## 4.3.5. Selection of test organisms - Eco plants

Author: J. Arie Vonk

Reviewers: Michiel Kraak, Gertie Arts, Sergi Sabater

Learning objectives:

You should be able to

- name the requirements for suitable laboratory ecotoxicity tests for primary producers
- list the most commonly used primary producers and endpoints in standardized ecotoxicity tests
- argue the need for selecting primary producers from different environmental compartments as test organisms for ecotoxicity tests

Key words:Test organism, standardized laboratory ecotoxicity test, primary producers, algae, plants, environmental compartment, photosynthesis, growth

Introduction

Photo-autotrophic primary producers use chlorophyll to convert CO2 and H2O into organic matter through photosynthesis under (sun)light. These primary producers are the basis of the food web and form an essential component of ecosystems. Besides serving as a food source, multicellular photo-autotrophs also form habitat for other primary producers (epiphytes) and many fauna species. Primary producers are a very diverse group, ranging from tiny unicellular pico-plankton up to gigantic trees. For standardized ecotoxicity tests, primary producers are represented by (micro)algae, aquatic macrophytes and terrestrial plants. Since herbicides are the largest group of pesticides used globally to maintain high crop production in agriculture, it is important to assess their impact on primary producers (Wang & Freemark, 1995). However, concerning testing intensity, primary producers are understudied in comparison to animals.

Standardized laboratory ecotoxicity tests with primary producers require good control over test conditions, standardized endpoints (Arts et al., 2008; see the Section on Endpoints) and growth in the controls (i.e. doubling of cell counts, length and/or biomass within the experimental period). Since the metabolism of primary producers is strongly influenced by light conditions, availability of water and inorganic carbon (CO2 and/or HCO3- and CO32-), temperature and dissolved nutrient concentrations, all these conditions should be monitored closely. The general criteria for selection of test organisms are described in the previous chapter (see the section on the Selection of ecotoxicity test organisms). For primary producers, the choice is mainly based on the available test guidelines, test species and the environmental compartment of concern.

Standardized ecotoxicity testing with primary producers

There are a number of ecotoxicity tests with a variety of primary producers standardized by different organizations including the OECD and the USEPA (Table 1). Characteristic for most primary producers is that they are growing in more than one environmental compartment (soil/sediment; water; air). As a result of this, toxicant uptake for these photo-autotrophs might be diverse, depending on the chemical and the compartment where exposure occurs (air, water, sediment/soil).

For both marine and freshwater ecosystems, standardized ecotoxicity tests are available for microalgae (unicellular micro-organisms sometimes forming larger colonies) including the prokaryotic Cyanobacteria (blue-green algae) and the eukaryotic Chlorophyta (green algae) and Bacillariophyceae (diatoms). Macrophytes (macroalgae and aquatic plants) are multicellular organisms, the latter consisting of differentiated tissues, with a number of species included in standardized ecotoxicity tests. While macroalgae grow in the water compartment only, aquatic plants are divided into groups related to their growth form (emergent; free-floating; submerged and sediment-rooted; floating and sediment-rooted) and can extend from the sediment (roots and root-stocks) through the water into the air. Both macroalgae and aquatic plants contain a wide range of taxa and are present in both marine and freshwater ecosystems.

Terrestrial higher plants are very diverse, ranging from small grasses to large trees. Plants included in standardized ecotoxicity tests consist of crop and non-crop species. An important distinction in terrestrial plants is reflected in dicots and monocots, since both groups differ in their metabolic pathways and might reflect a difference in sensitivity to contaminants.

Table 1. Open source standard guidelines for testing the effect of compounds on primary producers. All tests are performed in (micro)cosms except marked with *

Primary producer | Species | Compartment | Test number | Organisation
Microalgae & cyano-bacteria | various species | Freshwater | 201 | OECD 2011
Anabaena flos-aque | Freshwater | 850.4550 | USEPA 2012
Pseudokirchneriella subcapitata, Skeletonema costatum | Freshwater, Marine water | 850.4500 | USEPA 2012
Floating macrophytes | Lemna spp. | Freshwater | 221 | OECD 2006
Lemna spp. | Freshwater | 850.4400 | USEPA 2012
Submerged macrophytes | Myriophyllum spicatum | Freshwater | 238 | OECD 2014
Myriophyllum spicatum | Sediment (Freshwater) | 239 | OECD 2014
Aquatic plants* | not specified | Freshwater | 850.4450 | USEPA 2012
Terrestrial plants | wide variety of species | Air | 227 | OECD 2006
wide variety of species | Air | 850.4150 | USEPA 2012
wide variety of species (crops and non-crops) | Soil & Air | 850.4230 | USEPA 2012
legumes and rhizobium symbiont | Soil & Air | 850.4600 | USEPA 2012
wide variety of species (crops and non-crops) | Soil | 208 | OECD 2006
wide variety of species (crops and non-crops) | Soil | 850.4100 | USEPA 2012
various crop species | Soil & Air | 850.4800 | USEPA 2012
Terrestrial plants* | not specified | Terrestrial | 850.4300 | USEPA 2012

Representation of environmental compartments

Since primary producers can take up many compounds directly by cells and thalli (algae) or by their leaves, stems, roots and rhizomes (plants), different environmental compartments need to be included in ecotoxicity testing depending on the chemical characteristics of the contaminants. Moreover, the chemical characteristics of the compound under consideration determine if and how the compound might enter the primary producers and how it is transported through organisms.

For all aquatic primary producers, exposure through the water phase is relevant. Air exposure occurs in the emergent and floating aquatic plants, while rooting plants and algae with rhizoids might be exposed through sediment. Sediment exposure introduces additional challenges for standardized testing conditions, since changes in redox conditions and organic matter content of sediments can alter the behavior of compounds in this compartment.

All terrestrial plants are exposed through air, soil and water (soil moisture, rain, irrigation). Air exposure and water deposition (rain or spraying) directly exposes aboveground parts of terrestrial plants, while belowground plant parts and seeds are exposed through soil and soil moisture. Soil exposure introduces additional challenges for standardized testing conditions, since changes in water or sediment organic matter content of soils can alter the behavior of compounds in this compartment.

Test endpoints

Bioaccumulation after uptake and translocation to specific cell organelles or plant tissue can result in incorporation of compounds in primary producers. This has been observed for heavy metals, pesticides and other organic chemicals. The accumulated compounds in primary producers can then enter the food chain and be transferred to higher trophic levels (see the section on Biomagnification). Although concentrations in primary producers are indicative of the presence of bioavailable compounds, these concentrations do not necessarily imply adverse effects on these organisms. Bioaccumulation measurements can therefore be best combined with one or more of the following endpoint assessments.

Photosynthesis is the most essential metabolic pathway for primary producers. The mode of action of many herbicides is therefore photosynthesis inhibition, whereby different metabolic steps can be targeted (see the section on Herbicide toxicity). This endpoint is relevant for assessing acute effects on the chlorophyll electron transport using Pulse-Amplitude-Modulation (PAM) fluorometry or as a measure of oxygen or carbon production by primary producers.

Growth represents the accumulation of biomass (microalgae) or mass (multicellular primary producers). Growth inhibition is the most important endpoint in test with primary producers since this endpoint integrates responses of a wide range of metabolic effects into a whole organism or a population response of primary producers. However, it takes longer to assess, especially for larger primary producers. Cell counts, increase in size over time for either leaves, roots, or whole organisms, and (bio)mass (fresh weight and dry weight) are the growth endpoints mostly used.

Seedling emergence reflects the germination and early development of seedlings into plants. This endpoint is especially relevant for perennial and biannual plants depending on seed dispersal and successful germination to maintain healthy populations.

Other endpoints include elongation of different plant parts (e.g. roots), necrosis of leaves, or disturbances in plant-microbial symbiont relationships.

Current limitations and challenges for using primary producers in ecotoxicity tests

For terrestrial vascular plants, many crop and non-crop species can be used in standardized tests, however, for other environmental compartments (aquatic and marine) few species are available in standardized test guidelines. Also not all environmental compartments are currently covered by standardized tests for primary producers. In general, there are limited tests for aquatic sediments and there is a total lack of tests for marine sediments. Finally, not all major groups of primary producers are represented in standardized toxicity tests, for example mosses and some major groups of algae are absent.

Challenges to improve ecotoxicity tests with plants would be to include more sensitive and early response endpoints. For soil and sediment exposure of plants to contaminants, development of endpoints related to root morphology and root metabolism could provide insights into early impact of substances to exposed plant parts. Also the development of ecotoxicogenomic endpoints (e.g. metabolomics) (see the section on Metabolomics) in the field of plant toxicity tests would enable us to determine effects on a wider range of plant metabolic pathways.

References

Arts, G.H.P., Belgers, J.D.M., Hoekzema, C.H., Thissen, J.T.N.M. (2008). Sensitivity of submersed freshwater macrophytes and endpoints in laboratory toxicity tests. Environmental Pollution 153, 199-206.

Wang, W.C., Freemark, K. (1995) The use of plants for environmental monitoring and assessment. Ecotoxicology and Environmental Safety 30: 289-301.