## 4.3.6. Selection of test organisms - Microorganisms

Author: Patrick van Beelen

Reviewers: Kees van Gestel, Erland Bååth, Maria Niklinska

Learning objectives:

You should be able to

- describe the vital role of microorganisms in ecosystems.
- explain the difference between toxicity tests for protecting biodiversity and for protecting ecosystem services.
- explain why short-term microbial tests can be more sensitive than long-term ones.

Keywords: microorganisms, processes, nitrogen conversion, test methods

The importance of microorganisms

Most organisms are microorganisms, which means they are generally too small to see with the naked eye. Nevertheless, microorganisms affect almost all aspects of our lives. Viruses are the smallest of microorganisms, the prokaryotic bacteria and archaea are bigger (in the micrometer range), and the sizes of eukaryotic microorganisms range from three to hundred micrometers. The microscopic eukaryotes have larger cells with a nucleus and come in different shapes like green algae, protists and fungi.

Cyanobacteria and eukaryotic algae perform photosynthesis in the oceans, seas, brackish and freshwater ecosystems. They fix carbon dioxide into biomass and form the basis of the largest aquatic ecosystems. Bacteria and fungi degrade complex organic molecules into carbon dioxide and minerals, which are needed for plant growth.

Plants often live in symbiosis with specialized microorganisms on their roots, which facilitate their growth by enhancing uptake of water and nutrients, speeding up plant growth. Invertebrate and vertebrate animals, including humans, have bacteria and other microorganisms in their intestines to facilitate the digestion of food. Cows for example cannot digest grass without the microorganisms in their rumen. Also, termites would not be able to digest lignin, a hard to digest wood polymer, without the aid of gut fungi. Leaf cutter ants transport leaves into their nest to feed the fungi which they depend on. Also, humans consume many foodstuffs with yeasts, fungi or bacteria for preservation of the food and a pleasant taste. Beer, wine, cheese, yogurt, sauerkraut, vinegar, bread, tempeh, sausage and may other foodstuffs need the right type of microorganisms to be palatable. Having the right type of microorganisms is also vital for human health. Human mother’s milk contains oligosaccharides, which are indigestible for the newborn child. These serve as a major food source for the intestinal bacteria in the baby, which reduce the risk of dangerous infections.

This shows that the interaction between specific microorganisms and higher organisms are often highly specific. Marine viruses are very abundant and can limit algal blooms promoting a more diverse marine phytoplankton. Pathogenic viruses, bacteria, fungi and protists enhance the biodiversity of plants and animals by the following mechanism: The densest populations are more susceptible to diseases since the transmission of the disease becomes more frequent. When the most abundant species become less frequent, there is more room for the other species and biodiversity is enhanced. In agriculture, this enhanced biodiversity is unwanted since the livestock and crop are the most abundant species. That is why disease control becomes more important in high intensity livestock farming and in large monocultures of crops. Microorganisms are at the base of all ecosystems and are vital for human health and the environment.

The microbiological society has a nice video explaining why microbiology matters.

Protection goals

The functioning of natural ecosystems on earth is threatened by many factors, such as habitat loss, habitat fragmentation, global warming, species extinction, over fertilization, acidification and pollution. Natural and man-made chemicals can exhibit toxic effects on the different organisms in natural ecosystems. Toxic chemicals released in the environment may have negative effects on biodiversity or microbial processes. In the ecosystem strongly affected by such changes, the abundance of different species could be smaller. The loss of biodiversity of the species in a specific ecosystem can be used as a measure for the degradation of the ecosystem. Humans benefit from the presence of properly functioning ecosystems. These benefits can be quantified as ecosystem services. Microbial processes contribute heavily to many ecosystem services. Groundwater for example, is often a suitable source of drinking water since microorganisms have removed pollutants and pathogens from the infiltrating water. See Section on Ecosystem services and protection goals.

Environmental toxicity tests

Most environmental toxicity tests are single species tests. Such tests typically determine toxicity of a chemical to a specific biological species like for example the bioluminescence by the Allivibrio fisheri bacteria in the Microtox test or the growth inhibition test on freshwater algae and cyanobacteria (see Section on Selection of test organisms – Eco plants). These tests are relatively simple using a specific toxic chemical on a specific biological species in an optimal setting. The OECD guidelines for the testing of chemicals, section 2, effects on biotic systems gives a list of standard tests. Table 1 lists different tests with microorganisms standardized by the Organization for Economic Cooperation and Development (OECD).

Table 1. Generally accepted environmental toxicity tests using microorganisms, standardized by the Organization for Economic Cooperation and Development (OECD).

OECD test No | Title | Medium | Test type
201 | Freshwater algae and cyanobacteria, growth inhibition test (chapter reference) | Aquatic | Single species
209 | Activated sludge, respiration inhibition test | Sediment | Process
224 (draft guideline) | Determination of the inhibition of the activity of anaerobic bacteria | Sediment | Process
217 | Soil microorganisms: carbon transformation test | Soil | Process
216 | Soil microorganisms: nitrogen transformation test | Soil | Process

The outcome of these tests can be summarized as EC10 values (see Section on Concentration-response relationships), which can be used in risk assessment (see Sections on Predictive risk assessment approaches and tools and on Diagnostic risk assessment approaches and tools) Basically, there are three types of tests. Single species tests, community tests and tests using microbial processes.

Single species tests

The ecological relevance of a single species test can be a matter of debate. In most cases it is not practical to work with ecologically relevant species since these can be hard to maintain under laboratory conditions. Each ecosystem will also have its own ecologically relevant species, which would require an extremely large battery of different test species and tests, which are difficult to perform in a reproducible way. As a solution to these problems, the test species are assumed to exhibit similar sensitivity for toxicants as the ecological relevant species. This assumption was confirmed in a number of cases. If the sensitivity distribution of a given toxicant for a number of test species would be similar to the sensitivity distribution of the relevant species in a specific ecosystem, one could use a statistic method to estimate a safe concentration for most of the species.

Toxicity tests with short incubation times are often disputed since it takes time for toxicants to accumulate in the test animals. This is not a problem in microbial toxicity tests since the small size of the test organisms allows a rapid equilibrium of the concentrations of the toxicant in the water and in the test organism. On the contrary, long incubation times under conditions that promote growth, can lead to the occurrence of resistant mutants, which will decrease the apparent sensitivity of the test organism. This selection and growth of resistant mutants cannot, however, be regarded as a positive thing since these mutants are different from the parent strain and might also have different ecological properties. In fact, the selection of antibiotic resistant microorganisms in the environment is considered to be a problem since these might transfer to pathogenic (disease promoting) microorganisms which gives problems for patients treated with antibiotics.

The OECD test no 201, which uses freshwater algae and cyanobacteria, is a well-known and sensitive single species microbial ecotoxicity test. These are explained in more detail in the Section on Selection of test organisms – Eco plants.

Community tests

Microorganisms have a very wide range of metabolic diversity. This makes it more difficult to extrapolate from a single species test to all possible microbial species including fungi, protists, bacteria, archaea and viruses. One solution is to test a multitude of species (a whole community) exposed in a single toxicity experiment, it becomes more difficult to attribute the decline or increase of species to toxic effects. The rise and decline of species can also be caused by other factors, including species interactions. The method of Pollution-induced community tolerance is used for the detection of toxic effects on communities. Organisms survive in polluted environments only when they can tolerate toxic chemical concentrations in their habitat. During exposure to pollution the sensitive species become extinct and tolerant species take over their place and role in the ecosystem (Figure 1). This takeover can be monitored by very simple toxicity tests using a part of the community extracted from the environment. Some tests use the incorporation of building blocks for DNA (thymidine) and protein (leucine). Other tests use different substrates for microbial growth. The observation that this part of the community becomes more tolerant as measured by these simple toxicity tests reveals that the pollutant really affects the microbial community. This is especially helpful when complex and diverse environments like biofilms, sediments and soils are studied.

Tests using microbial processes

The protection of ecosystem services is fundamentally different from the protection of biodiversity. When one wants to protect biodiversity all species are equally important and are worth protecting. When one wants to protect ecosystem services only the species that perform the process have to be protected. Many contributing species can be intoxicated without having much impact on the process. An example is nitrogen transformation, which is tested by measuring the conversion of ammonium into nitrite and nitrate (see box).

Figure 1. The effect of growth on an intoxicated process performed by different species of microorganisms. The intoxication of some species may temporarily decrease process rate, but due to growth of the tolerant species this effect soon disappears and process rate is restored. Source: Patrick van Beelen.

The inactivation of the most sensitive species can be compensated by the prolonged activity or growth of less sensitive species. The test design of microbial process tests aims to protect the process and not the contributing species. Consequently, the process tests from Table 1 seldom play a decisive role in reducing the maximum tolerable concentration of a chemical. Reason is that the single species toxicity tests generally are more sensitive since they use a specific biological species as test organism instead of a process.

Box: Nitrogen transformation test The OECD test no. 216 Soil Microorganisms: Nitrogen Transformation Test is a very well-known toxicity test using the soil process of nitrogen transformation. The test for non-agrochemicals is designed to detect persistent adverse effects of a toxicant on the process of nitrogen transformation in soils. Powdered clover meal contains nitrogen mainly in the form of proteins which can be degraded and oxidized to produce nitrate. Soil is amended with clover meal and treated with different concentrations of a toxicant. The soil provides both the test organisms and the test medium. A sandy soil with a low organic carbon content is used to minimize sorption of the toxicant to the soil. Sorption can decrease the toxicity of a toxicant in soil. According to the guideline, the soil microorganisms should not be exposed to fertilizers, crop protection products, biological materials or accidental contaminations for at least three months before the soil is sampled. In addition, the soil microorganisms should at least form 1% of the soil organic carbon. This indicates that the microorganisms are still alive. The soil is incubated with clover meal and the toxicant under favorable growth conditions (optimal temperature, moisture) for the microorganisms. The quantities of nitrate formed are measured after 7 and 28 days of incubation. This allows for the growth of microorganisms resistant to the toxicant during the test, which can make the longer incubation time less sensitive. The nitrogen in the proteins of clover meal will be converted to ammonia by general degradation processes. The conversion of clover meal to ammonia can be performed by a multitude of species and is therefore not very sensitive to inhibition by toxic compounds. The conversion of ammonia to nitrate generally is performed in two steps. First, ammonia oxidizing bacteria or archaea, oxidize ammonia into nitrite. Second, nitrite is oxidized by nitrite oxidizing bacteria into nitrate. These latter two steps are generally much slower than ammonium production, since they require specialized microorganisms. These specialized microorganisms also have a lower growth rate than the common microorganisms involved in the general degradation of proteins into amino acids. This makes the nitrogen transformation test much more sensitive compared to the carbon transformation test, which uses more common microorganisms. Under the optimal conditions in the nitrogen transformation test some minor ammonia or nitrite oxidizing species might seem unimportant since they do not contribute much to the overall process. Nevertheless these minor species can become of major importance under less optimal conditions. Under acid conditions for example, only the archaea oxidize ammonia into nitrite while the ammonia oxidizing bacteria become inhibited. The nitrogen transformation test has a minimum duration of 28 days at 20°C under optimal moisture conditions, but can be prolonged to 100 days. Shorter incubation times would make the test more sensitive.