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New research by the Dutch National Institute for Public Health and the Environment (RIVM) indicates that the health risk of playing sports on synthetic turf pitches with an infill of rubber granulate is virtually negligible. Therefore, it is considered safe for people to play sports on such pitches. The research was conducted following public concerns prompted by the Dutch TV programme Zembla called 'Dangerous Play' in October 2016. RIVM hopes that the results of the research will help to answer questions from ministries, municipalities, sports clubs and parents. To evaluate the health risks of playing sports on rubber granulate, it is important to determine which hazardous substances are contained in the granulate and to what extent they may be released. Subsequently, it should be examined how people playing sports can come into contact with these substances and whether this can lead to health effects. Rubber granulate contains numerous substances, such as polycyclic aromatic hydrocarbons (PAHs), metals, plasticisers (phthalates) and bisphenol A (BPA). These substances were found to be released from the granulate in very low amounts. This is because the substances are more or less 'enclosed' in the granulate, which means that the effect of these substances on human health is virtually negligible. What did RIVM investigate? RIVM determined the substances in rubber granulate from 100 sports pitches that are representative of the synthetic turf pitches in the Netherlands. The institute further performed three types of laboratory tests to examine the release of substances from the granulate if a person playing sport comes into contact with them. These 'migration studies' were used to assess to which extent substances can enter the human body via the skin, via the gastrointestinal tract or via the lungs. This was used to calculate human exposure to the released substances and how this can affect health. In addition, RIVM studied the information available in the scientific literature on substances in rubber granulate, their properties and their health effects. Is there a link with leukaemia? No indications were found in the available literature of a link between playing sports on synthetic turf pitches with an infill of rubber granulate and the incidence of leukaemia and lymphoma. No international research has demonstrated this connection. Moreover, it is clear from the composition of the rubber granulate that the chemical substances that are capable of causing leukaemia or lymphoma are either not present (benzene, styrene and 1,3-butadiene) or are present in a very low amount (2-mercaptobenzothiazole). Since the 1980s, a slight rise has been observed in the number of people aged between 10 and 29 who get leukaemia. This trend has not changed since pitches made of synthetic turf were first used in the Netherlands in 2001. Nor does research conducted in the U.S. reveal any increase in the number of new cases of lymphoma in areas where there are relatively many pitches with synthetic turf with an infill of rubber granulate. Information from new American research will be available early 2017. As rubber granulate has been used on football pitches in the United States for a longer period of time (since 1997), it will be possible to analyse over a longer period whether a link exists between playing sports on synthetic turf pitches and getting leukaemia. RIVM is in contact with the researchers and is keeping a sharp eye on the research. Rubber granulate in the environment This research focuses on potential health risks for people who play sports on synthetic turf pitches with an infill of rubber granulate. The research confirms earlier insights showing that the rubber granulate contains metals capable of entering the environment. In particular, zinc was found to be released from the granulate. This metal is not hazardous to humans, but can have consequences for organisms in the soil or surface water. Does rubber granulate meet requirements? Rubber granulate needs to fulfil the regulatory requirements for 'mixtures'. This concentration limit prescribes the maximum permissible amount of certain substances allowed in rubber granulate (there is no limit for how much may be released). This concentration limit concerns substances that are carcinogenic (such as PAHs), hazardous for reproduction or that damage DNA. The amount of PAHs in rubber granulate easily satisfies this concentration limit. The concentration limit for consumer products is far more stringent: it allows far lower amounts of PAHs (100 to 1000 times lower) compared with the concentration limit for mixtures. The amount of PAH in rubber granulate is slightly higher than the concentration limit for consumer products. The European Chemicals Agency (ECHA) is currently conducting research to determine a suitable concentration limit for rubber granulate. RIVM recommends adjusting the concentration limit for rubber granulate to one that is closer to the concentration limit applicable to consumer products. Why is rubber granulate used for football pitches? Rubber granulate is finely crushed rubber particles that are usually made from old car tyres. When used as infill on pitches of synthetic turf, it gives the pitch properties similar to normal turf. It means the ball does not roll too fast and does not bounce too high, and makes the synthetic turf better suited to sliding tackles than it would be without the granulate. Synthetic turf pitches can be used intensively all year around and need less maintenance. A lot is invested nowadays in order to reuse old products as a raw material for new products. This also applies to car tyres. The questions that have arisen about the safety of rubber granulate show that tension may exist between the reuse of materials and concerns about the health risks of new products.

New research by the Dutch National Institute for Public Health and the Environment (RIVM) indicates that the health risk of playing sports on synthetic turf pitches with an infill of rubber granulate is virtually negligible. Therefore, it is considered safe for people to play sports on such pitches. The research was conducted following public concerns prompted by the Dutch TV programme Zembla called 'Dangerous Play' in October 2016. RIVM hopes that the results of the research will help to answer questions from ministries, municipalities, sports clubs and parents. To evaluate the health risks of playing sports on rubber granulate, it is important to determine which hazardous substances are contained in the granulate and to what extent they may be released. Subsequently, it should be examined how people playing sports can come into contact with these substances and whether this can lead to health effects. Rubber granulate contains numerous substances, such as polycyclic aromatic hydrocarbons (PAHs), metals, plasticisers (phthalates) and bisphenol A (BPA). These substances were found to be released from the granulate in very low amounts. This is because the substances are more or less 'enclosed' in the granulate, which means that the effect of these substances on human health is virtually negligible. What did RIVM investigate? RIVM determined the substances in rubber granulate from 100 sports pitches that are representative of the synthetic turf pitches in the Netherlands. The institute further performed three types of laboratory tests to examine the release of substances from the granulate if a person playing sport comes into contact with them. These 'migration studies' were used to assess to which extent substances can enter the human body via the skin, via the gastrointestinal tract or via the lungs. This was used to calculate human exposure to the released substances and how this can affect health. In addition, RIVM studied the information available in the scientific literature on substances in rubber granulate, their properties and their health effects. Is there a link with leukaemia? No indications were found in the available literature of a link between playing sports on synthetic turf pitches with an infill of rubber granulate and the incidence of leukaemia and lymphoma. No international research has demonstrated this connection. Moreover, it is clear from the composition of the rubber granulate that the chemical substances that are capable of causing leukaemia or lymphoma are either not present (benzene, styrene and 1,3-butadiene) or are present in a very low amount (2-mercaptobenzothiazole). Since the 1980s, a slight rise has been observed in the number of people aged between 10 and 29 who get leukaemia. This trend has not changed since pitches made of synthetic turf were first used in the Netherlands in 2001. Nor does research conducted in the U.S. reveal any increase in the number of new cases of lymphoma in areas where there are relatively many pitches with synthetic turf with an infill of rubber granulate. Information from new American research will be available early 2017. As rubber granulate has been used on football pitches in the United States for a longer period of time (since 1997), it will be possible to analyse over a longer period whether a link exists between playing sports on synthetic turf pitches and getting leukaemia. RIVM is in contact with the researchers and is keeping a sharp eye on the research. Rubber granulate in the environment This research focuses on potential health risks for people who play sports on synthetic turf pitches with an infill of rubber granulate. The research confirms earlier insights showing that the rubber granulate contains metals capable of entering the environment. In particular, zinc was found to be released from the granulate. This metal is not hazardous to humans, but can have consequences for organisms in the soil or surface water. Does rubber granulate meet requirements? Rubber granulate needs to fulfil the regulatory requirements for 'mixtures'. This concentration limit prescribes the maximum permissible amount of certain substances allowed in rubber granulate (there is no limit for how much may be released). This concentration limit concerns substances that are carcinogenic (such as PAHs), hazardous for reproduction or that damage DNA. The amount of PAHs in rubber granulate easily satisfies this concentration limit. The concentration limit for consumer products is far more stringent: it allows far lower amounts of PAHs (100 to 1000 times lower) compared with the concentration limit for mixtures. The amount of PAH in rubber granulate is slightly higher than the concentration limit for consumer products. The European Chemicals Agency (ECHA) is currently conducting research to determine a suitable concentration limit for rubber granulate. RIVM recommends adjusting the concentration limit for rubber granulate to one that is closer to the concentration limit applicable to consumer products. Why is rubber granulate used for football pitches? Rubber granulate is finely crushed rubber particles that are usually made from old car tyres. When used as infill on pitches of synthetic turf, it gives the pitch properties similar to normal turf. It means the ball does not roll too fast and does not bounce too high, and makes the synthetic turf better suited to sliding tackles than it would be without the granulate. Synthetic turf pitches can be used intensively all year around and need less maintenance. A lot is invested nowadays in order to reuse old products as a raw material for new products. This also applies to car tyres. The questions that have arisen about the safety of rubber granulate show that tension may exist between the reuse of materials and concerns about the health risks of new products.

In a study in which male rats have been exposed to 0,20, 60, 200, 600 and 2000 mg di(isononyl)phthalate (DINP)/kg diet for 2 weeks, body weight and liver weight and a number of enzyme parameters (palmitoyl coenzyme-A oxidase (PCO), enoyl coenzyme-A hydratase (ECH), carnitine acetyl transferase (CAT) and lauric acid hydroxylase (LAH)) have been determined in liver homogenates which are related with peroxisome proliferation. No effects have been observed on both liver and body weight. PCO, CAT and LAH, showed no effect at 200 mg DINP/kg diet, corresponding with 18.2 mg/kg b.w./day. ECH appeared to be the most sensitive parameter with a dose-without-effect of 60 mg DINP/kg diet, corresponding to 5.7 mg DINP/kg b.w./day. This value of 60 mg/kg diet has been defined as overall no-observed-effect-level for DINP in this experiment.

In a study in which male rats have been exposed to 0, 20, 60, 200, 600 and 2000 mg di(isodecyl)phthalate (DIDP)kg diet for 2 weeks, body weight and liver weight and a number of enzyme parameters (palmitoyl coenzyme-A oxidase (PCO), enoyl coenzyme-A hydratase (ECH), carnitine acetyl transferase (CAT) and lauric acid hydroxylase (LAH) have been determined in liver homogenates which are related with peroxisome proliferation. No effects have been observed on liver and body weight. CAT appeared to be the most sensitive parameter with a dose-without-effect (DWE) of 60 mg DIDP/kg diet, corresponding with 5.6 mg DIDP/kg b.w./day. The other enzyme parameters PCO, ECH and LAH showed no effect at 200 mg DIDP/kg diet, corresponding with 18.4 mg/kg b.w/day. Since CAT is considered to be not specific for peroxisome proliferation this latter value has been established as overall no-observed-effect-level.

In a study in which male rats have been exposed to 0, 200, 600, 2000, 6000 and 20000 mg benzylbutylphthalate (BBP)/kg diet for 2 weeks, body weight and liver weight and a number of enzyme parameters which are related with peroxisome proliferation (palmitoyl coenzyme-A oxidase (PCO), enoyl coenzyme-A hydratase (ECH), carnitine acetyl transferase (CAT) and lauric acid hydroxylase (LAH)) have been determined in liver homogenates. No effect has been observed on the liver weight. The body weight showed a significant decrease as the highest dose level. PCO and CAT showed no effect up to 600 mg BBP/kg diet. LAH was more sensitive with a dose-without-effect (DWE) of 200 mg BBP/kg diet, corresponding with 19.1 mg BBP/kg b.w./day. ECH appeared to be the most sensitive parameter with an effect in all dose levels used. Therefore, no overall no-observed effect level can be defined in this experiment.

In a study in which male rats have been exposed to 0, 200, 600, 2000, 6000 and 20000 mg diethylphthalate (DEP)/kg diet for 2 weeks, body weight and liver weight and a number of enzyme parameters which are related with peroxisome proliferation (palmitoyl coenzyme-A oxidase (PCO), enoyl coenzyme-A hydratase (ECH), carnitine acetyl transferase (CAT) and lauric acid hydroxylase (LAH) have been determined in liver homogenates. No effects have been observed on the liver and body weights. PCO and LAH showed no effects up to 2000 and 600 mg DEP/kg diet, respectively. ECH was more sensitive with a dose-without-effect (DWE) of 200 mg/kg diet. Cat was the most sensitive parameter with an effect in all dose groups. Although CAT turned out the most sensitive parameter, this enzyme was not considered in the establishment of an overall no-observed-effect-level for peroxisome proliferation since it is not specific for peroxisome proliferation. The second most sensitive parameter was the ECH activity. Therefore an overall no-observed-effect-level for peroxisome proliferation of 200 mg DEP/kg diet was established corresponding to 19 mg DEP/kg b.w./day.

Based on data from literature, an evaluation was made wether it is possible to monitore the exposure of weakeners to the general Dutch population as urinary metabolites. The exposure, which mainly occurred by food, could only be estimated with data from foreign countries and by a number of assumptions. Based on existing qualitative and partially estimated data, only DEHP and DEHA are suitable for monitoring from analytical standpoint. From toxicological point of view, only DEHA is possibly interesting for monitoring, because the exposure closely approaches the tolerable daily intake.

A derivatization procedure is described for the long chain fatty acid, lauric acid and metabolites using a fluorescent probe 4-(bromomethyl)-7-methoxycoumarin. The derivatives can be separated and detected in an isocratic high performance liquid chromatografic system using a fluorescence detector. The derivatization is rapid, simple and gives a good quantification by the use of an internal standard. The procedure has been applied on samples from a toxicity experiment with the plasticizer di (2-ethylhexyl)phthalate (DEHP). The activity of cytochrome P-450 IVA1 or lauric acid hydroxylase (LAH) in liver homogenates can be determined by the quantification of the 11- and 12-hydroxylated metabolites of lauric acid. The method turned out to be very sensitive and suitable to replace similar assays using radioactive compounds.

In a study in which male rats have been exposed to 0, 20, 60, 200, 600 and 2000 mg di(cyclohexyl)phthalate (DCHP)/kg diet for 2 weeks, body weight and liver weight and a number of enzym parameters which are related with peroxisome proliferation palmitoyl coenzyme-A oxidase (PCO), enoyl coenzyme-A hydratase (ECH), carnitine acetyl transferase (CAT) and lauric acid hydroxylase (LAH) have been determined in liver homogenates. No effects have been observed on the liver and body weights. CAT showed no effect up to 600 mg DCHP/kg diet. PCO and LAH were more sensitive with a dose-without-effect (DWE) of 200 mg DCHP/kg diet. ECH, an enzyme involved in the beta-oxidation, appeared to be the most sensitive parameter with a dose-without-effect of 60 mg DCHP/kg diet. Therefore an overall no-observed-effect-level of 60 mg DCHP/kg diet was defined corresponding to 5.8 mg DCHP/kg b.w./day.

The aim of the present thesis was to study the in vivo relevance of in vitro detected estrogenic effects of food associated compounds, with emphasis on prenatal exposure. The estrogenic potency of 21 food packaging-associated compounds was studied in ERα or ERβ transfected U2-OS (human osteoblasts devoid of endogenous estrogen receptors) cell lines. Six plasticizers and three anti-oxidants were slightly estrogenic in the ERα cells. BPA, NP, tris (2-ethylhexyl) trimellitate (TEHTM), propyl gallate and butylated hydroxy anisole (BHA) were estrogenic both in ERα and ERβ cells. These compounds appeared to be more estrogenic relative to estradiol (E2) in ERβ than in ERα cells. To study the in vivo relevance of these effects, in vivo biomarker-responses of BPA, NP, DEHA, DEHP, DIHP, p,p’-DDE and quercetin were studied in ER-Luc male mice. Of these seven compounds, BPA, DEHP and quercetin induced estrogenic effects after a single oral dosage at exposure levels 10-104 times higher than the established Tolerable Daily Intakes (TDI’s). It remains to be seen whether these margins are sufficiently high to allow the conclusion that these compounds are unlikely to represent a human health risk. As exposure to estrogenic compounds during developmental stages could be an important risk factor in developing hormone dependent cancers later in life, these seven compounds were studied in pregnant ER-Luc mice as well. After oral exposure of the mother animal the compounds were unable to significantly induce luc-activity in any of the tissues including fetuses. Unexpectedly however, NP, BPA and DIHP significantly lowered the placental luc-activity. The results indicate that at the current levels of exposure to food associated estrogenic compounds, direct estrogenic effects in the fetus are not expected. The mechanism and consequences of the significant luc-reduction in the placenta should be investigated to a further extent to elucidate its possible significance. Because of the absence of significant luc-induction in the fetuses, the fate and distribution of radioactively labeled E2, NP and p,p’-DDE were studied in pregnant C57black mice in order to investigate whether the compounds can actually reach the fetuses. E2 did not reach the fetus at a level above the detection limit, NP and p,p’-DDE levels were above the detection limit in fetuses exposed via the mother from GD8-16. Levels of E2 and NP detected in the placenta were significantly higher than those in the fetuses, up to five fold for E2 and three fold for NP, possibly due to prevention of transport of the compounds to the fetuses by placental binding proteins. Based on the findings of the present thesis it was concluded that in spite of the in vitro estrogenicity of various food-born estrogens, in vivo estrogenic effects are not likely expected as estrogenicity in vivo is shown at levels 10-104 fold higher than the TDI’s for humans and placental binding proteins may reduce fetal exposure. However, the estrogenic compounds were given as a single acute dose, whereas TDI values and also risks associated with dietary exposure can be expected to result from chronic combined exposure. Therefore, it remains to be seen whether the estimated margins are sufficiently high. Future experiments are therefore needed that focus on long term and combined exposure. Another factor that remains to be solved relates to the fact that only in in vitro assays the relative ability of compounds to differentially activate ERα or ERβ can be quantified. All together the results of the present thesis reveal that the extent at which in vitro estrogenic effects may result in in vivo estrogenicity may vary with the compound under study and should be evaluated on a case by case basis taking differences in absorption, distribution, metabolism and excretion, the intrinsic estrogenic potency of chemicals for either the ERα or ERβ and the differential levels of expression of these receptors in different tissues in vivo into account.

The aim of the present thesis was to study the in vivo relevance of in vitro detected estrogenic effects of food associated compounds, with emphasis on prenatal exposure. The estrogenic potency of 21 food packaging-associated compounds was studied in ERα or ERβ transfected U2-OS (human osteoblasts devoid of endogenous estrogen receptors) cell lines. Six plasticizers and three anti-oxidants were slightly estrogenic in the ERα cells. BPA, NP, tris (2-ethylhexyl) trimellitate (TEHTM), propyl gallate and butylated hydroxy anisole (BHA) were estrogenic both in ERα and ERβ cells. These compounds appeared to be more estrogenic relative to estradiol (E2) in ERβ than in ERα cells. To study the in vivo relevance of these effects, in vivo biomarker-responses of BPA, NP, DEHA, DEHP, DIHP, p,p’-DDE and quercetin were studied in ER-Luc male mice. Of these seven compounds, BPA, DEHP and quercetin induced estrogenic effects after a single oral dosage at exposure levels 10-104 times higher than the established Tolerable Daily Intakes (TDI’s). It remains to be seen whether these margins are sufficiently high to allow the conclusion that these compounds are unlikely to represent a human health risk. As exposure to estrogenic compounds during developmental stages could be an important risk factor in developing hormone dependent cancers later in life, these seven compounds were studied in pregnant ER-Luc mice as well. After oral exposure of the mother animal the compounds were unable to significantly induce luc-activity in any of the tissues including fetuses. Unexpectedly however, NP, BPA and DIHP significantly lowered the placental luc-activity. The results indicate that at the current levels of exposure to food associated estrogenic compounds, direct estrogenic effects in the fetus are not expected. The mechanism and consequences of the significant luc-reduction in the placenta should be investigated to a further extent to elucidate its possible significance. Because of the absence of significant luc-induction in the fetuses, the fate and distribution of radioactively labeled E2, NP and p,p’-DDE were studied in pregnant C57black mice in order to investigate whether the compounds can actually reach the fetuses. E2 did not reach the fetus at a level above the detection limit, NP and p,p’-DDE levels were above the detection limit in fetuses exposed via the mother from GD8-16. Levels of E2 and NP detected in the placenta were significantly higher than those in the fetuses, up to five fold for E2 and three fold for NP, possibly due to prevention of transport of the compounds to the fetuses by placental binding proteins. Based on the findings of the present thesis it was concluded that in spite of the in vitro estrogenicity of various food-born estrogens, in vivo estrogenic effects are not likely expected as estrogenicity in vivo is shown at levels 10-104 fold higher than the TDI’s for humans and placental binding proteins may reduce fetal exposure. However, the estrogenic compounds were given as a single acute dose, whereas TDI values and also risks associated with dietary exposure can be expected to result from chronic combined exposure. Therefore, it remains to be seen whether the estimated margins are sufficiently high. Future experiments are therefore needed that focus on long term and combined exposure. Another factor that remains to be solved relates to the fact that only in in vitro assays the relative ability of compounds to differentially activate ERα or ERβ can be quantified. All together the results of the present thesis reveal that the extent at which in vitro estrogenic effects may result in in vivo estrogenicity may vary with the compound under study and should be evaluated on a case by case basis taking differences in absorption, distribution, metabolism and excretion, the intrinsic estrogenic potency of chemicals for either the ERα or ERβ and the differential levels of expression of these receptors in different tissues in vivo into account.

Phthalic acid isomers (dicarboxy benzenes) play an important role in our human environment as constituents of polyester fibres, films, polyethylene terephthalate (PET) bottles and other plastics. Due to the use and generation of water during phthalic acid production from the corresponding xylenes, a concentrated wastewater is generated. The generated wastewater consists of a mixture of phthalic acid isomers, acetic acid, benzoic acid, and toluic acids. The aim of the work described in this thesis was to elucidate if anaerobic biological treatment may represent an attractive alternative for conventionally applied aerobic treatment methods. With regard to the anaerobic biodegradability of the phthalate isomers it was demonstrated that all three phthalate isomers could be degraded by two types of methanogenic granular sludge and digested sewage sludge. Lag-phases prior to degradation ranged from 17 to 156 days. More reduced aromatic analogues of the phthalates were not degraded or only at extremely low rates.Kinetic properties of the anaerobic degradation of the phthalate isomers were studied using enrichment cultures obtained from the biodegradability experiments or bioreactor biomass. The phthalate isomers grown cultures were capable of degrading only one of the phthalate isomers and degraded benzoate without a lag-period. A three species kinetic model enabled the dynamic description of intermediate acetate and molecular hydrogen accumulation and final formation of methane from the phthalate isomers and benzoate. It was shown that the syntrophic biomass cultivated had a low growth rate on the phthalate isomers (μ max≈ 0.09 day -1 ). The energetic efficiency for growth on the phthalate isomers was found to be significantly smaller when compared to growth on benzoate, suggesting that an energetic inefficiency prevails in the degradation pathway of the phthalate isomers. The cultures were furthermore strongly inhibited and even deactivated by co-incubation with acetate or benzoate, or a short period of a few hours without substrate.Despite these unfavourable microbiological characteristics, it was demonstrated that highly active terephthalate degrading biomass could be cultivated at high concentrations in both UASB (Upflow Anaerobic Sludge Bed) reactors and hybrid reactors, resulting in high terephthalate removal capacities (15-20 gCOD(Chemical Oxygen Demand) · l 1· day 1 ). High-rate terephthalate degradation in the UASB-reactors was strictly dependent on inoculation of the reactor with granular biomass.After demonstrating that terephthalate as sole substrate could be degraded at high-rates, we studied the feasibility of a two-stage reactor concept for the treatment of terephthalic acid production wastewater, consisting of a mixture of readily degradable substrates (acetate and benzoate) and slowly degradable substrates (terephthalate and para -toluate). It was demonstrated that through pre-removal of acetate and benzoate in the first stage the lag-phase prior terephthalate degradation in the second stage could be significantly reduced (from 300 to approximately 50 days) and the wastewater could be treated at high volumetric removal rates and short hydraulic retention times (25 gCOD · l 1· day 1 and 6 hours respectively). For start-up of a two-stage anaerobic bioreactor system for treatment of terephthalic acid production wastewater, a gradual transition from initial operation in parallel to operation in series is suggested.

Phthalic acid isomers (dicarboxy benzenes) play an important role in our human environment as constituents of polyester fibres, films, polyethylene terephthalate (PET) bottles and other plastics. Due to the use and generation of water during phthalic acid production from the corresponding xylenes, a concentrated wastewater is generated. The generated wastewater consists of a mixture of phthalic acid isomers, acetic acid, benzoic acid, and toluic acids. The aim of the work described in this thesis was to elucidate if anaerobic biological treatment may represent an attractive alternative for conventionally applied aerobic treatment methods. With regard to the anaerobic biodegradability of the phthalate isomers it was demonstrated that all three phthalate isomers could be degraded by two types of methanogenic granular sludge and digested sewage sludge. Lag-phases prior to degradation ranged from 17 to 156 days. More reduced aromatic analogues of the phthalates were not degraded or only at extremely low rates.Kinetic properties of the anaerobic degradation of the phthalate isomers were studied using enrichment cultures obtained from the biodegradability experiments or bioreactor biomass. The phthalate isomers grown cultures were capable of degrading only one of the phthalate isomers and degraded benzoate without a lag-period. A three species kinetic model enabled the dynamic description of intermediate acetate and molecular hydrogen accumulation and final formation of methane from the phthalate isomers and benzoate. It was shown that the syntrophic biomass cultivated had a low growth rate on the phthalate isomers (μ max≈ 0.09 day -1 ). The energetic efficiency for growth on the phthalate isomers was found to be significantly smaller when compared to growth on benzoate, suggesting that an energetic inefficiency prevails in the degradation pathway of the phthalate isomers. The cultures were furthermore strongly inhibited and even deactivated by co-incubation with acetate or benzoate, or a short period of a few hours without substrate.Despite these unfavourable microbiological characteristics, it was demonstrated that highly active terephthalate degrading biomass could be cultivated at high concentrations in both UASB (Upflow Anaerobic Sludge Bed) reactors and hybrid reactors, resulting in high terephthalate removal capacities (15-20 gCOD(Chemical Oxygen Demand) · l 1· day 1 ). High-rate terephthalate degradation in the UASB-reactors was strictly dependent on inoculation of the reactor with granular biomass.After demonstrating that terephthalate as sole substrate could be degraded at high-rates, we studied the feasibility of a two-stage reactor concept for the treatment of terephthalic acid production wastewater, consisting of a mixture of readily degradable substrates (acetate and benzoate) and slowly degradable substrates (terephthalate and para -toluate). It was demonstrated that through pre-removal of acetate and benzoate in the first stage the lag-phase prior terephthalate degradation in the second stage could be significantly reduced (from 300 to approximately 50 days) and the wastewater could be treated at high volumetric removal rates and short hydraulic retention times (25 gCOD · l 1· day 1 and 6 hours respectively). For start-up of a two-stage anaerobic bioreactor system for treatment of terephthalic acid production wastewater, a gradual transition from initial operation in parallel to operation in series is suggested.