Pesticides and Persistent Organic Pollutants
20.4 Persistent Organic Pollutants and Synthetic Environmental
Endocrine Disrupters
Chemical contaminants in food may consist of elements (e.g. metals) or different organic molecules. The latter can be present as a result of, for example, fungal activity (mycotoxins), appli- cation of pesticides or use of veterinary drugs directly in production of the food commodity, or they may come from the environment. In the lat- ter case especially compounds that are not easily degraded and maybe even (in addition) possess characteristics that favour their accumulation in living organisms, such as being easily absorbed after ingestion or being lipophilic and hence stored in metabolically less active tissues, are of major concern. Overall we often group such com- pounds under the designation POPs (persistent
organic pollutants).
POPs thus are organic compounds that are resistant to environmental degradation through
chemical, biological and photolytic processes. Therefore, they also are capable of long-range transport, and hence can bioaccumulate in animal and human tissues and biomagnify in food chains to be found in high concentrations even far away from their place of origin.
We have already touched upon some POPs. Thus, several of the earlier used chlorinated pesti- cides including DDT belong to this group. In 1995, the United Nations Environment Programme Governing Council (GC) began an investigation into POPs, starting with a shortlist comprising the following 12 compounds, known as the ‘dirty dozen’: aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, hexachlorobenzene, mirex, polychlorinated biphenyls, polychlorinated diben- zo-p-dioxins, polychlorinated dibenzofurans and toxaphene.
Of these 12 compounds two should be defined further, i.e. mirex and toxaphene (Fig. 20.20). Mirex is a chlorinated hydrocarbon commercial- ized as an insecticide and later banned because of its impact on the environment. It was popularized to control fire ants but by virtue of its chemical robustness and lipophilicity it was soon recog- nized as a bioaccumulative pollutant. The US EPA prohibited its use in 1976. Also, toxaphene is an insecticide which, from 1947 to 1980, was used primarily in the southern USA on cotton crops. Today widespread use of toxaphene has been banned in the USA.
Separately from the emerging focus on POPs, interest was additionally aroused in the so-called
environmental oestrogens (and anti-androgens) –
also grouped together as endocrine disrupters. To some extent the two groups proved to overlap, which is why here we treat them together as ‘POPs and synthetic environmental endocrine disrupters’. As our focus is on food safety, in the following we concentrate only on POPs, i.e. polychlorinated dibenzo-p-dioxins (dioxins) and polychlorinated
CH3 CI CI CI CI CI CI CI CI CI CI CI CI CH3 CH2 CIn (a) (b)
dibenzofurans together with PCBs (polychlorinated biphenyls), and supplement with information on nonylphenol.
Dioxins, furans and dioxin-like polychlorinated biphenyls
As already mentioned in the previous section when discussing the chlorophenoxy herbicides, members from the group of dioxins were formed as by- products (synthesis impurities) when synthesizing, for example, the herbicide 2,4,5-T. However, today we know that dioxins are formed in many reactions where organic and chlorine-containing substances (e.g. PVC) are heated (burnt) together, such as in incinerators. This is why many communities presently have strict rules about the temperature at which incinerators should oper- ate, in order to minimize the formation of dioxins.
Strictly taken, dioxins are polychlorinated diben- zo-p-dioxins (PCDDs). In the PCDDs, the chlorine atoms are bound at one or more of the eight free places on the molecule, i.e. at positions 1–4 and 6–9. In this way 75 different types of PCDD conge- ner come into existence (Fig. 20.21).
The toxicity of PCDDs depends on the number and positions of the chlorine atoms. Congeners that have chlorines at positions 2, 3, 7 and 8 have been found to possess significant toxicity in a number of contexts. However, in today’s risk assessment activities focusing on the toxic poten- tial of these compounds we most often merge them with relevant members of two other groups of chemical, namely the polychlorinated dibenzo- furans (PCDFs) and the so-called dioxin-like PCBs. This is due to the assumption that all 2,3,7,8- substituted PCDDs and PCDFs (Fig. 20.21), as well as the dioxin-like PCBs (Fig. 20.22), have the same mode of action, elicited by binding to the same receptor – the Ah receptor – and show com- parable qualitative effects, but with different potencies.
The dioxin-like PCBs include those where chlo- rines occupy: (i) usually no more than one of the
ortho positions; (ii) both para positions; (iii) at
least two meta positions; and where (iv) the struc- ture is not hindered from assuming the preferred planar configuration.
While the PCDFs – like the dioxins – are formed during heating/burning of different organochlo- rines (including PCBs) and mixed materials with a content of chlorine, the PCBs themselves have quite another origin. The first mixture of PCBs was synthesized in 1881 and later different such mixtures with physical characteristics ranging from light oily substances to greasy and waxy sub- stances were marketed and widely incorporated in a number of products, being used for a range of technical applications. These included the direct use as insulating fluids in the electrical industry, and the use as components in materials and prod- ucts such as plasticizers, paints, lubricants, insulat- ing tapes, fireproofing materials and hydraulic fluids. PCB production in general was terminated around 1980; however, at that time, more than half a billion kilograms had been produced in the USA alone.
Dioxins
The toxicity of the dioxins has been investigated over several decades with quite astonishing obser- vations gradually being made. First of all, these compounds show a very different acute toxicity depending on the actual number and placement of chlorine atoms. That is, we see a great varia- tion between the 75 different congeners, of which actually only the following seven are classified today as being toxic: 2,3,7,8-TCDD, 1,2,3,7, 8-PeCDD, 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-Hx- CDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-HpCDD and 1,2,3,4,6,7,8,9-OCDD (where T = tetra, Pe = penta, Hx = hexa and O = octa).
Second, it soon became clear that for the most acutely toxic compound (TCDD) the species
O
CIn CIm
O
Fig. 20.21. General structure of PCDDs where n and m can range from 0 to 4.
O5 3 2 4 3 2 1 9 8 7 6 1 1⬘ 2⬘ 3⬘ 4⬘ 5⬘ 6⬘ 5 6 4 (a) (b)
Fig. 20.22. Basic structure of (a) dibenzofuran (the skeleton of the PCDFs) and (b) biphenyl (the skeleton of the PCBs).
difference is indeed enormous. Thus, the oral LD50 values (Table 20.5) reported for a number of species vary from approximately 0.5 mg kg−1 BW for the guinea pig to about 1 mg kg−1 BW for the frog and hamster. This corresponds to a factor of 2000 between the most and the least sensitive species. Compared with the default factor of 10 normally used in risk assessment to reflect species differ- ences, this clearly is exceptional.
The high acute toxicity of TCDD and some other dioxins to a number of animal species prompted a great fear of these compounds, which of course led to much research as well as prompting attempts to regulate this area. Then the Seveso accident happened.
The 10th of July 1976 became a fatal day for the Italian town of Seveso, 15 miles from Milan. On that day the local chemical company Icmesa was struck by an accident. The alkaline hydrolysis of 1,2,3,4-tetrachlorobenzene to produce 2,4,5-T, to be used in the manufacture of herbicides, ran out of control. Overheating resulted in the forma- tion of dioxins and when a valve broke a little after noon that Saturday, close to 3000 kg of the reaction mixture was released into the air and spread over an area of about 1800 ha. The amount of dioxins involved is still not known, but has been estimated to be anywhere from about 100 g to 20 kg, or even more according to some sources. Three days afterwards dead animals were found among the wild fauna and soon approximately 5% of local farm animals also died. The rest, about 80,000 animals, were killed to prevent contamination of the food chain.
The authorities divided the area around the chemical plant into three zones depending on the concentration of dioxins in the topsoil. In zone A (87 ha) the average concentration was
235 mg m−2 with a maximum of approximately 5500 mg m−2. In this zone a quarter of the animals died immediately. Zone B (269 ha) showed an average of 3 mg m−2 with a maximum of 50 mg m−2. Zone C had sporadic contamination of up to about 5 mg m−2. A total of 36,000 inhabitants were evacuated from the area 2 weeks after the incident. Strikingly, no people died, only animals. The only symptom that was seen was chloracne, a severe skin rash. Out of 1300 exposed children, 1046 were hit by this effect. The blood dioxin concentration in the exposed victims as measured in 1976 ranged from 200 to 10,000 times higher than that of normal unexposed Italians. No strict correlation was found between blood dioxin concentrations and the occurrence/severity of chloracne.
The fact that no people died in Seveso and that only chloracne was observed as an acute effect, together with similar observations from expo- sures involving hundreds of workers at different industrial plants worldwide, led to the conclusion that man is less sensitive to the acute toxicity of dioxins than most animals. It also prompted dis- cussions about whether dioxins actually repre- sented a threat to humans at all in the longer perspective.
Today we know that dioxins are a threat. But let us start by going back to the people exposed in Seveso and look at their dioxin burden 20 years after the disaster. From the Seveso exposure scen- ario (together with other analysed scenarios) we learnt that the half-life for dioxins in man is high: on average 7.5 years, but with a sixfold variation between individuals for TCDD. Thus, it cannot be a surprise that the average blood dioxin concen- tration in the Seveso victims was still elevated 20 years after the exposure, with the highest con- centrations found in: (i) women; (ii) persons who had been eating locally grown vegetables; and (iii) obese persons. Twenty-five years after the exposure a large study was published that demon- strated a significant increase in breast cancer frequency and found individual serum TCDD concentration to be significantly related to breast cancer incidence among women in the Seveso Women’s Health Study Cohort.
Today IARC has concluded that 2,3,7,8-TCDD is carcinogenic to humans, based on being an unequivocal animal carcinogen, limited human information (epidemiological/other) and mechanis- tic plausibility; furthermore, that other dioxin-like Table 20.5. The acute toxicity measured as LD50
of TCDD for different species. Species LD50 Chicken 25μg kg−1 BW (0.025 mg kg−1 BW) Dog 1μg kg−1 BW (0.001 mg kg−1 BW) Frog 1 mg kg−1 BW Guinea pig 500 ng kg−1 BW (0.0005 mg kg−1 BW) Hamster 1157μg kg−1 BW (1.157 mg kg−1 BW) Mouse 114μg kg−1 BW (0.114 mg kg−1 BW) Rabbit 115μg kg−1 BW (0.115 mg kg−1 BW) Rat 20μg kg−1 BW (0.02 mg kg−1 BW)
compounds are likely to be carcinogenic and that complex environmental mixtures of such com- pounds therefore also are likely to be carcinogenic to man. On 25 March 2010, the US Department of Veterans Affairs published a proposed regulation that will establish B-cell leukaemias (such as hairy cell leukaemia), Parkinson’s disease and ischaemic heart disease to be associated with exposure to agent orange. Eligible Vietnam veterans may thus receive disability compensation for these diseases when the regulation becomes final.
The mechanism behind the carcinogenesis is not yet totally clear; however, it seems certain that it is elicited by binding to the Ah receptor.
The Ah receptor
The Ah (aryl hydrocarbon) receptor or AhR is a member of the so-called basic helix–loop–helix (bHLH)-Per-ARNT-Sim (PAS) family of transcrip- tional regulators controlling a number of develop- mental and physiological events, including neurogenesis, tracheal and salivary duct forma- tion, toxin metabolism, circadian rhythms, response to hypoxia, and hormone receptor function. AhR is a cytosolic factor responsive to both natural and man-made environmental com- pounds that is normally inactive, bound to several co-chaperones. Upon ligand binding (TCDD) the chaperones dissociate, resulting in AhR translo- cating into the nucleus and dimerizing with ARNT (AhR nuclear translocator), leading to changes in gene transcription. Much of our knowl- edge of AhR function stems from analyses of the mechanisms by which the ligand TCDD induces the transcription of CYP1A1. This gene encodes the microsomal enzyme cytochrome P4501A1, which oxygenates various xenobiotics as part of their stepwise detoxification. Additional routes of AhR-mediated actions have been proposed. Thus, it was demonstrated that TCDD induces changes in protein phosphorylation through the activation of protein tyrosine kinases within 10 min. This effect was shown to be AhR-dependent and occurred under cell-free conditions in the absence of a nucleus. Based on these results the TCDD- induced protein phosphorylation pathway may be considered a separate route of AhR signalling from the well-established nuclear translocation- dependent pathway. Figure 20.23 shows the two different signalling pathways of AhR upon ligand binding.
In the rat the liver is the main target organ for carcinogenesis, which seems not to be the case for humans. This might be explained by the fact that dioxins induce transcription of both CYP1A1 and
CYP1A2 in rats, whereas CYP1A1 transcription is
not induced in human liver.
Dioxins and dioxin-like compounds and food
Food in general accounts for about 95% of human exposure to dioxins (and dioxin-like compounds including dioxin-like PCBs). Since the compounds accumulate in the fat of animals, the highest sources in the human diet are beef, pork, lamb, fish, shell- fish, butter, cheese, processed meats, eggs, poultry and milk. Dioxins can also be taken up by plants; usually the concentrations are considerably lower, though. Dioxin levels tend to be highest in fatty fish from contaminated areas near industries that pro- duce dioxin. Generally, old, high-fat-content bot- tom fish, collected close to the contaminant source, have the highest levels, whereas lower-fat, non-sta- tionary fish have much lower concentrations, even in the vicinity of the contaminant source.
In the Annex to Commission Regulation (EC) No. 1881/2006, Section 5, we find that the EU defines and regulates dioxins and PCBs in the following way. For each group of commodities the maximum level is set for the content of ‘diox- ins’ as the sum of PCDDs and PCDFs (PCDD/F), expressed as WHO toxic equivalents (TEQ) using the WHO toxic equivalency factors (WHO- TEFs). In addition, another maximum level is set for the total sum of ‘dioxins and dioxin-like PCBs’ (sum of PCDDs, PCDFs and PCBs; PCDD/ F-PCB), again expressed as WHO-TEQ using the WHO-TEFs. A few examples are given below. ● Meat and meat products (excluding edible offal)
of bovine animals and sheep (sum of dioxins (in PCDD/F-TEQ)/sum of dioxins and dioxin-like PCBs (in PCDD/F-PCB-TEQ) ): 3.0 pg g−1 fat/ 4.5 pg g−1 fat.
● The corresponding maximum levels for muscle meat of eel (Anguilla anguilla) and products thereof are 4.0 pg g−1 wet weight/12.0 pg g−1 wet weight.
Nonylphenol
Nonylphenol originates principally from the deg- radation of nonylphenol ethoxylates, which are
widely used as industrial surfactants. It is classi- fied as an endocrine disrupter capable of interfer- ing with the hormonal system of numerous organisms. Nonylphenol ethoxylates reach sewage treatment works in substantial quantities where they biodegrade into several by-products includ- ing nonylphenol. Due to its physico-chemical characteristics, such as low solubility and high hydrophobicity, nonylphenol accumulates in envi- ronmental compartments that are characterized by high organic content, typically sewage sludge and river sediments, where it persists. The occur- rence of nonylphenol in the environment is clearly correlated with anthropogenic activities such as wastewater treatment, landfilling and sewage sludge recycling. Nonylphenol is found often in matrices such as sewage sludge, effluents from sewage treatment works, river water and sediments, soil and groundwater. The impacts of nonylphenol in the environment include feminiza- tion of aquatic organisms, a decrease in male fer- tility and the survival of juveniles at concentrations as low as 8.2 μg l−1. Owing to the harmful effects
of the degradation products of nonylphenol ethoxylates in the environment, the use and pro- duction of such compounds have been strictly monitored in many countries such as Canada and Japan and banned in EU countries. In July 2003 the EU passed Directive 2003/53/EC, which restricts the marketing and use in Europe of certain products and product formulations that contain more than 0.1% of nonylphenol ethoxy- lates or nonylphenol by weight. This applies to many industries, including the textile and leather industries among others.
Although it has been shown that the concentra- tion of nonylphenol in the environment is decreas- ing, it is still found at concentrations of 4.1 μg l−1 in river waters and 1 mg kg−1 in sediments. Nonylphenol has been referred to in the list of pri- ority substances in the Water Frame Directive and in the third draft Working Document on Sludge of the EU. Consequently there is currently a concern within some industries about the possibility of future regulations that may impose the removal of trace contaminants from contaminated effluents. Protein kinases AhR AhR ARNT Induced protein Differentiation and proliferation Altered protein phosphorylation AhR ligand mRNA
Fig. 20.23. Binding of the ligand (e.g. TCDD) to the AhR results in the release of associated proteins (chaperones, e.g. HSP90, heat shock protein 90) and translocation to the nucleus followed by dimerization with ARNT. The AhR–ARNT complex binds the xenobiotic response element (XRE) promoting target gene transcription. Ligands can also exert their effects in the cytoplasm through AhR-associated protein kinases to alter the function of a variety of proteins through a cascade of protein phosphorylation. From Pocar et al. (2005).6
The significance of upgrading sewage treatment works with advanced treatment technologies for removal of trace contaminants is discussed.
What are endocrine disrupters?
We have now discussed a number of pesticides used in food production, a number of POPs which are harmful due to their carcinogenicity and nonylphe- nol, which has been regulated due to being an endo- crine disrupter. But what is an endocrine disrupter and which compounds fall under this designation?
An endocrine disrupter is an exogenous sub- stance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations. Some chemicals can act on the endocrine system to disturb the homeostatic mech- anisms of the body or to initiate processes at abnormal times in the life cycle. The chemicals can exert their effects through a number of different mechanisms:
● They may mimic the biological activity of a hor- mone by binding to a cellular receptor, leading to an unwarranted response by initiating the cell’s normal response to the naturally occurring hormone at the wrong time or to an excessive extent (agonistic effect).
● They may bind to the receptor but not activate it. Instead the presence of the chemical on the receptor will prevent binding of the natural hormone (antagonistic effect).
● They may bind to transport proteins in the blood, thus altering the amounts of natural hormones that are present in the circulation. ● They may interfere with metabolic processes in
the body, affecting the synthesis or breakdown rates of the natural hormones.
Up to now, because of a series of observations in both humans and wildlife, the spotlight has focused on disruption to those hormones that play a major part in the control of reproduction and development. The main area of concern has been the steroid hormones produced by the gonads, which, in conjunction with some other hormones (particularly those produced by the pituitary), control processes such as reproduction and sexual behaviour, fetal differentiation and development, and maturation. They also influence the immune