## 3.1.7. Biota

(draft)

Author: Steven Droge,

Reviewer: Nico van der Brink, John Parsons

Learning objectives:

You should be able to:

- explain the effect of cell and body composition on toxicokinetics of compounds
- describe the role of biota in the environmental fate of chemicals

Keywords: cellular composition, body composition, exposure routes, absorption, distribution

Introduction

Just like soil, water, and air, the organic tissue of living organisms can also be regarded as a compartment of the ecosystem where chemical pollutants can accumulate or can be broken down. The internal concentration in living organisms provide important information on chemical exposure and ultimately determines the environmental risk of pollution, but it is important to understand the key features of tissue that influence chemical partitioning into organisms. Chemical accumulation in the tissue of living organisms is a series of chemical and biological processes, briefly based on:

- chemical uptake (mostly permeation from bulk media over certain membranes into cells);

- internal distribution (e.g. via blood flows through organs);

- metabolism (e.g. biotransformation processes in for instance the liver).

- excretion (e.g. through urine and feces, but also via gills, sweat, milk, or hairs)

These four processes are the basis of toxicokinetic modeling, and are often summarized as Absorption, Distribution, Metabolism, and Excretion, or “ADME”. These ADME processes can strongly vary for different polluting compounds due to the properties of the chemical structure. These ADME processes can also strongly vary for different organisms, because of:

- the physiological characteristics (e.g. having gills, lungs, or roots, availability of specific chemical uptake mechanisms, presence of specific metabolic enzymes, size-related properties like metabolic rate),

- the position in the polluted environment (flying birds or midge larvae living in sediment),

- the interaction with the polluted environment (living in soil or water, food choice, etc.)

- the behaviour in the polluted environment (being sessile or able to move (temporarily) away from a polluted spot).

More details of these toxicokinetic processes are presented in section 4.1 on Toxicokinetics and bioaccumulation. The current module aims to provide a summary of the key features of different tissue components that explain the internal distribution of chemicals (distribution), the different types of contact between pollutants and organisms (exposure-absorption), and temporal changes in physiology that may affect internal exposure (e.g. excretion, which includes examples such as release of POPs via lactation, and increasing POP concentrations during starvation). Before we discuss how chemicals are taken up into biota, it is important to first define the key chemical properties and the molecular composition of tissue that influence the way chemicals are absorbed from the surrounding environment and distributed throughout an organism.

Absorption-distribution: Tissue building blocks

All organisms are composed of cells, which are composed of a cell membrane, surrounding the largely watery solution filled with inner organelle membranes, protein structures, and DNA/RNA. Prokaryote organisms such as bacteria, but also algae, fungi and plants have reinforced membranes with cell walls to prevent water leaking by high osmotic pressures, and to protect the cell membrane. Metabolic energy is stored in large molecules such as fatty esters and sugars. Remarkably, for all existing living organism species, these tissue components are mostly structures made out of relatively simple and repetitive molecular building blocks, with minor variations on side chains. See examples in Figure 1. The composition of organs, as a collection of specific cells, in terms of the percentage of lipids, proteins and carbohydrates is important for the overall toxicokinetics of chemicals in the whole organism.

Figure 1. the key molecular components and partial building blocks of living tissue.

Cell walls are mostly made from highly polar polysaccharides, e.g.:

- cellulose, a polymer of sugary molecules, and chitin in fungi, which is highly polar and therefore permeable for water.
- peptidoglycan semi-crystal structures surrounding bacteria (90% of the dry weight of Gram-positive bacteria but only 10% of Gram-negative strains), a mixed polymer between N-acetylglucosamine (alike chitine) and short interconnecting 4 or 5 amino acid chains.
- lignin, a polar (~30% oxygen) but more hydrophobic supra-structure of polymerized phenolic molecules lining the main plant vessels that transport water.

The specific algae group of diatoms have a cell wall composed of biogenic silica (hydrated silicon dioxide), typically as two valves that overlap each other surrounding the unicellular species. Diatoms generate about 20 percent of the oxygen annually produced on the planet, and contribute nearly half of the organic material found in the oceans. With their specific cell wall structure, diatoms take in over 6.7 billion metric tons of silicon each year from the waters in which they live, which creates huge deposits when they die off.

Cell membranes are made up mostly of a phospholipid bilayer, with each phospholipid molecule basically having a polar and ionized headgroup connected to two long alkyl chains (Figure 1 example with POPC type phospholipid). The outer sides of a phospholipid bilayer are hydrophilic (water-loving), the inside is hydrophobic (water-fearing). Ions (inorganic salts, nutrients, metals, strong acids and ionized biomolecules) do not readily permeate through such a membrane passively, and require specific transport proteins that can transport as well as regulate ions in and out of the cell interior. Cholesterol molecules stabilize the fluidity of the membrane bilayers in cells of most organisms, but for example not in most Gram negative bacteria. Dissolved neutral chemicals may passively diffuse through phospholipid bilayers into and out of cells.

Proteins are chains of a variety of amino acids, 21 of which are known to be genetically coded, and of which humans can only produce 12. The other nine must be consumed, and are therefore called essential amino acids (coded H, I, L, K, M, F, T, W, V). Proteins form complex 3 dimensional structures that allow for enzymatic reactions to occur effectively and repeatedly. There are two amino acids with side chains that carry a positive charge at neutral pH: Arginine (pKa 12) and Lysine (pKa 10.6), and two amino acids with side chains that carry a negative charge at neutral pH: Aspartic acid (pKa 3.7) and Glutamic acid (pKa 4.1). Some amino acids carry typical hydrophobic side chains: amongst others Leucine and Phenylalanine. Cysteine has a thiol (SH) moiety that can form strong connective disulfide interactions with spatially nearby cysteine side groups in the 3D structure. The key blood transport protein albumin, for example, contains about 98 anionic amino acids, and 83 cationic amino acids, and about 35 cysteine residues.

DNA and other genetically encoding chains are composed of 4 different nucleotides that form a double helix of two opposing strands, held together by hydrogen bonds connecting the complementary bases: A and T (or A and U in RNA), and G and C. DNA can be densely packed around histone proteins, and is either or part of the cellular cytoplasm (in prokaryotic species) or separated within a membrane (in eukaryotic species). DNA is not a critical accumulation phase for chemicals, but of course it is a cellular structure where pollutants can strongly impact all kinds of cellular processes when they react with DNA components or affect the structural organisation otherwise.

Storage fat provides for many animals and fruits of plants an important energy reserve, but also insulates warm-blooded animals in cold climates, lubricates joints to move smoothly, and protects organs from shocks (e.g. eyes and kidneys). Seeds and nuts may contain up to 65% (walnuts) of fatty components, which of course provides energy for initial growth, but from which also oil can be pressed. Storage fat in most animals is present in the form of triglycerides, and as such neutral and very hydrophobic phases within tissue. Polyunsaturated fatty acid esters like omega-6 and omega-3 fatty acids are abundant in fish (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) and in seeds and plants (mostly alpha-linolenic acid (ALA), but algae also contain EPA and DHA). The high intake of algae by fish in aqueous food webs based on algae, results in the high EPA/DHA levels in many fish species, as they mostly cannot make it themselves (https://www.pufachain.eu). Humans can make some EPA and DHA from ALA.

The average composition of living organisms based on the key tissue components lipid, protein, and carbohydrate can range widely, as illustrated in Table 1.

Table 1. Tissue structure composition of the average dry weight of different organisms.

Organism | lipid % of d.w. | protein % of d.w. | carbohydrate % of d.w.
Grass | 0.5-4 | 15-25 | 60-84
Phytoplankton | 20 | 50 | 30
Zooplankton | 15-35 | 60-70 | 10
Oyster | 12 | 55 | 33
Midge larvae | 10 | 70 | 20
Army cutworms (moth larvae) | 72% of body |  |
Pike filet | 3.7 | 96.3 | 0
Lake trout | 14.4 | 85 | 0
Eel (farmed for 1.5y) | 65 | ~34 | 1
Deer game meat | 10 | 90 | 0

Table 2. Estimates on the tissue structure composition of a woman (BW= 60 kg, H = 163 cm, BMI = 22.6 kg/m2), (taken from Goss et al., 2018). Bones are not included.

Organ | Total organ volume (mL) | moisture content | phospholipid % of d.w. | storage lipid % of d.w. | protein % of d.w.
Adipose | 22076 | 26.5% | 0.3% | 93.6% | 6.1%
Brain | 1311 | 80.8% | 35.4% | 22.6% | 42.1%
Gut | 1223 | 81.8% | 9.9% | 22.0% | 68.1%
Heart | 343 | 77.3% | 19.1% | 17.7% | 63.2%
Kidneys | 427 | 82.4% | 16.5% | 5.9% | 77.7%
Liver | 1843 | 79.4% | 19.4% | 8.0% | 72.6%
Lung | 1034 | 94.5% | 13.1% | 13.7% | 73.2%
Muscle | 19114 | 83.7% | 2.6% | 2.5% | 95.0%
Skin | 3516 | 71.1% | 2.6% | 22.9% | 74.5%
Spleen | 231 | 83.5% | 5.6% | 2.4% | 92.0%
Gonads | 12 | 83.3% | 18.8% | 0.0% | 81.3%
Blood | 4800 | 83.0% | 2.5% | 2.4% | 95.1%
total | 55929 | 60.2% | 1.7% | 71.0% | 27.3%

Different organs in a single species can also largely differ in their composition, as well as their contribution to the overall body, as shown for a human in Table 2. Most organs have a moisture content >75%, but overall the moisture content is considerably lower, due to the low moisture content of bones and adipose tissue. Adipose tissue is by far the largest repository of lipids, but made up mostly of storage lipid, while the brain is also particularly rich in both lipids, but particularly enriched in phospholipids of cell membranes. Muscles and blood contain relatively high protein content.

Absorption-distribution: Chemical properties

The influence of chemical structure on the accumulation of chemicals in the biotic compartments is largely dependent on their bioavailability, as discussed in more detail in section 4.1 on Toxicokinetics and bioaccumulation, as well as on the basic binding properties as the results of the chemical’s hydrophobicity and volatility (section 3.4 on Partitioning and partitioning constants) and ionization state (section 2.2.6 on Ionogenic organic chemicals). In brief, the more non-polar the composition of a chemical, the more hydrophobic it is, the higher its affinity to partition from dissolved phases (both externally as well as internally) into poorly hydrated tissue phases such as storage fat and cell membranes. For this reason, the main issue with classical organic pollutants such as dioxins, DDT, and PCBs, is often their high hydrophobicity which results in strong accumulation in tissue. Such chemicals often take very long times to excrete from the tissue if they are not made less hydrophobic via biotransformation processes. This leads to foodweb accumulation and specific acute or chronic toxic effects at a certain organism level (section 4.1.6 on Food chain transfer). Proteins and sugary carbohydrates are mostly comprised of extended series of polar units and thus strongly hydrated, and bind hydrophobic chemicals to a much lower extent. Proteins may have three dimensional pockets that could fit either hydrophilic of hydrophobic chemicals, and as such act as transport proteins in blood throughout the body (transporting fatty acids for example), based on the specific binding affinity. Many protein based receptors are also based on a specific binding affinity, and in many cases this involves (combinations of) polar and electrostatic interactions that also have an optimum three-dimensional fitting space. Volatile chemicals are more abundantly present in the gas phase rather than being dissolved, and are more readily in contact with biota via gas-exchange on the extensive surfaces of lungs of animals and leaves of plants.

In order to be taken up into cells, or into organs, chemicals have to permeate through membranes. For most organic pollutants, the passive diffusion through phospholipid bilayers has an optimum at a certain hydrophobicity. The high accumulation in the membrane ensures desorption into the adjacent cellular solution. It is assumed that ionized chemicals have a passive permeation rates that are either negligible or at least orders of magnitude lower than that of corresponding neutral chemicals. For this reason, all kinds of molecular intra-extracellular gradients can be readily maintained, for example for protons (H+) or sodium-potassium (Na+/K+). The movement of very polar and ionic chemicals can be tightly regulated by transport proteins protruding the membrane bilayer. Specific molecules can be actively excreted from cells (e.g. certain drugs) or reabsorbed (e.g. in the kidneys back into the blood stream). This again is based on three dimensional fitting in the transport pocket and stepwise movement through the protein structure, and costs energy. For acids and bases with a very small fraction of neutral species at physiological pH, the passive permeation over membranes may still be dominated by the neutral species.

Exposure: Contact between biota and various environmental compartments

There are multiple routes by which chemicals can enter the tissue of biota, for example via respiratory organs, through digestion of contaminated food, or dermal contact. Most animals need to take in a more or less constant flux of oxygen and water, and periodically food, to release nutrients and energy from the food. Of course they also need to release CO2 (and other chemicals) as waste. Pollutant chemicals are taken up alongside these basic processes, and it depends on the chemical properties and the efficiency of the uptake route how much the organism will take in from these different exposure routes.

Plants need plenty of water and during daytime photosynthesis need CO2, but also require oxygen during the night. High algal densities can deplete the oxygen levels in shallow aquatic systems during the night, and replenish oxygen levels during daytime. Oxygen is plentiful in air (200,000 parts per million in the air), but it is considerably less accessible in water (15 parts per million in cool, flowing water), and often depleted below the first few mm of sediment. To obtain sufficient oxygen, water and food, aquatic organisms have to pass large volumes of waters through their gills. Sediment-dwelling organisms either have hemoglobin to bind oxygen, or constantly pump fresh overlying water through burrows created in sediment, often lined with mucus. Living organisms are thus constantly in contact with water dissolved pollutants, and air breathing organism are readily exposed to air pollutants. To simplify the domain of living organisms as part of this module about the biotic compartment and how they get into contact with chemicals relevant to Environmental Toxicology, they can for example be divided in:

- , which often take in large amounts of water from their surroundings via their roots, driven by the evaporation of water at the leaves and resulting internal flows.
- water breathing organisms, which pass large amounts of water through gills or gill like structures (tubules or other thin skin structures close to where water is passing) in order to take in enough oxygen and reduce the built up of CO2. Filter feeders like oysters and mussels, that populate enormous surfaces as reefs or banks, can turnover a huge volume of water on a daily basis, and thus allow dissolved chemicals get in close contact to the outer membranes.
- air breathing organisms, which can effectively exchange large quantities of volatile and gaseous chemicals with the air (oxygen but also organic compounds), but typically take in less/non-volatile chemicals via food and require active excretion and metabolism for the emission of less/non-volatile chemicals.

Figure 2. Left panel: Plant water transport (adapted from https://en.wikipedia.org/wiki/Xylem), a schematic view of the waxy upper layers on leaves (adapted from Moeckel et al. 2008)). Middle panel: water flushing along gill lamellae replenishes oxygen in blood. Top Right panel: Many benthic organisms living in anoxic sediments require overlying oxygen rich water, and feed on dissolved particles. Lower Right Panel: Earthworms take up oxygen via diffusion through their skin, but also metals and organic pollutants, as shown in studies where the mouth part has been glued shut during exposure (adapted from Vijver et al., 2003).

Plants

Nearly all plants have roots below ground, a sturdy structure of stem and branches above ground, and leaves. Along with soil pore water, soluble chemicals are readily transported from roots of the plant in the internal circulation stream through xylem cells, which are lined with water impenetrable lignin (see Figure 2). Moderately hydrophobic chemicals (Kow of 1-1000) are rapidly transported from roots to shoots to leaves, while hydrophobic chemicals may be strongly retained on the membranes and cell walls and mostly accumulate in root sections, limiting transport to above-ground plant tissues. Roots may also actively release considerable quantities of chemicals to influence the immediate surrounding media of the roots (rhizosphere), e.g. to stimulate microbial processes or pH in order to release nutrients. These plant root ‘exudates’ can be ions, small acids, amino acids, sterols, etc. Chemicals that enter plants via leaves, such as pesticides or semi-volatile organic pollutants, can be redistributed to other plant parts via the phloem streams.

The transport through xylem up to higher plant tissues occurs via capillary forces and is enhanced by three passive phenomena:

- the high sugar content in the phloem causing osmotic pressure to attract water from other parts.
- the evaporation of water creates surface tension on thousands of cells that pulls water from the soil through the xylem system.
- the osmotic pressure of root cells compared to soil pore water. Root pressure is highest in the morning before the stomata open and allow transpiration to begin.

As a result of the capillary forces needed to pull water up against gravity, and a certain maximum diameter of the vessels to do so, there is a maximum possible plant height of 122-130 m (Koch et al., 2004) which compares to Redwood trees (Sequoias) reaching a maximum height of 113 m.

Most leaves are covered with a waxy layer, to prevent damage and water evaporation. This wax layer may be 0.3-4.6 µm thick (Moeckel et al., 2008). Large forests provide enormous hydrophobic surfaces to which semi-volatile organic chemicals (SVOC) can bind out of air, which influences the global distribution of chemicals such as PCBs. Partitioning of SVOCs on the vegetation of extended grasslands contaminates the base of the food chain, as well as agricultural and cattle sectors used by humans. The grass/corn-cattle-milk/beef food chain accounts for the largest portion of background exposure of the European and North American population to many persistent SVOCs. The absorption rate of chemicals on the leaves often also depends on the air boundary layer surrounding leaves, which limits diffusion into the leave surfaces. Of course, all kinds of other factors such as wind speed, canopy formation and cuticle thickness also control exchange between leaves and gas phase (see also section 3.1.2 on the Atmosphere). Tiny openings or pores on the lower side of the leaves, called stomata, allow furthermore for gas exchange. In warm conditions, stomata can close to prevent water evaporation, but gas exchange is needed in many plant types to allow for CO2 to be metabolized and the release of O2 that is produced. The waxy layer on leaves can trap gaseous organic chemicals. Many plants, like coniferous trees, produce resins to provide effective defense against insects and diseases, and these resins release large amounts and structurally highly diverse organic volatiles such as terpenes and isoprenes (Michelozzi, 1999). These plant-produced volatiles can even contribute to ozone formation. Plants thus accumulate chemicals from their environment, but also release chemicals into the environment. It thus also matters for the exposure of grazing organisms to certain types of pollutants whether they eat roots, shoots, leaves, seeds or fruits of plants living in contaminated environments.

Organisms using dissolved oxygen

The ‘gill’ movements of water breathers create a constant flux of chemicals dissolved in bulk water along outer cell membranes (or mucus layers surrounding cell membranes) of gills. A 1 kg rainbow trout fish ventilates about 160 mL/min, so 230 L/day (Consoer et al., 2014). The total gill surface area in a fish depends on species behaviour and weight (active large fish require a lot of oxygen), and equals to about 1-6 cm2 per g fish (Palzenberger & Pohla, 1992). For a 1 kg fish of ~20 cm length, the ~1000 cm2 gill area compares to a ~500 cm2 outer body surface. This results in an effective partitioning of chemicals between water and cell membranes. Within the gills, the cells are in close contact with the blood system of the organism, and the build-up of chemical concentrations in the outer cells provides an effective exchange with the blood stream (or other internal fluids, Figure 2) flushing along that redistribute chemicals to the inner organs. The reverse equally occurs: chemicals dissolved in blood stream coming from organs will also rapidly exchange with bulk external water if concentrations are lower.

Of course, many pollutants can also enter water breathing organisms via food, but the gills-water exchange is very effective in controlling the distribution of chemicals. The salinity of water plays a strong role in the need of water breathing organisms to “drink” water, and hence take in contaminants via this route.

BOX 1. Osmoregulation (MSc level) Most aquatic vertebrate animals are osmoregulators: their cells contain a concentration of solutes that is different than the water around them. Fish living in freshwater typically have a cellular osmotic level (300 millOsmoles per Liter, mOsm/L) that is higher than the bulk fresh water (~20-40 mOsm/L), so a lot of water flows passively via the gills (not via the skin) into the tissue of fish. They are thus constantly taking in water (water molecules only) via the gills, which needs to be controlled, e.g. by strongly diluting the urine. They do also take in some water in their gastro-intestinal tract (GIT). Marine fish have similar cellular osmotic levels as freshwater fish, but the salty water (1000 mOsm/L) causes water to move out of the gill tissue through the linings of the fish’s gills by osmosis, which needs to be replenished by active intake of salty water, and separate excretion of the salts. Most invertebrate organisms in oceans have an internal overall concentration of dissolved compounds comparable to the water they live in, so that they don’t suffer from strong osmotic pressures on their soft tissue (osmoconformers).

A single adult oyster can cleanse about 200 liters of water per day (https://www.cbf.org/about-the-bay/more-than-just-the-bay/chesapeake-wildlife/eastern-oysters/oyster-fact-sheet.html). Plans to re-populate the harbour of New York with 1 billion oysters on artificial substrates can have enormous impacts on chemical redistribution. A single 2 cm zebra mussel (Dreissena polymorpha) that inhabits the shallow Lake IJsselmeer (6.05x1012 L) in can filter about 1 L per day, and the high densities of these and related species in this fresh water lake can turn over the lake volume once or twice per month (Reeders et al., 1989).

Even many soil organism are constantly in contact with wet soil surfaces, and contact between soil pore water and the outer surfaces (gills/soft skin areas) dominates the routes of chemical exchange for many chemicals. Earthworms, for example, do not have lungs and they exchange oxygen through their skin. Earthworms eat bacteria and fungi that grow on dead and decomposing organic matter, and thus act as major organic matter decomposers and recycling of nutrients. Earthworms dramatically alter soil structure, water movement, nutrient dynamics, and plant growth. It is estimated that earthworms turn over the top 15 cm of soil in ten to twenty years (LINK), and so they also are able to mix surface bound pollution into a substantial soil layer. In terms of biomass, earthworms dominate the world of soil invertebrates, including arthropods. In order to better understand how much contamination earthworms take in via food or via their skin, several studies have used earthworms in exposure tests with part of the organisms having their mouth parts sealed with surgical glue (Vijver et al., 2003). Uptake rates of the metals Cd, Cu and Pb in sealed and unsealed earthworms exposed to two contaminated field soils were similar (Vijver et al., 2003), indicating main uptake through the skin of the worms. The uptake rates as well as the maximum accumulation level for several organic contaminants from artificially contaminated soil were also comparable between sealed and non-sealed worms (Jager et al., 2003). The dermal route is thus a highly important uptake route for organic chemicals too. Dermal uptake by soil organisms is generally from the pool of chemicals in the soil pore water, hence the distribution of chemicals between solid particles and organic materials in the soil and the soil pore water is extremely important in driving the dermal uptake of chemicals by earthworms (See section 3.4 on Partitioning and partitioning constants, section 3.5 on Metal speciation and 3.6 on Availability and bioavailability).

Air breathing organisms

Air breathing organisms typically take in less/non-volatile chemicals via food and require active excretion and metabolism for the elimination of these chemicals via e.g. feces and urine. Dermal uptake is generally assumed to be negligible, while the intake of chemicals via air particles can be relatively high, for example of pollutants present in house dust, or contaminants on aerosols. The excretion via air particles is clearly not a dominant route. The chemicals in the food matrix inside the gastro-intestinal tract often first need to be fully dissolved in gut fluids, before they can pass the mucus layers and membranes lining the gastrointestinal tract and enter blood streams for redistribution. However, ‘endocytosis’ may also result in the uptake of (small) particulate chemicals in cells by first completely surrounding the particle by the membrane, after which the encapsulating membrane buds buds off inside the cell and forms a vesicle. Notwithstanding this endocytosis, chemical fractions of pollutants strongly sorbed to non-digestible parts may not always automatically be taken up from food. Grazing animals typically require microbial conversion in their gut to digest plant material like cellulose and lignin into chemical components that can be taken up as energy source.

Many aquatic foodwebs are structured such that they begin with aquatic plants being eaten by water breathing organisms, with air breathing marine animals or birds on top of the food chain. These air breathing top predators take in pollutants largely through their diet, but lack the effective blood-membrane-water exchange through gills. The blood-membrane-air partitioning in lungs is far less effective in removing chemicals via passive partitioning. For this reason, many top predators of foodwebs have the highest concentrations of pollutants. The chemical distribution in foodwebs will be discussed in more detail in section 4.1.6.

References

Palzenberger & Pohla 1992, Reviews in Fish Biology and Fisheries, 2, 187-216.

Jager, T.; Fleuren, R.H.L.J.; Hogendoorn, E.A.; de Korte, G. 2003. Elucidating the routes of exposure for organic chemicals in the earthworm, Eisenia andrei (Oligochaeta). Environ. Sci. Technol. 37 (15), 3399-3404

Koch et al. 2004, Nature (428) 851–854.

Moeckel et al. 2008, Environ. Sci. Technol. 42, 100–105.

Michelozzi 1999, Defensive roles of terpenoid mixtures in conifers, Acta Botanica Gallica 146 (1), 73-84.

Consoer et al. 2014, Aquatic Toxicology 156, 65–73.

Reeders et al. 1989, Freshwater Biology 22 (1), 133-141.

Vijver et al. 2003, Soil Biology and Biochemistry 35, 125-132.

Goss et al. 2018, Chemosphere 199, 174-181.