Biogeochemical Behaviour of Alkvlphenol Pol Vethoxvlates in the Aquatic Environment

BIOGEOCHEMICAL BEHAVIOUR OF ALKVLPHENOL POL VETHOXVLATES IN THE AQUATIC ENVIRONMENT

A dissertation submitted to the

UNIVERSITY OF ZAGREB INSTITUTE "RUDJER BO~KOVIC"

for the degree of DOCTOR OF NATURAL SCIENCES

presented by

MARIJAN AHEL

dipl. biotechnol., University of Zagreb M.Sc. Marine Sciences, University of Zagreb born on 3 October 1951 citizen of Zagreb, Croatia, Yugoslavia

Accepted on recommendation of Dr. Walter Giger Dr. Bo~ena Cosovic Dr. Nikola Bl~evic Dr. Sonja lskric Dr. Leo Klasinc

Zagreb 1987 The following parts of this dissertation have been published:

Section 3.1. Analytical Chemistry 1985, 57, 1577-1583.

Analytical Chemistry 1985, 57, 2584-2590.

Environmental Science & Technology 1987, 21, 697-703.

Section 3.3. Vom Wasser 1986, 67, 69-81.

Water Science & Technology 1987, 19, 449-460.

Gas-Wasser-Abwasser 1987, 67, 111-122. 'Io my sons I van, 'Ivrt{\g, Ja{\gv, and Juraj ACKNOWLEDGEMENTS

I am especially greatful to Dr. Walter Giger for his whole hearted support during this study. I have found in him an inspired teacher and a dear friend.

I owe my special thanks to Professor Werner Stumm, Director of the Swiss Federal Institute for Water Resources and Water Pollution Control, who made it possible for me to work in this renowned Institution.

I am also indebted to Dr. Bozena Cosovic for the encouragement and understanding and for useful advice while writing this thesis.

This dissertation would not have been possible without the support of many collegues and friends from the Chemistry Department of EAWAG and from the Center for Marine Research Zagreb of the Institute "Rudjer Boskovic" to whom I am indebted. - Christian Schaffner, for his expert technical assistance and invaluable advice in practical problems - Dr. James McEvoy for his expert advice in mass spectrometry, and for his help during sampling and analysing biological samples - Helga Ponusz for analysing the river water samples in my absence - Dr. Frank Scully, Dr. Bruce Faust, Dr. Verena Sturzenegger, and Professor JOrg Hoigne for their expert help during the investigation of the photochemical degradation - Dr. Theodor Conrad and Dr. Dubravka Hrsak for the help and advice while conducting experiments on biological degradation - Andrija Roman and Heidi Bolliger for the drawings - Nena Granic and ljiljana Cepulic for typing the manuscript - Dr. Damir Krznaric and Lela Krznari6 for helping translate the manuscript to english Table of Contents

PROBLEM STATEMENT AND SCOPE OF THE WORK ...... 1

1. INTRODUCTION ...... 2

1.1. Ecological Acceptability of Surfactants ...... 2 1.1.1. Definition, Classification and Characteristics of Surfactants ...... 2 1.1.2. Manufacture and Usage of Surfactants...... 3 1.1.3. Input, Behaviour and Harmful Effects on the Environment...... 4 1.1.4. Biodegradability of Surfactants ...... 5

1.2. Nonionic Surfactants...... 6 1.2.1. Types, Production and Usage...... 6 1 .2.2. Physico-chemical Properties ...... 8 1.2.3. Analytical Determination ...... 1o 1.2.4. Biological Degradation ...... 14 1.2.5. Occurrence in the Environment and Harmful Effects ...... 17

2. EXPERIMENT AL...... 19

2.1. Materials ...... 19 2.1.1. Chemicals ...... 19 2.1.2. Solutions and Reagents ...... •...... 20 2.1.3. Equipment...... 22

2.2. Analytical Procedures for the Determination of Alkylphenol Polyethoxylates and Their Metabolites ...... 23 2.2.1. Extraction and Purification of Extracts ...... 23 2.2.1.1. Alkylphenol Polyethoxylates ...... •...... •...... 23 2.2.1.2. Lipophilic Alkylphenol Ethoxylates and Alkylphenols ...... 25 2.2.1.3. Alkylphenoxy Carboxylic Acids ...... 26 2.2.2. Preparative Separation and Determination of Response Factors...... 28 2.2.2.1. Alkylphenol Ethoxylates ...... 28 2.2.2.2. Alkylphenoxy Carboxylic Acids ...... 29 2.2.3. Instrumental Methods...... 29 2.2.3.1. High-Performance Liquid Chromatography (HPLC) ...... 29 2.2.3.2. High-Resolution Gas Chromatography (HRGC) ...... 31 2.2.3.3. High-Resolution Gas Chromatography/Mass Spectrometry (HRGC/MS) ...... 31 2.2.3.4. Other lnstruments...... 32

2.3. Determination of Solubility In Water...... 32

2.4. Determination of Partition Coefficients between Water and Organic Solvents ...... 33

2.5. Determination of Biodegradability ...... 34

2.6. Determination of Photochemical Degradability...... 36

2. 7. Sampling...... 38 II

3. RESULTS AND DISCUSSION ...... 39

3.1. Determination of Alkylphenol Polyethoxylates and Their Metabolites ...... 39 3.1.1. Determination of Alkylphenol Ethoxylates and Alkylphenols ...... 39 3.1.1.1. Chromatographic Separation ...... 39 3.1.1.2. Response Factors of Alkylphenol and Alkylphenol Ethoxylates ...... 45 3.1.1.3. Extraction and Purification of Extracts...... 47 3.1.1.4. Accuracy, Precision and Sensitivity...... 50 3.1.1.5. Application ...... 52 3.1.2. Determination of Alkylphenoxy Carboxylic Acids ...... 60 3.1.2.1. Chromatographic Separation and Identification of Alkylphenoxy Carboxylic Acids ...... 60 3.1.2.2. Chromatographic Separation and Identification of Methyl Esters of Alkylphenoxy Carboxylic Acids ...... 63 3.1.2.3. Response Factors ...... 68 3.1.2.4. Extraction, Extract Purification and Derivatization ...... 69 3.1.2.5. Application ...... 73

3.2. Physlco-chemical and Biological Behaviour of Alkylphenol Polyethoxylates and Their Degradation Products...... 78 3.2.1. Solubility in Water ...... 78 3.2.1.1. Solubility of Pure APEO Oligomers ...... : ...... 78 3.2.1.2. Dependence of Solubility upon Temperature ...... 80 3.2.1.3. Solubility of APEO in Mixtures...... 82 3.2.2. Partition between Water and Organic Solvents ...... 84 3.2.3. Biological Degradation ...... 89 3.2.3.1. Biological Degradation of Alkylphenol Polyethoxylates ...... 89 3.2.3.2. Biodegradation of APEO and AP in Synthetic Sewage Using Shake Culture Test...... 90 3.2.3.3. Biodegradation of APEO and AP in Mineral Medium Using Shake Culture Test ...... 94 3.2.3.4. Biodegradation of APEO and AP in River Water Test ...... 97 3.2.3.5. Biodegradation of APEO and AP in Modified Closed Bottle Test...... 100 3.2.3.6. Degradation Products of APEO...... 102 3.2.4. Photochemical Degradation ...... 104 3.2.4.1. Direct Photolysis...... 104 3.2.4.2. Sensitized Photolysis...... 105 3.2.4.3. Photolysis in the Presence of Hydrogen Peroxide ...... 109 3.2.4.4. Photolysis by Sunlight...... 111

3.3. Occurrence and Behaviour of Alkylphenol Polyethoxylates and Their Degradation Products In Sewage Treatment Plants...... 115 3.3.1. Characteristics of Investigated Sewage Treatment Plants...... 115 3.3.2. Occurrence and Distribution in Raw Wastewaters and Effluents ...... 116 3.3.3. Diurnal and Daily Variations...... 122 3.3.4. Elimination of Alkylphenol Polyethoxylates and Their Metabolites During Biological Treatment...... 125 3.3.5. Role of Non biological Processes ...... 132 Ill

3.4. Occurrence and Behaviour of Alkylphenol Polyethoxylates and Their Degradation Products In Natural Waters .•••••••••••••••••.....•.••••••• 136 3.4.1. Glatt River ...... •...... 136 3.4.1.1. Characteristics of the Investigated Area ...... 136 3.4.1.2. Statistical Distribution of Concentrations ...... 136 3.4.1.3. Diurnal Dynamics of Concentration Variations ...... 138 3.4.1.4. Distribution of Concentrations and Mass Flows on the Longitudinal Profile and Seasonal Variations ...... 142 3.4.1.5. Analysis of Input and Output Mass Fluxes ...... 14 7 3.4.1.6. Sediments ...... 150 3.4.2. Comparison of Swiss Rivers ...... 151 3.4.2.1. Characteristics of Investigated Rivers ...... 151 3.3.2.2. Distribution of Concentrations ...... 154 2.4.2.3. Influence of Hydrological and Seasonal Conditions ...... 160 3.4.3. Lake Geneva ..•...... 164 3.4.3.1. Estimation of Input ...... 164 3.4.3.2. Distribution of Concentrations ...... 165 3.4.4. Infiltration of Riverwater into Groundwater...... 168 3.4.4.1. Characteristics of Investigated Locations ...... 168 3.4.4.2. Elimination of Nonylphenol Ethoxylates and Nonylphenol ...... 168 3.4.4.3. Elimination of Alkylphenoxy Carboxylic Acids ...... 173 3.4.5. Bioaccumulation in Freshwater Organisms...... 175

4. CONCLUSIONS...... 179

5. REFERENCES ...... 185

7. ABSTRACT ...... 199

Curriculum Vltae ...... 200 PROBLEM STATEMENT AND SCOPE OF THE WORK

Alkylphenol polyethoxylates (APnEO) are complex mixtures of oligomers, isomers, and homologues which are manufactured by addition of ethylene oxide to alkylphenols. Owing to their excellent surface active properties they are widely used as ingredients in many detergent formulations for household use, but have an even greater importance in various industrial applications. It has been well known that alkylphenol polyethoxylates are comparatively less biodegradable than the linear alcohol polyethoxylates. However, a decisive im- pulse for the revival of discussions on their environmental acceptability has been given just recently. Namely, newer investigations have certainly shown that biodegradation of alkylphenol polyethoxylates can lead to the formation of persistent metabolites, some of them being more toxic than the parent compounds itself.

The scope of this work was to develop analytical procedures for the determination of alkylphenol polyethoxylates and their degradation products using highly specific techniques such as high-performance liquid chromatography, high-resolution gas chromatography, and high-resolution gas chromatography/mass spectrometry and to apply them to studies of biogeochemical behaviour of these compounds in the aquatic environment. In order to achieve a better understanding of the behaviour and fate of alkylphenol polyethoxylates in the environment, laboratory investigations on their physico- chemical and biological properties will be combined with extensive field observations of these compounds in various environmental compartments. The results of this study should finally provide a basis for an assesment of the environmental acceptability of alkylphenol polyethoxylate surfactants. .2

1. INTRODUCTION

1.1. Ecological Acceptability of Surfactants

The basic element for an assessment of the ecological acceptability of a certain substance is its chemical structure. Namely, all physico-chemical and biological properties which determine the behaviour of a substance in the environment are determined by that structure. The investigations of the relationships between chemical structure and harmful effects upon the living world allowed the predic- tions of harmful behaviour of so far uninvestigated compounds (1 ). Besides the characteristics derived from the chemical structure, important criteria for the environmental assesment of a chemical are the data about the amounts produced, usage pattern and ways by which surfactants are introduced into environment.

1.1.1. Definition, Classlflcation and Characteristics of Surfactants

Surfactants represent highly variable and numerous groups of chemical substances. Their common structural characteristics is the presence of both hydrophobic and hydrophilic groups. The hydrophobic part of the surfactant molecule is usually an alkyl chain or alkylbenzene, while hydrophilic part can be very different. Surfactants are classified as anionic, cationic and amphotheric, depending on whether hydrophilic part of the molecule is neutral, negatively or positively charged. The consequence of the amphiphatic structure is the characteristic behaviour of surfactants in solutions and at Interfaces between different phases (2, 3, 4).

In highly diluted aqueous solutions, surfactants occur as real solutions. After reaching a certain concentration in solution, monomer surfactant molecules merge to form aggregates usually called micelles. The concentration at which micelles are formed is called critical micellar concentration (cmc). With ageing of the solutions, the water-air interface is enriched with surfactants forming monomolecular films whose hydrophilic part is solution oriented and the hydrophobic part is air oriented. Specific orientation of surfactant molecules is also retained in micelles, where hydrophilic parts are oriented towards solution and hydrophobic parts towards the center of micelle. Micelles are in cC>nstant dynamic equilibrium with the solution, and their size depends on the surfactant type (aggregation number). If the concentration of surfactant in the solution is increased even more, surfactants 3 form colloid solutions, while in highly concentrated solutions structural gels are formed (liquid crystals).

The consequence of surfactant adsorption on interfaces (liquid-gas or liquid-solid) is a decrease in surface tension and formation of an electric double layer. Such behaviour is the basis for many practical applications of surfactants (wetting, foaming, solubilization of slightly soluble substances, emulsifying, stabilizing of suspensions and colloids, dispersion, etc.). However, many important molecules in living organisms display also typical amphyphatic properties and can be considered natural surfactants (5). The most significant example is perhaps the lipoprotein cell membrane which allows exchange of the matter between the cell and its surroundings (6).

1.1.2. Manufacture and Usage of Surfactants

Washing and cleaning agents (detergents) are one of the most characteristic and most numerous products of modern civilization. In 1982 there were over 30 million tons of detergents produced globally for various industrial and domestic uses. There are significant differences in per capita consumption of detergents between specific geographical areas of the world. Consumption in developed countries of Western Europe and North America is several times greater than in Asian and African countries. With the annual consumption figure of 142.000 tons or 22.4 kg per year Switzerland is in the upper part of the consumption scale, somewhat higher than the West European average (21 6 kg per capita) (8). Production and consumption of detergents in Yugoslavia is experiencing fast growth (8.8 % per year). The estimated yearly production for 1985 is 496.000 tons which gives a significant consumption figure of 18.9 kg per capita per annum (9).

Modern detergents contain a great number of components which play different roles in the washing process (10), but the essential components are synthetic surface-active substances, so called surfactants.Total world production of surfactants for 1982 was 5 million tons (11, 12) and the majority (42 o/o) was used in the production of washing and cleaning agents. According to some authors (13) 60 o/o of the total world quantity of surfactants is anionic, 1o % cationic and about 30 % nonionic. 4

1.1.3. Input, Behaviour and Harmful Effects on the Environment

Surfactants are most often introduced to the environment through wastewaters. Scheme in Fig. 1 shows the ways of surfactant introduction into the environment, their distribution, and possible paths of harmful effects on living organisms.

81.UDGE WASTEWATERS

Figure 1. Inputs, distribution and behaviour of surfactants in the environment

If wastewaters enter the environment via sewage treatment plants the surfactants can be significantly eliminated before they enter natural waters due to the action of microorganisms. However, biological breakdown is not complete even in highly effective plants, leaving a certain quantity of surfactants undergraded. Moreover, microbial degradation proceeds not always to ultimate products, carbon dioxide and water, and can result in the formation of various degradation products which are more resistant to further microbial attack (14). Due to their biorefractory nature such degradation products can accumulate in the environment. The behaviour of surfactants and their degradation products is affected by various physico-chemical processes, primarily adsorption onto solid particles which can result in very high concentrations of surfactants in sludge (15). The disposal of sludge onto fields as 5

fertilizer introduces surfactants and their degradation products into arrable land. From there they can be taken up by the crop plants or they can reach ground water through soil leaching. In addition, wastewaters which usually contain surfactants are in some cases deliberately introduced into the groundwater. Namely, due to water shortages, there are efforts in many parts of the world to recycle treated wastewater; often it is firstly kept in accumulation ponds or underground (16) expecting spontaneous die-away of the present chemicals.

Following their introduction into natural waters surfactants can be further degraded by the present microorganisms, adsorbed onto solid particles and photochemically degraded. The surfactants present in natural waters influence numerous physico-chemical processes of organic (17) and inorganic compounds (18).

Because of the use of surfactants as washing and cleaning agents, and especially in personal care products, it is of the highest importance that they should not be harmful to human health. Generally, it can be stated that surfactant toxicity is rela· tively low for mammals, but can be considerable for aquatic organisms (11, 19, 20). Biorefractory degradation products, which can be even more toxic than surfactant itself, can reach the human population through polluted drinking water, or through bioaccumulation in edible water organisms.

1.1.4. Biodegradability of Surfactants

Biodegradation of surfactants, i.e. breaking down of complex molecules into sim· pier ones through the action of microorganisms, is one of the basic prerequisites of their environmental acceptability. The first negative consequences of surfactant application were already observed in the 1950's, through the use of the synthetic anionic surfactant tetrapropylenebenzene sulphonate (TBS). Foaming was ob· served in wastewater treatment facilities as well as in natural waters, which was the consequence of the surfactant resistance towards biodegradability (21 ). That was the reason for substitution of TBS with biodegradable linear alkylbenzene sulphonates. Fast and complete degradation of a certain surfactant simplifies the assessment of its environmental acceptability because it means that it will de· grade considerably in the wastewater treatment facilities. Furthermore, it means that its conc«?ntration in natural waters will be .lower than e:icpected from the figures on their discharge via nontreated wastewaters. The law regulations which deal 6

with the environmental acceptability of surfactants an~ actually based on thG rG- quirernent that applied surfactants must be at least 80 % biodegradable (22-26).

From the numerous methods for testing of the biodegradability (21) the most often used are the shake bottle test, the river water test and the method with activated sludge. The last method was officially accepted by all members of the Organization for Economic and Development (OECD) (27). According to that method, concentration of anionic surfactants is measured by the complex formation with methylene blue (MBAS method) (28), and concentration of nonionic surfactants by the method of complex formation with Dragendorff reagent (BiAS method) (29). Since the above mentioned analytical methods measure only the original surfactants and not their degradation products, these methods give insight only into the initial biodegradation. Recently there prevails the opinion that in the detergent formulations for wide use only those surfactants that can be degraded down to carbon dioxide and water should be applied. Therefore, valid law regulations on BO % degradability, which are based on MBAS and BIAS measurements, are no longer satisfactory.

1.2. Nonionic Surfactants

1.2.1. Types, Production and Usage

Nonionic surfactants are, together with anionic ones, the most important surfactants. While some older reports estimated that their share in the whole production and consumption was only 30 % (13), according to more recent reports (30) the usage of nonionic surfactants is strongly increasing and it is expected that they will soon become the dominant group. Nonionic surfactants today represent the most important group of surfactants for industrial applications (31 ). The increased use of nonionic surfactants is the consequence of their excellent surface-active (surface wetting, emulgation) and some special properties (weaker foaming) which make them acceptable for a number of applications other than as washing and cleaning agents. They are used in the textile, leather and paper industries, for the preparation of colours, varnishes and pesticides, as well as for flotation and for crude oil processing {32). Nonionic surfactants are the molecules whose hydrophilic group can not dissoci- ate to form ions, but it has to be sufficiently polar to ensure good solubility in water. There exists a considerable number of nonionic surfactant types (33), but the most important are those produced by the addition of ethylene oxide to different 7

hydrophobic molecules like long-chain alcohols, alkylphenols and fatty acids (34, 35). It should be pointed out that linear alcohol polyethoxylates (LAEO, Figure 2a) and alkylphenol polyethoxylates (APEO, Figure 2b) represent 80 % of all produced nonionic surfactants (13). The consumption of those nonionic surfactants in Western Europa, USA and Japan in 1982 was 500.000 tons of LAEO and 310.000 tons of APEO. The consumption for Switzerland can be estimated at about 5.000 tons/year (8).

During the synthesis of LAEO and APEO the oxygen atom of ethylen oxide trans- fers to hydroxyl group resulting in an electrophilic alkylation or more precisely in hydroxyethylation. The products thus formed should be consistently called oxethy- lates and not ethoxylates, commonly found in literature. However, in order to avoid confusion of the alternative use of different names for the same compounds, in this work the educts of ethylene oxide will be named ethoxylates. Oxethylation can be conducted with the use of acidic or basic catalysis which influences the composition of the formed reaction products; with basically catalysed synthesis the anion (alcoholate or phenolate) is formed first, which reacts subsequently with ethylene oxide through the nucleophilic reaction.

Li near Alcohol Polyethoxytates ( LAEO)

R-CH2-O-[CH2-CH2-01n -CH2-CH2-0H n::.0-20 R= C7-C1s (linear}

Alkyl phenol Pol yethoxylates (APEO)

n=0-20 R = c8• c9 (branched}

Figure 2. Structures, names, and abbreviations of the most important nonionic surfactants 8

The distribution of oligomeres formed by oxethylation should theoretically follow the Poisson statistical distribution, but due to the uneven reaction rate of resulting oligomeric compounds deviations from the ideal curve occur (3).

1.2.2. Physlco·chemical Properties

Lower molecular weight oligomers of LAEO and APEO are viscous liquids. With the increase of molecular weight they change to semisolid state {pastes).

The solubility of surfactants is thermodynamically determined by the value of free energy (.1.Go) neccessary for the transfer of molecules from the pure phase to the water solution. The value of .1.Go is obtained from the combination of contributions for hydrophilic (.1.Geo) and hydrophobic (.1.Gho) part of the molecule. These two contributions differ strongly:.1.Gho is positive (determined by the negative value of enthropy .1.Sho) and AGeo is negative and generally larger than AG ho. Since the solubility depends on the characteristics of the hydrophilic group of the surfactant, it is considerably different for ionic and nonionic surfactants; for nonionic surfactants it obviously depends on the number of polar groups forming the hydrophilic part of the molecule. It should be kept in mind that nonionic surfactants are as a rule complex mixtures of oligomers, isomers and homologues, so that their solubility is never considered as the solubility of pure chemical compounds. This is probably the reason why the exact data on the solubility of particular oligomers could not be found in literature. According to the manufacturers technical data (32) the lower oligomers of alkylphenol ethoxylates (nEO < 5) are described as "water insoluble", while higher oligomers form at 20 °C clear solutions; the behaviour in nonpolar organic solvents is quite opposite. Neither the concentrations nor the type of solutions for which these observations were made are stated. Some information about the solubility of the surfactants can be obtained from the data on their critical micellar concentration (cmc). The nonionic surfactants have much lower cmc than ionic surfactants with the same hydrophobic group. If nonionic surfactants alone are compared, it can be seen that cmc depends on both the hydrophilic and hydrophobic part of the molecule. Of decisive influence on cmc is the size of the hydrophobic part of the molecule, i.e. cmc decreases sharply with the increase of alkyl chain length. For example, cmc for OPEO (nE0==1 O) is 3.3 x 10-4 mol/L, and for NPEO with the same average number of EO groups is 7.5 x 10-s mol/L (36, 37). The introduction of aromatic ring into the hydrophobic part of the molecule has approximately the same effect as the increase 9

of alkyl chain length for 3.5 methylene groups (33). The aggregation number for micellar solutions of nonionic surfactants depends on the number of EO groups and varies in the wide range od 10-1000 (38). The solubility of nonionic surfactants in water is based on the hydratation of either functional groups through hydrogen bonds. Hydration decreases as the temperature increases and consequently the solubility of nonionic surfactants is decreasing and going towards higher temperatures. If the solution of nonionic surfactant is warmed, dehydratation occurs, resulting in the separation of phases at a temperature which is characteristic for the specific surfactant (turbidity point) (33). With the addition of electrolyte, turbidity point usually shifts towards lower temperatures, although certain electrolytes can increase turbidity point (39, 40). A positive correlation was found between the turbidity point and the hydrophilic- lipophilic balance (HLB) value, which is important for the choice of a surfactant as emulgator. According to Griffin's system (40, 41 ), HLB values for nonionic surfactants can be given on a scale from 0-20 according to the following equation:

HLB= E/5 (1)

where E is the share of oxyethylene chain in the molecule of surfactant. HLB value can be used for the judgement of surfactant hydrophobicity, but it has a more empirical than a theoretical base. From the fundamental point, the hydrophobic characteristics of the surfactant can be defined much better by the partition coefficient between the organic solvents and water (Kow) (43). Cratin (44) has shown that partition coefficients depend on the total energy needed for the transfer between two phases and can be considered as additive-constitutive property of a molecule. The total free energy of transfer µr can be given as the sum of free energies of transfer for lipophilic (µL) and n hydrophilic groups (µH) according to equations: µr(w) =µL(w) + n X µH(w) (2)

µr(o) = µL(o) + n x ~(o) (3) where w and o represent the water and organic phase. From the equation for equilibrium conditions

{4) equation (5) can be derived (44): 10

log Kow= µH/2.3 RT+ µi_/2.9 RT+ log V(w)N(o) (5) which shows that correlation between the logarithm of partition coefficient Kow and the number of hydrophilic groups n is linear. This was proved by direct measure- ment of partition coefficients for octylphenol polyethoxylate oligomers in the system isooctane/water (45) which can be shown by the following equation:

log Kiow = -0.442 nEO + 3.836 (6)

Partition coefficients can be used for the estimation of HLB values according to the following equation (43):

(HLB-7) =0.36 In (1/Kow) (7)

The distillation with water vapour is one of the less studied properties of nonionic surfactants, but it can play an important role by some technical applications (46) or by analytical determination of the surfactants. It was established that commercial APEO do not belong to steam-distillable substances.

1.2.3. Analytical Determination

The development of synthetic surface-active materials emphasized a need for analytical methods for their determination in various types of samples: .for quality control of raw materials and commercial products, for the investigation of biological degradation and for their determination in the environmental samples (47, 48).

Several reviews were published (49, 50, 51) showing that various techniques were applied for the determination of nonionic surfactants. The literature mentions the application of thin layer chromatography (52-55), paper chromatography (56), gas chromatography (57-60) and mass spectrometry (61-63). It appears that the best possibility for the analysis of nonionic surfactants is offered by high-performance liquid chromatography (64-71 ). The analysis of a mixture of homologues (reversed-phase system) and oligomers (normal-phase system) can be obtained in a relatively short time. During analysis, a refractometric detector can be used as a universal detector, but it has a rather low sensitivity. Aromatic nonionic: surfactants can be directly detected with high sensitivity using UV-spectrometry and spectrofluorometry, while nonaromatic nonionic surfactants must be first trans- 11

formed into respective derivatives (68). The other possibility is to apply the flame ionization detection {69).

A relationship between oxyethylene and oxypropylene in mixed polymers can be determined using NMR spectroscopy or by breaking down polyether molecules with HBr and subsequent gas chromatographic analysis of formed brominated products (72-74).

Although determination of small quantities of surfactants relies on above mentioned techniques, a need for increased specificity and sensitivity has brought about development of special methods for that purpose (75). Foremost, these methods are needed for investigating biological degradability, and for determining surfactants in wastewaters and in natural waters. In order to achieve a satisfactory level of sensitivity and to increase selectivity of the analytical procedure, several enrichment techniques such as evaporation till dryness, extraction by organic solvents (76-79), and adsorption on a solid phase (80-82) are applied prior to final determination.The most widely used technique is sublation of the water sample using an inert gas in the course of which the nonionic surfactants are enriched in overlying ethyl acetate (76).

Several methods are suggested for selective detection of traces of nonionic surfactants. One of the most commonly used techniques is based on the reaction with cobalt thiocyanate, resulting in complexes of blue colour which can be measured colourimetrically at 620 nm (CTAS method) (83-85). More recently, there have been attempts in automation of that method (86). The major shortcoming of the CTAS method is insufficient specificity in the presence of anionic and cationic surfactants (84). The use of potassium picrate as reagent for colourimetric determination (378 nm) of nonionic surfactants lowers interference of other types of surfactants, while very good sensitivity is achieved (87-89). A modified Wlckbold method (BiAS method) (90), which is based on the reaction of surfactant molecules with Dragendorff reagent has very wide application, especially in investigations of biological degradability of nonionic surfactants. The determination of nonionic surfactants with atomic absorption spectroscopy after the precipitation with molibdenphosphoric acid was also described (91 ). All complexing and precipitating reagents have two basic shortcomings: a) insufficient sensitivity to the lowest oligomers (nEO < 5), ·and b) determination is nonspecific and the contribution of various types of nonionic surfactants can not be differentiated. 12

Electrochgmical methods offer an interesting approach to the determination or nonionic surfactants in water samples by allowing direct determination at concentrations of mg/L (92, 93). These methods were successfully applied during investigations of biological degradability of several types of nonionic surfactants '(94) and have shown some advantages in comparison with the standard BIAS method (90). A very popular method in the investigations of biodegradability of nonionic surfactants is also IA-spectroscopy (95, 96). This method is very useful for qualitative identifications, but is not very suitable for quantitative work.

Otsuki and Shiraisi (97) have developed a very specific method for determination APEO at concentrations of µg/L with the combination of reversed-phase liquid chromatography and mass spectrometry. Some other authors have also described the applications of various mass spectrometric techniques for the analysis of traces of nonionic surfactants (98-102), but these methods can not be considered suitable for routine qualitative determinations. Although gas chromatography should represent an ideal technique for analysing complex oligomer mixtures, due to its high resolution power and high sensitivity, it is not suitable for the analysis of nonionic surfactants because of the low volatility and high polarity of higher oligomers. To avoid this limitation, Tobin and collaborators (103) have determined their breakdown products after cleavage of ether bonds with HBr. Other authors (104) have applied derivatlzation of primary alcohol group into trimethylsilylether to increase volatility. However, not even that technique can allow quantitative determination of the highest ollgomers (nEO > 12), which are otherwise important components in most often used commercial mixtures. However, the application of high-resolution gas chromatography was shown to be very useful for determinations of persistent biodegradable products of APEO (Fig. 3). Giger and collaborators (105-108) have successfully applied high-resolution gas chromatography in a combination with mass spectrometry or with a flame-ionization detector for determination of NP, NP1 EO and NP2EO. in secondary effluents (105-107) and activated sludge (108). Using the same techniques Reinhard and collaborators (109, 11 O) have identified and quantitatively determined ethoxylated and carboxylated degradation products of OPEO as well as their brominated and chlorinated derivatives in treated wastewaters. Another interesting possibility is determining LAEO and APEO by applying high-resolution gas chromatography in combination with mass spectrometry with chemical ionization (111, 112). Reversed-phase liquid chromatography with spectrophotometric and spectrofluo- 13 rometric detection have also been suggestad tor dt1tt1rmining NP In water and In sediments (113). Contrary to clear advantage of chromatographic and mass spectrometric techniques there still prevails the use of nonspecific collective methods for determining traces of specific types of nonionic surfactants. Brown and collaborators (114, 115) have published a comparison of improved BiAS method (116), HPLC determination, and a gas chromatographic determination after HBr cleavage for an investigation of nonionic surfactant behaviour during wastewater treatment. Good correlation of the results obtained by different methods was taken as proof for reliability of the BIAS method, while specific methods are considered only semi- quantitative at the present level of development. Only recently has an HPLC method been used for fully quantitative determination of alkylphenol polyethoxylates in real samples (117, 118).

A)°'cHfi~ AP1EO alkylphenol monoethoxylate R

R,er°'-<:HfH2-.~'cH~ AP2EO alkylphenol diethoxylate

· ok°'2~2 m'cH2 Ai APnEO alkylphenol polyethoxylate p {n==m+1)

,(rOH AP alkylphenol R 0 ~fOH APlEC alkylphenoxy acetic acid R R¢0ifH~~ AP2EC alkylphenoxyethoxy acetic acid

~fH2~~ APnEC alkylphenoxypolyethoxy acetic acid R (n•m+1)

R: CgH19 N nonyl R: CeH17 0 octyl

Figure 3. Structures, names, and abbreviations of the most important metabolites of alkylphenol polyethoxylates 14

1.2.4. Biological Degradation

Biological degradation of nonionic surfactants is determined by two elements of their chemical structure: a) the number of EO groups in the hydrophilic chain and b) by the structure of the hydrophobic group. A detailed survey of the research on the influence of chemical structure of nonionic surfactants on biological degradation was given by Swisher (21 ). Unfortunately, many older investigations were done without critical application of analytical methods so that many observations and conclusions are not quite reliable. Several authors (119-121) have already noticed that the most often used CTAS method is not suitable for a study of biodegradation of APEO and that limitations of this method must be taken into account when interpreting results. Furthermore, due to the lack of knowledge on the biodegradation mechanism of APEO neither the experimental results on the degree of biological degradations could be adequately explained. It was observed that the degradation of ethylene oxide aducts slowes down with the increase of EO chain, but this conclusion is valid only for LAEO, and not for APEO (21 ). This fact indicated at different mechanisms of LAEO and APEO degradation as fully proved in later research (122).

According to most authors LAEO are considered to be an easily degradable compounds. It was shown that these compounds are ultimately degradable (to C02 and H20) both in sewage treatment plants (122, 123) and in natural waters (124, 125). Using radioactively labelled compounds Steber and Wierich (126) have shown that the straight chain AEO with the length of the hydrophobic chain of 12- 18 C atoms and the number of EO groups 5-15 are ultimately degradable compounds. Some authors (127, 128) point out that the alkyl part of the molecule de- grades very fast, but that biodegradation of the hydrophilic chain (polyethyleneglycol) is much slower. Investigations of LAEO degradation mechanism (129, 130) have shown that in mixed cultures there are two simultaneous ways of initial degradation: a) intramolecular cleavage into hydrophilic and hy· drophobic part and b) <0· and p-oxidation of the alkyl chain. The alkyl chain is rapidly degraded to C02 and H20 and the degradation of the hydrophilic part of the LAEO molecule gives homologues series of neutral and acid polyglycol fragments which then gradually degrade further by hydrolythic or oxidative loss of C2 units.

According to Swisher (ref. 21, page 248) "assessment of the biodegradability of the alkylphenol ethoxylates has given rise to more disagreement, contradiction and controversy than any other area of surfactant degradation". As already men- 15 tioned some disagreements can b9 9XplaiMd by tn& uneritieal application of different analytical methods for determining APEO or by insufficient adaptation of applied bacterial cultures. The comparison of LAEO and APEO degradability shows that the presence of the alkylphenol group instead of the alkyl group lowers degradability, and this is especially noticeable if the alkyl chain of alkylphenols is highly branched (120, 121 ). This difference in biodegradability is caused by raw materials from which AEO and APEO are manufactured; AEO are synthesized from long-chain alcohols which are obtained either from natural sources or by Ziegler synthesis (131 ). Contrary to this, the hydrophobic part of APEO is obtained by petrochemical synthesis: phenol which is prepared from cumene is then alkylated by tripropylene or by diisobutylene during which the mixture of isomers with differently branched alkyl chain is formed. APEO degradability is much lower at lower temperatures (<10 °C) (132-143). The mechanism of biological degradation of APEO has been much less investigated than the mechanism of LAEO degradation. Majority of authors agree that degradation begins on the hydrophilic part of the molecule and according to Swisher (21) three mechanisms of degradation are possible (Fig. 4). According to the first proposed mechanism, the shortening of the hydrophobic part of the molecule occurs due to the hydrolytic loss of ethyleneglycol; according to the second, there first occurrs the oxidation of terminal OH group followed by hydrolytic loss of glycolic acid; the third mechanism also forsees the loss of glycolic acid, but through an oxidation at the inside C-atom to give a glycolic acid ester. According to the most recent publications (105-11 o, 135-137), only the metabolites which occur as a result of reactions I and II have been identified. Degradability of APEO Is rather fast for higher oligomers, but it decreases significantly with the shortening of the EO chain, and finally the products are formed which can be considered to be resistant to further biological degradation. Of these persistent metabolites NP2EO, NP1EO (135, 136) and NP2EC (137) were identified during model laboratory experiments. In addition to these metabolites in real sewage treatment plants a completely deetoxylated product NP (107, 108, 110) has also been identified. The hypothesis on biotransformation of APEO via oxydation of either alkyl chain or of benzene ring has not yet been proved. Degradation of APEO can be speeded up by combining microbiological and chemical treatment. Thus, it was observed that a more successful elimination of APEO is achieved with the application of ozone (138) or hydrogen peroxide (139). 16

! r 1 ~ f_ N' s ~e"ti .Y~ -:.. -~ ...Q) .i: ~i ~ ~ (f.) ~r - ~~~ s 2> ~ § 1 (\'! l ! ~ i ~ ! ~~ f ~' 8. cs i

~~ ~ !1U .,_ ()e i ~rY Q. ~ ~ } ~·Cl> ~ iii

I! "'::a .~ Li: 17

1.2.5. Occurrence In the Environment and Harmful Effects

For the assessment of ecotoxicological properties of polutants it is most important to evaluate their effect on man, but, since nonionic surfactants are typical water pollutants, it is particularly important to determine their effect on water organisms. Acute and chronic toxicity of LAEO and APEO for mammals is relatively small {lethal dose of NP9EO and C12H2s7EO for rats is 2600 mg/kg and 4150 mg/kg, respectively) (145). Based on present knowledge their cancerogenic effect has not yet been established. As for other types of surfactants, their toxicity for water organisms, which is caused by the interaction with cell membranes (6), is much more critical from the ecotoxicological point of view. Also, it was observed that an increased bioaccumulation of some toxic substances in fish could occur in the presence of nonionic surfactants (146). The toxicity of LAEO and APEO increases with the increasing length of the alkyl chain and is negatively correlated with the number of EO groups in the surfactant molecule (20, 140, 147). Since the toxicity for different water organisms varies very strongly (value of LCso ranges from 0.8-1000 mg/L, ref. 20), special attention should be given to the choice of the indicatory organism.

It is important to mention that the rule •greater the toxicity - better the degradability" which is valid for the majority surfactants, does not include APEO, and indicates the need for special care when determining the ecological acceptability of these surfactants. Namely, with LAEO • more lipophilic oligomers (shorter EO chain) show at the same time to have better degradabllity. The opposite behaviour was observed with the APEO (21 ). Degradation of LAEO produces polyethylene glycols which are significantly less toxic than the initial compounds (20, 148). On the contrary, during biodegradation of APEO more lipophilic are produced, therefore more toxic products are formed (107). Among them the most toxic metabolites of APEO are alkylphenols. Mc Leese and coworkers (149, 150) have investigated the toxicity of various alkylphenols for water organisms and have found that toxicity increases with the increasing length of the alkyl chain, i.e. with increasing lipophilicity. The nonylphenol has shown high toxicity for shrimp (LCso =0.3 mg/L) and for salmon (LCso= 0.13-0.19 mg/L). So far there are no published figures about the toxicity of APEC, but it can be expected that their toxicity is lower than that of AP and APEO due to the lower lipophilicity. Apart from showing the direct toxic effect on water organisms it has also been shown that alkylphenols can accumulate in water organisms (145, 150). 18

High concentrations of nonionic surfactants (1-10 mg/L) which could, according to the above mentioned data, directly endanger water organisms. have only been recorded in wastewater (114). Real concentrations in natural waters range from 0.01 to 0.1 mg/L (140, 141) which is significantly lower than the level determined as dangerous for water organisms. However, it must be stressed that the mentioned concentration levels of nonionic surfactants have been measured using BiAS method, and do not, therefore, show what part in the total concentration belongs to the aromatic nonionic surfactants (APEO) or what part belongs to the aliphatic nonionic surfactants (LAEO). Moreover, these measurements do not in· clude persistent metabolites of APEO, since they can not be detected using the BiAS method.

The importance of persistent degradation products of APEO, some of which are more toxic than nonionic surfactants themselves, was definitely pointed out only just recently and so far there is very limited data on their concentration in wastewaters and especially in natural waters. Sheldon and Hites (142, 143) have identified some APEO metabolites in the Delaware river but have not explained the sources of their input. Giger and coworkers (105, 107) have pointed out the immediate source of input for these constituents into the rivers, having determined high concentrations of NP, NP1 EO and NP2EO in the secondary effluents of municipal sewage treatment plants (<10-220 µg/L). Similar concentrations of brominated and chlorinated lipophilic APEO and APEC have been measured by Reinhard and coworkers (109, 11 O) in secondary effluents which have been ad· ditionally chlorinated. In several sewage treatment plants which release their effluent into Lake Geneva it was found that concentrations of NP, NP1 EO and NP2EO range from 106- 440 µg/L, and in the lake itself from 3.6-12.4 µg/L (144). 19

2. EXPERIMENTAL

2.1. Materials

2.1.1. Chemicals

In this work a number of commercial mixtures of alkylphenol polyethoxylates was used which differed in both hydrophobic (nonyl and octyl) and hydrophilic part of the molecule (average number of etoxy groups):

Mar1ophen 810: mixture of nonylphenol polyethoxylates (nEO = 1-18); average number of EO groups= 10; Chemische Werke HOls, Marl, FR Germany; Marlophen 83: mixture of nonylphenol ethoxylates (nEO = 1-6); average number of EO groups = 3.15; Chemische Werke HOls, Mar1, FR Germany; lmbetin N/7A: mixture of nonylphenol ethoxylates (nEO = 1-3); average number of EO groups= 1-2; Dr. W. Kolb AG, Hedingen, Switzerland; Synperonic OP10: mixture of octylphenol polyethoxylates (nEO = 1-18); average number of EO groups= 10; Imperial Chemical Industries, Petrochemical Division, Middlesborough, U.K.; 4-tert-octylphenol-2Aeo: mixture of octylphenol ethoxylates (nEO = 1-4); average number of EO groups= 2; Dr. W. Kolb AG, Hedingen, Switzerland. Pure oligomers NPEO were obtained from the mentioned commercial products by preparative liquid chromatography (section 2.2.2.).

The following alkylphenols were used: 4-nonylphenol, technical, Fluka, Buchs, Switzerland; mixture of isomers with differently branched nonyl chain; main impurities are 2-nonylphenol (ortho isomer), decylphenol and dinonylphenol (together about 10 %); purified 4- nonylphenol was obtained by preparative HPLC; 4-octylphenol, purum (95 % 1, 1,3,3-tetramethylbutylphenol), Fluka, Buchs, Switzerland; purified by recristalization from pentane; 2,4,6-trimethylphenol, 99 %, EGA-Chemie, Steinhelm, FR Germany; 2,4-di-tert-butylphenol, 97 %, Fluka AG, Buchs, Switzerland; 2-tert-butyl-6-methylphenol, 99 o/o, Fluka AG, Buchs, Switzerland.

Alkylphenoxy carboxylic acids (NP1 ec~NP4EC and OP1EC) were obtained by synthesis and purified by preparative HPLC as described in section 2.2.2 .• 20

The following chemicals were used as purchased (p.a., Merck, Darmstadt, FR Germany and Fluka AG, Buchs, Switzerland), without additional purification: nonylbenzene, sodium chloride, sodium bicarbonate, potassium hydroxide, hydrochloric acid (cone.), sulphuric acid (cone.), formaldehide (36 %), mercury(ll) chloride, rose Bengal dye and hydrogen peroxide.

Sodium sulphate (anhydrous), p.a., Merck, Darmstadt, FR Germany, was heated before use for 12 hours at 800 °C.

The following adsorbents were used: aluminium oxide: 90, neutral, activity I, 0.063-0.2 mm, Merck, Darmstadt, FR Germany; before use it was deactivated by addition of distilled water (1.5- 5 %); silica gel: Kiesegel 40, 0.063-0.2 mm; Merck, Darmstadt, FR Germany; before use silica gel was activated at 200 °C and then deactivated by the addition of 15 % distilled water; Florisll: 0.063-0.16 mm, Fluka AG, Buchs, Switzerland; porous glass powder: WHB 345; BA; 1690 A; 0.71g/cm·3;9 m2/g; 0.10· 0.32 mm, NBS, USA.

Following organic solvents of high purity (p.a., "nanograde" or "HPLC grade"; Mallinckrodt, St. Luis, USA; Merck, Darmstadt, FR Germany; Fluka AG, Buchs, Switzerland) were used without additional purification: dichloromethane, chloroform, pentane, n-hexane, cyclohexane, 2-propanol, methanol, ethanol, acetonitrile, ethylacetate; 1-octanol: purum, Merck, Darmstadt, FR Germany; water: "carbon free", Mallinckrodt, St. Luis, USA or doubly distilled in the laboratory.

2.1.2. Solutions and Reagents

The initial solutions of higher NPEO (Marlophen 810) and OPEC (Synperonic OP10) were prepared in ethylacetate at concentration of 100 µgJJi.L The standard test solutions for normal-phase liquid chromatography were prepared In n-hexane or cyclohexane, and rarely in other more polar solvents. Since higher APEO are poorely soluble in n-hexane, standard solutions injected into liquid chromatograph (1-5 µg/µL) were prepared by diluting the initial solution in the mixture of n-hexane and 2-propanol in the ratio of 9:1. The initial solution of Marlophen 83 containing 21

lower NPEO. which are solubl9 in nonpolar solvents, was prepared In cyclohexane (10 µg/µL). The same solvent was used in preparation of the solutions of nonylphenol (5-10 µg/µL), octylphenol (10 µg/µL) and internal standards 2,4,6- trimethylphenol (5.2 µg/µL), 2,4-dHert-butylphenol (5 µg/µL) and 2-tert-butyl-6- methylphenol (5 µg/µL). A typical test solution used for daily testing of normal-phase chromatographic system, was prepared by dilution of standard solutions in n-hexane or cyclohexane so that the concentration of specific components was 1.5 µg/µL Marlophen 83, 0.15 µg/µL nonylphenol and 0.12 µg/µL 2,4,6-trimethylphenol. If the spectrofluorometric detection was used, the concentration of such a test solution was up to 1o times lower. The solution used for testing the reversed-phase chromatographic system was prepared from the standard solutions in methanol and acetonitrile (10 µg/µL). The concentrations of the diluted solutions injected on the column were 0.1 µg/µL for NP and 1 µg/µL for OPEO and NPEO.

The solution of octylphenoxy acetic acid (1 o µg/µL) was prepared in methanol or dlchloromethane. Preparation of solutions in dichloromethane or chloroform is advisable to avoid the spontaneous methylation in methanol solutions. The standard solutions of methylester APEC (NP1 EC-NP4EC and OP 1 EC) were prepared with n-hexane.

The solution of nonylbenzene was prepared in cyclohexane at the concentration of 4.5 µg/µL.

The water solutions of AP and APEO, used for the bio- and photochemical degradability determinations, were prepared by dilution of the saturated water solutions to those compounds. The saturated solutions were prepared as described in the chapter 3.2.1 ..

During the investigation of the biodegradability, the growth media according to Husmann (151) (synthetic sewage), and Horvath, and Kott (152) (mineral medium) were applied. The substrate by Husmann contained the following constituents: sodium chloride (p.a, Merck, Darmstadt, FR Germany), 7 mg/L; calcium chloride (CaCl2 x 2H~. Merck, Darmstadt, FR Germany), 4 mg/L; magnesium sulphate (MgS04 x 7H20, Merck, Darmstadt, FR Germany), 2 mg/L; potassium hydrogenphosphate (K2HP04, p.a., Kemika, Zagreb), 28 mg/L; 22

pepton (Torlak, Belgrade}, 160 mg/l; meat extract (Torlak, Belgrade), 11 O mg/L.

The growth medium by Horvath and Koft was prepared dissolving the following components in distilled water (filled up to 1 l): ammonium chloride (p.a. Merck, Darmstadt, FR Germany), 1 g; sodium hydrogenphosphate (Na2HP04 x 12H20, p.a., Kemika Zagreb), 1 g; potassium chloride (p.a., Merck, Darmstadt, FR Germany}, 0.5 g; magnesium chloride (MgCl2 x 6H~. Alkaloid, Skopje), 0.18 g.

Mercury(ll) chloride (1 % solution in distilled water) was used as an antibacterial agent.

The methylating agent (1 N solution of HCI in methanol), was prepared by the introduction of hydrogen chloride (generated by dropping concentrated sulphuric acid onto sodium chloride in a three-necked flask) into methanol. Jones reagent, which was used for the oxidation of APEO during the synthesis of APEC, was prepared by dissolving 33.6 g of potassium dichromate in 2 M sulphuric acid (22.2 ml of concentrated acid was diluted with water up to 200 ml).

2.1.3. Equipment

All equipment and laboratory glassware used for the experiments were made of glass, and whenever possible the use of plastic materials was avoided except for teflon. The clean glassware was washed with organic solvent before it was used to diminish the danger of contamination.

Evaporation of extracts with a larger volume (>10 ml) was performed in a rotary evaporator (BOchi, Switzerland) and for the extracts with a smaller volume (<5 ml), the evaporation under a stream of nitrogen was used. The glass column used for the purification of extracts by liquid chromatography had an intemal diameter of 1Omm and the top of the column contained a reser- voir for elution solvents (capacity of 50 ml). Microsyringes Hamilton, S.G.E. or Rheodyne of various volumes (5-500 µl), were used for the injection and the dilution. 23

2.2. Analytical Procedures for the Determination of Alkylphenol Polyethoxylates and Their Metabolites

Alkylphenol polyethoxylates are rather complex mixtures of oligomers, isomers and homologues, which together with their most important degradation products, form a numerous group of aromatic compounds. Since specific oligomers of APEO, and especially their degradation products, can differ considerably in their physico-.chemical characteristics the determination of these compounds, which are of interest during the investigation of biogeochemical behaviour of APEO, was not possible by a single analytical procedure. Three methods were developed during the investigations: a) the method for the determination of nonionic surfactant itself (APnEO, nEO"' 3-20); b) the method for the determination of lipophilic alkylphenol ethoxylates (AP1 EO. AP2EO) and alkylphenol (AP) and c) the method for the determination of alkylphenoxy carboxylic acids (APEC). Table 1 presents a survey of techniques and procedures is shown for each of the mentioned analyti· cal methods in different types of samples.

2.2.1. Extraction and Purification of Extracts

2.2. 1. 1. Alkylphenol Polyethoxylates

The water samples of various origin (untreated and treated wastewaters, natural waters) were extracted by a standard extraction procedure for surfactants according to Wickbold (76). One liter of the sample to which 40 g of sodium chloride and 5 g of sodium bicarbonate was previously added, was placed into the extrac· tion apparatus and was overlayed by 60 ml of ethyl acetate. The enrichment of surfactant in ethyl acetate was performed by bubbling nitrogen through the water sample at the rate of 30 Umin in duration of 5 min. The obtained extract was collected in a dean funnel and 60 ml of fresh ethyl acetate was added to the extraction apparatus. The sample was extracted in the same manner once again. The combined extracts were then transferred into a separatory funnel to remove the excess of water. After drying it with sodium sulphate. the ethyl acetate extract was evaporated to a small volume (1-2 ml) under reduced pressure using a rotary evaporator (temperature about 40 °C). The concentrated extract was then transferred to a small vial (3-5 ml) sealed with a screw cap and was washed with several small portions of dichloromethane. The solvent was then completely evaporated under a stream of nitrogen, and the sample was redissolved in approximately 1 ml of dichloromethane. TABLE 1. Overview of Analytical Methods Applied tor the Determination of Alkylpheno/ Polyethoxylates and Their Metabolites in Environmental Samples

Compounds Sample Enrichment Purification Separation Detection Aemar1

APnEO wastewaters sublation with N2 Al20:! (1.5 % H20) nonnal-phase 1 HPLC uv,.2nnm separation of : natural waters into ethyl acetate reversed-ptiase2 HPLC Fluor.=277/300 nm 1oligomers (nE0=3-20) (rel. 76) 2homologues

wastewaters normal-phase HPLC UV=2n nm AP, AP1EO, l\l natural waters Fluor.=2n1aoo nm AP2EO solid samples steam-distillation I HRGC Fto3 3 extract purification """ (sludges, sedi- extraction (rel. 153) necessary ments, biota)

~~AP, .artificial samples extraction with normal-phase HPLC uv.. 2n nm 4 samples of 10-1 oo AP1 EO-AP5EO (tab. experiments) n-hexane (1 :20)4 Ftuor.=2771300 nm ml~~

~~wastewaters extraction with normal-phase HPLC uv-2nnm s emulsion formation natural waters chloroform5 HAGC Fluor.,.2771300 nm when extracting FID primary effluents AP1EC,AP2EC 5102 (15% H2Q)~~

~~sludge6 Soxhlet extraction normal-phase HPLC Fluor .•2n/300 nm 6 problems with the determin.of AP1 EC 25~~

The cleaning of the so preparod oxtraet was performed in a column of alumlnlum oxide deactivated with 1.5 % water. Aluminium oxide (about 4 g) was added into the glass column, with an internal diameter of 10 mm as a suspension in dichloromethane. The excess of dichloromethane was drained from the filled column, and the extract was transferred by doing several rinses in the bottle with the ext,ract. Nonpolar components are eluted with 35 ml of dichloromethane and this fraction was discarded. ~fter that, alkylphenol polyethoxylates were eluted with 25 ml of methanol. This methanol fraction was concentrated in a rotary evaporator to a small volume (1-2 ml) and was transferred to a 3 ml srew cap vial. The concentrated methanol eluate was evaporated to dryness under a stream of nitrogen and the residue was redissolved in the exact volume (usually 500 µl) of the n-hexane and 2-propanol mixture in ratio 9:1. Aliquots of a such purified were then analyzed by normal-phase HPlC. If reversed-phase chromatography is used, it is better to have the purified extract in the methanol solution. Therefore, in such a case it was not necessary to evaporate methanol eluate to a dryness and to transfer the residue into nonpolar solvent.

~~2.2.1.2. Lipophilic Alkylphenol Ethoxylates and Alkylphenols~~

For the extraction of lipophilic AP and APEO two methods were used. For the analysis of environmental sa!Jlples (water, sediment, activated sludge) a method was used which enables simultaneous exhaustive, steam-distillation, and continuous extraction into an organic solvent (153). The analysis of water samples was carried out as follows: to the water sample (for natural waters the usual volume is 1-2 l), 20 g of sodium chloride was added and pH value was adjusted to 7.0-7.5. A prepared sample was transferred into the extraction apparatus and brought to boiling. The sample was kept under reflux for 3 h using cyclohexane (1-2 ml) as the extracting solvent through which the distillate percolated. In such manner, the lipophilic AP and APEO, which are distillable with steam were enriched in organic solvent in the ratio 1 :1000. The same technique was applied for the analysis of sewage sludge, water sediments and soil, so that the solid sample (usually 10- 15 g) was first suspended in 1.5 l of deionized water. After the extraction was completed (3 h) the cyclohexane layer was put into a clean 10 ml glass tube. The walls of the extraction apparatus were rinsed with several mLs of distilled water, and the rinslngs were added to the extract in the glass tube put. Into the.above (cyclohexane) layerthe known amount of internal standard, In the form of solution in cyclohexane (3-20 µl), was added. For the quantitative determinations by HPlC 2,4,6-trimethylphenol was used as the inter- 26 nal standard, and for the gas chromatographic determinations, nonylbenzene was used. The cyclohexane extract was then transferred into a glass vial with a Teflon- lined screw cap, and dried by the addition of a small amount of anhydrous sodium sulphate put into the vial. The aliquot of a prepared extract can be directly analysed by HPlC for all types of samples. When gas chromatography was used it was often neccessary, for certain types of samples (untreated wastewaters, sewage sludge), to include a supplementary purification of the extract in order to remove coextracted organic compounds which interferred in analyses. This was especially neccessary if nonselective flame-ionization of detection was used. For that purpose, the same type of adsorption column, as previously described with alkylphenol polyethoxylates (chapter 2.2.1.) was used. Nonpolar compounds were eluted with 30 ml of dichloromethane, and the other fraction containing AP and lipophilic APEO was obtained by elution with 25 ml of dichloromethane/methanol mixture in the ratio 1 :1. This eluate was concentrated in a rotary evaporator to a small volume (0.5-2 ml), and was transferred to a glass vial with a Teflon-lined screw cap using the rinsing with dichloromethane. When purifying the extracts, it is advisable to add a second internal standard (for example 2,4-di- tert-butylphenol) into the final extract, before the gas chromatographic analysis, in order to determine the losses which possibly occurred in this step of the sample treatment. For the analysis of water samples concerning investigation of biodegradability, physico-chemical properties, and photochemical degradability of lipophilic AP and APEO in model laboratory experiments, a simple method of extraction by shaking the organic solvent lighter than water; mainly n-hexane, was used. The extraction was carried out in the graduated vials (10-50 ml) by using a solvent to a sample ratio of 1 :20 and 1 min of shaking. After separation of the phases the known amount of internal standard (usually 2,4,6-trimethylphenol) was added into the hexane phase. If during the shaking stabile emulsion was formed, the phase separation was hastened by a 2 min centrifugation at 2000-3000 rpm. The prepared extract was analysed by one of the applied instrumental methods, taking aliquots directly from the vial in which extraction took place.

~~2.2.1.3. Alkylphenoxy Garboxylic Acids~~

Alkylphenol carboxylic acids were extracted from the water samples by two techniques: a) batch extraction with organic solvent in a separatory funnel and b) gaseous stripping with nitrogen in an apparatus according to Wickbold (76). During the extraction in the separatory funnel, 1 l of water sample was acidified to 27 pH 2 by the addition of dilutad H!!S04 (60 91..) and o>!traet&d twice with 50 and 20 ml of chloroform. Before the extraction in Wickbold apparatus, the pH of the sample was adjusted to 2, and 40 g of sodium chloride was added. The nitrogen was purged through the sample, which was overlayed by ethyl acetate (60 ml) at 30 Umin during 5 min. The procedure was repeated with a fresh portion of the solvent. The combined extracts were then transferred into a separatory funnel to remove the excess of water. The extracts obtained by either techniques a) orb) were further identically treated . Before the concentration to a small volume done by the rotary evaporation, extracts were dried by filtration through the column of sodium sulphate. The concentrated extracts were transferred into small vials with Teflon-lined screw caps and were evaporated to dryness under a stream of nitrogen. The residue was redissolved in dichloromethane and subjected to chromatography on a silica gel column in order to remove the coextracted compounds which could hinder the analysis. A glass column of 10 mm internal diameter was packed with 4 ml of silica gel, deactivated with 15 o/o of water and in the form of slurry in dichloromethane. After the extract was added to the column and after the repeated rinsing of the vial with the sample using small portions of dichloromethane, the nonpolar compounds were eluted with dichloromethane (25 ml). This portion does not contain APEC and can be discarded. The second fraction, containing polar compounds including APEC, was eluted with 25 ml of methanol. This fraction was evaporated to dryness and the residue was redissolved in 2 ml of methylating agent (1 O % solution of boron trifluoride or 1 N solution of hydrogen chloride in methanol). Methylation was carried out in a water bath at 80 °C for 30 min. The reaction mixture was cooled down and 0.5 ml of distilled water was added. The methylated acids were extracted from the reaction mixtur$S by shaking with exactly 1 ml of n-hexane. In the later phase of the method development, the extraction with chloroform was used (1+1 ml), and before extraction 1.5 ml of water was added to the extraction mixture. Aliquot of the hexane extract can be analysed with the normal-phase HPlC directly from the reaction mixture (upper layer), while the chloroform extract must be first evaporated to dryness and the rest transferred into an exact amount of n-hexane. For the quantitative determination by normal-phase HPlC octylphenoxy acetic acid was used as the external standard, and nonylbenzene was used as the internal standard for the gas chromatographic determinations. During the later phase of the wort< 2-terl-butyl-6- methylphenol was used as the internal standard and it was added to the treated extract in the form of a solution in n-hexane. 28

~~2.2.2. Preparative Separation and Determination of Response Factors~~

~~2.2.2. 1. Alkylphenol Ethoxylates~~

Pure oligomers of APEO are not commercially available because the common APEO products on the market are purchased in the form of complex mixtures. Specific oligomers, which were needed during the method development or for the study of physico-chemical properties, had to be separated using preparative liquid chromatography from different commercial products. Single oligomers of nonylphenol ethoxylates with 1-17 ethoxy groups in the molecule were preparatively separated from the commercial mixtures Marlophen 810, and Marlophen 83. For this purpose, normal-phase HPLC using a preparative column (250 x 8 mm) packed with irregularly shaped amino-silica (Lichrosorb NH2, 1o µm) was applied. Elution conditions were much like those described for analytical column with the same stationary phase (chapter 3.3.1.) except for the proportionally increased flow of the mobile phase. Specific fractions containing separated oligomers were collected, evaporated to dryness under the stream of nitrogen, and the residue was redissolved in exactly 1 ml of solvent.

The concentration of APEO oligomers, in the solutions, was determined by the weighing of aliquots (20 µL) on the Cahn microbalance (Cahn Electrobalance, Model 4100). It should be pointed out that oligomers of p-NPEO, preparatively separated from Marlophen 81 o, can contain up to 10 % of impurities, especially o- NPEO and decylphenol ethoxylates.

Diluted solutions in a corresponding solvent were prepared from the initial standard solutions, and were analysed by liquid chromatography, gas chromatography and HRGC/MS in order to verity the identity and purity of the specific fractions. After the addition of internal standard (2,4,6-trimethylphenol or nonylbenzene), the response factors for the NPEO were determined according to the following equation:

~~RFn == Ais /An x mn/mis (8)~~

~~RFn - response factor for oligomer n; An. Ais - the peak area for oligomer n and internal standard; mn. mis - the injected amount of oligomer n and internal standard. 29~~

~~The response factors for higher oligomers (n E0>5) were determined versus 2,4,6-trimethylphenol as external standard according to the equation (8).~~

~~2.2.2.2. Alky/phenoxy Carboxylic Acids~~

In contrast to the APEO, alkylphenoxy carboxylic acids could not be obtained on the market even in the form of commercial mixtures, and it was therefore neccessary to synthesize them first. The starting material for the preparation of APEC was the commercial mixture of APEO (Marlophen 83, lmbetin Nf7A). The corresponding APEC were obtained by oxidation with Jones reagent (109), or by the reaction of the corresponding alkylphenol with the chloroacetic acid .. Further purification of the synthesized APEC and their preparative separation was done after their transformation into mett)ylesters. The aliquots of the hexane solutions of APEC methylesters were analysed by normal-phase HPLC (Lichrosorb NH2, 1 O µm; 250 x 4.6 mm; chapter 3.2.1.) with the preparative collection of fractions. In this way, the pure oligomers of NPEC {n == 1-4) as well as OP1EC In the form of methylester were obtained. The solutions of the known concentrations were prepared as already described for APEO. The identity of a specific fraction was determined by HRGC/MS, and In addition, the ultraviolet and fluorescent spectra were taken. A known amount of internal standard (2-tert-butyl-6-methyl-phenol) was added to the solutions and the response factors were calculated according to the equation (8).

~~2.2.3. Instrumental Methods~~

~~2.2.3.1. High-Performance Liquid Chromatography (HPLC)~~

During the work, several types and configurations of the liquid chromatographs were used. The major part of the analyses in this work was done by a liquid chromatograph containing two high-pressure pumps (Waters Inc., Model 6000 A, or Model 45). a system for the gradient elution with high-pressure solvent mixing (Waters Solvent Programmer, Model 660), and an injector (Waters, Model U6K). In the work with isocratic conditions very often a simple configuration was used, which consisted of one pump and one injector. For the detection ofthe investigated compounds, the spectrophotometric detectors with variable detection wavelengths were used (Perkin Elmer, Model LC-50 30 and Kratos Spectroflow 773), as well as a spectrofluorometrlc detector {Kontron SFM 22).

During the later phase of the investigation, a more advanced liquid chromatograph {Perkin Elmer Series 4) equipped with a Rheodyne syringe loading sample injector (Model 7125) was employed. This chromatograph was connected to a spectrophotometric detector. (Kratos Spectroflow 773) or to a more advanced spectrofluorometric detector {Perkin Elmer LS-3). Alkylphenol polyethoxylates and their degradation products, were detected either spectrophotometrically at 277 nm (maximum in the apsorption spectrum) or spec- trofluorimetrically using excitation wavelength of 277 nm and emission wavelength of 300 nm.

The choice of the elution solvent depended on the type of chromatographic process desired for the analysis. In the normal-phase liquid chromatography n-hexane was applied as the basic eluent, and 2-propanol was added in order to modi- fy the polarity. In some cases of gradient elution the eluent B contained up to 10 % of water. The mobile phase flow was in the range 1-2 mUmin. In the reversed-phase chromatography the mixtures of methanol and water or acetonitrile and water in different ratios were used. The mobile phase flow was 0.5-1.0 mUmin.

In the course of this work, a number of commercial chromatographic columns (Dr. H. Knauer, Berlin, FR Germany) filled with chemically modified silica were used. Stationary phase applied for the normal-phase chromatography was aminosilica whereas for the reversed-phase chromatography octylsilica or octadecylsilica were used. The majority of the analyses using normal-phase system was carried out with the columns (250 x 4.6 mm) filled with irregularly shaped particles of 1o µm (Llchrosorb NH2, Merck, Darmstadt), or with the columns (100 x 4 mm) filled with the spherical particles of 3 µm (Hypersyl, APS). In the reversed-phase system, the columns (250 x 3 mm) packed with irregularly shaped particles with octylsillca of 10 µm (Hibar RP-8, Merck, Darmstadt), or the columns (100 x 4 mm) with spherical particles of 5 µm were used.

~~Peak areas were determined by electronic integration (Hewlett Packard 3390 A). 31~~

~~2.2.3.2. High-Resolution Gas Chromatography (HRGCJ~~

The gas chromatographic analyses were performed with the Carlo Erba (Italy) instruments equipped with glass capillary columns, injector after Grob (154), and the flame ionization detector. During the usual procedure, 1-2 µLa sample (extract) was injected into the chromatograph at room temperature without the splitting of the carrier gas stream. After 30 s the split valve was opened allowing the septum and injection part to be purged. After the major part of the solvent eluted from the column, the oven tern· perature was raised to 2 °C/min from 50-300 °C. In certain cases even faster temperature programmes were used (up to 6 °C/min). The injector and detector temperature were usually 250 °C and 300 °C respectively. Hydrogen was used as the carrier gas with the total flow of 30·40 mUmin resulting in the linear flow velocity on the column of 1.4 mis at 25 °C. The chromatographic separations were performed on persilylated glass capillary columns (19-22 m long and 0.3 mm Ld.) coated with a thin film (0.15 µm) of immobilized stationary phase (SE-54, PS 255) (155). The characteristics of the columns were tested by standard procedure according to Grob (156). For the quantitative wor1<, the detector response was integrated electronically (Hewlett Packard 3390 A).

~~2.2.3.3. High-Resolution Gas Chromatography/Mass Spectrometry (HRGC!MS)~~

For the analysis with the HRGC/MS system, a Finnigan mass spectrometer (Model 4021 C) interfaced with the data processing system (INCOS 2000) was used. Gas chromatograph (Carlo Erba, Model 4160) supplied with the Grob injector (154) and a glass capillary column (SE-54, 22 m), was connected with the ionic source through fused silica capillary. Aliquots of the extracts (1-2 µL) were injec· ted into gas chromatograph at room temperature. The valve for splitting of the carrier gas stream (helium, 30 mUmin) was opened after 30 seconds. After the solvent was eluted, temperature programming started at the speed of 2-4 °C/min up to maximal temperature of 270 °C. The setting of the gas spectrometer was as follows: ionization energy 70 eV; temperature of ion source, 270 °C; analyser pressure, 1.1 x 10-e or; sensitivity, 10-1 ampN; mass range, 45-480; scanning time, 1 s.

~~The identification of compounds was done by the comparison of the mass spectra of the sample with those of authentic reference compounds as well as with the 32~~

mass spectra published in handbooks and scientific papers. For the quantitative determination of NP, NP1 EO and NP2EO by HRGC/MS, selected ion monitoring method was applied using the sum of the characteristic ions m/e 220; 264; 308; 135; 149; 179; 191; 193.

~~2.2.3.4. Other Instruments~~

~~The spectra in UV and visible range were recorded on a Kontron Spectrophoto- meter (Uvikon 810) using quartz cells with the optical path length of 1 cm.~~

~~The emission and excitation spectra were taken with a Perkin Elmer instrument using the same cells. The slit width of the monochromator varied between 2- 1O nm, and the scan speed was 60 nm/min.~~

~~IR spectra were recorded in the range of wave numbers of 4000-600 cm-1 on a Perkin Elmer instrument (Model 297) by preparing the sample with potassium bromide.~~

~~A Cahn Microbalance (Model 4100) with the weighing precision of 0.1 µg, was used for the determination of the exact concentrations of oligomers in solution.~~

~~2.3. Determination of Solubility in Water~~

In order to obtain more reliable results, several techniques for the solubility determinations of AP and APEO were investigated. The equilibration of distilled water with the pure substance (about 300 mg/L) was performed under slow mixing with a magnetic stirrer. An aliquot (usually 1O ml) of the formed solution was taken during certain time intervals for the analysis, taking care that no drops of the substance in excess get into it.

Using the generation column technique (157, 158), two types of carriers were investigated: florisil and porous glass powder. The generation column was prepared by coating support material of a thin film ofthe investigated substance. The coating was performed by dissolving a known amount of the investigated substance (about 1 %of carrier amount) in organic solvent (usually chloroform), and by the subsequent addition of the weighed amount of the carrier. The solvent was slowly evaporated in a rotary evaporator so that the investigated substance remained on 33

the carrier particles in the form of a thin layer. Thus, prepared material was placed in a glass column of internal diameter 6 or 1o mm, and was eluted with distilled water. The first part of eluate (about 50 ml) was discarded because the stable equilibrium was not yet established. The following eluates represent saturated solution of the investigated substance and are suitable for the solubility determinations. They were collected in 1O ml graduated vials later used for extraction. The elution rate was in the range of 2-5 mUmin. The concentration of the compound was determined by one of the described methods, usually by normal-phase HPlC.

~~2.4. Determination of Partition Coefficients between Water and Organic Solvents~~

The partition coefficients between octanol and water, as well as hexane and water, were determined by the CECO method (159). Firstly, the mutually saturated solutions of the organic solvent and water were prepared by vigorous shaking in the separatory funnel. AP or APEO were added into the organic solvent saturated with water, so that the concentration was 1 x 1o·3 mof/L. A volume of 5, 10 and 15 ml of this solution was placed into the separatory funnel (100 ml} and subsequently 45, 40 and 35 ml of water saturated with organic solvent were added (total volume 50 ml; ratio of organic water phase 9:1, 4:1 and 7:3) and shaked. The phase separation was accelerated by centrifuging (2 min, 2000-3000 rpm). An aliquot of the water phase was taken out by pipette and analysed. The concentration of the investigated compound in the water phase was determined by the normal-phase HPlC after the simple extraction with n-hexane in a graduated bottle of 25 ml. The concentration In the organic phase was determined after dilution of the aliquot with n- hexane in the ratio 1:100. Partition coefficients (Kow) are calculated as the ratio of concentrations of the investigated compound in organic phase (Co) and water phase (Cw):

~~Kow= Co/Cw (9) 34~~

~~2.5. Determination of Biodegradability~~

Biodegradation of alkylphenol polyethoxylates and alkylphenols was investigated, under laboratory conditions, applying several methods (160): shake culture test, river water test, closed bottle test, coupled unit test. The shake culture test was performed in two different modifications: a) on the synthetic sewage which contained, along the APEO and AP, the additional carbon source (Husmann's medium) and b) on mineral medium (growth medium by Horvath and Koft) in which the investigated compounds were the only source of the organic carbon.

Several mixed bacterial cultures were used: spontaneous bacterial culture formed in the solutions of APEO and AP during their preparation (no addition of inoculum); bacterial culture isolated from three different sources: river waters (Orava at Osijek), wastewater of the detergent production plant, and soil (Kopacki rit). The exact description of the isolated mixed bacterial cultures is given elsewhere (161 ). After isolation, the experimental cultures were kept in 40 % solution of glycerol, also containing 0.33 g/L of meat extract and about 7.5 mg/l of commercial mixture of nonylphenol polyethoxylates (average number of EO groups 9, Hechst AG, Frankfurt, FR Germany) at -16 °C. The revival of the stored cultures was done by repetitive inoculation into synthetic sewage using the shake culture method. The solutions of AP and APEO were prepared by the equilibration of the excess of these substances (1 g) with 5 l of water and with slow mixing. From the formed concentrated solutions, the appropriate dilutions were made so that the concentration of AP or APEO in the medium, ranged from 0.5-3 mg/L. A volume of 200 ml of synthetic sewage, or mineral medium, which also contained the known concentration of the investigated compound was added to the sterile Erlenmeyer flask and inoculated with the mixed bacterial culture. No inoculum was added to the first flask (it contained only spontaneous bacterial culture), while to the last one, after the addition of inoculum, 10 ml of 1 % solution of mercury(ll) chloride was added. This flask served as a sterile control. For each experiment two parallel series of identical samples were prepared. The flasks were placed on a shaker at 23.5 °C, and in defined time intervals the sample was taken (1 O ml) for chemical (determination of the remaining AP and APEO concentration) and bacteriological analysis (determination of the number 35 and type of bacteria). The chemical analyses were performed by techniques described in this work, and a number of the bacteria was determined by the standard method of counting the developed colonies on the Petri dishes with nutrient agar. When the major part of the investigated compound was degraded, the content of the parallel sample was extracted in order to identify the formed degradation products. At the end of experiment, the walls of the flasks were rinsed with n-hexane in order to determine the amount of adsorbed AP and APEO.

In the investigation of AP and APEO degradation the use of the river water test was done in two modifications: a) no stirring (static method) and b) with stirring (modified river water test). The degradation was examined at increased concentrations of AP and APEO (about 1 mg/l), and at the usual concentrations for natural waters. The major part of the experiments was performed at room temperature (20±2 °C) and the minor part at lower temperatures (4 °C).

Artificially polluted samples were prepared by the addition of excess of investigated substance into river water (Sava river at Micevec, March, 1985; BOD = 3 mg/l; dissolved oxygen =8 mg/l). The obtained concentrated solution was diluted with the same river water to the neccessary concentration. Degradation measurements were performed in dark bottles of 1.3 l containing 1 l of sample. 1o ml aliquots were taken during certain time intervals, and the remaining concentration of the investigated compounds was determined by HPlC.

For the investigations of AP and APEO biodegradability in naturally polluted samples, a larger volume of the sample (20 l) of secondary effluent was taken from the sewage treatment plant of the town DObendorf, Switzerland. The samples, to which 1 % solution of formaldehyde was added served as sterile control.

The closed bottle test was modified so that, a short period after the experiment begun (addition of inoculum), anaerobic conditions were established. This was achieved .by preparing the solution of the investigated substance in the synthetic sewage which it contained easily degradable organic compounds. Such a solution was added into the Winkler bottles of 115-135 ml volume. The bacterial culture present in the solution degraded first the pepton and meat extract, and by doing that used up all the dissolved oxygen within only a few hours (this was proved by determination of the dissolved oxygen). During more than two months, the samples were taken from the bottles and analysed In orderto determine the remaining concentration of APEO. The samples for the bacteriological analyses were taken as well. 36

The coupled unit test (160) resembles the procedure which is most commonly used for the biological wastewater treatment. The experimental conditions, used for the investigation of APnEO degradation, are described in more detail elsewhere (136).

~~2.6. Determination of Photochemical Degradability~~

~~The photochemical degradability of AP and APEO was investigated under model laboratory conditions as well as exposing the solutions to sunlight.~~

The experiments in the laboratory were performed by the use of a merry-go-round reactor (MGRR) which can be supplied with various light sources (mercury lamp, wolfram-halogen lamp). The apparatus has a cooling system as well as a rotating system, so that radiation could be uniform for all samples. The function and construction of the apparatus are described elsewhere in more detail (162). The water solutions of AP and APEO were exposed to light in quartz or glass (Jena) tubes of 50 ml. After a specified irradiation time the tubes containing the sample were taken out from the apparatus and the concentration of the investigated compound was determined. For these analyses a simple extraction with n-hexane and quantitative determination by normal-phase HPLC.

The photochemical degradation of NP and NPEO by sunlight was investigated in two ways: a) the solution of investigated compounds was exposed to sunlight in quartz tubes, immersed into a shallow vessel filled with tap water, and b) In tubes immersed in a brook at the depth of 20-25 cm. The solutions of NP and NPEO were prepared in filtered (0.45 µm) lake water (Greifensee, 25th April, 1985; DOC= 4 mg/L; pH= 8.4). The spectra of lake water, in which solutions were prepared, and waters of brook Chriesbach showed no appreciable difference in UV and visible range. During the experiment the brook water was clear and the temperature varied between 14.5-17 °C, depending on the time of day. The temperature in shallow vessel was adjusted (addition of ice) to be similar to that of the brook (17±3 °C). At the same time with the photolysis of the investigated compounds, it was determined, by actinometry with p-nitroanisol (pNA), that the intensity of the irradiation in the shallow vessel (kpNA =0.12 h·1) was about three times higher than in the brook at the depth at which tubes were placed (kpNA =0.038 h·1). 37

The experiments were performed twice: on 11th S&ptQmbQr. 1Q85 (from 1120 tc 1720 h Middle-European time; total duration 351 min) and on 18th September, 1985 (from 1105 to 1505 h; total duration 288 min). The average intensity of sun irradiation during the experiment were O. 705 kWh/m2h (11th September) and 0.706 kWh/m2h (18th September). The first experiment was performed at the original pH value of lake water (8.4), while in the second it was adjusted to 9.4 by the addition of NaOH. How~ver, during the second experiment pH value decreased spontaneously to 8.7 with sedimentation of, most probably, calcium car- bonate. The solutions exposed to sunlight were prepared from the saturated solutions obtained by generation columns filled with porous glass powder (chapter 2.5.): NP of 0.39 and 0.43 µmol/L, OP at 0.48 µmol/L and NP1 EO at 0.33 µmol/L. Since the intensity of sunlight irradiation varies during the day, the presentation of the results in time scale is not very appropriate. Therefore, the results were presented in dependence of the total irradiation dose. The intensity and amount of sunlight irradiation were measured by the apparatus which was placed in the vicinity of the place at which the samples were exposed. The total irradiation in a certain time period was calculated by integrating the values which were recorded in time intervals of 10 min.

~~The rate constants of the photolysis (kp) were calculated by pressuming that the reactions were of the first order (163, 164) according to the equation:~~

kp == In (Co/C)lt (10) where Co and C are initial concentration and concentration at the moment t, respectively. It follows the above expression that the half-time of degradation can be calculated from:

~~tt12 =0.693/kp (11) 38~~

~~2.7. Sampling~~

The water samples were collected using quite different techniques. A greater number of river and wastewater samples was collected with a plastic .can (grab samples). The composite samples of waste and river water were taken with the help of a special sampling apparatus working in two ways: a) equal water volumes were taken in regular time intervals (time proportional sampling) or b) always the same portion of the total flow was taken {flow proportional sampling). The second method was applied at the majority of stations for the continuous monitoring of physico-chemical characteristics of swiss rivers {165). Lake water from different depths was collected by Van Dom sampler. Groundwaters were sampled with a special imersive pump (Mikropumpe 20, Wilhelm Keller, FR Germany).

Activated sewage sludge was collected with a can by taking the wastewater from the airation tank of a sewage treatment plant. The water phase was decanted and the dense suspension of the sewage sludge was taken to be analysed. Anaerobically stabilized sewage sludge was taken with a handy vessel or directly from the tap of the anaerobic fermentor.

~~River sediments were collected by hand from the bank and were stored at -20 °C until analysis.~~

~~Fresh water algae were also collected by hand and stored in deep-freeze until analysed.~~

~~Fish were caught by hand and dissected immediately. Specific organs and tissue were wrapped in aluminium foil and were stored in a deep-freeze (-20 °C). 39~~

~~3. RESULTS AND DISCUSSION~~

~~3.1. Determination of Alkylphenol Polyethoxylates and Their Metabolites~~

~~3.1.1. Determination of Alkylphenol Ethoxylates and Alkylphenols~~

~~3. 1. 1. 1. Chromatographic Separation~~

~~Figures Sa and Sb represent the chromatograms obtained by separation of -components of the commercial mixtures of Marlophen 810 and Synperonic OP10 by normal-phase HPLC (aminosilica).~~

~~11 8 9 10 12 B 7 13 6 14 5~~

~~0 6 12 18 24 30 36 42 48 54 60 ml 0 10 20 30 40 min~~

Figure 5. Normal-phase high-performance liquid chromatograms of the nonionic surfactants of APEO type: (A and C) Marlophen 810 (NPnEO); (8) Synperonic OP10 (OPEO). The peak numbers refer to numbers.of ethoxy groups. The HPLC columns were 250 mm x 4.6 mm i.d. , 10 µm Lichrosorb NH2 (A, 8) and 100 mm x 4 mm·l.d., 3 µm Hypersil APS (C). 40

All oligomers in the range from 10-18 EO groups were successfully separated in a relatively short time, approximately 40 min. A similar separation of APEO was obtained by other authois (48, 65), but using tetrahydroturane in eluent A. By the change of elution conditions the higher oligomers (nEO > 20) can be separated as well. During the work with gradient elution, no problems were observed which would indicate the deactivation of the column. After a short equilibration with the mobile phase of initial composition in the duration of S-10 minutes, the column was ready for the new injection.

Nonyl- and octylphenol ethoxylates, with the same number of ethoxy groups, are eluted in a very similar time. This indicates that the shortening of the hydrophobic part of the molecule for a methylene group does not considerably influence the chromatographic behaviour of the APEO in the system with a normal-phase. However, it can be observed that the peaks of the oligomers on the chromatograms for the commercial mixture of the Synperonic OP10 (Figure Sb) are much sharper than the peaks of the Marlophen 81 o (Figure Sa), which resemble the insufficiently separated double peaks. This difference could be explained by the composition of the technical alkylphenols which are used for the synthesis of these two nonionic surfactants. In the technical 4-octylphenol generally only one isomer prevails (95 % 1, 1,3,3-tetramethyl-butylphenol), because it is obtained by synthesis from the phenol and isobutylene. Technical nonylphenol is produced from the phenol and isopropylene, and due to that a complex mixture of isomers with differently branched nonyl chain is formed (105).

Under the conditions described for the separation of APEO oligomers in a wide range, simultaneous separation of AP1 EO and AP2EO from the fully deethoxylated component (AP) was not achieved. In order to do that, the chromatographic conditions were adjusted in such a way that the maximal resolution was in the range of the lipophilic APEO. In Fig. 6 the separation of the components of the commercial mixture of Marlophen 83 (nEO = 1-5) is presented. The technical nonylphenol and 2,4,6-trimethylphenol, as internal standard, were added to the mixture. Besides the main components, which belong to p-isomers of NPEO and NP, in Figure 6 smaller peaks can be observed. On the basis of the preparatively separated fractions and HRGC analysis they were attributed to the o-isomers of these compounds. The presence of o-isomers can be explained by the fact that technical NP used for their synthesis contained 5-1 O % of a-isomers, and therefore a similar percentage should be expected in NPEO as well. In the applied normal-phase chromatographic system ortho-isomers of NPEO were eluted with shorter retention times than the corresponding para-isomers, which it most 41

~~TMP NP2EO A NP~~

~~NP3EO~~

~~NP4EO NP5EO~~

~~B TMP~~

~~NP2EO NP NP3EO~~

~~TMP NP2EO c NP NP3EO NPIEO~~

~~0 8 16 24 32 40 48 56 64 ml 0 10 20 30 min~~

Figure 6. Normal-phase high-performance liquid chromatograms of the reference mixtures containing Marlophen 83, technical 4-nonylphenol, and 2,4,6,- trimethylphenol. Three different aminosilica columns (A, 8, and CJ were used as described in the text. A and B: Lichrosorb NH2, irregularly shaped 10 µm, 250 mm x 4.6 mm i.d.. C; Hypersil APS, spherfcal 3 µm, 60 mm x 4 mm i.d.• NP: 4-nonylphenol, ·NP1 EO-NP5EO: 4-nonylphenol ·monoethoxylate to 4-nonylphenol pentaethoxylate, TMP: 2,4,6-trimethylphenol. 42

probably reflects the ortho-shielding effect of the alkyl chain on the polar part of APEO molecule. Figures 6a and 6b show different elution sequences to NP, NP1EO and NP2EO, although columns of the same dimensions, filled with the same stationary phase (Uchrosorb NH2), and bought from the same supplier were used. The observed differences could be explained with the different extent of coverage of the silica surface by the amino groups. Phenolic OH group has different characteristics (weak acid) as the alcoholic OH group of APEO and therefore it is very probable that the variable shares of the silanol and amino (weak base) groups in the stationary phase could cause different chromatographic behaviour. If the HPLC columns filled with regularly shaped aminosilica of 3 µm (Hypersil APS) were used, the time needed for elution can be saved without influencing the efficiency of oligomer separation (Figures 5c and 6c). The great advantage Is that with the use of such columns of 100 mm in length, the separation of lipophilic APEO and AP by isocratic elution (Figure 13) can be achieved, which considerably simplifies their determination. It should be mentioned that the chromatographic conditions are rather different when Hypersil APS stationary phase is used from those cited for Uchrosorb NH2. The elution of APEO on Hypersyl APS was achieved at much greater nonpolar conditions.

In contrast to the normal-phase HPLC the separation of octyl and nonyl homologues of AP and APEO can be achieved by high-resolution gas chromatography (107). However, due to low volatility of the higher oligomers, this technique is limited to lower oligomers (nE0<10). The higher oligomers can be separated by the use of reversed-phase HPLC (Figure 7). The commercial mixture of nonylphenol polyethoxylates (Marlophen 810) is eluted in two separated peaks (Figure 7a), while octylphenol polyethoxylates are eluted in one peak (Figure 7b). By the preparative separation of the mentioned NPnEO peaks and the analysis of the collected fractions by high-resolution gas chromatography, it was concluded that they are most probably two groups of isomers with differently branched nonyl chain (60), and not the compounds of the octylphenol polyethoxylate type. It was shown, in the same manner, that the observed peaks do not represent the separated o- and p-isomers of NPEO.

On the used column, alkylphenols elute with very similar retention time as the corresponding APEO (Figure 7c). Besides the peaks of OP and NP, which are the main components of the standard mixture,· an incompletely separated peak of decylphenol{DP, Figure 7c), which is present as impurity ill the technical NP, as 43

~~well as the peak of decylphenol polyethoxylates (OPEC, Figure 7a) can be observed.~~

~~NPEO A ~~~

~~8 OPEO~~

~~OP c NP~~

~~0 2 6 8 ml 0 5 min~~

Figure 7. Reversed-phase high-performance liquid chromatograms of alkylphenols and nonionic surfactants of the APEO type: (A) Marlophen 810 (NPEO); (8) Synperonic OP10 (OPEO); (C) 4-octylphenol (OP) and 4-nonylphenol (NP). The HPLC column was 250 mm x 3 mm i.d., 10 µm Hibar RP-8. Injected samples contained 10 µL of methanol solutions containing 1 µglµL of Marlophen 810 (A), 1 µglµL of Synperonic OPt o (8), and 0.1 µglµL of both 4-octyl- and 4-nonylphenol (C).

~~A similar chromatographic separation as in Figure 7 can be achieved by using a column packed with octadecyl silica~~

Coelution of APEO oligomers in a wide range of molecular weights (nEO = 1-20) in the chromatographic system with reversed-phase HPLC was quite unexpected considering a significant differences in the polarity and solubility of the oligomers 44

(for some details about this problem see chapter 3.2.2.). A successful separation of their alkyl homologues of APEO with coelution of all the oligomers indicates a dominant influence of the alkyl group on the chromatographic behaviour of these compounds in reversed-phase systems. However, through the application of a larger number of the reversed-phase columns, it was observed that the characteristics of the same type of the stationary phases can vary considerably, which is probably the consequence of the different coverage of polar silanol groups by the nonpolar alkyl chains during the stationary phase preparation (166). For example, a column filled with regular spheric particles of octylsilica (5 µm, Merck, Darmstadt, FR Germany) gave very good separation of the alkyl homologues of APEO, but in contrast to the already described columns it also separated single oligomers in the sequence which is characteristic for normal-phase chromatography (retention time increases with the number of ethoxy groups in a molecule) (Figure 8).

~~NP2EO~~

~~3 9 IS 21 27 33 39 ml 2 6 10 14 18 22 26 min~~

Figure 8. Reversed-phase high-performance liquid chromatogram of nonylphenol ethoxylates (Marlophen 83). The HPLC column was 100 mm x 4 mmi.d., 5 µm Spherisorb RP-8; eluent: methanol!H2') 65135; 1.5 mUmin; detection 277 nm.

This indicates that, apart from the predominant hydrophobic interaction of the alkyl chain, the chromatographic behaviour of the oligomers was also influenced by the interaction of the polar part of the APEO molecule with the free silanol groups of the stationary phase. 45

~~3. 1. 1.2. Response Factors of Alkylphenol and Alkylphenol Ethoxylates~~

By the analysis of solutions containing the known concentrations of the previously preparatively separated oligomers of NPnEO, the response factors were determined for each used instrumental technique. The method for the response factor calculation is shown in the experimental part (chapter 2.2.2.1.), and the results are given below in Table 2. ·

~~TABLE2. HPLC Response Factors of Alkylpheno/s and Alky/phenol Po/yethoxy/ates~~

~~HPLC (UV 277 nm) HPLC Fluor. HRGCIFID HRGC/MS NoolEO (2771300 nm) units per 8 8 8 8 molecule NPE0 NPEOb OPE Ob NPE0 NPE0 NPEOc NPE0~~

0 1.7 1.6 1.5 1.55 1.1 1.6 1.0 1 2.6 2.4 2.3 1.B 1.5 2.0 2.7 2 2.9 2.8 2.7 2.1 1.7 2.3 4.5 3 3.2 3.2 3.1 n.d. 1.9 2.6 n.d. 4 3.5 3.6 3.6 2.3 3.2 5 4.0 4.0 3.9 2.6 3.6 6 4.2 4.4 4.3 n.d. n.d. 7 4.6 4.8 4.7 8 5.2 5.2 5.1 9 5.6 5.6 5.5 10 5.9 6.0 5.9 11 6.3 6.4 6.3 12 6.6 6.8 6.7 13 7.0 7.2 7.1 14 7.8 7.6 7.5 15 7.9 7.9 7.B 16 8.4 8.4 8.3 17 8.8 8.7 18 9.2 9.1 a Related to 2,4,6-trlmethylphenol (TMP) as described in the Section 2.2.2.1.; b Stoichiometrlcally calculated based on the RF of NP3EO equal to 3.2 corresponding to a molar response factor related to TMP of 1.2; c Related to nonylbenzene as an Internal standard; n.d.: not determined.

The values of tt:ie response factors are expressed in respect to 2,4.6-trimethylphenol (TMP), which served as the internal and/or external standard in the HPLC determinations. The response factors for the gas chromatographic determinations are given in respect to nonylbenzene as the internal standard. It should be pointed out that the experimentally determined response factors belong to the p-isomers 46

of AP and APEO. Regression analysis showed a linear relationship between the response factors and the number of ethoxy groups in the APEO molecule for all the applied methods. Experimentally determined dependence of the response factor values on the number of EO groups (n = 1-16) in normal-phase HPLC with UV-spectrophotometric detection can be represented by the following equation: RF(nEO) = 0.39 nEO + 2.0 (r = 0.9977). The response factor value for NP (1.7) is considerably lower than the value obtained by the introduction of nEO = O into the above equation, which indicates that AP have higher absorptivity than APEO. The regular increase of the response factor with the increase of the number of EO groups is the result of the relative decrease of the share of the aromatic part of the molecule with the increasing molecular weight of APEO. The molar response factors versus TMP are the same for all the oligomers of APEO (1.25), while the molar response factor for the alkylphenols is 1. The response factor values obtained by the measurements are compared with the values calculated on the basis of molecular weight relationships, taking the response factor of NP3EO (3.2) as the referent value. Very good agreement was obtained (Table 2). The response factors for the OPEO oligomers were calculated stoichiometrically, on the basis of the confirmed dependence of the response factor on the molecular weight of APEO. Although, on the basis of the discussed facts, one could make the extrapolation even for the higher oligomers (nE0>20), in Table 2 the response factors are given for the oligomers in the range of 1-19 EO groups per molecule which are most commonly applied in the detergent formulations and therefore are expected to be found in the environment.

The response factors for spectrofluorometric detection (277/300 nm) have shown dependence on the molecular weight of the APEO which is similar to the one already described for the UV-spectrophotometric detection. The only difference in the response factors, the alkylphenols are on the same correlation curve as the APEO oligomers. Fluorescence of the alkylphenols, however, depends on the share of water in the solvent (water quenches fluorescence; ref. 167) and therefore, response factors in the reversed-phase chromatography should be applied with special care.

The relationship of the response factors to the number of EO groups, for the gas chromatographic determinations with flame ionization detection, can be expressed as RF(nEO) = 0.29 nEO + 1.13 (r = 0.9937; nEO = 0-5). Included are only the lower oligomers which can be quantitatively analysed by HRGC/FID technique without derivatization .. 47

When the gas chromatographic determinations, and the mass specific detection was applied, the sensitivity decreased sharply (response factors increase) with the increase of the number of EO groups according to: RF(nEO) = 1.75 nEO + -0.98 (r = 0.9998). The reason for this sensitivity loss is a shift in the mass spectra of higher oligomers to lower fragment ions (3) which were not monitored by the employed mass chromatograms. Namely, since these ions are not sufficiently specific, they were not taken into consideration for the quantitative determination of APEO.

~~3.1.1.3. Extraction and Purification of Extracts~~

The extraction yield of the mixture of NPnEO (n = 1-18) was determined after the addition of 1 mg of Marlophen 810 to distilled water (10 µg/µL in ethyl acetate). The result of the triplicate determination is given in Table 3. The total yield was relatively high with very good reproducibility (87.4±1.3 %). The distribution of the oligomers after extraction and extract treatment did not differ considerably from the distribution of the oligomers in the original mixture. The yield of the analytical procedure for specific oligomers was 90-100 % in the range form NP3EO to NP11 EO and then it slowly decreased to 70 % for the highest oligomers (NP17EO). Somewhat lower extraction yield for higher oligomers is most probably the result of their unfavourable partition between ethyl acetate and water. By the real samples analysis, the extraction of APEO by the Wickbold method alone is not sufficiently selective. The nonpolar compounds (aromatic hydrocarbons, phtalates) present in the extract interfere during the determination of the lower oligomers (NP1 EO and NP2EO). Therefore, it was neccessary to purify the extracts on the column of aluminium oxide before HPLC analysis.

Since the mentioned lipophilic oligomers represent the most toxic and therefore ecotoxicologically most important APEO compounds, it was neccessary to develop specific techniques of the extraction and the chromatographic separation which would enable realiable and specific determination of these compounds in the different environmental samples.

Two different techniques were applied for the extraction. The water samples with low AP and APEO concentrations and the samples containing considerable amounts of other organic matter along with the investigated compounds were extracted by a relatively time consuming (3 h) technique in a special apparatus which involved both distillation and simultaneous extractions (153). 48

~~TABLE3. Recovery and Reproducibility of the Determination of Alkylpheno/ Polyethoxylates in Water and Wastewater~~

~~Concentration (µg/L)a No. of ethoxy units per fortified distilled synthetic wastewater<: raw municipal molecule waterb wastewater~~

1 6.3 ± 2.2 (35) 143± 54 {38) 11.0 ± 1.4 (13) 2 7.1 ± 1.0 (14) 152± 10 (7) 35.5± 0.7 (2) 3 10.3 ± 0.7 (7) 269± 10 (4) 38.0± 1.4 (4) 4 18.6 ± 0.5 (3) 606± 12 (2) 69.5± 3.5 (5) 5 28.7 ± 1.4 (5) 1200± 58 (5) 118 ± 2.0 (2) 6 40.9 ± 1.6 (4) 1820± 56 (3) 162 ± 5.0 (3) 7 64.2 ± 2.0 (3) 2440± 110 (5) 215 ±15 (7) 8 72.8 ± 4.8 (7) 2930± 180 (6) 287 ± 10 (3) 9 101 ± 3.0 (3) 3140± 120 (4) 268 ± 19 (7) 10 144 ± 8.0 (6) 2930± 180 (6) 261 ± 23 (9) 11 123 ± 4.0 (3) 2470± 170 (7) 215 ± 13 (6) 12 93.0 ± 1.0 (1) 1860± 110 (6) 153 ± 5.0 (3) 13 62.8 ± 2.7 (4) 1310± 54 (4) 106 ± 11 (10) 14 42.2±1.3 (3) 883± 16 (2) 91.5± 6.4 (7) 15 32.5 ± 1.4 (4) 581± 55 (9) 66.5± 3.5 (5) 16 19 ± 3.0 (16) 370± 58 (16) 40.0± 0.0 (0) 17 7.8 ± 1.0 (13) 132± 21 (16) 27.5± 4.9 (18)

Total 874 ± 11 (1.3) 23200± 1200 (5.2) 2160 ± 65 (3.0) a Arithmetic averages and standard deviations are given. Numbers in parentheses are relative standard deviations in %; b Triplicate analysis of one liter of double distilled water spiked with 1 mg of Martophen 810 by adding 10 µl of a solution containing 100 mg of Marlophen 810 In 1 ml of ethyl acetate; c Triplicate analysis of a synthetic wastewater containing Synperonic OP1 O; d Duplicate analysis of a sample of raw municipal wastewater from the treatment plant at Fananden.

NP and NPEO determination in artificially polluted water samples (125 µg/L Marlophen 83 and 21.2 µg/L technical NP), employing the described extraction technique, show very high yields for NP (95 %), NP1 EO (105 %) and NP2EO (82 %), while the extraction yield for NP3EO is already very low (15 %) (Table 4). Lower yield of the higher oligomers (nE0>2) can be explained by their weaker steam distillation (46) and it cannot be increased even by the prolonged extraction procedure (after additional three hours of extraction only 1o % more of NP3EO was obtained). High extraction yield was proven for the real samples as well (Table 4). The two consecutive extractions of the secondary effluent sample, lasting 3 hours each, have shown that the first extraction yielded more than 95 % of NP, NP1 EO and NP2EO. Such a selective extraction proved to be especially useful. for the extraction of AP and APEO form solid samples containing a high amount of organic matter like sewage sludge and river sediments. 49

~~TABLE 4. Recovery of the Extraction of Upophilic Nonylpheno/s and Nonylphenol Ethoxylates (%)~~

~~Method 18 Method 2b Compound Fortified water Secondary Fortified soil Fortified water samplec effluentd samplee samplef~~

~~NP 105 97 95 96 NP1EO 94 96 n.d. 96 NP2EO 82 95 n.d. 95 NP3EO 15 n.d. n.d. 95 NP4EO n.d. n.d. n.d. 94 NP5EO n.d. n.d. n.d. 91~~

a Samples were extracted by the closed-loop steam-distillation and solvent extraction method (3h, pH 7.0-7.5); b Extraction with n-hexane (n-hexane/sample = 1/20); c Two liters of deionized water were fortified with 200 µg of Marlophen 83 and 42.4 µg of NP; d Effluent from a mechanical biological sewage treatment plant containing 9.2, 44 and 52 µg/L of NP, NP1EO and NP2EO, respectively; e soil samples (10-30 g) fortified with 1-50 µgig of NP; f Distilled water sample (25 ml) containing 1-10 mg/L of NPEO; the relative yield of the first of two consecutive extractions is given n.d.: not determined

For the soil samples (1 o and 30 g), which were artificially polluted by nonylphenol (added previously to the stabilized sewage sludge) at the concentration level 1, 10, and 50 µg/g, the extraction yield was found to be 95 % (Table 4). Similar extraction yield values from sewage sludge and stabilized sewage sludge (93- 105 %) were given by Giger et al. (108).

High extraction yields of AP and APEO from solid samples indicate that, through the application of the described technique for water sample analysis, both the molecules in the dissolved phase and adsorbed on the suspended particles can be successfully extracted.

Continuous extraction allows a high enrichment factors of over 1000 times (2 L of water sample against 1-2 ml of extract) is attained without the subsequent evaporation of the extract. The use of the apparatus is very simple and the danger of laboratory contamination of the sample is minimized. Furthermore, very high selectivity is achieved through the applied technique because only those lipophilic compounds are extracted which were distilled by steam. Cyclohexane proved to be a very suitable extraction solvent and it is a appropriate solvent for the lipophilic AP and· APEO and is very soluble in water. By the transport of the distil- 50 late through the layer of the cyclohexsne no significant losses ooour, like in the case with some greater polar solvents (ethyl acetate). If high enrichment factors are not neccessary, a simple and fast extraction of lipophilic AP and APEO from water samples by shaking with n-hexane (20:1) can be applied. Due to the high distribution coefficients (chapter 3.2.2.) the yield of a single extraction is very high for lower oligomers of APEO (nE0<5) (Table 4). This method of extraction is not as selective as the one which also includes the steam distillation step, and is not suitable for the direct extraction of solid samples. The direct extraction of small water samples (10-50 ml) with n-hexane proved to be very useful during laboratory investigations of physico-chemical characteristics as well as biological and photochemical degradability of AP and APEO, because during these investigations it was often neccessary to analyse more than 20 samples a day, which would have not been feasible by applying time-consuming extraction.

In the case that some extract, obtained by one of the described methods, is loaded with coextracted substances which could hamper the analysis, it is purified on the aluminium oxide column (chapter 2.2.1.). By this purification procedure the interferring nonpolar substances (because they elute in the vicinity of AP and APEO) as well as the strongly polar substances (which accumulate on HPL'C and HRGC columns thus changing their activity and causing their fast deterioration), are removed.

~~3.1.1.4. Accuracy, Precision and Sensitivity~~

Table 3 shows reproducibility for the determination of the APEO oligomers in the model solutions of known concentration (Marlophen 81 o, 1000 µg/L), in synthetic wastewater, and in real municipal waste water. The relative standard deviation (RSD) for determination of the main oligomers (nEO = 3-15) was 1-7 %. A smaller precision of determination was reached for the lowest oligomers (RSDNP1EO = 25 % and RSDNP2EO == 14 %) and for the highest oligomers (RSDNP16EO • 16 %). This could partly be attributed to the fact that they are the minor components of the APEO mixture which are present at very low concentrations. A similar precision of determination was obtained for the analysis of synthetic waste water (which contained commercial mixture of OPEO, pepton, meat extract and nutrient salts) as well as for the real waste water. RSO for the determination of APEO in real waste water sample was 1-1 o % for the greatest number of single oligomers and for the total concentration of APEO was only 3 %. 51

The limit of detection based on the sample of 1 L depends on the detection method and it is different for the specific oligomers if it is expressed in weight units (response factors, Table 2). When UV-spectrophotometric detection was applied (277 nm), the detection limit was 1 µg/L for the lower oligomers and 3 µg/L for the highest oligomers. A better sensitivity can be attained by the spectrofluorometric detection (277 nm/300 nm), especially with the more modern detectors (about 5- 10 times). The reliability of the determination of low concentrations of NP1 EO and NP2EO is sometimes diminished by the procedural blank (up to 3 µg/L) when they are determined after the Wickbold extraction together with the whole range of the oligomers nEO = 1-18. In order to enable reliable determination of the lowest oligomers it was sometimes necessary to repeat the extract purification. The problem with the remaining coextracted impurities are far less expressed if spectrofluorometric detection is used.

The problems with determination of low concentrations of AP1 EO and AP2EO are completely eliminated with the analytical procedure devoted to the selective determination of the lipophilic APEO and AP (chapter 2.2.2.). The reproducibility of the determination of NP, NP1 EO and NP2EO in river water and stabilized sewage sludge is shown in Table 5. The relative standard deviation obtained for the analysis of river water at fairly low concentrations was small (3.0-4.4 %). Similar values were obtained for the analysis of sewage sludge (3.7-7 %).

~~TABLE 5. Precision of the Determinations of Nonylphenol and Nonylphenol Ethoxylates by Normal-phase HPLC~~

~~Sample NP NP1EO NP2EO~~

~~River water'- mean concentration ( µg/L) 3.9 23.4 9.4 relative slandard devlalion (%) 4.4 3.2 3.0~~

Digested sewage sludgeb mean concenlralion (glkg) 1.61 0.33 n.d. relalive slandard devialion (%) 3.7 7 n.d. a Triplicate analyses of a sample from the Glatt River; no corrections for recovery; b Triplicale analyses of a sample from lhe lrealment plant Glatt of the city of ZOrich; no corrections for recovery; n.d.: not delermined.

~~The detection limit for the lipophilic AP and APEO, based on 2 L sample, is 0.1 µg/L (0.3 nmol/L) for the spectrophotometric detection and about 1O times ------52~~

lower (0.01 µg/L or 0.03 nmol/L) for the spectrofluorometric detection. With gas chromatography, under the same conditions, somewhat lower sensitivity is obtained (0.1-0.5 µg/L. However, this sensitivity is considerably higher than the one obtained by Stephanou and Giger (10 µg/L; ref. 107).

~~For solid samples (1 O g) the limit of detection lower than 0.1 µg/g was achieved.~~

Injecting standard solutions of NP and NPEO into liquid chromatograph it was found that both spectrophotometric and spectrofluorometric detection show a wide range of linear of response (1 ng - 1 µg). The possible variations of the sensitivity are compensated by the application of the internal standard technique during the majority of quantitative determinations.

~~3.1.1.5. Application~~

The examples for the chromatographic determinations of alkylphenol polyethoxylates (nEO = 3-20) in waste and natural waters are given in Figures 9, 10 and 11. By the use of normal-phase HPLC technique (Figures 9 and 1O) the quantitative determination for each specific oligomer was made possible. The reversed-phase HPLC technique (Figure 11 a) can be recommended as a fast and simple method for the determination of the total concentration, unless it is not neccessary to know the distribution of oligomers in the sample.

The reliability of the determination of alkylphenol polyethoxylates in real water samples was proved by the analysis of the extracts with the combination of the preparative HPLC and HAGC techniques (Figure 11 ). Prior to their HRGC analysis, APEO were derivatized from the trimethylsilyl ethers.

The additional identification of NPEO, extracted from waste water, was performed by UV- and IA-spectra. UV-spectra showed maximum at 277 nm and a smaller peak at 283 nm, which corresponds to the characteristics recorded for the pure compounds. IA-spectra contained all the characteristics bands: at 1260 cm-1 for arylethers, at 1115 cm-1 for alkylethers and for the aromatic ring at 1630 and 1515 cm-1. 53

~~A INFLUENT 8 9 10 6 7~~

~~2 B EFFLUENT~~

~~0 6 12 18 24 30 36 ml 0 10. 20 min~~

Figure 9. Normal-phase high-performance liquid chromatograms of NPnEO extracted from a synthetic wastewater (A) and the effluent from a laboratory-scale biological activated sludge treatment (B). Peak numbers refer to numbers of ethoxy units. The HPLC column was 125 mm x 3 mm i.d., 5 µm Spherisorb NH2.

It should be stressed that the developed method is specific for the aromatic nonionic surfactants. Although, nonionic surfactants of aliphatic type (linear alcohol polyethoxylates) are extracted as well by the applied extraction technique, the specific method of detection enables a selective determination of aromatic surfactants. For the determination of AP, AP1 EO and AP2EO. ecotoxicologically important degradation products of the alkylphenol polyethoxylates, in the environmental 54

~~8 A 6 5 7 4 9 10 11~~

~~1 2 B~~

~~2 c~~

~~0 6 12 18 24 30 36 42 ml 0 10 20 30 min~~

Figure 10. Normal-phase high-performance liquid chromatograms of NPnEO extracted from municipal wastewaters and river water: (AJ mechanically treated municipal wastewater; (BJ biologically treated municipal wastewater; {C, DJ water from the Glatt River downstream (CJ and upstream (DJ from the discharge of treated sewage effluents. The wastewater samples are from the sewage treatment plant Glatt of the City of Zurich. Peak numbers refer to the number of ethoxy units. The HPLC column is described in Figure 9. 55

~~8 9~~

Figure 11. Reversed-phase high-performance liquid chromatogram and high- resolution gas chromatogram of the NPEO extracted from raw municipal wastewater. HPLC conditions are similar to those given in Figure 7. Collected HPLC fraction was susequently analysed by HRGC.

samples the most useful method proved to be the method which includes continuous steam distillation/extraction and determination by normal-phase HPLC using the spectrophotometric or the spectrofluorometric detection. This method allows efficient and selective enrichment of the investigated compounds, their specific and sensitive detection has a simplicity which makes it suitable for the analysis of a large number of samples. Chromatograms in Figure 12 and 13 show that the concentration relationships between AP, AP1 EO and AP2EO in various environmental samples is very different, which stresses the need for a method which enables a reliable determination of each of these compounds.

In order to verify the reliability of a simple HPLC method for the AP, AP1 EO, and AP2EO determination the method was compared to the HRGC method with the flame-ionization and the mass-specific detection. The results of the AP and APEO determinations, with the previously mentioned methods, in treated wastewater and river water (Table 6) show to be in agreement. From a practical point of view, the HPLC method was the fastest and the most simple technique. An especially important fact is that the extracts of the different environmental samples can be analysed directly, without any purification. 56

~~TMP RAW TMP ACTIVATED WASTEWATER SLUDGE A D l'f>1EO NP~~

~~TMP f'PIEO TREATED ThP DIGESTED NP ~ WASTE- SLUDGE WATER B E NP2EO~~

~~TMP TMP NP RIVER DIGESTE WATER EFFLUENT c F NP1EO NP2EO NPIEO NP NP2EO~~

~~0 8 16 24mL ·o 8 16 24 ml 0 10 min 0 10 min~~

Figure 12. Normal-phase high-performance liquid chromatograms of steam- distillation extracts obtained from raw wastewater (A), biologically treated wastewater (B), river water (C}, activated sludge (D), digested sludge (E), and di- gester effluent (F). Grab samples were taken from the treatment plant of the City of Zurich. The chromatographic conditions are described in Fig. 6. TMP: 2,4,6,- trimethylphenol (internal standard) 57

~~-TMP -TMP A B c~~

~~NP1EO ( -NP NP1EO ( -NP ....NP2EO~~

~~-TMP -TMP -NP1EO D E TMP F~~

~~,,NP /NP2EO (NP1EO ,...... NP CPIEO,...NP ,.....NP2EO ,...NP2EO~~

~~O 4 8 12 ml 0 4 8 12 ml 0 4 8 12 ml 0 2 4 6min 0 2 4 6 min 0 2 4 6 min~~

Figure 13. Normal-phase high-performance liquid chromattograms of steam- distillation extracts obtained from sediments (A, 0), algae (B, E) and fish (C, F) using spectrophotometric ( A, B, C) and spectrofluorometric detection. The chromatographic conditions are described in Fig. 6. TMP: 2,4,6,-trimethylphenol (internal standard) TABLE 6. Nonylphenol and Nonylphenol Ethoxylates in Treated Wastewaters and in River Water Determined by Different Techniques

~~Concentration (µg/L)a Sample and location NP NP1EO NP2EO~~

~~HPLC HAGC HRGCIMS HPLC HRGC HRGC/MS HPLC HRGC HRGCIMS~~

~~Treated wastewaterb~~

~~Glatt RiverC FlUlanden <0.5 0:5 <0.5 <0.5 1 <0.5 <0.5 <0.5 <0.5 Hagenholz 1 1 1 6.5 6.5 6 3 2 2 ROmlang 2 2 1.5 15 18 16 14 16 11 Rheinsfelden 0.5 1 0.5 6 5 6 7 7 7~~

a No corrections for recovery; b Grab safll)les of the secondary effluents from mechanical biological treatment plants; c Grab samples from points at increasing flow distance from the outflow of the. Greifensee. 59

The specific chromatographic behaviour of NP and NPEO, as well as the selective detection, ~277 nm) contribute to the selectivity. Even greater selectivity is obtained by the spectrofluorimetric detection, which is illustrated in Figure 13. In using the spectrofluorimetric detection a considerably higher sensitivity is obtained, and therefore the method can also be successfully used for the analysis of ground waters. The coextracted components like alkanes, aromatic hydrocarbons, phtalates and fatty acids often hamper HRGC determination of lipophilic APEO if nonselective flame-ionization detection is used. The necessary selectivity is achievable through the application of mass-specific detection, but with loaded extracts there is a great danger of the fast deteriorating of the sensitive capillary columns through the accumulation of the nonvolatile polar substances.

Since octyl and decyl homologues (108, 109) were identified in the environment, besides NPEO, the shortcoming of the normal-phase HPLC method, in comparison with HRGC, is insufficient resolution power, so that neither specific alkyl isomers nor homologues can be specifically determined. However, since the differences in ecotoxicological characteristics between NP and OP homologues and isomers are relatively small (150), and their origin is the same, the mentioned shortcoming is not an obstacle for its appplication. Still, in order to avoid mistakes in interpretation, it is occasionally neccessary to verify the obtained HPLC determinations with the identification techniques (HRGC/MS). If only HPLC is available, the confirmation of the type of alkyl chain in the APEO molecule should be obtained on the basis of the reversed-phase chromatography. Figure 11 shows that the share of OPEO in swiss waters is negligible. Secondly, a disadvantage of the HPLC methods (both normal- and reversed-phase) is caused by their lower separation power compared to the HRGC techniques and there is an increased danger of overlooking the interference of the coextracted unknown compounds. Thus, during the analysis of the stabilized sewage sludge, it was observed that, skatol (3-methylindol) formed as a degradation product of triptophane can interfere with the determination of NP1 EO. In many other examples no serious problems were observed with substances which could interfere during determinations.

The lower resolution, which was mentioned as an drawback of the HPLC determinations, shows from the practical point of view, a certain advantage because of the simple form of the chromatogram. The integration of the peaks and calculation of the concentrations is, in this case, much simpler than with the HRGC determinations. 60

~~3.1.2. Determination of Alkylphenoxy Carboxylic Acida~~

~~3. 1.2. 1. Chromatographic Separation and Identification of Alkylphenoxy Carboxylic Acids~~

By transformation of alcoholic group of APEO into carboxylic group of APEC the characteristics, which determine the chromatographic behaviour of the formed compounds, are changed considerably. While elution of APEO, from the columns filled with aminosilica could be performed with relatively nonpolar mobile phase (chapter 3.1.1.1.), APEC could not be eluted, even with 100 % 2-propanol. The reason for that was probably from a very strong acid-base interaction of the carboxylic group of the APEC with amino group from the stationary phase.

~~Separation of APEC formed by oxidation of the Marlophen 83 was possible with the gas chromatography by applying a high quality persilylated glass capillary~~

~~t 1~1EC I j j I! I I, 1111l1fru I I I I >- ~I I I-u; "''' z w m/e'.22 I- 1 ~ I ·""":;r- I- -~~. z w a: a: m/e'.~ :::> 1 ,., 'EEC () ' I I z Q NPIEC lTIC r----i ¢':. I .~, 20 24 28 32 36 40 44 TIME~~

Figure 14. Total ion current and mass chromatograms of nonylphenoxy carboxylic acids. Chromatograms reconstructed by monitoring the total ion current (TIC) and molecular ions (mlz 278, 322, and 366); column: glass capillary 19 m x 0.3 mm i.d. coated with PS-255 stationary phase; temperature programming 50-280 °C, 4 °C/min. 61

columns with immobiliwd stationary phasG (166) (Figuro 14 ). Tne peal< of AP~C (NP1 EC-NP4EC) are rather deformed, which is the consequence of unsuitability of the nonpolar stationary phase for the determination of these relatively polar compounds. Still, in addition to good separation of the oligomers, specific nonyl isomers were separated as well. It should be pointed out that this technique cannot be recommended for the rutine work because, after only a few injections, an ir- reversible deterioration of the column takes place. However, HRGC separations allowed a more detailed description of mass spectra for the nonmethylated NPEC isomers which were, according to our knowledge, not described in literature.

Mass spectra were recorded for each separated peak. In Figure 15 the chosen characteristic mass spectra of NP1 EC, NP2EC and NP3EC are shown, on the basis of which fragmentation of these compounds in the ion source can be explained. Some basic characteristics of their fragmentation could have been presumed on the basis of knowledge about mass spectrometry of the NPEO (105). The molecular ions of NPEC (m/z 27B+n x 44) are of very low intensity, but are still visible on the spectra and can be successfully applied for identification and re- construction of the mass chromatograms (Figure 14). The base peak of the greatest number of NPEC isomers is formed by a cleavage of the benzylic bond and by the loss of the longer alkyl chain (169). Due to the different chain branching, the mass spectra for the different isomers are (like for NPEO) different (105). The series of ions are formed which can be described by the expressions 165+n x 14 for NP1 EC, 209+n x 14 for NP2EC and 253+n x 14 for NP3EC. The most frequent benzylic ion of the APEC oligomers is m/z (M-85)+, which is formed by the loss of the hexyl chain from the quarternary C-atom. The fragmentation mechanism, which includes the cleavage of both benzylic and ether bond, with the transfer of hydrogen on to the phenolic ion is also important for all NPEC oligomers. By this pattern the series of ions are formed which could be described by the expression m/z 107 +n x 14. Their further fragmentation produces a number of smaller ions including m/z 91 which belongs to the tropilium ion. Along the mentioned mechanisms, which are common to all oligomers, there are some by which specific oligomers are differentiated. Ion m/z 45, with the formula O=C-OH+, is weakly represented in NP1 EC, but with the increase of the ethoxy chain length it becomes more intensive. For NP3EC, it represents the most dominant peak in the mass spectra. Ions m/z 133, 147, 161 and 175 have low intensity, but they indicate fragmentation which includes simultaneous cleavage of 62

~~193 193 100 A NP1EC 100 B NP1EC~~

~~207~~

~~50 50 179~~

~~185 221~~

~~55 249 278 249 278~~

~~50 100 150 200 250 rn'e 50 100 150 200 250 rn'e~~

~~237 100 C NP2EC oo D NP2EC 237 251 107 135 265 135 45 121 50 50 149~~

~~163 193 223 279 322 193 223 251265 295 322 in 201~~

~~50 100 150 200 250 300 rn'e 50 100 150 200 250 300 rn'e~~

~~45 100 E NP3EC 100 F NP3EC~~

~~50 50 237 189 223~~

100 150 200 rn'e 2 309 1: 5 ...... o-__._1 ...so_ ...... __20...... -o-__.[ _~_,.l,__._.._l_~~ 366 ~~~, 1-250( - '·· • • • _-r----' 300 350 400 rn'e r ---...... ,250 300 ...... -...._...- 350 ...... 400 rn'e

Figure 15. Typical mass spectra of isomers of the free nonylphenoxy carboxylic acids. NPt EC: (nonylphenoxy)acetic acids; NP2EC: [(nonylphenoxy)ethoxy]acetic acids; NP3EC: [{nonylphenoxy)dietoxy]acetic acids 63

alkyl and ethoxy carboxylic part of the molecule but in a different manner than in the case of ionic series m/z 107+n x 14. The primary ion, from which the mentioned ions could be formed, is m/z 233 ion which is, despite of a very low intensity, still visible on the mass spectra. The collective empirical formula for the m/z 133+n x 14 series could be probably written as CnH2n-1-C4H5-0+=CH2, but since the mass spectra recorded low resolution conditions, so far we do not posses reliable facts about their elemental composition. An important difference of higher oligomers to NP1 EC is a diagnostically characteristic ion m/z 103 which is formed by the cleavage of the ethoxy carboxylic chain with a formula C2H40+-CH2-COOH. This ion is analogous to the ion m/z 89 by higher APEO (105). The intensity of this fragment also increases sharply towards higher oligomers. A further difference, which is caused by a different structure of the ethoxy carboxylic part of NP1EC molecule and higher oligomers, relates to the loss of a neutral fragment of mass 44 from the primary benzylic ion. In this way diagnostic ions of higher NPEC (m/z 193 and 207 for NP2EC and m/z 223, 237 and 251 for NP3EC) are formed.

On the basis of these considerations it is obvious that the basic mechanisms for APEO and APEC fragmentation are very similar, but the differences between specific oligomers are more pronounced with NPEC.

~~3. 1.2.2. Chromatographic Separation and Identification of Methyl Esters of Alkylphenoxy Carboxy/ic Acids~~

Chromatographic separation of APEC is made considerably easier by their transformation into the methyl esters (APECMe). Oligomers of NPECMe, obtained by oxidation of Mar1ophen 83 (nEO = 1-5) and by methylation of the formed products, can be separated in analogy to the oligomers of NPEO, according to the number of EO moieties in the molecule (Figure 16a) using normal-phase HPLC (aminosilica). With the reversed-phase HPLC (octyl silica) all oligomers are eluted with approximatelly the same retention time (Figure 16b).

Methyl esters of APEC are also very suitable for the HRGC determination of using columns with nonpolar stationary phases. Their retention times are somewhat longer than the retention times of the corresponding APEO. Since transformation into methyl esters the polar group of APEC is blocked, APECMe show very good chromatographic characteristics and it can be applied even to the columns which 64

~~NP2EC NP1EC A NP3EC~~

~~0 4 12 20 28 ml 0 2 6 10 14 min~~

~~~---_}L_-P_1E-C~---8~~-~~

~~NP4EC~~

~~0 3 9 15 21 ml 0 2 6 10 14 min~~

Figure 16. Normal-phase (A) and reversed-phase (B) high-performance liquid chromatograms of methylated nonylphenoxy carboxylic acids. (A) column: Li- chrosorb NH2, 10 µm,250 mm x 4.6 mm i.d.; eluent A: n-hexane, eluent B n-hexane/2-propanol 111, gradient: 2-30 % Bin 30 min, 2 mUmin; (B) column: ODS II 85, 5µm, 250 x 3 mm i.d.; eluent: acetonitrilelwater 812; 1.5 mUmin; NP1EC-NP4EC: (nonylphenoxy)acetic acid to [(nonylphenoxy)triethoxy]acetic acid 65

~~cannot be used any more for the determination of APEO. Besides methylation, the transformation of the APEC into trimethylsilyl esters (111, 137) has been also applied for HRGC determination.~~

The first successful identification of NPEC in the real samples was achieved by HRGC/MS analysis of the previously methylated extract of the secondary effluent from the sewage treatment plant of the town of DObendorf. The corresponding chromatograms reconstructed on the basis of the total ion current and on the basis of selected ions are shown in Figure 17. Two major groups of peaks,

~~E I..,., I~~

~~D 207 ~ l . l"'' I I I .. Il[" c~~

~~i''.NPIEC c16 ~C,18:1 A C4 OP1EC Cl8~~

~~I f I j ~A , I I • .. ,. I I I '1 200 400 600 800 1000 1200 1400 1600 SCAN 4:00 8:00 12:00 16:00 20:00 24:00 28:00 32:00 TIME~~

Figure 17. Total ion current and mass chromatograms of methylated alkylphenoxy carboxylic acids isolated from the secondary effluent of the sewage treatment plant at Dubendorf. Chromatograms reconstructed by monitoring the total ion current (TIC) and characteristic ions (mlz 292, 336, 207, and 117). NP1 EC: (nonylphenoxy)acetic acids; NP2EC: [(nonylphenoxy)ethoxy]acetic acids; OP1 EC: (octylphenoxy)acetic acid; C14, C16, C1B, and C1B:1: common fatty acids. showing a distribution typical for the nonyl isomers of NPEO, occur on the chromatograms (105, 107). These two peak groups were assigned to the compounds with molecular ions at m/z 292 and 336, which correspond to the molecular 66 weights of NP1 ECMe and NP2ECMe, respectively. No higher ollgomers of NPEC were identified in this sample. In some other extracts NP3EC and NP4EC were sometimes detected, but always in considerably lower concentrations than NP1 EC and NP2EC. The extract also contained traces of methyl ester of octylphenoxy acetic acid (OP1 ECMe). The compounds with carboxylated alkyl chain, as suggested by Schober! et al. (122), were not detected. The remaining peaks, which could be observed on the chromatogram around the peak group attributed to NP1 ECMe and marked as C14, C15, C1a and C1a:1. belong to the methyl esters of common fatty acids. Although none of these compounds, which are otherwise quite abundant in waste waters (170), interfere directly in the HRGC determination of NPEC, the identification of specific NP1 ECMe peaks would be insufficiently reliable if it would be based on the total ion current response only. The mass chromatograms reconstructed on the basis of molecular ions of NP1 ECMe and NP2ECMe (Figure 17b and 17c) allow the selective detection of these compounds in the secondary effluent. In the chromatograms about 10 peaks, which certainly belong to NP1 EC Me and NP2ECMe, can be observed. The major part of the identified NPEC belong to the 4-NPEC (p-isomers), but there have also been observed peaks of the same molecular mass which elute with somewhat shorter retention times and could be attributed to a-isomers.

Figure 18 shows typical mass spectra for NP1 ECMe and NP2ECMe isolated from wastewater. On the basis of the shown mass spectra, the main characteristics of the fragmentation of methyl esters of APEC can be discussed. Some important characteristics of the mass spectra for the methylated APEC were described by Reinhard et al. (109) on the basis of mass spectra of OP1 ECMe and OP2ECMe. In comparison to OPECMe, the interpretation of mass spectra for NPECMe is more complicated due to the isomeric structure of the alkyl chain. Although the mass spectra of specific NPECMe isomers can vary considerably (Figure 18), on the basis of the mass spectra only, no complete identification of single isomers can be done. Like nonmetylated NPEC their methyl esters exibit also a very low molecular ion. The formation of ion series at m/z 179+n x 14 for NP1 EC Me and at m/z 223+n x 14 can be explained by the rupture of the benzylic bond with th loss of the longest alkyl moiety (169). The base peak of most NP1 ECMe, m/z 207, which arises from the loss of the hexyl group (M-85+), can be successfully used for the selective detection of NP1 ECMe in real extracts (Figure 17). 67

~~A NP1EC 'JJJ7 C NP2EC 100 100 117~~

~~50 59~~

~~163~~

~~50 100 150 200 279~~

~~193 ~ 91 117 147 179 I 334 I I I I f 50 100 150 200 250 300 'T~ 300 350 400~~

~~100 'JJJ7 100 D NP2EC B NP1EC 117~~

~~50 59 221 149~~

~~50 100 150 193 200~~

~~M+ 307 M+ 292 336 I I f 50 100 150 200 250 300 :i~~

~~161 147 150 200 200~~

~~360 I ·:rI I I I I f 250 r300 350 400 m'e 400 m'e~~

Figure 18. Typical mass spectra of the methyl esters of nonylphenoxy carboxylic acids isolated from a secondary effluent. NP1 EC: (nonylphenoxy)acetic acid; NP2EC: [(nonylphenoxy)ethoxy]acetic acid; NP3EC: [(nonylphenoxy)diethoxy] acetic acid 68

The other ions (m/z 233, 133, 147, 161and91) in the mass spectra of NP1ECMe have low abundances. The small diagnostic ion m/z 233 is probably formed by the loss of a radical O=C'-0-CHa thus corresponding to m/z (M-59)+ and can be represented by the formula C9H19-C5H4-0+=CH2. The fragment ions m/z 133, 147, and 161 represent a homologous series presumably corresponding to CnH2n-1- C5H4-0+=CH2, which arise from further fragmentation of the ion m/z 233. This pattern involves the loss of an alkyl radical (benzylic cleavage) from the alkyl side chain as well as a subsequent loss of an H radical. Further fragmentation of ions mentioned above leads to the formation of ions at m/z 119 and 105 (neutral loss of CO), ions at m/z 117 and 103 (loss of CH2=0), and the fragment at m/z 91.

The main fragmentation path for the isomers of NP2ECMe and higher oligomers differs considerably from the fragmentation of NP1 ECMe. The base peak for all isomers of NP2ECMe is m/z 117, which is produced by cleavage of the aromatic ether bond yielding the fragment C2H40+-CH2-COOCH3. This fragment is analogous to the fragments rn/z 89 of NP2EO and to m/z 103 of NP2EC, and could serve well for the specific detection of the NP2ECMe in the real extracts (Figure 17e). The intensity ratio of ions m/z 59 and m/z (M-59)+ for NP2ECMe is a reverse of that to NP1 ECMe. The ion rn/z 59, which is formed by a-cleavage, is one of the most intensive ions in the mass spectra, while the ion (M-59)+ is very weak. The diagnostic ions rn/z 107, 121, 135, 149 and 163 represent a homologous series of ions (mlz 107+n x 14), and are formed in a complete analogy to the NPEO (105) by the simultaneous cleavage of ether and benzylic bonds with the hydrogen transfer to the phenolic ion.

~~3. 1.2.3. Response Factors~~

By the analysis of the solutions of pure methyl esters of APEC (chapter 2.2.2.) the response factors for the specific oligomers related to 2-tert-butyl-6-methylphenol, which served as the internal standard during HPLC determinations, were determined. The response factor of OP1 ECMe was determined to be related to the nonylbenzene, which was used as the internal standard during the HRGC determinations. The results given in Table 7 show that the stoichiometric dependence of the response factors on the share of the aromatic part of the molecule, which is described for APEO (Table 2) is also valid for APECMe, if the UV-spectrophotometric 69

detection at 277 nm was applied. The mentioned dependence, for the oligomers in the range NP1 ECMe-NP4ECMe, can be described by the expression: RF= 0.155 nEO + 1.525 {r =0.9928). When spectrofluorometric detection (277 nm/300 nm) was used, the direct increase of the response factor with the molecular weight is valid only for oligomers higher than NP1 ECMe. The response factor for NP1 ECMe is 20 times

~~TABLE 7. Response Factors (RF) of Methyl Esters of A/ky/phenoxy Carboxy/ic Acids~~

~~Compound HPLC/UV (2n nm)8 HPLC/Fluor. (2n/300 nm)a HRGC/FIOb~~

~~OP1ECMe 1.6 n.d. 1.7 NP1ECMe 1.7 16.3 n.d. NP2ECMe 1.8 0.7 n.d. NP3ECMe 2.0 0.8 n.d. NP4ECMe 2.15 0.9 n.d.~~

a Related to 2-tett-butyl-4-methylphenol (BMP) as an internal standard; b Related to nonylbenzene as an Internal standard. n.d. : not determined larger than for NP2ECMe. A drastic drop of the fluorescence intensity for NP1 ECMe can be attributed to the effect of the polar carboxylic group in the vicinity of the aromatic ring. In all higher oligomers there is at least one ethoxy group (-CH2-CH2-0-) between the carboxylic group and the benzene ring, and therefore, the influence of carboxylic group is not felt any more. These compounds have intensive fluorescence and are very suitable for spectrofluorometric detection.

The response factor of OP1 ECMe related to nonylbenzene was 1. 7. The response factors for NPECMe oligomers in HRGC determinations were not experimentally determined, but the same value was presumed for them as the one obtained for OP1 ECMe. In fact, in the analogy to APEO, the response factors for the higher oligomers (>NP2ECMe) are probably somewhat higher.

~~3.1.2.4. Extraction, Extract Purification and Derivatization~~

During the preliminary experiments, several methods of extraction of APEC from water (enrichment on anion exchanger; ref. 171; modified method of continuous extraction with simultaneous distillation, ref. .153) were tried, but they had no suc- cess. The good results were obtained by a simple batch extraction in a separatory funnel and by sublation of a water sample with N2 (76). The results on the extrac- 70 tion efficiency from the water samples by the application of th& mentionod teol'lni· ques and using two methods of quantitative determinations (HPLC and HRGC) are shown in Table 8. It is obvious that even with a single extraction of acidified water sample (pH = 2) in a separatory funnel (chloroform to sample ratio 1 :20), high yields {>80 %) were achieved. By the application of a double extraction the quantitative yield of OP1 EC was obtained (106 ± 6 %). Somewhat lower extraction yield (65 %) was obtained by the application of the Wickbold method.

Both methods of extraction and quantitative determination enable a good precision (relative standard deviation 3-5 %). Results on the reproducibility of APEC determinations in river water are given in Table 8. The quantitative determinations. were again performed with two methods (HPLC and HRGC) and it can be seen that with both methods similar results were obtained. It can be concluded that HPLC (RSD = 7.6 %) and HRGC determinations (RSD =6.4 %) show good precision for NPEC determinations in real samples. During the batch extraction of the untreated sewage samples in a separatory funnel, very stable emulsions were formed. These emulsions were destroyed by the addition of a larger amount {20 g) of anhydrous sodium sulphate into the extract itself. Formation of emulsions can be avoided, by the application of the Wickbold method for the extraction.

~~TABLE 8. Accuracy and Precision of the Determination of Alkylphenoxy Carboxylic Acids~~

~~Recoverya~~

~~Compound HPLCb HPLCC HRGCC~~

~~OP1EC 65± 1 83±1 82±3~~

~~Preclslond~~

~~Compound HPLC HRGC~~

NP1EC n.d.9 14.3 ± 0.5 (3.5) NP2EC 12.9 ± 1.0 (7.6) 15.8± 1.0(6.4) a Triplicate determinations; b Extraction by gaseous stripping into ethyl acetate according to Wickbold (17) of bldlstilled water samples (pH 2) spiked with 250 µg/l OP1 EC; c Liquid-liquid extraction of bidlstilled water samples spiked with 100 µg/L OP1 EC; One-lier samples were once extracted in a separatory runnel with 50 ml of chloroform at pH 2; d Triplicate analysis of a water sample from the Chriesbach, a small creek at OObendorf. Extractions were performed by llquld-Hquld partitioning. Concentrations and standard deviations are given in µgll. Relative standard deviations In percent are given in parenthesis; e not detectable. 71

The best method to avoid any emulsion formation would be to use the continuous liquid extraction described by Stephanou and Giger (107). However, since the concentrations of the APEC in untreated waste waters are low, the mentioned extraction apparatus, which usually contains only 250 ml of sample, should be modified for larger samples.

The extraction of the APEC from solid samples was not investigated in detail. The solid samples (sewage sludge) were extracted either in the apparatus by Soxhlet, or in the ultrasonic bath, using methanol, or a mixture of methanol and dichloromethane, as extraction solvent.

The derivatization of free APEC into their methyl esters was performed by the use of two reagents: 1O % solution of BF3 in methanol and 1 N solution of HCI in methanol. Both methylating reagents proved very efficient, but 1 N HCI in methanol was better. The reason was that commercial reagent solution of BF3 in methanol, contained impurities which interfered with NP1 ECMe in HPlC determination. These impurities could not be removed even after the extraction of reagent by n-hexane. After derivatization it is neccessary to extract APECMe from the reaction mixture. Before the extraction it is important to add water into the reaction mixture (0.5- 1 ml into 2 ml of reaction mixture) in order to improve the extraction efficiency. Several solvents for the extraction of APECMe were examined and the results are given below in Table 9.

~~TABLE 9. Extraction Recovery of the Methyl Esters of Alkylphenoxy Carboxylic Acids from the Reaction Mixture after Derivatization~~

~~No of the consecutive Recovery (%)a extraction n-hexane dichloromethane chloroform~~

~~1 80 91 97 2 16 9 3 3 4 <1 <1 a Recoveries are given for three consecutive extractions assuming that 100% of the analytes is recovered after 3 extractions.~~

The single extraction by the chlorinated solvents dichloromethane and chloroform is much more effective (>90 %) than the extraction with n-hexane (80 %). Double extraction with n-hexane yielded over 95 % of APECMe whereas the extraction with a chlorinated solvent gave quantitative recovery. From the practical point of 72

view, hexane has advantages over chlorinated solvents, because the extracts can be injected directly into the HPLC taking an aliquot of the hexane (upper) phase from the bottle inside which both derivatization and extraction were performed. When chlorinated solvents are used, it is neccessary to separate the extract, evaporate the solvent to dryness and to dissolve APECMe in n-hexane. The internal standard 2-tert-butyl-6-methylphenol is added into the final hexane extract, because its reextraction from the reaction mixture is incomplete.

When analysing extracts which are highly loaded with organic substances (nontreated waste water, primary effluents, sewage sludge etc.) the purification using silica column has to be performed before the analysis (chapter 2.2.1.3.). Model experiments with pure solutions of OP1 EC show~d that quantitative yield (97 %) of this compound is obtained in the second (methanolic) fraction by the applied procedure. Possible influence of the coextracted organic matter of the purification procedure on silica was examined by dividing the extract of the secondary effluent in two parts. One part was analysed directly, and the other after the applied purification. The extracts were analysed by HPLC using both UV-spectrophotometric and spectrofluorometric detection (Table 10).

~~TABLE 10. Recovery of the Extract Cleanup Procedure on Si02 Column Used in the Determination of Alkylphenoxy Carboxylic Acids~~

~~HPLC/UV HPLC/Fluor.~~

~~Compound Concentration ( µg/L) Recoverya Concentration (µg/L) Recoverya~~

~~prior after prior after (%) (%) cleanup cleanup cleanup cleanup~~

NP1EC 39 37.5 96 42 37 88 NP2EC 45 47 104 47.5 45 97 a The total extract of a secondary effluent was divided into two parts and analysed both with and without application of cleanup procedure on Si02 column. Recovery was determined as follows: Recovery (%) = (concentration after cleanup) I (concentration prior cleanup) x 100

The concentration of NP2EC was identical for both the procedures, and the concentration of NP1 EC was somewhat lower after the purification. This small difference could be explained by the fact that purification removes certain compounds which interfere in the HPLC determination of the NP1 EC. Moreover, it is often not possible to carry out the determination of the NP1 EC in sewage sludge because of the impurities present which cannot be effectively removed by the applied purification method. 73

The limit of determination depends upon the applied detection method, as well as on the type and size of the sample. Based on the sample of 1 L volume sensitivity is better than 1 µg/L for all of the techniques of quantitative determinations mentioned in this work. Detection limit for the determination of NP2EC, using spectrofluorimetric detection is lower than 0.1 µg/L (0.3 nmol/L).

~~3. 1.2.5. Application~~

Figures 19 and 20 show the results of HPLC and HRGC analysis of nontreated sewage (A), secondary effluent (B) river water downstream from the sewage outlet (C) and upstream from the outlet (D). Both HPLC and HRGC methods could be used successfully for the analysis of secondary effluent and river water. However, the determination of NPEC in untreated sewage was possible only by the HPLC method. The HRGC determination was made quite impossible by the presence of other coextracted matter, in spite of the fact that purification on the silica was performed. The identification and quantitative determination by the HRGC technique was possible only when using mass specific detection (Figure 17) or after the additional purification of the extract by the preparative HPLC, as shown in Figure 21. This comparison of HPLC and HRGC presents a good illustration of the advantages of the selective detection at 277 nm in this type of analyses. Even more selective detection is obtained by the application of the spectrofluorometric detector (277/300 nm). In Figure 22 are shown the HPLC chromatograms for the extracts of river water, primary effluent and sewage sludge with the application of the spectrophotometric and spectrofluorimetric detection. The superior selectivity of the spectrofluorimetric detection is especially obvious when analysing the primary sewage effluent and sewage sludge extracts. Unfortunately, the low fluorescence of NP1 ECMe (high response factor) considerably diminishes the advantages of the spectrofluorimetric detection for this particular oligomer. In further work, a more selective extraction and purification method should be found in order to solve the problems of the determination of AP1 EC in sewage sludge. Beside selectivity, it is important to mention that, from the practical point of view, HPLC determination of the APEC is simpler and faster than HRGC determination. This is especially true because in real extracts mainly lower oligomers of APEC (NP1 EC and NP2EC) are found, while in higher oligomers (nE0>2) either they are detected or are present at very low concentrations. When HPLC analysis is directed only towards the determination of NP1 EC and NP2EC, it is possible to 74

~~NP2EC B~~

~~m ~ ~ ~~ ELUTION TEMPERATURE~~

Figure 19. HRGC!FID determinations of alkylphenoxy carboxylic acids in (A) nontreated sewage, (8) secondary sewage effluent, (C) river water downstream of the secondary effluent outlet, and (0) river water upstream of the secondary effluent outlet: NP1EC: (nonylphenoxy)acetic acids; NP2EC:{(nonylphenoxy)ethoxy] acetic acids; NP3EC: [(nonylphenoxy)diethoxy]acetic acids. Gas chromatographic conditions are similar as in Figure 17. carry out chromatography with isocratic elution and the analysis lasts only 10 min. Since nonyl isomers of each oligomer elute as one peak, the integration and quantitative determination are very simple. This simplicity is partly the result of 75

~~lower resolution in comparison to HRGC method, and at the same time is one of the drawbacks of the HLPC determination. However, due to very similar physico-~~

~~NP2EC I~~

~~D NP2EC ~ 4 12 20 ml I I I I .. 2 6 10 min~~

Figure 20. HPLC determinations of alkylphenoxy carboxylic acids in (A) nontreated sewage, (8) secondary sewage effluent, (C) river water downstream of the secondary effluent outlet, and (D) river water upstream of the secondary effluent outlet: NP1 EC: (nonylphenoxy)acetic acids; NP2EC:[(nonylphenoxy)ethoxy]acetic acids; HPLC conditions: column Lichrosorb NH2, 10. µm, 250 x4.6 mm i.d., eluent: 0.25 % of 2-propanol in n-hexane, 2 mUmin; detection: 277 nm. 76

chemical and ecotoxicological characteristics of isomers, their differentiation, for the routine APEC determination, is not so important. An occasional application of the HRGC method, especially in connection with the mass spectrometry, is important as the control of HPLC method, in order to make a positive identification as well as to exclude the possibility of any overlooked interferences.

~~Detailed considerations about the occurrence and behaviour of APEC in the sewage treatment plants and natural waters are given in other chapters (3.3. and 3.4.).~~

~~w (/) z 0 D.. B (/) w a:~~

~~I I I 150 200 2so •c~~

Figure 21. Combined HPLCIHRGC determination of the alkylphenoxy carboxylic acids isolated from a primary sewage effluent. For HPLC and HRGC conditions see Figures 19 and 20; s.fr.:sampled fraction; NP1EC: (nonylphenoxy) acetic acids; NP2EC:[(nony/phenoxy)ethoxy]acetic acids 77

~~BMP BMP NP1EC A B~~

~~BMP BMP c D NP1EC~~

~~NP2EC~~

~~0 4 12 20 ml 0 4 12 20 ml I I I I JI I I f I ,. 0 2 6 10 min 0 2 6 10 min~~

Figure 22. Normal-phase high-performance liquid chromatograms of the nonylphenoxy carboxylic acids extracted from the river water (A; 8), primary effluent (C, DJ, and activated sewage sludge (E, F). The same extracts were analysed using spectrophotometric (A, C, E) and spectrofluorometric (8, D, F) detection. HPLC conditions are very similar to those in Figure 20. NP1 EC: (nony/phenoxy) acetic acids; NP2EC:[(nonylphenoxy)ethoxy]acetic acids; BMP: 2~tert-butyl-4- methylphenol. 78

~~3.2. Physico-chemical and Biological Behaviour of Alkylphenol Polyethoxylates and Their Degradation Products~~

~~3.2.1. Solubility in Water~~

~~3.2.1.1. Solubility of Pure APEO Oligomers~~

The results of solubility determinations for specific p-APEO oligomers (isolated by preparative normal-phase HPLC) at 20.5 °C expressed in weight (mg/L} and molar units (µmol/L}, are given in Table 11.

~~TABLE 11. Solubility of Pure Oligomers of Alkylphenol Ethoxylates and Alkylphenols in Water (t = 20.5 °C)~~

~~Compound Solubility8~~

~~(mg.IL)b (µmoVL)b n RSD(%)~~

NP 5.43 ± 0.17 24.7 ± 0.77 4 3.1 NP1EO 3.02 ± 0.07 11.4 ± 0.27 5 2.2 NP2EO 3.38 ± 0.12 11.0 ± 0.39 5 3.6 NP3EO 5.88 ± 0.13 16.7 ± 0.34 3 2.2 NP4EO 7.65 ± 0.29 19.3 ± 0.73 3 3.8 NP5EO 9.48 ± 0.49 21.5 ± 1.11 3 5.2

OP 12.6 ± 0.50 61.2 ± 2.43 3 4.0 OP1EO 8.0 ±0.18 32.0 ± 0.72 3 2.2 OP2EO 13.2 ± 0.21 44.9 ± 0.71 3 1.6 OP3EO 18.4 ± 0.55 54.4 ± 1.63 3 3.0 OP4EO 24.5 ± 0.89 64.1 ± 2.33 3 3.6 a Solubility was detellTlined by generation column method; b arithmetic mean and relative standard deviation are given; n: number of determinations; RSD: relative standard deviation(%).

Values reported in Table 11 represent arithmetic mean of 3-5 determinations and show good reproducibility (<5 %}. By the elution of substances from the generation column, the content of the investigated matter on the support material (porous glass) decreases. Some authors suggested that for reliable determination of the solubility the decrease should not be more than 2 % of the substance originally present in the column (172). However, in our investigations it was shown that even the loss of 15 % does not influence the reliability of the determination. This is very important, because it enables a greater number of measurements with the same generation column which was very useful for the so- 79

lubility investigations at various conditions (for example temperature dependence), as well as for generating solutions of constant concentration for the biodegradation experiments. The solubility of NPEO oligomers (nEO = 1-5) ranges between 11.0-21.5 µmol/L (3.0-9.5 mgn), and the solubility of OPEC oligomers (nEO = 1-4) between 32.0- 64.1 µmol/L (8.0-24.5 mgl). Figure 23 shows the relationship between solubility of APEO and the number of EO groups in the molecule. The solubility for both OPEC and NPEO increases linearly with the increase of the number of EO groups with a high degree of correlation {r = 0.9971 and 0.9993). The increase of solubility, due to the addition of one EO group. is 2.6 µmol/L and 10.6 µmol/L for NPEO and OPEC respectively.

~~70 A OPEO o NPEO 60 OP~~

~~:J 50 0 s =10.58 n + 22.4 E r =0.9971 ..3- 40 >- ~ :0 30 ::J 0 (/') NP 20~~

~~S= 2.5Sn +8.95 10 r =0.9993~~

~~0 2 3 4 s Number of EO groups~~

Figure 23. Dependence of the solubility of alkylphenol ethoxylates and alkylphenols in water upon the number of ethoxy units in the molecule; NPEO: nonylphenol ethoxylates; OPEO: octylphenol ethoxylates, NP:nonylphenol; OP: octylphenol

The experimentally determined solubility value for p-NP2EO (11.0 µmof/L) does not fit well into the described relationship and was not included in the calculation of the linear regression. The solubility value for p-NP2EO, which follows the re- 80 gression equation, would be 14.0 µmol/L. Since such a behaviour was not observed with OP2EO the observed deviation reflects very probably an error during the determination (for example the influence of impurities which were still present in preparatively isolated p-NP2EO), rather than an unusual behaviour of this compound.

The solubilities of p-NP (24.7 µmol/L or 5.4 mg/L) and of p-OP (61.2 µmol/L or 12.6 mg/L) are much higher than the solubilities of the corresponding AP1 EO. The effect of the decreased solubility of AP1 EO versus AP, in spite of the fact that AP1 EO molecule is larger for one polar (EO) group, should be attributed to the different behaviour of the OH group of alkylphenol and alcoholic group of AP1EO.

Solubility of OP and OPEO is much higher than the solubility of their nonyl homologues, which shows the influence of the additional methylene group on the hydrophobicity of these compounds. The increase of solubility due to the shortening of the alkyl chain for one methylene group is considerably larger than the increase of the solubility due to the extending of the hydrophilic part of the molecule for one EO group. For example, the solubility difference between OP1EO and NP1EO in 20.6 µmol/L and between OP1EO and OP2EO is 12.9 µmol/L. Moreover, it can be seen in Figure 23 that the straight lines repre- senting the dependence of the solubility for OPEO and NPEO on the number of EO groups are not parallel. Thus, increase of solubility, due to the addition of one EO group is 2.6 µmol/L and 10.6 µmol/L for NPEO and OPEO respectively.

It is obvious that additional EO group influences the solubility more when the alkylphenol moiety is more hydrophilic. Therefore, it could be expected that for lower homologues the correlation straight-lines would be even steeper than for OPEO. Although the solubility ratios between OP- and NP-homologues, in the investigated range of the oligomers, are not constant, they are around the value of 3 which is a typical value due to the influence of an additional methylene group on the hydrophobicity of chemical substances (43).

~~3.2.1.2. Dependence of Solubility upon Temperature~~

The influence of temperature on the solubility of NP and NPEO was investigated in the range which is of interest for the investigations of natural waters (2-25 °C). The results are shown in Table 12. 81

~~It can be dearly seen from Figure 24 that for both NP and NP1 EQ solubility increases towards higher temperatures, although the increase is faster for NP.~~

~~TABLE 12. Solubility of Nonylphenol and Nonylphenol Ethoxylates in Water at Different Temperatures~~

~~Tempera -tu rec Solubilitya~~

~~(oC) (mg/L)b (µmoVL)b NP NP1EO NP2EO NP NP1EO NP2EO~~

2 4.60 ± 0.10 2.77 ± 0.02 3.64 ± 0.18 20.9 ± 0.45 10.5 ± 0.08 11.8 ±0.58 10 4.92 ± 0.31 2.81±0.08 3.38 ± 0.09 22.3 ± 1.41 10.6 ± 0.03 10.8 ± 0.56 14 5.24± 0.11 2.84 ± 0.10 3.00 ±0.25 23.8 ± 0.86 10.8 ± 0.38 9.7 ±0.81 20.5 5.43 ± 0.17 3.02 ± 0.07 3.38 ± 0.12 24.7 ± 0.77 11.4 ± 0.27 11.0 ± 0.36 25 6.35 ± 0.10 3.37 ± 0.06 3.69 ± 0.15 28.9 ± 0.45 12.3 ± 0.23 12.0 ± 0.49

~~a Determined using generation column method; b Arithmetic mean and standard deviation are given {n-3-5); c Accuracy± 0.5 °C.~~

~~30 o NP fl NP1EO v 25 NP2EO~~

~~::J 0 20 -E 2- >- ~ 15~~

~~:.a::I 0 (/) 10~~

~~o1...-~---!5~~--:'10=--~--:,5:--~-:2~0~~~2~5-~~

~~Temperature (°C)~~

~~Figure 24. Dependence of the solubility of nonylphenol ethoxylates (NP1 EO, NP2EO) and of nonylphenol (NP) in water upon temperature. 82~~

The solubility of NP at 25 °C (28.9 µmol/L) is 1.4 times higher than at 2 °C (20.9 µmol/L), while the increase for NP1 EO is only 1.2 times (12.8 versus 10.5 µmol/L). For NP2EO the unusual behaviour was observed, so that the lowest solubility was at 14 °C (9.7 µmol/L). The solubility increased towards lower and higher temperatures and at 2 °C it was 11.8 µmol/L, while at 25 °C it was 12.0 µmol/L. The ratio of the lowest and highest solubility, in the observed temperature range, was only 1.2, the same as with NP1 EO. Although it is usual for the solubility of the hydrophobic molecules to increase with the increase of the temperature, in accordance with the endothermic reaction of solubilization for such substances {43), the similar behaviour as for NP2EO was observed also for some polycyclic aromatic hydrocarbons (173).

~~It can be concluded that the variations of temperatures (summer-winter), which are characteristic for natural waters, will relatively little influence solubility of APEO, and somewhat more that of AP.~~

~~3.2.1.3. Solubility of APEO in Mixtures~~

It should be stressed that all solubility determinations were performed with the compounds which were collected by the preparative separation of specific oligomers from the commercial mixtures of the APEO by normal-phase liquid chromatography. However, completely pure compounds were not collected, because each fraction, especially in the case of NPEO, contains a number of isomers which have a differently branched alkyl chain. Therefore, measured solubility values for NP homologues cannot be considered, in a strict sense, as physico-chemical parameters of the defined chemical compounds. Still, it can be presumed that the variations due to the different chain branching are small in comparison with the contribution of EO group and that the determined solubilities represent a good average value for all isomers.

Solubility of the mixture of the APEO oligomers depends, presuming an ideal behaviour, on the composition of the mixture (molar fraction of specific components) and on the solubility of each oligomer as a pure substance {174), which is described by the expression:

~~(12) 83~~

where Ss is the solubility of APEO mixture (µmol/L), and Xn and Sn are the molar fraction in the original mixture and the solubility of the n-th component. The concentration ratio of the specific components in the solution is changed, in comparison with the original mixture, in proportion to their solubilities as pure substances.

Table 13 gives the results for the determination of the solubilities of two commercial mixtures, Marlophen 83 and lmbetin N/7A, which are obtained either by direct measurements or are calculated on the basis of the known solubilities of pure oligomers '(Table 11) based on the equation (12). The concentrations of single oligomers in the solution of mixture are much lower than their solubilities as pure substances. Similar observations were given by some other authors (175) concerning the complex mixtures of the polychlorinated biphenyls. However, while the mentioned authors claim that the original composition of the mixture does not change through solubilization and transfer into the water phase, it can be seen from Table 13 that the composition of the APEO oligomers significantly changes so as to increase the share of the more soluble higher oligomers.

~~TABLE 13. Solubility of Mixtures of Nonylphenol Ethoxylates~~

~~Concentration (µmoVL)~~

~~Compound Marlophen 53a lmbetin N/7Ab exp. calc. exp. exp. calc.~~

~~1. NP1EO 2.1 2.0 8.0 7.6 8.6 2. NP2EO 5.0 5.6 2.4 2.5 2.8 3. NP3EO 4.7 4.6 0.5 0.7 0.8 4. NP4EO 2.1 2.7 n.d. n.d. n.d.~~

Sum 1-4: 13.9 14.9 10.9 10.8 12.2 a mol fractions of oligomers in the original mixture: NP1EO =0.174; NP2EO = 0.401; NP3EO = 0.281; NP4EO = 0.142; b mol fractions of oligomer in the original mixture: NP1 EO = o. 75; NP2EO ~ 0.20; NP3EO • 0.05; c Determined using generation column method; d Determined using slow stirring method; 8 Calculated from the expression c = l: Xi Si ,where Xi is the mol fraction of single oligomer in the original mixture and Si water solubility of the pure oligomer (Table 11 ).

A very good agreement between the experimental and calculated values was obtained. The experimental values are somewhat lower, which could be explained by the fact that only p-oligomers were considered. If the concentration is 84 corrected by 1o % (the approximate contribution of o-isomers) then excellent agreement is obtained for both the total concentrations and the concentrations of single oligomers. It was shown that the mixtures of NPEO behave as ideal solutions and, on the basis of the known solubilities for the individual oligomers, the solubility and the mixture composition in the dissolved phase can be obtained. This indicates that the simultaneous presence of the different oligomers does not cause spontaneous formation of aggregates (micelles) as was pressumed by some authors concerning polychlorinated biphenyls (175). They are formed only after the introduction of (mechanical) energy into the heterogeneous system which is in equilibrium. This conclusion is, however, valid for the range of the oligomers investigated in this work (nE0<5). For higher oligomers (nE0>5) the deviations from the observed ideal behaviour could not be excluded, because they have a considerably higher surface activity and therefore can possibly increase the solubility of the substances co-present in the solution.

~~3.2.2. Partition between Water and Organic Solvents~~

The partition coefficients for AP and lipophilic APEO were determined for systems octanol/water (Kow) and n-hexane/water (Khw) by the direct method of shaking the organic solvent with water (159). The values of partition coefficients for NP (log Kow = 4.5) and OP (log Kow = 4.1) presented in Table 14 are somewhat higher than the literature values (150).

~~TABLE 14. Partition Coefficients of Alkylphenols and Alkylphenol Ethoxylates between Water and Organic Solvents~~

~~Compound log Kawa log Khw log Kowc~~

~~NP 4.48 ± 0.12 3.7±0.04 4.2 NP1EO 4.17±0.15 3.4 NP2EO 4.21 ± 0.18 3.3 NP3EO 4.20 ± 0.11 OP 4.12±0.10 3.0 ±0.04 3.7~~

~~Kow: Partition coefficient between n-octanol and water; Khw: Partilion coefficient between n-hexane and water; a Arithmetic mean and standard deviation; c Literature values (ref. 150).~~

The partition coefficients of NP1 EO and NP2EO are almost identical (log Kow = 4.2 and log Khw = 3.3-3.4), which proves that the additional ethoxy group had a small influence on this physico-chemical property. However, log Kow and log 85

Khw values obtained for these compounds are lower (0.3 units) than the partition coefficients of NP. This was unexpected considering the greater solubility of NP. Namely, it is expected that the partition coefficients should be in a negative correlation with the solubility in water (179, 180). The estimation of the partition coefficients of OP and NP by reversed-phase liquid chromatography (176· 178) is shown in Figure 25.

~~__ .!_o~-~o~_N!_~~~------6 __ .!_Ojl~.Q'!!~~:_S.:.5 ______---- ___ _~~

~~! log K w=3.111 log RT'+ 1.4 I 2 0 I r :0.9992 I I I I I I~~

~~logRT'OP 1 1ogRTNP 1 \i 1 \if 0'-----02'----~0A.._ __~n~s---='os-=---~1n:---- 71.2::-'...._71A:----~1£::-'..... log RT' (min)~~

Figure 25. Estimation of partition coefficients between n-octanol and water (Kow) for long chain alkylphenols using reversed-phase high-performance liquid chromatography. NP:nonylphenol; OP: octylphenol; P: phenol; oC: 2-methylphe· no/; EtP: 4-ethylphenol; TMP: 2,4,6-trimethylphenol; tBP: 4-tert-butylphenol; HPLC column: Spherisorb S5, ODS II, 5 µm, 100 x 4 mm; eluent: methanol/water (70130).

High correlation was obtained (log Kow == 3.14 log Rr + 1.4; r =0.9992) between the retention time (RT') and the value of the partition coefficients (Kow) for the lower alkylphenols which were used for calibration. If it ls presumed that the ob· tained correlation is also valid for the higher alkylphenols, the logarithms of the partition coefficients would be 5.5 for OP and 6.3 for NP, which in comparison with direct determinations (Table 14) the values are too high. Moreover, since in the mentioned chromatographic system all APEOs elute in the same retention 86 time together with the corresponding AP (chapter 3.1.1.) it should be concluded that they have the same partition coefficients. However, due to large difference in the solubilities of specific oligomers (chapter 3.2.1.) that assumption seems to be very unlikely. Furthermore, it was observed that during the chromatography of APEO, with the reversed-phase system, the interaction with free silanol groups can occur, and in some cases the elution sequence for the APEO oligomers was obtained which was contrary to the expected one (Figure 8, chapter 3.1.1.). This indicates that the applied system is not quite suitable for the estimation of the partition coefficients of more polar substances like APEO.

In this work the partition coefficients of higher oligomers of APEO, were not experimentally determined, because none of the used techniques was quite appli- cable. Certain comments about the non-applicability of the reversed-phase liquid chromatography were already made. The direct method, which involves vigorous shaking of the organic solvent with water, is not suitable for the substances which tend to form micelles or foam (159). The best method would be long equilibration on two phases with very careful mixing. Due to the lack of the experimental determination, an attempt was done to estimate partition coefficients for higher oligomers on the basis of certain correlations from the literature.

~~For OPEO (45) the following dependence of the partition coefficients on the number of hydrophilic (EO) groups in the isooctane/water (Kiow) system was described:~~

~~log Kiow = -0.442 nEO + 3.836 (6)~~

~~Presuming that log Kiow changes, with the addition of a methylene group, approximately for 0.5 units (43), the expression is obtained which describes the same dependence for NPnEO:~~

~~log Kiow = -0.442 nEO + 4.3 (13)~~

~~The equation (6) and (13) are valid for the isooctane/water system. Using the relationship between partition coefficients in hydrocarbon solvent and octanol (181):~~

~~log Kow = 0.78 log Khw + 0.59 (14) they can be transferred into the form which is valid for the octanol/water system: 87~~

~~log Kow = -0.34 nEO + 4.1 for NPEO (15) log Kow = -0.34 nEO + 3.6 for OPEO (16)~~

~~The partition coefficients can be also estiamted from the solubility values according to the expression (182):~~

~~log K0 w = -0.747 log S + 0.73 (17) where S is the solubility of a specific oligomer.~~

According to the equations (15), (16) sand (17) the values of log Kow were calculated for the oligomers of the APEO in the range nEO = 0-10 (Table 15). The solubility values, needed for the calculation of the partition coefficients according to the equation (17) were taken from Table 11 (chapter 3.2.1.). For the alkylphenols, relatively similar values were obtained to those directly measured (Table 14), and somewhat better agreements were obtained using the partition coefficient/solubility relationship (17). The same was observed for lipophilic APEO except that the difference between the two calculation methods was much more pronounced. The range of log Kow values calculated from the dependence upon the number of EO groups for NPnEO (nEO = 1-10) is 0.7-3.76, and on the

~~TABLE 15. Estimation of the Octanol/Water Partition Coefficients for Alkylpheno/ Polyethoxylates~~

~~No of EO groups OPnEO NPnEO per molecule Method 1a Method 2b Method 1a Method 2b~~

0 3.6 3.86 4.1 4.17 1 3.26 4.08 3.76 4.41 2 2.92 3.98 3.42 4.35 3 2.58 3.92 3.08 4.29 4 2.24 3.86 2.74 4.25 5 1.90 3.81 2.40 4.21 6 1.56 3.77 2.06 4.17 7 1.22 3.73 1.72 4.14 8 0.88 3.70 1.38 4.11 9 0.54 3.67 1.04 4.08 10 0.20 3.64 0.70 4.06

a Calculated according to the expressions: log Kaw= -0.34 nEO + 3.6 (OPnEO) log Kaw a -0.34 nEO + 4.1 (NPnEO); b Calculated from the solubility using expression: log Kaw• -0.747 log S + 0.73; solubility values are taken from the Table 11 and Figure 23. 88

basis of correlation with the solubility is only 4.06-4.4. The acceptance of one or the other estimation of the partition coefficients of the NPEO leads to the completely different conclusions about the behaviour of higher APEO oligomers in aqueous systems. If the estimation based on the expressions (15) and (16) is

accepted, the partition coefficients for the oligomers with 10 EO groups (log K0 w = 0.2 and 0.7) would indicate very hydrophilic molecules. However, the estimation based on the solubility (log Kow = 3.64 and 4.08) indicates that, in spite of a considerable share of the hydrophilic groups, the basic lipophilic character of these compounds was preserved. Although expressions for the calculation of the partition coefficients contain many approximations which could lead to considerable inaccuracies, the observed differences are so pronounced that they can hardly be explained in this way.

It is interesting to mention that the similarity of the partition coefficients, estimated on the solubility basis, can explain the coelution of all the APEO oligomers (nEO = 1-20) in the reversed-phase system. Fast decrease of the partition coefficients as calculated in the dependence on the increased number of EO groups is perhaps the result of the fact that the mentioned relationships was originally determined in the hydrocarbon solvent (isooctane). Namely, in this solvent, solubility decreases considerably with the increase of the number of EO groups and that could be one of the reasons for obtaining lower partition coefficients for higher oligomers (181 ). This is also indicated by the data given in Table 14 where, even for lower oligomers, the difference between log Kaw and log Khw is 0.8-1.1 units. Their observations should be, however, proved by the direct measurements, which would include higher oligomers for which the two mentioned esti- mations of the partition coefficients vary considerably. 89

~~3.2.3. Biological Degradation~~

~~3.2.3.1. Biological Degradation of Alkylphenol Polyethoxylates~~

The degradation of commercial alkylphenol polyethoxylates, with an average number of EO groups of 1O and with different length of alkyl chain (Cs and Cg) was investigated using standard biodegradation apparatus according to the OECD coupled unit test for the simulating aerobic sewage treatment (160). The results of the analysis of the synthetic wastewater (influent) and effluent from the apparatus are given in Table 16.

~~TABLE 16. Biodegradation of Alkylphenol Polyethoxylates in the Confirmatory Test after OECD (160)~~

~~Concentration (mg/L)~~

~~Noo!EO Synperonic OP10 Marlophen 810 groups synthetic effluent sludge a synthetic effluent sludge a sewage sewage~~

1 0.25 0.3 6.29 0.16 0.13 1.1 2 0.20 2.45 23.4 0.13 0.59 4.4 3 0.29 <0.02b n.d. 0.27 0.21 n.d. 4 0.63 0.50 <0.02b 5 1.20 0.81 6 1.94 1.10 7 2.63 1.44 8 3.15 1.93 9 3.32 2.11 10 3.10 2.41 11 2.64 2.35 12 2.08 2.10 13 1.41 1.69 14 0.97 1.34 15 0.58 0.88 16 0.40 0.48 17 0.12 0.24 a concentration, µgig; b concentration of all higher isomers is lower than the actual detection limit (0.02 mg/L); n.d.: not determined

It can be observed that higher oligomers of APEO (nE0>3) were very efficiently (>95 %) removed from the original mixture, and only AP1 EO and AP2EO remained (Figure 9). Fully deetoxylated products, alkylphenols, could not be identified and very probably were not formed at all under the applied conditions. The concentration of NP2EO in effluent is 10 time larger than in the influent solution, 90

which shows that this oligomers is not only resistant to biodegradation but is actually formed by biodegradation of higher NPEO. It is interesting that the dominant lipophilic degradation product of APnEO is AP2EO. AP1 EO, which was present in influents at similar concentration as AP2EO was found in effluents at several times lower concentration than AP2EO. Alkylphenoxy carboxylic acids were also identified in effluents but no quantitative determinations were undertaken in the described experiments. In addition to the high concentrations in the effluent significant amounts of lipophilic APEO were adsorbed on the sewage sludge (Table 16). It can be concluded that higher oligomers can be very successfully eliminated by biodegradation. However, during that process some stable degradation products are formed. The same results were obtained in model experiments by some other authors (135-137), but on the basis of our results it is for the first time possible to make these conclusions based on the specific quantitative measurements of the single oligomers in a wide range (nEO = 1-18).

~~3.2.3.2. Biodegradation of APEO and AP in Synthetic Sewage Using Shake Culture Test~~

It is obvious that the main problem concerning the biodegradability of alkylphenol polyethoxylates consists in the persistence of their degradation products, but there is almost no information in the literature about this issue. The knowledge on the persistence of the lipophilic metabolites is especially important for the assessment of ecological acceptability, because they are much more toxic than the original APnEO (107).

The biodegradation of NP1 EO and NP2EO (added in the form of commercial mixture lmbetin N/7A) in the synthetic sewage using shake culture test is shown in Figure 26a. Degradation started very soon and 50 % of the degradation was achieved after 3-5 days, depending on the culture used as inocculum. After 8 days the remaining concentration of the NP1 EO and NP2EO was 5-19 %, and after 15 days only traces could be detected. The bacterial cultures from the river water and wastewater showed similar degradation curves, while the culture isolated from soil proved somewhat less effective. Degradation was the fastest in a sample containing only spontaneous bacterial culture which was formed during the preparation and standing of the solution of lmbetin N/7 A. Obviously, the spontaneous culture was best adapted to the degradation of NP1 EO and NP2EO. The other microbial cultures were, namely, isolated and kept in the 91

~~A NP1EO NP2EO~~

~~c 0 :; 60 ~ c -~ 40 c 0 u 20~~

~~0 I 2 3 4 5 6 7 8 9 W IB Time (days>~~

~~NPlEO B NP2EO~~

~~c 0 60 :; ~ 'E 40 ~ c 0 20 u~~

~~L..1'--'2--'-3-'-,-'-s-6..__?.___.8~1->...:....:;.:;!!1!1.21--- o·L-~1~2..._.3_....__.__._~::.i<..~~~~-- Tlme (days> (days>~~

Figure 26. Biodegradation of lipophilic nonylphenol ethoxylates in synthetic sewage using shake culture test. lnoculum: (x) no inoculum added (spontaneous bacterial culture); (o) bacterial culture from river water; (Li} bacterial culture from the wastewater from a detergent manufacturing chemical plant; (0) bacterial culture from a forest soil;(•) sterile control (500 mg/L HgCl2added); Initial number of bacteria: (A) 1()6/mL, (B) 104/mL; NP1EO: nonylpheno/ monoethoxylate; NP2EO: nonylphenol diethoxylate; presence of alkylphenol polyethoxylates and were not as successful in the degradation of AP1 EO and AP2EO. The initial number of bacteria in the NPEO solutions used for the substrate preparation (spontaneous culture) in the first experiment, was rather high (106/ml), which resulted in a short lag phase (less 92

than 1 day). In an identical experiment, when the initial number of the bacteria in the spontaneous culture was smaller (104/ml), degradation started only after 24- 48 h (Figure 26b). The relations between the degradation curves for different bacterial cultures in this experiment were also very similar to those described in Figure 26a. The remaining concentration after 8 days (end of experimental phase) was 1-25 %, with NP2EO having slightly lower values than NP1 EO.

Figure 27 shows the degradation of the octylphenol ethoxylates (nEO = 1-3) in synthetic sewage, also with the application of the shake culture method. On the basis of comparison with the results shown in Figure 26b, it can be concluded that there was no significant difference in the degradation rate of the octyl and nonyl homologes when the same bacterial cultures are used; after eight days the remaining concentration of OPEO was 1-20 %, and it can be seen that the degradation efficiency slowly decreases from OP3EO towards OP1 EO. This is especially apparent in the experiment with the bacterial culture isolated from the soil: the degradation of OP3EO was similarly to the other bacterial cultures, already with OP2EO lagging is visible whereas complete stagnation in the OP1 EO elimination is observed between the second and the fifth day. This could be explained by assuming that a bacterial culture tries first to degrade the easier degradable OP3EO and OP2EO, and only then OP1 EO. Besides that, it is possible that OP1 EO can be formed from higher oligomers. The degradation of NP by the shake culture method (Figure 28) has shown that this compound behaves differently from NP1 EO and NP2EO. The remaining concentration in the flasks with any of the applied bacterial cultures was higher than 90 % after 4 days, and after 10 days higher than 60 %. After 45 days the remaining concentration was less than 50 %, but was still quite high (25-41 %). It is important to observe that the same concentration decrease was noticed in the bottles to which mercury(ll) chloride was added, and therefore it can be concluded that the concentration decrease was not the consequence of biodegradation but from the abiotic elimination. This was also proven by the determination of the number of bacteria that were not present in the bottle to which mercury(ll) chloride was added. Nonylphenol is a very lipophilic compound and can be adsorbed on the container walls. By washing the walls of the flaks which contained the substrate with n-hexane it was determined that about 10-20 % of the totally present NP was in the adsorbed state. Besides that, it was noticed that adsorption was stronger in the sterile culture than in the· bottles containing greater number of bacteria; Abiotic decrease of NP concentration could be the consequence of evaporation from the bottles which were plugged with cotton plugs. It was determined that in our experiment 93

~~OPlEO OP2EO 100 ~ 80 c 0 :;::: ...Ill 60 c -Q) 0 c 40 0 () 20~~

~~0 I 2 3 4 5 6 7 8 I 2 F1789l023 Time Time (daysl~~

~~OP3EO Bacterial culture: ( x l spontaneous (o) from river water (Al from waste water (o) from forest soil ( •) sterile~~

Figure 27. Biodegradation of lipophilic octylphenol ethoxylates in synthetic sewage using shake culture test. Conditions are identical with those in Figure 268; OP1 EO: octylphenol monoethoxylate; OP2EO: octylphenol diethoxylate; OP3EO: octylphenol triethoxylate evaporation of water with the rate of 1-2 mUday occured. As it is known NP dis- tils with water vapour (chapter 3.1.1.), this type of elimination could represent one of the possible explanations. Photochemical degradation could be excluded because the experiments were performed in the dark. If abiotic elimination is taken into consideration, on the basis of the performed experiment it can be concluded that the degradation of NP, in the shake culture test, did not start even after 50 days. 94

~~NP NP 1.5mg/L 100~~

~~~BO BO~~

~~§ 60 60 ~ ~ 1.0 1.0 0 c: 0 0 20 20~~

~~o'--~1_,_2___.3__,1,.__~5~10__..~___.30~w'---w_._~w- 0 .__~,_,_2__.3~",___~s~"~1b----'~'--~~"~o__._sb__ • Time (days) Time (days)~~

Figure 28. Biodegradation of nony/phenol in synthetic sewage using shake culture test: (x) no inoculum added (spontaneous bacterial culture); (.1) bacterial culture from the wastewater from a detergent manufacturing chemical plant; (•) sterile control (500 mg/L HgC/2 added)

~~3.2.3.3. Biodegradation of APEO and AP in Mineral Medium Using Shake Culture Test~~

~~In order to investigate whether APEO can be used as the sole source of organic carbon, the experiments should be performed in completely mineral medium (according Horvath and Koft}.~~

The degradation curves of the NP1 EO and NP2EO are shown in Figure 29. The applied bacterial cultures were identical to those from the experiments with the synthetic sewage. Very similar kinetics of the degradation were obtained with the spontaneous bacterial culture and the bacterial cultures isolated from the wastewater and river water, while cultures isolated from the soil degraded the investigated compounds significantly slower. For more effective cultures 50 % degradability was achieved after 8-11 days, while the culture from the soil degradation was, even after 18 days, smaller than 50 %. Very similar results to those for the NPEO were obtained for OPEO (Figure 30). The lowest efficiency was obtained by the bacterial culture isolated from the soil (Figure 30) whereas all the other bacterial cultures have shown similar kinetics of degradation. The 95

~~degradation of OPEO was somewhat faster than that of the NPEO (Figure 29); 50 % degradation of the OPEO was reached after 6-9 days.~~

~~NP!EO~~

~~#. 80 # 80 -c: -c: .2 60 .2 60 ...«I -...... «I ...... i 40 ~ 40 0 0 c: c: 0 0 0 20 0 20~~

~~0 0 2 4 6 8 10 l2 l4 16 18 20 2 4 6 8 10 12 14 16 18 20 Time (days> Time (days>~~

Figure 29. Biodegradation of /ipophilic nonylphenol ethoxylates in mineral medium using shake culture test. lnoculum: (x) no inoculum added (spontaneous bacterial culture); (o) bacterial culture from river water; (.1) bacterial culture from the wastewater from a detergent manufacturing chemical plant; (0) bacterial culture from a forest soil; (e) sterile control (500 mg/L HgC/2 added); NP1 EO: nonylphenol monoethoxylate; NP2EO: nonylphenol diethoxylate;

According to the presented results it can be concluded that the applied bacterial cultures can use APEO as the sole source of organic carbon. However, the kinetics of such biodegradation is much slower than the degradation in synthetic sewage.

The experiments with the degradation of nonylphenol in the mineral medium have shown that after a longer time the same elimination of this compound takes place (Figure 31 ). However, this elimination cannot be attributed to the biological degradation, because the same elimination was observed in the bottles containing mercury(ll) chloride (sterile control). Namely, in the experiments with the mineral medium the number of bacteria per ml was much smaller than in the experiments with the synthetic sewage and consequently the contribution of the adsorption of NP on the flask walls was more pronounced. In the analogy with the discussion concerning the experiments with the synthetic sewage there is no 96 unambiguous indication that the investigated bacterial cultures have capability for the biological elimination (transformation) of NP in the mineral medium.

~~~ 80 ~ 60 c c .2 60 .2 60 iii.. iii.. 40 cQ) 5i 40 (.) -(.) c c 8 20 8 20~~

~~0 0 '---'2'--,'--'s'--'s--'"10-'-12_,_~_._~_._m_2~0- 2 , s e 10 12 " 1s 18 20 Time Cdaysl Time (days> OP3EO~~

~~Bacterial culture: (x l spontaneous~~

~~0''--~2-,...._s...._e,__10,__12'--'"'-"'-'~---"'s'--'20~ Time Cdaysl~~

Figure 30. Biodegradation of Jipophi/ic octylphenol ethoxylates in mineral medium using shake culture test: {x) no inoculum added (spontaneous bacterial culture); (o) bacterial culture from river water; (Li) bacterial culture from the wastewater from a detergent manufacturing chemical plant; (0) bacterial culture from a forest soil;(•) sterile control (500 mg!L HgCl2 added); OPtEO: octylphenol monoethoxylate; OP2EO: octylphenol diethoxylate; OP3EO: octylphenol triethoxylate 97

~~NP 100~~

~~-;.~~

~~c 0 :;::: Ill... c CD 40 -(.) c 0 0 20~~

~~0 2 4 6 8 10 12 14 16 18 20 Time~~

Figure 31. Biodegradation of nonylphenol in mineral medium using shake culture test: (x) no inoculum added (spontaneous bacterial culture); (o) bacterial culture from river water; ('1) bacterial culture from the wastewater from a detergent manufacturing chemical plant; (0) bacterial culture from a forest soil; (•) sterile control (500 mg/L HgCl2 added);

~~3.2.3.4. Biodegradation of APEO and AP in River Water Test~~

In order to have a better assessment of the APEO and AP degradation in natural waters, their degradation was examined by the river water test (static) and the modified river water test (with stirring). It can be seen in Figure 32 that at higher concentrations (NP = 500 µg/L; NP1 EO = 800 µg/L; NP2EO = 300 µg/L) a relatively effective elimination of NP1 EO and NP2EO occurs, while the elimination of NP was much slower. Already after 2 days 50 % elimination of the NP1 EO and NP2EO was reached, and at the same time the elimination of the NP did not even start; after 9 days the remaining concentration of the NPEO was only 12 % of the initial one, compared to 68 % for the NP. In the experiment with stirring (modified river water test) the elimination for all investigated compounds was considerably faster. Already after 3 days the remaining concentration of the NPEO was lower than 3 %. This could be explained by the fact that stirring accelerates the exchange of matter in the solution. On the basis of previous results using shake culture test the observed elimination of the NPEO can 98

~~be considered as a result of biodegradation, but for the NP it is not definitely clear whether it was biological or abiotic elimination.~~

~~100 o NP ti NPlEO A 80 ~0 v NP2EO~~

~~c: 0 :;:; 60 cu ...... c: CD (.') 40 c: 0 () 20~~

~~0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (days> 100 o NP ti NPlEO 8~~

~~~0 v NP2EO~~

~~c: .Q..... 60 cu ...... c: CD (.') 40 c: 0 () 20~~

~~0~-'-~~~===:!!!!!!!!~:;;;;;;;:;:=.,...t._..- 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (days>~~

~~Figure 32. Biodegradation of nonylphenol (NP), nonylphenol mono- (NP1 EO), and diethoxylate (NP2EO) in the river water test: (A) static, (B) with stirring; initial concentration about 1 mg/L.~~

Biodegradation of NP and NPEO. at the concentrations similar to those in the polluted natural waters was also examined. For the initial sample the secondary effluent of the sewage treatment plant was taken (Figure 33). The experiment was performed at two different temperatures (4 and 20 °C). A sample, to which 99

~~4 ° C. l°lo HCHO~~

~~§ 60 ~.... +-' ~ c: NPlEO Cl> 40 0 c: 0 ; NP2EO 0 20~~

~~0'----L--'---'~-'---'--'----''--....__.___._~..____._~~~~- 23456789101112 Time (days) (Cl>)~~

~~~ 80~~

~~c: .Q 60 (ti .::: o NP,4°C c: ~ 40 • 1°/.,HCHO c: NP+ 0 o CD NP.20°c 20~~

~~0'---L--'---L~-'---'--'---''---'--L---L~....__.._ __ 2 3 4 S 6 7 8 9 10 II 12 Time (days)~~

Figure 33. Biodegradation of nonylphenol (NP), nonylphenol mono- (NP1 ED) and diethoxylate (NP2EO) in the river water test: at different temperatures,· Initial concentration: NP, 13 µg!L; NP1EO, 90 µg/L; NP2EO, 64 µg/L 100

1 % of formaldehyde was added, served as a sterile control. At 4 °C and after 12 days there was only a small decrease in the concentration of the NP1 EO and NP2EO (10-20 %), and the concentration of the NP remained unchanged. The decrease of the NP1 EO and NP2EO concentration it was more pronounced at 20 °C (50-60 % after 12 days), while the concentration of the NP remained almost constant, even at the increased temperature. The unchanged concentrations of NPEO and NP during the 12 days, in the samples to which formaldehyde was added, show that this is an effective way for sample conservation (i.e. collecting composite samples during longer period).

~~3.2.3.5. Biodegradation of APEO and AP in Modified Closed Bottle Test~~

The closed bottle test was used in the attempt to show the influence of anaerobic conditions on the biodegradation of the investigated compounds. Through the degradation of easily degradable organic substances present in the synthetic sewage (in the Winkler bottle) the anaerobic conditions in the sample after a few hours degradation of NP1 EO and NP2EO did not even start. The counting of the bacteria showed that their number had suddenly increased and the composition of the bacterial culture used as inocculum had changed as well. Although, according to the previous experiences, the number of bacteria used was sufficient for an effective degradation of NPEO, that did not happen even after 60 days (Figure 34). In order to determine if the lack of oxygen (<1 mg/L) was respon- sible for the stagnation in the degradation, the samples were analysed from the bottles which had already been opened once for taking a sample, and which air had entered. By the analysis of such samples in various periods, it was observed that the degradation started after the air had entered. After 20 days 82 % of NP2EO and 68 % of NP1 EO was degraded. Although this experiment is not sufficient proof that APEO degradation does not take place in anaerobic conditions, it does prove the importance of the anaerobic conditions for the biodegradation of APEO. It should be stressed that the formation of nonylphenol, as the degradation product of NPEO, was not observed in this experiment. However, in a similar experiment performed with real samples of untreated wastewater (wastewater sample was kept closed for 2-3 months) it was observed that in this way the conditions in the sample can be created which favour the formation of NP. The analysis of samples, which initially contained 1 mg/L of nonylphenol polyethoxylates and less than 50 µg/L of NP, showed after storage to have high concentrations of NP (80-580 µg/L) together with low concentrations of NP2EO 101

~~100~~

~~--- 80 IJ. closed bottle '#- NPlEO • after opening c 0 60 v closed bottle :;:; NP2EO 'after opening .....as c -CJ) 40 0 c 0 (.) 20~~

~~0 10 20 30 40 so 60 Time (days)~~

~~Figure 34. Blodegradation of llpophilic nonylphenol ethoxylates in the modified closed bottle test~~

~~TABLE 17. Formation of Nonylphenol in Primary Effluent Samples Containing Nonylphenol Polyethoxylatesa after a Storage in Closed Bottles~~

~~Timeof Concentration (µg/L) Sample/Date storage (months) NP NP1EO NP2EO~~

ZOrich-Glattb, Dec. 1982 2 580 240 <1 Nlederglattc, Dec. 1982 2 560 170 <1 DObendortd, Dec. 1982 2 360 190 <1 ZOrich-Glattb, Nov 1982 3 130 50 <1 Nlederglattc, Nov. 1982 3 80 250 <1 a concentration about 1 mgtl; b typical concentration: NP· 16 µgll; NP1EO = 93 µgtl; NP2EO • 65 µgtl (0611211983); c typical concentration: NP-26µg/L;NP1EO = 85 µgtl; NP2E0·117 µgtl{0611211983); d typical concentration: NP =38 µgll; NP1 EO • 61 µg/l; NP2EO = 57 µgtl (10106/1983). 102

(<1 µg/L) (Table 17). This concentration relationship between NP and NP2EO is very unusual for untreated sewage and is quite similar to the distribution of nonylphenolic compounds in the effluents from the anaerobic sludge treatment. Although the oxygen content in the analysed samples was not measured, it can be assured that anaerobic conditions existed is quite acceptable due to the presence of the considerable amounts of organic matter in the sewage (chapter 3.3.).

~~3.2.3.6. Degradation Products of APEO~~

After the end of the exponential phase of APEO degradation (8-10 days after the start of experiment) the content of the samples, were extracted. The obtained chloroform extracts were analysed by the HRGC/MS method. It was found that alkylphenoxy carboxylic acids were the dominant constituents of the extracts and they could be considered as the products of biotransformation of APEO. Figures 35a and 35b show the chromatograms of typical samples. The mass spectra of the identified components (APEC) are discussed in detail in chapter 3.1.2. In the experiment of NPEO degradation, NP1 EC and NP2EC were identified corresponding to NP1 EO and NP2EO which were originally present in the medium. Similarly, in the experiment with OPEO, OP1 EC, OP2EC, OP3EC and OP4EC were identified. This data indicate that the mechanism of oxydative deethoxylati- on (21) is a very likely mechanism of the initial degradation of APEO. During the biodegradation experiments, the simultaneous quantitative determination of APEO and APEC was not done, but was in some samples at the end of the exponential phase. The results of quantitative analyses have shown that APEC were, in comparison to the initial concentrations of specific APEO oligomers (100-1000 µg/L), present in considerable concentrations (260-380 µg/L NP1 EC, 17-67 µg/L NP2EC, 170-580 µg/L OP1 EC, 340-850 µg/L OP2EC and 110-500 µg/L OP3EC). Thus, it can be concluded that carboxylation is probably the most important mechanism of the aerobic transformation of APEO. Moreover, the presence of high concentrations of APEC in the medium indicates their high persistence towards further biodegradation under aerobic conditions. 103

~~100 A~~

~~NP1EC~~

~~(I) Chc: 0 C1s- -C1e:1 0. so 0 (I) a:~~

~~0 16 24 32 Time~~

~~-OPIEC -OP3EC (I) Chc: a.0 so Ch (J) a:~~

~~0 10 20 30 Time (min)~~

Figure 35. Total ion current chromatograms of methylated alkylphenoxy carboxylic acids extracted from the synthetic sewage which originally contajned alkylphenol ethoxylates (8 days after beginning the biodegradation experiment); NP1EC-NP2EC: nonylphenoxy carboxylic acids; OP1EC-OP4EC: octylphenoxy carboxylic acids. C16, C1s:1: common fatty acids. 104

~~3.2.4. Photochemical Degradation~~

~~3.2.4. 1. Direct Photolysis~~

Direct photolysis was investigated by the addition of NP (1.13 µmol/L) and NPEO (1 µmol/L lmbetin N/7A) to the distilled water. In the irradiation apparatus (MGRR) a mercury lamp (700 W) and a glass filter Solidex (A.>280 nm) were used. The samples of the prepared solution were irradiated in either quartz or glass (Jena) tubes. The results are presented in Figure 36. Rate constants of the direct photolysis (kp) and half-!ifes (t112} in quartz tubes are kp = 0.199 h·1 and t1 12 = 3.48 for NP, and kp = 0.057 h·1 and t112 = 12.5 h for NP1 EO (Table 18). It can be also noted that photolysis of NP is faster in quartz tubes (kp = 0.199 h·1) than in the glass ones (kp = 0.171 h·1; t112 = 4.05 h), which is the consequence of better transmittance of quartz to UV-light.

~~0.8l.Or~~;::~:------A.._ 0.6~~

~~0 0.4 o NP, quartz ~ (.) ® NP, glass A NP1EO, quartz 0.2~~

~~O.I ._____ 6..... 0 ____ 12.._0 ___ ...... 180 ____ 2..... 40 __~~

~~nme~~

~~Figure 36. Direct photolysis of nonylphenol (NP) and nonylphenol mono· ethoxylate (NP1 EO); Hg-lamp 700 W.~~

~~TABLE 18. Rate Constants and Half-fifes of the Direct Photolysis of Nonylpheno/ and Nonylphenol Monoethoxylate~~

~~Compound Tubes 1112 (h)~~

~~NP quartz 0.199 3.48 0.9700 NP glass 0.171 4.05 0.9959 NP1EO quartz 0.057 12.2 0.9372 105~~

It needed to be stressed that direct photolysis by sunlight (/·.>295 nm) has to be much slower than direct photolysis by applied laboratory apparatus. Namely, according to the absorption spectra of AP and APEO (A.max = 277 nm} there is a sharp decrease in the absorption of NP between 280 nm (wavelength cutoff of applied Solidex filter) and 295 nm (wavelength cutoff by ozone adsorption).

~~The products of direct photolysis were not isolated and identified, but it can be presumed that degradation starts with the oxydation of the aromatic ring (164).~~

~~3.2.4.2. Sensitized Photolysis~~

It is known that photochemical degradation for many specific organic compounds can be much faster in the presence of other dissolved substances (183), which is particularly important in natural waters where humic and fulvic acids represent a dominant part of the total dissolved organic carbon (DOC).

Photochemical degradation of NP and NP1 EO was investigated in filtered (Millipore, 0.45 µm) lake water (Greifensee, August 1984, DOC = 4 mg/L) and in the solutions containing 5 mg/L of rose bengal dye. Rose bengal dye is considered an excellent sensitizer of photochemical reactions and is especially known as an effective source of singlet oxygen (184). The conditions of irradiation were identical to that of direct photolysis. The results given in Figures 37 and Table 19 show that photolysis, in the presnece of natural organic matter, is much faster for both NP (kp = 0.923 h·1; t112 = 0.75 h) and NP1EO (kp = 0.515 h·1; t1/2 = 1.35 h) than the direct photolysis. In the presence of rose bengal dye photolysis becomes even several times faster (kp = 6.42 h·1; t112 = 0.11 h).

In a preliminary experiment the photolysis of the mixture of higher oligomers of NPEO (nEO = 3-18) was investigated and it was observed that they degraded even slower than NP1 EO; no difference in the photolysis rate was observed between specific oligomers in the range NP6EO - NP17EO (kp = 0.026 h·1; t112 = 26.7 h).

~~The alkylphenols are weak acids which can be present in water solutions either in the nondisssociated form or in the form of phenolate:~~

~~(18) 106~~

~~1.0 0.8 0.6~~

~~0.4~~

~~0.2~~

~~<..? ;::; 0.1 0.08 0.06~~

~~004 A NP1EO. lake water.quarz Cll NP, lake water.glass o NP, lake water.quartz e NP, distilled water + 5mg/L rose bengal 0.02~~

~~0.01 0 30 60 90 120 150 180 Time (mini~~

~~Figure 37. Photochemical degradation of nonylphenol (NP) and nonylphenol monoethoxylate (NP1EO) in the presence of dissolved organic matter (sensibilized photolysis); Hg-lamp, 700 W;~~

~~TABLE 19. Rate Constants and Half-fifes of the Photolysis in the Presence of DOC for NP and NP1 EO~~

~~Compound Conditions kp (h-1) t112 (h)~~

NP Lake water. glass8 0.857 0.81 0.9986 NP Lake water, quartz8 0.923 0.75 0.9947 NP Distilled water + 5 mg/L 6.42 0.11 0.9175 rose benga1b, quartz NP1EO Lake water, quartz& 0.515 1.35 0.9972 a filtered 0.45 µm lake water (Greifensee, DOC 4 mg/L; pH 8.4); b 3',4',5',6'-tetrachloro-2,4,5,7-tetraiodofluorescein, disodium salt (C20H2Cl4l4Na~5). 107

In the analogy with chlorophenols (184) it can be presumed that undissociated AP and phenolate do not react photochemically in the same way. The share of the dissociated form depends on the dissociation constant (i.e. on pK value) and on the pH of water solution. The dependence of the photolysis rate of NP on pH of the solution was investigated in the range pH = 7.05-10.7 (Figure 38; Table 20).

~~1.0 0.8 0.6~~

~~o0.4 ...... u o pH 7.05 u ® pH 9.0 e pH 10.7 0.2~~

~~O.I 0 10 30 so 70 90 110 Time (min)~~

~~Figure 38. Photochemical degradation of nonylphenol at different pH values; 5 mg!L rose bengal dye added; tungsten-halogen lamp, 60 V~~

~~TABLE 20. Rate Constants (kp) and Half-fifes (t112) of the Sensibilized Photolysis of Nonylphenol at Different pH Valuesa~~

~~Compound pH t112(h)~~

~~NP 7.05 0.445 1.56 0.9999 NP 9.0 0.499 1.39 0.9821 NP 10.7 0.920 0.75 0.9888 a MGRR; tungsten-halogen lamp, 60 V; 5 mg/L of rose bengal dye added.~~

The experiments were performed in the same apparatus as previously described, but tungsten-halogen lamp {60 V) was used instead of the mercury lamp. The solution of potassium dichromate was used as the filter solution (A.>530 nm). Into the NP solution (about 1 µmol/L) 5 mg/L of rose bengal dye was added as a sensitizer. The photochemical degradation of NP varies very 108

slightly between pH 7.05 and pH 9.0 (trn? - 1.66 and 1.39 h), while th& photolysis at pH 10.7 is much faster (kp = 0.920 h-1; t112 = 0.75 h). This effect could be explained by the higher rate of the photochemical degradation of phenolate. Namely, it can be estimated that pK value for long-chain alkylphenols {Ca. Cg) is somewhat larger than 10 (185). Therefore at pH 10.7 a considerable part of AP is in dissociated form and that could explain the increase of the photolysis rate in comparison with the lower pH values. However, at pH values typical of natural waters (pH 6-9) almost all dissolved AP is in undissociated form.

It was shown in literature (184) that the photoxydation mechanism via singlet oxygen (102) can play an important role in the photolysis of phenolates. Since the half-life of singlet oxygen in heavy water (D20) is much longer than in ordi- nary water (H20) (186), the comparison of photolysis rates for these two solvents can indicate relative importance of the mentioned mechanism. Figure 39 and Table 21 give the comparison of photolysis in water and heavy water containing 10 % of H20.

1.0 • 08. \ \ O' ...... \ u-....-0 0.6 \ ...... '\ ...... 0,4 ...... uo ...... '@ ...... u \ .... ' o NP in HiO 0.2 '\ ' ® NPin90°1o0i0 '\;> pH=9.0 - --- pH= 10.7 '' O.I 0 10 30 so 70 90 110 Time (min)

~~Figure 39. Photochemical degradation of nonylphenol in H20 and D20; 5 mg/L rose bengal dye added; tungsten-halogen lamp, 60 V;~~

The results indicate that the photolysis via the singlet oxygen is important for alkyl phenols only at higher pH values (pH > 10). At pH 9.0 the ratio of photolysis rates in H20 (kp = 0.362 h-1) and D20 (kp = 0.680 h-1) is only 1.0, while at pH 10.7 it is significantly higher (4.6). On the basis of the given data, it seems that 109 the singlet oxygen mechanism is not very significant for the photochemical degradation of AP in natural waters (pH = 6-9). TABLE 21. Rate Constants (kp) and Half-lites (t112) of the Nonylphenol Photolysis in H20 and D20 (10 % H2o;a

~~Compound Conditions t112 (h)~~

~~NP H20.pH ,.9 0.362 1.91 0.9719 NP D20,pH=9 0.680 1.02 0.9990 NP H20, pH= 10.7 0.806 0.86 0.9441 NP D20. pH= 10.7 3.70 0.19 0.9804 a MGRR; tungsten-halogen lamp, 60 V; 5 mg/l of rose bengal dye added.~~

~~3.2.4.3. Photolysis in the Presence of Hydrogen Peroxide~~

Hydrogen peroxide is one of the well known agents for the oxydation of organic matter and is, in combination with irradiation with UV-light, used for the removal of DOC from the samples of natural water in many laboratory procedures. Concentrations of hydrogen peroxide in natural waters can reach significant values of 10-7 mol/L (187, 188). The concentrations in the surface layer of the swiss lake Greifensee ranges between 0.1-0.2 µmol/L (189). Because of that the photolysis of NP in the presence of the H202 was examined. The same irradiation apparatus with mercury lamp (700 W), as described previously, was used. In order to observe the effect of the present H202 the direct photolysis was performed simultaneously as a control reaction (solution of NP in bidistilled water). In addition, the reaction between H202 and NP in the dark was examined. In this experiment concentrationss of both NP and H202 were determined (190).The results are given in Figure 40, and the calculated rate constants of specific reactions and degradation half-fifes in Table 22. In two independent experiments with essentially different concentration ratios of NP and H202 (a: 12 µmol/L H202 and 0.48 µmol/L NP; b: 6 µmol/L H202 and 3 µmol/L NP) quite similar constants of the rate of photolysis (kp = 0.369 h·1 and 0.314 h-1) were obtained. The reaction between H202 and NP takes place even in the dark, but it is much slower (kr = 0.053 h·1; t112 = 13.1 h) than the photolysis in the presence of H202. Direct photolysis is also considerably slower (kp = 0.225 and 0.219 h·1) than in the presence of H202. It should be mentioned that these values of kp for direct photolysis are very similar to those in Table 18, which indicates good reproducibility of kp determination in the applied apparatus. 110

~~1.0 0.8 0.6 J> u 0.4~~

~~0.2~~

~~0 KXl Time (min) 200 1.0 0.8 0.6~~

~~u0 0.4 B -u NP+HzOz.M 0.2~~

~~IOOT.1me (')200mm~~

~~Figure 40. Photochemical degradation of nonylphenol (NP) in the presence of H202: (A) 0.48 µmo/IL NP and 12 µmo/IL H202; (BJ 3 µmo/IL NP and 6 µmo/IL H202; Hg-lamp 700 W;~~

~~TABLE 22. Rate Constants (kp) and Half-fifes (t112) of the Nonylpheno/ Photolysis in Presence of Hydrogen Peroxydea~~

~~Conditions kp (h"1) t112 (h)~~

0.48 µmol/L NP, h v 0.215 3.22 0.9864 0.48 µmollL NP+ 12 µmoVL H,P2, hv 0.369 1.88 0.9921 3 µmol/L NP + hV 0.225 3.08 0.9966 3 µmol/L NP + 6 µmol/L H20i!. hv 0.314 2.21 0.9988 3 µmol/L NP + 6 µmol/L H20i!. dark 0.053 13.1 0.9729 a Hg-lamp, 700 W. 111

~~Through quantitative considerations it was observed that the photolysis of NP in the presence of H202 could be represented as the sum of direct photolysis and the reaction of those substances in dark:~~

~~(19)~~

However, this estimation is only apparently the result of a simple sum of two independent reactions, direct photolysis, and the reaction of the NP with H202. By the irradiation of solution, H202 is also photolytically degraded and its real concentration available for the reaction with NP should be less than nominal. Contrary to that, it was observed that photolysis of NP produces H202 in the molar ratio 1 :0.6, which greatly substitutes photolytically degraded H202. This resulted in the measured netto effect that the decrease ot H202 concentration was negligible. An additional degradation of NP due to the reaction with the concurrently formed H202 has to be taken into account even when measuring the photolysis in bidistilled water.

The photolysis of NP sensitized by organic matter in the presence of H202 was not investigated, but it seems that this mechanism could play a role in the photochemical degradation of NP in natural waters. Namely, the concentration ratio of NP (0.05 µmol/L; chapter 3.4.) and H202 (0.3 µmol/L; ref. 189) in natural waters is very similar to the one applied in our laboratory experiments, only the concentrations of both were about ten times lower.

~~3.2.4.4. Photolysis by Sunlight~~

Using the information that the irradiation applied in the laboratory (mercury lamp) was about 10 times more intensive than the sunlight irradiation during a sunny summer day (162) a rough estimate could be obtained about the importance of the photochemical degradation for the behaviour of the NP in natural waters. However, in order to obtain a more realistic view of the photochemical reactivity of a certain compound in natural conditions, the experiments should simulate natural conditions as much as possible.

The results of the photolysis of NP by sunlight are given in Figures 41a and 41b, and the rate constants of the reaction as well as calculated half-lifes in Table 23. Although the photolysis by sunlight was much slower than under laboratory conditions (mercury lamp), the results show that in one day a considerable 112

~~0.81.0 t""~~~;::::::~= o.6 A Lake water, pH=8.4 0 } o NP in a flat-bottom container ~ 0.4 h. NPlEO immersed u ~ NP } Lake water, pH=8.4 '1 NPIEO immersed in a brook~~

~~0.2~~

~~0 2 3 4 irradiation (kWh/m2)~~

1.0 0.8 0.6 8 0 NP }Lake water, pH 9.3-8.7 immersed in a flat-bottom container uo 0.4 0 OP ....u 0 NP }Lake water, pH 9.3-8.7 8 OP immersed in a brook e NP } distilled water 0.2 Iii OP immersed in a flat-bottom container h. NPlEO Lake water, pH 9.3-8.7 immersed in a flat-bottom container

~~0.10 2 3 4 irradiation (kWh/m2)~~

~~Figure 41. Sunlight photolysis of nonylphenol (NP), octylphenol (OP), and nonylpheno/ monoethoxylate (NP1EO) in lake water: (A) 1110911985, (B) 1810911985 {for details see chapter 2.8.) 113~~

degradation of NP can happen (~40 %1). On the contrary, for NP1 EO possible concentration decrease was not detectable. The photolysis of NP in tubes immersed in a water-filled flat-bottom container was much faster (kp = 0.091 and 0.092 h-1) than the tubes immersed into the brook (kp = 0.064 and 0.051 h-1). This could be attributed to the decrease of light intensity in the brook, which was proved by the actinometry with pNA. It should be mentioned that in the second experiment (18th Sept) photolysis stopped after 3 hours, which could perhaps be explained by the dispersion of sunlight on the air bubbles which were formed on the outer walls of the tubes immersed in the brook.

~~TABLE 23. Rate Constants (kp) and Half-lites (t112) of the Sunlight Photolysis of Alkylphenols and Alkylphenol Ethoxylates in Lake Watet6~~

~~Conditions kp (kWh-1 m2) 1112 (kWh m·2) t112(h)~~

NP, pH= 8.4 0.091 7.6 10.7 0.9972 NP, pH =8.4 0.064 10.8 15.3 0.9421 NP, pH=9.3b 0.092 7.53 9.9 0.9831 NP, pH=9.3b 0.051 13.6 17.8 0.9279 OP, pH =9.3b 0.102 6.79 8.92 0.9947 OP, pH•9.3b 0.046 15.1 19.8 0.9316 NP1 EO, pH = 8.4 n.d. n.d. n.d. n.d. NP1 EO, pH = 8.4 n.d. n.d. n.d. n.d. NP1 EO, pH • 9.3b n.d. n.d. n.d. n.d. a Solutions of NP, OP and NP1 EO were prepared in filtered water from Greilensee (DOC - 4 mg/L); rate constant for the sunlight photolysis in distilled water was 0.02 kW h-1m2; b pH value decreased during the experiment from 9.4 to 8.7; n.d.: not determined because photolysis was to slow.

By increasing the pH value to 9.4 the rate of photolysis did not increase, which was in agreement with the observations from the laboratory experiments (Figure 38). It should also be stressed, that during the experiment, pH decreased spontaneously to 8.7.

~~The rate of photolysis of OP (kp = 0.102 and 0.046 h-1) does not differ from that determined for NP.~~

The sunlight of AP in bidistilled water was performed under the same conditions as described for the samples which were prepared in filtered lake water. The essentially slower reaction (kp = 0,020 h-1) shows that in the case of the photolysis by sunlight as well the main contribution derives from the sensitized photolysis, and only a smaller one from direct photolysis. The photolysis rate for NP in 114 the laboratory conditions (mercury lamp, 700 W) was approxlmatelly fO limes higher than sunlight photolysis for both direct and sensitized photolysis. This is in agreement with the ratio obtained on the basis of the photolysis for the other compounds (162).

The half-life of the photochemical degradation was estimated from the value of the irradiated dose needed for 50 % degradation (t112) base done the data of average irradiation intensity during the experiment (0.708 kWh/m2h for 11th Sept. and 0.761 kWh/m2h for 18th Sept.). The values oft112 range between 10- 20 hours (Table 23). Since the results were obtained in late summer, It is probable that the values of t112 for June, July and August, when the sunlight irradiation is stronger {about 1000 kWh/m2h; ref. 162) were considerably lower (about 1o hours). On the basis of this estimation, it could be expected that in clear and shallow waters photochemical degradation could play a role in elimination of AP. Contrary to this photochemical degradation of APEO seems to be insignificant. The possible photolysis products of NP were not established. 115

~~3.3. Occurrence and Behaviour of Alkylphenol Polyethoxylates and Their Degradation Products in Sewage Treatment Plants~~

~~3.3.1. Characteristics of Investigated Sewage Treatment Plants~~

~~A large number of sewage treatment plants located in the Cantone of Zurich, Switzerland, which included different treatment methods and a large range of capacities (Table 24) was investigated.~~

~~TABLE 24. Technical Data of the Investigated Sewage Treatment Plants~~

~~Treatment plant PE ca Samplingb Operational types of wastewater and sludge treatment~~

Bassersdorf 22 500 primary clarifier/aeratiOn tank/secondary clarifier; digestion BOlach 34 000 primary clarifier/aeration tank/secondary clarifier; digestion Dietikon 67 000 primary clarifier/aeration tank/secondary clarifier; digestion Dobendorf 41 000 primary clarifier with chemical flocculation/aeration tank/secondary clarifier; centrifugation Fallanden 50 000 primary clarifier/aeration tank/secondary clarifier; digestion Flaa ch 4 000 primary clarifier/aeration tank/secondary clarifier; digestion Nanikon 12 000 primary clarifier/aeration tank/secondary clarifier;c Niederglatt 37 750 first aeration tankJlirst clarifier/second aeration tank/ secondary clarifier; digestion Opfikon 34 000 primary clarifier with chemical flocculation/aeration tank/secondary clarifier; digestion Pfungen 33 000 primary clarifier/aeration tank/secondary clarifier; digestion Regensdorf 15 000 primary clarifier with chemical flocculation/aeration tank/secondary clarifier; digestion Uster 60 000 primary clarifier/aeration tank/secondary clarifier/filtration; digestion ZOrich-Glatt 240 000 primary clarifier/aeration tank/secondary clarifier; digestion a PEC: population equivalent capacily; b I: flow proportional, t: time proportional; c external sludge treatment.

~~A typical plant contained a primary clarifier, aeration tank and a secondary clarifier for mechanical and biological sewage treatment (Figure 42). 116~~

~~VOLATILIZATION ?~~

~~SLUDGE DISPOSAL~~

~~Figure 42. Scheme of a mechanical-biological sewage treatment plant~~

~~3.3.2. Occurrence and Distribution in Raw Wastewaters and Effluents~~

The distribution of APEO oligomers in the samples of raw wastewater, primary and secondary effluents from the sewage treatment plant in Uster is given in Figure 43. The distribution of oligomers in the commercial mixture of NPnEO (Marlophen 810) is also shown in the same Figure. The majority of alkylphenol polyethoxylates in the analysed samples (Figure 43) belong to 4-nonylphenol ethoxylates, but 2-nonyl-, 4-decyl, and 4-octylphenol ethoxylates were also present, although in much smaller amounts (<5 %). Moreover, in addition to the APEO oligomers some carboxylated compounds (AP1 EC, AP2EC) were also determined in the samples (they are not included in Figure 43). The commercial mixtures of APnEO, which are the most often used in household detergent formulations show the Poisson distribution of oligomers centering at AP9EO-AP10EO whereas the share of NP1EO and NP2EO is quite small (less than 1 %). Completely deetoxylated product of NP was not detected ln the original commercial mixtures. (Chapter 3 ..1 .). For that reason the sum of oligomers, in the range of ethoxy groups nE0=3-20, was used to express the concentration of the original surfactant in analysed water samples. 117

~~o nontreated wastewater A mechanically treated wastewater CJ biologically treated wastewater X commercial NPnEO mixture~~

~~c: 0 ~.... ~ (.) c: 0 ()~~

~~7 8 9 10 I! 12 13 14 IS 16 17 18 number of EO groups per molecule~~

Figure 43. Distribution of individual NPnEO oligomers in raw sewage, primary effluent, and secondary effluent of the sewage treatment plant at Uster. For the comparison of oligomer distn·bution in commercial mixture Marlophen 810 is given.

The statistical distribution of the total NPnEO concentrations (nE0=3-20) in non· treated sewage and primary effluent samples is shown in Figure 44. Ranges and average concentration values, calculated as arithmetic mean, are given in the Table 25. According to our knowledge, these are the first results on the concentrations of aromatic nonionic surfactants in wastewater obtained by the specific analytical method. The concentrations of NPnEO in nontreated sewage and pri· mary effluents were very similar and ranged between 400-2200 mg/m3. The most frequent values were between 800-1000 mg/m3. The distribution of the NPnEO oligomers in nontreated sewage and primary effluents is also very similar (Figure 43). Therefore, the same ratio (1.07) is obtained by the comparison of their average molar concentrations (µmoltm3) and by the comparison of their weight concentrations (mg/m3).The comparison of nontreate.d sewage and secondary effluents gives a completely different picture. The average weight concentration in the nontreated sewage is 17 times lower, and the average molar 118 concentration Is only 12.8 times lower. This can be explained by the greater share of lower oligomers {nE0=3-10) in the secondary effluents.

~~20 primary effluents ,,,..s 0- 15 n = 39~~

~~01--r-..,....,~~4""1L+'l'f..qG.<4--f'~~+"'l'"'f"'-l'+-- o 500 1000 1500 2000 NPnEO concentration (mg I m3)~~

~~Figure 44. Frequency distribution of the total concentrations of nonylphenol polyethoxylates (NPnEO, n=3-20) in raw sewage and primary effluents~~

~~TABLE 25. Concentrations of Nonylphenol Polyethoxylate Surfactants in Nontreated Wastewaters and Sewage Effluents from Sewage Treatment Plants in the Glatt Valley, Switzerland~~

~~Concentrations~~

~~Sample (mglm3) (µmoVm3)~~

~~Range xb s Range xb s n~~

~~Nontreated 730-2170 1140 520 1.29-3.61 1.93 0.86 6 wastewater~~

~~Primary 520-2080 1064 402 0.87-3.47 1.87 0.69 21 effluent~~

~~Secondary 3·320 63 75 0.01-0.64 0.14 0.15 24 effluent a determined as the sum of oligomers in the range nE0--3-20; b arithmetic mean: s: standard deviation; n: number of samples. 119~~

Contrary to the unimodal distribution of oligomers in the commercial APEO mixtures, the extracts of nontreated sewage and primary effluents show characteristics bimodal distribution of the oligomers. The greatest part of the distribution curves (nE0=3-18) for the nontreated sewage and primary effluent is almost identical (Figure 43). The maximal concentration of a single oligomers was reached for NP8EO. which is very similar to the distribution described for the commercial mixture. However, in the wastewater samples another maximum is observed in the region of lower oligomers (nE0<3), which is not present in the original commercial mixture. This is very probably the result of the transformation of APnEO which already started in the sewer system and in the primary clarifier. However, the most drastical change of the composition of APnEO occurs during the activated sludge treatment as it can be seen from the curve displaying the oligomer distribution in secondary effluent. The largest part of the higher oligomers disappeared, and only traces of oligomers in the range NP3EO- NP9EO remained. The maximum concentration of the single oligomer is reached for NP2EO. This is in agreement with some previous reports on the biodegradation of APnEO in the laboratory experiments (135, 136). However, in the real- scale sewage treatment NP was also determined at significant concentrations which was not the case with the mentioned laboratory experiments.

Nonylphenolic compounds found in sewage effluents which are not present in the original commercial APEO mixtures, such as NP and NPEC, must be regarded as their metabolic products. Contrary to this, each nonylphenol ethoxylate oligomer could be regarded both as parent compound as as metabolic product. From practical reasons only those ollgomers which show comparatively increased persistence against further microbial transformation (NP1 EO and NP2EO) were considered as the. metabolic products, whereas all higher oligomers (NPnEO, n=3-20) are arbitrarily considered as the parent compounds.

The relative abundance of the parent compounds and the metabolic products in the primary and secondary sewage effluents is given in Fig. 45. The contribution of each nonylphenolic compound class is obtained as the average composition on weight basis for 11 STP's. Nonytphenol polyethoxylates (n=3-20) with 82.4 % are, as expected, the most abundant class in the primary effluents. However, considerable contribution (17.6 %) belongs to the various metabolic products of NPnEO. Among them, the most abundant were NP1 EO+NP2EO with 11.5 % while NP and NPEC contributed by only 3.0 and 3.1 % respectively. The secondary effluents showed completely different feature. The metabolic products of NPnEO contributed to the total concentration by 71.2 %, the most 120

abundant class being NPEC with 46.1 %. The lipophllic metabolites NP and NP1 EO+NP2EO achieved the share of 3.9 % and 21.8 % respectively. It is obvious that a successful study of nonylphenol polyethoxylates surfactants in STP, requires the survey of a full range of different nonylphenolic compounds. Only in that way is it possible to get a precise insight into the role of the various processes which determine the fate of these surfactants in sewage treatment.

~~PRIMARY EFFLUENTS SECONDARY EFFLUENTS~~

~~26%~~

~~5 °lo~~

~~~ 20% NPnEO [:·.::::::'.::) NP1EO+NP2EO ~ NP ~ NP1EC + NP2EC~~

~~2.97 m mol /m3 1.18 m mol/m3~~

~~Figure 45. Relative abundance of nonylphenol polyethoxylates and their metabolites in primary and secodary effluents (average value of 11 STP in the Glatt Valley, Switzerland)~~

The wastewaters examined in this study varied significantly with respect to their content of total organic matter as well with respect to the concentrations of APnEO and their metabolites. The data given in Table 26 present the concentrations of the nonylphenolic compounds along with the concentrations of common organic parameters (DOC, COD, BOD). The total concentration of nonylphenolic compounds calculated as the sum of concentrations of NPnEO, n=3-20, NP1 EO+NP2EO, NP, and NP1 EC+NP2EC varied in the ranges 1090-2064 µg/L (x = 1438±348) and 240-760 µg/L (x = 397±174) for primary and secondary effluents respectively. As shown in Table 26 there is no clear correlation between the values of common organic parameters and summed concentrations of the nonylphenolic compounds. The lack of such correlations suggests relatively variable origin of the wastewaters and reflected perhaps different contributions of the domestic and industrial sources. The concentration of nonylphenolic TABLE 26. Concentrations of Surfactant Derived Nonylpheno/ic Compounds and of Common Organic Parameters in Primary and Secondary Effluents of 11 Sewage Treatment Plants in the Glatt Valley, Switzerland (August 1984)

~~STP Primary effluents Secondary effluents~~

COD BOD DOC NP-c (NP-c/DOC)a COD BOD ooc NP-c (NP-C/DOC)a Nanikon 240 101 18 1120 4.0 56 10.8. 7.3 430 4.3 Fallanden 210 77 15.7 1390 5.8 37 3.2 6.3 300 3.5 Bassersdorf 250 83 14 2060 9.6 50 4.6 6.3 270 3.1 DObendorf 250 96 18.5 1120 4.0 79 7.5 5.3 460 8.0 ZOrich-Glatt 230 73 13 1200 6.0 66 10.2 7.3 240 3.2 I\') Opfikon 280 143 24.6 2020 5.3 80 26 9.3 760 6.0 Niederglatt 250 101 14.8 1440 6.3 52 14 8.7 240 2.0 BOia ch 240 108 16.4 1640 6.5 77 14.7 6.8 370 4.0 S1adel 180 64 15.4 1090 4.6 64 21 6.3 530 6.1 Glaltfelden 270 122 13.1 1220 6.0 61 28 7.0 300 3.1 Rheinsfelden 290 101 33 1530 3.0 57 4.6 8.2 300 2.7

~~x 244 97 17.8 1440 5.5 62 13.1 7.2 400 4.1 s 31 23 5.9 350 1.8 13 8.6 1.2 170 1.9~~

COD: chemical oxygen demand (KMn04); mg/L; BOD: biological oxygen demand, mg/L; DOC: dissolved organic carbon; mg/L NP-c: sum of nonylpheno6c compounds, µg/L; NP-C/DOC: concentration of NP-c expressed as percent of DOC,%; a correction for the carbon content of NP-c being a) 65% in primary effluents and b) 73% in secondary eflhJents was applied. 122

compounds relative to the concentration of DOC varied for the primary effluents in the range of 3.9-9.6 %, which could be considered as a fairly high contribution. This contribution was somewhat lower in secondary effluents (2.0- 8.0 %).

~~3.3.3. Diurnal and Dally Variations~~

Diurnal and daily variations of the alkylphenolic compounds in STP's can be very pronounced. Figure 46 represents the daily variations of the concentrations and mass fluxes in the STP Zurich Glatt. The influent load of this STP with nonylphenolic compounds was 1.6 times higher during workdays (about 50 kg/day) than during the weekend (30 kg/day). The same trend was observed for DOC load (780-1300 kg/day). The DOC load in secondary effluent was clearly related to the load in the primary effluent and showed a reduction of 67-71 %. Contrary to this, the load of nonylphenolic compounds in the secondary effluent did not fit well to the changes in the primary effluents. With exception of somewhat to the decreased load on Friday, the load of the sum of nonylphenolic compounds in the secondary effluents was rather constant (about 10 kg/day). Among the different nonylphenolic compounds only parent NPnEO exhibited typical loading pattern like DOC. Such regularly occurrence was not observed for either NP1 EO+NP2EO or NPEC (Figure 46b). The concentration profile of NPEC in the secondary effluents suggested even a negative correlation with the concentrations of NPnEO in the primary effluents.

The variability of NPnEO concentrations during the day was even more expressed than that during the week (Figure 47). The maximum was observed in the late afternoon followed by a minimum at night. The concentrations of NP and NP1 EO+NP2EO in the primary effluent generally followed the concentration changes of the parent compounds (Fig. 47a). However, the concentrations of NPEC showed as a opposite trend. Since APEC are typical products of the aerobic transformation of APnEO, this could be a consequence of decreasing oxygen content caused by the increased load of the sewage by BOD substances entering STP in the period of diurnal maximum. As already mentioned discus- sing daily variations, even the very strong changes of the input concentrations of the metabolic products were not reflected on their concentration changes in secondary effluents (Figure 47b). The concentrations of the two most abundant metabolic product classes, NPEC and NP1 EO+NP2EO, have been surprisingly constant during the day. 123

~~-:!? ~20 0 tg 10 1000 zCl. w 5 500 oPE •SE t NPEO + NPEC ----...... ' ,• 2 200 OPE DOC •SE~~

~~~~..._ __.____.~_,_~_.__, 100 16. 17. 18. 19. 20. Thu Fri Sat Sun Mo June 1983~~

~~IOOO~~

c? 500 E Cl -§ 200 I"---· ...... c .... ,' .... 0 100 , l\ PE SE NPnEO(n:3-20). :;._ A .... V PE 5i 50 --..- ... .. SENPIEO+NP2EO 0 ----- ...... T c 0 CJPENPEC (.) 20 , , 6 •SE 10L-..1-~:..._~==:~~l....-- 1s. 11 10. t9. 20. Thu Fri Sat Sun MO June 1983

Figure 46. Daily variations of the mass fluxes (A) and concentrations (B) of nonylpheno/ polyethoxylates (NPnEO) and their metabolic products compared with the variations of mass flux for DOC in the sewage treatment plant Zurich- Glatt; PE: primary effluent; SE: secondary effluent 124

~~a NPnEO v NP1EO +NP2EO 5000 o NP ...... C') D E NP2EC -;; 2000 [ 1000~~

~~-c:: 0 :;:: ...ca c:: (J) -(.) c:: 0 0 408 12 20 24 04 08 Time (h)~~

~~.._ 0 E :::i -c .Q -ns..... c (J) -(.) c:: 0 0~~

Figure 47. Diurnal variations of the concentrations of nonylphenol polyethoxylates (NPnEO) and their metabolites in (A) primary effluents and in (8) secondary effluents of the sewage treatment plant Zurich-Glatt 125

~~3.3.4. Elimination of Alkylphenol Polyethoxylates and Their Metabolites During Biological Treatment~~

Table 27 presents the data on elimination of nonylphenolic compounds by activated sludge treatment compared with the elimination of common organic parameters (BOD, COD, DOC). It has to be stressed at this point that the term elimination expresses every loss from the aqueous phase regardless whether it was caused by biological or nonbiological processes.

~~TABLE 27. Elimination of Nonylphenolic Compounds, BOD, COD and DOC during Activated Sludge Treatment in Several Sewage Treatment Plants in the Glatt Valley, Switzerland, August 1984~~

~~Sewage Ellmlnatlon (%)a treatment plant BOD COD DOC NP-cb NP-cc~~

Na.nikon 89 77 59 61 36 Fallanden 96 82 60 78 69 Bassersdorf 94 80 55 87 79 DObendorf 92 68 69 43 26 Zurich-Glatt 86 71 44 80 71 Oplikon 82 71 62 62 47 Niederglatt 86 79 41 84 78 BOlach 86 68 59 77 66 Stadel 67 64 59 52 42 Glattfelden 77 77 47 75 59 Rheinsfelden 95 80 75 80 73

~~x±s 86±9 74±6 57±10 70±15 59±18~~

aEliminatlon =(CpE- CPE) I CpE x 100 (%); CpE: primary ettluent; CsE: secondary effluent; bElimination on the basis of weight concentrations; celimination on the basis of molar concentrations; NP-c: Sum of nonylphenol polyethoxylates and their degradation products.

The average elimination rate for all nonylphenolic compounds (70±15 %) was significantly lower than the elimination of BOD (86±9 %) indicating that nonylphenolic compounds do not belong to the most biodegradable fraction of the DOC in the sewage. However, the weight based elimination of the parent NPnEO (n=3-20) only was very similar (90±7 %) to that of BOD. It is interesting to note that the considerable part of the DOC in the primary effluents are biorefractory organic materials which can be seen from the average elimination rate of only 57±1 o %. This is significantly lower than the elimination of BOD (86±9 %), COD (74±6 %) and of the sum of nonylphenolic compounds on the weight basis (70±15 %), but it is almost equal to the elimination of the 126

~~nonylphenolic compounds on the molar basis (69±18 OJ..). Thg latAst figure is the consequence of the biorefractory nature of the NPnEO metabolites which still contain an alkylbenzene moiety.~~

The specific methods applied in this study allowed a precise insight into the behaviour of each single NPnEO oligomer during the biological treatment. Result of such an analysis is presented for two typical STP's in the Table 28.

~~TABLE 28. Occurrence and Elimination Efficiency of Single Oligomers of Nonylphenol Polyethoxylates in STP Opfikona and BassersdorfS (Glatt Valley, Switzerland, August 1984)~~

~~No of EO Oplikon Bassersdorf groups peb sec ed peb sec ed~~

0 259 164 37 109 25 77 1 417 428 -3 110 76 31 2 221 231 -5 373 32 91 3 202 159 21 270 31 89 4 197 104 47 270 25 91 5 214 34 84 270 10 96 6 335 74 78 347 8 98 7 369 85 77 362 17 95 8 341 37 89 343 8 98 9 315 34 89 329 7 98 10 280 30 89 289 n.d. >99 11 232 24 90 241 n.d. 12 182 19 90 186 n.d. 13 130 14 89 131 n.d. 14 85 10 88 85 n.d 15 48 5 90 49 n.d. 16 24 3 88 29 n.d. 17 14 3 79 17 n.d. a For treatment conditions see in Table 30; b Concentration in primary effluent (PE), µmoVm3; c Concentration In secondary effluent (SE, µmoVm3; d Efimination (E). (Cpe- Cse l 1Cpe x 100 (o/o). n.d.: non detectable

STP Basserdorf operated at low loading nitrifying conditions and STP Opfikon at high loading non-nitrifying conditions (see in Table 27}. The elimination of the higher NPnEO, n>6 in STP Basserdorf was very efficient (98 %). The elimination rate of lower oligomers gradually decreased, being still high for NP2EO (about 89 %}, but was much less efficient for NP1 EO and NP (31 % and 77 %). A fairly different feature was obtained for STP Opfikon. The higher oligomers nE0=8-17 were eliminated by a uniformely high rate of 88-90 %. The interme· diate NPnEO, n=3-7 were eliminated to a very different extent, from 78 % for 127

NP6EO to only 21 % for NP3EO. Finally, NP1 EO and NP:;!EO showed no net elimination indicating that their formation during activated sludge treatment was faster than their degradation. Presented data suggest that, even in the highly loaded STP's, the elimination rate of higher oligomers of NPnEO is satisfactory, mostly at over 90 %. However, this elimination cannot be interpreted in terms of ultimate biodegradation, because it leads to the formation of the products which are more persistent to further microbial attack. Both examples presented in Table 28, especially that for less efficiently working STP Opfikon, clearly indicate that the biorefractory behaviour of the lower NPEO is caused by their increased lipophilicity.

Table 29 presents the data on the occurrence of 4 different classes of nonylphenolic compounds in the primary and secondary effluents of 11 STP's. The concentration of each compound class is expressed in molar units, which allows more exact comparison between them. As already mentioned, the elimination of the parent NPnEO was very efficient in the majority of the investigated STP's showing no larger variations (90±7 %). Only in STP's of DObendorf, Stadel, and Opfikon the elimination was slightly lower than 80 % (76-79 %). However, abundances and elimination rates of the persistent metabolic products of NPnEO varied in the very broad ranges. For example, the elimination of NP1 EO+NP2f;O ranged from -19 % (net formation) to 80 % (STP Zurich-Glatt). Similarly, NP occurring at 110-430 µmol!m3, was eliminated very differently, from 9 % (STP Nanikon) to 94 % {STP Fallanden). The most interesting behaviour during aerobic biological treatment showed NPEC which were formed at very high rates in all the investigated STP's. Their concentrations in secondary effluents were 2.1-7.6 times higher than in primary effluents. It is therefore obvious that NPEC represent the dominant persistent metabolic products of NPnEO formed under aerobic conditions. Their high abundance in secondary effluents is the main cause for the low overall elimination of the surfactant derived nonylphenolic compounds (26-79 %, x = 59±18 %).

Although the applied methods do not enable a direct insight into the ultimate degradation of NPnEO, on the basis of the obtained results it can be presumed that at least a part of the eliminated nonylphenolic compounds are degraded to water and carbon dioxide. Namely, if the results shown in Table 29 are compared with the results of Krawetz et al. (121, 123), who investigated biodegradation of NPnEO under real conditions using radioactively labelled compounds (3H and 14C), a very good agreement can be observed. The distribution of the radio- activity of tritium in the secondary effluents showed that TABLE 29. Occurrence and Elimination of Nonylphenol Polyethoxylates and Their Metabolites in 11 Sewage Treatment Plants in the Glatt Valley, Switzerland (August 1984)

~~Sewage treatment plant Nan F!ll Bass Diib Zh·G Opf Nied Bill Stad Glat Rhei~~

Primary effluentsa 1. NPnEO, n-3-20 1480 1980 3220 1570 1690 2980 1890 2390 1310 1730 2170 2. NP1EO+NP2EO 510 310 480 660 450 640 790 530 830 390 840 3. NP1 EC+NP2EC 150 180 170 160 140 170 90 130 100 80 270 4.NP 220 170 11 0 160 140 260 270 170 430 130 11 0 5. Sum 1·4 2360 2640 3980 2550 2420 4050 3040 3220 2670 2330 3390

Secondary effluentsa I\) 1. NPnEO, n=3·20 210 140 11 0 380 140 640 160 280 280 130 130 o:> 2. NP1 EO+NP2EO 490 70 1 0 470 90 760 170 270 620 140 200 3. NP1 EC+NP2EC 610 600 580 930 440 590 290 490 580 610 560 4.NP 200 1 0 30 100 20 160 40 30 70 60 30 5. Sum 1·4 1510 820 830 1880 690 2150 660 1070 1550 940 920

Eliminationb 1. NPnEO, n=3·20 86 93 97 76 92 78 92 88 79 92 94 2. NP1 EO+NP2EO 4 77 77 29 80 - 1 9 78 49 25 64 76 3. NP1 EC+NP2EC -310 -230 -240 -480 -21 0 -2 50 -2 20 -280 -4 80 ·6 6 0 - 11 0 4. NP 9 94 73 37 86 38 85 82 84 54 73 5. Sum 1-4 36 69 79 26 71 47 78 67 42 60 73

Nan - Nanikon; Fill - Fa!landen; Bass - Bassersdorf; Diib - Dubendorf; Zh-G - Zilrich-Glatt; Opf - Opfikon; Nied - Niderglatt; Bill - Billach; Siad - Stadel; Glat - Glattfelden; Rhei - Rheinsfelden a Concentrations in µmol/m3; b Elimination = (CpE· CsE)/ CpE x 100 (%); CpE, CsE: concentrations in primary and secondary effluent 129

about 25 % of NPnEO was ultimatgly dGgradgd into wator. ThQ romainino ra- dioactivity of tritium was divided 33 % in the biomass and 33 % in the dissolved organic compound fraction. That gives the value for elimination of nonylphenolic compounds from the water phase of 58 %, and that value is in very good agreement with our value of 59±18 % (Table 27). Furthermore, Krawetz et al. (121, 123) determined also the loss of hydrophilic part of the NPnEO molecule, using the compounds with the uniformly labelled ethoxy chain (14C) being 91 %. This value completely corresponds to the value of the degradation of original NPnEO by the shortening of the ethoxy chain, which can be determined from our work. For example, weight eliminations of nonylphenolic compounds due to the loss of ethoxy chain, calculated for two typical plants Bassersdorf and Opfikon (Table 28), were 95 and 78 % respectively.

In order to explain the differences in the behaviour of nonylphenolic compounds in various sewage treatment plants, the elimination of the specific groups was compared with the operating conditions in the plant. Special attention was given to the total and specific load of the plant, sludge loading rate and ammonium elimination. As expected, the more efficient elimination was attained in the plants with low sludge loading rate and in those which operated at nitrifying conditions (Table 30). The remaining concentration of nonylphenolic compounds in the secondary effluents was in a good correlation with a very simple measure of the plant load, i.e. with the ratio between its actual load and its designed capacity (Figure 48).

~~~ 0 ig 100 zll.. w 0 80 y • 0.77K • 15.3 c: t :0.9035 0 ~ 60~~

~~EQ) 0 § 40 0 g> ·2 20 "iii E £ O.__..._..._..._...._....__.__.__.__.__.__.__.__ JO 20 30 40 50 60 70 80 90 I()() 110 120 Relative load of STP (%)~~

~~Figure 48. Correlation of the elimination of nonylphenol polyethoxylate surfactants and relative load of investigated sewage treatment plants 130~~

The correlation of the elimination rate of nonylphenolic compounds with nitrifying conditions, i.e. with elimination of ammonia, was very instructive. Positive correlations were obtained for the parent NPnEO, NP1 EO and NP (Figure 49), but not for NP2EO and NPEC. The data for the STP Nanikon do not fit well with the obtained regression lines and this could be explained by the special composition of the influent waste water in this STP which contains a great portion of the wastewaters from the detergent manufacturing chemical plant. Apparently, good correlations were found only for those nonylphenolic compounds which showed no (NPnEO, NP) or limited (NP1 EO) formation under aerobic conditions.

Mann and Reid (132) have shown that degradation of NPnEO depends on the temperature. The elimination of these surfactants in the real sewage treatment plants, determined by the nonspecific methods was considerably greater in the summer than in winter. Elimination rates for NP, NP1EO and NP2EO in the STP Zurich-Glatt in January, June and August are given in Table 31. For all types of compounds the elimination was better in summer (NP = 75-82 %; NP1 EO = 40- 72 %; NP2EO = 25-47 %) than in winter (65 %, 18 %, -2 %, respectively). Obviously, even in the plant which is very effective during the summer months, the elimination can be very low during the winter.

~~TABLE 30. Correlation of Elimination of Nonylphenolic Compounds with Operating Conditions in Sewage Treatment Plants~~

~~~ Sludge treatment NP-c load Elimination (%) loading plant N03· ratec total3 specificb NP-c NHafNH4+ (mg/L)~~

Nttnikon 2.3 0.38 36 95 1 7 0.30 Fttllanden 9.9 0.39 69 98 24 0.26 Bassersdorf 7.3 0.73 79 99 14.5 0.13 DObendorf 14.9 0.32 26 6 1.2 0.30 ZOrich-Glatl 49.6 0.29 71 7 0.20 Opfikon 23.2 0.86 47 1 6 0.4 0.52 Niederglatt 13.2 0.53 78 99 15.5 0.28 BOlach 9.8 0.47 66 97 1 7 0.58 Stadel 0.4 0.30 42 83 15.5 d Glattfelden 59 61 2.5 Rheinsfelden 73 99 1 5 a Total load (kg/day) .. CpE (mgtrn3 x Q (rW/day); b Specific load (kg/PEQ x day) .. total load (kg/day)/ PEO; c sludge loading rate = kg BOD/kg MLSS x day; PEO: Population equivalents; d: sewage treatment plant with 1rickling finer. 131

~~r NPnEO NPnEO, n=3-20 100 y = 0.40x + 30.4 100 0 r =0,8339 0 0-,Jf- !:: 80 80 :00-- c: .Q 60 60 0 - 40 0 40 ·=E (o) 9 y =0.17x + 76.5 [jj 20 20 r =0.9078~~

~~0 20 40 60 80 100 0 20 40 60 80 100 NH /NH~ Elimination 3 l°lol Elimination NH3/NH4 (°lo)~~

~~100 100 NP1EO 0 NP ~ 80 y = 0.7x -2.7 80 r = 0.8400 ·.;:::;g 60 60 0 ·= 40 E 0 y = 0.61 + 27.6 [jj 20 r = 0.9488 o foJ OL....LD.1---1.~-'---L-~J--· 20 40 60 80 100 Elimination NH3 INHZ (°lo!~~

~~Figure 49. Correlation of the elimination of nonylphenol polyethoxylates and their metabolites with the elimination of ammonia in the secondary treatment.~~

TABLE 31. Dependence of the Elimination Efficiency of Nonylphenol (NP), Nonylphenol Monoethoxylate (NP1 EO) and Nonylphenol Diethoxylate (NP2EO) in the Sewage Treatment Plant Zurich-Glatt upon Temperature (Season)

~~Date Temperature (°C) Elimination (%)a NP NP1EO NP2EO~~

31/01/1984b 10-13 65 18 -2 20/06/1983b 18 75 40 25 27/08/1984b 20 82 72 47 a Elimination= (CpE- CsE) I CpE x 100 (%); CPE, CsE: concentrations in primary and secondary effluent b 24-h composite sample. 132

~~3.3.5. Role of Nonbiologlcal Processes~~

Beside the biological processes the behaviour of the organic constituents in the sewage treatment plants is influenced by the physico-chemical processes, especially by the partition between water and solid phase and the exchange of matter with the atmosphere (191).

In the case of NPnEO and their degradalion products the most probable nonbiological mechanism is the adsorption on solid particles. The distribution of the main groups of nonylphenolic compounds in the effluents and sludge of the STP is shown in Figure 50. The mechanical treatment influences very little in the con- centralion of the parent NPnEO, n=3-20, but decreases considerably in the concentration of the lipophilic NP, NP1 EO and NP2EO. Since, the concentration of the most degradable compounds remained almost unchanged, the elimination of the lipophilic degradation products can only be explained by a nonbiological process i.e. by adsorption on the primary sludge. Physico-chemical elimination by lipophilic adsorption was observed also during the secondary (activated sludge) and tertiary (flocculation for the removal of P) treatment. Concentrations of the lipophilic NP, NP1EO and NP2EO are considerably lower after tertiary treatment, while the concentrations of more hydrophilic degradation products (NPEC) decrease only slightly. The importance of the adsorption of nonylphenolic compounds onto sewage sludge can be expressed by the ratios of their concentrations in the secondary effluent and the sewage sludge itself. The strongest one was the adsorption of NP (kNP = 10500 I/kg). The affinity towards the solid phase for other metabolites was significantly lower (kNP1EO = 1800 Ukg, kNP2EO = 900 Ukg, kNP2EC = 500 Ukg). The higher adsorbability of NP in comparison to other metabolites cannot be completely explained by its higher partition coefficient between water and organic solvents (chapter 3.2.2.), nor by its solubility (chapter 3.2.1.) which is even higher than for NP1 EO, NP2EO, or NP2EC. Additional experiments would be needed in order to explain the adsorption behaviour of the lipophilic alkylphenolic compounds on the sludge. The adsorption characteristics of the lipophilic nonylphenolic compounds influence considerably their distribution and mass flow in the sewage treatment plants. It should be kept in mind that nonylphenolic compounds leave a plant in two ways: in the liquid phase (secondary effluents) and in the solid phase (sewage sludge). 133

1393 I I f 1000 NPnEO ( n= 3-20) I I 500 I 500 I f I M I I E en I -""' I -0 <20 I <10 <10 <20 E 100 100 0 :::i. - I E :::i.. NP1 EO I NP2EO 500 500 c: c: 0 297 0 194 C\'I ...... -C\'I ijJ)\f,l~ ...... c: !)~~ 87 25 90 100 Q.I 100 2630 c: v _m. Q.I c:: v 0 500 c: v 500 NP 0 v 259 Q> "'::I 0 168 en -0 Q.I ::I ::I c:r 7 < 100 16 100 (/) NP1 EC, NP2EC 500 500 effluents sludges

~~250~~

~~...... ___23__ __. 100 100 ...___R__t~--p--2~--- SE TE AS OS~~

Figure 50. Distribution of nonylphenolic compounds in effluents and sludges of the sewage treatment plant at Uster. RS: raw sewage; PE: primary effluent; SE: secondary effluent; TE .tertiary effluent; AS: activated sludge; DS: digested sludge. 134

~~TABLE 32. Mass Fluxes of Nonylphenol, Nonylphenol Monoethoxylate and Nonylphenol Diethoxylate in Secondary Treatment~~

~~Sewage treatment Mass flux (gld) plant NP NP1EO NP2EO~~

~~SE 360 2210 1980 Zurich-Glatta ASC 280 390 135 AS/SE 0.78 0.18 0.07 SE 44 69 240 Usterb ASC 41 11 17 AS/SE 0.94 0.16 0.07~~

SE: secondary effluent; AS: activated sludge; a 30/06/1983, Q = 45000 m31d; activated sludge• 2200 kgtd; b 10/08/1983, Q = 12600 m3td; activated sludge =1100 kgtd; c Grab sample from the aeration tank.

Table 32 represent the mass fluxes of NP, NP1 EO and NP2EO resulting from a secondary treatment. Surprisingly the similar figure for the contribution of nonylphenolic compounds adsorbed on the sludge to the total mass flux was obtained for two different STP's. Almost half of NP (44-48 %) was released from the secondary treatment in adsorbed form. The contribution of adsorbed form was significantly lower for NP1EO (14-15 %) and especially for NP2EO (6-7 %). Obviously, in assessment of the efficiency of the solely biological elimination during the secondary treatment, the elimination via adsorption on the sludge should be taken into account. The calculation of the elimination rate by dividing the difference between the concentrations in primary and secondary effluent with the concentration in primary effluent (Tables 27-30) gives as the result an over- estimation of the biodegradation efficiency, especially for the lipophilic nonylphenolic compounds. For NP itself, biodegradation could be even a minor part of the total elimination. Table 33 presents the mass fluxes of nonylphenolic compounds for the entire sewage treatment procedure: nontreated sewage is taken as the input and secondary effluent and digested sludge output. The calculations are carried out on both molar and weight basis. As it can be seen, the significant part of the nonylphenolic compounds (about 36 o/o on molar basis) is discharged from STP via digested sludge. This is especially strongly pronounced for the NP of which 92~96 o/o were in. digested sludge and only 4-8 % in secondary effluents. 135

~~TABLE 33. Mass Fluxes of Nonylphenol Polyethoxylates and Their Metabolites in Sewage Treatment Plants~~

~~Sewage Mass flux Com- treatment plant pounds (kg/d) (moVd) RS SE DS DS+SE RS SE DS DS+SE~~

~~Ustera NP-c 14.2 3.7 1.4 5.1 30.2 11.6 6.4 18.0 NP 0.7 0.05 1.2 1.25 3.3 0.2 5.8 6.0~~

~~NP·c ZOrich-Glattb 540 11.7 4.8 16.5 94.5 39.6 21.8 61.4 NP 0.65 0.4 4.4 4.8 2.9 1.6 20.4 22.0~~

RS: raw sewage; SE: secondary effluent; DS: anaerobically digested sludge; NP-c: sum of nonylphenol polyethoxylates and their degradation products; NP: nonylphenol; a a = 12600 m3/d; dry matter flux in digested sludge =2200 kg/d; b a= 45000 m31d; dry matter flux in digested sludge - 4400 kg/d.

Adsorption of the NP on sewage sludge and preferable formation of this meta- bolite from other nonylphenolic precursors during the anaerobic stabilization of the sludge (108, 192) leads to the extremely high concentration of NP in anaerobically digested sewage sludge. Thus, the output flux of NP in the STP ZOrich Glatt was 7.5 times higher than the input flux. Only a smaller part of the mass flux of NP in anaerobically digested sewage sludge could be explained by the amounts of NP and their precursors present in the activated (secondary) sludge. This fact suggests the importance of the lipophilic adsorption on the primary sludge. Similar pattern of the surfactant distribution in STP was observed for linear alkylbenzene sulphonates (LAS) (193). NP and their precursors ad· sorbed on the primary sludge are not processed through the aerobic treatment and the possibility for their biodegradatlon in STP is considerably diminished. 136

~~3.4. Occurrence and Behaviour of Alkylphenol Polyethoxylates and Their Degradation Products in Natural Waters~~

~~3.4.1. Glatt River~~

~~3.4. 1. 1. Characteristics of the Investigated Area~~

The Glatt Valley is a very densely populated region in the northern part of Switzerland which includes a part of the agglomeration of Zurich. In the region covering an area of 260 km2 lives about 240,000 inhabitants, and also significant industrial capacities are located there (194). In Figure 51 showing a map of the Glatt Valley, with the locations of sewage treatment plants as well as sampling locations on the Glatt River are indicated. The Glatt River is the outflow of the Greifensee and is a tributary to the Rhine River. The discharge rate of the Glatt River fluctuates in the range of 3-9 m3/s, and on its short journey from the source to the mouth (35 km) it receives considerable amounts of treated wastewaters from about ten sewage treatment plants which include both mechanical and biological treatment (chapter 3.3.). In the lower part of the river up to 15-20 % of the water are treated wastewaters (194). The most important tributaries are Chimlibach and Chriesbach, the relatively small creeks (0.03-0.4 m2/s) which themselves have a higher share of treated wastewaters in their total flow. The Greifensee is highly eutrophic lake with a maximum depth of 32 m. It is stratified from spring to fall and mixed during winter. The Greifensee receives effluents from several sewage treatment plants.

~~3.4.1.2. Statistical Distribution of Concentrations~~

The summary of the determination of the nonylphenolic compounds in various surface waters of the Glatt Valley is given in Table 34. The Greifensee waters contained very low concentrations of NP, NP1 EO and NP2EO (<0.5 µg /L ), whereas the concentrations of these compounds in the Glatt River and the creeks Chriesbach and Chimlibach were much higher and varied in a wide range. The share of nonylphenolic compounds in the total dissolved organic carbon in the water varied strongly (0.03-1.01 %), and it was more uniform in the polluted part of the river (0.44-1.01 %}. The given values, especially those for the polluted part of the river, can be considered very high. 137

~~GLATT VALLEY SWITZERLAND~~

~~.... ,.l ······...... :~ ..l ...... f.urlch-Glatt ·········.. --@sampling station ...... • sewage treatment plant \ ...~ ···•·· 0 5 10 km '======'~~

Figure 51. Map of the Glatt Valley, Switzerland; Glatt River at : (A) Fii.llanden, o km; (8) Hagenholz, 9 km; (C) ROmlang, 15 km; (D) Rheinsfelden, 35 km; E: Chimlibach; F: Chriesbach; G: Greifensee; The diameters of the black dots correspond to the sizes of the sewage treatment plants: H: STP Zurich-Glatt;

Statistical distributions of the concentrations of different nonylphenolic compounds in the surface waters of the Glatt Valley are shown in Figure 52. Presented distributions include the results obtained from different locations and sampling times (season, part of day). Therefore, the concentrations of specific compounds vary in a large range: <0.3-45 µg/L for NP, <0.3-69 µg/L for NP1EO, <0.3-30 µg/L for NP2EO, <1-45 µg/L for NP1EC and 2-71 µg/L for NP2EC. Only 4 % of NP concentration values are higher than 5 µg/L in comparison to 55 °/o of NP1 EO, 36 % of NP2EO, 85 % of NP1 EC and 98 % of NP2EC. From these data it is obvious that carboxylated degradation products of 138

~~TABLE 34. Determination of Nonylphenol Polyethoxylates and Their Metabolites in the Surface Waters of the Glatt Valley, Switzerland~~

~~Compound Concentration (µgll)a Glatt Chriesbach ChimUbach Greifensee~~

~~NPnEO, n-3-20 <1.0-7.1 n.d. n.d. n.d. NP <0.3. 7.9 2.5. 6.7 1.0. 45 <0.2 NP1EO <0.3. 20 2.3 -69 1.2 - 15 <0.2 NP2EO <0.3. 21 3.3 ·26 3.0 - 30 <0.2 NP1EC <1.0. 29 45 <1.0 n.d. NP2EC 2.0 -59 71 56 n.d.~~

~~a Concentration range; n.d.: not determined.~~

NPnEO are more abundant than the ethoxylated ones, while alkylphenols are even less abundant. It should be mentioned that higher oligomers of NPEO were also detected in the samples (1-7.1 µg/L). The concentrations of the specific oligomers with 3-5 EO groups were very low (about 1 µg/L in the polluted part of the river), while the higher oligomers (nE0>6) could not even be detected (<1 µg/L ).

The ratio of the specific degradation products of NPnEO in the surface waters of the Glatt Valley reflects the composition of these components in the treated wastewaters (chapter 3.3.) which are discharged into them in considerable amounts.

~~3.4.1.3. Diurnal Dynamics of Concentration Variations~~

Diurnal dynamics of the concentration variations in the Glatt River reflects diurnal dynamics in the wastewater treatment plants (chapter 3.3.). Diurnal variations of the concentrations for all sampling stations and for different types of nonylphenolic compounds are shown in Figures 53 and 54. A typical dynamics with the minimal concentrations during the night is obvious for all nonylphenolic compounds on the stations situated downstream from the larger sewage treatment plants (Hagenholzbrilcke and Rumlang). The stations which are not under the direct influence of secondary effluents (FAllanden), or are far from the dominant discharge points (Rheinsfelden), no regularity was observed. Puring the measurements of the diurnal dynamics, sampling was done in such a manner that the 139 river flow velocity was considered, and it can be said thGt from all tho stations the same "water wave" was sampled. In spite of that, diurnal dynamics on the

~~40 NP n= 110 > 30 *u c CD & 20 NPIEC ....CD 40 n:48 LL 10 ~ 30 > 0 u I 3 5 7 9 11 13 15 rl 19 45 ~ 20 Concentration lµg/Ll C'" CD.... LL 10 NPIEO 20 n = 110 > 0 *u c CD :I C'" (I) NP2EC it 30 n = 48~~

~~>20 *u i 'ii- NP2EO & 10 20 n= 110 CD > .... u LL c 0 ~ 10 C'" CD LL.... 0~~

Figure 52. Statistical distribution of nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), (nonylphenoxy) acetic acid (NP1 EC), and [(nonylphenoxy)ethoxy}acetic acid (NP2EC) concentrations in analysed samples from the Glatt River. 140

~~~·b~~

~~NP1EO 8~~

~~8 NP2EO~~

~~c 5~~

~~I(.) : 2 d~~

~~0 10 1214 1 1 28/08/1984 29/08/1984 Time~~

Figure 53. Diurnal dynamics of nonylphenol (NP), nonylphenol monoethoxylate (NP1 EO), and nonylphenol diethoxylate (NP2EO) concentrations in the Glatt River at diffrent locations: (a) Fallanden, (b) Hagenholzbrucke, (c) Rumlang, and (d) Rheinsfelden 141

~~NP1EC~~

~~c 0 :::::ca 20 ...... d c ~ 10 c:~~

~~8 o~~!;==;~~;:::::J;;::::;;;;::=;~~-=-=~a::--"!::--:-:-~~"'::-:'--'----10 12 14 16 18 20 22 24 02 04 06 OB 10 12 14 16 18 20 22 24 20/oa/1954 29/00/1904 Time~~

~~60 NP2EC :::; ..... ~ 50 ~ c: d 0 40 ::::: lU ...... c: 30 CD () c: b 0 20 (.)~~

~~10 u-= C> --0- o a 0'--~~~,2~14~~,.-,1~8-2~0-2~2__..24__.02__.04,__06.._0~8~IO~l2__,14,__~..__1~6__..20__,,22___,2~4~-...-- 20/08/1984 29/06/1984 Time~~

Figure 54. Diurnal dynamics of (nony/phenoxy)acetic acid (NP1 EC) and [(nony/phenoxy)ethoxy]acetic acid (NP2EC) concentrations in the Glatt River at diffrent locations: (a) Ftillanden, (b) HagenholzbrOcke, (c) Rlimlang, and (d) Rheinsfelden 142 station at Rhelnsfelden does not show the regularity observed from the upstream stations. This indicates that hydrodynamic conditions (longitudinal and transver· sal dispersion; ref. 195) as well as physico-chemical and biological processes can considerably influence the distribution of nonylphenolic compounds in the river.

In spite of the diurnal concentration variations of the nonylphenolic compounds, the measured values from the ROmlang station are always higher than from the other locations, because it is located immediately after the largest sewage treat· ment plants in the Glatt Valley, Zorich-Glatt, and Opfikon. The lowest concentrations were always recorded from the location at Fallanden which does not directly receive any secondary effluent, but actually has the characteristics of the lake (Greifensee) from which it flows.

~~3.4. 1.4. Distribution of Concentrations and Mass Flows on the Longitudinal Profile and Seasonal Variations~~

The concentration levels of NP, NP1EO and NP2EO in various locations and seasons, are shown in Figure 55. As expected, maximal concentration was always recorded from the location at ROmlang and minimal at Fi:illanden. The concentrations were, especially for NP1 EO and NP2EO, higher in winter (November, December, January) than in summer {July, August). The observed seasonal variations could be explained by the lower input of the nonylphenolic compounds via treated wastewaters, by the variations in river flow (dilution), and by the seasonally dependent biological and physico-chemical processes (biodegradation and photochemical degradation). Since the flow of the Glatt River in summer months is usually very low with low concentrations for this time of the year could not be explained by the dilution. Besides that, the decrease of the concentration during summer is not the same for all nonylphenolic compounds: it is much less pronounced for NP than for NP1 EO and NP2EO which could be explained by better biodegradability of the last two. Based on the same process, a greater efficiency of the sewage treatment plant could be presumed {chapter 3.3.) which would contribute to the lower load of the river in the summer period.

Some of the mentioned mechanisms which determine the concentrations of the nonylphenolic compounds in the river can be documented by the results presented in Figure 56 which show the distribution of concentrations and mass fluxes of 143

~~NP Rumtang c 8 0 ~ 6 Hagtnholzbri.icke Rheinsftldtn !: 4 c ~ 2 a oL.-.L.L'""!""~:;i_-1,:L.J..,.l....L::!--L--1x,,l...J~,LI:-:cx1!--1..___iJ...l::-1-.Jl.J.-l--· 0 QWQ QWU Season (month)~~

20 NP1 Ri.imlang :J.._ 18 Cl 16 ::i. ~ 14 HagenholzbJUcke Rhcinslelden § 12 ~ 10 !: 8 c ~ 6 c Falonden 0 0 2 oL-.L.1.""""...c::J'-'-...l....LL..L.L.L..l.__lLJXLLILLXLl.l_~XJ..JWILLXILL.....,. XII VII XII XU VII XII Season

22 NP2EO RUmlang :J 20 e;, 18 2- 16 c 14 0 .. Rheinsfcldtn ~ 12 ~ ... 10 Hagenholzbriicke i e 0 - § 6 0 4 ~a:n:n - r o1--ix-'-'~,-xw1L.L-LX.L..l.IJ...lXWILI-...l...X.LLl...L.lXJl....L.....l...XL.l..l.L.LX~l..L-- Xll VII XII XII VII Xii XII VII XII XII VII XII Season !month>

~~Figure 55. Dependence of nonylphenol (NP), nonylphenol monoethoxylate (NP1 EO), and nonylphenol diethoxylate (NP2EO) concentrations in the Glatt River upon location and season (grab samples). 144~~

~~20 _ 18 ~ o NP 0,16 e,. ::::i.. NP1EO -- 14 v NP2EO § 12 :;::: ~ 10 16 8 -0 § 6 () 4~~

~~9 15 Distance Ckm) 35~~

~~o NP 10 t:. NP1EO 9 v NP2EO~~

~~-"O 8 0, 7 =6~~

~~0 I 15 Distance (km) 35~~

Figure 56. Longitudinal concentration and load profiles of nonylphenol (NP), nonylphenol monoethoxylate (NP1 EO), and nonylphenol diethoxylate (NP2EO) in the Glatt River. Arithmetic mean and the range of one standard deviation for five determinations froin the winter period are presented. 145

NP, NP1 EO and NP2EO on tho longitudinal profll& of th«) Glatt River. Cach point was obtained as an arithmetic mean of five determinations in the winter period and the range of one standard deviation is marked. The already mentioned variations in concentration levels, for different locations, can be clearly observed. The water of the Glatt River, which at its beginning (Fallanden) is characterized by low concentrations of nonylphenolic compounds, receives considerable amounts of secondary effluents from sewage treatment plants (Fallanden, Nani- kon, Bassersdorf and DObendorf) so that the concentrations of the determined compounds increase 3-6 times. A further increase in concentrations of all investigated compounds occurs after the inflow of the secondary effluents from the sewage treatment plants Zurich-Glatt and Opfikon (location at Rumlang). In the lower part of the river it receives secondary effluents from 4 more sewage treatment plants (Niederglatt, BOlach, Stadel and Glattfelden), but the concentrations of the nonylphenolic compounds are lower at the Rheinsfelden than at ROmlang. Since the mass flows of NP1 EO and NP2EO also show a decrease in the lower part of the river, it must be obviously a result of an elimination process. The concentration profiles and mass flow profiles for NP1 EO and NP2EO are almost the same, which indicates that their transformation in the aquatic environment is very similar. This similarity in the behaviour of NP1 EO and NP2EO was observed also in the sewage treatment plants (chapter 3.3.). Contrary to this, the mass flow distribution curve for NP indicates that the elimination of this compound, in the lower part of the river, was much smaller.

The behaviour of nonylphenolic compounds in different seasons was investigated in more detail on the basis of composite samples collected in February and August of 1984. Water temperature in February and in August were 4.8-5.6 °C and 18-20 °C, respectively. Each concentration value shown in Figure 57 represents arithmetic mean of 12 determinations (12 successive two-hour composite samples) and the range of one standard deviation is given. The longitudinal concentration profiles of the nonylphenolic compounds obtained on the basis of this sampling (Figure 57) were the same as those described for grab samples (Figure 56). The mass flow profiles of nonylphenolic compounds are presented on the lower part of the Figure 57. The results of winter sampling have shown the increase of mass flows for all determined degradation products of the NPnEO on the whole river profile including the critical river section from ROmlang (15 km) to RheinsteJden (35 km). The mass flow of NP, NP1 EO and NP2EO at Rheinsfelden during winter was about 15 % higher than at ROmlang. Contrary to that the mass flows of NP, NP1EO and NP2EO on the same river section have 146

~~shown a decrease during summer {30-35 %), Indicating the much hlgh1:1r efficiency of elimination processes.~~

~~70 Summer~~

~~24 60 22 Winter I :J 20 ate :I. o NP -16 A NP1EO + NP2EO s 14 cNPEC :; 12 ::c •o oNP 8 8 A NP1EO + NP2EO 6 6 (.) 4~~

~~0 I 35~~

~~22 Summer 20 Winter 20 - 18 18 .....,, 16 0) ~ 16 Cl oNP ~ 14 ~ 14 A NPIEO + NP2 EO 12 12 x )( c NPEC ~ 10 ~ 10~~

~~9 15 35 15 35 Distance (km) Distance (km)~~

Figure 57. Longitudinal concentration and load profiles of nonylphenol (NP), nonylphenol ethoxylates (NP1EO+NP2EO), and nonylphenoxy carboxylic acids (NP1 EC+NP2EC) in the Glatt River tor the winter (left) and summer (right) period. Each point represents an average of 12 2-hours composite samples.

It can also be observed that the mass flows of NP and NP1 EO+NP2EO at the most loaded location (Rumlang) are significantly lower during summer (0.9 and 3 kg/day) than in winter (1.3 and 12.3 kg/day). However, it is obvious that this difference is much less pronounced for the biologically more easily degradable NP1 EO and NP2EO than for NP. 147

The data for the concentrations of NPEC are lacklng ror the winter period, and therefore no seasonal comparison for those important degradation products can be done. However, summer profiles of concentration and mass flow for NPEC clearly show that they are most resistant degradation products of NPnEO in the river. While in the summer period mass flows of all remaining degradation products of NPnEO, between ROmlang and Rheinsfelden decrease, the flow of NPEC increases from 20.5 to 23.5 kg/day (increase of 15 %).

~~3.4.1.5. Analysis of Input and Output Mass Fluxes~~

On the basis of composite sampling during the summer of 1984 a study of mass fluxes of the nonylphenolic compounds considering various sources were made. During this analysis, the nonylphenolic compounds were divided into several groups according to the characteristic behaviour in aquatic environment as it was done in Chapter 3.3., while the Glatt River was considered as a large chemical and biological reactor.

A detailed analysis of 11 wastewater treatment plants in the Glatt Valley, which represent the main sources of the NPnEO and their degradation products, has given the insight into the input of the investigated compounds from these specific sources (Chapter 3.3.). The sampling of one-day composite samples from the wastewater treatment plants was performed in August 1984 with the simultaneous gathering of the composite samples from the Glatt River (Chapter 3.4.1.5.). The source of the Glatt River, lake Greifensee (Glatt-F~llanden), was considered as another specific source of the nonylphenolic compounds. The output mass flow from the Glatt River was determined from the results at the location of the Rheinsfelden which is located near the mouth of the Glatt River where it joins the Rhine River. The results shown in Table 35 indicate that the main part (over 95 %) of nonylphenolic compounds enter the river by direct discharge of secondary effluents, and only a small part originates from the Greifensee. All the results were calculated in mol/day units, because it enables a direct comparison of different nonylphenolic compounds. The loads of specific nonylphenolic compounds ranged from 5.1 mol/day (4.9 %) for NP, 24.3 mof/day (22.5 %} for NP1EO + NP2EO, 55.2 mof/day (51 %) for NP1 EC + NP2EC to 23.4 mof/day (21.6 %) for NPnEO. It follows that the total input was 108.2 mol/day. 148

~~TABLE 35. Mass Fluxes of Nonylphenol Polyethoxylates and Their Metabolites in the Glatt River, Switzerland~~

~~Location Mass flux (moVd) NPnEO NP1EO+ NP NP1EC+ NP-cC (n-3-20) NP2EO NP2EC~~

Input: 1. Nltnikon 0.4 1.0 0.4 1.3 3.1 2. FlUlanden 1.0 0.5 0.1 4.3 5.9 3. Bassersdorf 0.4 0.4 0.1 2.1 3.0 4. DObendorf 5.1 6.3 1.3 12.4 25.1 5. Zurich-Glatt 5.8 3.7 0.8 18.2 28.5 6. Opflkon 7.4 8.7 1.8 6.8 24.7 7. Niederglatt 1.5 1.6 0.4 2.7 6.2 8. BOlach 1.7 1.6 0.2 2.9 6.4 9. Stadel 0.1 0.2 0.03 0.2 0.53 Sum1-9a: 23.4 24.0 5.1 50.9 103.4

~~10. Glatt-Fallanden <0.1 0.3 0.2 4.3 4.8~~

~~Total Input (1-10): 23.4 24.3 5.3 55.2 108.2~~

~~Output: 1. Glatt-Rheinsfelden 2.8 7.2 2.0 70.1 82~~

~~Elimination (%)b: 88 70 62 -27 24~~

~~a Secondary effluents from sewage treatment plants; b Elimination• (Minput ·Moutput) I Minput x100 (%) c Sum of nonylphenol polyethoxylates and their metabolites.~~

The sources of input of the NPnEO and their degradation products on the longitudinal profiles are not evenly distributed. About 75 o/o of the total input enters the river in the middle part, between 7th and 12th kilometers (sewage treatment plants DObendort, Zurich-Glatt and Opfikon). On the basis of this data it can be estimated that the hydraulic retention time, for water containing the main part of the nonylphenolic compounds, is about 10-15 hours. This time could be suffi· cient for various physico-chemical and biological processes (for example, hydraulic retention time in the sewage treatment plant ZOrich-Glatt is about 6 hours). The total output of nonylphenolic compounds from the observed system is 82 mol/day. The comparison of this value with the total input amount (108.2 mol/day) shows that the elimination of 24 o/o occurred. This is much lower than in the sewage treatment plants where the elimination of the nonylphenolic compounds was 26-79 o/o (Chapter 3.3.). However, it should be mentioned that the composition of the nonylphenolic compounds in the river was rather different 149 from that in the wastewater before the biological treatmsnt: the majority of the more easily biodegradable compounds (NPnEO) were already eliminated in the sewage treatment plant, and the remaining compounds belong to the group which is more resistant to biodegradation. These are the very compounds which enter the river. If specific group of nonylphenolic compounds is considered sepa- rately, a much clearer picture is obtained. The composition of nonylphenolic compounds for the input and output are given in Figure 58.

~~INPUT OUTPUT~~

~~2,S°lo 9%~~

~~~ NPnEO ln=J-20, -NP ~::::::;:;:;::} NPlEO + NP2EO - NP1EC+NP2EC~~

Figure 58. Relative abundance of nonylphenol po/yethoxylates (NPnEO) and their degradation products at the input and output from the Glatt River (composite samples from August 1984); nonylphenol (NP), nonylphenol ethoxylates (NP1 EO+NP2EO), and nonylphenoxy carboxy/ic acids (NP1 EC+NP2EC).

The biggest change was observed for NPnEO (decrease from 21 % to 3.5 o/o) and for NPEC (increase from 51 to 85 o/o). The elimination for NP, NP1 EO + NP2EO, and NPnEO in the Glatt River was 62 %, 70 %, and 88 o/o , respectively, which is very similar to the values obtained in the sewage treatment plants (Chapter 3.3.). Contrary to this, a negative elimination, i.e. formation in the river, was observed with earboxylated compounds (NP1 EO + NP2EO); the mass flow at the output (70.1 mol/day) was 27 % higher than the total input amounts (55.2 mol/day). The explanation for the elimination of NPEO and the formation 150

of NPEC is biotransformation. Transformation of NPEO into the oorrHpondlng NPEC is a very probable mechanism (Chapter 3.3.), especially under aerobic, nitrificating conditions. The measurements of dissolved oxygen, nitrates and ammonia during the investigation showed that a favourable conditions existed for such transformations.

~~3.4.1.6. Sediments~~

In order to get a better idea about the distribution of nonylphenolic compounds in the Glatt River the concentrations of the lipophilic degradation products (NP, NP1 EO and NP2EO) in the sediment were measured (Table 36). ·.. The samples were collected mostly from the same locations as the water samples (Figure 51) and they show a very similar distribution of the concentrations on the longitudinal profile. The only unexpected result was a relatively high concentration in the sample from Fallanden (1.02 µg/g). However, it should be mentioned that the samples were collected with a relatively primitive technique (mud collected manualy near the bank) which makes the comparison of specific locations less reliable. The highest concentrations of NP, NP1 EO and NP2EO were determined at the locations of ROmlang and OberhOri (> 10 µgig).

~~TABLE 36. Concentrations of Nonylphenol, Nonylphenol Monoethoxylate and Nonylphenol Diethoxylate in Sediments~~

~~Sample/Location Concentration (µglg)a NP NP1 EO NP2EO~~

Chriesbachb, rrud 2.83 0.29 n.d. Chriesbachb, sand 0.19 0.10 0.08 Fallandenc, mud 1.02 0.66 0.30 HagenholzbrGckec, mud 0.51 0.52 0.24 ROmlangC, mud 5.61 6.28 2.72 Rheinsfeldenc, mud 1.38 1.21 0.52 OberhOrid, mud 13.1 8.85 2.4 a concentration Is expressed on the dry weight of the sample; b 20/09/1984, EAWAG; c August, 1984; d August, 1984

River mud rich in organic matter contained considerably higher concentrations of NP, NP1 EO and NP2EO (Table 36) than sand collected from the same location (Chriesbach, DObendorf). The difference is much more pronounced if concentrations of NP were compared (factor 15) than the concentrations of NP1 EO (Factor 151

3). This could be explained by better adsorption ol thtl NP onto the pardcles rlcll in organic matter due to its greater lipophilicity (Chapter 3.2.2.), as already observed for sewage sludge, and perhaps also by greater persistence towards biodegradation which could take place on particle surfaces. In the other sediments collected in Glatt River (mostly muds rich in organic matter) NP dominated over NP1 EO and NP2EO, although their ratio in water was reverse. Ratios between the concentrations in water and sediments ranged as follows: NP= 364-5100, NP1EO = 158-2200 and NP2EO = 141-3000. However, it should be stressed that given values do not represent the partition coefficients between water and suspended matter, because the concentrations in water were measured in nonfiltered samples and thus included both dissolved and adsorbed part of the determined compounds. Unfortunately, we have not at our disposal the measurements of dissolved NP, NP1EO and NP2EO, but it can be expected that the real enrichment factors are greater than the given ones.

~~3.4.2. Comparison of Swiss Rivers~~

It was shown in the previous chapter that AP and APEO represent an important class of organic pollutants in the highly polluted Glatt River. However, for a more complete assessment of their ecotoxicofogical importance it was necessary to investigate the occurrence of these compounds in different types of rivers. The investigations described in this chapter were carried out within the frame- work of continuous analytical surveillance of swiss freshwaters (Oas nazionale Programm fOr die analytische Daueruntersuchung der schweizerischen Fliess- wasser - NAOUF) in the period 1983-1984 (165).

~~3.4.2. 1. Characteristics of Investigated Rivers~~

The monitoring of concentrations of NP, NP1 EO and NP2EO was performed by the analysis of weekly composite samples on several rivers of different type (Inn, Aare, Limmat, Rhine, Birs, Kleine, Emme, Glatt and Thur; Figure 59). The basic characteristics of the investigated rivers at the sampling stations for which measurements were performed in a longer continuous period of over one year (May 1983 - June 1984) are given in Table 37. 152

~~Figure 59. Map of Switzerland with indicated sampling stations on the different rivers~~

~~TABLE 37. Catchment Areas and Flows of the Investigated Swiss Rivers (ref. 165)~~

~~River/Location Catchment No of River flow (m3/s) area inhabitants (km2) (in 1000) average 01s 0$47~~

Rhine-Laufenburg 34100 5900 1146 1889 619 Limmat-Gebensdorl 2396 800 106 219 48.9 lnn-Martinsbruck 1945 21 57.3 164 8.22 Birs-MOnchenstein 911 141 15.1 44.9 3.03 Glatt-Rheinsfelden 416 319 9.36 20.3 4.11

~~01 a:R!ver flow achieved less than 18 days/year; Qs47:River flow achieved at least 347 days/year.~~

The Inn belongs to the bigger swiss rivers and its catchment area is predominantly of alpine character, which means that this river has large flow variations during the year. The Inn catchment area includes a high-altitude alpine valley which is relatively thinly populated and that results in low load of the river (370 inhabitants/(m3/s)). The Rhine River at Laufenburg (Q = 1141 m3/s) has a very 153

large catchmAnt arga (34100 km2) ltR hydrologiogl r()gimo I~ ~trongly influonood by the left tributaries Aare, Lim mat, and Reuss. In. comparison to other swiss rivers, the Rhine River at Laufenburg can be classified as an averagely polluted river (5150 inhabitants/(m3/s)). The River Ummat flows through a densely populated area (Zurich-Baden) and receives a relatively high anthropogenic input. Its hydrological regime is determined by the fact that it flows out of Lake Zurich so that the flow does not show great seasonal variations. The Glatt and Birs represent smaller rivers (Q = <20 m3/s) characterized by large loads from different anthropogenic sources. This is especially valid for the Glatt River which is with a load of 34,000 inhabitants/(m3/s), the most polluted swiss river. Besides that, the Glatt River flows out of the Greifensee, a small euthrophic lake, with a high DOC concentrations. Therefore, the concentrations of DOC in the Glatt River are often larger than 2 mg/L which is according to swiss regulations the maximum allowed for natural waters (165). Hydrological regime of the smaller rivers, especially river Birs, is characterized by extremely high flow variations (during the rain storms the flow could increase 5-10 times in a very short period of time).

The average share of wastewaters in the total flow of the swiss rivers is about 2 %, but for the highly polluted rivers like the Glatt River and Birs this percentage could increase up to 20 %. Therefore, it is important to know in each particular case which is the percentage of the wastewaters which are treated in the sewage treatment plants before being discharged into the river. It can be seen from Table 38 that the highest percentage of treated wastewaters is received from the most polluted river (Glatt). This significantly diminishes the expected extremely high organic load of the Glatt River. The least favourable ratio between the specific load (inhabitants/(m3/s)) and percentage of treated wastewaters is the Birs River.

~~TABLE 38. Anthropogenic Load of Investigated Swiss Rivers (ref. 165)~~

~~River/Location Specific loada Wastewater DOC (mg/L) treatment!> (o/o)~~

Rhine-Laufenburg 5150 83 2.1 Limmat-Gebensdorl 7550 94 1.9 lnn-Martlnsbruck 370 70 0.8 Birs-MOnchenstein 9350 61.5 2.2 Glatt-Rheinslelden 34000 98.4 3.8 a specific load • (no. of inhabitants) I (river flow); (inh. Sfm3>; b o/o of wastewaters Included In sewage treatment; DOC: dissolved organic carbon. 154

~~3.4.2.2. Distribution of Concentrations~~

The results of NP, NP1 EO and NP2EO determinations in the five mentioned swiss rivers are presented in Table 39 and Figures 60, 61 and 62. In order to get a better idea about the concentration relationships of specific alkylphenolic compounds, the results are expressed in molar units (nmol/L}, and for simpler comparison with literature data concentrations in µg/L are also given. In the literature there are very few results about the quantitative determinations of AP and APEO in natural waters (144). Taking into consideration their abundance in the so far investigated freshwaters as well as their ecotoxicological importance the lack of such determinations is not justified (108). These toxic compounds are not even on the EPA list of the priority pollutants, although some less toxic alkylphenols are listed (196). The concentration values of NP, NP1 EO, and NP2EO found in Table 39 prove the fact that these compounds are present in all the swiss rivers in measurable (>0.1 µg/L) and sometimes in very high concentrations. The range of measured concentrations is very large, from 0.5 nmol/L (0.1 µg/L) to over 250 nmol/L (55 µg/L}. Similar concentrations were also measured in the river Sava, near Zagreb where it was also shown that AP and APEO represent the dominant group of anthropogenic phenolic compounds (197).

The statistical distribution of concentrations for all the rivers shows departure from normal distribution (Figure 60-62) so that the arithmetic mean is not the best measure of the average value. Occasionally very high results have decisive influence on the arithmetic mean and the dispersion of results (relative standard deviation) is very high. In such cases a much better measure ment of the average value is the median, and the comparison of the rivers was made on the basis of this statistical value. The relationships for the concentrations of NP and NPEO in the different rivers could be presented by the following decreasing series:

~~NP: Rhine > Inn > Glatt > Limmat > Birs~~

~~NP1 EO and NP2EO: Glatt> Limmat > Birs > Rhine> Inn TABLE 39. Concentration of Nonyfphenol, Nonylphenol Monoethoxylate and Nonylphenol Diethoxylate in Swiss Rivers, 198311984~~

~~River/Location Compound Noof (nmollL) (µg/L) samples8 Range x (s)b Median Range x (s)b Median~~

Rhine River- NP 59 <0.45-252 57.0 (54.9) 37.3 <0.1-55.4 12.5 (12.1) 8.21 Laufenburg NP1EO 58 <0.38-36.0 5.76 (6.10) 3.98 <0.1-9.50 1.52 (1.61) 1.05 NP2EO 59 <0.32-20.8 3.54 (4.48) 2.27 <0.1-6.41 1.09 (1.38) 0.70

Ummat• NP 61 <0.45-125 17.5 (22.0) 10.0 <0.1-27.5 3.85 (4.84) 8.20 (]I Gebensdorf NP1EO 61 <0.38-54.5 11.7 (9.90) 9.46 <0.1-14.4 3.09 (2.61) 2.50 (]I NP2EO 61 <0.32-119 9.2 (15.5) 5.19 <0.1-36.7 2.83 (4.n) 1.60

Inn- NP 68 <0.45-171 39.7 (35.5) 22.5 <0.1-37.6 8.73 (7.81) 4.95 Martlnsbruck NP1EO 65 <0.38-65.5 4.11 (10.2) 0.76 <0.1-17.3 1.09 (2.69) 0.20 NP2EO 65 <0.32-121 4.62 (16.2) 0.32 <0.1-37.2 1.42 (4.99) 0.10

Birs- NP 61 <0.45-36.8 10.0 (7, 10) 8.63 <0.1-8.10 2.20 (9.56) 1.90 Munchenstein NP1EO 61 <0.38-39.8 6.81 (7.27) 4.92 <0.1-10.5 1.80 (1.92) 1.30 NP2EO 61 <0.32-92.9 6.67 (11.9) 4.22 <0.1-28.6 2.05 (3.67) 1.30

Glatt· NP 61 <0.45-94.5 16.1 (15.9) 12.7 <0.1-20.8 3.54 (3.50) 2.79 Rheinsfelden NP1EO 61 2.65-122 21.1 (18.7) 15.9 0.7-32.2 5.57 (4.94) 4.20 NP2EO 61 3.25-103 18.5 (16.6) 13.6 1.0-31.7 5.69 (5.11) 4.19

~~a weakly composite samples; b arithmetic mean and standard deviation; 156~~

100 -"------:;:::.::::~;;;.:.;,:;,,..... - 100 ".. ---·-·--·.:;.:::.::::.::.:-"="-~~'"''"'"""-· -:;. ;•"" .,,. 75 -- --.. -- ·;.;"''"''=::: ...... -- . -· ------. - 75 ·-·····/···--···········---·-··•"••·········-· ~ 50 _____ ,:______>- • g 50 -···/····---·····---·-·-··-············· Q) : • 1 !C" 25 -' 25 1 :ti-r·-----·------··-···--· 5-Q) '-;-!-···········-····················: i : itG> ·Iirli• .cl .::L it lill. .... 0 64 128 191 255 0 25 so 75 tOO Concentration (nmol/Ll Concentration (nmol/Ll

_ 100 -----······-~::::;:;.;.;.;.;.;.;.;:.;.:m:.;.-;"'""''" 100 --·······-·-····-·-·;:.:.::.:.:;:::.::.:=-· .,,. /~· - 75 ----··r·-----···--······-·-··-----·---··- *- 75 ··-··-----·/------·-·------/ >- : i >- .... ~ so 11·---···-·-···-·--·------···--·· ~ 50 ·-···1------·------c- 25 U,.,.------·· 5- 25 ····ri·r··-·············-············· ~ ip Q) u.. .d:l1L.. . . U: V:'di : i .111.1: 0 33 65 130 0 10 20 30 40 Concentration (nmol/Ll Concentration (nmol/Ll

~~100 ..... -·- .. --·-·····-;:::.:.::.;:... ::.:":.:.;;.,,..-~~

*- 75 ··············r·-··-··-·············-··.,.,.. .. .,,.,. /- > ,.,,.·"' g so ···-1--···--········-·····------·· ~ ;·. . ~ 25 ml·-----··:·:·------·------LL. ·:::h:il:! ... . 0 44 88 131 175 Concentration (nmol/ll

~~Figure 60. Statistical distribution of nonylphenol concentrations in the samples from different swiss rivers: (AJ Rhine, (BJ Ummat, (CJ Inn, (DJ Glatt, (EJ Birs 157~~

10 ----·--·-----·;.:.::.:.::.:.::.:::::::.::=.::.:.:::.:::.::... • ~ ~·/ - 75 ------/------>- (,) § 5 ----1------!CT ...G> g':s 25 "" ·1··············-·-·o········· -···- ·· . IJ,. it f 1111. . . 0 30 IJJ 0 31 63 94 125 Concentration Cnmol/U Concentration Cnmol/U

100 • ------,,,,,,--·.,,,... ·-----;;.:.:.::.:.::.:.::.:.::~· - 100 ---.----·--··:;.:::.:.;_::..:::.:::.::=::t:.:....--• (/.- ,,/' ti. F- 75 ------7------75 ·-----1------50 ___ i)' 5 •••.• / ...... f l ______

CT 25 • ······························---·- I 25 --V.1-1------·- G> it VI l1i1 • ••• . it I II 0 15 30 45 60 o n 23 34 .45 Concentration Cnmol/Ll Concentration Cnmol/U

~~_ 100 r--·-;.::.:.::;:.:.:.:::;.;.::.•.:.::::.:.:.::.•.::•.:::.::•-·~~

~~: 75 ~I(~:----·------·-- g 50 ------G> :s : G>CT 25 ··························----·--···---• it d.. . . . 0 18 35 53 70 Concentration Cnmol/U~~

~~Figure 61. Statistical distribution of nonylphenol monoethoxylate concentrations in the samples from different swiss rivers: (A) Rhine, (B) Limmat, (C) Inn, (D) Glatt, (E) Birs 158~~

~~~ I:: .-:::::::;:~:~~~~:~~~~:~~~~~:~:~ >. / ~ 50 ----{------~~

~~[ 25 ill·;-1------i' I I • . • • . . . 0 6 13 19 25 0 26 53 79 105 Concentration (nmoD Concentration (nmoll~~

~~100 ·------_:.:;:.:.=::.:.;::.:.:.:.:.:.:.::.:.;.::"".::::>.. - 100 -----7:.:.:.:.:.:.:.:.:.:.:.:.:.::.:::.::.:.:.:.:.::.::.::-~~

~ 75 ·vi--~:------*: 75 :11------··- / (,) .. (,) 50 : ------~ 50 v;------· ~ ~ i g 25 ~-i------...g 25 1-1------. I at i i i : : 11. d ! 0 31 63 94 125 0 24 48 71 95 Concentration (nmoD Concentration (nmoD

~~0 31 63 94 125 Concentration (nmoll~~

~~Figure 62. Statistical distribution of nonylphenol diethoxylate concentrations in the samples from different swiss rivers: (A) Rhine, (8) Limmat, (C) Inn, (D) Glatt, (E) Birs 159~~

~~If molar concentrations of all m8asur8d nonylph8nolic compounds :;iro s.ummod, the following relationships can be written:~~

~~Rhine > Glatt > Limmat > Inn > Birs.~~

The relationship between NP and NPEO concentrations in the different rivers is not constant. Only in the Glatt River was NP less abundant than NP1 EO and NP2EO, while in the other rivers it was the dominant alkylphenolic compound. The ratio of NP/NP1 EO was for Glatt 0.8, for Limmat 1.06, for Bi rs 1.75, for Rhine 9.4, and for Inn even 29.6. It is obvious that the ratio of NP/NP1 EO/NP2EO for the Inn and Rhine Rivers is basically different from that for the Birs, Glatt and Limmat. The reason for the observed difference could be the specific source of input (higher contribution of effluents from anaerobic fermenter} or the specific transformation of nonylphenolic compounds, which probably takes place in the river itself. It should be kept in mind that NP shows much higher persistence towards biodegradation than NPEO (Chapter 3.2.3.). A special surprise were the high concentrations of the NP measured in the river with the smallest specific load (Inn). The Inn River flows through a rather low populated alpine region without any significant industry, and so far no explanation can be found for the high concentrations of NP in this river.

Besides NP, NP1 EO and NP2EO other degradation products of the NPnEO were also found in the analysed samples, but they were not measured systematically. In several samples the concentrations of NP1 EC and NP2EC were determined and they were about 1 µg/L. In river Sava, near Zagreb, the measured concentrations of NP2EC were in the range of 0.1-5 µg/L.

The measured concentrations of NP have a great ecotoxicological importance, because they are only one order of magnitude lower than the concentration at which toxic effects on the freshwater organisms were observed (107, 150). The fact that the concentrations of NP were determined in weekly composite samples gives the reason for the assumption that in certain shorter periods river water could have contained considerably higher concentrations (>100 µg/l). These are the values which are very close to the concentrations at which lethal toxicity for the freshwater organisms was observed (150). For comparison, the concentrations of very toxic polychlorinated phenols, which were determined within the same monitoring programme (165) in the almost identical samples, never ex- ceed 1 µg/L, and the usual concentrations ranged between 0.1-0.3 µg/L. This is 10-100 times lower than the concentration of the NP. It follows that the ratio be- 160 tween the measured concentration and the one at which the toxic effects on me freshwater organisms were observed is much critical for NP than for the somewhat more toxic polychlorinated phenols.

~~If the investigated rivers were compared according to the content of dissolved organic carbon the following relationship would be obtained (165):~~

~~Glatt > Birs > Rhine > Limmat > Inn~~

This relationship differs considerably from the one that results from the comparison of concentrations of the NP and the NPEO. Keeping in mind the previous discussion about the ecotoxicological importance of the measured concentrations of NP, it clearly demonstrates that DOC cannot be accepted as the only criterium for assessing the organic pollution of the investigated rivers. The share of the determined nonylphenolic compounds in the total dissolved organic carbon varied for particular rivers, from 0.3 % for Birs to 1.4 % for Inn.

The lack of positive correlation between specific load of a river (inhabitants/(m3/s)) and concentration of nonylphenolic compounds, could indicate either the importance of some specific sources of their input or it could indicate their different behaviour (chemical and biological transformation} in various freshwaters.

~~3.4.2.3. Influence of Hydrological and Seasonal Conditions~~

The decrease of the pollutant concentration through dilution depends on the ratio of wastewater flow and the river flow. If it is presumed that the input of a certain substance is constant in a given period of time, the dependence of the concentration upon the river flow can be expressed as:

C=Co+Aw/O (20} where Co and C represent the concentration of the substance before and after the input of wastewater, Aw is the flux of the substance discharged into the river (g/s) and Q is the river flow (m3/s). A simple dilution effect given by the above equation can be expected only with the conservative parameters (for example chloride; ref. 165), while with biologically and chemically reactive substances it is difficult to observe the expected 161

correlation. This is well illustrated in Fig. 63, which shows the dependence of NP concentration on the flow rate for the Limmat, Bi rs, Rhine and Inn.Rivers. NP was chosen to examin this correlation because it is, in comparison with other determined substances, the most persistent towards biodegradation, and it was expected that it should have a more conservative behaviour. However, it can be seen that the expected correlations are low for all rivers. For the river Inn the dispersion of points is so wide that it is difficult to observe any regularity. This shows that in the river Inn dilution is not the main factor which influences the concentration variations. In this case irregular input dynamics as well as the influence of the season has to be presumed. The expected dependence of NP concentration upon the river flow is perhaps better pronounced for the river Limmat (Figure 638), but dispersion of points is still rather wide. Separation of results according to the season would probably give a much clearer dependence (165). The relationship between the concentration of NP and flow rate for the Rhine River at Laufenburg (Figure 630) suggests partially positive correlation with the river flow, especially for high flow rates. This could be probably explained by resuspension of sediments containing high concentrations of NP (Chapter 3.4.1., Table 36). Namely, the applied analytical method allowed indeed the measure- ment of the total (adsorbed+dissolved) concentration of NP and NPEO (Chapter 3.1.1.). The dependence of concentration upon temperature (season) is clearly expressed for all nonylphenolic compounds in the rivers Limmat and Glatt (Figure 64A-C), and in the Rhine River for NP2EO only. The concentrations of the determined compounds are higher during the periods of low water temperature (winter), and lower in summer. However, it should be kept in mind that the efficiency of the sewage treatment plants is also better in the summer than in the winter (Chapter 3.3.), so that part of the concentration decrease in the summer period could be attributed to that fact. In the river itself, biodegradation and photochemical degradation are the major transformation processes of the nonylphenolic compounds. For NPEO biodegradation i.e. transformation into NPEC is the most probable mechanism (Chaper 3.2.3.), while for NP photochemical degradation should be also considered (Chaper 3.2.4.). 162

175 • 150 A B ~140 120 • s0 • ~ E • E ..S.105 • ..s. 90 c ., •• c • ~ 70 • ~ 60 • c: ·" J • • E • • g •• • • • § • • ••• • 8 35 • • • 30 . • • • • 8 •• ••• :· •• JI". ••• • • •• • • ··..;.~· ..·- ••••• 0 ...... 0 • 100 150 200 250 50 River flow (m3/s)

50 I . . 300 c D ~250 ~40 • 0 s E • 0 • c E 200 -30 • _..s. • c 0 "lij .~ 150 • ~ 20 • • • • • (I) -~ • •• • • g fJ•.. ~ 100 • 8 10 8 ••• ) '•' • 50 • • .. "'·~ ,. • ~··• • . ..·.. . 0 ·~ ! 0 """ ...· . 25 00 75 100 125 0.5 1.0 1.5 2.0 2.5 River flow (m3/s) Riverflow (m3/s)

~~Figure 63. Dependence of the nonylphenol concentrations upon river flow for different swiss rivers: (A) Inn, (B) Limmat, (C) Birs, and (D} Rhine 163~~

60 NP ::r:::::. 0 E .s 40- • • ,gc • g 20- • • .... c •• ~ • • c •• 8 0 ••• .,-},;,,· 0 5 10 - 15 - 2b 25 Temperature (°C) 75 NP1EO ::r:::::. 0 .[so ,_ c 0 :;:>' • • g 25- • • .... c •• • • ~ c • 0 • • • (.) •••• ' 0 '-::• 1,. 0 5 10 15 20 25 Temperature (°C) 75 ~ NP2EO 0 .[so .... c 0 :;:> jg 25- • r- c ,,. •• ~ • c 0 • (.) •• • I..' • 0 .··' 0 5 10 15 .. ·-'-20 25 Temperature (°C)

~~Figure 64. Dependence of nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), and nonylphenol diethoxylate (NP2EO) concentrations in the Glatt River upon temperature (season) 164~~

~~3.4.3. Lake Geneva~~

~~3.4.3.1. Estimation of Input~~

There are two main inputs of nonylphenolic compounds into the Lake Geneva (Figure 65): wastewaters which are predominantly biologically treated (199) and the river Rhone. The concentrations of NP, NP1 EO and NP2EO, measured in September 1984 in the secondary effluent of the sewage treatment plant LausanneNidy were 37, 80, and 43 µg/L respectively. Similar concentrations were determined by Stephanou (144) in secondary effluents from the settle- ments around the lake. On the basis of the mentioned figures, which are very similar to those found for the effluents in the Glatt Valley (Chapter 3.3.), a rough estimation was made that about 90 kg/day (32 t/year) of nonylphenolic compounds enter the Lake Geneva via secondary effluents. The measurements of concentrations of NP, NP1 EO and NP2EO in the river Rhone are so far very scarce. According to the data from September 1984, the concentrations of the mentioned compounds immediately before the entrance to the lake (Pont Scex) were 0.16, 0.41 and 0.21 µg/L respectively. Presuming that the given values are the average ones, it can be estimated that the mass flux of the nonylphenolic compounds entering the lake from this particular source {Q = 181 m3/s; ref. 165) amounts 13.1 kg/day (5.9 t/year).

~~GENEVA~~

~~Figure 65. Map of the Lake Geneva with indicated sampling stations 165~~

~~3.4.3.2. Distribution of Concentrations~~

Concentrations of the nonylphenolic compounds were determined in the samples from two typical vertical profiles (SHL-2 and Vidy-4) (Figure 65). The measured values were in the range of 0.03-5.3 µg/L for NP, <0.1-5.8 µg/L for NP1EO, 0.06-6.7 µg/L for NP2EO and <0.1-2.2 µg/L for NP2EC (Table 40). The concentrations were much higher on location Vidy-4 than on the location SHL-2, which can be easily explained by the influence of the treated wastewaters from STP Lausanne-Vidy which enter the lake there at the depth of 12 m. Vertical concentration profiles of nonylphenolic compounds on the station Vidy-4 are shown in Fig. 66. The concentration profiles obtained in summer 1984 and summer 1985 are very similar, which is the consequence of the similar hydro- graphic conditions during the sampling. The lake was, in both cases, stratified with well defined thermocline. The maximal concentrations of nonylphenolic compounds were observed at the depths of 7.5-10 m, which corresponds to the depth of the thermocline, and not in the bottom layer into which wastewater are introduced. However, this is in accordance with the tendency of wastewaters in stratified water column to spread in the layer of the same density as the wastewater itself (200). The concentrations of nonylphenolic compounds in the bottom layer and on the surface were surprisingly low (<1 µg/L) considering the immediate vicinity of the outlet of secondary effluents. Exception is the increased concentration of the NP in the bottom layer, which could be attributed perhaps to the higher concentration of suspended particles. In the winter period a very irregular vertical concentration profile was recorded (Fig. 66) which was difficult to interpret because different specific nonylphenolic compounds were differently distributed.

The vertical profile on station SHL-2, which is located in the middle of the lake (Fig. 65), several kilometers from the coast, was very similar to the one recorded at the station near the coast. The maximal concentration was also measured at the thermocline with the highest values being about 1 µg/L. These values are rather high considering the distance between the sampling location and the known source of investigated compounds, so that there was some doubt about the possible laboratory contamination. However, according to Imboden and Lerman (201 ), the horizontal mixing in the majority of swiss lakes is very fast, so that a homogeneous distribution of matter in one particular layer through the whole lake can occur. This assumption could be supported perhaps by the concurrently performed measurements of tetrachlorethylene and p-dichlorobenzene 166

~~TABLE 40. Concentrations of the Metabolites of Nonylphenol Po/yethoxylates in Lake Geneva~~

~~Location/ Depth Tempe- Concentrationa (µg/L) Date rature (m) (oC) NP NP1EO NP2EO NP1EC NP2EC~~

~~0 20.3 0.1 0.1 0.1 1 20.3 0.3 0.3 0.4 Vidy-4 5 19.5 1.3 2.4 2.9 (20/08184) 7.5 19.2 2.7 5.8 6.7 10 18.1 0.9 1.3 0.6 13.5 15.4 1.9 0.8 0.2~~

~~1 0.8 1.1 0.5 5 5.3 1.5 <0.1 Vidy-4 10 0.2 0.5 <0.1 (10/12/84) 12 <0.1 0.9 <0.1 15 0.2 1.4 0.5 20 1.3 4.2 0.7 30 1.0 1.0 5.4~~

~~Vidy-4 0 0.08 0.17 0.13 <0.1 0.5 (23/09/85) 5 0.16 0.38 0.07 <0.1 0.4 10 0.41 2.08 0.77 <0.1 2.2 16 0.44 0.73 0.29 <0.1 0.4~~

~~0 19.9 0.2 0.3 <0.1 1 19.9 <0.1 0.5 <0.1 SHL-2 5 18.7 <0.1 <0.1 <0.1 (20/08/84) 7.5 17.5 <0.1 0.3 <0.1 10 16.3 <0.1 0.3 <0.1 15 14.5 0.4 0.2 <0.1 17.5 12.4 0.4 0.2 0.2~~

0 0.17 0.64 0.28 <0.1b o.8b SHL-2 5 0.03 0.13 0.06 (23/09/85) 10 0.10 0.17 0.07 20 0.21 0.95 0.32 <0.1c <0.1c 300 0.03 0.32 0.14 a concentrations in the samples from 20/08/84 and 10/12184 were determined by the normal- phase HPLC using spectrophotometric detection whereas the samples from 23/09185 were determined using spectrofluorometric detection; b composite sample from the depths 0.1 and 5 m; c composite sample from the depths 10. 20 and 300 m.

(202) which also show a surprisingly small difference of concentrations at Vidy-4 and SHL-2 stations. An example of dispersion of chlorinated hydrocarbon pollution in the Lake Zorich in a relatively thin layer was shown by Schwarzenbach et al. (203). In order to obtain better support for the conclusions. about the distribution of nonylphenolic compounds on the locations further from the coast, additional 167

~~measurements with more detailed sampling on the vertical profile should be made.~~

~~Concentration <µg/L) 0 2 3 4 s 6 7 20/08/1984 -E .s:: 10 a. -Q) 0 20~~

~~Concentration <µg/L) 2 3 4 5 6 20/12/1984~~

~~10 -E~~

~~20 50. Q) 0 30~~

~~Concentration (µg/U 0 2 3 23/09/1985 E o NP -- 1> .s:: 10 NPlEO 0.~~

Figure 66. Vertical concentration profiles of nonylphenol (NP), nonylphenol monoethoxy/ate (NP1EO), nonylphenol diethoxytate (NP2EO), and [(nonylphenoxy)ethoxy]acetic acid in the Lake Geneva (sampling station Vidy-4) 168

~~3.4.4. Infiltration of Riverwater into Groundwater~~

~~3.4.4.1. Characteristics of Investigated Locations~~

The behaviour of nonylphenolic compounds during the infiltration of river water into groundwater was investigated at two locations: Glatt River near Glattfelden and Sitter River near Engelburg (St. Gallen), The first station is located in the lower part of the Glatt Valley where this river infiltrates into fluvioglacial aquifer. According to the previous investigations (204, 205) it can be assumed that at a chosen location a constant infiltration of the riverwater into groundwater through the saturated zone takes place and that the direction of the groundwater flow is approximately perpendicular to the direction of the river flow. The observation wells were located at different distances from the river bed (2.5-120 m) in the direction of the groundwater flow. Samples of the river water and groundwater for the analysis of nonylphenolic compounds were collected during the period of one year, approximately in monthly intervals (17 times). The Sitter River flows through the northeastern part of Switzerland and, according to the organic pollution, belongs to the moderately polluted swiss rivers (206). On the location at Silberbach near Engelburg (St. Gallen) there is a small pumping station only 15 m from the river bed. Under pumping conditions, the time neccessary for river water to reach the pumping station is very short (2-4 hours). Therefore, that pumping station is very sensitive towards the pollution which can be carried by the river. An investigation is performed in order to establish whether the concentration changes of the pollutants present in the river were reflected in the groundwater. Concentrations of NP, NP1 EO and NP2EO were monitored during 24 hours by taking grab samples from the river and of groundwater each 2 hours.

~~3.4.4.2. Elimination of Nonylphenol Ethoxylates and Nonylphenol~~

Average values and ranges of concentrations of NP, NP1 EO and NP2EO obtained during one year study at the location of Glattfelden are shown in Table 41. The concentrations in the river varied in the range of 0.7-26 µg/L for NP, 2.0- 20 µg/L for NP1 EO and 0.8-21 µg/L for NP2EO, and the respective ranges in groundwaters were <0.1-28 µg/L for NP, <0.1-4.8 µg/L for NP1 EO and <0.1- 1.6 µg/L for NP2EO. It can be seen that concentrations in both river and ground 169

~~TABLE 41. Concentrations of the Metabolites of Nonylphenol Polyethoxy/ates in the Glatt River and Groundwaters at the Location Glattfelden~~

~~Compound Concentration (µg/L) Glatt River P1 P2 P3 P4~~

~~NPa 4.1 1.0 0.5 0.5 0.3 (0.7-26) (0.14·3.1) (<0.1-1.4) (<0.1-1.5) (<0.1-1.1)~~

~~NP1Eoa 7.5 1.0 0.3 0.2 0.1 (2.0-20} (<0.1·4.8} (<0.1-1.5) (<0.1-0.3} (<0.1-0.2}~~

~~NP2Eca 8.2 0.4 0.3 0.2 0.1 (0.8-21) (<0.1-1.6} (<0.1-1.1} (<0.1-0.4) (<0.1-0.2}~~

~~NP1ECb 14.3 10.9 9.7 2.9 4.5 (8.4-20.1) (8.7-13.1) (9.5-9.8} (2.8-3.0) (<0.1-8.9)~~

~~NP2ECb 24.7 22.4 15.3 14.5 5.1 (20.6-28.7) (21.6-32.2) (10.6-20.0) (12.7-16.3) (<0.1-10.1)~~

~~Distance from 0 2.5 5 7 13 the river bed (m)~~

~~P1-P4: observation wells; a arithmetic mean and range of 17 determinations; b arithmetic mean and range of 2 determinations.~~

water varied in a relatively wide range, but were considerably lower in the groundwaters. Figure 67 shows that a fast decrease of concentrations of the investigated compounds with the increased distance of the observation well from the river occured. Already in the first three meters of the aquifer the major part {over 90 %) of NP, NP1 EO and NP2EO was eliminated, while the further concentration de~ crease was much slower. Therefore, it can happen that, in spite of the initially very effective elimination, NP reaches locations further than 100 m from the river bed. Thus measurable concentrations of NP (>0.1 µg/L) were determined in the pumping station ROtteli. Although all of the investigated compounds seem to be effectively eliminated, some differences can be observed. Elimination efficiency decreases as follows: NP2EO > NP1 EO > NP. The most probable mechanism of the elimination is biodegradation (Chapter 3.2.3.). Together with the investigated compounds, the concentrations of some other pollutants (pentachlorophenol and nitrilotriacetate) were determined In the same samples (207). In comparison with these organic pollutants, NP1 EO and NP2EO were eliminated as effective as the biodegradable nitrilotriacetic acid, while the elimination of pentachlorophenol was much lower (207). 170

25 20 NP2EO 15 10 5 E: 0 0 3 4 5 6 7 8 9 10 11 12 13 ~ 25 c: 0 20 NP1EO +:I ~ 15 c: 10 Q) -(.) c: 5 0 1 1 1 11 11 11 11 1 11 0 0 +--F'~""""-·'f' .. '""f ' .... ( '.Y '1' 'f ' ( 0 1 2 3 4 5 6 7 8 9 10 11 12 13 30 25 NP 20 15 10 5 ~----i.- l>'>n'7•"'•"''(-''t'_,.,..,...... f''''('"' 1 2 3 ...... 4 5 6 7 8 9 10111213 Distance [m]

Figure 67. Concentration profiles of nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), and nonylphenol diethoxylate (NP2EO) during infiltration of river water into groundwater at the location Glattfelden

Concentrations of nonylphenolic compounds in the Sitter River varied depending on the season and time of day in following ranges: 0.25-2.7 µg/L for NP, 0.40-3.2 µg/L for NP1 EO and <0.1-3.3 µg/L for NP2EO (206). The concentration relationships for NP, NP1 EO and NP2EO are typical for the secondary effluent of the sewage treatment plant. The concentration changes observed at the location Silberbach did not show typical daily dynamics as it was observed for some locations on the Glatt River (Chapter 3.4.1.). The variations of concentrations in the river were not followed by the same changes in the groundwater (Figure 68). Similarly as on the location of Glattfelden, the results indicate a very successful elimination {>90 %) of NP, NP1 EO and NP2EO, so that their concentrations in groundwater were very low, mainly in the range 0.01-0.1 µg/L. 171

~~3.0 3.5~~

~~2.5 3.0~~

~~~2.5 2.0 i a,. 2.0 c: c: 0 1.5 0 ~ c !!! 1.5 1.0 c 8c: 8c: 1.0 8 8 0.5 0.5~~

~~10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 lime (h) lime (h)~~

~~3.5~~

~~3.0~~

~~~ 2.5 a c: ·e0 2.0 c 1.5 c:8 8 1.0 0.5~~

~~10 12 14 16 18 20 22 24 2 4 6 8 lime (h)~~

Figure 68. Diurnal variations of nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), and nony/phenol diethoxylate (NP2EO) concentrations in the Sit- ter River and groundwaters at the location Enge/burg.

It was shown in Chapters 3.4.1. and 3.4.2. that NP, NP1 EO, and NP2EO concentrations in the rivers depend upon the season, and it could be reflected in the groundwaters as well. Seasonal variations of the concentrations of NP, NP1 EO and NP2EO in the Glatt River and groundwaters at the location of Glattfelden are shown in Fig. 69. The results were obtained by the analysis of grab samples so that large variations of concentrations are observed, especially in the river itself. However, the increase of concentrations during the winter period (November- April; t = 3-10 °C) and the decrease during the summer (May-October; t • 10- 20 °C), is well expressed for all three of the investigated compounds, both in the 172

18 o river Gatt 16 li ground water NP2EO :J .... 14 Cl 2- 12 c: 0 10 -as... 8 c: -Q) 6 (.) c: 0 4 () 2 0 08 09 10 11 12 01 02 03 04 05 06 07 08 09 1984 1985 Time (month) 16 :J.... 14 NP1EO g_ 12 -5 10 -as... 8 c: 6 -Q) (.) c: 4 0 () 2 0 08 09 10 11 12 01 02 03 04 05 06 07 08 09 1984 1985 j 28.1 Time (monthl 26.1 :J.... 14 Cl 12 :i. NP -;; 10 0 8 as -c: 6 -Q) (.) c: 4 0 () 2 0 08 09 10 11 12 01 02 03 04 05 06 07 08 09 1984 1985 Time (monthl

Figure 69. Seasonal variation of nonylphenol (NP}, nonylphenol monoethoxylate (NP1EO}, and nonylphenol diethoxylate (NP2EO) concentrations in the Glatt River and in groundwater (distance from the river bed 2.5 m) 173 river and groundwater. Also, there is a good agreement between the concentration changes in the river and in the groundwater. The efficiency of elimination of NP in the first 2.5 m of aquifer was significantly smaller in winter than in summer (Figure 69C), which possibly suggests a biological character of the ongoing elimination process. These results show that winter period represents the most critical part of the year in respect to the danger of polluting groundwaters which are used for the public water supply by the investigated compounds. Similar behaviour like NP, although not as pronounced, was apparent for NP1 EO. Contrary to that, NP2EO was rather efficiently eliminated even during the colder part of the year, which could be explained by its better biodegradability (Chapter 3.2.3.).

~~3.4.4.3. Elimination of Alkylphenoxy Carboxylic Acids~~

According to the investigations shown in Chapter 3.4.1., APEC are the dominant degradation products of the NPnEO found in the Glatt River and therefore it was important to investigate their behaviour during the infiltration of riverwater into the groundwater. Unfortunately, determination of NPEC was not included in the investigations which were carried out for the period of a whole year, so that we have at our disposal only two series of measurements of these compounds.(Fig. 70). The determinations in the riverwater proved that NPEC are the dominant nonylphenolic compounds (NP1 EC = 8.4-20.1 µg/L; NP2EC = 20.6-28. 7 µg/L). Furthermore, in contrast to the fast elimination of NP and NPEO in the first several meters of aquifer, NPEC showed a slower elimination (Fig. 70). However, the results from the second sampling (20th May .1986.) has suggested that in spite of the low elimination of NPEC in the first part of the aquifer, later elimination of NPEC may take place. Additional investigations are necessary to make final conclusions about the behaviour of NPEC during the infiltration of riverwater into the groundwater. A better solubility of NPEC as well as the resistance to biodegradation in aerobic conditions speak in favour of their higher mobility in the aquifer compared to NP1 EO and NP2EO. 174

48 44 a NPEC 40 ll NPEO 14/05/86 -....J 36 0) 20/05/86 -3- 32 ---q_ c 28 \ .Q ' \ ~ 24 ' -..... ' c 20 ' ' -Q) t:l., (..) c 16 ' 0 ' () 12 ' ' ', ' 8 ' ' ' ' ' 4 ' ', ------' ~"A 2.5 5 7 13 Distance (m)

Figure 70. Concentration profiles of (nonylphenoxy)acetic acid (NP1EC) and [(nonylphenoxy)ethoxy]acetic acid (NP2EC) during infiltration of river water into groundwater at the location of Glattfe/den. 175

~~3.4.5. Bloaccumulatlon in Freshwater Organisms~~

Some degradation products of NPnEO have a pronounced lipophilic character. Already the data on concentrations of NP, NP1 EO, and NP2EO in sediments (Chapter 3.4.1.) and sewage sludge (Chapter 3.3.) have shown that their interaction with organic rich solid phase can be significant. The lipophilicity of an organic compound, expressed as the value of partition coefficient between organic solvent and water (Chapter 3.2.2.) is usually in a very good correlation with the bioaccumulation in aquatic organisms (208) so that some bioaccumulation of the investigated compounds has also been expected.

~~The analyses of aquatic organisms in the surface water of the Glatt Valley were performed at different trophic levels: in macrophytic algae, fish and birds.~~

The analyses of several types of algae, which are the most frequent in the Glatt River (209), have shown a significant bioaccumulation of NP, NP1 EO and NP2EO (Table 42). The concentrations varied in the range of 2.48-38.4 µg/g for NP, 0.9-80.0 µg/g for NP1 EO and 0.59-28.7 µg/g for NP2EO. Considerable differences were observed between the different species of algae collected on the same location (creek Chriesbach). The species Cladofora glomerta shows higher accumulation than the species Fontinalis antipyretica and Potamogeton crispus. It should be stressed that abundance and qualitative composition of algae on the longitudinal profile of the Glatt River have many variables (209), and depend upon several factors, first of all upon the input of the wastewaters and insolation of the river (presence of the bank vegetation). The coverage of the river bed by macrophytic algae is smaller than 1o % in the section between Greifensee and the first outlets from the sewage treatment plants (Figure 51), and is often almost 100 % in the section the downstream from the outlet of the sewage treatment plant Zurich-Glatt. The dominant species in the most polluted part of the river is Cladofora glomerata (209). The relationship between the specific nonylphenolic compounds in algae is not the same as in water. The increase of NP and NP1 EO concentration in comparison to the concentration NP2EO can be observed. High concentrations of NP and NPEO in algae, especially in Cladofora glomerata, are important for the understanding of the fate of these compounds for two reasons. First, the mentioned algae represents, during the summer months, a considerable part of the biomass of living organisms In the river. In order to su- press their growth each year they are mechanically removed and the problem of 176

their final disposal should be considered. The data about the algal biomass which is removed from the river, as well as the fate of the nonylphenolic compounds after the accumulation into algae are not available.

~~TABLE 42. Concentrations of Nonylphenol and Nonylphenol Ethoxylates in Freshwater Algae~~

~~Species Location/Date Concentration (µg/g)a NP NP1EO NP2EO~~

C/adofora glomerata Chriesbach, 38.4 4.74 4.32 20/09/1984 FontinaHs antipyretica Chriesbach, 4.22 0.90 0.59 20/0911984 Potamogeton crispus Chriesbach, 2.48 1.14 1.93 20109/1984 C/adofora glomerata Glatt, wastewater 5.48 10.0 3.12 outletb, 1011984 Cladofora glomerata Glatt, ROmlarigC• 3.52 1.80 1.31 10/1984 Cladofora glomerata Glatt, ROmlangd, 5.07 11.0 2.38 10/1984 Ctadofora glomerata Glatt, ROmlangd, 25.4 80.0 28.7 0811985 Cladofora glomerata Glatt, OberhOri, 6.42 10.8 6.80 08/1985 C/adofora glomerata Glatt, Glattlelden, 18.0 23.7 8.90 05109/1984 Cladofora glomerata Glatt, Glattlelden, 7.21 14.1 4.67 0811985

a Concentration expressed on the dry weight of the sample; b Outlet of the secondary effluents from sewage treatment plant ZOrich-Glatt; c 14th km of the Glatt River (Figure 51); d 1sth km of the Glatt River (Figure 51).

Algae growing in the river serve as food for the other freshwater organisms and it can therefore be assumed that biomagnification of nonylphenolic compounds in higher food chain levels, especially fish, can occur. In order to examin this suposition several species of fish were analysed (Barbus barbus L.,Squalius cephalus Heck and Sa/mo gairdnen) which are characteristic for the area of the investigation and the results are given in Table 43. It is apparent that concentrations are lower in fish than in algae (NP: <0.03-1.59 µgig; NP1 EO: 0.06-7.02 µg/g; NP2EO: <0.03-3.07 µgig) so that it can be concluded that biomagnification through the food chain did not take place. 177

~~TABLE 43. Concentration of Nony/phenols and Nony/phenol Ethoxylates in Freshwater Fish~~

~~Species Tissue Location/Date Concentration (µgig) NP NP1EO NP2EO~~

Squalius muscle Chriesbach, 0.18 0.18 0.13 cephalusb gut 20/09/84 1.22 0.87 0.13 Heck. liver 1.03 1.82 1.35 gills 1.41 1.59 1.13 Squalius gut Chriesbach, 0.46 0.34 <0.03 cephalusc liver 28/09/84 1.39 1.01 <0.03 Heck. gills 0.98 0.51 <0.01 Barbus barbus d muscle Chriesbach, 0.38 3.12 2.30 L. gut 28/09/84 0.05 0.17 0.05 liver 0.98 0.88 0.14 gills <0.03 0.06 <0.03 heart 0.30 0.15 <0.03 roe 0.09 0.15 <0.03 Sa/mo muscle Chriesbach, 0.15 0.42 0.05 gairdneri 8 gut 28/09/84 1.59 7.02 3.07 Sa/mo muscle Neffbach, 0.78 1.24 gairdneri f gut 20/09/84 1.50 0.87

Composite brain Chriesbach, 0.14 0.08 sample9 28/09/84 a Concentrations are expressed on the dry weight basis; b Composite sample from 4 specimens (16-18 cm; 46-111 g); c Composite sample from 3 specimens (12-22 cm; 16-120 g); d Composite sample from 2 specimens (48-49 cm; 670-1080 g); e 1 specimen (19 cm; 68 g); f 1 specimen (17 cm; 49 g); 9 Composite sample from specimens described under c,d,e.

The analysis of NP, NP1 EO and NP2EO in various organs and tissues has shown their presence in the whole organism, but have not indicated any stronger predominance in the distribution which could suggest the dominant way of intake (gills, diet). The number of performed analyses is so far insufficient to conclude wether there is a connection between the fish size (age) and the accumulated amount of investigated compounds and whether there is a difference between the specific species of fish. The concentration of investigated compounds in the edible parts of the fish (muscles) varied in the range of 0.2-3.12 µg/g. It is difficult to give an ecotoxicological assessment of the determined concentrations. However, the fact that bioaccumulation occurs in fish indicates a possibility that these, so far insufficiently, investigated compounds reach man. Presuming an average concentra- 178

tion of NP in the creek Chriesbach of 4.6 µg/L, a bioaccumulation factor of 209 is obtained for the species Squa/ius cepha/us Heck. A very similar value for the bioaccumulation factor was obtained by Mcleese et al. (150) in the laboratory experiments with salmon, while the bioaccumulation factor for mussels was only about 1O (210). According to our knowledge, there is no known data in the literature about the bioaccumulation of lipophilic APEO, on the basis of the concentration in the water (Chapter 3.4.1.) and species Squa/ius cephalus Heck. From the creek Chriesbach, the bioaccumulation factors for NP1 EO were calculated to be 50-70, which is much lower than for NP. The difference between NP and NP1 EO is in agreement with the somewhat lower Kow values of the NP1 EO (Chapter 3.2.2.).

Lipophilic degradation products of NPnEO were also found in all the tissues and organs of wild ducks which lived on the banks of the creek Chriesbach (Table 44) where the majority of fish were sampled. The highest concentrations of NP and NPEO were measured in the muscle tissue (1.15 µg/g NP; 2.06 µg/g NP1 EO; 0.35 µg/g NP2EO). The majority of the tissues concentrations of NP and NP1 EO were considerably higher than in the concentration of NP2EO. In comparison with the concentrations found in the fish, no significant differences were observed.

~~TABLE 44. Concentrations of Nonylphenol and Nonylphenol Ethoxylates in the Tissues of the Wild Duck~~

~~Location/Date Tissue Concentration (µg/g) 8 NP NP1EO NP2EO~~

muscle 1.15 2.06 0.35 liver 0.10 0.16 <0.03 Chriesbach, guts 0.54 0.39 <0.03 28/09/1984 stomach ·0.24 0.05 <0.03 stomachb 0.19 0.08 <0.03 heart <0.03 <0.03 <0.03 brain 0.19 0.18 a Concentrations are expressed on the dry weight of the sample; b sediment from the stomach. 179 4. CONCLUSIONS

The first prerequisite for successful investigation on biogeochemical behaviour of alkylphenol polyethoxylates was the availability of analytical methods. It was necessary to develop several highly specific methods which allowed, not only the determination the parent compounds but also the qualitative and quantitative determination of the compounds which are formed by their transformation. By a combination of various techniques of sample extraction, extract treatment and instrumental analysis, three different analytical procedures were developed which included: (a) determination of wide range of alkylphenol polyethoxylate oligomers (nE0=3-20), (b) determination of lipophilic alkylphenol ethoxylates and alkylphenols, and (c) determination of alkylphenoxy carboxylic acids. The basic analytical technique was high-performance liquid chromatography with the application of spectrophotometric or spectrofluorimetric detection. The separation of oligomers was achieved by the application of normal-phase liquid chromatography (Figures 5 and 6), and separation of homologues by the application of reversed-phase liquid chromatography (Figure 7). High-resolution gas chromatography/mass spectrometry was used for identification purposes as well as for the confirmation of the results obtained from routine measurements using HPLC techniques.

By model laboratory experiments it was shown how the change of hydrophilic and/or hydrophobic part of the molecule affects the properties of alkylphenol ethoxylates and alkylphenols which determine their behaviour in aqueous solutions such as solubility and partition between water and organic solvents. The solubility of alkylphenol ethoxylates linearly decreases with decreasing number of hydrophilic (ethoxy) groups in the molecule (Figure 23), but the solubility value of the fully deethoxylated compound, alkylphenol, is considerably higher than the value which may be derived from this linear relationship. An additional methylene group in the hydrophobic chain Jowers the solubility much more than an additional ethoxy group increases it. Solubilization of the mixtures of lower oligomers of alkylphenol ethoxylates in water follows a very simple rule according to which the concentration of a single oligomer in the saturated solution can be expressed as the product of mol fraction of the oligomer in the original mixture and its solubility as pure compound (Table 13).

Alkylphenols and alkylphenol ethoxylates (nE0=1-2) are characterized by high partition coefficients in the system octanol/water (log Kow about 4; Table 14) which indicates their pronounced lipophilic character. The difference between 180 the partition coefficients for octanol/water and n-hexane/water systems suggests that these compounds are rather polar molecules.

Biological degradation is the main path for the transformation of alkylphenol polyethoxylates in aquatic environments. The experiments with adapted bacterial cultures showed that the biodegradation of higher oligomers, which is carried out by shortening of the hydrophilic part of the molecule, is a fast and efficient process. However, with increasing lipophilicity {caused by the loss of hydrophilic fragments) degradation becomes much slower and thus the accumulation of alkylphenol diethoxylates and alkylphenol monoethoxylates occurs. This observation was already put forward by some other authors (135, 136), but in our work the conclusions were supported for the first time by specific quantitative determinations of single oligomers present in synthetic wastewater as well as in the effluent and sludge from the laboratory apparatus for wastewater treatment (Table 16). Biodegradation of the comparatively persistent lipophilic alkylphenol ethoxylates was also studied in laboratory experiments, and it was shown that their further biological transformation proceeds via formation of corresponding alkylphenoxy carboxylic acids (Figures 26 and 27). This transformation occurs even when AP1 EO and AP2EO are the sole source of organic carbon (Figures 29 and 30). Contrary to this, nonylphenol proved to be very persistent to microbial attack under identical experimental conditions (Figures 28 and 31 ). Modified closed bottle test showed that aerobic conditions are necessary for the biotransformation of APEO into alkylphenoxy carboxylic acids (Figure 34), while anaerobic conditions favour the formation of alkylphenols (ref. 108; Table 17).

The photochemical degradation of alkylphenol polyethoxylates is very slow. However, this process can be considered as significant for alkylphenol transformation. It was shown that dissolved organic matter present in natural waters increases photochemical degradation considerably in comparison to direct photolysis (Tables 18 and 19). The mechanism of photochemical degradation via singlet oxygen (102) (Table 21) appears to be rather insignificant in natural waters (pH 6-9), because in this pH range alkylphenols are virtually nondissoci- ated, and the mentioned mechanism is effective only if alkylphenols are available in the form of phenolate ions (184). Photochemical degradation of NP is accelerated in the presence of hydrogen peroxide (Table 22) which can be found in natural waters at considerable concentrations as the product of photochemical reactions. It was estimated that the half-life, which is attributed to the 181 photolysis only, of NP and OP, in shallow and clear natural waters during a sunny summer day, is about 1O hours.

It was established that nonylphenol ethoxylates occur in high concentrations in the nontreated sewage and primary treated effluents (0.4-2.2 mg/L) (Table 25). To our knowledge these are the first quantitative results on the occurrence of aromatic nonionic surfactants in the aquatic environment. The share of nonylphenol polyethoxylates in the total dissolved organic carbon was significant and, for the analysed samples, it reached the values of 3.0-9.6 %.

Elimination of alkylphenol polyethoxylates during the mechanical treatment is negligible, but it is very efficient (up to more than 90 %) during biological treatment with activated sludge (Table 27). However, the observed elimination cannot be interpreted in terms of efficient ultimate degradation of alkylphenol polyethoxylates to carbon dioxide and water, but rather as a transformation to the metabolites which are much more persistent to further microbial attack. Formation of a specific type of metabolic product depends on the conditions during the treatment. Although all details of the complex biotransformation path of the alkylphenol polyethoxylates, are not yet completely understood, it may be summarized that the alkylphenols are the typical product of their anaerobic transformation, while the alkylphenoxy carboxylic acids are the product of aerobic transformation. Apart from biological processes, the behaviour and fate of the alkylphenol polyethoxylates and their degradation products is affected also by the physico-chemical processes, particularly by partition between the aqueous and solid phase. In considering mass fluxes of the investigated substances in the sewage treatment plants (Tables 32 and 33), it was established that a significant part of the lipophilic degradation products (AP, AP1 EO, AP2EO) is eliminated from aqueous phase by adsorption on the sludge. The lipophilic adsorption on the sludge and the additional formation from the present precursors during anaerobic sludge treatment are the cause of the extremely high concentrations of nonylphenols in the anaerobically stabilized sludges (108).

The residual concentration of alkylphenol polyethoxylates in secondary effluents is in general comparatively low and therefore the concentrations levels in the re- cepient natural waters do not reach high values (usually <10 µg/L). Contrary to this, concentrations of the persistent degradation products of the alkylphenol polyethoxylates, which represent the major fraction of alkylphenolic compounds in the secondary effluents (about 80 %; Figure 45), can reach very high values (up to 100 µg/L) in natural waters. Biodegradation which has started in sewage 182

treatment plant continues in natural waters so that the concentration and retatfve contribution of alkylphenol polyethoxylates further decrease. Calculation of input and output mass fluxes of the different types of alkylphenolic compounds in the Glatt River (Table 35), has shown that the transformation of alkylphenol polyethoxylates into alkylphenoxy carboxylic acids occurs in the river. Even 85 % of the mass flux of nonylphenolic compounds at the output profile was in the form of nonylphenoxy carboxylic acids (Figure 58).

Biodegradation products of nonylphenol polyethoxylates were determined at considerable concentrations in other swiss rivers as well (Table 39). In contrast to the Glatt River in some of the other investigated rivers, the clearly dominant alkylphenolic compound was nonylphenol. It has been observed that the behaviour and distribution of the nonylphenolic compounds are influenced by hydrological and seasonal (temperature, sunlight intensity) conditions. The concentration of the nonylphenolic compounds generally showed a negative correlation with the river flow, but examples of positive correlation were also noted. The second case could be explained as the consequence of the resuspension of sediments which can contain a considerable concentrations of the lipophilic degradation products of APnEO. The effect of the season was manifested through the increased elimination of the investigated substances in the summer period. Based upon the results of the model experiments the elimination of alkylphenol polyethoxylates should be attributed in the first place to the biodegradation, while the alkylphenols photochemical degradation should also be taken into consideration.

The degradation products of alkylphenol polyethoxylates show, like many other organic compounds, a typical distribution in the water column of the stratified lakes with maximum concentrations at the thermocline (Figure 66).

It was established that biodegradation products of alkylphenol polyethoxylates can contaminate ground waters by infiltration of polluted river water. The behaviour of various specific alkylphenolic compounds during infiltration was different. Alkylphenol ethoxylates (NP1 EO and NP2EO) are eliminated very successfully in the first several meters of aquifer (Figures 67 and 69), and the most probable mechanism is biodegradation. Somewhat poorer elimination, especially in the colder part of the year, was observed for nonylphenol. The greatest mobility in the aquifer was shown by alkylphenoxy carboxylic acids (Figure 70), which should be attributed to their greater hydrophilicity and resistance towards biodegradation under aerobic conditions. 183

A significant bioaccumulation of the lipophilic degradation products of the nonylphenol polyethoxylates in aquatic organisms was observed {Tables 42, 43 and 44). The bioconcentration factors of NP for algae were exceptionally high {even above 1000), while for fish and water birds it was much lower {about 200).

The results mentioned above indicate a number of negative ecotoxicological characteristics of alkylphenol polyethoxylates because of which these compounds could not be considered ecotoxicologically acceptable surfactants. If the existing law regulations on 80 % biodegradability of surfactants are interpreted in terms of an ultimate degradation to carbon dioxide and water, then alkylphenol polyethoxylates do not fulfill even the basic requirement for their ecological acceptability. Namely, primary biodegradation of APnEO results in the formation of various persistent metabolites, and thus the elimination efficiency of APnEO does not meet legal constraint even in the most efficient sewage treatment plants. Of special importance for the assessment of the ecotoxicological acceptability of alkylphenol polyethoxylates is the fact that, by their biodegradation, such metabolites are formed which are several times more toxic than the original compounds. This makes the most important difference between APnEO and other aromatic surfactants. Namely, biodegradation of linear alkylbenzene sulphonates, which also contain alkylbenzene as hydrophobic part of the molecule, starts by carboxylation of the alkyl chain during which more hydrophilic and less toxic degradation products are formed. Since alkylphenol polyethoxylates have highly branched alkyl chain, degradation cannot start on the hydrophobic part of the molecule, but instead it starts on the hydrophilic part (polyoxyethylene chain). The consequence is a decrease of hydrophilicity {solubility) of the formed degradation products and simultaneous increase of the lipophilicity which is positively correlated with toxicity to the aquatic organisms. The increase of lipophilicity, by shortening of the hydrophilic part of the molecule is partly diminished by the biotransformation of terminal alcoholic group into the carboxylic one. Therefore, aerobic biotransformation into the alkylphenol carboxylic acids could be considered as a ecotoxicologically favourable process. It was shown that the most lipophilic and the most toxic degradation products are formed under anaerobic conditions. This was clearly shown in the case of anaerobic stabilization of sludge (108). However, it seems that the conditions favourable for such transformations are possible in the nontreated wastewaters themselves {Table 17), and very probably also in the natural waters. The most critical degradation products of APnEO are alkylphenols, both due to their toxicity to water organisms and bioaccumulation as well as due to their 184

persistence towards further biodegradation. Alkylphenols, in a certain way, represent the final product of biotransformation of alkylphenol polyethoxylates. Namely, any of other stable degradation product (AP1 EO, AP2EO, AP1 EC, AP2EC) can be further transformed by a reaction taking place on the hydrophilic part of the molecule (carboxylation, loss of ethylen glycol or glycolic acid), and under certain conditions all those compounds can be precursors for the formation of alkylphenol. Contrary to this, further transformation of alkylphenol can be done only by the change of the hydrophobic part of the molecule. Hydrophobic part of the molecule represents, however, a branched chain of tripropylene substituted on benzene ring, a structure known for a long time for its exceptional persistence towards biodegradation (for example, tetrapropylene benzene sulphonates). Additional investigations should be undertaken in order to explain the conditions and paths of the possible further biological degradation of alkylphenols with the special emphasize on its degradation in natural waters and soils. 185 5. REFERENCES

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Inputs, distribution, and transformation of alkylphenol polyethoxylates in the aquatic environment were investigated using highly specific methods which included normal-phase and reversed-phase high-performance liquid chromato- · graphy as well as the high-resolution gas chromatography and mass spectrometry. Alkylphenol polyethoxylates, which are important household and industry nonionic surfactants, were found at considerable concentrations (0.4- 2.2 mg/L) in municipal wastewaters. It has been established that in wastewater canals, and especially in sewage treatment plants, the original composition of alkylphenol polyethoxylates changes significantly. Depending on conditions in the sewage treatment plant, three different types of persistent metabolites are formed: (a) lipophilic alkylphenol ethoxylates, (b) alkylphenoxy carboxylic acids, and (c) alkylphenols. Secondary effluents and sludges resulting from the biological treatment predominatly contain biodegradation products of alkylphenol polyethoxylates. In the receiving waters (rivers and lakes), the biotransformation processes proceed further and lead to almost complete elimination of parent compounds. However, some of the metabolites (alkylphenols, alkylphenoxy carboxylic acids) showed a very persistent behaviour and could be found in the polluted freshwaters at rather high concentrations (up to 0.1 mg/L). It should be pointed out that the lipophilic metabolites are much more toxic for the aquatic life than the parent compounds. The infiltration of the biodegradation products from the polluted rivers into the groundwaters as well as accumulation of the lipophilic metabolites into aquatic organisms has been observed. Behaviour and fate of alkylphenol polyethoxylates in the environment have been explained based on model laboratory experiments in which physico-chemical properties, biological degradation, and photochemical degradation of both parent compounds and their degradation products were studied. 200

~~Curriculum Vitae~~

~~Marijan Ahal~~

3 October 1951 Born in Bjelovar, Croatia, Yugoslavia 1958-1966 Primary School in Bjelovar and Zagreb 1966-1970 I Gymnasium in Zagreb 1970-1975 Undergraduate studies at the Faculty of Biotechnology, University of Zagreb 1975 Diploma in Biotechnology 1975-1987 Research Assistant in the Center for Marine Research Zagreb, Institute "Rudjer Boskovic", Zagreb 1976-1979 Postgraduate studies in Marine Sciences, University of Zagreb 1982-1983 Graduate student fellowship in the Department of Chemistry (Organic Group) at the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG) 1983 MS in Marine Sciences (Thesis "Investigation of the Petroleum Hydrocarbon Pollution in the Rijeka Bay") 1982-1987 Doctoral studies in the Swiss Federal Institute for Water Resources and Water Pollution Control, DObendorf and in the Center for Marine Research Zagreb, Institute "Rudjer Boskovic", zagreb 1987-1989 Senior Research Assistant, Center for Marine Research Zagreb, Institute "Rudjer Boskovi6", Zagreb 1989- Research Associate, Center for Marine Research Zagreb, Institute "Rudjer Boskovic", zagreb