constituent of the yolk in oocytes. Male fish possess the gene for vitellogenin, but normally do not express it due to low concentrations of estrogen in their systems. Treatment of male fish with estrogen, or chemicals with estrogen-like activity, will induce vitellogenin production and blood concentrations of vitellogenin will increase rapidly. Because this vitellogenin response is directly related to the binding of chemicals to the estrogen receptor, it may be used as a biomarker for estrogenicity (Bjerregaard et al. 1998).

Following intraperitoneal injections of nonylphenol (at concentrations ranging from 10 to 200mg·kg -1 

### body weight), elevated vitellogenin levels (as much as several hundred-fold and as high as 8000 mg·L-1 

### ) have been detected in the blood plasma of various fish species, including rainbow trout (Christiansen et al. 1998a), flounder ( Platichthys flesus; Bjerregaard et al. 1998), Atlantic salmon (Yadetie et al. 1999), and eelpouts ( Zoarces viviparus; Christiansen et al. 1998b). Elevation of vitellogenin levels from control levels of 30 mg·L-1 

### to 40000 mg·L-1 

### of plasma have

### 31

also been observed in eelpouts exposed to nonylphenol (at 1000 μg·L-1 

### ) through the water column (Korsgaard and Pedersen 1998). The increase in vitellogenin level observed in the plasma shows a dose-dependent relationship with the amount of nonylphenol administered (Bjerregaard et al. 1998; Korsgaard and Pedersen 1998; Yadetie et al. 1999).

Arukwe et al. (1997b) have suggested that eggshell zona radiata proteins (Zr p) are even more sensitive than vitellogenin as biomarkers of estrogenic activity by nonylphenol in fish. Like vitellogenin, Zr

p are produced in the liver in response to estrogen stimulation, and are transported to the ovary where they form eggshells, while vitellogenin forms the yolks. Juvenile Atlantic salmon injected intraperitoneally with nonylphenol have shown significantly elevated levels of Zr p as well as vitellogenin (Arukwe et al. 1998; Yadetie et al. 1999).

Estrogenicity of nonylphenolic compounds has also been investigated through in vitro assays in which vitellogenin induction in fish hepatocytes has been assessed. Jobling and Sumpter (1993) assessed the estrogenic capacities of NP, NP1EC, NP2EO, NP9EO, and NP40EO using an in vitro bioassay with cultures of male rainbow trout hepatocytes. Treatment with NP2EO and NP1EC resulted in a vitellogenin production 40-50 times higher than that of controls with mean ED 50

### s of 17.27 μM (5326 μg·L-1 

### ) and 15.25 μM (4916 μg·L-1 

### ), respectively. NP and NP9EO caused an 18-20 fold increase in vitellogenin production with mean ED 50

### s of 16.15μM (3558μg·L-1 

### ) and 82.31 μM (50800μg·L-1 

### ), respectively. All of these substances became cytotoxic at concentrations between 50 and 200 μM, with NP being more toxic than the ethoxylates. The concentration of vitellogenin found in the NP40EO treatment was not significantly different from the control. The authors noted that the potency of the polyethoxylate compounds seems to decrease with increasing length of the ethoxylate chain. When NP was added to rainbow trout hepatocytes with Tamoxifen (which inhibits binding to the estrogen receptor site), the vitellogenin production was significantly lower than when NP was added alone. This result further confirms that NP also acts via the estrogen receptor in a manner similar to estradiol itself (Jobling and Sumpter 1993).

Rainbow trout hepatocytes were treated with estradiol in both the presence and absence of nonylphenol to assess the effects on induction of vitellogenin mRNA and estrogen receptor mRNA (Flouriot et al. 1995). No synergistic, additive, or repressive effects were observed. Knudsen et al. (1998) also found no evidence of synergism in the induction of zona radiata proteins or estrogen receptor modulation when octylphenol and estradiol were administered simultaneously to rainbow trout through intraperitoneal injections. When five estrogenic chemicals (nonylphenol, octylphenol, o,p’

-DDT, Aroclor 1221, and bisphenol A) were administered together to rainbow trout hepatocytes, a greater induction of vitellogenin was observed when compared with the effects of each chemical when administered singly (Sumpter and Jobling 1995).

Aside from vitellogenesis, other estrogenic effects have also been observed in fish. Intraperitoneal injection of male eelpouts with 10 and 100 μg·g -1 

### body weight per week of nonylphenol resulted in reduced gonadosomatic indices and severe histological effects on the testicular structure (Christiansen et al. 1998b).

In a study comparing historical pesticide application data with recreational catch data from Atlantic Canada, epidemiological evidence showed a relationship between use of Matacil 1.8D (an insecticide formulated with NP) and a decline in catches of Atlantic salmon, Salmo salar (Fairchild et al. 1999). Similar declines were not observed with the application of other Matacil that did not contain NP, indicating that NP was the causal agent for the reduced fish abundance. Because the timing of the pesticide spraying coincided with the final stages of salmon smoltification, the authors speculated that endocrine disruption of smoltification may have led the lower catches (Fairchild et al. 1999). Laboratory studies with juvenile Atlantic salmon have also indicated that NP may affect the smolting and osmoregulatory processes of anadromous fish when they are exposed to seawater (Madsen et al. 1997; Brown et al. 1998).

### 32

Endocrine disruption and estrogenic effects of NP, NP2EO, NP1EC and NP2EC have also been observed in a variety of organisms other than fish. Exposure to nonylphenol has been found to increase the production of cypris major protein in larvae of the marine barnacle Balanus amphitrite (Billinghurst et al. 2000). Cypris major protein is required for successful larval settlement and juvenile development.

Immature female rats showed dose-dependent increases in uterine mass, increased protein and DNA contents of the uterus, and increased levels of the uterine peroxidase enzyme when injected intraperitoneally with nonylphenol at concentrations greater than 1 mg per animal (Lee and Lee 1996). This estrogenic response due to nonylphenol exposure was approximately 1x10 -3

### to 2x10 -3 

### times as potent as that elicited by estradiol. Administration of an estrogen antagonist blocked the effects of NP, confirming that the action of nonylphenol in rats is through the estrogen receptor (Lee and Lee 1996). The authors also found that there is direct competition between NP and estradiol. Because NP is less efficient than estradiol, its presence reduces the estrogenic effects of estradiol. Chapin et al. (1999) examined effects of NP administered through the diet on several generations of rats. Effects indicative of an estrogenic response included vaginal opening at a younger age when exposed to NP at 650 mg·kg -1 

### food, and increased estrous cycle length at an NP concentration of 2000 mg·kg -1 

### food.

Using a recombinant yeast strain, Routledge and Sumpter (1996) examined the ability of nonylphenolic compounds to stimulate transcriptional activity of the human estrogen receptor (hER). NP, NP1EC, NP2EC and NP2EO were all found to produce dose-dependent elevations of estrogenic activity. This yeast estrogen screening assay was repeated by Metcalfe et al. (2001), confirming the estrogenic responses to NP, NP1EO and NP2EO. W ith NP1EC and NP2EC, however, Metcalfe et al. (2001) observed no estrogenic response. Similar results were obtained in assays with Japanese medaka ( Oryzias latipes) in which a mixture of NP1EO and NP2EO was observed to alter gonadal development, while no evidence of estrogenicity was observed for a mixture of NP1EC and NP2EC (Metcalfe et al. 2001). The contradiction between these two studies indicates that further work is needed to confirm whether or not NPECs produce estrogenic responses.
White et al. (1994) found that NP, NP2EO and NP1EC were estrogenic in mammalian (mouse, human) and avian (chicken) cells. Lutz and Kloas (1999) compared the estrogenic binding affinities for several compounds by examining the competitive displacement of estradiol in cytosolic fractions from the livers of the amphibian Xenopus laevis. They found that NP had a higher binding affinity than octylphenol. Fifty percent displacement of 18.9 nM estradiol occurred at 7424 μg·L-1 

### NP and at 16153 μg·L-1 

### OP.

Besides acting as an estrogen agonist, it appears that nonylphenol may also be capable of interacting with androgen receptors, though with a lower affinity than for estrogen receptors (Gray and Kelce 1996). Both NP and NPEs have been found to reduce the elimination of glucose- and sulphate-conjugated testosterone metabolites while increasing the elimination of oxido-reduced derivatives of testosterone and increasing metabolic androgenization in daphnids (Baldwin et al. 1997, 1998).

A brief discussion of the ecological relevance of endocrine disruption, and whether such endpoints should be used in the development of environmental quality guidelines, is presented in Annex I.

Physical Surfactancy Apart from interactions with cellular membranes and membrane transport proteins, or endocrinal effects (as discussed in the next section), the only other mode of toxicity for nonylphenol mentioned in the literature deals with its physical surfactancy. For insects with an aquatic larval stage, such as mayflies and caddisflies, decreased adult emergence has been observed during and immediately after NP application to water. The decreased emergence has been attributed to the surfactant properties of NP that may have rendered the insects “wettable”, thus preventing them from escaping the surface tension (Schmude et al. 1999). Effects due to physical

### 33

surfactancy are most likely to occur if concentrations of NP or NPEs are sufficiently high for micelles to form. Typical environmental concentrations in Canada are generally below critical micelle concentrations for these substances, and therefore this mode of toxicity is not likely to occur often (see the list of critical micelle concentrations in Chapter 1 and the discussion of Canadian environmental concentrations in Chapter 2).

Toxic Effects Toxicity of nonylphenol and nonylphenol ethoxylates has been observed in diverse biological taxa including bacteria, fungi, protozoans, algae, aquatic macrophytes, terrestrial plants, crustaceans, insects, molluscs, oligochaetes, annelids, rotifers, nematodes, collembolans, fish, amphibians, and mammals. Researchers have also determined the toxicity of NP and NPEs in many of these organisms at various developmental stages. Observed toxic effects include mortality, growth inhibition, reduced fecundity, reduced germination, reduced abundance, reduced egg viability, reduced larval settlement, reduced emergence, reduced hatch rate, delayed maturity, reduced photosynthesis, immobilization, lethargy, loss of equilibrium, membrane disruption, induced vitellogenesis, zona radiata induction, modified sex ratios, induction of testis-ova, and changes in soil nutrient cycling (e.g., nitrification, nitrogen-fixation by bacteria).

Comprehensive lists of all fresh water, marine water, sediment, and soil toxicity data found in the literature have been compiled in Appendices I, II, III, and IV, respectively. W ithin each of these appendices, the data have been organized under broad taxonomic headings (e.g., microorganisms, plants, invertebrates, fish, amphibians, etc.). The data have also been grouped by compound (i.e., NP, NPEs, NPECs). Toxicity data relevant to the derivation of the water, sediment, and soil quality guidelines are discussed within Chapters 5, 6 and 7, respectively. Below is a brief summary of the major, over-arching trends in the toxicity data.

Comparing LC 50

### and EC 50

### values across the various taxonomic groupings, there appears to be a great deal of overlap. Toxic NP concentrations are quite comparable for most invertebrates and fish, while plants and algae, in general, are slightly less sensitive. Comparing across media, NP is approximately one hundred times more toxic in water (either freshwater or marine) than it is in sediment or soil. The bulk of toxic responses in water fall within the range of 0.1 to 10 ppm (i.e., 100 to 10000 μg·L-1 

### ; see Appendix I and II) while most toxic responses in sediment and soil are within the range of 10 to 1000 ppm (i.e., 10 to 1000 mg·kg -1 

### dry weight; see Appendix III and IV). This difference likely reflects the lower bioavailability of the compounds in soil and sediment due to particle adsorption. There are large differences in toxicity among the various nonylphenolic ethoxamers. For example, LC 50

### s for freshwater exposure of Japanese killifish, Oryzias latipes, to NP, NP1EO, NP6.4EO, NP9EO, and NP16.6EO are 1400, 3000, 5400, 12000, and 110000 μg·L-1



### , respectively (Yoshimura 1986). In general, toxicity decreases with increasing ethoxylation.

It appears that the concentrations of NP required to elicit estrogenic effects are comparable to concentrations at which other chronic toxic effects are observed. Estimates of NP chronic toxicity in freshwater fish, for conventional endpoints, range from 6.0 μg·L-1 

### ( Oncorhynchus mykiss 91 -day NOEC growth; Brooke 1993a) to 460 μg·L-1 

### ( Oryzias latipes 17-day LC 50

### embryotoxicity; Gray and Metcalfe 1997). For estrogenicity endpoints, the chronic toxicity estimates are of a similar magnitude, ranging from 10 μg·L-1 

### ( Oryzias latipes 3-month NOEC for testis-ova induction; Gray and Metcalfe 1997; also Oncorhynchus mykiss 72-hour LOEC vitellogenin induction; Lech et al. 1996 ) to 100 μg·L-1 

### ( Oryzias latipes 3-month LOEC for sex ratio change; Gray and Metcalfe 1997). These data indicate that guidelines for nonylphenolic substances set at concentrations that protect against conventional toxicity endpoints are likely to protect against most endocrine disrupting effects as well.

### 34

Mixtures and the TEQ Approach Nonylphenol, nonylphenol ethoxylates, and nonylphenol ethoxycarboxylates typically occur together in the aquatic environment as mixtures. Therefore, for the purpose of assessing acceptable levels of these substances in water, sediment or soil, it is more useful to develop guidelines that consider their combined effects. This can be accomplished through the use of a toxic equivalency (TEQ) approach.

The TEQ approach is used to characterize the toxicity of a mixture of compounds by expressing the toxicities of each individual compound in common terms, and summing them. This method is commonly applied to PCBs, dioxins, and furans, and has previously been used in the development of Canadian Environmental Quality Guidelines for these chemicals (Environment Canada 2000a,b). Application of the TEQ approach requires that all components of a mixture have a common mode of toxicity such that their effects are additive. Nonylphenolic substances meet this requirement. At typical environmental concentrations, and for conventional toxicity endpoints, the mode of toxicity for nonylphenol and for at least the lower ethoxylate chain length NPEs and NPECs is narcosis (see previous discussion). If these substances are all acting through narcosis, then additivity of their effects (as opposed to synergism or antagonism) is most likely (Servos et al. 2000).

To apply a TEQ approach, toxic equivalency factors (TEFs) are determined for each individual compound in a mixture by expressing the potency of each compound relative to the most toxic compound in the mixture. For PCBs, dioxins, and furans, TEFs are measured relative to the highly toxic 2,3,7,8-TCDD (van den Berg et al. 1998). Each compound’s concentration in the mixture is then multiplied by its TEF to obtain its toxic equivalent (TEQ) for that sample, relative to 2,3,7,8-TCDD. The overall toxicity of the mixture is then calculated by summing the TEQs for all compounds contained in the mixture.

We propose that a similar TEQ approach be applied towards mixtures of nonylphenolic substances. We are not recommending the incorporation of nonylphenolic substances into the same TEQ system used for PCBs, dioxins, and furans; the mode of toxicity for these two groups of substances are different, so their toxicities cannot be compared (Roe et al. 2000; Environment Canada 2000a). Rather we are suggesting that a separate set of nonylphenol TEF values (NP TEFs) be used. Several researchers have studied the relative toxicity of various nonylphenol ethoxylates and found that toxicity increases with decreasing length of the ethoxylate chain (e.g., Maxwell and Piper 1968; Macek and Krzeminski 1975; Yoshimura 1986; Miossec and Bocquéné 1986; Hall et al. 1989). Therefore the degradation products of NPEs (e.g., NP, NP1EO, NP2EO) are more toxic than the parent compounds. In the application of a TEQ approach to these compounds then, potency should be expressed relative to nonylphenol, the most toxic substance.

Relative toxicity values, or NP TEFs, for various nonylphenolic compounds were calculated for the CEPA assessment of nonylphenol (Servos et al. 2000) and are presented in Table 4. These values were derived based on a broad dataset that included both acute and chronic studies on a range of vertebrate and invertebrate species. Reported toxic concentrations for the various nonylphenolic compounds were matched up against similar endpoints for nonylphenol with the same species, and, where possible, from the same laboratory; then a relative toxicity ratio was calculated. From the resulting list of relative toxicity values for each group of compounds, a mean relative toxicity value (TEF) was calculated, with more weight given to those studies deemed to be of higher quality.

These TEFs represent an initial attempt to define the relative toxicity of groups of nonylphenolic compounds, rather than for each individual ethoxamer. Future work to develop and validate more specific TEF values is needed, but, in the interim, it is recommended that these more general TEFs be applied. Also, TEF values for NPEs containing 3 to 8 ethoxylate groups were not estimated in the CEPA assessment because there have been very few toxicity tests conducted with these substances. Instead, we have provided our own estimate. As toxicity is known to generally decrease with increasing number of ethoxylate groups, the relative toxicity of NP3EO-

### 35

NP8EO likely lies somewhere between the TEFs of 0.5 (for NP2EO) and 0.005 (for NP nEO where

n Ú 9). Therefore, for NPEs with 3 to 8 EO groups, until sufficient toxicity data is available to estimate relative potencies, it is recommended that a conservative TEF of 0.5 be adopted, with the caveat that the use of this TEF may overestimate the toxicity of a mixture.

The total concentration of NP and NP-equivalents in a sample can be calculated using the equation: Total concentration = ∑(Cx 

### x TEF)

where, Cx 

### = concentration of each nonylphenolic compound TEF = toxic equivalency factor for each particular nonylphenolic compound.

In the assessment of a mixture of nonylphenolic compounds, the environmental concentrations of NPEs and NPECs should be expressed in terms of NP equivalents, and the sum of these concentrations should then be compared against the guidelines developed in this document.

The NP TEF values determined in the CEPA assessment (Servos et al. 2000) were based on toxicity to aquatic species which reside in the water column, but they may also be applied towards the soil and sediment guidelines for NP and NPEs. Narcosis appears to be a common mode of toxicity for nonylphenolic substances across all taxonomic groups (see earlier discussion under “Modes of Toxicity”), so the NP TEF values should be applicable to plants, invertebrates, fish, mammals, etc., regardless of whether they are water-, sediment-, or soil-dwelling species.

If octylphenol, octylphenol ethoxylates, or octylphenol ethoxycarboxylates are present in environmental samples, it is recommended that these substances also be considered in the calculation of total NP-equivalents. OP and OPEs are very similar in structure and behaviour to NP and NPEs, and likely act through a similar toxic mode of action. Servos et al. (2000) have suggested TEF values relative to NP for OP, OP1EC and OP2EC at 1.0, 0.005, and 0.005, respectively. We would recommend that for OP nEOs, the same TEF values for the corresponding NP nEO analogs could be used.

The TEQ approach has not been previously applied towards NP and NPEs, so the guidelines currently available from other jurisdictions, as described in Chapter 4, are either based solely on a single ethoxamer (e.g. NP or NP9EO), or they are based on the straight sum of NP and NPE concentrations with no regard for relative toxicities among the ethoxamers. The Canadian Environmental Quality Guidelines for NP and NPEs, developed in Chapters 5, 6, and 7, are expressed on a TEQ basis.

### 36

Table 4. Summary of relative toxicity of nonylphenolic and related compounds.

Chemical Toxic equivalency factors (TEFs) relative to NP NP 1 NP1EO 0.5 NP2EO 0.5 NP nEO (3 Û n Û8) 0.5* NP nEO ( n Ú9) 0.005 NP1EC 0.005 NP2EC 0.005

OP 1 OP nEO (1 Û n Û8) 0.5* OP nEO ( n Ú9) 0.005* OP1EC 0.005 OP2EC 0.005

Adapted from Servos et al. (2000) except where otherwise noted. * conservative estimation by the authors of this document

### 37

CHAPTER 4. GUIDELINES FROM OTHER JURISDICTIONS

Water

Freshwater Tables 5 and 6 provide summaries of existing freshwater guidelines, objectives, criteria, standards, and environmental concern levels for nonylphenol (NP) and nonylphenol ethoxylates (NPEs), respectively, from various jurisdictions both within and outside of Canada.

In Canada, the Quebec provincial government has established interim water quality criteria for both nonylphenol (CAS 25154-52-3) and nonylphenol ethoxylates (CAS 9016-45-9). For nonylphenol, the interim criteria (developed in 1996) for the protection of aquatic life from acute and chronic toxicity are 6.8 μg·L-1 

### and 0.3 μg·L-1 

### , respectively. The interim criteria for nonylphenol ethoxylates (established in 1991) for the protection of aquatic life from acute and chronic toxicity are 470 μg·L-1 

### and 21 μg·L-1 

### , respectively (MEF 1998). These criteria have recently undergone review, and new criteria are scheduled for release in 2000 (Guay 2000). For nonylphenol, new acute and chronic final criteria will be 29 μg·L-1 

### and 6.0 μg·L-1 

### , respectively. For NP9EO, provisional acute and chronic criteria will be 280 μg·L-1 

### and 12 μg·L-1 

### , respectively. For NP1EC, provisional acute and chronic criteria will be 100 μg·L-1 

### and 4 μg·L-1 

### , respectively. It was decided that there is currently insufficient data available to derive criteria for NP1EO, NP1.5EO, NP2EO, NP10EO, and NP2EC.

The province of Ontario has an interim provincial water quality objective for nonylphenol of 0.04μg·L-1 

### . This Ontario objective was based on the 48h LC 50

### of 100 μg·L-1 

### for Daphnia pulex (EPS 1981 as cited in NRCC 1982), that was then divided by a highly conservative uncertainty factor of 2332 due to the scarcity of toxicological data at the time (OMOE 1991). Many more aquatic toxicity data are now available.

Outside of Canada, Denmark has an environmental quality standard for freshwater concentrations of NP and NPEs combined. Australia and New Zealand have an environmental concern level for nonylphenol in fresh water, and the U.S.A. has developed draft numbers for both acute and chronic ambient aquatic life water quality criteria for nonylphenol.

Marine/Estuarine A summary of existing marine and estuarine water quality guidelines for NP from other jurisdictions are provided in Table 7. There are currently no jurisdictions in Canada with marine water quality guidelines for NP or NPEs. Australia and New Zealand have an environmental concern level for NP in marine waters, while the U.S.A. has both acute and chronic ambient aquatic life water quality criteria for NP in marine waters.

Sediment There are no jurisdictions within Canada, nor outside of Canada, which have guidelines for NP or NPEs in either freshwater or marine sediment.

### 38

Soil No guidelines for nonylphenol in soil currently exist for other jurisdictions within Canada. Outside of Canada, only Denmark has a soil quality criterion for nonylphenol in soil (Table 8). Denmark also has a guideline for the total concentration of NP, NP1EO, and NP2EO that may be present in sludge which is intended to be applied to agricultural soils. Sweden also has a guideline on the allowable concentration of NP in sludge used as an agricultural fertilizer (see Table 8).

Table 5. Freshwater quality guidelines for nonylphenol from other jurisdictions.

JURISDICTION GUIDELINE (μg·L-1 

### ) LEVEL OF PROTECTION REFERENCE Ontario, Canada 0.04 interim provincial water quality objective for the protection of aquatic life OMEE 1994

Australia / New Zealand 1 

### 0.2 environmental concern level See footnote

Quebec, Canada 2 

### 0.3 interim water quality criteria for the protection of aquatic life (chronic exposure) MEF 1998

Denmark 1.0 (NP and NPEs) environmental quality

standard Renner 1997

U.S.A. 3 

### 5.9 (4-d average) ambient aquatic life water

quality criteria (draft stage) Brooke and Thursby 2000 Quebec, Canada4 

### 6.0 water quality criteria for the protection of aquatic life (chronic exposure) Guay 2000

Quebec, Canada 2 

### 6.8 interim water quality criteria for the protection of aquatic life (acute exposure) MEF 1998

U.S.A. 3 

### 27.9 (1-h average) ambient aquatic life water

quality criteria (draft stage) Brooke and Thursby 2000 Quebec, Canada4 

### 29.0 water quality criteria for the protection of aquatic life (acute exposure) Guay 2000

1 

### Australian and New Zealand Environment and Conservation Council, Agriculture and Resource Management Council of Australia and New Zealand 1999
2

### These criteria have recently undergone review, and will likely be updated in 2000 (Guay 2000).
3

### These average concentrations should not be exceeded more than once every three years, on average. 4

### These criteria are still at the draft stage and have not yet been officially approved or implemented (Guay 2000).

### 39

Table 6. Freshwater quality guidelines for nonylphenol ethoxylates and ethoxycarboxylates from other jurisdictions.

JURISDICTION GUIDELINE (μg·L-1 

### ) CHEMICAL LEVEL OF PROTECTION REFERENCE

Denmark 1.0 NP and NPEs environmental quality standard Renner 1997

Quebec, Canada 2 

### 4.0 NP1EC provisional water quality criteria for the protection of aquatic life (chronic exposure) Guay 2000

Quebec, Canada 2 

### 12.0 NP9EO provisional water quality criteria for the protection of aquatic life (chronic exposure) Guay 2000

Quebec, Canada 1 

### 21.0 NPEs interim water quality criteria for the protection of aquatic life (chronic exposure) MEF 1998

Quebec, Canada 2 

### 100.0 NP1EC provisional water quality criteria for the protection of aquatic life (acute exposure) Guay 2000

Quebec, Canada 2 

### 280.0 NP9EO provisional water quality criteria for the protection of aquatic life (acute exposure) Guay 2000

Quebec, Canada 1 

### 470.0 NPEs interim water quality criteria for the protection of aquatic life (acute exposure) MEF 1998

1 

### These criteria have recently undergone review, and will likely be updated in 2000 (Guay 2000).
2 

### These criteria are still at the draft stage and have not yet been officially approved or implemented (Guay 2000).

### 40

Table 7. Marine water quality guidelines for nonylphenol from other jurisdictions.

JURISDICTION GUIDELINE (μg·L-1 

### ) LEVEL OF PROTECTION REFERENCE Australia / New Zealand 1 

### 1.2 environmental concern level See footnote

U.S.A. 2 

### 1.4 (4-d average) ambient aquatic life water

quality criteria (draft stage) Brooke and Thursby 2000 U.S.A. 2 

### 6.7 (1-h average) ambient aquatic life water

quality criteria (draft stage) Brooke and Thursby 2000

1 

### Australian and New Zealand Environment and Conservation Council, Agriculture and Resource Management Council of Australia and New Zealand 1999
2

### These average concentrations should not be exceeded more than once every three years, on average.

Table 8. Soil and sludge guidelines from other jurisdictions.

JURISDICTION GUIDELINE

(mg·kg -1 

### ) CHEMICAL LEVEL OF PROTECTION REFERENCE

Denmark 0.01 NP soil quality criterion Jensen et al. 1997 Denmark 1 

### 10 4-NP, NP1EO and NP2EO guideline for application of

sludge to agricultural soils European Commission 1999 Sweden 50 NP allowable dry weight concentration in sludge for use as agricultural fertilizer Bennie et al. 1998

1 

### Danish Ministerial Order No. 823, Sept. 16, 1996; guideline takes effect as of June 2000

### 41

CHAPTER 5. DEVELOPMENT OF CANADIAN WATER QUALITY GUIDELINES

Canadian Water Quality Guidelines (WQGs) are developed using the methods outlined in “A protocol for the derivation of water quality guidelines for the protection of aquatic life” (CCME 1991; CCME 1999). This protocol was created with the intent that guidelines be protective of all forms of aquatic life at all life stages during an indefinite period of exposure in the water column. A brief summary of the protocol is provided in Annex 2, including a definition of primary data.

Freshwater

Toxicity Data A large number of studies describing the toxic effects of nonylphenol and its ethoxylates on freshwater aquatic life exist in the scientific literature. A comprehensive list of all freshwater toxicity data for nonylphenol that was consulted has been compiled in Appendix I. Rather than giving an exhaustive review of these studies, a brief description of the toxicity data relevant to the derivation of Canadian Water Quality Guidelines is provided below. This information reflects the data requirements set forth in the protocol (CCME 1991) and is presented by taxonomic groups.

Fish From the primary toxicity dataset for nonylphenol (Table 9), the most sensitive freshwater fish species is the rainbow trout, Oncorhynchus mykiss, with a 91-day LOEC for growth of 10.3 μg·L-1 

### (Brooke 1993a). A comparable value of 14 μg·L-1 

### was reported as the 33-day LOEC for survival with fathead minnows, Pimephales promelas (Ward and Boeri 1991b). The other primary toxicity endpoints are generally an order of magnitude higher. For example, 28-day and 96-hour LOECs for survival have been reported for Lepomis macrochirus at 126 and 211 μg·L-1 

### , respectively, and for P. promelas

at 193 and 230 μg·L-1 

### , respectively (Brooke 1993b). Median lethal concentrations (96h LC 50

### s) for P.promelas are cited at 128 μg·L-1 

### (Brooke 1993a) and 135 μg·L-1 

### (Holcombe et al. 1984), and values of 209 and 221 μg·L-1 

### are cited for L. macrochirus and O. mykiss

, respectively (Brooke 1993a).

Invertebrates The most sensitive primary data for a freshwater invertebrate is the 96h LC 50

### of 20.7 μg·L-1 

### for Hyalella azteca

(Brooke 1993a). England and Bussard (1995) also report a 96h LC 50

### of 170μg·L-1 

### and a 96h EC 50

### for immobilization of 150 μg·L-1 

### for H. azteca. For Daphnia magna

, concentrations of 84.8, 100, and 215 μg·L-1 

### have been reported for a 48h immobilization EC 50 

### (Brooke et al. 1993a), a 21-day LC 50

### (Brooke et al. 1993a), and a 21-day LOEC for reproduction (Comber et al. 1993), respectively. Numerous primary datapoints exist for the midge, Chironomus tentans, including 14-day EC 50

### s of 95 μg·L-1 

### for both total adverse effects and larval growth (England and Bussard 1993), a 14-day LC 50

### of 119 μg·L-1 

### (England and Bussard 1993), a 20-day LOEC for survival of 91 μg·L-1 

### (Kahl et al. 1997), and a 14-day LOEC for larval growth of 150μg·L-1 

### (England and Bussard 1993). Primary data is only available for two non-arthropod invertebrates. Brooke (1993a) reported 96h LC 50

### s of 342 and 774 μg·L-1 

### for the annelid Lumbriculus variegatus and the snail Physella virgata , respectively.

Plants/Algae A limited amount of primary data is available for toxicity of nonylphenol to algae and aquatic macrophytes. The macrophyte Lemna minor exhibits reduced frond production after 96 hours of exposure to nonylphenol at a concentration of 2080μg·L-1 

### (Brooke 1993a). The green algae Selenastrum capricornutum experiences growth reduction after 96 hours of exposure to nonylphenol concentrations as low as 190.0 μg·L-1 

### , and the corresponding median effective concentration (EC 50

### ) is 410 μg·L-1 

### (Ward and Boeri 1990a).

### 42

Guideline Derivation Primary data which were selected as acceptable for use in guideline derivation (i.e., that met the screening criteria as described in CCME 1991 and in Annex 2) are shown in Table 9. This dataset is sufficient to meet the minimum requirements for derivation of a full guideline for nonylphenol in fresh water.

According to the protocol, water quality guidelines for a chemical are preferably derived from the chronic study that gives the most sensitive non-lethal lowest-observable-effect level (LOEL) for an ecologically relevant response. The study must have demonstrated a clear dose-response relationship, and the LOEL must be statistically significant..

A chronic toxicity test with rainbow trout, Oncorhynchus mykiss, by Brooke (1993a) was selected as the critical study. This study produced the most sensitive acceptable endpoint, a 91d LOEC for reduced growth in rainbow trout at 10.3 μg·L-1 

### . Rainbow trout were exposed, under flow-through conditions, to two replicates each of five concentrations (ranging from 6 to 114 μg·L -1 

### ) of nonylphenol, plus a control. Each replicate originally contained 100 embryos, and after approximately 45 days, 15 fry from each replicate were randomly chosen for completion of the assay. The total length of the exposure period was 91 days. Temperature, dissolved oxygen, total alkalinity, total hardness, pH, and conductivity were measured regularly through the exposure period and remained within acceptable levels (Brooke 1993a). Water samples from the exposure chambers were collected and analyzed twice per week, throughout the duration of the test. Concentrations of nonylphenol were anaylzed using HPLC. One-way analysis of variance and Dunnett’s test were used to determine NOECs and LOECs. In this study, appropriate experimental methods and conditions were used, and all relevant information was reported in the article. Growth is an ecologically relevant response, and the LOEC reported was statistically significant.

Because of possible variations in toxicity among species, among endpoints, and between lab and field exposures, it is recommended that a safety factor be used. Therefore, the value of 10.3μg·L-1 

### (from Brooke 1993a) was multiplied by a safety factor of 0.1 (CCME 1991) to yield 1.03 μg·L-1 

### . Rounding off, the freshwater guideline value is 1.0 μg·L-1 

### . The effects diagram in Figure 3 shows the guideline value and the distribution of the primary toxicity data that was used to derive it.

### 43

Table 9. Data used in derivation of guideline for toxicity of nonylphenol to freshwater aquatic life.

Species (Common name) Life cycle stage Duration Endpoint Effect Concentration (μg·L-1 

### ) Reference

PRIMARY DATA Algae/Macrophytes Lemna minor (duckweed) 2-frond stage 96h LOEC frond production 2080 Brooke 1993a

Selenastrum capricornutum

(algae) 4-7 days 96h LOEC growth (cell counts) 190 Ward and Boeri 1990a 4-7 days 96h EC 50 

### growth (cell count) 410 Ward and Boeri 1990a

Invertebrates Chironomus tentans larva 20d LOEC survival 91 Kahl et al. 1997 (midge) 2 nd

### instar larva 14d EC 50 

### lethargy, paleness, reduced size 95 England and Bussard 1993

2 nd

### instar larva 14d EC 50 

### larval growth 95 England and Bussard 1993 2 nd

### instar larva 14d LC 50 

### mortality 119 England and Bussard 1993 2 nd

### instar larva 14d LOEC larval growth 150 England and Bussard 1993 Daphnia magna neonate 48h EC 50 

### immobilization 84.8 Brooke 1993a (water flea) neonate 21d LC 50 

### mortality 100 Comber et al. 1993 neonate 21d LOEC reproduction 215 Brooke 1993a Hyalella azteca juvenile 96h LC 50 

### mortality 20.7 Brooke 1993a (amphipod) juvenile 96h EC 50 

### lethargy/ immobility 150 England and Bussard 1995b juvenile 96h LC 50 

### mortality 170 England and Bussard 1995b Lumbriculus variegatus (annelid) adult 96h LC 50 

### mortality 342 Brooke 1993a

Physella virgata (snail) adult 96h LC 50 

### mortality 774 Brooke 1993a

Fish Lepomis macrochirus (bluegill sunfish) 11-13 weeks 28d LOEC survival 126 Brooke 1993b

juvenile 96h LC 50 

### mortality 209 Brooke 1993a 13-15 weeks 96h LOEC survival 211 Brooke 1993b

Oncorhynchus mykiss (rainbow trout) embryo 91d LOEC growth 10.3 Brooke 1993a

45 days 96h LC 50 

### mortality 221 Brooke 1993a

### 44

Table 9 (continued). Data used in derivation of guideline for toxicity of nonylphenol to freshwater aquatic life.

Species (Common name) Life cycle stage Duration Endpoint Effect Concentration (μg·L-1 

### ) Reference

Pimephales promelas (Fathead minnow) embryo 33d LOEC survival 14 Ward and Boeri 1991b 25-35 days 96h LC 50 

### mortality 128 Brooke 1993a

31-35 days 96h LC 50 

### mortality 135 Holcombe et al. 1984 4 weeks 28d LOEC survival 193 Brooke 1993b 4 weeks 96h LOEC survival 230 Brooke 1993b

LOEC = lowest observed effect concentration LC 50

### = median lethal concentration EC 50

### = median effective concentration

Note: The critical study used in the guideline derivation is shaded.

### 45VertebratesInvertebratesAcuteChronic 

### Toxicity information Species Toxicity endpoint Concentration (μg·L-1 

### )

Toxicity endpoints: Canadian Water Quality Guideline:

1.0 μg·L -1



### primary critical valueInvertebratesPlantsVertebrates



### Canadian Guideline 10 0 

### 10 1 

### 10 2 

### 10 3 

### 10 4 

### P. promelas P. promelas P. promelas O. mykiss L. macrochirus L. macrochirus P. virgata L. variegatus H. azteca H. azteca H. azteca D. magna
L. minor S. capricornutum S. capricornutum

P. promelas P. promelas L. macrochirus O. mykiss
D. magna D. magna C. tentans C. tentans C. tentans C. tentans C. tentans 96-h LOEC

96-h LC50 

### 96-h LC50 

### 96-h LC50 

### 96-h LOEC 96-h LC50 

### 96-h LC 50 

### 96-h LC 50 

### 96-h LC 50 

### 96-h EC50 

### 96-h LC 50 

### 48-h EC50 

### 96-h LOEC 96-h LOEC 96-h EC50



### 28-d LOEC 33-d LOEC 28-d LOEC 91-d LOEC

21-d LOEC 21-d LC 50 

### 14-d LOEC 14-d LC 50 

### 14-d EC50 

### 14-d EC50 

### 20-d LOEC

Figure 3. Select freshwater toxicity data for nonylphenol.

### 46

Marine/Estuarine Water

Toxicity Data A large number of studies describing the toxic effects of nonylphenol and its ethoxylates on marine aquatic life exist in the scientific literature. A comprehensive list of all marine toxicity data for nonylphenol that was consulted has been compiled in Appendix II. Rather than giving an exhaustive review of these studies, a brief description of the toxicity data relevant to the derivation of Canadian Water Quality Guidelines is provided below. This information reflects the data requirements set forth in the protocol (CCME 1991) and is presented by taxonomic groups.

Fish From the nonylphenol toxicity dataset, three acceptable (secondary) chronic toxicity studies were available for marine fish. A 21-day LOEC of 24.5 μg·L-1 

### was reported for increased hepatosomatic index in flounder, Platichthys flesus (Allen et al. 1999). A 21-day LOEC of 100μg·L-1 

### was reported for increased plasma concentrations of vitellogenin in eelpout, Zoarces viviparus (Korsgaard and Pedersen 1998). A 10-day LOEC of 2203 μg·L-1 

### for the induction of sublethal abnormalities in embryos of the killifish, Fundulus heteroclitus, was also reported (Kelly and Di Giulio 2000). Six acceptable acute toxicity endpoints have been reported for marine fish. The 96h LC 50

### for sheepshead minnow, Cyprinodon variegatus, has been estimated at 310.0μg·L-1 

### (Ward and Boeri 1990c) and 142.0 μg·L-1 

### (Lussier et al. 2000). For inland silversides, Menidia beryllina , a 96h LC 50

### of 70.0 μg·L-1 

### has been cited (Lussier et al. 2000). A 96h LC 50

### of 17μg·L-1 

### has been measured in winter flounder, Pleuronectes americanus (Lussier et al. 2000). In F. heteroclitus, 96h LC 50

### s have been reported for embryos and for 2-week larvae at 5441 and 209 μg·L-1 

### , respectively (Kelly and Di Giulio 2000).

Invertebrates The lowest reported endpoint for a marine invertebrate came from a secondary study by Billinghurst et al. (1998) in which the 48h LOEC for reduced settlement in larvae of the barnacle Balanus amphitrite

was 0.06 μg·L-1 

### . Several studies indicate that the mysid Americamysis bahia (recently renamed from Mysidopsis bahia) is also a sensitive marine invertebrate. Ward and Boeri (1991a) reported reductions in the growth of A.bahia after a 28-day exposure to nonylphenol concentrations as low as 6.7 μg·L-1 

### and reduced reproduction and survival were both observed at concentrations as low as 9.1 μg·L-1 

### . Ward and Boeri (1990b) also reported a 96h LC 50

### for A. bahia

of 43.0 μg·L-1 

### , and Lussier et al. (2000) reported a 96h LC 50

### of 60.6 μg·L-1 

### for this same species. Slightly higher 96h LC 50

### s of 59.4, 61.6, 71.0, and >195 μg·L-1 

### are reported for the grass shrimp Paleomonetes vulgaris, the amphipod Leptocheirus plumulosus, the lobster Homarus americanus, and the mudcrab Dyspanopeus sayii, respectively (Lussier et al. 2000). Lussier et al. (2000) also reported a 48h EC 50

### of 37.9 μg·L-1 

### for inhibition of fertilization in the coot clam Mulinia lateralis. From a 53-day study on the copepod Tisbe battagliai , a LOEC of 41.0μg·L-1 

### has been reported for egg production, offspring survival, and intrinsic rate of increase (Bechmann 1999). For this same species, the 96h LC 50

### s for nauplii and for adults are 31 -62μg·L-1 

### and 500-1000 μg·L-1 

### , respectively (Bechmann 1999). For larvae of the Pacific oyster, Crassostrea gigas, exposure to 100 μg·L-1 

### resulted in both a 48h LOEC for various deformities, and a 72h LOEC for delayed development (Nice et al. 2000).

Algae The marine alga Skeletonema costatum has a 96 hour EC 50

### for growth inhibition of 27.0 μg·L-1 

### (Ward and Boeri 1990d). This is the only acceptable toxicity study available for marine algae or plants.

### 47

Guideline Derivation Primary data that were selected as acceptable for use in guideline derivation (i.e., that met the screening criteria as described in CCME 1991 and in Annex 2) are shown in Table 10. These data are insufficient to meet the minimum requirements for derivation of a full guideline for the protection of aquatic marine life. Three additional primary studies are needed for a full guideline. Of these studies, two must include chronic endpoints for two different temperate, marine fish species. The third study must have a chronic endpoint for a temperate invertebrate that belongs to a class other than the Crustacea.

When the minimum data requirements for derivation of a full guideline cannot be met, it may still be possible to derive an interim guideline. Secondary studies, as well as primary studies, may be used to meet the minimum requirements for an interim guideline. Studies which were considered of secondary quality, and therefore could be used to meet the minimum data requirements for an interim guideline, are also listed in Table 10. Using both the primary and secondary studies, there are sufficient data to meet the minimum requirements for deriving an interim guideline for nonylphenol.

As for freshwater, marine water quality guidelines are preferably derived from the chronic study that gives the most sensitive nonlethal LOEL (CCME 1991). The LOEL is then multiplied by a safety factor of 0.1 to arrive at the final guideline value.

The LOEC for reduced settlement in barnacles, Balanus amphitrite, was the most sensitive endpoint reported (48h LOEC at 0.06 μg·L-1 

### ; Billinghurst et al. 1998). However, this study was of secondary quality, involved a short exposure time, and used an unconventional endpoint. Also, this LOEC value was two orders of magnitude lower than any other endpoints reported in the literature. This suggests that either larval settlement is much more sensitive than other endpoints, or that this particular LOEC is an outlier that is not reproducible. Unfortunately, there have been no other studies conducted on barnacle larval settlement with exposure to nonylphenol with which to compare these results. The LOECs reported for larval settlement of Balanus amphitrite exposed to cadmium and phenol (Wu et al. 1997) are higher than the LOECs for many other endpoints that have been reported for these substances in marine aquatic life (see CCME 1999). This suggests that larval settlement is not a particularly sensitive endpoint. Due to the suspicion cast by these various sources of uncertainty, confidence in the study by Billinghurst et al. (1998) was not high enough to base the guideline value on this LOEC.

The chronic assay with the mysid, Americamysis bahia , by Ward and Boeri (1991a) was selected as the critical study. This study reported a 28-day LOEC for reduced growth at 6.7 μg·L-1 

### (Ward and Boeri 1991a). The study was evaluated as being of primary quality because appropriate experimental methods and conditions were used, and all relevant information was reported. The test protocol used was based on U.S. EPA test guidelines. The study involved exposure of mysids, under flow-through conditions, to five concentrations of nonylphenol (ranging from 3.9 to 21 μg·L-1 

### ), plus a dilution water control and solvent control (acetone). Eight replicates of each test concentration were used, with five mysids per replicate. The total length of the exposure period was 28 days. Temperature, dissolved oxygen, pH, and salinity were measured and recorded daily throughout the exposure period and remained within acceptable levels. Water samples from the test vessels were sampled weekly, with nonylphenol concentrations anaylzed by HPLC with fluorescence detection. One-way analysis of variance and Dunnett’s test were used to test for treatment effects. Growth is an ecologically relevant response, and the LOEC reported was statistically significant.

Therefore, the LOEC value of 6.7 μg·L-1 

### (from Ward and Boeri 1991a) was multiplied by a safety factor of 0.1 (CCME 1991), and rounded off, to obtain the interim guideline of 0.7 μg·L-1 

### for the protection of marine life against the adverse effects of nonylphenol. The effects diagram in Figure 4 shows the interim guideline value and the distribution of the toxicity data that was used to derive it.

### 48

Table 10. Data used in derivation of guideline for toxicity of nonylphenol to marine aquatic life.

Species (Common name) Life cycle

stage Duration Endpoint Effect Concentration (μg·L-1 

### ) Reference

PRIMARY DATA Algae Skeletonema costatum (algae) NR 96h EC 50 

### growth inhibition (cell count) 27 Ward and Boeri 1990d

Invertebrates Americamysis bahia (mysid) neonates 28d LOEC mean total length 6.7 Ward and Boeri 1991a
neonates 28d LOEC survival 9.1 Ward and Boeri 1991a
neonates 28d LOEC number of young per surviving female 9.1 Ward and Boeri 1991a

neonates 96h LC 50 

### mortality 43 Ward and Boeri 1990b post-larvae 1d 96h LC 50 

### mortality 60.6 Lussier et al. 2000

Leptocheirus plumulosus (amphipod) adult 96h LC 50 

### mortality 61.6 Lussier et al. 2000

Paleomonetes vulgaris (grass shrimp) 2d larvae 96h LC 50 

### mortality 59.4 Lussier et al. 2000

Tisbe battagliai (copepod) nauplii 53d LOEC egg production, offspring survival, intrinsic rate of increase 41 Bechmann 1999

Fish Cyprinodon variegatus (sheepshead minnow) juveniles 96h LC 50 

### mortality 142 Lussier et al. 2000

juveniles 96h LC 50 

### mortality; lethargy, bloating, loss of equilibrium 310 Ward and Boeri 1990c

Menidia beryllina (inland silversides) juveniles 96h LC 50 

### mortality 70 Lussier et al. 2000

### 49

Table 10 (continued). Data used in derivation of guideline for toxicity of nonylphenol to marine aquatic life.

Species (Common name) Life cycle

stage Duration Endpoint Effect Concentration (μg·L-1 

### ) Reference

SECONDARY DATA Invertebrates Balanus amphitrite (barnacle) cyprids

(larvae) 48h LOEC reduced settlement 0.06 Billinghurst et al. 1998 Crassostrea gigas (Pacific oyster) larvae 48h LOEC deformity 100 Nice et al. 2000

larvae 72h LOEC delayeddevelopment 100 Nice et al. 2000

Dyspanopeus sayii (mudcrab) 4th

### and 5th



### stage zoea 96h LC 50 

### mortality > 195 Lussier et al. 2000

Homarus americanus (American lobster) first stage

zoea 96h LC 50 

### mortality 71 Lussier et al. 2000

Mulinia lateralis (Coot clam) embryolarval 48h EC 50 

### inhibition of fertilization 37.9 Lussier et al. 2000

Tisbe battagliai (copepod) <1 day

nauplii 96h LC 50 

### mortality 31-62 Bechmann 1999

adult 96h LC 50 

### mortality 500-1000 Bechmann 1999

Fish Fundulus heteroclitus (killifish) 2 wk larvae 96h LC 50 

### mortality 209 Kelly and Di Giulio 2000

embryos 10d LOEC sublethalabnormalities 2203 Kelly and Di Giulio 2000

embryos 96h LC 50 

### mortality 5441 Kelly and Di Giulio 2000 Platichthys flesus (flounder) adults 21d LOEC increasedhepatosomatic index 24.5 Allen et al. 1999

Pleuronectes americanus (winter flounder) 2d larvae 96h LC 50 

### mortality 17 Lussier et al. 2000

Zoarces viviparus (eelpout) pregnant

females 21d LOEC increased plasma levels of vitellogenin 100 Korsgaard and Pedersen 1998

LOEC = lowest observed effect concentration LC 50

### = median lethal concentration EC 50

### = median effective concentration NR = not reported Note: The critical study used in the guideline derivation is shaded.

### 50VertebratesInvertebratesAcute 

### Toxicity information Species Toxicity endpoint Concentration (μg·L-1 

### )

F. heteroclitus F. heteroclitus C. variegatus C. variegatus M. beryllina P. americanus

T. battagliai T. battagliai D. sayii C. gigas C. gigas H. americanus L. plumulosus A. bahia A. bahia P. vulgaris M. lateralis B. amphitrite 96-h LC 50



### 96-h LC 50 

### 96-h LC 50 

### 96-h LC 50 

### 96-h LC 50 

### 96-h LC 50



### 96-h LC 50 

### 96-h LC 50 

### 96-h LC 50 

### 48-h LOEC 72-h LOEC 96-h LC 50 

### 96-h LC 50 

### 96-h LC 50 

### 96-h LC 50 

### 96-h LC 50 

### 48-h EC50 

### 48-h LOECChronicInvertebratesPlantsVertebrates



### Toxicity endpoints: Canadian Interim Water Quality Guideline:

0.7 μg·L -1



### primary secondary critical value Canadian Guideline 10 -2 

### 10 0 

### 10 2 

### 10 4 

### S. costatum

F. heteroclitus Z. viviparus P. flesus

T. battagliai A. bahia A. bahia A. bahia 96-h EC50



### 10-d LOEC 21-d LOEC 21-d LOEC

53-d LOEC 28-d LOEC 28-d LOEC 28-d LOEC

Figure 4. Select marine toxicity data for nonylphenol.

### 51

Recommended Canadian Water Quality Guidelines

Guideline for the Protection of Freshwater Life A full guideline of 1.0 μg·L -1 

### , expressed in nonylphenol toxic equivalency units (NP TEQs), is recommended for the protection of aquatic freshwater life from the adverse effects of nonylphenol and its ethoxylates.

There were sufficient toxicity data available to derive a full freshwater guideline. This guideline should be applied to the total concentration (in NP TEQs) of all nonylphenolic substances occurring in a water sample. The total concentration should be calculated using the nonylphenol toxic equivalency factors provided in Chapter 3 (Table 4). Further guidance and recommendations for the implementation of this guideline are provided in Chapter 9.

The freshwater guideline for nonylphenol and its ethoxylates proposed in this document is the same as the environmental quality standard of 1.0 μg·L-1 

### that exists in Denmark (see Table 5). It is lower than both the acute and chronic U.S. criteria for nonylphenol (at 27.9 μg·L-1 

### and 5.9μg·L-1 

### , respectively), but is higher than the environmental concern level of 0.2 μg·L-1 

### proposed by Australia/New Zealand (see Table 5). It is also higher than Quebec’s interim water quality criteria for chronic exposure of 0.3 μg·L-1 

### , although recommendations have been made to increase this value to 6.0 μg·L-1 

### (Guay 2000). The Ontario provincial objective, at 0.04 μg·L-1 

### , is more than an order of magnitude lower than the Canadian water quality guideline proposed here. It should be noted that the Ontario objective was calculated using an extremely conservative safety factor because at the time of its development, in 1991, there were much fewer toxicity data available.

Guideline for the Protection of Marine Life An interim guideline of 0.7 μg·L -1 

### , expressed in NP TEQs, is recommended for the protection of aquatic marine life from the adverse effects of nonylphenol and its ethoxylates.

This guideline is expressed in nonylphenol toxic equivalency (NP TEQ) units and may be applied to mixtures of nonylphenol and nonylphenol ethoxylates. The total concentration of nonylphenolic substances in a water sample (in NP TEQs) should be calculated using the nonylphenol toxic equivalency factors provided in Chapter 3 (Table 4). Further guidance and recommendations for the implementation of this guideline are provided in Chapter 9.

The guideline proposed here is lower than both the environmental concern level for nonylphenol of Australia/New Zealand, at 1.2 μg·L-1 

### , and the U.S. acute and chronic criteria of 6.7 μg·L-1 

### and 1.4μg·L-1 

### , respectively (see Table 7).

### 52

CHAPTER 6. DEVELOPMENT OF CANADIAN SEDIMENT QUALITY GUIDELINES

Canadian Sediment Quality Guidelines are developed using the methods outlined in “Protocol for the derivation of Canadian sediment quality guidelines for the protection of aquatic life” (CCME 1995, 1999). This protocol was created with the intent that guidelines be protective of all forms of aquatic life and all aspects of their aquatic life cycles during an indefinite period of exposure to substances associated with bed sediments.

Toxicity Data Data on the toxicity of nonylphenol in sediments to aquatic life were only available for invertebrates and amphibians, and were quite limited. These data are also presented in Appendix III.

Invertebrates Toxicity of nonylphenol in sediment has been determined for a single freshwater invertebrate species, the midge, Chironomus tentans(England and Bussard 1993). The study used spikedsediment toxicity testing to assess both lethal and sublethal effects of nonylphenol on the midge larvae over a 14 day exposure period. Acceptable spiking procedures were used and a standard test protocol was followed. For both mortality and growth, NOEC values were reported at 20.1 mg·kg -1 

### dry weight (England and Bussard 1993). A LOEC of 34.2 mg·kg -1 

### (dry weight) was observed for both mortality and growth, with 31% mortality and a growth reduction of 27% occurring at that concentration.

Two studies have examined the toxicity of nonylphenol to marine invertebrates, both through spiked-sediment toxicity testing. One study evaluated nonylphenol lethality to the amphipod Ampelisca abdita

(Fay et al. 2000). Standard sediment spiking and test procedures were followed. A ten day LC 50

### of 98.7 mg·kg -1 

### was observed, with 100% mortality occurring at a concentration of 243 mg·kg -1 

### . The other study evaluated sub-lethal effects of nonylphenol on the polychaete Capitella

sp. I over a 78 day exposure period (Hansen et al. 1999). Sub-lethal endpoints examined included time to first reproduction, time between reproductive events, number of broods per reproductive individual, number of eggs per individual and per reproductive individual, population growth rate, and size at maturity. For all of these endpoints, the NOEC was 52 mg·kg -1 

### and the LOEC was 174 mg·kg -1 

### (Hansen et al. 1999). The results of this study, however, should be treated with caution because standard sediment spiking procedures were not followed.

Amphibians Ward and Boeri (1992) used a 30 day spiked-sediment toxicity test to examine toxicity of nonylphenol to tadpoles of the bullfrog, Rana catesbiana . Standard sediment spiking and test procedures were followed. Reported endpoints for mortality included a NOEC of 155 mg·kg -1 

### and a LOEC of 390 mg·kg -1 

### with a calculated LC 50

### of 260 mg·kg -1 

### . The authors also examined sublethal endpoints such as growth, lethargy, haemorrhaging, and occurrence of tail lesions. For all of these sub-lethal effects, the NOECs were 155 mg·kg -1 

### , the LOECs were 390 mg·kg -1 

### , and EC 50

### s were calculated at 220 mg·kg -1 

### (Ward and Boeri 1992).

Protocol Approaches The protocol relies on the modified National Status and Trends Program (NSTP) approach and the Spiked Sediment Toxicity Test (SSTT) approach. If sufficient information exists to support both methods, a full sediment quality guideline may be recommended. If information is only available to support one of the approaches, then an interim sediment quality guideline is recommended. In cases where there is insufficient information to derive interim guidelines using either the modified NSTP approach or the SSTT approach, provisional guidelines may be

### 53

recommended by adopting guidelines from other jurisdictions. Further discussion of these three approaches is provided in Annex 3 of this document. At present, insufficient information exists to derive sediment quality guidelines for nonylphenol using any of the three approaches described in the protocol.

The protocol (CCME 1995) does not provide guidance on other possible options if there is insufficient data to meet the minimum data requirements for the NSTP and SSTT approaches, and if there are no acceptable sediment quality guidelines from other jurisdictions that can be adopted. One possibility would be to not derive a guideline at all. While acknowledging the need for toxicity data of the highest quality, we believe that guidelines based on the best available data and protocol approaches are still more practical, scientifically defensible, and protective of the environment than the absence of guidelines, or than guidelines that are not risk-based. Therefore, we are recommending the use of an equilibrium partitioning approach for the development of provisional Canadian interim sediment quality guidelines for nonylphenol and its ethoxylates.

Equilibrium Partitioning Approach The water/sediment equilibrium partitioning approach is used in the United States for development of sediment quality guidelines (MacDonald et al. 1992) and is also supported by the Ontario Ministry of the Environment (OMOE 1993). This approach establishes a guideline based on the concentration of a substance in sediments that ensures that the concentration in the interstitial water does not exceed the water quality guideline for that chemical at equilibrium (USEPA 1992). It is based on the assumption that continuous equilibrium exchange of a contaminant between sediment and interstitial water occurs (MacDonald et al. 1992). For non-polar organic substances, such as nonylphenol, it is known that concentrations in the interstitial water are dependent on concentrations in the sediment phase with the partitioning largely controlled by the total organic carbon (TOC) content of the sediment (MacDonald et al 1992). This appears to be true for NP and NPEs. Studies have shown that organic content is an important determining factor in the adsorption of at least the short-chain NPEs to sediments (Urano 1984; John et al. 2000). Johnson et al. (1998) found a positive relationship between amounts of octylphenol sorbed to sediments and the amount of TOC in the sediments; one would expect NP to behave similarly. Through spiked-sediment bioaccumulation tests with benthic amphipods, Hecht et al. (2000) found that higher concentrations of NP moved into the pore water of those sediments containing lower TOC concentrations. Based on similar studies, Gunnarsson et al. (2000) also found that pore water appears to be the major route of exposure to NP for benthic amphipods, and they concluded that the equilibrium partitioning theory is valid for nonylphenol.

As the protocol for the development of Canadian sediment quality guidelines (CCME 1995) allows for the adoption of guidelines from other jurisdictions that were derived using the equilibrium partitioning approach, it seems reasonable, and in keeping with the spirit of the protocol, that this approach could be used to derive sediment quality guidelines based on the Canadian water quality guidelines for nonylphenol and its ethoxylates. Sediment quality assessment values based on partitioning are less ecologically relevant and scientifically defensible than effect-based guidelines that are derived from direct sediment exposure studies (CCME 1995). The equilibrium partitioning method assumes that sediment-dwelling organisms and water column organisms are equally sensitive to nonylphenol and that the concentration of NP in sediment, interstitial water, and benthic organisms is at thermodynamic equilibrium. However, because there are insufficient data available for derivation of sediment quality guideline values by other methods, the equilibrium partitioning approach will be used to develop provisional ISQGs.

### 54

Guideline Derivation Using the equilibrium partitioning approach, provisional ISQGs were derived from the Canadian water quality guidelines for nonylphenol and its ethoxylates, developed in Chapter 5, using the following equation (Di Toro et al. 1991; MacDonald et al. 1992):

ISQG = WQG · K p 

### Equation 1 Where: WQG = water quality guideline (in μg·L-1 

### ); Kp 

### = sediment/water partition coefficient ( in L·kg -1 

### )

For non-polar organic contaminants such as nonylphenol, the organic carbon content of the sediment is a major factor controlling the partitioning between water and sediment (MacDonald et al. 1992). The sediment/water partition coefficient can therefore be related to the organic carbon/water partition coefficient by the following equation (Di Toro et al. 1991):

Kp

### = Koc

### ·foc 

### Equation 2 Where: Koc 

### = organic carbon/water partition coefficient (in L·kg oc-1 

### ); foc 

### = fractio n of organic carbon

Therefore, by substituting Equation 2 into Equation 1, the ISQG may be calculated as:

ISQG = WQG · Koc

### · foc 

### Equation 3

Sekela et al. (1999) estimated that the log K oc

### for nonylphenol ranges from 4.7 to 5.6. Taking the anti-log of these two values, and then calculating the geometric mean, yields a Koc

### of 141 254L·kg oc-1 

### . The freshwater Canadian WQG for nonylphenol, derived herein, is 1.0 μg·L-1



### . Substituting these values into Equation 3 yielded a freshwater ISQG of 141 mg NP per kg of organic carbon. This value was adjusted in order to express the guideline as dry weight of sediment, so that the guideline would be consistent with other Canadian SQGs. Therefore, by normalizing to 1% TOC (i.e., foc

### = 0.01), the freshwater ISQG of 1.4 mg·kg -1 

### dry weight was derived. It is recommended that the guideline be adjusted, as described in Chapter 9, based on local levels of TOC to obtain a site-specific objective.

Similarly for marine sediments, an ISQG was derived by substituting into Equation 3 the Koc

### of 141 254L·kg oc-1 

### , and the marine Canadian WQG of 0.7 μg·L -1 

### . This resulted in an ISQG of 99mgNP per kg of organic carbon. The value was expressed as dry weight of sediment by normalizing to 1% TOC, to give a marine ISQG of 1.0 mg·kg -1 

### dry weight. Again, it is recommended that the guideline be adjusted, as described in Chapter 9, based on local levels of TOC to obtain a site-specific objective.

Comparing these guideline values with the available sediment toxicity data, it would appear that they are likely to be protective of most sediment-dwelling organisms. The lowest effect concentrations observed in freshwater and marine sediment toxicity tests with nonylphenol were 34.2 mg·kg -1 

### and 174 mg·kg -1 

### , respectively. The guidelines are lower than these effect concentrations by more than a factor of 20, allowing for a large margin of safety.

It should be noted that because these guidelines were derived using an equilibrium partitioning approach, they represent sediment concentrations that will be protective of sediment-dwelling biota when the sediment and interstitial water are in equilibrium. Their use requires the assumptions that there are no changes in the thermodynamics of the system (i.e., constant temperature and pressure with no input/output of energy or mass), and that there has been sufficient time for the NP and/or NPEs to distribute throughout the system (Spacie et al. 1995). It is possible that non-equilibrium conditions could exist in some cases, such as immediately

### 55

following a spill, in river channels where the sediments are unstable, or in active dredge disposal areas (NYSDEC 1999). The USEPA SAB (1992) cautions against the use of guidelines based on equilibrium partitioning in areas of rapid sediment deposition or erosion. Where partitioning of nonylphenolic compounds between the sediment and interstitial water is not in equilibrium, it is possible that the sediment guidelines may not be protective against adverse effects to benthos.

Recommended Canadian Sediment Quality Guidelines

Freshwater Sediments Insufficient toxicological data exist for freshwater sediments to develop a freshwater TEL and PEL using the modified NSTP approach, or to calculate a freshwater SSTT value using the SSTT approach. There are also no sediment quality guidelines that currently exist in other jurisdictions that could be adopted. Therefore, a provisional freshwater ISQG was calculated from the Canadian freshwater WQG for nonylphenol proposed in this document using the equilibrium partitioning approach.

A provisional interim sediment quality guideline of 1.4 mg·kg -1

### dry weight, expressed in nonylphenol toxic equivalent units (NP TEQs) and normalized to 1% TOC, is recommended for the protection of freshwater life from the adverse effects of nonylphenol and its ethoxylates.

This guideline is recommended for concentrations of nonylphenolic substances in freshwater bulk surficial sediments (i.e., top 5cm). The guideline is expressed in nonylphenol toxic equivalency (NP TEQ) units and may be applied to mixtures of nonylphenol and nonylphenol ethoxylates. The total concentration of nonylphenolic substances in a sediment sample (in NP TEQs) should be calculated using the nonylphenol toxic equivalency factors provided in Chapter 3 (Table 4). The guideline should be adjusted for site-specific levels of total organic carbon. Further guidance and recommendations for the implementation of this guideline are provided in Chapter 9.

The distribution of the toxicity data for nonylphenol in freshwater sediments, as compared with the provisional ISQG is shown in Figure 5. There are no effects concentrations below the provisional freshwater ISQG; therefore, it is thought to be protective of aquatic organisms.

Marine Sediments As was the case for freshwater sediments, insufficient toxicological data exist for marine sediments to develop a marine TEL and PEL using the modified NSTP approach, or to calculate a freshwater SSTT value using the SSTT approach. Also, there are no marine sediment quality guidelines that currently exist for nonylphenol in any other jurisdictions. Therefore, a provisional marine ISQG was calculated from the Canadian marine WQG for nonylphenol proposed in this document by using the equilibrium partitioning approach.

A provisional interim sediment quality guideline of 1.0 mg·kg -1

### dry weight, expressed in NP TEQs and normalized for 1% TOC, is recommended for the protection of marine life from the adverse effects of nonylphenol and its ethoxylates.

This guideline is recommended for concentrations of nonylphenolic substances in marine bulk surficial sediments (i.e., top 5cm). The guideline is expressed in toxic equivalency (TEQ) units and may be applied to mixtures of nonylphenol and nonylphenol ethoxylates. The total concentration of nonylphenolic substances in a sediment sample (in NP TEQs) should be calculated using the nonylphenol toxic equivalency factors provided in Chapter 3 (Table 4). The guideline should be adjusted based on site-specific levels of total organic carbon. Further guidance and recommendations for the implementation of this guideline are provided in Chapter 9.

### 56

The distribution of the toxicity data for nonylphenol in marine sediments, as compared with the provisional ISQG is shown in Figure 6. There are no effects concentrations below the provisional marine ISQG; therefore, it is thought to be protective of aquatic organisms.

### 57 

# 1 10 100 1000 

# Nonylphenol (mg·kg -1 

# )Frequency 

## SQG = 1.4



# 0.1

Figure 5. Distribution of nonylphenol concentrations in freshwater sediments that are associated with adverse biological effects ( l ) and no adverse effects ( m ). The provisionally recommended Canadian freshwater interim sediment quality guideline (SQG) is indicated by the dotted line.



# 1 10 100 1000



# Nonylphenol (mg·kg -1 

# )Frequency 

## SQG = 1.0



# 0.1

Figure 6. Distribution of nonylphenol concentrations in marine and estuarine sediments that are associated with adverse biological effects ( l ) and no adverse effects ( m ). The provisionally recommended Canadian marine interim sediment quality guideline (SQG) is indicated by the dotted line.

### 58

CHAPTER 7. DEVELOPMENT OF CANADIAN SOIL QUALITY GUIDELINES

Canadian soil quality guidelines are developed using the methods outlined in “A protocol for the derivation of environmental and human health soil quality guidelines” (CCME 1996). This protocol was created with the intent that guidelines be protective of terrestrial biota and ensure a healthy functioning ecosystem capable of sustaining current and possible future uses of a site by ecological receptors and humans. Human health guidelines have not been developed for nonylphenol, therefore, this chapter discusses only the environmental health component of the Canadian soil quality guidelines for nonylphenol. According to the protocol, soil quality guidelines for each chemical are developed for four different land use categories: agricultural, residential/parkland, commercial, and industrial. A flow chart illustrating the steps that were taken in developing the environmental soil quality guidelines for nonylphenol is provided in Figure 7. The primary procedure for derivation of environmental soil quality guidelines is through the use of direct soil contact toxicological data. A second procedure based on soil and food ingestion is also applied in the case of agricultural land uses (if sufficient data is available), with adoption of the lowest of the two values as the guideline for agricultural land use.

Toxicity Data The first stage in development of environmental soil quality guidelines is the compilation of toxicity data that is evaluated based on the acceptability of experimental design, laboratory procedures, and documentation of relevant experimental conditions and parameters. Studies that provide high quality data are classified as “accepted” and this toxicity data may be used in deriving the guideline. Studies in which key information is missing, or in which experimental methods or conditions are questionable, are classified as “consulted” and are not used in the derivation of the guideline value, though their results may be taken into consideration. A comprehensive list of all soil toxicity data that was consulted has been compiled in Appendix IV. Studies were excluded from use because of one or more of the following reasons: 1. the test was not conducted using soil or artificial soil (e.g., conducted in nutrient media or on agar) 2. the test soil was amended with sewage sludge or a mixture of toxicants, such that effects could not be conclusively attributed to nonylphenol 3. important details of the experimental procedure were not described (e.g., number of NP concentrations tested, analytical methodology used, etc.) The primary data selected as acceptable for use in guideline derivation are shown in Table 11. Effective concentration data in this table were considered to be biologically significant in addition to statistically significant.

A brief description of the toxicity data relevant to the derivation of Canadian Soil Quality Guidelines is provided below. This information reflects the data requirements set forth in the protocol (CCME 1996) and is presented by taxonomic groups.

Microorganisms A number of studies have looked at the toxic effects of nonylphenol on microorganisms in artificial nutrient media, but only toxicity tests conducted with soil are described here. The effects of nonylphenol on the respiration of soil microorganisms, as measured through CO 2

### evolution, have been studied by two sets of researchers. In a 40 day test, Trocmé et al. (1988) observed no effect of CO 2

### evolution when nonylphenol was present in the soil at a concentration of 100mg·kg -1 

### , but at 1000 mg·kg -1 

### there was a significant reduction in CO 2

### evolution of 64%. In a three month assay, Kirchmann et al. (1991) observed no effect on CO 2 

### evolution at a nonylphenol concentration of 10 mg·kg -1 

### . At 500 mg·kg -1 

### Kirchmann et al. (1991) did see an effect, but in contrast with the results of Trocmé et al. (1988), the effect was an increase in CO 2 

### evolution by 26%. Kirchmann et al. (1991) also examined effects of nonylphenol on the nitrogen-cycling of microorganisms. No effects on nitrification were observed at a nonylphenol concentration of

### 59

10mg·kg -1 

### . With 500 mg·kg -1 

### , no effect on nitrogen mineralization occurred, but a 35% decrease in nitrification was observed.

Plants Toxicity studies have been conducted with a wide range of plants, or plant cell cultures, grown in culture medium (see Appendix IVc). These studies cannot be used in the derivation of a soil quality guideline, and therefore will not be discussed. Nonylphenol toxicity tests using soil have been conducted for five crop species. From a 14 day test with the turnip, Brassica rapa , an EC 5 

### for growth reduction of 10 mg·kg -1 

### was calculated (Günther and Pestemer 1992). Other reported endpoints are one to two orders of magnitude higher. In 21 day tests with nonylphenol, soybean ( Glycine max

), sunflower ( Helianthrus rodeo), and sorghum ( Sorghum bicolor ) all showed no growth effects at 100 mg·kg -1 

### , but had EC 50

### s for growth reduction at 1000 mg·kg -1 

### (Windeatt and Tapp 1986). EC 50

### s for growth reduction have also been reported for lettuce ( Lactuca sativa) at 559 mg·kg -1 

### after seven days of exposure, and at 625 mg·kg -1 

### after 14 days of exposure (Hulzebos et al. 1993).

Invertebrates Toxicity tests with nonylphenol have been conducted for three soil-dwelling invertebrates. From a 28 day exposure of the springtail Folsomia candida , Madsen et al. (1998) reported that the EC 50 

### for reproduction falls between 16 and 30 mg·kg -1 

### , and the median lethal concentration is greater than 30 mg·kg -1 

### . Researchers have also studied survival and reproduction in another species of springtail, Folsomia fimetaria . In 21 day exposures with this second species, Krogh et al. (1996) reported an LC 10

### of 75.1 mg·kg -1 

### and a LC 50

### of 151.3 mg·kg -1 

### . For reproduction in F. fimetaria

, Krogh et al. (1996) reported a 21 day EC 10

### of 23.6 mg·kg -1 

### , while Holm (as cited in Jensen et al. 1997) reported EC 10

### s of 27 mg·kg -1 

### and 48 mg·kg -1 

### . The EC 50

### s for reproduction in F. fimetaria were reported at 65.5 mg·kg -1 

### (Krogh et al. 1996) and 39 mg·kg -1 

### and 59 mg·kg -1 

### (Holm, as cited in Jensen et al. 1997). The effects of nonylphenol on the earthworm Apporectodea calignosa in a 21 day exposure was also studied (Krogh et al. 1996). The median lethal concentration occurred at greater than 40 mg·kg -1 

### . The median effective concentration for growth reduction was 23.9mg·kg -1 

### . The authors calculated the EC 10

### and EC 50

### for effects on reproduction as 3.44mg·kg -1 

### and 13.7 mg·kg -1 

### , respectively.

Mammals The only mammals which have been tested for nonylphenol toxicity through ingestion are laboratory rats. In a multigenerational study, Chapin et al. (1999) examined the effects of nonylphenol administered through dosed food on Sprague Dawley rats ( Rattus norvegicus). At a diet concentration of 650 mg·kg -1 

### (i.e., a dose of 30-108 mg·kg -1 

### body weight) vaginal opening at an earlier age was observed in the F1 generation. Significant effects observed at a diet concentration of 2000 mg·kg -1 

### (i.e., a dose of 100-360 mg·kg -1 

### body weight) included increased relative kidney weights and decreased adult ovary weights in the F1 generation, and increased estrous cycle length in both the F1 and F2 generations. Laws et al. (2000) reported LOECs for increased uterine weight and earlier vaginal opening both at a diet concentration of 50 mg·kg -1



### , and disruption of estrous cyclicity at a diet concentrations of 100 mg·kg -1 

### . A LOEC of 75 mg·kg -1 

### body weight per day for increased uterine weight in ovariectomized female rats has been reported by Odum et al. (1999).

### 60 AGRICULTURAL RESIDENTIAL/PARKLAN D COMMERCIAL & I NDUSTRIAL

SOIL SOIL AND FOOD SOIL SOIL CONTACT INGESTION CONTACT CONTACT

Calculate TEC Calculate DTED* Calculate TEC Calculate ECL

 ̊

WOE LOEC ME WOE LOEC ME WOE LOEC Method Method Method Method Method Method Method Method  ̧  ̧  ̧

Nutrient and Energy Nutrient and Ene rgy Nutrient and Energy Cycling Check* Cycling Check* Cycling Check*

 ̊  ̊  ̊

SQG SC 

### SQG I 

### * SQG SC 

### SQG SC



###  ̧  ̊  ̧  ̧

Take lowest of two values

SQG E 

### SQG E 

### SQG E



###  ̧  ̧  ̧

Groundwater Groundwater Groundwater Check* Check* Check*

 ̧  ̧  ̧

Figure 7. Overview of steps leading to the development of final Environmental Soil Quality Guidelines for nonylphenol. TEC = threshold effects concentration; DTED = daily threshold effects dose; ECL = effects concentration low; WOE = weight of evidence; LOEC = lowest observed effect concentration; ME = median effects; SQG SC

### = soil quality guideline for soil contact; SQG I

### = soil quality guideline for soil and food ingestion; SQG E

### = final environmental soil quality guideline  ̧Indicates steps used in the derivation of guidelines for nonylphenol.

 ̊Indicates values that were not calculated in the derivation of guidelines for nonylphenol due to insufficient data. * Indicates steps that are not mandatory for deriving a guideline if insufficient data is available.

### 61

Table 11. Primary data available for toxicity of nonylphenol though direct soil contact.

Species (Common name) Duration Endpoint Effect Concentration (mg·kg -1 

### ) Reference

Plants Brassica rapa (turnip) 14d EC 5 

### growth 10 Günther and Pestemer 1992 Glycine max (soybean) 21d NOEC growth 100 W indeatt and Tapp 1986 21d EC 50 

### growth 1000 W indeatt and Tapp 1986
Helianthrus rodeo (sunflower) 21d NOEC growth 100 W indeatt and Tapp 1986 21d EC 50 

### growth 1000 W indeatt and Tapp 1986
Lactuca sativa (lettuce) 7d EC 50 

### growth 559 Hulzebos et al. 1993

14d EC 50 

### growth 625 Hulzebos et al. 1993 Sorghum bicolor (sorghum) 21d NOEC growth 100 W indeatt and Tapp 1986 21d EC 50 

### growth 1000 W indeatt and Tapp 1986

Invertebrates Apporectodea 21d EC 10 

### reproduction 3.44 Krogh et al. 1996 calignosa 21d EC 50 

### reproduction 13.7 Krogh et al. 1996 (earthworm) 21d EC 50 

### growth 23.9 Krogh et al. 1996 21d EC 10 

### mortality >40 Krogh et al. 1996 21d EC 50 

### mortality >40 Krogh et al. 1996
Folsomia candida 28d EC 50 

### reproduction 16-30 Madsen et al. 1998 (springtail) 28d LC 50 

### survival >30 Madsen et al. 1998
Folsomia fimetaria 21d LC 10 

### survival 75.1 Krogh et al. 1996 (springtail) 21d LC 50 

### survival 151.3 Krogh et al. 1996 21d EC 10 

### reproduction 23.6 Krogh et al. 1996 21d EC 10 

### reproduction 27 Holm* 21d EC 10 

### reproduction 48 Holm* 21d EC 50 

### reproduction 65.5 Krogh et al. 1996 21d EC 50 

### reproduction 39 Holm* 21d EC 50 

### reproduction 59 Holm*

* As cited in Jensen et al. 1997

NOEC = no observed effect concentration ECx 

### = concentration at which effects are observed in x% of the population LCx 

### = concentration that is lethal to x% of the population

### 62

Agricultural and Residential/Parkland Land Uses

Soil Contact There are slight differences in the derivation procedures for soil contact guidelines (SQG SC

### ) among land uses such that a higher level of protection is provided for agricultural and residential/parkland soils than for commercial and industrial soils. For agricultural and residential/parkland land use, derivation of the threshold effect concentration (TEC) due to direct soil contact may proceed by one of three possible methods (weight of evidence method, lowest observed effect concentration method, median effects method), depending on the toxicity data available. A nutrient and energy cycling check is performed (when sufficient data are available) and compared with the TEC. The lower of these two values is adopted as the soil quality guideline for soil contact.

The preferred approach for derivation of the soil contact guideline is to use a weight of evidence method that considers a range of acceptable endpoints. The minimum requirements for this approach are 10 data points from at least three studies, with at least two soil invertebrate species and at least two plant species represented. According to Section 7.5.2.2 of the protocol (CCME 1996), the geometric mean should be used when multiple data are available for the same endpoint with the same species. For the nonylphenol data, geometric means have been applied to the three EC 10

### values and the three EC 50

### values reported for reproduction in springtails (Krogh et al. 1996; Holm as cited in Jensen et al. 1997). The soil toxicity data for nonylphenol satisfied the minimum data requirements with a total of 20 data points (after geometric means were taken for multiple data) from 6 studies representing 5 plant species and 3 soil invertebrate species (see Table 11).

For agricultural and residential/parkland land uses, if the minimum data requirements are met, then the TEC may be calculated by taking the 25th

### percentile of the frequency distribution (i.e., the “no potential effects” range) and dividing it by an uncertainty factor (if deemed necessary).

Therefore,

TEC = NPER ÷ UF

where, TEC = threshold effects concentration (mg·kg -1 

### soil) NPER = no potential effects range (25 th

### percentile of distribution) UF = uncertainty factor (if needed)

Using rank percentile and linear reqression methods, the 25 th

### percentile of the soil contact toxicity data was calculated as 17.15 mg·kg -1 

### soil (Figure 8).

Although there were ample data available representing a wide range of taxonomic groups, the use of an uncertainty factor was deemed necessary. There was a preponderance of median lethal and median effective concentration data that may have introduced some bias into the dataset. Also, more than 25% of the data below the 25th

### percentile were definitive effects data (i.e., LCx 

### and ECx



### data, as opposed to LOECs and NOECs). Therefore, as recommended in the protocol (CCME 1996), an uncertainty factor of 3 was chosen.

Thus, TEC = 17.15 ÷ 3 = 5.72 mg·kg -1 

### soil ≈5.7 mg·kg -1 

### soil

### 63 

# y = 38.25 [Log(x)] - 22.22 R 2

# = 0.95

0 10 20 30 40 50 60 70 80 90 100

1 10 100 1000
Nonylph enol Concentr ation ( mg·kg -1 

# )Rank Percentile



# 25 th

# percentile

17.15

Figure 8. Distribution of soil contact toxicity data used in the guideline derivation for agricultural and residential/parkland land uses. The no potential effects range (i.e., the 25 th

### percentile of the distribution) is indicated by the dotted line.

### 64

Nutrient and Energy Cycling Check Once a soil contact TEC has been calculated, it is then checked against a microbial (nutrient and energy cycling) value. Microorganisms are an essential component of healthy, functioning soils. The ability of soil to support plant life depends on the coordinated activities of microorganisms that mediate processes such as decomposition, respiration, and organic nutrient cycles. The rates of these processes may be affected by contaminants, therefore, the microbial check provides an additional tool for assessing the toxicological impact of soil-associated contaminants on terrestrial life.

The microbial check may be calculated by weight of evidence, LOEC, or median effects methods, depending on available data. Preferred endpoints are nitrogen fixation and nitrification, although carbon cycling and nitrogen mineralization data may also be considered.

The data that were available for microbial responses to nonylphenol are presented in Appendix IVa. Acceptable endpoints included a NOEC and a LOEC for nitrification, a NOEC for nitrogen mineralization, and two NOECs and two LOEC for respiration (Trocmé et al. 1988; Kirchmann et al. 1991). These data were insufficient to meet the minimum requirements for the weight of evidence, LOEC, or median effects approaches, so a nutrient and energy cycling check could not be calculated. It appears though, on the basis of the acceptable LOEC values that are available for microbial processes (ranging from 500 to 1000 mg·kg -1 

### ), that microorganisms are generally less sensitive to nonylphenol than other terrestrial organisms. Hence, it is unlikely that omission of this check will result in a lack of protection for microbial processes. To calculate the nutrient and energy cycling check using the LOEC method, at least one more study reporting a LOEC value would be required. For the weight of evidence approach, several studies reporting median effects data would be required, preferably with endpoints of nitrogen fixation and nitrification.

As a nutrient and energy cycling check could not be calculated, the soil contact TEC of 5.7mg·kg -1 

### was adopted as the soil quality guideline for soil contact (SQG SC

### ) for both agricultural and residential/parkland land uses.

Soil and Food Ingestion The soil quality guideline for soil and food ingestion (SQG I 

### ) applies only to agricultural land use. Derivation of the soil and food ingestion guideline first involves calculating the daily threshold effects dose (DTED). A minimum of three studies is required, at least one of which is an oral avian study, and two of which are oral mammalian studies (no more than one laboratory rodent study, and preferably including an ungulate study). The DTED is calculated as the lowest observed adverse effect level (LOAEL) from the most sensitive species divided by an uncertainty factor. The soil quality guideline for ingestion is then calculated such that the ingestion of soil and plants growing on the soil does not result in the most sensitive species receiving more than 75% of the DTED. More detailed information on these calculations is provided in the protocol (CCME 1996).

There is a lack of data on the toxicity of nonylphenol to grazing livestock and wildlife through ingestion. The few studies that have been conducted have all examined effects on laboratory rodents (Appendix IVd).

The minimum data requirements cannot be met for calculating a soil quality guideline for ingestion (SQG I 

### ). Rough calculations based on the data available suggest that an SQG I

### likely would not be lower than the SQG SC 

### ; however, due to the high uncertainty involved, no conclusions concerning ingestion can be made at this time.

### 65

Commercial and Industrial Land Uses

Soil Contact For commercial and industrial land use, the effects concentration low (ECL) due to direct soil contact may be calculated using either the weight of evidence method or LOEC method, depending on the toxicity data available. A nutrient and energy cycling check is performed (when sufficient data are available) and the resulting microbial value is compared with the ECL.

As with the agricultural and residential/parkland land uses, the weight of evidence approach was used because the minimum data requirements for this method were met. For commercial and industrial land uses, if the minimum data requirements are met, then the ECL may be calculated by taking the 25th

### percentile of the “effects” data distribution only (rather than the 25th

### percentile of the entire data distribution, as is done for the NPER). At this percentile, called the effects range low (ERL), it is expected that some effects will be incurred by soil-dependent biota, but not at the level of median lethality in the population(s). Less stringent protection levels are required than for residential/parkland and agricultural land because the primary land use activities of commercial and industrial lands are not directly dependent on the need to sustain a high level of ecological processes. For this same reason, it is not desirable to achieve lower estimates of the ECL using uncertainty factors.

Therefore, the effects concentration low (ECL) for nonylphenol was calculated as

ECL = ERL

where, ECL = effects concentration low (mg·kg -1 

### soil) ERL = effects range low (25 th

### percentile of effects data distribution, in mg·kg -1 

### soil)

Using ranked percentile and linear reqression methods, the 25 th

### percentile of the soil contact effects data was calculated as 14.16 mg·kg -1 

### soil (Figure 9).

Thus, ECL = 14.16 mg·kg -1 

### soil ≈14 mg·kg -1 

### soil

### 66 

## y = 34.863 [Log(x)] - 15.126 R 2

## = 0.94

0 10 20 30 40 50 60 70 80 90 100

1 10 100 1000
Nonylph enol Concentr ation ( mg·kg -1 

## )Rank Percentile



# 25th

# percentile

14.16

Figure 9. Distribution of soil contact effects toxicity data used in guideline derivation for commercial and industrial land uses. The effects range low (i.e., the 25 th

### percentile of the effects data distribution) is indicated by the dotted line.

### 67

Nutrient and Energy Cycling Check Once a soil contact ECL has been calculated, it is then checked against a microbial (nutrient and energy cycling) value. The microbial check may also be calculated by either the weight of evidence method or the LOEC method, depending on available data. Preferred endpoints are nitrogen fixation and nitrification, although carbon cycling and nitrogen mineralization data may also be considered. If the microbial value is lower than the ECL, then significant adverse effects may be experienced by microorganisms controlling nutrient and energy cycling processes. In this case, the geometric mean of the two values is taken and used as the soil quality guidelines for soil contact (SQG SC

### ) for commercial and industrial land use. However, if the microbial value is above the ECL, then a level of effects consistent with the objective of this procedure is expected and the ECL is adopted directly as the SQG SC

### for commercial and industrial land use.

The data that were available for microbial responses to nonylphenol are presented in Appendix IVa. These data were insufficient to meet the minimum requirements for either the weight of evidence method or the LOEC method. To calculate the nutrient and energy cycling check using the LOEC method, at least one more study reporting a LOEC value would be required. For the weight of evidence approach, several studies reporting median effects data would be required, preferably with endpoints of nitrogen fixation and nitrification.

As a nutrient and energy cycling check could not be calculated, the soil contact ECL of 14 mg·kg -1 

### was adopted as the soil quality guideline for soil contact (SQG SC

### ) for both commercial and industrial land uses.

Groundwater Check A major concern with soil contamination is that it can lead to contamination of groundwater. Soil quality guidelines for all four classes of land use must be designed to account for possible transfers to groundwater such that concentrations of a substance in groundwater do not exceed the water quality guidelines for the protection of freshwater aquatic life. To prevent such an exceedence, a groundwater check is calculated based on equilibrium partitioning between solid and aqueous phases. The applicability of equilibrium partitioning to nonylphenol is discussed in Chapter 6. For non-ionic organics, such as nonylphenol, the following equation (CCME 1996) is used:

Y = DF · Cwa 

### · (Kd 

### + 

## θ m

### )

where, Y = concentration of nonylphenol in soil (mg NP·kg -1 

### dry soil) DF = dilution factor = 50 (default) C wa 

### = water protection value = Canadian water q uality guideline (mg·L-1 

### ) Kd 

### = distribution coefficient 

## θ m 

### = soil moisture content = 0.1 (default)

The distribution coefficient (Kd

### ) is directly related to the organic matter content and can be derived using the sorption partition coefficient (Koc

### ) and the organic fraction of dry soil (foc

### ) as described by the equation:

Kd

### = Koc

### · foc 

### A default value of 0.3% (or 0.003) is used for foc

### (CCME 1996). The log Koc

### for nonylphenol has been estimated to range from 4.7 to 5.6 (Sekela et al. 1999), so, a mean Koc

### can be determined by taking the anti-log of these two values and then calculating the geometric mean.

Therefore,

### 68

Koc 

### = 141 254 L·kg oc-1 

### Kd 

### = 423.8 L·kg soil -1



### For the protection of freshwater aquatic life, a water quality guideline of 1.0 μg·L-1 

### (or 0.001 mg·L -1 

### ) was developed for nonylphenol in Chapter 5. Therefore:

Y = (50) (0.001) (423.8 + 0.1)

= 21.2 mg NP·kg -1 

### dry soil

The groundwater check indicates that for the protection of freshwater aquatic life, the level of nonylphenol in soil should not exceed 21 mg·kg -1



### .

Off-site Migration Check Contaminated soil can be transferred from one property to another through erosion by water and wind with subsequent deposition. This transfer of contaminants can result in unacceptable degradation of an adjacent property that was previously uncontaminated, or used for a more sensitive land use. Therefore, an off-site migration check was calculated to determine the concentration of nonylphenol in soil from an industrial site that could, through erosion, hypothetically result in levels on an adjacent residential/parkland property that exceed the residential/parkland SQG E 

### . The check value was calculated using the following equation (CCME 1996):

C i

### = (14.3 · SQG RP

### ) - (13.3 · BSC)

where, C i 

### = concentration of nonylphenol in the eroded soil (mgNP·kg -1 

### ) SQG RP 

### = soil quality guideline for residential/parkland land use BSC = background concentration of nonylphenol in the receiving soil (mgNP·kg -1 

### )

The soil quality guideline for soil contact for residential/parkland land uses is 5.7 mg·kg -1 

### , and the background concentration of nonylphenol is assumed to be zero. Substituting these values into the equation yields the following off-site migration check value:

C i

### = 82 mgNP·kg -1



### The off-site migration check indicates that for the protection of adjacent properties, the level of nonylphenol in soil on industrial sites should not exceed 82 mg·kg -1



### .

Environmental Soil Quality Guideline Derivation (SQG E

### ) The final environmental soil quality guideline for agricultural land use is determined by taking the lower of either the soil contact guideline (SQG SC

### ) or the soil ingestion guideline (SQG I 

### ), if both are available. If there are not enough data to calculate a soil ingestion guideline, then the soil contact guideline is taken as the final environmental guideline; however, the final environmental guideline cannot be derived if there is insufficient data for the soil contact guideline. For all other soil uses, the soil contact guideline is used as the final environmental soil quality guideline.

Because no data were available on toxicity of nonylphenol through ingestion of food or soil, the SQG SC 

### of 5.7 mg·kg -1 

### will be taken as the final SQG E

### for agricultural land use. For the other three land uses, the SQG SC

### is used as the SQG E 

### also. Therefore, the SQG E 

### s for residential/parkland, commercial, and industrial land uses are 5.7 mg·kg -1 

### soil, 14 mg·kg -1 

### soil, and 14 mg·kg -1 

### soil, respectively. The groundwater check value was higher than all of the SQG E

### values, indicating that these guidelines are sufficiently low to protect against contamination of groundwater. The off-site migration check value was higher than the SQG E

### for industrial land uses, indicating that this

### 69

guideline is sufficiently low to protect against degradation of adjacent lands through deposition of eroded soil.

Recommended Canadian Soil Quality Guidelines The final Canadian Soil Quality Guidelines for a substance are determined as the lower of the environmental soil quality guideline (SQG E 

### ) and the human health soil quality guideline (SQG HH 

### ) for each land use. Because human health soil quality guidelines have not been developed for NP and NPEs, the environmental guidelines will be taken as the final Canadian Soil Quality Guidelines for nonylphenol and its ethoxylates. A summary of the soil quality guideline values for each land use type is provided in Table 12. These guidelines are expressed in nonylphenol toxic equivalency units (NP TEQs) and may be applied to mixtures of nonylphenol and nonylphenol ethoxylates. The total concentration of nonylphenolic substances in a soil sample (in NP TEQs) should be calculated using the nonylphenol toxic equivalency factors provided in Chapter 3 (Table 4). Further guidance and recommendations for the implementation of these guidelines are provided in Chapter 9.

The Canadian environmental soil quality guidelines recommended for agricultural and residential/parkland land use are higher than the Danish soil quality criterion for nonylphenol (see Table 8). The Danish criterion was based on the 21 day EC 50

### for reduced reproduction in earthworms at 13.7 mg·kg -1 

### as determined by Krogh et al. (1996). This endpoint, which represented the lowest toxicity value in their data set, was divided by an application factor of 1000 and rounded off to 0.01 mg·kg -1 

### . It is due to this extremely conservative application factor that the Danish soil criterion is so much lower than the Canadian soil guideline. Both Sweden and Denmark have also set limits for NP in sludge used as agricultural fertilizer (Table 8). The Danish guidelines refer to the total concentration of 4-NP, NP1EO and NP2EO in sludge (European Commission 1999; Danish Ministerial Order No. 823, Sept. 16, 1996). The higher guideline values for sludge, compared with soil, reflect the fact that the nonylphenol concentrations in the sludge will be effectively diluted through the application of the sludge over a large area of soil.

### 70

Table 12. Summary of soil quality guideline derivation for nonylphenol. *

Land use
Guideline (mg·kg -1 

### ) Agricultural Residential/ Parkland Commercial Industrial

Final Canadian SQG 5.7 5.7 14 14
SQG E 

### 5.7 5.7 14 14
SQG SC 

### 5.7 5.7 14 14 Nutrient and energy cycling NC NC NC NC

SQG I 

### NC NA NA NA

Groundwater check (freshwater aquatic life) 21 21 21 21

Off-site migration check NA NA NA 82
SQG HH 

### NC NC NC NC

* All guideline values are expressed in toxic equivalent (TEQ) units. SQG E

### = soil quality guideline for environmental health; SQG SC

### = soil quality guideline for soil contact; SQG I

### = soil quality guideline for soil and food ingestion; SQG HH

### = soil quality guideline for human health NC = not calculated; NA = not applicable

### 71

CHAPTER 8. AREAS FOR FUTURE RESEARCH

The guidelines developed in this document are based on the best available data. In the process of deriving these guidelines, however, it was noted that there are certain areas of study in which limited data are available for nonylphenol and its ethoxylates. This chapter highlights some of the gaps in our knowledge of these substances with the hope that it will provide stimulation and direction for further research.

Concentrations in Canada Historically, concentrations of NP and NPEs have not been routinely monitored in the Canadian environment. Consequently, there is limited data concerning the distribution and levels of these substances that occur in Canada. In particular, there is either no data, or very limited data, that exist for Canadian marine and estuarine waters, marine sediments, groundwaters, soils, and industrial effluents and sludges. There is also a lack of data on levels of NPECs and NP nEOs ( n≥3) in freshwater sediments.

Fate and Behaviour

There is a need for further studies into the fate and behaviour of NP and NPEs in the environment. In particular, more field studies on the fate of these substances in sediments are needed because most work to date has been conducted in a laboratory setting. There does not appear to have been any work done on the effects of photolysis on NP/NPEs present on soil surfaces. There also has been no research into the effects of varying pH on the aquatic toxicity or bioavailability of nonylphenol. Further investigation into the biological fate of NP and NPEs is also needed. Specifically, the data available on uptake and elimination rates in invertebrates, uptake by aquatic plants, and uptake by terrestrial animals are limited.

Toxicity Testing An area of research that has been largely unexplored is the toxic effects of NP and NPEs on mammalian and avian consumers of aquatic life. There are very limited toxicity data available for sediment-dwelling organisms. Toxicity testing has also not been conducted for terrestrial wildlife and grazing livestock.

The toxicity data that does exist for aquatic algae, plants, invertebrates and vertebrates is largely for NP, NP1-2EO, NP9-10EO, and, to a lesser degree, NP1-2EC. There is a need for more toxicity data on other NPE oligomers, particularly those containing between 3 and 8 ethoxylate groups.
Further investigation into the mode of toxicity for these substances would also be useful. At present, the exact mechanisms of toxicity via narcosis are unclear. There is also some controversy over the exact mechanisms involved in the endocrine disrupting effects produced by these substances.

Guideline Derivation and Implementation When more toxicity data becomes available, it may be possible to upgrade the status of the marine interim WQG developed in this document. A full marine WQG could be derived if more chronic toxicity data becomes available for marine fish and for marine invertebrates other than crustaceans.

An unconventional approach (equilibrium partitioning) was used to derive the sediment quality guidelines in this document due to a lack of sediment toxicity data. In the future, if more sediment toxicity data becomes available for nonylphenol it may be possible to derive guidelines which are based on approaches in the CCME protocol (CCME 1995). In order to use the NSTP approach,

### 72

12 more entries in the effect data set and 3 more entries in the no-effect data set would be required for the freshwater SQG; 11 more entries in the effect data set and 6 more entries in the no-effect data set would be required for the marine SQG. Alternatively, the freshwater SQG could be derived using the SSTT approach if three more studies on sediment-resident invertebrates areconducted, including at least one study on a benthic amphipod, and at least one partial or full lifecycle test that reports ecologically relevant sub-lethal endpoints. A marine SQG could be derived using the SSTT approach if at least two more studies on sediment-resident invertebrates are conducted, at least one of which is a partial or full life-cycle test with sub-lethal endpoints.

The soil quality guidelines in this document were developed without the calculation of either a soil and food ingestion guideline, or a nutrient and energy cycling check. The soil and food ingestion guideline could be calculated in future if at least one oral avian study was conducted, and at least one oral mammalian study was conducted with a mammal other than a laboratory rodent, but preferably with an ungulate species. To calculate a nutrient and energy cycling check, at least one more study reporting a LOEC value would be required, or several studies reporting median effects data for endpoints such as nitrogen fixation and nitrification.

This document recommends the use of TEF values that were developed by Servos et al. (2000). Further work is needed to verify and refine these TEF values. For example, a single TEF is currently given for the entire group of NPEs ranging from NP9EO and up. It may be possible to develop oligomer-specific TEFs if variations in toxicity are known to occur for different ethoxylate chain lengths. Due to a lack of toxicity data for NPEs with 3 to 8 EO groups, a conservative TEF value was estimated from the other TEFs. Further work is need to develop TEFs for this group of ethoxamers.

At present, we are recommending that OP, OPEs, and OPECs be included in the TEQ approach for nonylphenolic substances. Further investigation is needed to confirm whether these substances should be grouped together, or whether they warrant separate guidelines. As with NPEs, further refinement and validation of the TEF values for OPEs is needed.

### 73

CHAPTER 9. GUIDANCE ON IMPLEMENTATION

General Guidance for all Guidelines The legislative authority for implementation of Canadian Environmental Quality Guidelines (EQGs) lies with each provincial and territorial jurisdiction (with the exception of federal lands). The provincial and territorial governments may develop their own science-based criteria, guidelines, objectives and standards, but often the Canadian EQGs form the scientific basis for these (CCME 1999). Canadian EQGs may also form the basis for federal use-specific guidelines and objectives.

The Canadian EQGs developed in this document for nonylphenol should be used along with all other relevant information in making practical and informed decisions regarding water, sediment, and soil quality. It is widely recognized that no single assessment tool can always be used to predict whether adverse biological effects will occur as a result of contaminant exposure. Rather, the appropriate use of several different tools provides the most useful information (Chapman 1995). Apart from EQGs, other scientific tools available to assist in the protection and management of water, sediment, or soil quality include other assessment values (such as the PEL for sediment), biological assessments, or guidelines for other media.

It should also be noted that socio-economic or technological factors that may influence the application of Canadian EQGs are not considered in their development process. However, suchfactors may play a varying role in the application of EQGs (and/or in the development of sitespecific objectives) within the decision-making framework of different jurisdictions and programs (Gaudet et al. 1995).

Negligible risk to biota, their functions, or any interactions that are integral to sustaining the health of ecosystems and the designated resource uses they support, should result when nonylphenol (and other nonylphenolic substances) are present in the ambient environment at the concentrations recommended by the Canadian EQGs developed in this document. It should be noted, however, that for ecosystems of superior quality, impairment to these guideline concentrations is not advocated.

Additional general information on the use of Canadian EQGs is available in the introductory chapter of the publication Canadian Environmental Quality Guidelines (CCME 1999). More specific guidance, relevant to the guidelines for nonylphenol and its ethoxylates, is provided below.

Application of the TEQ Approach to EQGs When comparing environmental samples to the guidelines developed in this document, it is important that concentrations of NP and all NPEs and NPECs in the sample are measured, converted to nonylphenol toxic equivalents (NP TEQs), and then summed. It is also recommended that OP, OPEs and OPECs, if present in the sample, be included in the calculation of the total NP TEQ. Toxic equivalency factors (TEFs) are provided in Table 4 of Chapter 3. The following example illustrates the proper use of the TEQ approach and its application towards the Canadian Environmental Quality Guidelines for nonylphenol and its ethoxylates.

Consider a water sample collected from a freshwater stream that has been analysed for all possible nonylphenolic substances. The sample was found to contain 0.1 μg·L-1 

### of NP, 1.0 μg·L-1 

### of NP1EO, 0.2 μg·L-1 

### of NP3EO, and 2.0 μg·L-1 

### of NP1EC. The TEF values for toxicity of NP, NP1EO and NP1EC are 1, 0.5, and 0.005, respectively, and we will assume a relative potency of 0.5 for NP3EO (see Table 4). Therefore, using the equation

Total concentration = ∑(Cx 

### x TEF),

### 74

and plugging in the concentrations from our water sample, we get:

Total concentration = (0.1 μg·L-1 

### x 1) + (1.0 μg·L-1 

### x 0.5) + (0.2 μg·L-1 

### x 0.5) + (2.0 μg·L-1 

### x 0.005)

= 0.71 μg·L-1



### Therefore, our water sample contains 0.71 μg·L-1 

### of nonylphenolic substances, as expressed in NP TEQ units. This total concentration 0.71 μg·L-1 

### is the value that should then be compared against the nonylphenol Canadian Water Quality Guideline for the protection of freshwater aquatic life (i.e., 1.0 μg·L-1 

### ). In this example then, the water sample does not exceed the freshwater guideline for nonylphenol and its ethoxylates and adverse effects to aquatic life are not expected to occur in this water.

These same NP TEF values should be used to determine total NP TEQs for sediment and soil samples before comparing them to the respective guideline values. Narcosis is a common mode of toxicity for nonylphenolic substances across all taxonomic groups (see earlier discussion under “Modes of Toxicity” in Chapter 3), so the NP TEF values should be applicable to plants,invertebrates, fish, mammals, etc., regardless of whether they are water-, sediment-, or soildwelling species.

Water Quality Guidelines The toxicological data upon which these guidelines are based were conducted within the pH ranges of 5.6 to 8.9 and 7.3 to 9.6 for fresh and marine waters, respectively. Most natural waters will have pH values within these ranges. However, site-specific considerations may be needed for those locations where the water pH falls outside of these ranges. In particular, these guidelines may not be sufficiently protective for highly alkaline waters. The acid dissociation constant (pKa

### ) of nonylphenol is 10.7, indicating that in most natural waters NP will remain predominantly in the undissociated form. In waters with a pH greater than 10.7, the dissociated form, which is more soluble and may differ in bioavailability or toxicity, will be present. It is currently unknown whether pH has any effect on the aquatic toxicity or bioavailability of NP and NPEs.

The presence or absence of sensitive species at a local site is another factor that might play a role in the development of site-specific objectives.

These guidelines refer to concentrations in unfiltered samples.

Sediment Quality Guidelines The interim sediment quality guidelines presented here represent the concentrations ofnonylphenol or nonylphenol equivalents in sediments below which adverse effects in sedimentdwelling aquatic biota are not expected to occur. Although these interim sediment quality guidelines are considered provisional at this time, they should not be used differently than they would be if they were full sediment quality guidelines. The revision and re-assessment of these provisional ISQGs will be possible when sufficient data become available to support the use of the modified NSTP or SSTT approaches as described in the formal protocol (CCME 1995).

Using the equilibrium partitioning approach, the provisional interim sediment quality guidelines were normalized to 1% TOC (foc

### = 0.01) in order to express them in terms of sediment dry weight. In jurisdictions where the percentage of organic carbon in sediments is known to be consistently higher or lower than 1%, local estimates of TOC may be used to set site-specific sediment objectives. This is done by multiplying the ISQG by the percentage of organic carbon. For example, for freshwater sediments with 2% TOC, the freshwater ISQG of 1.4 mg·kg -1 

### would be multiplied by 2 to obtain a site-specific objective of 2.8 mg·kg -1 

### . With lower levels of organic carbon there will be less binding of contaminants to the sediments, and consequently greater bioavailability. Therefore, it is important that the foc

### value used is based on the lowest sediment organic carbon concentration that might possibly occur, such that all aquatic life in the designated

### 75

jurisdiction will be protected. It is not recommended that foc

### values outside of the range 0.002 to 0.12 be used in deriving site-specific objectives. For sediments that contain less than 0.2% or greater than 12% TOC, other factors that the equilibrium partitioning method does not account for may influence the contaminant partitioning (USEPA SAB 1992). Further generic guidance regarding the implementation of Canadian sediment quality guidelines is provided in CCME (1995).

Soil Quality Guidelines The toxicological studies upon which these guidelines are based did not report soil pH in many cases; however, where reported, the studies were conducted within the pH range of 5.8 to 7.5. Therefore, site-specific consideration may be required for soils that fall outside of this pH range.

In particular, these guidelines may not be sufficiently protective for sites with highly alkaline soils (i.e., with a pH greater than 10). The high acid dissociation constant of nonylphenol (pKa

### = 10.7) indicates that in most natural environments NP will remain predominantly in the undissociated form. Extremely high pHs may create dissociated forms that are less readily sorbed than the parent nonylphenol, and much more mobile in soil. In these cases, the groundwater check as utilized in this document will not be applicable (see CCME 1996), and the development of more conservative site-specific guidelines than presented here may be necessary to ensure adequate protection.

### 76

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Ward, T.J. and R.L. Boeri. 1992. Toxicity of nonylphenol to the tadpole Rana catesbiana . Report No. 8981-CMA. Envirosystems Division, Resource Analysts Inc. Washington, D.C., Chemical Manufacturers Association. 78 pp.

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ANNEX I. ECOLOGICAL RELEVANCE OF ENDOCRINE DISRUPTION

Environmental guidelines must be based on toxicity data that have some ecological relevance. Conventional toxicity endpoints, such as mortality, growth inhibition, and reduced reproductive output are widely acknowledged as ecologically-relevant effects that can cause impacts at organizational levels higher than the individual (i.e., population, community and ecosystem levels). There has recently been much debate over the ecological relevance of effects caused by endocrine disrupters. Endocrine disrupting chemicals (EDCs) work at various biochemical levels, such as affecting the synthesis of hormones, hindering movement of hormones through the blood by hormone transporting proteins, upsetting the metabolism of hormones, or by directly interfering with cellular hormone receptors (Bjerregaard et al. 1998). However, even though an EDC may cause biological and physiological changes within an organism, whether or not these changes affect whole organism performance in terms of survival, reproduction or growth will depend on the hormone affected, the degree of impairment, the timing of the exposure in terms of the organism’s life stage, and the relative sensitivity of the particular species (Van Der Kraak 1998).

Evidence of endocrine disruption, in various forms, has been extensively documented in vertebrates such as fish, marine mammals, predatory birds, and even alligators (Fry and Toone 1981; Reijnders 1986; Brouwer et al. 1989; Munkittrick et al. 1991, 1992; Jobling and Sumpter 1993; Purdom et al. 1994; Fry 1995; Guillette et al. 1994, 1995, 1996; Harries et al. 1997). There has been less documentation of endocrine disruption in invertebrates species; most cases have involved daphnids or marine molluscs (Taylor et al. 1999). Although there is a wealth of data describing the effects of endocrine disrupters at the cellular and biochemical levels, very few studies have investigated the long-term ramifications of these effects at the population or community level (Hutchinson and Matthiessen 1999). In some cases, although population-level changes have been observed, the effects have not been linked to a specific chemical.

Ankley and Giesy (1998) have developed a weight of evidence approach for conclusively demonstrating ecologically relevant effects of an EDC. First, there must be documentation of effects in individuals and of adverse effects in populations. Second, there must be a connection between the effects observed in populations and those observed in individuals. Third, a plausible mode of action must be identified that is consistent with the effects observed in the individuals. Fourth, there must be positive identification of specific contaminants operating through this mode of action. Fifth, the exposure metrics must be reasonable in terms of individual-level effects occurring via the mode of action under consideration. And finally, there should be evidence that the population or individuals will recover upon removal of the chemical stressor.

For NP, short-chain NPEs and NPECs, there is extensive documentation of effects at the individual level, and the mechanism of action is generally understood. However, how these effects on individuals may translate into adverse effects at the population level is not well understood.

In the case of fish, to determine the significance for populations of endocrine disrupter effects such as vitellogenin induction and intersex states, it will first be necessary to establish their impacts on individual fitness (Taylor et al. 1999). It is important to establish possible links between the vitellogenin response and ecologically important parameters that are relevant to population dynamics, such as semen quality, testicular development, histological changes and offspring sex ratio and development (Bjerregaard et al. 1998). A few studies have examined the effects of xenobiotically-induced vitellogenesis on the individual fitness of fish. Carragher and Sumpter (1991) found that when they induced vitellogenin synthesis in juvenile rainbow trout with 17ß-estradiol, there was a significant decrease in the amount of calcium in the fish scales, and a significant increase in plasma calcium levels (possibly for incorporation into vitellogenin). The authors suggested that at higher demands for calcium due to vitellogenesis, the scales could become loosened, thus compromising the fish’s defenses. Therefore, there may be a link between vitellogenesis and fish survival rates (Carragher and Sumpter 1991). Herman and

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Kincaid (1988) found that when rainbow trout underwent prolonged vitellogenin induction due to ßestradiol exposure, mortality rates increased, and necrosis and damage to the kidney and liver tissues were severe. Induction of vitellogenin in male fish has also been correlated to decreases in testicular growth and serum testosterone levels (Jobling et al. 1996; Folmar et al. 1996). These effects might have a bearing on reproductive success.

In their multigenerational study with rats, Chapin et al. (1999) observed increased estrous cycle length in those individuals exposed to NP. This estrogenic response could have adverse effects at the population level because lengthened estrous cycles have been associated with reduced fecundity (Chapin et al. 1997).

It seems likely that these estrogenic effects of NP and short-chain NPEs and NPECs could result in population-level effects. However, at this point such effects are largely speculative and actual documentation of population-level effects in the field are needed.

In some cases, population-level effects have been observed due to NP, but further research is needed to confirm that these effects are the result of endocrine disruption. For example, adverse effects of NP on the smoltification of juvenile Atlantic salmon ( Salmo salar ) have been observed in laboratory studies (Madsen et al. 1997; Brown et al. 1998), and resultant decreases in population sizes have been documented in the field (Fairchild et al. 1999). Although the authors suspect endocrine disruption, the mechanism by which NP affects smoltification is unknown.

In an attempt to address the current data gaps concerning the ecological impacts of EDCs, there has been a call for more multigenerational toxicity tests with suspected endocrine disrupters (Taylor et al. 1999). Studies examining population parameters such as offspring survival and recruitment are needed to assess the effects of long-term exposure to EDCs. The OECD is currently working to develop and validate test methods for assessing the potential of new and existing chemicals for endocrine disruption (Taylor et al. 1999).

Endocrine disruption endpoints were considered in the development of the guidelines in this document. In the aquatic toxicity dataset, the range of concentrations at which EDC effects were observed were comparable with the range of concentrations for more conventional endpoints. The lowest primary toxicity data available for both the freshwater and marine Water Quality Guidelines used conventional endpoints, so, there was no need to decide whether a guideline could be based on endpoints related to endocrine disruption, and whether these endpoints are ecologically relevant.

If future research concludes that EDC effects due to NP and short-chain NPEs do result in ecologically relevant impacts on populations, then there may be a need to re-examine the toxic equivalency factors that are recommended in this document. Servos et al. (2000) calculated relative estrogenicity values for various alkylphenolic compounds, relative to NP. These relative estrogenicity values for NP1EO, NP2EO, NP nEO ( n≥9), NP1EC, NP2EC, OP, OP1EC and OP2EC are 0.67, 0.67, 0, 0.63, 0.63, 4.1, 0.63 and 0.63, respectively. These relative estrogenicities differ from the TEF values based on conventional toxicity endpoints. For example, although NPECs have a low relative conventional toxicity, with TEFs of 0.005, they are considerably more estrogenic, with relative estrogenicity values estimated at 0.63. Octylphenol, though similar to NP in toxicity, is about four times more estrogenic. Therefore, environmental samples which do not exceed the guidelines based on conventional toxicity TEFs, could still presumably cause adverse effects due to estrogenic effects, particularly if they contain significant quantities of OP, OPECs, or NPECs. In future, this is an issue that will need to be resolved as more and more information on EDCs is coming to light.

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ANNEX 2. DATASET REQUIREMENTS FOR DERIVING WATER QUALITY GUIDELINES

Water quality guidelines are based on a single, ecologically-relevant, toxicity endpoint for the most sensitive life stage of the most sensitive aquatic species investigated. To determine the concentration that represents the most sensitive and protective toxicity endpoint, toxicological data are evaluated according to a number of screening criteria. The screening criteria (e.g., acceptable experimental design, laboratory procedures, documentation of relevant experimental conditions and parameters) have been described in detail in the protocol (CCME 1991; CCME 1999). Toxicity studies are classified as primary, secondary, or unacceptable. Only primary studies may be used in the actual derivation of the WQG. Secondary studies, however, may be used to help meet the minimum toxicological data set requirements.

Studies which were identified as being of primary quality, and therefore could be used in guideline derivation, met all of the following criteria: 1. flow-through or renewal test conditions were used (rather than static) 2. the study was conducted under controlled laboratory conditions rather than in the field 3. water parameters, such as temperature, pH, and hardness, were measured and reported 4. sufficient replication was used 5. more than a single concentration of NP was tested 6. concentrations of NP in the water were measured (i.e., did not just report nominal concentrations) 7. conventional endpoints with documented ecological relevance were used 8. dose-dependent concentration responses were observed 9. experimental conditions and/or analytical methods were described sufficiently to evaluate the validity of the study (note: data obtained from published abstracts, citations in other articles/reports, or from articles published in foreign languages were considered unacceptable)

The following discussion summarizes the minimum data set requirements for development of freshwater and marine WQGs.

Freshwater

For a full freshwater life guideline, the toxicological data set of primary studies must minimally include the following:

• at least three studies on three or more freshwater fish species resident in North America, including at least one cold-water species (e.g., trout) and one warm-waterspecies (e.g., fathead minnow), and at least two must be chronic (partial or full lifecycle) studies

• at least two chronic (partial or full lifecycle) studies on two or more invertebrate species from different classes, one of which includes a planktonic species resident in North America (e.g., daphnid).

• at least one study of a freshwater vascular plant or freshwater algal species resident in North America (note: for highly phytotoxic variables, four acute and /or chronic studies on nontarget freshwater plant or algal species are required)

The primary data available for freshwater toxicity due to nonylphenol met all of the minimum dataset requirements.

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Marine

The minimum requirements for a full marine guideline are the same as those for freshwater life except that the studies must include temperate marine fish, invertebrates and plants/algae. Also, the invertebrate studies must include two species from different classes, but it is not necessary that one of those species be planktonic.

The marine toxicity dataset for nonylphenol was unable to meet these minimum requirements, and therefore a full marine guideline could not be derived.

When the minimum data requirements for derivation of a full guideline cannot be met, it may still be possible to derive an interim guideline. The minimum data set requirements for an interim marine life guideline are:

• at least two acute and/or chronic studies on two or more marine fish species, one of which is a temperate species

• at least two acute and/or chronic studies on two or more marine invertebrate species from different classes, one of which is a temperate species Secondary studies, as well as primary studies, may be used to meet the minimum requirements for an interim guideline.

Studies which were considered of secondary quality, and therefore could be used to meet the minimum data requirements for an interim guideline, met the same criteria as the primary studies, except for one or more of the following characteristics : 1. test conditions were static (rather than flow-through or renewal) 2. only nominal (rather than measured) concentrations of NP in water were reported 3. ecologically relevant endpoints other than survival, reproduction or growth were reported

There were sufficient marine toxicity data to meet the minimum requirements for the derivation of an interim guideline.

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ANNEX 3. SUMMARY OF SEDIMENT QUALITY GUIDELINE PROTOCOL

There are three approaches to deriving sediment quality guidelines that are recommended in the CCME protocol: the National Status and Trends Program Approach, the Spiked-Sediment Toxicity Test Approach, and adoption of guidelines from other jurisdictions (CCME 1995). The data available for nonylphenol did not support the use of any of these three approaches, and therefore they were not used in the final derivation of Canadian Sediment Quality Guidelines for nonylphenol and its ethoxylates. This annex provides a brief summary of the three protocol approaches and includes a discussion of the nonylphenol sediment toxicity data that would be needed to support the use of these guideline derivation methods.

The Modified National Status and Trends Program (NSTP) Approach

The NSTP approach to deriving sediment quality assessment values was originally developed by Long and Morgan (1990). This approach was adopted, with some modifications, for use in developing sediment quality guidelines in Canada (CCME 1995) and is therefore referred to in this document as the modified NSTP approach. Modifications to this approach include the separate evaluation of information for freshwater and marine systems, an expansion of the information originally compiled, and the use of derivation procedures that consider all of the compiled information (see also Long and MacDonald 1992; MacDonald 1994; CCME 1995; and MacDonald et al. 1996 for more detailed explanations of these modifications).

The NSTP approach primarily relies on the use of field-collected data, in which chemical mixtures occur, to derive sediment quality assessment values (Long & Morgan 1990; Long 1992; Long and MacDonald 1992; MacDonald 1994; CCME 1995; Long et al. 1995). Data for individual studies are evaluated to establish an association between the concentration of each of the chemicals measured in the sediments and any adverse biological effect observed. Cause-and-effect relationships between the concentration of individual chemicals and the observed adverse biological effect cannot be inferred from this evaluation.

Other sediment chemical and biological data may also be used, including models of equilibrium partitioning in sediments, sediment quality assessment values from other jurisdictions (e.g., those derived using the apparent effects threshold approach and the screening level concentration approach), spiked-sediment toxicity tests, and field studies (including acute and chronic toxicity results from sediment bioassays and analyses of benthic community composition). Candidate studies are critically evaluated according to a number of screening criteria to ensure that high quality data sets are used. These screening criteria (e.g., appropriatesediment handling procedures, acceptable toxicity test procedures) have been described in more detail elsewhere (MacDonald 1994; CCME 1995; Long et al. 1995; Environment Canada 1996).

Acceptable data is sorted by sediment type (i.e., freshwaterversus marine) and arranged in ascending order of the chemical's concentration to produce separate ascending data tables , or guideline derivation tables , for individual chemicals and sediment types (CCME 1995; MacDonald 1994; Long et al. 1995). These tables summarize the compiled information that associates a chemical's concentration with either adverse effects or no adverse effects on aquatic organisms (also referred to as the effect data set and the no-effect data set, respectively).Concentrations are expressed as the total concentrations of chemicals in sediments on a dryweight basis. Many of the previous Canadian sediment quality guidelines were derived based on ascending data tables produced from a large central database called the Biological Effects Database for Sediments (BEDS). However, the guideline derivation tables for nonylphenol did not originate from the BEDS database, but instead were independently compiled.

The guideline derivation tables for individual chemicals (e.g., nonylphenol; Tables 13 and 14) consist of a number of entries, each of which belongs to either the effect data set(concentrations associated with an adverse biological effect) or the no-effect data set(concentrations not

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associated with an adverse biological effect), that are sorted according to ascending chemical concentrations. Entries in the guideline derivation tables were designated as being associated with an effect (an asterisk in the 'Hit' column) if an adverse biological effect was reported. These effects included acute or chronic toxicity observed during a controlled spiked-sediment test, apparent effect thresholds (concentrations above which specific biological effects would always be expected), and predicted toxicity based on equilibrium partitioning theory (which determines a sediment chemical concentration that ensures the concentration of the chemical in the interstitial water does not exceed the water quality guideline for that chemical at equilibrium). An entry was also assigned an asterisk (effect descriptor) if concordance was apparent between the observed biological response and the measured chemical concentration in a field (i.e., co-occurrence) study. Concentrations of individual chemicals reported in field studies were considered to be associated with the observed toxic response (i.e., concordancewas apparent) if the mean concentration at sites at which significant adverse effects were observed was a factor of two or more greater than the mean concentration at sites at which effects were not observed (i.e., at toxic versus non toxic sites) (Long et al. 1995). A factor of two was chosen to ensure that the difference in the response (i.e., adverse effect) was associated with a signifi cant difference in the chemical concentration. For each chemical, all of the entries designated by an asterisk, as described above, are collectively referred to as the effect data set

. All entries in the guideline derivation tables other than those designated by an asterisk are collectively referred to as the no-effect data set , and are represented by those entries for which chemical concentrations were not associated with adverse biological effects. These entries include those associated with non-toxic, reference, or control conditions (i.e., no effects; NE).

Individual entries in the guideline derivation tables consist of the chemical concentration in the sediment, an indication of whether this concentration was part of the effect data set (i.e., *) o r the no-effect data set (i.e., NE), location of the study, analysis type (or approach used), test duration, toxicological end-point measured, species and life stage tested, information on the characteristics of the sediments if available (e.g., concentrat ions of TOC, physical classification of the sediments), and the study reference. In most cases, information for individual entries (i.e., chemical concentrations and sediment chemical and physical characteristics) represents the means of several samples. Standard deviations of these means are provided wherever possible. The following discussion summarizes the information contained in the freshwater and marine guideline derivation tables for nonylphenol (Tables 13 and 14).

Guideline Derivation Tables for Nonylphenol

The freshwater guideline derivation table for nonylphenol (Table 13) contains 25 entries, all of which are results from spiked-sediment toxicity tests (SSTT). The freshwater s edimentassociated nonylphenol concentrations in the table range from 2.34 to 680mg·kg -1 

### dw. A number of adverse biological effects are associated with nonylphenol concentrations ranging from 34.2 to 680mg·kg -1 

### dw.

Chironomids ( Chironomus tentans) and frogs ( Rana catesbiana) are the only freshwater taxa for which toxicological information was obtained and included in the freshwater guideline derivation table. Effects on survival and growth are the most common indicators of adverse biological effects measured for sediment bioassays with laboratory-spiked sediments. Other chronic toxicological endpoints tested include lethargy, haemorrhaging, and tail lesions.

Information on total organic carbon (TOC), pH of the sediment and/or overlying water column, water temperature, conductivity, chemical oxygen demand (COD), biological oxygen demand (BOD), cation exchange capacity (CEC), and the physical classifications of the bed sediments is also included for each entry in the freshwater guideline derivation table, when available (Table 13). The mean TOC content of sediments ranges from 0.05 to 1.27%. The mean proportions of sand, silt, and clay in the freshwater sediments represented in the ascending data table range from 26.4 to 99.4%, <0.7 to 52%, and <0.7 to 21.6%, respectively.

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The marine guideline derivation table (Table 14) contains 23 entries, all of which are results from spiked-sediment toxicity tests (SSTT). Concentrations of sediment-associated nonylphenol contained in the table range from 14 to 243 mg·kg -1 

### dw (Table 14). Adverse biological effects associated with nonylphenol in marine sediments occur at concentrations of 98.7 to 243 mg·kg -1 

### dw.

Only two taxa, the amphipod Ampelisca abdita and the polychaete Capitella sp., are represented in the marine guideline derivation table. Most of the observed adverse biological effects were measurements of various reproductive effects, both at the individual and population level. Size of the organisms at maturity and survival were other endpoints examined.

Information on total organic carbon (TOC), temperature, and salinity were also included for each entry in the marine guideline derivation table (Table 14). The mean TOC content of sediments was 7.4%. The grain size composition of the sediments was not reported in either study.

Derivation of TELs and PELs

As is described in CCME (1995), the derivation procedures used to calculate two assessment values in the original NSTP approach (Long and Morgan 1990) were modified to consider both the effect and no-effect data compiled in the guideline derivation tables. In the modified NSTP approach, the lower value, referred to as the threshold effect level(TEL), represents the concentration below which adverse biological effects are expected to occur rarely. The upper value, referred to as the probable effect level(PEL), defines the level above which adverse effects are expected to occur frequently. Concentrations that fall in the range between the TEL and the PEL are occasionally expected to be associated with adverse biological effects. The definition of these ranges (also referred to as the minimal, possible, and probable effect ranges) is based on the assumption that the potential for observing toxicity of sediment-associated chemicals increases with increasing chemical concentrations (Long et al. 1995).

Minimum toxicological data requirements have been set to ensure that the TELs calculated from the guideline derivation tables provide adequate protection of aquatic life and that these values are supported by a weight of evidence for a broad range of sediment types and characteristics. For a given guideline derivation table, both the effect data set and the no-effect data set must contain at least 20 entries (CCME 1995). In the case of nonylphenol, the freshwater guideline derivation table contained 8 entries in the effect data set and 17 entries in the no-effect data set. For marine sediments, there were 9 entries in the effect data set and 14 entries in the no-effect data set. Therefore, both the freshwater and marine datasets contained insufficient data to derive TELs or PELs for nonylphenol. To derive a freshwater sediment quality guideline using the NSTP approach, 12 more entries in the effect data set and 3 more entries in the no-effect data set would be required. For derivation of a marine sediment quality guideline using the NSTP approach, 11 more entries in the effect data set and 6 more entries in the no-effect data set would be required to meet the minimum data requirements.

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The Spiked-Sediment Toxicity Test (SSTT) Approach

The SSTT approach, which involves an independent evaluation of information from spikedsediment toxicity tests (that are included in the guideline derivation tables described above), is a complementary procedure to the modified NSTP approach for estimating the concentration of a chemical in sediments below which adverse effects are not expected to occur (CCME 1995). In contrast to the modified NSTP approach, which is used to derive TELs and PELs using information on associations between chemical concentrations in sediments and effects on sediment-associated organisms, the SSTT approach is used to derive SSTT values using data from controlled laboratory tests in which organisms are exposed to sediments that have beenspiked with known concentrations of a chemical. Such studies provide quantifiable cause-andeffect relationships between the concentration of a chemical in sediments and the observed biological response (e.g., survival, reproductive success, growth). Spiked-sediment toxicity tests are generally used to evaluate the toxic effect of a single chemical, or a specific mixture of chemicals, to exposed organisms. They may also be used to determine the extent to which environmental conditions modify the bioavailability of a chemical, and ultimately the response of organisms exposed to the spiked-sediments.

Data from spiked-sediment toxicity tests must be evaluated and determined to be acceptable before it can be used in the SSTT approach (CCME 1995). In acceptable studies, information on the analytical chemistry of the sediments and overlying water (particularly characteristics that might influence bioavailability of sediment-associated chemicals) should be reported for both the beginning and end of the test. Variables that should be measured in the overlying water include pH, dissolved oxygen, total suspended solids, suspended and dissolved organic carbon, and water hardness or salinity. In the sediment, reported variables should include total organic carbon, particle size distribution, acid volatile sulphide, pH, redox conditions, and sediment type. Generally accepted laboratory practices of exposure and environmental controls should be employed and tests that follow standardized guides or protocols are preferable. Information on the health and survival of the test species for at least a week prior to the test, and repeated evaluation of the condition of the test species throughout the test, should be reported. Where significant mortalities have occurred prior to the test period, the data should not be used. Concentrations of the chemical in sediment must be measured – nominal concentrations are not acceptable. Also, appropriate statistical procedures should be used and reported in detail. Further discussion of data evaluation in provided in the protocol (CCME 1995).

Minimum toxicological data requirements have been set for the SSTT approach to ensure that SSTT values derived using spiked-sediment toxicity information provide adequate protection to aquatic organisms (CCME 1995). Data requirements include at least four independent studies on two or more sediment-resident invertebrate species that occur in North American waters. For freshwater sediments, at least one species must be a benthic crustacean and one must be a benthic arthropod (other than a crustacean). For marine sediments, at least one species must be a benthic amphipod. SSTT values can be derived from studies conducted on sensitive species (e.g., fish, aquatic plants, protozoa, fungi, bacteria) provided that the minimum data set requirements are met. An additional requirement is that at least two of the studies must be partial or full life cycle tests that consider ecologically-relevant endpoints (e.g., growth, reproductive success, developmental effects). In addition to these requirements, the procedures used to generate spiked-sediment toxicity data must be evaluated and determined to be appropriate before they can be used to develop SSTT values. Although methods (e.g., spikingprocedures, equilibration periods) for spiked-sediment toxicity tests are currently not standardized (Environment Canada 1995), information provided in individual studies (e.g., equilibriumtime, stability of concentrations over the test duration, responses in control treatments) can be used to evaluate the influence of the use of specific methods on test results.

Two independent studies have examined the toxicity of nonylphenol to North American freshwater organisms in spiked-sediment toxicity tests (Appendix IIIa). These studies evaluated nonylphenol toxicity (lethality) to the midge, Chironomus tentans (England and Bussard 1993), and to the

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amphibian, Rana catesbiana (Ward and Boeri 1992). England and Bussard (1993) also assessed sub-lethal endpoints (i.e., growthand reproduction) for Chironomus tentans, and Ward and Boeri (1992) examined sub-lethal endpoints for Rana catesbiana such as growth, lethargy, haemorrhaging, and occurrence of tail lesions. W ith only two independent studies, insufficient data are available to derive an SSTT value for freshwater sediments. At least three additional independent studies on North American freshwater sediment-resident invertebrates, at least one of which is a benthic amphipod, are required. Also, at least one of the studies must be a partial or full life-cycle test that reports ecologically relevant sub-lethal endpoints (e.g., growth, reproduction, developmental effects) in order to fulfill the minimum data requirements for the derivation of a freshwater SSTT value.

Two independent studies examined the toxicity of nonylphenol to a marine organism in spikedsediment toxicity tests (Appendix IIIb). One study evaluated nonylphenol toxicity (lethality) to the amphipod Ampelisca abdita (Fay et al. 2000). The other study evaluated sub-lethal effects of nonylphenol on the polychaete Capitella sp. (Hansen et al. 1999). Sub-lethal endpoints examined included time to first reproduction, time between reproductive events, number of broods per reproductive individual, number of eggs per individual and per reproductive individual, population growth rate, and size at maturity. These data, however, are insufficient to derive an SSTT value for marine sediments. At least two additional independent studies on North American marine sediment-resident invertebrates are required. Also, at least one of the studies must be a partial or full life-cycle test that reports ecologically relevant sub-lethal endpoints (e.g., growth, reproduction, developmental effects) in order to fulfill the minimum data requirements for the derivation of a marine SSTT value.

Adoption of Guidelines from Other Jurisdictions

When insufficient information is available to derive ISQGs using either the modified NSTP or SSTT approaches, sediment quality assessment values from other jurisdictions are evaluated and, if appropriate, adopted provisionally as ISQGs (CCME 1995). This adoption process gives preference to biological-effects based sediment quality assessment values that have been derived using methods that are philosophically consistent with the protocol’s guiding principles. Guidelines that have been derived using effects-based approaches are considered to be the most ecologically relevant and scientifically defensible. Using this approach, the lowest of theguidelines that incorporate data on the effects of sediment-associated substances on sedimentdwelling organisms is selected. For organic contaminants, such as nonylphenol, guidelines derived using the equilibrium partitioning approach can also be considered for adoption.

No sediment quality guidelines for NP or NPEs, either for freshwater or marine sediments, currently exist in any other jurisdiction. Therefore, the approach of adopting sediment guidelines from another jurisdiction cannot be used to develop Canadian sediment quality guidelines.

### 111

### 112

Table 13. Summary of the available biological effects and related physicochemical data for

NP (mg·kg -1 ) Hit Analysis

Type Test

Type Endpoint Measured Species Life Stage TOC

(%) pH sediment 2.34 NE SSTT 14d not toxic (7% mortality) Chironomus tentans (freshwater midge) larvae 1.27 5.5

2.34 NE SSTT 14d not toxic (13% growth increase) Chironomus tentans (freshwater midge) larvae 1.27 5.5

4.79 NE SSTT 14d not toxic (11% mortality) Chironomus tentans (freshwater midge) larvae 1.27 5.5

4.79 NE SSTT 14d not toxic (10% growth increase) Chironomus tentans (freshwater midge) larvae 1.27 5.5

9.51 NE SSTT 14d not toxic (8% mortality) Chironomus tentans (freshwater midge) larvae 1.27 5.5

9.51 NE SSTT 14d not toxic (13% growth increase) Chironomus tentans (freshwater midge) larvae 1.27 5.5

20.1 NE SSTT 14d not toxic (10% mortality) Chironomus tentans (freshwater midge) larvae 1.27 5.5

20.1 NE SSTT 14d not toxic (6% growth reduction) Chironomus tentans (freshwater midge) larvae 1.27 5.5

34.2 * SSTT 14d toxic (31% mortality) Chironomus tentans (freshwater midge) larvae 1.27 5.5

34.2 * SSTT 14d toxic (27% growth reduction) Chironomus tentans (freshwater midge) larvae 1.27 5.5

36 NE SSTT 30d not toxic (0% mortality) Rana catesbiana (bullfrog) tadpole 0.05 NR

36 NE SSTT 30d not toxic (5% sublethal effects) Rana catesbiana (bullfrog) tadpole 0.05 NR

36 NE SSTT 30d not toxic (6% growth reduction) Rana catesbiana (bullfrog) tadpole 0.05 NR

57 NE SSTT 30d not toxic (0% mortality) Rana catesbiana (bullfrog) tadpole 0.05 NR

57 NE SSTT 30d not toxic (0% sublethal effects) Rana catesbiana (bullfrog) tadpole 0.05 NR

57 NE SSTT 30d not toxic (7.5% growth reduction) Rana catesbiana (bullfrog) tadpole 0.05 NR

155 NE SSTT 30d not toxic (10% mortality) Rana catesbiana (bullfrog) tadpole 0.05 NR

155 NE SSTT 30d not toxic (10% sublethal effects) Rana catesbiana (bullfrog) tadpole 0.05 NR

155 NE SSTT 30d not toxic (25% growth reduction) Rana catesbiana (bullfrog) tadpole 0.05 NR

220 * SSTT 30d toxic (sublethal effects and growth) Rana catesbiana (bullfrog) tadpole 0.05 NR

260 * SSTT 30d toxic (50% mortality) Rana catesbiana (bullfrog) tadpole 0.05 NR

390 * SSTT 30d toxic (85% mortality) Rana catesbiana (bullfrog) tadpole 0.05 NR

390 * SSTT 30d toxic (100% sublethal effects - lethargy, haemorrhaging, tail lesions) Rana catesbiana (bullfrog) tadpole 0.05 NR

390 * SSTT 30d toxic (29% growth reduction) Rana catesbiana (bullfrog) tadpole 0.05 NR

680 * SSTT 30d toxic (100% mortality) Rana catesbiana (bullfrog) tadpole 0.05 NR

TOC = total organic carbo n Temp. = temperature Cond. = conductivity measured in μMhos·cm -1 COD = chemical oxygen demand BOD = biological oxygen demand CEC = cation exchange capacity NE = no effect * = effect d = days NR = not reported

### 113

sediment-associated nonylphenol (mg·kg -1 

### dry weight) in freshwater ecosystems.

pH water Temp.

(°C) Cond. COD

(mg·L-1 ) BOD

(mg·L-1 ) CEC

(meq·L-1 ) Sand

(%) Silt (%) Clay (%) Area Reference

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

7.2-8.5 19-21 320 352.5 9.2 23.9 26.4 52 21.6 Laboratory England and Bussard 1993

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

6.9-8.3 17.1-18.8 740 620 μg/g 45 μg/g 2.3 meq/100g 99.4 <0.7 <0.7 Laboratory Ward and Boeri 1992

### 114

Table 14. Summary of the available biological effects and related physicochemical data for

NP (mg·kg -1 ) Hit Analysis

Type Test

Type Endpoint Measured Species Life Stage TOC

(%) pH sediment 14 NE SSTT 78d not toxic (1% decrease in time to first reproduction) Capitella sp. I (polychaete) larvae to adult 7.4 NR

14 NE SSTT 78d not toxic (7% increase in time between reproductive events) Capitella sp. I (polychaete) larvae to adult 7.4 NR

14 NE SSTT 78d not toxic (2% decrease in number of broods per reproductive individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

14 NE SSTT 78d not toxic (12% increase in number of eggs per reproductive individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

14 NE SSTT 78d not toxic (25% decrease in number of eggs per individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

14 NE SSTT 78d not toxic (11% increase in size at maturity) Capitella sp. I (polychaete) larvae to adult 7.4 NR

14 NE SSTT 78d not toxic (6% increase in population growth rate) Capitella sp. I (polychaete) larvae to adult 7.4 NR

52 NE SSTT 78d not toxic (0% change in time to first reproduction) Capitella sp. I (polychaete) larvae to adult 7.4 NR

52 NE SSTT 78d not toxic (3% increase in time between reproductive events) Capitella sp. I (polychaete) larvae to adult 7.4 NR

52 NE SSTT 78d not toxic (2% increase in number of broods per reproductive individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

52 NE SSTT 78d not toxic (3% decrease in number of eggs per reproductive individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

52 NE SSTT 78d not toxic (3% decrease in number of eggs per individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

52 NE SSTT 78d not toxic (16% decrease in size at maturity) Capitella sp. I (polychaete) larvae to adult 7.4 NR

52 NE SSTT 78d not toxic (0% change in population growth rate) Capitella sp. I (polychaete) larvae to adult 7.4 NR

98.7 * SSTT 10d toxic (50% mortality) Ampelisca abdita (amphipod) juvenile NR NR

174 * SSTT 78d toxic (17% increase in time to first reproduction) Capitella sp. I (polychaete) larvae to adult 7.4 NR

174 * SSTT 78d toxic (46% increase in time between reproductive events) Capitella sp. I (polychaete) larvae to adult 7.4 NR

174 * SSTT 78d toxic (44% decrease in number of broods per reproductive individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

174 * SSTT 78d toxic (78% decrease in number of eggs per reproductive individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

174 * SSTT 78d toxic (76% decrease in number of eggs per individual) Capitella sp. I (polychaete) larvae to adult 7.4 NR

174 * SSTT 78d toxic (33% decrease in size at maturity) Capitella sp. I (polychaete) larvae to adult 7.4 NR

174 * SSTT 78d toxic (24% decrease in population growth rate) Capitella sp. I (polychaete) larvae to adult 7.4 NR

243 * SSTT 10d toxic (100% mortality) Ampelisca abdita (amphipod) juvenile NR NR

TOC = total organic carbon Temp. = temperature NE = no effect * = effect d = days NR = not reported

### 115

sediment-associated nonylphenol (mg·kg -1 

### dry weight) in marine ecosystems.

Temp. (°C) Water Salinity

(g·L-1 ) Sand

(%) Silt (%) Clay (%) Area Reference

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 29-32 NR NR NR Laboratory Fay et al. 2000

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 26-27 NR NR NR Laboratory Hansen et al. 1999

20 29-32 NR NR NR Laboratory Fay et al. 2000

### 116 

## APPENDIX I

Toxicity of nonylphenol and its ethoxylates to freshwater life

through water column exposure

### APPENDIX 1a: Toxicity of nonylphenol to freshwater algae and macrophytes through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hard.

ness** Temp.

(°C) Evaluation

Rank Reference

Ceratophyllum demersum (coontail) NR 21d S, N bleaching EC 100 6250 6.8 NR NR 23 U Weinberger and Greenhalgh 1984

Chlamydomonas reinhardii NR 1h S, N membrane disruption NR 10 NR NR NR 22 U Weinberger & Rea 1981

(flagellate algae) NR 1h S, NR inhibition of photosynthesis ICF 100 500-750 6.7 NR NR 23 U Moody et al. 1983

NR 21d S, N growth (biomass) LOEC 1000 NR NR NR 23 U Weinberger & Greenhalgh 1984

NR 21d S, N algistasis LOEC 6250-1000 NR NR NR 23 U Weinberger & Greenhalgh 1984 9d old culture 24h S, N ATP reduction EC 75 EC20 6250

2500 NR NR NR 23 U Weinberger & Greenhalgh 1984

Chlamydomonas segnis (algae) 4 life cycle phases 24h S, N algistatic or algicidal NR 2500 6.8 NR NR 23 2 Weinberger et al. 1987

NR 24h NR,NR mortality LC 50 1500 NR NR NR NR U Weinberger 1984 1
NR 24h NR,NR inhibition of photosynthesis NR 500-750 NR NR NR NR U Weinberger 1984 1

Chlorella pyrenoidosa (algae) NR 24h S, N mortality LC 50 LC 100 1500

25000 6.8 NR NR 22 U Weinberger & Rea 1981, 1982

Lemna minor (duckweed) 2-4 fronds 4d R, N growth (dry matter production) LOEC 125 NR NR NR 20 U Prasad 1989

2-4 fronds 6d S, N growth (frond production) LOEC 500 NR NR NR 20 U Prasad 1989

2-4 fronds 4d S, N growth (dry matter production) LOEC 500 NR NR NR 20 U Prasad 1989

NR 7d S, N growth (new frond production) LOEC 1250 5.6 NR NR 25 U Weinberger and Iyengar 1983

NR 7d S, N growth (fresh and dry mass) LOEC >1250 >2500 5.6 NR NR 25 U Weinberger and Iyengar 1983

NR 7d S, N chlorophyll content LOEC 1600 5.6 NR NR 25 U Weinberger and Iyengar 1983

2-frond stage 96h F, M frond production NOEC LOEC MATC 901

2080 1369 8.0-8.7 6.5-8.5 108-210 22.8- 24.2 1 Brooke 1993a

NR 7d S, N photosynthesis (CO2fixation) LOEC 2500 5.6 NR NR 25 U Weinberger and Iyengar 1983

### APPENDIX 1a (continued): Toxicity of nonylphenol to freshwater algae and macrophytes through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Lemna minor (duckweed) NR 24h S, N ATP content measurement LOEC 2500 5.6 NR NR 25 U Weinberger and Iyengar 1983

NR 24h NR, NR total ATP LC 50 2500 NR NR NR NR U Weinberger 1984 1
NR 96h S, N growth inhibition EC 50 5500 5.6 NR NR 25 U Weinberger and Iyengar 1983 NR 21d S, N bleaching EC 100 6250 6.8 NR NR 23 U Weinberger and Greenhalgh 1984 Potamogeton

sp. and Chara sp. primarily (macrophytes) NR 21d R, M survival NOEC LOEC MATC 243

>243 >243 7.7 7.3 122 19.5 1 Liber et al. 1999b

Salvinia molesta (aquatic fern) 2-4 fronds 9d S, N growth (frond production) LOEC 2500 NR NR NR 20 U Prasad 1989

Scenedesmus subspicatus (green algae) NR 72h NR,NR growth EC 50 1300 NR NR NR NR U Hüls 1996 2

Selenastrum capricornutum (green algae) 4-7d 96h S, M growth (cell counts) EC 50

NOEC LOEC MATC 410

92 190 132 7.4-8.9 NR NR 23.2- 23.7 1 Ward & Boeri 1990a

NR 96h S, M cell production NOEC LOEC MATC 694

1480 1013 7.3-7.7 NR 16 25.2- 26.0 1 Brooke 1993a

NR = not reported * duration reported in hours (h) or days (d) Test type: S = static, R = static-renewal, F = flow-through (continuous or intermittent)

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration D.O. = dissolved oxygen ** hardness reported in mg CaCO 3 ·L-1 Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable ICF 100= concentration causing 100% inhibition of fluorescence response

1 As cited in Servos et al. 2000 2As cited in OECD 1997 (as cited in Servos et al. 2000)

### APPENDIX Ib. Toxicity of nonylphenol to freshwater invertebrates through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Crustacea Acanthocyclops

sp. (copepod) nauplii to adult 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Acroperus

sp. (cladoceran) NR 92d R, M abundance NOEC LOEC 23

76 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Alona sp (Cladocera) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Bosmina sp. (cladoceran) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Calanoida (copepod) nauplii to adult 92d R, M abundance NOEC LOEC MATC 5

23 11 7.8 8.97 118 17.3 U O'Halloran et al. 1999; Liber et al. 1999b

Ceriodaphnia dubia (water flea) NEO 7d R, N survival NOEC LOEC MATC 3

10 5.5 8.4-8.8 6.9-8.0

(85-101% saturation) 134 -

166 24-25 2 England 1995

NEO 7d R, N total adverse effects of mortality and delayed maturity EC 50 27 8.4-8.8 6.9-8.0 (85-101% saturation) 134 -

166 24-25 1 England 1995

NR 96h F, NR NR EC 50 69 NR NR NR NR U Weeks et al. 1996 NR 7d NR,NR NR EC 50 99 NR NR NR NR U Weeks et al. 1996 NEO 7d R, N reproduction (time to first brood, # young/ adult reprod. days NOEC

LOEC MATC 100

300 170 8.4-8.8 6.9-8.0

(85-101% saturation) 134 -

166 24-25 1 England 1995

NR 7d F, NR reproduction MATC 134 NR NR NR NR U Weeks et al. 1996 NR 7d NR,NR mortality LC 50 258 NR NR NR NR U Weeks et al. 1996 NR 96h NR,NR mortality LC 50 276 NR NR NR NR U Weeks et al. 1996 NR 7d NR,NR NR MATC 276 NR NR NR NR U Weeks et al. 1996) NEO 7d R, N mortality LC 50 >300 8.4-8.8 6.9-8.0 (85-101% saturation) 134 -

166 24-25 2 England 1995

### APPENDIX Ib (continued). Toxicity of nonylphenol to freshwater invertebrates through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Ceriodaphnia dubia (water flea) NEO 96h R, N mortality, total adverse effects LC 50

EC 50 both >300 8.4-8.8 6.9-8.0 (85-101% saturation) 134 -

166 24-25 2 England 1995

NR 48h S, N mortality LC 50 470 (mean of 340, 710, 430, 410) 8 NR NR 25 U Ankley et al. 1990

Ceriodaphnia

sp. (water flea) NR 92d R, M abundance NOEC LOEC 23

76 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Chydorus

sp. (Cladocera) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Cladocera (taxa combined) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Copepoda (taxa combined) nauplii to adult 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Daphnia galeata mendotae (cladoceran zooplankton) NEO 30d R, N deformity LOEC 10 NR NR NR 20 2 Shurin & Dodson 1997 NEO 30d R, N sex determination (more female offspring) LOEC 50 NR NR NR 20 U Shurin & Dodson 1997

Daphnia magna (water flea) NEO 21d R, M reproduction NOEC 24 8.25 NR 180 20 1 Comber et al. 1993

NR chronic NR, NR reproduction rate NOEC

LOEC MATC 24

39 31 NR NR NR NR U Naylor 1995

NEO 21d R, M length of parent NOEC 39 8.25 NR 180 20 1 Comber et al. 1993

NEO 21d R, N fecundity MATC 71 8 NR NR 22 2 Baldwin et al. 1997 NR 48h NR, NR acute toxicity NOEC LOEC MATC 77

160 110 NR NR NR NR U Naylor 1995

NEO 48h R, M mortality EC 50 84.8 7.66-8.04 6.3-8.2 175.6- 195.1 21-23 1 Brooke 1993a

10d adult females 48h S, N inc. androgen (from inhibition of testost'one metab. elim.) LOEC 100 8 NR NR 22 U Baldwin et al. 1997

NR 21d NR,NR reproduction NOEC 100 NR NR NR NR U Hüls 1992 2

### APPENDIX Ib (continued). Toxicity of nonylphenol to freshwater invertebrates through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Daphnia magna (water flea) NEO 21d R, M reproduction NOEC LOEC MATC 116

215 157.9 8.14-8.65 7.5-9.6 156- 188 19.7-21.8 1 Brooke 1993a

NEO 21d R, M survival of parent (mortality) LC 50 100 8.25 NR 180 20 1 Comber et al. 1993

9-10d adult females 96h R, N immobilization or mortality NOEC 0.617 μM (136 μg·L-1 ) 7.8-8.4 >60% saturation 227 20 2 Gerritsen et al. 1998

NR 48h NR,NR mortality LC 50 140 NR NR NR NR U Hüls 1992 2
NR 48h S, N immobilization EC 50 180 NR NR NR NR U Bringmann and Kühn 1982 NEO 48h NR, NR mortality LC 50 190 NR NR NR 20 or 25 U Naylor 1995 NEO 48h S, M immobilization EC 50 190 8.25 NR 180 20 1 Comber et al. 1993 NEO 96h R, N immobilization or mortality NOEC 1.37 μM (302 μg·L-1 ) 7.8-8.4 >60% saturation 227 20 2 Gerritsen et al. 1998

NR 48h NR,NR mortality LC 50 440 NR NR NR NR U Monsanto 1985b 1 NEO 48h NR, NR reproduction EC 50 440 NR NR NR 20 or 25 U Naylor 1995 NEO 48h S, N immobilization EC 50 NOEC LOEC 440

250 500 7.5-8.7 6.7-8.4 264- 320 22.1 U Calvert & Adams 1981

Daphnia pulex (water flea) JUV 48h S, N mortality LC 50 140, 176, 190 NR NR 27.1 23-27 U Ernst et al. 1980

Diacyclops

sp. (copepod) nauplii to adult 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Eucyclops

sp. (copepod) nauplii to adult 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Harpacticoida (copepod) nauplii to adult 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Hyalella azteca (amphipod) JUV (2 mm) 96h F, M mortality LC 50 20.7 7.69-8.01 7.6-8.8 46-60 23.7-25.6 1 Brooke 1993a

JUV (2 mm) 96h F, M immobilization EC 50 20.7 7.69-8.01 7.6-8.8 46-60 23.7-25.6 1 Brooke 1993a

NR 96h F, NR NR EC 50 MATC 150

123 NR NR NR NR U Weeks et al. 1996

JUV 96h F, M lethargy and immobility EC 50 150 7.9 - 8.7 1.4 - 8.0 152 - 158 21 1 England & Bussard 1995 JUV 96h F, M mortality LC 50 170 7.9 - 8.7 1.4 - 8.0 152 - 158 21 1 England & Bussard 1995

### APPENDIX Ib (continued). Toxicity of nonylphenol to freshwater invertebrates through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Kurzia sp. (cladoceran) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Macrocyclops

sp. (copepod) nauplii to adult 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Mesocyclops

sp. (copepod) nauplii to adult 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Ostracoda (ostracod) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Paracyclops

sp. (copepod) nauplii to adult 92d R, M abundance NOEC LOEC 5

23 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Pleuroxus

sp. (Cladocera) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Simocephalus

sp. (cladoceran) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

Insecta Chironomini (chironomid) NR 21d R, M survival NOEC LOEC MATC 243

>243 >243 7.7 7.3 122 19.5 1 Liber et al. 1999b; Schmude et al. 1999

Chironomus tentans (freshwater midge) LAR (24h to 4th instar) 20d R, M survival NOEC LOEC 42

91 7.73 7.04 NR 23 1 Kahl et al. 1997

LAR-ADT 53d R, M emergence, reproduction, sex ratio NOEC 45 7.73 7.04 NR 23 2 Kahl et al. 1997

LAR (2nd instar) 14d F, M total adverse eff. (lethargy, small size, pale) EC 50 95 7.6-8.6 0.5-7.5 130- 154 19-21 1 England and Bussard 1993

LAR (2nd instar) 14d F, M larval weight (growth) EC 50

LOEC NOEC MATC 95

150 76 107 7.6-8.6 0.5-7.5 130- 154 19-21 1 England and Bussard 1993

LAR (2nd instar) 14d F, M mortality (survival) LC 50

LOEC NOEC MATC 119

150 76 107 7.6-8.6 0.5-7.5 130- 154 19-21 1 England & Bussard 1993

LAR 96h NR,NR mortality LC 50 MATC 119

107 NR NR NR NR U Weeks et al. 1996, Naylor 1995 NR 96h F, NR NR EC 50 MATC 160

97 NR NR NR NR U Weeks et al. 1996

### APPENDIX Ib (continued). Toxicity of nonylphenol to freshwater invertebrates through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Ophiogomphus

sp. (dragonfly) NYM (285 mg) 96h F, M loss of equilibrium, posterior of abdomen flexed over back EC 50 596 7.82-8.52 7.7-8.9 50.7- 54.6 21.5-25.0 1 Brooke 1993a

NYM (285 mg) 96h F, M mortality LC 50 >768 7.82-8.52 7.7-8.9 50.7- 54.6 21.5-25.0 1 Brooke 1993a

Tanytarsini (chironomid) LAR 21d R, M survival NOEC LOEC 76

243 7.7 7.3 122 19.5 U Schmude et al. 1999

Mollusca Anodonta cataractae (freshwater clam) 15g 144h R, M mortality LC 50 1700 NR NR NR 10 U McLeese et al. 1980a

Elliptio complanata (mussel) excised gills 50 min S, N metabolic heat rate inhibition LOEC 70000 NR NR NR 30 U Levine and Cheney 2000 excised gills 50 min S, N respiratory inhibition LOEC 220300 NR NR NR 25 U Levine and Cheney 2000 Gastropoda (snails, mostly Planorbidae) NR 21d R, M survival NOEC LOEC 76

243 7.7 7.3 122 19.5 U Schmude et al. 1999

Physella virgata (snail) ADT (476 mg) 96h F, M mortality LC 50 774 7.73-8.02 7.8-8.2 50-60 22.8-24.5 1 Brooke 1993a

ADT (476 mg) 96h F, M unable to retract into shell with prodding EC 50 378 7.73-8.02 7.8-8.2 50-60 22.8-24.5 1 Brooke 1993a

Pisidium sp. (bivalve) NR 21d R, M survival NOEC LOEC MATC 23

76 42 7.7 7.3 122 19.5 U Schmude et al. 1999; Liber et al. 1999b

Oligochaeta Naididae (oligochaete) NR 21d R, M survival NOEC LOEC 23

76 7.7 7.3 122 19.5 U Schmude et al. 1999

Tubificidae (oligochaete) NR 21d R, M survival NOEC LOEC 243

>243 7.7 7.3 122 19.5 U Schmude et al. 1999

Annelida Lumbriculus variegatus (annelid) ADT (5 mg) 96h F, M mortality LC 50 342 6.63-6.9 7.2-8.5 50.7- 54.6 22.2-25.9 1 Brooke 1993a

ADT (5 mg) 96h F, M inactivity EC 50 268 6.63-6.9 7.2-8.5 50.7- 54.6 22.2-25.9 1 Brooke 1993a

### APPENDIX Ib (continued). Toxicity of nonylphenol to freshwater invertebrates through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Rotifera Rotifera (taxa combined) NR 92d R, M abundance NOEC LOEC MATC 243

>243 >243 7.8 8.97 118 17.3 U O'Halloran et al. 1999; Liber et al. 1999b

Brachionus

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 243

>243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Colurella sp. (rotifer) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Conochilus

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 243

>243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Euchlanis

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Keratella sp. (rotifer) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Lecane sp. (rotifer) NR 92d R, M abundance NOEC LOEC 243

>243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Lepadella

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 243

>243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Monostyla

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 243

>243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Mytilina sp. (rotifer) NR 92d R, M abundance NOEC LOEC 243

>243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Notholca sp. (rotifer) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Platyias sp. (rotifer) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Pleosomasp. (rotifer) NR 92d R, M abundance NOEC LOEC 243

>243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Polyarthra

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 243

>243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Testudinella

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Trichocerca

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 23

76 7.8 8.97 118 17.3 U O'Halloran et al. 1999 Trichotria

sp. (rotifer) NR 92d R, M abundance NOEC LOEC 76

243 7.8 8.97 118 17.3 U O'Halloran et al. 1999

### APPENDIX Ib (continued). Toxicity of nonylphenol to freshwater invertebrates through water column exposure

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Miscellaneous Macroinvertebrates (taxa combined) NR 21d R, M survival NOEC LOEC MATC 76

243 136 7.7 7.3 122 19.5 U Liber et al. 1999b

Periphyton (taxa combined) NR 92d R, M survival NOEC LOEC MATC 243

>243 >243 7.8 8.97 118 17.3 U O'Halloran et al. 1999; Liber et al. 1999b Tetrahymena pyriformis (freshwater protozoan) NR 24h S, N relative growth rate EC 50 460 NR NR NR 30 U Yoshioka et al. 1985

Zooplankton (taxa combined) NR 92d R, M abundance NOEC LOEC MATC 76

243 136 7.8 8.97 118 17.3 U O'Halloran et al. 1999; Liber et al. 1999b

NR = not reported Life Stage (at start of assay): ADT = adult, JUV = juvenile, PUP = pupae, LAR = larvae, NYM = nymph, NEO = neonates * duration reported in minutes (min), hours (h), or days (d) Test type: S = static, R = static-renewal, F = flow-through (continuous or intermittent)

M = measured concentrations throughout experiment, N = n ominal concentration Conc. = concentration D.O. = dissolved oxygen ** hardness reported in mg CaCO 3 ·L-1 Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable

1 As cited in Etnier 1985 as cited in Talmage 1994 2As cited in Servos et al. 2000

### APPENDIX Ic. Toxicity of nonylphenol to freshwater fish through water column exposure

Species (Common name) Life stage Sex Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Culaea inconstans (5-7-spined stickleback) NR NR 21d R, M survival NOEC LOEC MATC 243

>243 >243 7.7 7.3 122 19.5 1 Liber et al. 1999b

Gasterosteus aculeatus (stickleback) NR NR 96h NR,NR mortality LC 50 370 NR NR NR NR U Granmo et al. 1991a

Gila elegans (bonytail chub) 0.29- 0.52 g NR 96h S, N mortality LC 50 290 8.35 low values 173 22 2 Dwyer et al. 1995

Ictalurus punctatus (channel catfish) JUV (65- 95g) NR 7d IPS, N vitellogenesis LOEC 237 mg·kg -1 (1) NR NR NR NR U Nimrod & Benson 1996hepatocytes from adult fish 400- 500g male 4-6d R, N vitellogenin induction LOEC

NOEC 10 μM

(2203 μg·L-1 ) 1 μM (220 μg·L-1 ) NR NR NR NR 2 Monteverdi and Di Giulio 1999

hepatocytes from 2 yr old fish (300- 400g) female 5h S, NR inhibition of estradiol binding to estrogen receptor site IC 50 4531 nM (998 μg·L-1 ) NR NR NR room temp U Nimrod and Benson

1997

Lebistes reticulatus (guppy) NR NR 24h F, NR NR NR NR NR NR NR NR U Swedmark 1968

Lepomis macrochirus (bluegill sunfish) 11-13 wks old NR 28d F, M mortality NOEC LOEC 59.5

126 6.80-

7.78 4.4-7.4 46.8-

51.2 23.3-

26.0 1 Brooke 1993b

JUV (9 cm, 12 g) NR 21d R, M survival NOEC LOEC MATC 76

243 136 7.7 7.3 122 19.5 1 Liber et al. 1999a

13-15 wks old NR 96h F, M mortality NOEC LOEC 86.5

211 7.51-

7.99 5.2-8.0 49.4-

50.8 25.3-

26.6 1 Brooke 1993b

JUV (<1 year old), 23 mm, 294 mg NR 96h F, M loss of equilibrium, loss of schooling ability EC 50 203 7.26- 7.79 7.5-8.2 50.7-

54.6 21.5-

23.8 1 Brooke 1993a

### APPENDIX Ic (continued). Toxicity of nonylphenol to freshwater fish through water column exposure

Species (Common name) Life stage Sex Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Lepomis macrochirus (bluegill sunfish) JUV (<1 yr old), 23 mm, 294 mg NR 96h F, M mortality LC 50 209 7.26- 7.79 7.5-8.2 50.7-

54.6 21.5-

23.8 1 Brooke 1993a

Leuciscus idus (golden orfe) NR NR 48h NR,NR mortality LC 50 950 NR NR NR NR U Hüls 1987

Oncorhynchus apache (Apache trout) 0.38-0.85 g NR 96h S, N mortality LC 50 170 8.24 low values 169 12 2 Dwyer et al. 1995

Oncorhynchus clarki henshawi (Lahontan cutthroat trout) 0.31 g NR 96h S, N mortality LC 50 180, 170 8.24 low values 169 12 2 Dwyer et al. 1995

Oncorhynchus clarki stomias (greenback cutthroat trout) 0.34-0.57 g NR 96h S, N mortality LC 50 150, 156 8.24 low values 169 12 2 Dwyer et al. 1995

Oncorhynchus mykiss (rainbow trout) JUV female 22d,35d F, N growth inhibition LOEC 1 6.5 NR 12.5 7-13 U Ashfield et al. 1998

eggs male 12 mo. NR,NR vitellogenin induction LOEC 1 NR NR NR NR U Fent et al. 1999

eggs male 12 mo. NR,NR Vg mRNA induction LOEC 1 NR NR NR NR U Fent et al. 1999

EMB NR 91d F, M growth NOEC LOEC MATC 6

10.3 7.861 6.34-

7.84 7.7-

11.7 39.0-

66.3 8.9-14.4 1 Brooke 1993a

ADT and JUV male

and female 72h F, N vitellogenin induction LOEC 10 NR NR NR 10-15 U Lech et al. 1996

ADT and JUV male

and female 72h F, N

(calc) estrogenic (vitellogenin induction) EC 50 14.14 NR NR NR 10-15 U Lech et al. 1996

2 yr male 21d F, M elevation of plasma vitellogenin LOEC 20.3 NR NR NR NR 2 Jobling et al. 1996

hepatocytes male 24h S, N Vg mRNA induction; rtER mRNA induction NOEC

LOEC 10 -7M

(22 μg·L-1 ) 10 -6M (220 μg·L-1 ) NR NR NR NR U Flouriot et al. 1995

### APPENDIX Ic (continued). Toxicity of nonylphenol to freshwater fish through water column exposure

Species (Common name) Life stage Sex Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Oncorhynchus mykiss (rainbow trout) JUV (sexually immature 35-50g) NR 21d R, N vitellogenin induction NOEL

LOEL 25

50 8.0-8.4 >90%

saturation NR 13 2 Tremblay and Van Der Kraak 1998

2 yr male 21d F, M 500 fold increase in vitellogenin NR 37 NR NR NR NR U Jobling et al. 1996

JUV (46- 174g) NR 9d IPS, N vitellogenin induction NOEC 50 mg·kg -1 NR NR NR NR U Andersen et al. 1999

JUV (103- 168g) NR 12d IPR, NR vitellogenin induction LOEC (only

conc tested) 50 mg·kg -1 NR NR NR NR U Pedersen et al. 1999

JUV (103- 168g) NR 12d IPR, NR vitellogenin induction NOEC (only

conc tested) 50 mg·kg -1 NR NR NR NR U Pedersen et al. 1999

JUV (100- 200g) NR NR F, N induction of vitellogenin mRNA significant

effect 100 NR NR NR 10-15 U Ren et al. 1996

45d, 27.2 mm, 241 mg NR 96h F, M loss of equilibrium, poor fright response EC 50 109 6.53- 6.98 9.3-10.4 54.6-

58.5 11.3-

12.6 1 Brooke 1993a

JUV (103- 168g) NR 9d F, M vitellogenin induction LOEC 75 NR NR NR NR U Pedersen et al. 1999

JUV (103- 168g) NR 9d F, M vitellogenin induction NOEC 109 NR NR NR NR U Pedersen et al. 1999

0.27- 1.25 g NR 96h S, N mortality LC 50 190, 250 8.24 low values 169 12 2 Dwyer et al. 1995

ADT and JUV (50- 200g) male

and female 72h F, N mortality LC 50 193.65 NR NR NR 10-15 U Lech et al. 1996

45d, 27.2 mm, 241 mg NR 96h F, M mortality LC 50 221 6.53- 6.98 9.3-10.4 54.6-

58.5 11.3-

12.6 1 Brooke 1993a

JUV NR 96h NR,NR mortality LC 50 230 NR NR NR NR U Armstrong and Kingsbury 1979 (2)

### APPENDIX Ic (continued). Toxicity of nonylphenol to freshwater fish through water column exposure

Species (Common name) Life stage Sex Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Oncorhynchus mykiss (rainbow trout) 42- 60mm, 0.5-2.7g NR 96h S, N mortality LC 50 230 NR NR NR 9 U Holmes and Kingsbury 1980

15-30d NR 96h NR,NR mortality LC 50 230 NR >6 NR 12 U Naylor 1995 EMB NR acute24h exp + 48h post-exp S, M mortality LC 50 484 NR NR 27.1 9.5-28.5 U Ernst et al. 1980

JUV (1.4g, 5.2 cm) NR 96h S, N mortality LC 50 920, 560 6.2-8.3 4.6-10.6 27.1 15-16 U Ernst et al. 1980

hepatocytes from JUV fish males 48h S, N vitellogenin production ED 50 16.15 μM (3558 μg·L-1 ) NR NR NR NR U Jobling and Sumpter 1993

Oryzias latipes (Japanese medaka) 5-8d old, larval swim-up stage NR 28d

(& 1 mo. in clean water) F, M sex ratio,

fecundity, egg viability or hatchability NOEC 1.93 NR NR NR 25 2 Nimrod and Benson 1998

hatch to 3 mths NR 3 mo. R, N induction of testis-ova NOEC

LOEC 10

50 7.4-7.8 NR 80-100 20 U Gray & Metcalfe 1997 hatch to 3 mths NR 3 mo. R, N sex ratio NOEC LOEC 50

100 7.4-7.8 NR 80-100 20 U Gray & Metcalfe 1997 EMB NR 17d S, N embryotoxicity LC 50 460 NR NR NR 25 U Gray and Metcalfe 1997 2 cm, 0.2 g NR 48h S, NR mortality LC 50 1400 7.2 NR 50 16.2 U Yoshimura 1986

Pimephales promelas (fathead minnow) ADT NR 42d F, M histologic lesions LOEC

NOEC 1.6 - 3.4

0.4 - 0.93 NR NR NR 25-26 2 Miles-Richardson et al. 1999 EMB NR 33d F, M survival NOEL LOEL MATC 7.4

14 10.2 7.1-8.2 6.3-9.7 160-180 23.4- 26.3 1 Ward and Boeri

1991b

early life stage NR 30d F, NR NR MATC 10.2 NR NR NR NR U Weeks et al. 1996

EMB NR 33d F, M # embryos hatched, time to hatch, time to first feeding, total length, weight NOEC

LOEC MATC for all

endpoints: 23 >23 >23 7.1-8.2 6.3-9.7 160-180 23.4- 26.3 1 Ward and Boeri

1991b

### APPENDIX Ic (continued). Toxicity of nonylphenol to freshwater fish through water column exposure

Species (Common name) Life stage Sex Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Pimephales promelas (fathead minnow) 4 wks old NR 28d F, M mortality NOEC LOEC 77.5

193 6.91-

7.92 4.4-7.7 45.8-

52.2 23.2- 26.3 1 Brooke 1993b

4 wks old NR 96h F, M mortality NOEC LOEC 83.1

230 7.50-

7.84 5.0-6.7 46.2-

48.4 23.0-

25.6 1 Brooke 1993b

25-35d old, 81.6g, 17.1 mm NR 96h F, M loss of equilibrium, poor schooling and fright responses EC 50 96 6.77- 7.58 6.6-8.2 53.6-

63.4 21.3- 23.1 1 Brooke 1993a

31-35d, 220 mg NR 96h F, M loss of equilibrium LOEC 98 6.9-7.7 7.4 44.9 24.6 2 Holcombe et al. 1984

25-35d old, 81.6g, 17.1 mm NR 96h F, M mortality LC 50 128 6.77- 7.58 6.6-8.2 53.6-

63.4 21.3- 23.1 1 Brooke 1993a

31-35d, 220 mg NR 96h F, M mortality LC 50 135 6.9-7.7 7.4 44.9 24.6 1 Holcombe et al. 1984

32d NR 96h F, M mortality LC 50 140 7.29 7.1 45.1 24.5 1 Geiger et al. 1985

31-35d NR 96h F, M haemorrhaging LOEC 187 6.9-7.7 7.4 44.9 24.6 2 Holcombe et al. 1984

31-35d NR 96h F, M bodies swollen with fluid LOEC 187 6.9-7.7 7.4 44.9 24.6 2 Holcombe et al. 1984

31-35d, 220 mg NR 96h F, M lethargic LOEC 187 6.9-7.7 7.4 44.9 24.6 2 Holcombe et al. 1984

0.32- 0.56g NR 96h S, N mortality LC 50 270, 320 8.35 low values 173 22 2 Dwyer et al. 1995

0.21g, 26

mm NR 96h S, N mortality LC 50 NOEC 300

180 7 9.2 45 22 2 Analytical BioChemistry Laboratories, Ltd. 1981 Ptychocheilus lucius (Colorado squawfish) 0.32- 0.34 g NR 96h S, N mortality LC 50 260, 240 8.35 low values 173 22 2 Dwyer et al. 1995

Salmo salar (Atlantic salmon) 1 yr, immatur e 23g NR 30d IPR,

N smoltification vitellogenesis sig. effect 3 mg NP in 100μL oil NR NR NR 8-12 U Madsen et al. 1997

1 yr, immatur e23g NR 30d IPR,

N osmoregulation sig. effect 3 mg NP in 100μL oil NR NR NR 8-12 U Madsen et al. 1997

### APPENDIX Ic (continued). Toxicity of nonylphenol to freshwater fish through water column exposure

Species (Common name) Life stage Sex Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Salvelinus fontinalis (brook trout) 36-55mm, 0.5-1.4g NR 96h S, N mortality LC 50 145 6.5-7.0 NR 51.6(3) ambient stream temp. U Holmes and Kingsbury

1980

NR NR 96h NR,NR mortality LC 50 145 NR NR NR NR U Armstrong and Kingsbury 1979 1 Salvelinus namaycush Walbaum (lake trout) 54-92mm, 0.6-60g NR 35d S, N mortality LC 50 >40 NR NR NR 12.5 U Holmes and Kingsbury 1980

Xiphophorus maculatus (platyfish) ADT 0.6-1.2 g males 28d R, N survival NOEC LOEC 320

640 8.1 NR NR 28 U Kinnberg et al. 2000

ADT 0.6-1.2 g males 28d R, N growth NOEC 1280 8.1 NR NR 28 U Kinnberg et al. 2000

Xyrauchen texanus (razorback sucker) 0.31-0.32 g NR 96h S, N mortality LC 50 170, 200 8.35 low values 173 22 2 Dwyer et al. 1995

NR = not reported Life Stage (at start of assay): ADT = adult, JUV = juvenile, NEO = neonates, EMB = embryo * duration reported in hours (h), days (d), or months (mo.) Test type: S = static, R = static-renewal, F = flow-through (continuous or intermittent), IPS = single i ntraperitoneal injection, IPR = repeated intraperitoneal injections

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration D.O. = dissolved oxygen ** hardness reported in mg CaCO 3 ·L-1 Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable Critical study for freshwater Canadian Water Quality Guideline appears in bold

(1)Units are in mg·kg -1 because the nonylphenol was administered as an agar suspension that was injected intraperitoneally (2)As cited in McLeese et al. 1981 (3)In Holmes and Kingsbury (1980) this value was given as 3 GPG (grains per gallon), but has been converted here to mg·L-1 CaCO 3(by multiplying by a factor of 17.2).

### APPENDIX Id. Toxicity of nonylphenol ethoxylates to freshwater algae and macrophytes through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Algae Scenedesmus

sp. (green algae) NR NP4EO NR NR,NR mortality lethal threshold 6000 NR NR NR NR U Janicke et al. 1969

Scenedesmus

sp. (green algae) NR NP6EO NR NR,NR mortality lethal threshold 10000 NR NR NR NR U Janicke et al. 1969

Selenastrum capricornutum (green algae) NR NP6EO 21d S, N growth NOEC 500000 NR NR NR 25 U Nyberg 1988

Scenedesmus

sp. (green algae) NR NP7EO NR NR,NR mortality lethal threshold 16000 NR NR NR NR U Janicke et al. 1969

Selenastrum capricornutum (green algae) NR NP7EO 96h NR,NR growth EC 50 >1000000 NR NR NR NR U Markarian et al. 1989 1

, 1990

Selenastrum capricornutum (green algae) NR NP8EO 48h S, N growth inhibition EC 50 20000 7.4 NR NR 24 2 Yamane et al. 1984

Selenastrum capricornutum (green algae) NR NP9EO 96h NR,NR growth EC 50 50000 NR NR NR NR U Lewis 1986

4-7d NP9EO 96h S, M cell count (growth) EC 50

NOEC LOEC MATC 12000

8000 16000 11300 7-7.5 2-5 150 22-25 1 Dorn et al. 1993

NR NP9EO 21d S, N growth NOEC 500000 NR NR NR 25 U Nyberg 1988 NR NP9EO 48h S, N growth inhibition EC 50 50000 7.4 NR NR 24 2 Yamane et al. 1984

green and blue-green algae NR NP9.5EO NR NR,NR growth inhibition NR 20000 NR NR NR NR U Davis & Gloyna 19672

Scenedesmus

sp. (green algae) NR NP10EO NR NR,NR mortality lethal threshold 31000 NR NR NR NR U Janicke et al. 1969

Scenedesmus sp. (green algae) NR NP20EO NR NR,NR mortality lethal threshold 125000 NR NR NR NR U Janicke et al. 1969

### APPENDIX Id (continued). Toxicity of nonylphenol ethoxylates to freshwater algae and macrophytes through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluatio

n Rank Reference

Selenastrum capricornutum Printz (green algae) NR NP30EO 21d S, N growth NOEC 500000 NR NR NR 25 U Nyberg 1988

Scenedesmus

sp. (green algae) NR NP30EO NR NR,NR mortality lethal threshold 5000000 NR NR NR NR U Janicke et al. 1969

Macrophytes Egeria densa (macrophyte) leaflets removed from plants NP4EO several hours S, NR depolarization

of membrane potential LOEC 10000 7.3 NR NR NR U Linsel et al. 1990

Egeria densa (macrophyte) leaflets removed from plants NP20EO several hours S, NR depolarization

of membrane potential LOEC 100000 7.3 NR NR NR U Linsel et al. 1990

NR = not reported * duration reported in hours (h) or days (d) Test type: S = stat ic, R = static-renewal, F = flow-through (continuous or intermittent)

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration D.O. = dissolved oxygen ** hardness reported in mg CaCO 3 ·L-1 Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable

1 As cited in Servos et al. 2000 2As cited in Talmage 1994

### APPENDIX Ie. Toxicity of nonylphenol ethoxylates to freshwater invertebrates through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Crustacea Hyalella azteca (amphipod) 2-4d mixture of APEs 96h S, N mortality LC 50 450 7.4-8.1 6.3-8.2 40 25 U Collyard et al. 1994

0-2d mixture of

APEs 96h S, N mortality LC 50 600 7.4-8.1 6.3-8.2 40 25 U Collyard et al. 1994

6-8d mixture of

APEs 96h S, N mortality LC 50 750 7.4-8.1 6.3-8.2 40 25 U Collyard et al. 1994

4-6d mixture of

APEs 96h S, N mortality LC 50 850 7.4-8.1 6.3-8.2 40 25 U Collyard et al. 1994

16-18d mixture of

APEs 96h S, N mortality LC 50 870 7.4-8.1 6.3-8.2 40 25 U Collyard et al. 1994

10-12d mixture of

APEs 96h S, N mortality LC 50 900 7.4-8.1 6.3-8.2 40 25 U Collyard et al. 1994

20-22d mixture of

APEs 96h S, N mortality LC 50 1250 7.4-8.1 6.3-8.2 40 25 U Collyard et al. 1994

24-26d mixture of

APEs 96h S, N mortality LC 50 1520 7.4-8.1 6.3-8.2 40 25 U Collyard et al. 1994

Ceriodaphnia dubia (water flea) NEO NP1.5EO 7d R, M survival and reproduction NOEC

LOEC MATC 285

886 503 8.0 - 8.9 4.0-8.5

(49-106% saturation ) 140 - 154 23-25 1 England & Downing 1995

NEO NP1.5EO 7d R, M total adverse effects EC 50 319 8.0 - 8.9 4.0-8.5 (49-106% saturation ) 140 - 154 23-25 1 England & Downing 1995

NR NP1.5EO 7d NR,NR NR EC 50 319 NR NR NR NR U Weeks et al. 1996 NR NP1.5EO 7d NR,NR reproduction MATC 503 NR NR NR NR U Weeks et al. 1996 NR NP1.5EO 96h NR,NR NR EC 50 626 NR NR NR NR U Weeks et al. 1996 NEO NP1.5EO 96h R, M total adverse effects - mortality, small size, pale appearance EC 50 626 8.0 - 8.9 4.0-8.5 (49-106% saturation ) 140 - 154 23-25 1 England & Downing 1995

NEO NP1.5EO 7d R, M mortality LC 50 886 8.0 - 8.9 4.0-8.5 (49-106% saturation ) 140 - 154 23-25 1 England & Downing 1995

### APPENDIX Ie (continued). Toxicity of nonylphenol ethoxylates to freshwater invertebrates through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Ceriodaphnia dubia (water flea) NR NP1.5EO 7d NR,NR mortality LC 50 886 NR NR NR NR U Weeks et al. 1996

NEO NP1.5EO 96h R, M mortality LC 50 1016 8.0 - 8.9 4.0-8.5 (49-106% saturation ) 140 - 154 23-25 1 England & Downing 1995

NR NP1.5EO 96h NR,NR mortality LC 50 1016 NR NR NR NR U Weeks et al. 1996 NR NP1.5EO 48h S, N mortality LC 50 1040 8 NR NR 25 2 Ankley et al. 1990

Daphnia magna (water flea) NEO NP2EO 48h S, NNR mortality LC 50 67 NR NR hard 20 U Maki et al. 1998

NEO NP2EO 48h S, NR mortality LC 50 148 NR NR hard 20 U Maki et al. 1998

Daphnia magna (water flea) NR NP7EO 96h NR,NR mortality LC 50 4100 U Markarian et al. 1989 1

Daphnia magna (water flea) NEO NP9EO 7d R, M mortality LC 50 NOEC LOEC MATC 9000

10000 20000 14000 7-7.5 2-5 150 22-25 1 Dorn et al. 1993

NEO NP9EO

(Igepal CO-630, GAF Corp.) 7d R, M carapace length (growth) NOEC

LOEC MATC 10000

>10000 >10000 7-7.5 2-5 150 22-25 1 Dorn et al. 1993 (also Kravetz et al. 1991)

NEO NP9EO 48h R, M mortality LC 50 14000 7-7.5 2-5 150 25 1 Dorn et al. 1993 (also Kravetz et al. 1991) Daphnia pulex (water flea) NR NP9EO 48h NR,NR immobilization EC 50 2870 NR NR NR NR U Salanitro et al. 1988

Daphnia magna (water flea) NR NP10EO 24h NR,NR mortality LC 50 44200 NR NR NR NR U Unilever Research Laboratories 19772 Daphnia pulex (water flea) NR NP10EO 48h S, NR mortality LC 50 12520 7.0-8.0 3.7-7.5 25-40 20-21 U Moore et al. 1987

Daphnia magna (water flea) NEO NPnEO 21d R, N fecundity NOEC 5000 8.0 NR NR 22 Baldwin et al. 1998

### APPENDIX Ie (continued). Toxicity of nonylphenol ethoxylates to freshwater invertebrates through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Daphnia magna (water flea) NEO NPnEO 21d, then 16h withtestosterone R, N metabolic

elimination of testosterone EC 100 5000 8.0 NR NR 20 U Baldwin et al. 1998

10d females NPnEO 64h (16 withtestosterone) S, N metabolic

elimination of testosterone NOEC 5000 8.0 NR NR 22 U Baldwin et al. 1998

NEO NPnEO 21d R, N reproduction chronic value 5700 8.0 NR NR 22 U Baldwin et al. 1998 NEO NPnEO 48h NR, N mortality LC 50 8600 8.0 NR NR 22 U Baldwin et al. 1998

Daphnia magna (water flea) NR NP1EC 48h NR,NR mortality LC 50 NOEC 14000

11000 NR NR NR NR U Naylor et al. 1997

Ceriodaphnia dubia (water flea) NR NP1EC 7d NR,NR reproduction NOEC 2200 NR NR NR NR U Naylor et al. 1997

NR NP1EC 7d NR,NR mortality NOEC 8400 NR NR NR NR U Naylor et al. 1997 NR NP1EC 96h NR,NR mortality LC 50 17000 NR NR NR NR U Naylor et al. 1997

Daphnia magna (water flea) NEO NP2EC 48h S, NR mortality LC 50 141 NR NR hard 20 U Maki et al. 1998

NEO NP2EC 48h S, NR mortality LC 50 990 NR NR hard 20 U Maki et al. 1998

Insecta Culex pipiens (southern house mosquito) PUP NP1EO NR S, NR emergence EC 50 80000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Culex pipiens (mosquito) PUP NP1.5EO NR S, NR emergence EC 50 67000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Culex pipiens (mosquito) PUP NP4EO NR S, NR emergence EC 50 6000 - 8000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Culex pipiens (mosquito) PUP NP6EO NR S, NR emergence EC 50 6000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

### APPENDIX Ie (continued). Toxicity of nonylphenol ethoxylates to freshwater invertebrates through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Culex pipiens (mosquito) PUP NP9EO NR S, NR emergence EC 50 10000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Culex pipiens (mosquito) PUP NP9.5EO NR S, NR emergence EC 50 7000 - 19000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Culex pipiens (mosquito) PUP NP10EO NR S, NR emergence EC 50 19000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Aëdes aegypti (mosquito) LAR (4th stage) NP11EO 10d NR, NR pupae mortality LC 50 50000 NR NR NR 25 U van Emden et al. 1974 LAR (2 nd & 3 rd stage) NP11EO 24h NR, NR mortality LC 50 LC 100 500000

1000000 NR NR NR 25 U van Emden et al. 1974

EGG NP11EO 10d NR, NR hatch rate EC 50 500000 NR NR NR 25 U van Emden et al. 1974

Culex pipiens (mosquito) PUP NP15EO NR S, NR emergence EC 50 9000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Culex pipiens (mosquito) PUP NP20EO NR S, NR emergence EC 50 28000, 120000, >200000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Culex pipiens (mosquito) PUP NP30EO NR S, NR emergence EC 50 >400000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

Culex pipiens (mosquito) PUP NP50EO NR S, NR emergence EC 50 >400000 7.5-8.0 NR NR 78°F U Maxwell and Piper 1968

### APPENDIX Ie (continued). Toxicity of nonylphenol ethoxylates to freshwater invertebrates through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Mollusca Elliptio complanata (mussel) excised gills NP1.5EO 50 min S, N metabolic heat rate inhibition LOEC 9000 NR NR NR 30 U Levine and Cheney 2000

Biomphalaria glabrata (snail) 1.5-2 cm NP11EO 24h NR, NR mortality LC 100 23000 NR NR NR 25 U van Emden et al. 1974

Cnidaria Hydra littoralis (hydra) mixture of budded, unbudded NPnEO 11d R, NR growth thresholdconcentration 640 NR NR NR NR U Stephenson 1979

NR = not reported Life Stage (at start of assay): ADT = adult, JUV = juvenile, PUP = pupae, LAR = larvae, NYM = nymph, NEO = neonates * duration reported in minutes (min), hours (h), or days (d) Test type: S = static, R = static-renewal, F = fl ow-through (continuous or intermittent)

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration D.O. = dissolved oxygen ** hardness reported in mg CaCO 3 ·L-1 Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable

1 As cited in Servos et al. 2000 2As cited in Anonymous 1977 as cited in Talmage 1994

### APPENDIX If. Toxicity of nonylphenol ethoxylates to freshwater fish through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP1EO 48h S, NR mortality LC 50 3000 7.2 NR 50 16.2 U Yoshimura 1986

Oryzias latipes (Japanese medaka) 1 day posthatch NP1.5EO 90d R, N growth NOEC 100 NR NR NR 22-24 2 Metcalfe et al. 20011 day posthatch NP1.5EO 90d R, N sex ratio NOEC 100 NR NR NR 22-24 2 Metcalfe et al. 20011 day posthatch NP1.5EO 90d R, N testis-ova NOEC LOEC 50

100 NR NR NR 22-24 U Metcalfe et al. 2001

Oncorhynchus mykiss (rainbow trout) JUV (females) NP2EO 22d,35d F, N growth inhibition LOEC 1 6.5 NR 12.5 7-13 U Ashfield et al. 1998 2 yr-old males NP2EO 21d F, M 200 fold increase in vitellogenin NR 38 NR NR NR NR U Jobling et al. 1996

hepatocytes from JUV males NP2EO 48h S, N vitellogenin production ED 50 17.27 μM (5326 μg·L-1 ) NR NR NR NR U Jobling and Sumpter 1993

Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP3.3EO 48h S, NR mortality LC 50 2500 7.2 NR 50 16.2 U Yoshimura 1986

Lepomis macrochirus (bluegill sunfish) NR NP4EO 96h S, M mortality LC 50 1300 7.1 9.0-5.1 NR 18 U Macek and Krzeminski 1975

Lepomis macrochirus (bluegill sunfish) NR NP5EO 96h S, M mortality LC 50 2400 - 2800 7.1 9.0-5.1 NR 18 U Macek and Krzeminski 1975 Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP5EO 48h S, NR mortality LC 50 3600 7.2 NR 50 16.2 U Yoshimura 1986

Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP6.4EO 48h S, NR mortality LC 50 5400 7.2 NR 50 16.2 U Yoshimura 1986

Pimephales promelas (fathead minnows) NR NP7EO 48h NR,NR mortality LC 50 3200 NR NR NR NR U Markarian et al. 1989 1

### APPENDIX If (continued). Toxicity of nonylphenol ethoxylates to freshwater fish through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Oncorhynchus mykiss (rainbow trout) 12-16 cm NP8EO 14d F, N mortality LC 50 4250 7.3-7.4 >70% saturation 290-310 15-15.6 U Calamari and Marchetti 1973 12-16 cm NP8EO 96h F, N mortality LC 50 4700 7.3-7.4 >70% saturation 290-310 15-15.6 U Calamari and Marchetti 1973

Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP8.4EO 48h S, NR mortality LC 50 11600 7.2 NR 50 16.2 U Yoshimura 1986

Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP8.9EO 48h S, NR mortality LC 50 11200 7.2 NR 50 16.2 U Yoshimura 1986

Carassius auratus (goldfish) NR NP9EO 48h NR,NR mortality LC 50 18000 NR NR NR NR U Tomiyama 1974

Lepomis macrochirus (bluegill sunfish) NR NP9EO 192h F, N mortality LC 50 6300 7.1 9.4-8.9 38 21 U Macek and Krzeminski 1975 NR NP9EO 96h S, M mortality LC 50 7900 7.1 9.0-5.1 NR 18 U Macek and Krzeminski 1975 Leuciscus idus (golden orfe) NR NP9EO 48h NR,NR mortality LC 50 7000 NR NR NR NR U Fischer 19732

Oncorhynchus mykiss (rainbow trout) hepatocytes from JUV males NP9EO 48h S, N vitellogenin production ED 50 82.31 μM (50800 μg·L-1 ) NR NR NR NR U Jobling and Sumpter 1993

Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP9EO 48h S, NR mortality LC 50 12000 7 NR 3400 16.2 U Yoshimura 1986

2 cm, 0.2 g NP9EO 48h S, NR mortality LC 50 14000 7 NR 25 16.2 U Yoshimura 1986

Pimephales promelas (fathead minnow) 1d NP9EO 7d R, M weight at 7d (growth) NOEC

LOEC MATC 1000

2000 1400 7-7.5 2-5 150 22-25 1 Dorn et al. 1993 (also Kravetz et al. 1991) NR NP9EO 96h NR,NR mortality LC 50 1620 NR NR NR NR U Salanitro et al. 1988 1d NP9EO 7d R, M mortality LC 50 NOEC LOEC MATC 2900

1800 2000 1400 7-7.5 2-5 150 22-25 1 Dorn et al. 1993

7-27d NP9EO 96h R, M mortality LC 50 4600 7-7.5 2-5 150 22-25 1 Dorn et al. 1993 NR NP9EO 96h NR,NR mortality LC 50 6600 NR NR NR NR U Williams et al. 1996 1

### APPENDIX If (continued). Toxicity of nonylphenol ethoxylates to freshwater fish through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Carassius aurtus (goldfish) 5-6 cm NP9.5EO 6h S, NR mortality LC 50 6900 NR NR 100 20 U Reiff et al. 1979

Rasbora heteromorpha (harlequin fish) 1.3-3.0

cm NP9.5EO 96h F, NR mortality LC 50 8600 NR NR 20 20 U Reiff et al. 1979

Lepomis macrochirus (bluegill sunfish) NR NP9.5EO 96h S, M mortality LC 50 7600 7.1 9.0-5.1 NR 18 U Macek and Krzeminski 1975 Leuciscus idus4 (golden orfe) 5-7 cm NP9.5EO 48h S, NR mortality LC 50 4900 NR NR 150 20 U Reiff et al. 1979

5-7 cm NP9.5EO 96h F, NR mortality LC 50 7000 NR NR 268 20 U Reiff et al. 1979 5-7 cm NP9.5EO 96h S, NR mortality LC 50 11200 NR NR 268 20 U Reiff et al. 1979 Oncorhynchus mykiss (rainbow trout) 1 yr old, 9.4 cm, 8.3g NP9.5EO 24h S, NR loss of equilibrium EC 50 2800 7.5-8.1 >90%saturation 114 15 U Muller 1980

NR NP9.5EO 96h NR,NR mortality LC 50 7500- 12500 4500- 5900 NR NR NR NR U Unilever Research Laboratories 1977 3

1 yr old, 9.4 cm, 8.3g NP9.5EO 24h S, NR mortality LC 50 8500 7.5-8.1 >90%saturation 114 15 U Muller 1980

Salmo trutta (brown trout) 2.8 or 5.8

cm NP9.5EO 96h F, NR mortality LC 50 1000 NR NR 26-30 15 U Reiff et al. 1979

Carassius auratus (goldfish) 5cm NP10EO 48h NR, N mortality LC 50 5400 NR NR NR NR U Kurata et al. 1977

NR NP10EO 48h NR,NR mortality LC 50 13800 NR NR NR NR U Unilever Research Laboratories 1977 3 Rasbora heteromorpha (harlequin fish) NR NP10EO 48h NR,NR mortality LC 50 11300 NR NR NR NR U Unilever Research Laboratories 1977 3
Leuciscus idus (golden orfe) NR NP10EO 48h NR,NR mortality LC 50 7400, 9500 NR NR NR NR U Unilever Research Laboratories 1977 3 Oncorhynchus mykiss (rainbow trout) perfused head of fish 160- 220 g NP10EO 1h F, N change in gill resistence; sodium net flux NOEC

LOEC 87.6

(+0.9 Cd) 63000 (+905 Cd) 7 measured NR 10 U Pärt et al. 1985

### APPENDIX If (continued). Toxicity of nonylphenol ethoxylates to freshwater fish through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Oncorhynchus mykiss (rainbow trout) 23 days after hatching NP10EO 6h S, N mortality LC 50 2100 7.3-7.4 5 240-260 10-14 U Marchetti 1965

fry 25 days after hatching NP10EO 6h S, N mortality LC 50 2300 7.3-7.4 5 240-260 10-14 U Marchetti 1965

40d old (fry) NP10EO 6h S, N mortality LC 50 5200 7.3-7.4 5 240-260 10-14 U Marchetti 1965

210d old(fingerling) NP10EO 6h S, N mortality LC 50 5200 7.3-7.4 5 240-260 10-14 U Marchetti 1965

alevin 12 days after hatching NP10EO 6h S, N mortality LC 50 5200 7.3-7.4 5 240-260 10-14 U Marchetti 1965

alevin 19 days after hatching NP10EO 6h S, N mortality LC 50 5200 7.3-7.4 5 240-260 10-14 U Marchetti 1965

3-10 cm NP10EO 96h NR,NR mortality LC 50 7500- 11700 NR NR 20-300 10 and 15 U Unilever Research

Laboratories 1977 3 alevin 6 days after hatching NP10EO 6h S, N mortality LC 50 22000 7.3-7.4 5 240-260 10-14 U Marchetti 1965

alevinimmediately after hatching NP10EO 6h S, N mortality LC 50 42000 7.3-7.4 5 240-260 10-14 U Marchetti 1965

perfused head of fish 160- 220 g NP10EO 1h F, N change in gill resistence & O 2transfer factor; Na+ net flux significantly

different from control 63000 7 measured NR 10 U Pärt et al. 1985

Phoxinus phoxinus (minnow) NR NP10EO 48h NR,NR mortality LC 50 8800 NR NR NR NR U Unilever Research Laboratories 1977 3 Salmo trutta (brown trout) NR NP10EO 48h NR,NR mortality LC 50 2700 NR NR NR NR U Unilever Research Laboratories 1977 3

### APPENDIX If (continued). Toxicity of nonylphenol ethoxylates to freshwater fish through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Lebistes reticulatus (guppy) 14d JUV (0.007g, 0.7cm) NP11EO 24h NR, NR mortality LC 100 52000 NR NR NR 25 U van Emden et al. 1974

Lebistes reticulatus (guppy) females (0.12- 0.38g, 2 cm) NP11EO 24h NR, NR mortality LC 100 57000 NR NR NR 25 U van Emden et al. 1974

Lebistes reticulatus (guppy) males (0.05- 0.08g, 2 cm) NP11EO 24h NR, NR mortality LC 100 64000 NR NR NR 25 U van Emden et al. 1974

Oncorhynchus mykiss (rainbow trout) JUV (46- 174g) NP12EO 9d IPS, N vitellogenin induction NOEC 50 mg·kg -1 NR NR NR NR U Andersen et al. 1999 Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP13.1EO 48h S, NR mortality LC 50 48000 7.2 NR 50 16.2 U Yoshimura 1986

Oryzias latipes (Japanese killifish) 2 cm, 0.2 g NP16.6EO 48h S, NR mortality LC 50 110000 7.2 NR 50 16.2 U Yoshimura 1986

Lepomis macrochirus (bluegill sunfish) NR NP30EO 96h S, M mortality LC 50 >1 x 10 6 7.1 9.0-5.1 NR 18 U Macek and Krzeminski 1975

Pimephales promelas (fathead minnow) sexually mature NP7-11EO 42d F, M histologic lesions NOEC 10 NR NR NR 25-26 2 Miles-Richardson et al. 1999

Oncorhynchus mykiss (rainbow trout) JUV (females) NP1EC 22d,35d F, N growth inhibition LOEC 10 6.5 NR 12.5 7-13 U Ashfield et al. 1998 2yr males NP1EC 21d F, M 100-fold increase in vitellogenin NR 32 NR NR NR NR U Jobling et al. 1996

hepatocytes from immature males NP1EC 48h S, N vitellogenin production ED 50 15.25 μM (4916 μg·L-1 ) NR NR NR NR U Jobling and Sumpter 1993

Pimephales promelas (fathead minnow) NR NP1EC 96h NR,NR mortality LC 50 NOEC 2000

1000 NR NR NR NR U Williams et al. 1996 1

### APPENDIX If (continued). Toxicity of nonylphenol ethoxylates to freshwater fish through water column exposure

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Hardness** Temp.

(°C) Evaluation

Rank Reference

Oryzias latipes (Japanese medaka) 1d posthatch NP1.5EC 90d R, N growth NOEC 100 NR NR NR 22-24 2 Metcalfe et al. 20011d posthatch NP1.5EC 90d R, N sex ratio NOEC 100 NR NR NR 22-24 2 Metcalfe et al. 20011d posthatch NP1.5EC 90d R, N testis-ova NOEC 100 NR NR NR 22-24 2 Metcalfe et al. 2001

Oryzias latipes (Japanese killifish) 2 cm, 0.2 g sodium 4-

NP2EC 48h S, NR mortality LC 50 8900 7.2 NR 50 16.2 U Yoshimura 1986

2 cm, 0.2 g sodium 4-

NP1EC 48h S, NR mortality LC 50 9600 7.2 NR 50 16.2 U Yoshimura 1986

NR = not reported Life Stage (at start of assay): ADT = adult, JUV = juvenile, NEO = neonates, EMB = embryo * duration reported in hours (h) or days (d) Test type: S = static, R = static-renewal, F = flow-through (continuous or intermittent), IPS = single intraperitoneal injection, IPR = repeated intraperitoneal injections

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration D.O. = dissolved oxygen ** hardness reported in mg CaCO 3 ·L-1 Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable

1 As cited in Servos et al. 2000 2As cited in Gloxhuber 1974 3As cited in Anonymous 1977 as cited in Talmage 1994 4Although reported asIdus idus in the original article, this species has since been renamed as Leuciscus idus

## APPENDIX II

Toxicity of nonylphenol and its ethoxylates to marine aquatic life

through water column exposure

### APPENDIX IIa. Toxicity of nonylphenol to marine algae through water column exposure.

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp.

(°C) Evaluation

Rank Reference

Skeletonema costatum (algae) NR 96h S, M growth inhibition (cell count) EC 50

NOEC 27

10 7.9-9.6 NR 30 21-22 1 Ward and Boeri 1990d

NR 96h F, NR NR EC 50 NOEC LOEC MATC 27

10 20 14.1 NR NR NR NR U Weeks et al. 1996 (also cited in Naylor 1995)

NR = not reported * duration reported in hours (h) Test type: S = static, F = flow-through (continuous or intermittent)

M = measured concentrations throughout experiment Conc. = concentration D.O. = dissolved oxygen Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable

### APPENDIX IIb. Toxicity of nonylphenol to marine invertebrates through water column exposure.

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp.

(°C) Evaluation

Rank Reference

Crustaceans Acartia tonsa (calanoid copepod) ADT (10-12d) 48h S, N mortality LC 50 LC 50 LC 50 190

280 360 NR NR 18 20 U Kusk and Wollenberger 1999

Americamysis bahia1 (mysid) NEO 28d F, M mean total length NOEL

LOEL MATC 3.9

6.7 5.1 7.4-8.3 5.0-8.5 20-21 23.2-26.4 1 Ward and Boeri 1991a

NR 28d F, NR length MATC NOEC LOEC 5.1

3.9 6.7 NR NR NR NR U Weeks et al. 1996 (also cited in Naylor 1995)

NEO 28d F, M number of young per surviving female NOEL

LOEL MATC 6.7

9.1 7.8 7.4-8.3 5.0-8.5 20-21 23.2-26.4 1 Ward and Boeri 1991a

NEO 28d F, M survival NOEL LOEL MATC LC 50 6.7

9.1 7.8 >21 7.4-8.3 5.0-8.5 20-21 23.2-26.4 1 Ward and Boeri 1991a

NR 28d F, NR reproduction MATC NOEC LOEC 7.8

6.7 9.1 NR NR NR NR U Weeks et al. 1996 (also cited in Naylor 1995)

NR 28d F, NR survival MATC NOEC LOEC 7.8

6.7 9.1 NR NR NR NR U Weeks et al. 1996 (also cited in Naylor 1995)

NEO (2.8 mg) 96h F, M mortality LC 50 NOEC 43

18 7.3-8.2 6.5-7.8 20 23.8-25.3 1 Ward and Boeri 1990b

NR 96h F, NR mortality LC 50 43 NR NR NR NR U Weeks et al. 1996 (also cited in Naylor 1995)postlarvae 1-day 96h F, M mortality LC 50 60.6 7.8-8.2 NR 30-31 24-25 1 Lussier et al. 2000

Balanus amphitrite (barnacle) LAR 1-6d R, M cypris major protein production 50%

increase 0.023 NR NR NR 28 U Billinghurst et al. 2000

LAR (day-3 cypris) 48h R, M reduced settlement LOEC 0.059 NR NR NR 28 2 Billinghurst et al. 1998

LAR 1-6d R, M cypris major protein production 2-fold

increase 0.693 NR NR NR 28 U Billinghurst et al. 2000

### APPENDIX IIb (continued). Toxicity of nonylphenol to marine invertebrates through water column exposure.

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp.

(°C) Evaluation

Rank Reference

Balanus amphitrite (barnacle) LAR (day2 cypris) 48h in

NP, 48h in clean water R, M reduced

settlement LOEC 0.693 NR NR NR 25 U Billinghurst et al. 1998

Corophium volutator Pallas (amphipod) <5d JUV

mean 1.02 mm 120d S , N density and population growth LOEC 10 NR NR 25 15 U Brown et al. 1999

<5d JUV

mean 1.02 mm 120d S , N time to maturity; length; sex ratio NOEC 10 NR NR 25 15 U Brown et al. 1999

mean 4.38 mm 42d S , N mortality 96h LC 50 10d LC 50 30d LC 50 1670

620 270 NR NR 25 15 U Brown et al. 1999

Crangon crangon (brown shrimp) NR 7d NR, NR mortality LC 50 340 NR NR NR NR U Waldock & Thain 1991 2
NR 96h NR,

NR mortality LC 50 420 NR NR NR NR U Waldock & Thain 1991 2
NR 96h R,NR mortality LC 50 600 NR NR 32 17 U Granmo et al. 1991a Crangon septemspinosa (sand shrimp) 2 g each 96h R, M mortality LC 50 300 NR NR NR 10 U McLeese et al. 1981

1.3g 96h R, M mortality LC 50 300 NR NR NR 10 U McLeese et al. 1980a Crassostrea gigas (Pacific oyster) LAR 72h S, N delayed larval development LOEC 100 measure d NR 35 20 2 Nice et al. 2000

LAR 48h S, N deformity LOEC 100 measure d NR 35 20 2 Nice et al. 2000

Dyspanopeus sayii (mudcrab) zoea 4th& 5thstage 96h F, M mortality LC 50 >195 7.8-8.2 NR 30-31 24-25 2 Lussier et al. 2000

Homarus americanus (American lobster) zoea first stage 96h R, N mortality LC 50 71 7.8-8.2 NR 30-31 16-17 2 Lussier et al. 2000

NR (450g) 96h R, M mortality LC 50 170 NR NR NR 10 U McLeese et al. 1980a Leptocheirus plumulosus (amphipod) ADT 96h F, M mortality LC 50 61.6 7.8-8.2 NR 30-31 24-25 1 Lussier et al. 2000

Nitocra spinipes (harpacticoid) NR 96h NR, NR mortality LC 50 118, 139 NR NR NR NR U Bergström 1984 3

Paleomonetes vulgaris (grass shrimp) LAR (2- day) 96h F, M mortality LC 50 59.4 7.8-8.2 NR 30-31 24-25 1 Lussier et al. 2000

### APPENDIX IIb (continued). Toxicity of nonylphenol to marine invertebrates through water column exposure.

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp.

(°C) Evaluation

Rank Reference

Tisbe battagliai (copepod) nauplii <1 day old 53d R, M reduced survival of offspring, intrinsic rate of increase, egg product-ion NOEC LOEC 20

41 NR NR 25 10 1 Bechmann 1999

nauplii <1 day old 96h S, N mortality LC 50 31-62 NR NR 25 10 2 Bechmann 1999

ADT 96h S, N mortality LC 50 500- 1000 NR NR 25 10 2 Bechmann 1999

Molluscs Mulinia lateralis (coot clam) EMB - LAR 48h S, N inhibition of fertilization EC 50 37.9 7.8-8.2 NR 32 18.3 2 Lussier et al. 2000

Mya arenaria (soft-shelled clam) NR 144h R, M mortality NOEC >700 NR NR NR 10 U McLeese et al. 1980a

NR (20g) 360h R, M mortality NOEC >1000 NR NR NR 10 U McLeese et al. 1980a Mytilus edulis (common blue mussel) ADT (40- 50mm) 30d F, N byssus strength NOEC LOEC 32

56 NR NR 32 10 U Granmo et al. 1989

NR 32d NR,

NR growth & strength LOEC 56 NR NR NR NR U Naylor 1995

NR 30d R,NR byssus strength LOEC 56 NR NR 32 17 U Granmo et al. 1991a NR 850h R,NR mortality LC 50 140 NR NR 32 17 U Granmo et al. 1991a eggs to veligers 72h NR,

N fertilization, development NOEC 200 NR NR 32 17 U Granmo et al. 1989

NR 96h NR,

NR mortality LC 50 2600 NR NR NR NR U Naylor 1995

ADT (40- 50mm) 32d R, N mortality 96h LC 50 360h 850h 3000

500 140 NR NR 32 17 U Granmo et al. 1989

NR 96h R,NR mortality LC 50 3000 NR NR 32 17 U Granmo et al. 1991a NR = not reported Life Stage (at start of assay): ADT = adult, JUV = juvenile, NEO = neonates, EMB = embryo * duration reported in hours (h) or days (d) Test type: S = static, R = stati c-renewal, F = flow-through (continuous or intermittent)

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration; D.O. = dissolved oxygen; Temp. = temperature

Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable Critical study for marine Canadian Water Quality Guideline appears in bold

1 This species was formerly named Mysidopsis bahia 2As cited in Servos et al. 2000 3As cited in Wahlberg et al. 1990

### APPENDIX IIc. Toxicity of nonylphenol to marine fish through water column exposure.

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp.

(°C) Evaluation

Rank Reference

Agonus cataphractus (hook nose) NR 7d NR, NR mortality LC 50 360 NR NR NR NR U Waldock and Thain 1991 1

NR 96h NR, NR mortality LC 50 510 NR NR NR NR U Waldock and Thain 1991 1 Cyprinodon bovinus (Leon Springs pupfish) NR 96h NR, NR NR LC 50 480 NR NR NR NR U Brecken-Folse et al. 1995

Cyprinodon variegatus (sheepshead minnow) JUV 96h F, M mortality LC 50 142 7.8- 8.2 NR 30-31 24-25 1 Lussier et al. 2000

JUV (mean 0.55g ww, 26.3 mm) 96h F, M mortality; bloating, lethargy, etc. LC 50

NOEC 310

240 7.4-

8.1 7.0-8.8 (60-105% saturation) NR 22 (21.4- 23.0) 1 Ward and Boeri 1990c

NR 96h NR, NR NR LC 50 460 NR NR NR NR U Brecken-Folse et al. 1995 Fundulus heteroclitus (killifish) LAR (2 weeks old) 96h R, N mortality LC 50 209 NR NR 20 20 2 Kelly and Di Giulio 2000

EMB 10d R, N sublethal abnormalitie

s LOEC 2203 NR NR 20 20 2 Kelly and Di Giulio 2000

EMB 96h R, N mortality LC 50 5441 NR NR 20 20 2 Kelly and Di Giulio 2000 Gadus morhua (cod) NR 21d F, NR mortality median tolerance limit <100 NR NR NR 16 U Swedmark 1968

NR 96h F, NR mortality median tolerance limit 300 NR NR NR 16 U Swedmark 1968

NR 96h NR, NR mortality LC 50 370 NR NR 32 NR U Granmo et al. 1991a Gasterosteus aculeatus (3-spined stickleback) NR 96h R, NR mortality LC 50 370 NR NR 32 17 U Granmo et al. 1991a

Menidia beryllina (inland silversides) JUV 96h F, M mortality LC 50 70 7.8- 8.2 NR 30-31 24-25 1 Lussier et al. 2000

Platichthys flesus (flounder) ADT (male) 15d IPR, N dosedependent increase in vitellogenin observed NR 10 - 200 mg·kg -1

· week-1 NR NR NR NR U Bjerregaard et al. 1998

### APPENDIX IIc (continued). Toxicity of nonylphenol to marine fish through water column exposure.

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp.

(°C) Evaluation

Rank Reference

Platichthys flesus (flounder) ADT 21d F, M vitellogenin induction NOEC 24.5 NR >80% 34 10 2 Allen et al. 1999

ADT 21d F, M effect on gonadosoma -tic index NOEC 24.5 NR >80% 34 10 2 Allen et al. 1999

ADT 21d F, M increasedhepatosomatic index LOEC 24.5 NR >80% 34 10 2 Allen et al. 1999

Pleuronectes americanus (winter flounder) LAR (2-day) 96h S, M mortality LC 50 17 7.8- 8.2 NR 30-31 5 2 Lussier et al. 2000

Salmo salar (Atlantic Salmon) JUV (8 g each) 96h F, M mortality LC 50 130, 160 NR NR NR 10 U McLeese et al. 1981

JUV (8 g each) 96h R, M mortality LC 50 190 NR NR NR 10 U McLeese et al. 1981

hepatocytes from males or juveniles 48h R, N induction of zona radiata proteins NOEC

LOEC 1 μM (220.3

μg·L-1 ) 5 μM (1100 μg·L-1 ) NR NR NR 10 U Celius et al. 1999

JUV (4g) 96h R, NR mortality LC 50 900 NR NR NR 10 U McLeese et al. 1980a hepatocytes from males or juveniles 96h R, N vitellogenin induction NOEC

LOEC 5 μM (1100

μg·L-1 ) 10 μM (2203 μg·L-1 ) NR NR NR 10 U Celius et al. 1999

JUV (ca. 1 year old) 167g 14d IPS, N increased vitellogenin and zona radiata protein levels significant

effect 25 mg·kg -1 NR NR 34 10 U Arukwe et al. 2000a

JUV (ca. 1 year old) 75-120g 14d F

(water); S (NP), N decreased

EROD activity LOEC

NOEC 125 mg·kg -1

fish 25 mg·kg -1 fish NR NR NR 10 U Arukwe et al. 1997a

### APPENDIX IIc (continued). Toxicity of nonylphenol to marine fish through water column exposure.

Species (Common name) Life stage Dur.* Test

type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp.

(°C) Evaluation

Rank Reference

Zoarces viviparus (eelpout) pregnant females (70g) 21d R, N increased vitellogenin

conc. LOEC

NOEC 100

50 NR NR 20 11 2 Korsgaard and Pedersen 1998

EMB 21d R, N increased vitellogenin

conc. LOEC 1000 NR NR 20 NR U Korsgaard and Pedersen 1998

males 21d R, N vitellogenin increased 10000-fold NR 1000 NR NR NR 12 U Christiansen et al. 1998b,c

ADT (male) 25d IPR, N vitellogenin increased 10000-fold NR 0.1 mg·g -1

· week-1 NR NR NR 12 U Christiansen et al. 1998b,c

males 25d IPR, N vitellogenin increased 1000-fold NR (70.1 mg·g -1

· week-1 NR NR NR 12 U Christiansen et al. 1998b,c

NR = not reported Life Stage (at start of assay): ADT = adult, JUV = juvenile, NEO = neonates, EMB = embryo * duration reported in hours (h) or days (d) Test type: S = static, R = static-renewal, F = flow-through (continuous or intermittent), IPS = single intraperitoneal injection, IPR = repeated intraperitoneal injection s

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration D.O. = dissolved oxygen Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable 1 As cited in Servos et al. 2000

### APPENDIX IId. Toxicity of nonylphenol ethoxylates to marine algae and macrophytes through water column exposure.

Species (Common name) Life

stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp. (°C) Evaluation

Rank Reference

12 species marine phytoplankton NR NP9EO 14d NR, NR growth inhibition EC 50 10000 NR NR NR NR U Ukeles 1965

NR NP30EO 14d NR, NR growth inhibition EC 50 1000000 NR NR NR NR U Ukeles 1965

NR = not reported * duration reported in days (d) Conc. = concentration D.O. = dissolved oxygen Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable

### APPENDIX IIe. Toxicity of nonylphenol ethoxylates to marine invertebrates through water column exposure.

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Salinity

(‰) Temp. (°C) Evaluation

Rank Reference

Crustaceans Americamysis bahia 1 (mysid) 3-8 days old NP1.5EO 48h R, M mortality LC 50 110 7.7-8.0 >90% saturation NR 25 2 Hall et al. 1989

Americamysis bahia 1 (mysid) NR NP2EO 48h R, NR mortality LC 50 ca. 150 NR NR NR NR U Patoczka and Pulliam 1990

Americamysis bahia 1 (mysid) 3-8 days old NP9EO 48h R, N mortality LC 50 900 - 2000 710 - 1560 7.7-8.0 >90% saturation NR 25 2 Hall et al. 1989

NR NP9EO 48h R, NR mortality LC 50 1230 NR NR NR NR U Patoczka and Pulliam 1990 NR NP9EOPO 3 -2 48h R, NR mortality LC 50 4600 NR NR NR NR U Patoczka and Pulliam 1990 3-8 days old NP9EO

phosphated 48h R, N mortality LC 50 5230 7.7-8.0 >90% saturation NR 25 2 Hall et al. 1989

3-8 days old NP9EO

sulphonated 48h R, N mortality LC 50 29600 24300 7.7-8.0 >90% saturation NR 25 2 Hall et al. 1989

NR NP9EOSO 3 - 48h R, NR mortality LC 50 29600 NR NR NR NR U Patoczka and Pulliam 1990

Balanus balanoides (common barnacle) LAR (stage II nauplius) NP10EO 96h R, N mortality LC 50 1500 NR NR NR 6-8 U Swedmark et al. 1971

ADT NP10EO 96h F, N mortality LC 50 <25000 NR NR NR 6-8, 15-17 U Swedmark et al. 1971

Carcinus maenas

. (shore crab) ADT NP10EO 96h F, N mortality LC 50 >100000 NR NR NR 6-8 U Swedmark et al. 1971

Eupagurus bernhardus (hermit crab) ADT NP10EO 96h F, N mortality LC 50 >100000 NR NR NR 6-8 U Swedmark et al. 1971

Hyas araneus (spider crab) LAR (stage 1 zoea) NP10EO 96h S, N mortality LC 50 10000 NR NR NR 6-8 U Swedmark et al. 1971

ADT NP10EO 96h F, N mortality LC 50 >100000 NR NR NR 6-8 U Swedmark et al. 1971 Leander adspersus (decapod) postmoult phase NP10EO 96h F, N mortality LC 50 10000 NR NR NR 15-17 U Swedmark et al. 1971

intermoult phase NP10EO 96h F, N mortality LC 50 50000 NR NR NR 15-17 U Swedmark et al. 1971

ADT NP10EO 96h F, N mortality LC 50 >100000 NR NR NR 6-8 U Swedmark et al. 1971 Leander squilla (decapod) ADT NP10EO 96h F, N mortality LC 50 >100000 NR NR NR 6-8 U Swedmark et al. 1971

### APPENDIX IIe (continued). Toxicity of nonylphenol ethoxylates to marine invertebrates through water column exposure.

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Salinity

(‰) Temp. (°C) Evaluation

Rank Reference

Crangon crangon (brown shrimp) NR NP12EO 48h NR, NR mortality LC 50 89500 NR NR NR NR U Portmann and Wilson 1971 2 Carcinus maenus (shore crab) NR NP12EO 48h NR, NR mortality LC 50 >100000 NR NR NR NR U Portmann and Wilson 1971 2
NR NP12EO 48h NR, NR mortality LC 50 >100000 NR NR NR NR U Waldock and Thain 1991 3 Pandalus montagui (pink shrimp) NR NP12EO 48h NR, NR mortality LC 50 19300 NR NR NR NR U Portmann and Wilson 1971 2

Americamysis bahia 1 (mysid) NR NP15EO 48h R, NR mortality LC 50 ca. 2000 NR NR NR NR U Patoczka and Pulliam 1990 3-8 days old tp-NP15EO 48h R, N mortality LC 50 2570 7.7-8.0 >90% saturation NR 25 2 Hall et al. 1989

Americamysis bahia 1 (mysid) 3-8 days old tp-NP40EO 48h R, N mortality LC 50 >100000 7.7-8.0 >90% saturation NR 25 2 Hall et al. 1989

NR NP40EO 48h R, NR mortality LC 50 ca. 100000 NR NR NR NR U Patoczka and Pulliam 1990

Americamysis bahia 1 (mysid) NR NP50EO 48h R, NR mortality LC 50 ca. 4000000 NR NR NR NR U Patoczka and Pulliam 1990 3-8 days old tp-NP50EO 48h R, N mortality LC 50 >4110000 7.7-8.0 >90% saturation NR 25 2 Hall et al. 1989

Americamysis bahia 1 (mysid) NR NP1EC 96h NR, NR mortality LC 50

NOEC 9400

4100 NR NR NR NR U Naylor et al. 1997

Mollusca Monodonta turbiformis (snail) NR NP9.5EO 14d NR, NR differential survival of allozyme genotypes NR 7000- 117000 8.3 NR NR 22 U Lavie et al. 1984

Monodonta turbinata (snail) NR NP9.5EO 14d NR, NR differential survival of allozyme genotypes NR 6740- 67400 8.3 NR NR 22 U Lavie et al. 1984

### APPENDIX IIe (continued). Toxicity of nonylphenol ethoxylates to marine invertebrates through water column exposure.

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O. (mg·L-1 ) Salinity

(‰) Temp. (°C) Evaluation

Rank Reference

Cardium edule (cockle) ADT NP10EO 96h F, N mortality LC 50 5000 NR NR NR 6-8 U Swedmark et al. 1971 JUV NP10EO 96h F, N mortality LC 50 <<10000 NR NR NR 15-17 U Swedmark et al. 1971 Mya arenaria (clam) ADT NP10EO 96h F, N mortality LC 50 <10000 NR NR NR 15-17 U Swedmark et al. 1971 ADT NP10EO 96h F, N mortality LC 50 18000 NR NR NR 6-8 U Swedmark et al. 1971 Mytilus edulis (common blue mussel) ADT NP10EO 96h F, N mortality LC 50 <10000 NR NR NR 15-17 U Swedmark et al. 1971 ADT NP10EO 96h F, N mortality LC 50 12000 NR NR NR 6-8 U Swedmark et al. 1971 Pecten maximus (scallop) ADT NP10EO 96h F, N mortality LC 50 <<5000 NR NR NR 15-17 U Swedmark et al. 1971 Pecten opercularis ADT NP10EO 96h F, N mortality LC 50 <<10000 NR NR NR 15-17 U Swedmark et al. 1971

Cardium edule (cockle) NR NP12EO 48h NR, NR mortality LC 50 92500 NR NR NR NR U Portmann and Wilson 1971 2

NR = not reported Life Stage (at start of assay): ADT = adult, JUV = juvenile, NEO = neonates, EMB = embryo * duration reported in hours (h) or days (d) Test type: S = static, R = static-re newal, F = flow-through (continuous or intermittent), IPS = single intraperitoneal injection, IPR = repeated intraperitoneal injections

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration D.O. = dissolved oxygen Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable

1 This species was formerly named Mysidopsis bahia 2As cited in Talmage 1994 3As cited in Servos et al. 2000

### APPENDIX IIf. Toxicity of nonylphenol ethoxylates to marine fish through water column exposure.

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp. (°C) Evaluation

Rank Reference

Anguilla anguilla (European eel) - spawn in saltwater post-LAR (elvers) (70.7 mm) NP5EO 24h exposure, 24h recovery S, M mortality LC 50 1200 7.92 95-98% saturation NR 12 2 Miossec and Bocquéné 1986

Anguilla anguilla (European eel) - spawn in saltwater post-LAR (elvers) (70.7 mm) NP8EO 24h exposure, 24h recovery S, M mortality LC 50 3000 7.78 94-104% saturation NR 12 2 Miossec and Bocquéné 1986

Anguilla anguilla (European eel) - spawn in saltwater post-LAR (elvers) (70.7 mm) NP10EO 24h exposure, 24h recovery S, M mortality LC 50 3300 7.89 94-95% saturation NR 12 2 Miossec and Bocquéné 1986

Gadus morhua (cod) NR NP10EO NR NR, NR avoidance behaviour >20 NR NR NR NR U Hoglund 1976 1

ADT (30 cm) NP10EO 96h F, N mortality LC 50 2500 NR NR NR 15-17 U Swedmark et al. 1971 ADT (30 cm) NP10EO 96h F, N mortality LC 50 6000 NR NR NR 6-8 U Swedmark et al. 1971 Pleuronectes flesus (flounder) ADT NP10EO 96h F, N mortality LC 50 3000 NR NR NR 15-17 U Swedmark et al. 1971

Anguilla anguilla (European eel) - spawn in saltwater post-LAR (elvers) (70.7 mm) NP12EO 24h exposure, 24h recovery S, M mortality LC 50 7500 7.95 93-94% saturation NR 12 2 Miossec and Bocquéné 1986

Anguilla anguilla (European eel) - spawn in saltwater post-LAR (elvers) (70.7 mm) NP17EO 24h exposure, 24h recovery S, M mortality LC 50 95000 7.84 93-96% saturation NR 12 2 Miossec and Bocquéné 1986

Anguilla anguilla (European eel) - spawn in saltwater post-LAR (elvers) (70.7 mm) NP20EO 24h exposure, 24h recovery S, M mortality LC 50 970000 7.9 96-98% saturation NR 12 2 Miossec and Bocquéné 1986

### APPENDIX IIf (continued). Toxicity of nonylphenol ethoxylates to marine fish through water column exposure.

Species (Common name) Life stage Chemical Dur.* Test type Effect Endpoint Conc. (μg·L-1 ) pH D.O.

(mg·L-1 ) Salinity

(‰) Temp. (°C) Evaluation

Rank Reference

Anguilla anguilla (European eel) - spawn in saltwater post-LAR (elvers) (70.7 mm) NP30EO 24h exposure, 24h recovery S, M mortality LC 50 >1 x 10 7 NR NR NR 12 2 Miossec and Bocquéné 1986

NR = not reported Life Stage (at start of assay): ADT = adult, JUV = juvenile, NEO = neonates, EMB = embryo * duration reported in hours (h) Test type: S = static, R = static-renewal, F = flow-through (continuous or intermittent) , IPS = single intraperitoneal injection, IPR = repeated intraperitoneal injections

M = measured concentrations throughout experiment, N = nominal concentration Conc. = concentration; D.O. = dissolved oxygen; Temp. = temperature Evaluation Rank: 1 = primary, 2 = secondary, U = unacceptable 1 As cited in Servos et al. 2000

## APPENDIX III

Toxicity of nonylphenol to sediment-dwelling life

### Appendix IIIa. Toxicity of nonylphenol to freshwater life through spiked-sediment exposure.

Organism Life Stage Endpoint NP (mg·kg -1 dw) Temperature (°C)

pH % TOC Study Conditions Reference

LETHAL TESTS Chironomus tentans (midge) larvae 14-d exposure, 10% mortality, not toxic (NOEC) 20.1 19 to 21 

### 5.5 (sediment) 1.27 - based on standard ASTM and USEPA test

methods - clean, flow-through water - system equilibrated for 1 day England and Bussard 1993

larvae 14-d exposure, 31%

mortality, toxic (LOEC) 34.2 19 to 21 

### 5.5 (sediment) 1.27 - based on standard ASTM and USEPA test

methods - clean, flow-through water - system equilibrated for 1 day England and Bussard 1993

Rana catesbiana (bullfrog) tadpole 30-d exposure, 10% mortality, not toxic (NOEC) 155.0 

### 17.1 to 18.8 

### 6.9 to 8.3 (water) 0.05 - based on USEPA test methods

- clean, flow-through water - system equilibrated for 2.5 hrs Ward and Boeri 1992

tadpole 30-d LC 50 , (toxic) 260.0 

### 17.1 to 18.8 

### 6.9 to 8.3 (water) 0.05 - based on USEPA test methods

- clean, flow-through water - system equilibrated for 2.5 hrs Ward and Boeri 1992

tadpole 30-d exposure, 85%

mortality, toxic (LOEC) 390.0 

### 17.1 to 18.8 

### 6.9 to 8.3 (water) 0.05 - based on USEPA test methods

- clean, flow-through water - system equilibrated for 2.5 hrs Ward and Boeri 1992

SUB-LETHAL TESTS

Chironomus tentans (midge) larvae 14-d exposure, 6% growth reduction, not toxic (NOEC) 20.1 19 to 21 5.5 (sediment) - based on standard ASTM and USEPA test

methods - clean, flow-through water - system equilibrated for 1 day England and Bussard 1993

larvae 14-d exposure, 27%

growth reduction, toxic (LOEC) 34.2 19 to 21 5.5 (sediment) - based on standard ASTM and USEPA test

methods - clean, flow-through water - system equilibrated for 1 day England and Bussard 1993

### Appendix IIIa (continued). Toxicity of nonylphenol to freshwater life through spiked-sediment exposure.

Organism Life Stage Endpoint NP (mg·kg -1 dw) Temperature (°C)

pH % TOC Study Conditions Reference

Rana catesbiana (bullfrog) tadpole 30-d exposure, 10% sublethal effects (lethargy, hemmorhaging, tail lesions), not toxic (NOEC) 155.0 

### 17.1 to 18.8 

### 6.9 to 8.3 (water) 0.05 - based on USEPA test methods

- clean, flow-through water - system equilibrated for 2.5 hrs Ward and Boeri 1992

tadpole 30-d exposure, 25%

growth reduction, not toxic (NOEC) 155.0 

### 17.1 to 18.8 

### 6.9 to 8.3 (water) 0.05 - based on USEPA test methods

- clean, flow-through water - system equilibrated for 2.5 hrs Ward and Boeri 1992

tadpole 30-d EC50for growth and

other sublethal effects 220.0 

### 17.1 to 18.8 

### 6.9 to 8.3 (water) 0.05 - based on USEPA test methods

- clean, flow-through water - system equilibrated for 2.5 hrs Ward and Boeri 1992

tadpole 30-d exposure, 29%

growth reduction, toxic (LOEC) 390.0 

### 17.1 to 18.8 

### 6.9 to 8.3 (water) 0.05 - based on USEPA test methods

- clean, flow-through water - system equilibrated for 2.5 hrs Ward and Boeri 1992

tadpole 30-d exposure, 100%

sublethal effects (lethargy, hemmorhaging, tail lesions), toxic (LOEC) 390.0 

### 17.1 to 18.8 

### 6.9 to 8.3 (water) 0.05 - based on USEPA test methods

- clean, flow-through water - system equilibrated for 2.5 hrs Ward and Boeri 1992

NOEC = no observed effect concentration LOEC = lowest observed effect concentration LC 50= median lethal concentration EC 50= median effective concentration dw = dry weight

### Appendix IIIb. Toxicity of nonylphenol to marine life through spiked-sediment exposure.

Organism Life Stage Endpoint NP (mg·kg -1 dw) Temperature

(°C) pH % TOC Study Conditions Reference

LETHAL TESTS Ampelisca abdita (amphipod) juvenile 0.7-1.2

mm 10-d LC 50 (toxic) 98.7 20 NR NR - based on USEPA test methods

- 29 to 32 g·L-1 seawater - aeration of water column Fay et al. 2000

juvenile 0.7-1.2

mm 10-d LC 100(toxic) 243 20 NR NR - based on USEPA test methods

- 29 to 32 g·L-1 seawater - aeration of water column Fay et al. 2000

SUB-LETHAL TESTS Capitella sp. I (polychaete) larvae to adult 78-d exposure, 17% delay in time to

first reproduction, toxic (LOEC) 174 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 0% difference in time to

first reproduction, not toxic (NOEC) 52 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 46% increase in time

between reproductive events, toxic (LOEC) 174 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 3% increase in time

between reproductive events, not toxic (NOEC) 52 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 44% fewer broods per

reproductive individual, toxic (LOEC) 174 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 2% more broods per

reproductive individual, not toxic (NOEC) 52 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 78% fewer eggs per

reproductive individual, toxic (LOEC) 174 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 3% fewer eggs per

reproductive individual, not toxic (NOEC) 52 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 76% fewer eggs per

individual, toxic (LOEC) 174 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

### Appendix IIIb (continued). Toxicity of nonylphenol to marine life through spiked-sediment exposure.

Organism Life Stage Endpoint NP (mg·kg -1 dw) Temperature (°C) pH % TOC Study Conditions Reference

Capitella sp. I (polychaete) larvae to adult 78-d exposure, 3% fewer eggs per

individual, not toxic (NOEC) 52 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 33% decrease in size at

maturity, toxic (LOEC) 174 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 16% decrease in size at

maturity, not toxic (NOEC) 52 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 24% decrease in

population growth rate, toxic (LOEC) 174 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

larvae to adult 78-d exposure, 0% decrease in

population growth rate, not toxic (NOEC) 52 20 NR 7.4 - 26 to 27 g·L-1 seawater

- renewal of water and sediment weekly Hansen et al. 1999

NR = not reported NOEC = no observed effect concentration LOEC = lowest observed effect concentration dw = dry weight

## APPENDIX IV

Toxicity of nonylphenol and its ethoxylates to terrestrial life

### APPENDIX IVa. Toxicity of nonylphenol to soil microbial processes.

Organisms / Effect Endpoint Concentration (mg·kg -1 ) Duration* Soil pH Test Substrate Reference

Selected CO2evolution CO2evolution (-64%) NOEC LOEC 100

1000 40d 6.8 mixture of sewage sludge compost (digested sludge and sawdust matured for 9 months) & Permian sandstone (75% sand, 13% clay, 12% silt) in ratio 1:2 (dw); adjusted to 60% field moisture capacity with demineralized water Trocmé et al. 1988

CO2evolution CO2evolution (+26%) NOEC LOEC 10

500 3 mo. 7.3 silt loam, Udic Haploboroll, Calcaric Phaeozem, 13% clay, 42% silt, 55% sand; dried, sieved, stored for 2 years; 3.5% CaCO 3 Kirchmann et al. 1991

N-mineralization NOEC 500 3 mo. 7.3 silt loam, Udic Haploboroll, Calcaric Phaeozem, 13% clay, 42% silt, 55% sand; dried, sieved, stored for 2 years; 3.5% CaCO 3 Kirchmann et al. 1991

(nitrite accumulation) Nitrification Nitrification (-35%) NOEC LOEC 10

500 7d 7.3 silt loam, Udic Haploboroll, Calcaric Phaeozem, 13% clay, 42% silt, 55% sand; dried, sieved, stored for 2 years; 3.5% CaCO 3 Kirchmann et al. 1991

Consulted Azotobacter

spp. (bacteria) growth growth (-95%) NOEC LOEC 18.8 mg·L-1

37.6 mg·L-1 72h NR medium: CaCl 2 , FeSO 4· 7 H 20, K2 HP04 , MgSO4· 7 H 20, MnCl 2 , Na2 MoO4 , mannitol, deionized water Mårtensson and Torstensson 1996

Azotobacter

spp. (bacteria) N 2 -fixation N 2 -fixation NOEC LOEC 18.8 mg·L-1

37.6 mg·L-1 72h NR medium: CaCl 2 , FeSO 4· 7 H 20, K2 HP04 , MgSO4· 7 H 20, MnCl 2 , Na2 MoO4 , mannitol, deionized water Mårtensson and Torstensson 1996

Biomass ATP evolution NOEC 1000 40d 6.8 mixture of sewage sludge compost (digested sludge and sawdust matured for 9 months) & Permian sandstone (75% sand, 13% clay, 12% silt) in ratio 1:2 (dw); adjusted to 60% field moisture capacity with demineralized water Trocmé et al. 1988

Candida albicans

(yeast), decrease in growth rate EC 50 EC 98 28 μM (6.2 mg·L-1 )

100 μM (22 mg·L-1 ) 8h NR yeast nitrogen base medium; incubated on orbital shaker Karley et al. 1997

Neurospora crassa growth inhibition growth inhibition EC 50 EC 100 6 μM (1.3 mg·L-1 )

100 μM (22 mg·L-1 ) 168h (7d) NR Vogel’s minimal media plus 2% (wt/vol) sucrose; incubated on

orbital shaker Karley et al. 1997

Neurospora crassa oxygen uptake rate inhibition oxygen uptake rate inhibition EC 50 EC 60 23 μM (5.1 mg·L-1 )

100 μM (22 mg·L-1 ) 10 min. 5.8 buffer: 3,3-dimethylglutarate, CaCl 2 , glucose, KOH Karley et al. 1997

### APPENDIX IVa (continued). Toxicity of nonylphenol to soil microbial processes.

Organisms / Effect Endpoint Concentration (mg·kg -1 ) Duration

* Soil pH Test Substrate Reference

Nitrification NOEC 10 g·kg -1 sludge dw (concentration of sludge/soil not reported) 6h NR sandy loam from Flakkebjerg, Denmark; sieved (2 mm); stored at - 20C; activated sludge aerobically stabilized to remove organic contaminants, spiked with NP, mixed with soil Madsen et al. 1998

Photobacterium phosphoreum

(bacteria), bioluminescence EC 50 5.6 μmol·L-1 (1.2 mg·L-1 ) 30 min. NR NR (Microtox test) Carlson-Ekvall and Morrison 1995

NR = not reported dw = dry weight * duration reported in minutes (min.), hours (h), days (d), or months (mo.)

### APPENDIX IVb. Toxicity of nonylphenol to soil invertebrates.

Organism Life stage Duration* Effect (% decrease) Endpoint Conc. (mg·kg -1 ) Soil

pH Test substrate Reference

Selected Apporectodea calignosa (earthworm) ADT 21d reproduction (cocoon production) EC 10

EC 50 3.44

13.7 NR sandy soil from Lundgård site, 89.7% sand,

3.8% silt, 4.3% clay, 2.2% humus, moisture set to 16% of dw Krogh et al. 1996

JUV 21d growth EC 50 23.9 NR sandy soil from Lundgård site, 89.7% sand, 3.8% silt, 4.3% clay, 2.2% humus, moisture set to 16% of dw Krogh et al. 1996

ADT 21d mortality EC 10 EC 50 >40

>40 NR sandy soil from Lundgård site, 89.7% sand,

3.8% silt, 4.3% clay, 2.2% humus, moisture set to 16% of dw Krogh et al. 1996

Folsomia candida (springtail) JUV 28d reproduction EC 50 16 - 30 NR loamy sand from Jyndevad, Denmark; sieved (2 mm); defaunated; mixed with spiked sludge Madsen et al. 1998

JUV 28d adult survival LC 50 >30 NR loamy sand from Jyndevad, Denmark; sieved (2 mm); defaunated; mixed with spiked sludge Madsen et al. 1998

Folsomia fimetaria (springtail) 23-26 day 21d reproduction EC 10 EC 50 23.6

65.5 NR LUFA sand (German standard soil), 89.4%

sand, 5.6% silt, 5.1% clay, 3.9% humus Krogh et al. 1996

Folsomia fimetaria (springtail) NR 21d reproduction EC 10 EC 50 27

39 NR sandy soil Holm as cited in Jensen et al. 1997 Folsomia fimetaria (springtail) NR 21d reproduction EC 10 EC 50 48

59 NR sandy soil, mixed with sludge Holm as cited in Jensen et al. 1997 Folsomia fimetaria (springtail) 23-26 day 21d adult survival LC 10 LC 50 75.1

151.3 NR LUFA sand (German standard soil), 89.4%

sand, 5.6% silt, 5.1% clay, 3.9% humus Krogh et al. 1996

Consulted Folsomia fimetaria (springtail) 23-26 day 21d reproduction EC 50 EC 10 44.2

27.2 NR Lundgård soil, 89-92% sand, 3-4% silt, 3-4%

clay, 1-2% humus Krogh et al. 1997

Folsomia fimetaria (springtail) 23-26 day 21d reproduction EC 50 EC 10 58.4

48.7 NR Lundgård soil, 89-92% sand, 3-4% silt, 3-4%

clay, 1-2% humus; mixed with spiked sludge Krogh et al. 1997

Folsomia fimetaria (springtail) 23-26 day 21d adult survival LC 50 LC 10 85.9

42.7 NR Lundgård soil, 89-92% sand, 3-4% silt, 3-4%

clay, 1-2% humus Krogh et al. 1997

Folsomia fimetaria (springtail) 23-26 day 21d adult survival LC 50 >100 NR Lundgård soil, 89-92% sand, 3-4% silt, 3-4% clay, 1-2% humus; mixed with spiked sludge Krogh et al. 1997

NR = not reported Life stage (at start of assay): ADT = adult, JUV = juvenile * duration reported in days (d) dw = dry weight

### APPENDIX IVc. Toxicity of nonylphenol to terrestrial plants.

Organism Life stage Duration* Effect (% decrease) Endpoint Conc. (mg·kg -1 ) Soil

pH Test substrate Reference

Selected Brassica rapa ssp. rapa (turnip) seedling 14d growth inhibition EC 5 10 6.1 sandy loam soil, 54.3% sand, 35.7% silt, 9.9% clay; max water-holding capacity 23.2g·100g -1 soil) Günther and Pestemer 1992

Glycine max (soybean) seedling 21d growth NOEC EC 50 100

1000 5.8 grit/loam soil Windeatt and Tapp 1986

Helianthrus rodeo (sunflower) seedling 21d growth NOEC EC 50 100

1000 5.8 grit/loam soil Windeatt and Tapp 1986

Lactuca sativa (lettuce) seed to seedling 7d

14d growth

growth EC 5 0

EC 50 559

625 7.5 agricultural loam, 24% clay, 1.8%

organic matter, sieved (4 mm), 25-30% moisture content (i.e. 80% waterholding capacity) Hulzebos et al. 1993

Sorghum bicolor (sorghum) seedling 21d growth NOEC EC 50 100

1000 5.8 grit/loam soil Windeatt and Tapp 1986

Consulted Atriplex hortensis (garden orache) intact plant 4d reduced dry weight yield EC 50 0.01-0.05 NR grown aseptically in nutrient medium Bokern et al. 1998

cell culture 7-14d reduction of plant cell growth EC 50 22-110 mg·L-1 (100-500 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

Beta vulgaris var. altissima L. cv. Bush Mono Leaves (sugarbeet) cell culture 7-14d reduction of plant cell growth EC 50 2.2-11 mg·L-1 (10-50 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

Beta vulgaris var. esculenta L. cv. Monogold (beet) cell culture 7-14d reduction of plant cell growth EC 50 22-110 mg·L-1 (100-500 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

Betula papyrifera

Marsh (white birch) seedling roots 9-11d electrical

conductivity (ion leakage) EC 50 5 mg·L-1 NR media bathing roots Weinberger and Greenhalgh 1984

seeds 21d reduction in sprout length EC 85 5-10 mg·L-1 NR no soil – seeds in petri dishes with NP Weinberger and Greenhalgh 1984

seeds 6d germination NOEC 10 mg·L-1 NR no soil – seeds in petri dishes with NP Weinberger and Greenhalgh 1984 seedlings 21d reduced sprout growth EC 80 20 mg·L-1 NR no soil -- seeds in petri dishes with NP Weinberger and Vladut 1981

seeds 21d enhanced germination EC26 20 mg·L-1 NR no soil -- seeds in petri dishes with NP Weinberger and Vladut 1981

seeds 21d reduction in percent germination EC26 100 mg·L-1 NR no soil -- seeds in petri dishes with NP Weinberger and Vladut 1981

### APPENDIX IVc (continued). Toxicity of nonylphenol to terrestrial plants.

Organism Life stage Duration* Effect (% decrease) Endpoint Conc. (mg·kg -1 ) Soil

pH Test substrate Reference

Chenopodium quinoa (quinoa) cell culture 7-14d reduction of plant cell growth EC 50 2.2-11 mg·L-1 (10-50 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

Chenopodium rubrum (red goosefoot) cell culture 7-14d reduction of plant cell growth EC 50 11-22 mg·L-1 (50-100 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

Daucus carota (carrot) cell culture 48h growth (dry weight) NOEC 1 mM NR cell culture medium Harms 1992

cell culture 7-14d reduction of plant cell growth EC 50 >220 mg·L-1 (>1000 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

Glycine max (soybean) cell culture 7-14d reduction of plant cell growth EC 50 >220 mg·L-1 (>1000 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

Hordeum vulgare (barley) cell culture 48h growth (dry weight) LOEC

NOEC 0.5 mM

0.1 mM NR cell culture medium Harms 1992

cell culture 7-14d reduction of plant cell growth EC 50 22-110 mg·L-1 (100-500 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

Lactuca sativa (lettuce) seed to seedling 21d growth EC 50 >0.1, <0.32 NR for germination: perlite saturated with nutrient solution; after germination 5 seedlings with roots >3cm long transferred to nutrient solution only for

growth test Hulzebos et al. 1993

cell culture 10d reduction of plant cell growth EC 50 110-220 mg·L-1 (500- 1000 μM) NR MS medium + 2 mg·L-1 2,4-D + 0.25 mg·L-1 kinetine + 1 mg·L-1 6- benzylaminopurine; sucrose 20 g·L-1 Bokern and Harms 1997

Lepidium sativum (garden cress) seeds 72h inhibition of radicle elongation EC 50 6400 mg·L-1 NR on filter paper in petri dishes Günther and Pestemer 1992

Lupinus hartwegii (Hartweg’s bluebonnet) cell culture 7d reduction of plant cell growth EC 50 11-22 mg·L-1 (50-100 μM) NR MS medium + 2 mg·L-1 2,4-D + 0.25 mg·L-1 kinetine + 1 mM KH 2 PO 4 Bokern and Harms 1997

root culture 7d growth inhibition EC 50 0.1 mM NR MS medium + 2 mg·L-1 2,4-D + 0.25 mg·L-1 kinetine + 1 mM KH 2 PO 4 ; cultured aseptically Bokern et al. 1998

### APPENDIX IVc (continued). Toxicity of nonylphenol to terrestrial plants.

Organism Life stage Duration* Effect (% decrease) Endpoint Conc. (mg·kg -1 ) Soil

pH Test substrate Reference

Lupinus polyphyllus (bigleaf lupine) root culture 7d growth inhibition EC 50 >1 mM NR MS medium + 2 mg·L-1 2,4-D + 0.25 mg·L-1 kinetine + 1 mM KH 2 PO 4 ; cultured with microorganisms Bokern et al. 1998

cell culture 7d reduction of plant cell growth EC 50 >220 mg·L-1 (>1000 μM) NR MS medium + 2 mg·L-1 2,4-D + 0.25 mg·L-1 kinetine + 1 mM KH 2 PO 4 Bokern and Harms 1997

root culture 7d growth inhibition EC 50 >1 mM NR MS medium + 2 mg·L-1 2,4-D + 0.25 mg·L-1 kinetine + 1 mM KH 2 PO 4 ; cultured aseptically Bokern et al. 1998

Lycopersicon esculentum (tomato) cell culture 7-14d reduction of plant cell growth EC 50 11-22 mg·L-1 (50-100 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

cell culture 48h growth (dry weight) LOEC

NOEC 0.1 mM

0.05 mM NR cell culture medium Harms 1992

Pennisetum americanum Rich. (pearl millet) cell culture 9d reduction of plant cell growth EC 50 11-22 mg·L-1 (50-100 μM) NR MS medium + 5 mg·L-1 2,4- dichlorophenoxyacetic acid Bokern and Harms 1997

Pinus banksiana (jack pine) seeds 14d reduction in sprout length EC 60 5-10 mg·L-1 NR no soil – seeds in petri dishes with NP Weinberger and Greenhalgh 1984 seeds NR germination NOEC 10 mg·L-1 NR no soil – seeds in petri dishes with NP Weinberger and Greenhalgh 1984 seedling roots 9-11d electrical

conductivity (ion leakage) EC 50 10 mg·L-1 NR media bathing roots Weinberger and Greenhalgh 1984

seeds 14d reduction in percent germination EC 60 100 mg·L-1 NR no soil -- seeds in petri dishes with NP Weinberger and Vladut 1981

seedlings 14d reduced sprout growth EC20 100 mg·L-1 NR no soil -- seeds in petri dishes with NP Weinberger and Vladut 1981

Triticum aestivum (winter wheat) cell culture 7-14d reduction of plant cell growth EC 50 22-110 mg·L-1 (100-500 μM) NR B-5 or MS medium; cultivated on shaker at 120 rpm Bokern and Harms 1997

NR = not reported * duration reported in hours (h) or days (d) Conc. = concentration

### APPENDIX IVd. Toxicity of nonylphenol to terrestrial vertebrates.

Organism Effect (% effect relative to control) Endpoint Diet Concentration (mg·kg -1 ) Average Dose

(mg·kg -1 BW·d -1 ) Exposure Period Reference

Selected Rattus norvegicus Sprague Dawley (Norway rat) F0 generation: decreased litter size (9%) increased male kidney weight (7%) decreased female body weight (9%) F1 generation: decreased female body weight (7%) earlier vaginal opening (47%) increased pup vagina/uterus relative weight (14%) increased estrous cycle length (14%) increased male relative kidney weight (20%) increased female relative kidney weight (11%) decreased adult ovary weight (19%) decreased adult testis weight (6%) decreased adult male body weight (9%) F2 generation: increased gestation length (2%) sooner to vaginal opening decreased sperm density (8%) increased male relative kidney weight (9%) decreased ovary weight (12%) decreased adult male body weight (8%) increased estrous cycle length (18%) increased male pup weight adjusted for litter size decreased number of spermatids (13%) increased relative kidney weight in female pups (9%) decreased adult female body weight (10%) F3 generation: decreased adult female body weight (10%) sooner to vaginal opening decreased adult epididymis weight (13%) decreased adult male body weight (7%) decreased adult ovary weight reproductive effects EC EC EC

EC EC EC EC EC EC EC EC EC

EC EC EC EC EC EC EC EC EC EC EC

EC EC EC EC EC NOAEL 2000 650 2000

650 650 650 2000 2000 2000 2000 2000 2000

200 650 650 650 650 650 2000 2000 2000 2000 2000

650 650 650 2000 2000 200 88-185

28-63 88-185

30-108 30-108 30-108 100-353 100-353 100-353 100-353 100-353 100-353

10-35 31-115 31-115 31-115 31-115 31-115 109-360 109-360 109-360 109-360 109-360

NR NR NR NR NR NR 8 weeks

11weeks 11 weeks

21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks

21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks

21 weeks 21 weeks 21 weeks 21 weeks 21 weeks 21 weeks/ generation Chapin et al. 1999

### APPENDIX IVd (continued). Toxicity of nonylphenol to terrestrial vertebrates.

Organism Effect (% effect relative to control) Endpoint Diet Concentration (mg·kg -1 ) Average Dose

(mg·kg -1 BW·d -1 ) Exposure Period Reference

Long Evans rat increased uterine weight in prepubertal females (10%) increased uterine weight in prepubertal females (65%) increased uterine weight in ovariectomized females (10%) increased uterine weight in ovariectomized females (50%) sooner to vaginal opening (5%) sooner to vaginal opening (16%) disruption of estrous cyclicity (29%) disruption of estrous cyclicity (35%) NOEC LOEC NOEC LOEC NOEC LOEC NOEC LOEC 25 50 10 50 25 50 50 100 NR NR NR NR NR NR NR NR 3 days

3 days 3 days 3 days 15 days 15 days 25 days 25 days Laws et al. 2000

Noble rat increased uterine weight in ovariectomized females LOEC NR 75 3 days Odum et al. 1999

Consulted Rattus norvegicus Sprague Dawley (Norway rat) reduced body weight (7%) reduced body weight gain (10-16%) mortality increased male kidney weight all adverse subchronic effects EC EC NOEC LOEC NOAEL 2000 2000 2000 2000 650 129-149

129-149 129-149 129-149 45-50 90 days Cunny et al. 1997

decreased seminiferous tubule diameter decreased epididymal mass and ratio decreased testicular mass, testis ratio, and sperm count LOEC LOEC LOEC 100 250 400 NR NR NR 10 weeks

10 weeks 10 weeks De Jager et al.

1999a

decreased body mass, testicular mass, seminiferous tubule diameter of offspring increased testis ratio, decreased epididymal mass, decreased sperm count in offspring LOEC

LOEC 100

250 NR

NR prenatal to 10

weeks after weaning De Jager et al.

1999b

NR = not reported

### APPENDIX IVe. Toxicity of nonylphenol ethoxylates to terrestrial organisms.

Organism Life Stage Chemical (trade name) Duration

* Effect (%

decrease) Endpoint Concentration Soil pH Test substrate Reference

Bacteria/Fungi Candida albicans (unicellular fungus) yeast form NP1EO 8h decrease in growth rate NOEC

LOEC 100 μM

200μM NR yeast nitrogen base medium; incubated on orbital shaker Karley et al. 1997

Nectria galligena NR t-NP9.5EO (Triton N101) NR growth inhibition EC42 500 μg·L-1 NR nutrient media Hislop et al. 1977

Trichoderma viride NR t-NP9.5EO (Triton N101) NR growth inhibition EC 80 500 μg·L-1 NR nutrient media Hislop et al. 1977

Rhizobium trifolii (N-fixing bacteria) NR 4-t-NPnEO (PP 222) 35d growth (reduced colony diameter) EC 50 1000 mg·L-1 6.8 agar medium Fisher et al. 1978

Plants Phaseolus vulgaris (green beans) started from seed NPnEO

(DME) 56d reduced dry weight yield NOEC 12.3 g·1.8 kg -1 soil (=0.00683 mg·kg -1 ) 6.2 Dagor silt loam; 10% clay; fine-loamy, mixed, mesic Cumulic Haploxerolls formed from calcareous sandstones; top 50 cm; air-dried, sieved (0.5 cm) Miller et al. 1980

Zea mays

var. saccharata (sweet corn) started from seed NPnEO

(DME) 56d reduced dry weight yield NOEC 12.3 g·1.8 kg -1 soil (=0.00683 mg·kg -1 ) 6.2 Dagor silt loam; 10% clay; fine-loamy, mixed, mesic Cumulic Haploxerolls formed from calcareous sandstones; top 50 cm; air-dried, sieved (0.5 cm) Miller et al. 1980

Glycine max (soya) seedling NP2EO 21d growth NOEC EC 50 10

1000 5.8 grit/loam soil Windeatt and Tapp 1986

Helianthus rodeo (sunflower) seedling NP2EO 21d growth NOEC EC 50 100

1000 5.8 grit/loam soil Windeatt and Tapp 1986

Sorghum bicolor (sorghum) seedling NP2EO 21d growth NOEC EC 50 10

1000 5.8 grit/loam soil Windeatt and Tapp 1986

### APPENDIX IVe (continued). Toxicity of nonylphenol ethoxylates to terrestrial organisms.

Organism Life Stage Chemical (trade name) Duration

* Effect (%

decrease) Endpoint Concentration Soil pH Test substrate Reference

Vicia faba

cv. Maris Bead (bean) leaf discs (3rd & 4th leaf pair), 21- 28d plants NP4EO

(Synperonic) 24h potassium leakage ca. 22% of

total 0.1% w/v NR leaf discs exposed to toxicant solution only Silcox and Holloway 1986

Vicia faba

cv. Maris Bead (bean) leaf discs (3rd & 4th leaf pair), 21- 28d plants NP6EO

(Synperonic) 24h potassium leakage ca. 58% of

total 0.1% w/v NR leaf discs exposed to toxicant solution only Silcox and Holloway 1986

Vicia faba

cv. Maris Bead (bean) leaf discs (3rd & 4th leaf pair), 21- 28d plants NP8EO

(Synperonic) 24h potassium leakage ca. 82% of

total 0.1% w/v NR leaf discs exposed to toxicant solution only Silcox and Holloway 1986

Vicia faba

cv. Maris Bead (bean) leaf discs (3rd & 4th leaf pair), 21- 28d plants NP10EO

(Synperonic) 24h potassium leakage ca. 80% of

total 0.1% w/v NR leaf discs exposed to toxicant solution only Silcox and Holloway 1986

Vicia faba

cv. Maris Bead (bean) leaf discs (3rd & 4th leaf pair), 21- 28d plants NP12EO

(Synperonic) 24h potassium leakage ca. 78% of

total 0.1% w/v NR leaf discs exposed to toxicant solution only Silcox and Holloway 1986

Vicia faba

cv. Maris Bead (bean) leaf discs (3 rd& 4th leaf pair), 21- 28d plants NP20EO

(Synperonic) 24h potassium leakage ca. 47% of

total 0.1% w/v NR leaf discs exposed to toxicant solution only Silcox and Holloway 1986

Trifolium repens (white clover) seedling 4-t-NPnEO (PP 222) 84d growth (plant weight) LOEC

NOEC 5000

1000 7 sterile John Innes compost, innoculated with Rhizobium trifolii Fisher et al. 1978

NR = not reported * duration reported in hours (d) or days (d) w/v = weight per volume