Dioxins and dioxin-like compounds

Dioxins and dioxin-like compounds (DLC) are by-products of various industrial processes, and are commonly regarded as highly toxic compounds that are environmental pollutants and persistent organic pollutants (POPs). They include:
 * Polychlorinated dibenzo-p-dioxins (PCDDs), or simply, but inaccurately, dioxins. Technically PCDDs are derivatives of dibenzo-p-dioxin. There are 75 PCDDs, and seven of them are specifically toxic.
 * Polychlorinated dibenzofurans (PCDFs), or simply furans. Technically PCDFs are derivatives of dibenzofuran. There are 135 congeners (derivatives differing only in the number and location of chlorine atoms). Whilst they strictly speaking are not dioxins, ten of them have "dioxin-like" properties.
 * Polychlorinated biphenyls (PCBs), which also are not dioxins, but twelve of them have "dioxin-like" properties. Under certain conditions PCBs may form more toxic dibenzofurans through partial oxidation.
 * Finally, dioxin may refer to dioxin proper, the basic chemical unit of the more complex dioxins. This simple compound is not persistent and has no PCDD-like toxicity.

Because dioxins refer to such a broad class of compounds that vary widely in toxicity, the concept of toxic equivalence (TEQ) has been developed to facilitate risk assessment and regulatory control. Toxic equivalence factors (TEFs) exist for seven congeners of dioxins, ten furans and twelve PCBs. The reference congener is the most toxic dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which per definition has a TEF of one.

In reference to their importance as environmental toxicants the term dioxins is used almost exclusively to refer to the sum of compounds (as TEQ) from the above groups which demonstrate the same specific toxic mode of action associated with TCDD. These include 17 PCDD/Fs and 12 PCBs. Incidents of contamination with PCBs are also often reported as dioxin contamination incidents since it is this toxic characteristic which is of most public and regulatory concern.

Mechanism of toxicity
The toxic effects of dioxins are measured in fractional equivalencies of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), the most toxic and best studied member of its class (see TCDD for more detailed description of the mechanism). The toxicity is mediated through the interaction with a specific intracellular protein, the aryl hydrocarbon (AH) receptor, a transcriptional enhancer, affecting a number of other regulatory proteins. This receptor is a transcription factor which is involved in expression of many genes. TCDD binding to the AH receptor induces the cytochrome P450 1A class of enzymes which function to break down toxic compounds, e.g., carcinogenic polycyclic hydrocarbons such as benzo(a)pyrene.

While the affinity of dioxins and related industrial toxicants to this receptor may not fully explain all their toxic effects including immunotoxicity, endocrine effects and tumor promotion, toxic responses appear to be typically dose-dependent within certain concentration ranges. A multiphasic dose-response relationship has also been reported, leading to uncertainty and debate about the true role of dioxins in cancer rates. The endocrine disrupting activity of dioxins is thought to occur as a down-stream function of AH receptor activation, with thyroid status in particular being a sensitive marker of exposure. It is important to note that TCDD, along with the other PCDDs, PCDFs and dioxin-like coplanar PCBs are not direct agonists or antagonists of hormones, and are not active in assays which directly screen for these activities such as ER-CALUX and AR-CALUX. These compounds have also not been shown to have any direct mutagenic or genotoxic activity. Their main action in causing cancer is cancer promotion. A mixture of PCBs such as Aroclor may contain PCB compounds which are known estrogen agonists, but on the other hand are not classified as dioxin-like in terms of toxicity. Mutagenic effects have been established for some lower chlorinated chemicals such as 3-chlorodibenzofuran, which is neither persistent nor an AH receptor agonist.

Toxicity in animals
The symptoms reported to be associated with dioxin toxicity in animal studies are incredibly wide ranging, both in the scope of the biological systems affected and in the range of dosage needed to bring these about. Acute effects of single high dose dioxin exposure include wasting syndrome, and typically a delayed death of the animal in 1 to 6 weeks. By far most toxicity studies have been performed using 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The of TCDD varies wildly between species and even strains of the same species, with the most notable disparity being between the seemingly similar species of hamster and guinea pig. The oral for guinea pigs is as low as 0.5 to 2 μg/Kg body weight, whereas the oral  for hamsters can be as high as 1 to 5 mg/Kg body weight. Even between different mouse or rat strains there may be tenfold to thousandfold differences in acute toxicity. Many pathological findings are seen in the liver, thymus and other organs.

Some chronic and sub-chronic exposures can be harmful at much lower levels, especially at particular developmental stages including foetal, neonatal and pubescent stages. Well established developmental effects are cleft palate, hydronephrosis, disturbances in tooth development and sexual development as well as endocrine effects.

Human toxicity
Dioxins have been considered highly toxic and able to cause reproductive and developmental problems, damage the immune system, interfere with hormones and also cause cancer. This is based on animal studies. Unequivocal evidence of the toxic effects on dioxins on human beings have been shown by surprisingly few studies. The best proven is chloracne. Even in poisonings with huge doses of TCDD, the only persistent effects after the initial malaise have been chloracne and amenorrhea. In occupational settings many symptoms have been seen, but exposures have always been to a multitude of chemicals including chlorophenols, chlorophenoxy acid herbicides, and solvents. Therefore proof of dioxins as causative factors has been difficult. The suspected effects in adults are liver damage, and alterations in heme metabolism, serum lipid levels, thyroid functions, as well as diabetes and immunological effects.

In line with animal studies, developmental effects may be much more important than effects in adults. These include disturbances of tooth development, and of sexual development. An example of the variation in responses is clearly seen in a study following the Seveso disaster indicating that sperm count and motility were affected in different ways in exposed males, depending on whether they were exposed before, during or after puberty.

Carcinogenicity
Dioxins are well established carcinogens in animal studies, although the precise mechanistic role is not clear. Dioxins are not mutagenic or genotoxic. The United States Environmental Protection Agency has categorised dioxin, and the mixture of substances associated with sources of dioxin toxicity as a "likely human carcinogen". The International Agency for Research on Cancer has classified TCDD as a human carcinogen (class 1) on the basis of clear animal carcinogenicity and limited human data, but was not able to classify other dioxins. It is thought that the presence of dioxin can accelerate the formation of tumours and adversely affect the normal mechanisms for inhibiting tumour growth, without actually instigating the carcinogenic event.

As with all toxic endpoints of dioxin, a clear dose-response relationship is very difficult to establish. After accidental or high occupational exposures there is evidence on human carcinogenicity. There is much controversy especially on cancer risk at low population levels of dioxins. Among fishermen with high dioxin concentrations in their bodies, cancer deaths were decreased rather than increased. Some researchers have also proposed that dioxin induces cancer progression through a very different mitochondrial pathway.

Risk assessment
The uncertainty and variability in the dose-response relationship of dioxins in terms of their toxicity, as well as the ability of dioxins to bioaccumulate mean that the tolerable daily intake (TDI) of dioxin has been set very low, 1-4 pg/Kg body weight per day, i.e. 0.000 000 000 07 to 0.000 000 000 28 g per 70-kg person per day, to allow for this uncertainty and ensure public safety in all instances. Specifically, the TDI has been assessed based on the safety of children born to mothers exposed all their lifetime prior to pregnancy to such a daily intake of dioxins. It is likely that the TDI for other population groups could be somewhat higher. The most important cause for differences in different assessments is carcinogenicity. If the dose-response of TCDD in causing cancer is linear, it might be a true risk. If the dose-response is of a threshold-type or J-shape, there is little or no risk at the present concentrations. Understanding the mechanisms of toxicity better is hoped to increase the reliability of risk assessment.

Human intake and levels
Most intake of dioxin-like chemicals is from food of animal origin: meat, dairy products or fish predominate depending on the country. The daily intake of dioxins and dioxin-like PCBs as TEQ is of the order of 100 pg/day, i.e. 1-2 pg/kg/day. In many countries both the absolute and relative significance of dairy products and meat have decreased due to strict emission controls, and brought about the decrease of total intake. E.g. in the United Kingdom the total intake of PCDD/F in 1982 was 239 pg/day and in 2001 only 21 pg/day (WHO-TEQ). Since the half-lives are very long (for e.g. TCDD 7–8 years), the body burden will increase almost over the whole lifetime. Therefore the concentrations may increase five- to tenfold from age 20 to age 60. For the same reason, short term higher intake such as after food contamination incidents, is not crucial unless it is extremely high or lasts for several months or years.

The highest body burdens were found in Western Europe in the 1970s and early 1980s, and the trends have been similar in the U.S. The most useful measure of time trends is concentration in breast milk measured over decades. In many countries the concentrations have decreased to about one tenth of those in 1970s, and the total TEQ concentrations are now of the order of 10-30 pg/g fat (please note the units, pg/g is the same as ng/kg, or the non-standard expression ppt used sometimes in America). The decrease is due to strict emission controls and also to the control of concentrations in food. In the U.S. young adult female population (age group 20-39), the concentration was 9.7 pg/g lipid in 2001-2002 (geometric mean).

Certain professions such as subsistence fishermen in some areas are exposed to exceptionally high amounts of dioxins and related substances. This along with high industrial exposures may be the most valuable source of information on the health risks of dioxins.

Common uses
According to the Agency of Toxic Substances & Disease Registry :


 * Dioxins are not intentionally produced and have no known use. They are the by-products of various industrial processes (i.e., bleaching paper pulp, and chemical and pesticide manufacture) and combustion activities (i.e., burning household trash, forest fires, and waste incineration). The defoliant Agent Orange, used during the Vietnam War, contained dioxins. Dioxins are found at low levels throughout the world in air, soil, water, sediment, and in foods such as meats, dairy, fish, and shellfish. The highest levels of dioxins are usually found in soil, sediment, and in the fatty tissues of animals. Much lower levels are found in air and water.


 * CDDs are not manufactured commercially in the United States except on a small scale for use in chemical and toxicological research. They are unique among the large number of organochlorine compounds of environmental interest in that they were never produced intentionally as desired commercial products.

Dioxins belong to the persistent organic chemicals ("dirty dozen") the production and use of which was banned by the Stockholm Convention in 2001.

Environmental sources
PCB-compounds, always containing low concentrations dioxin-like PCBs and PCDFs, were synthesized at large amounts for various technical purposes (see Polychlorinated biphenyls). They have entered the environment through accidents such as fires or leaks from transformers or heat exchangers, or from PCB-containing products in waste landfills or during incineration. Because PCBs are somewhat volatile, they have also been transported long distances by air leading to global distribution including the Arctic.

PCDD/F-compounds were never synthesized for any purpose, except for small quantities for scientific research. Small amounts of PCDD/Fs are formed whenever carbon, oxygen and chlorine are available at suitable temperatures. This is augmented by metal catalysts such as copper. The optimal temperatures are 400 to 700 °C. This means that formation is highest when organic material is burned at poor burning conditions. The most important sources of PCDD/Fs have been incineration of mixed municipal or hospital waste at too low temperatures as well as metal smelting and refining. Chlorine bleaching of pulp has been an important source of PCDD/Fs to waterways. PCDD/Fs are also formed as synthesis side products of several chemicals, especially PCBs, chlorophenols, chlorophenoxy acid herbicides and hexachlorophene. A poorly appreciated but important production of dioxins is “backyard barrel burning” of waste.

When the problems were realized, both industries and municipal authorities have worked to decrease the emissions. In waste incineration this is based on basically two improvements, increase of burning temperatures to over 1000 °C at a long enough residence time for burning gases, and flue gas cleaning and filtering techniques. This has been very important in Europe, because most of municipal mixed waste is incinerated. Then dioxins spread around the incinerator, and the fallout on pastures caused accumulation in farm animals, and high concentrations in dairy and meat products. Therefore European Union set in 2000 very strict exhaust limit values for dioxins, 0.1 ng/Nm³ (TEQ in exhaust gases).

Both in Europe and in U.S.A. the emissions have decreased dramatically since the 1980s, by even 90 %. This has also led to decreases in human body burdens, which is neatly demonstrated by the decrease of dioxin concentrations in breast milk.

Unfortunately the private small-scale burning (“backyard barrel burning”) has not decreased effectively, and in the U.S. it is now the most important source of dioxins. Total annual emissions decreased from 14 kg in 1987 to 1.4 kg in 2000. However, backyard barrel burning decreased only modestly from 0.6 kg to 0.5 kg, resulting in over one third of all dioxins coming in the year 2000 from backyard burning alone.

Low concentrations of dioxins have been found in some soils without any anthropogenic contamination. A puzzling case of milk contamination was detected in Germany. The reason was found out to be kaolin added to animal feed. Dioxins have been repeatedly detected in clays from Europe and USA since 1996. Contamination of clay is assumed to be the result of ancient forest fires or similar natural reasons, and enrichment during clay sedimentation.

Environmental persistence and bioaccumulation
All groups of dioxin-like compounds are persistent in the environment. Neither soil microbes nor animals are able to break down effectively the PCDD/Fs with lateral chlorines (positions 2,3,7, and 8). This causes very slow elimination. Ultraviolet light is able to slowly break down these compounds. Lipophilicity (tendency to seek for fat-like environments) and very poor water solubility make these compounds move from water environment to living organisms having lipid cell structures. This is called bioaccumulation. Increase in chlorination increases both stability and lipophilicity. The compounds with the very highest chlorine numbers (e.g. octachlorodibenzo-p-dioxin) are, however, so poorly soluble that this hinders their bioaccumulation. Bioaccumulation is followed by biomagnification. Lipid soluble compounds are first accumulated to microscopic organisms such as phytoplankton (plankton of plant character, e.g. algae). Phytoplankton is consumed by animal plankton, this by invertebrates such as insects, these by small fish, and further by large fish and seals. At every stage or trophic level the concentration is higher, because the persistent chemicals are not “burned off” when the higher organism uses the fat of the prey organism to produce energy.

Due to bioaccumulation and biomagnification the species at the top of the trophic pyramid are most vulnerable to dioxin-like compounds. In Europe the white-tailed eagle and some species of seals have been close to extinct due to poisoning of persistent organic pollutants. Likewise in America the populations of bald eagle declined because of POPs causing thinning of eggs and other reproductive problems. Usually the failure has been attributed mostly to DDT, but dioxins are also a possible cause of reproductive effects. Both in America and in Europe many waterfowl have high concentrations of dioxins, but usually not high enough to disturb their reproductive success Due to supplementary winter feeding and other measures also the white-tailed eagle is recovering (see White-tailed eagle). Also ringed seals in the Baltic Sea are recovering.

Human being is also at the top of the trophic pyramid, but due to a multitude of food sources as compared with seals and eagles feeding almost exclusively on fish, human concentrations are much less, 10-100 pg/g, compared with 9000 to 340,000 pg/g (TEQ in lipid) in eagles.

Because of different physicochemical properties, not all congeners of dioxin-like compounds find their routes to human beings equally well. Measured as TEQs, the dominant congeners in human tissues are 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD and 2,3,4,7,8-PeCDF. This is very different from most sources where hepta- and octa-congeners may predominate. The WHO panel re-evaluating the TEF values in 2005 expressed their concern that emissions should not be uncritically measured as TEQs, because all congeners are not equally important. They stated that “when a human risk assessment is to be done from abiotic matrices, factors such as fate, transport, and bioavailability from each matrix be specifically considered”.

All POPs are poorly water soluble, especially dioxins. Therefore ground water contamination has not been a problem even in cases of severe contamination due to the main chemicals such as chlorophenols. In surface waters dioxins are bound to organic and inorganic particles.

Fate of dioxins in human body
The same features causing persistence of dioxins in the environment, also cause very slow elimination in humans and animals. Because of low water solubility, kidneys are not able to secrete them in urine as such. They should be metabolised to more water-soluble metabolites, but also metabolism especially in humans is extremely slow. This results in biological half-lives of several years for all dioxins. That of TCDD is estimated to be 7 to 8 years, and for other PCDD/Fs from 1.4 to 13 years, PCDFs on average slightly shorter than PCDDs.

Dioxins are absorbed well from the digestive tract, if they are dissolved in fats or oils (e.g. in fish or meat). On the other hand, dioxins tend to adsorb tightly to soil particles, and absorption may be quite low: 13.8 % of the given dose of TEQs in contaminated soil was absorbed.

In mammalian organisms dioxins are found mostly in fat. Concentrations in fat seem to be relatively similar, be it serum fat, adipose tissue fat or milk fat. This gives the possibility to measure body burden of dioxins by analysing breast milk. Initially, however, at least in laboratory animals, after a single dose high concentrations are found in the liver, but in a few days adipose tissue will predominate. In rat liver, however, high doses cause induction of CYP1A2 enzyme, and this binds dioxins. Thus depending on the dose, the ratio of fat and liver tissue concentrations may vary considerably in rodents.

Sources of human exposure
The most important source of human exposure is fatty food of animal origin (see Human intake, above). There is quite a lot of variation between different countries as to the most important items. In U.S. and Central Europe milk and dairy products and meat have been by far the most important sources. In some countries, notably in Finland and to some extent in Sweden, fish is important due to contaminated Baltic fish and very low intake from any other sources. In most countries a significant decrease of dioxin intake has occurred due to stricter controls during the last 20 years.

Historically occupational exposure to dioxins has been a major problem. Dioxins are formed as important toxic side products in the production of PCBs, chlorophenols, chlorophenoxy acid herbicides, and other chlorinated organic chemicals. This caused very high exposures to workers in poorly controlled hygienic conditions. Many workers had chloracne. In a NIOSH study in the U.S., the average concentration of TCDD in exposed persons was 233 ng/kg (in serum lipid) while it was 7 ng/kg in unexposed workers, even though the exposure had been 15-37 years earlier. This indicates a huge previous exposure, in fact the exact back-calculation is debated, and the concentrations may have been even several times higher than originally estimated.

Handling and spraying of chlorophenoxy acid herbicides may also cause quite high exposures, as clearly demonstrated by the users of agent orange in Vietnam War. The highest concentrations were detected in nonflying enlisted personnel (e.g. filling the tanks of planes), although the variation was huge, 0 to 618 ng/kg TCDD (mean 23.6 ng/kg). Other occupational exposures (working at paper and pulp mills, steel mills and incinerators) have been remarkably lower.

Accidental exposures have been huge in some cases. The highest concentrations in people after the Seveso accident were 56,000 ng/kg, and the highest exposure ever recorded was found in Austria in 1998, 144,000 ng/kg (see  TCDD). This is equivalent to a dose of 20 to 30 μg/kg TCDD, a dose that would be lethal to guinea pigs and some rat strains.

Exposure from contaminated soil is possible when children eat dirt or if dioxins are blown up in dust. This was clearly demonstrated in Missouri, when waste oils were used as dust suppressant in horse arenas. Many horses and other animals were killed due to poisoning. Dioxins are neither volatile nor water soluble, and therefore exposure of human beings depends on direct eating of soil or production of dust which carries the chemical. Contamination of ground water or breathing vapour of the chemical are not likely to cause a significant exposure. Currently, in the United States, there are 126 Superfund sites (with a completed exposure pathway) contaminated with dioxins.

TEF values
All dioxin-like compounds share a common mechanism of action via the aryl hydrocarbon receptor (AHR), but their potencies are very different. This means that similar effects are caused by all of them, but much larger doses of some of them are needed than of TCDD. Binding to the AHR as well as persistence in the environment and in the organism depends on the presence of so called "lateral chlorines", in case of dioxins and furans, chlorine substitutes in positions 2,3,7, and 8. Each additional chlorine decreases the potency, but qualitatively the effects remain similar. Therefore a simple sum of different dioxin congeners is not a meaningful measure of toxicity. To compare the toxicities of various congeners and to render it possible to make a toxicologically meaningful sum of a mixture, a toxicity equivalency (TEQ) concept was created.

Each congener has been given a toxicity equivalence factor (TEF). This indicates its relative toxicity as compared with TCDD. Most TEFs have been extracted from in vivo toxicity data on animals, but if these are missing (e.g. in case of some PCBs), less reliable in vitro data have been used. After multiplying the actual amount or concentration of a congener by its TEF, the product is the virtual amount or concentration of TCDD having effects of the same magnitude as the compound in question. This multiplication is done for all compounds in a mixture, and these "equivalents of TCDD" can then simply be added, resulting in TEQ, the amount or concentration of TCDD toxicologically equivalent to the mixture.

The TEQ conversion makes it possible to use all studies on the best studied TCDD to assess the toxicity of a mixture. This resembles the common measure of all alcoholic drinks: beer, wine and whiskey can be added together as absolute alcohol, and this sum gives the toxicologically meaningful measure of the total impact.

The TEQ only applies to dioxin-like effects mediated by the AHR. Some toxic effects (especially of PCBs) may be independent of the AHR, and those are not taken into account by using TEQs.

TEFs are also approximations with certain amount of scientific judgement rather than scientific facts. Therefore they may be re-evaluated from time to time. There have been several TEF versions since the 1980s. The most recent re-assessment was by an expert group of the World Health organization in 2005.



WHO Toxic Equivalence Factors (WHO-TEF) for the dioxin-like congeners of concern

(T = tetra, Pe = penta, Hx = hexa, Hp = hepta, O = octa)