_Lead image: Sprouting lettuce seeds, [Rasbak](https://commons.wikimedia.org/wiki/File:Sla-kiemend.jpg), [CC BY SA](https://creativecommons.org/licenses/by-sa/3.0/deed.en)_
## What’s a bioassay and how does it work? **A bioassay is a method that uses plants, animals, or other living things to measure the harmful effects of environmental pollution.** This method works by exposing living things to different environmental samples, like soil or water, that vary in how polluted they are. Polluting chemicals in soil or water can affect how that living thing grows or functions. For example, contaminated soil can stunt a plant’s growth (see the image below) or prevent plant seeds from sprouting. So, measuring and comparing growth in living things exposed to different environmental samples can indicate the relative toxicity of the sample. You can find a good introduction to bioassays with a short **list of environmental monitoring studies that have used bioassays** at the [Environmental Inquiry](http://ei.cornell.edu/toxicology/bioassays/Uses.html) page from Cornell University.
## DIY bioassay methods ### Lettuce seed bioassay for soil or water This method uses common lettuce seeds to test for toxicity in soil or water samples. Contaminants in the soil or water can prevent seeds from germinating (sprouting) or slow down root growth. lettuce seed bioassay _Image: A lettuce seed bioassay showing longer roots in seeds that sprouted in distilled water (DW) compared to shorter roots in seeds exposed to different concentrations of ethyl acetate, a chemical that negatively affects growth. From [Waqas et al. 2013](https://doi.org/10.3390/molecules181215519), [CC BY](http://creativecommons.org/licenses/by/3.0/)_
**How to use this method:** 1. On the [Environmental Inquiry](http://ei.cornell.edu/toxicology/bioassays/lettuce/index.html) page, you’ll find bioassay procedures and worksheets originally developed for high school classrooms. + Instructions on [how to carry out the lettuce seed bioassay](http://ei.cornell.edu/toxicology/bioassays/lettuce/EnvSample.html) on soil samples + [How to collect and interpret your data](http://ei.cornell.edu/toxicology/bioassays/lettuce/data.html)
2. The US EPA [Protocols For Short Term Toxicity Screening Of Hazardous Waste Sites](https://nepis.epa.gov/Exe/ZyPDF.cgi/2000HUXX.PDF?Dockey=2000HUXX.PDF) document is a more detailed guide providing background information, guidance on sampling and data analysis, and hypothetical case studies, plus procedures. + Recommended step-by-step protocols for **lettuce seed germination** (section A.8.6, pg. 84) and **lettuce root elongation** (section A.8.7, pg. 90). These protocols are more involved and require more equipment than the “Environmental Inquiry” procedure linked above, so maybe less DIY.
**Basic info about this method:** + _How easy is it to use?_ Beginner + _How easy is it to make?_ Beginner + _Cost upfront?_ + [~$6 USD for a pack of 1000 seeds ](https://www.burpee.com/vegetables/lettuce/lettuce-buttercrunch-organic-prod000736.html) (make sure the seeds aren’t advertised as “treated”) + ~$1 USD for a gallon (3.8 L) of distilled water + [~$10 USD for qualitative filter paper](https://scientificfilters.com/filter-papers-cellulose-filters-qualitative-filter-papers-1001-070). Note from Environmental Inquiry guide: _“Absorbent paper towels or coffee filters can be substituted for the filter paper, as long as they are first shown to be nontoxic. (Bleached paper may contain dyes or chlorine.)”_ + More materials and specialized equipment are needed for the EPA recommended protocols. + _Time investment:_ 5 days for seed germination A particular variety of lettuce called “Buttercrunch” (Latin species name _Lactuca sativa_) is the standard variety typically used in bioassays and recommended by the US EPA.
### Duckweed bioassay for water Duckweeds are tiny floating plants that live in freshwater ponds, lakes, and wetlands. They grow quickly by adding leaf-like fronds. The bioassay compares growth rates by counting the number of new fronds over time. duckweed _Image: Flowering duckweed, [rjp](https://www.flickr.com/photos/zimpenfish/26959842/in/photostream/), [CC BY SA](https://creativecommons.org/licenses/by-sa/2.0/)_
**How to use this method:** The [Environmental Inquiry](http://ei.cornell.edu/toxicology/bioassays/lettuce/index.html) page is again a great resource. On the [duckweed bioassay page](http://ei.cornell.edu/toxicology/bioassays/Duckweed/default.html), you’ll find links for guidance on culturing/keeping duckweed, instructions for performing the bioassay, and guidance for analyzing your results.

## Not-quite-DIY and commercial bioassay methods ### Bioluminescence inhibition assay The “bioluminescence inhibition assay” is a widely-used and standardized method that uses bioluminescent (*_glowing_*) marine bacteria to indicate toxicity of environmental samples, including soil, sediment, and water. Pollutants in the sample interfere with the bacteria’s functioning, and that reduces (inhibits) the amount of visible light they produce. The change in their light output is measured and correlated with toxicity levels. In general, the more polluted the sample, the dimmer the bacteria get. The bacteria most commonly used is _Aliivibrio fischeri_ (a.k.a _Vibrio fischeri_), which is widely found in oceans around the world. Hawaiian bobtail squid with glowing bacteria _Image: A Hawaiian bobtail squid whose glowing skin comes from bioluminescent marine bacteria. The bioluminescence inhibition assay uses the bacteria, not the squid. [Margaret McFall-Ngai](https://commons.wikimedia.org/wiki/File:Hawaiian_Bobtail_squid.tiff), [CC BY](https://creativecommons.org/licenses/by/4.0/deed.en)_ **How to use this method:** A portable commercial unit with all the equipment needed from the [Microtox company](https://www.thomassci.com/Laboratory-Supplies/Water-Quality-Test-Kits/_/Microtox-FX-Portable-Toxicity-Luminometer?q=microtox) costs about $10k USD. Here’s a [rapid test kit from another company](https://www.biotoxicity.com/index.php/ebpi-toxicity-tests/aliivibrio-fischeri-toxicity-tests/biotox-lumostix-instant-kit) for $160 USD.

## Uses for bioassays + Determining the immediate toxicity of a potentially polluted soil or water sample. + Evaluating whether remediation of a contaminated site is working (comparing bioassay results before, during, and after remediation).

## Limitations + Bioassays won’t tell you what specific pollutants are present in an environmental sample. + A single test organism / bioassay may not respond to all of the polluting chemicals you might be concerned about (Bitton and Koopman 1992, [link to book chapter](https://link.springer.com/chapter/10.1007%2F978-1-4612-2890-5_1)). + The specific organism responses measured in the bioassay might not always be sensitive enough to indicate contamination. In the lettuce seed bioassay, for example, seed germination after 5 days is a common “endpoint” measurement that’s used to compare different samples. But some researchers have found that this measurement alone doesn’t always indicate toxic effects, and that comparing germination rates daily might be a more sensitive test (Da Silva Júnior et al. 2013, [link to paper](https://www.researchgate.net/publication/237079078_Assessment_of_a_Soil_with_Moderate_Level_of_Contamination_using_Lettuce_Seed_Assay_and_Terrestrial_Isopods_Assimilation_Assay)).
## Questions [questions:bioassay]
## Research notes [notes:bioassay]
## Activities [activities:bioassay]

## Further reading and resources ### Guides and protocols + **[Protocols For Short Term Toxicity Screening Of Hazardous Waste Sites](https://nepis.epa.gov/Exe/ZyPDF.cgi/2000HUXX.PDF?Dockey=2000HUXX.PDF)**, by the US EPA: includes background information, guidance on sampling and data analysis, hypothetical case studies, plus detailed protocols for various bioassays (earthworm, lettuce seed, Daphnia, algae) + **[Soil Toxicity and Bioassessment Test Methods for Ecological Risk Assessment](https://oehha.ca.gov/media/downloads/ecotoxicology/document/soiltox120208.pdf)**, by the [California Office of Environmental Health Hazard Assessment](https://oehha.ca.gov/ecotoxicology/general-info/toxicity-testing-guidance-documents): information on test methods and protocols, often standardized, for bioassays using various microorganisms, plants, and other organisms.
### Published articles + Valerio M.E., García J.F., and Peinado F.M. 2007. **Determination of phytotoxicity of soluble elements in soils, based on a bioassay with lettuce (Lactuca sativa L.)**. _Science of The Total Environment_, 378(1–2): 63-66. [LINK to abstract](https://doi.org/10.1016/j.scitotenv.2007.01.007). + Da Silva Júnior F.M.R., Garcia E.M., Baisch R.M., Mirlean N., and Muccillo-Baisch A.L. 2013. **Assessment of a soil with moderate level of contamination using lettuce seed assay and terrestrial isopods assimilation assay**. _Soil & Water Research_, 8: 56–62. [LINK to full-text](https://www.researchgate.net/publication/237079078_Assessment_of_a_Soil_with_Moderate_Level_of_Contamination_using_Lettuce_Seed_Assay_and_Terrestrial_Isopods_Assimilation_Assay). + Bierkens J, Klein G., Corbisier P., Van Den Heuvel R., Verschaeve L., Weltens R., and Schoeters, G. 1998. **Comparative sensitivity of 20 bioassays for soil quality**. _Chemosphere_, 37(14–15): 2935-2947. [LINK to full-text](https://d1wqtxts1xzle7.cloudfront.net/48112784/s0045-6535_2898_2900334-820160817-6584-adx7dt.pdf?1471423411=&response-content-disposition=inline%3B+filename%3DComparative_sensitivity_of_20_bioassays.pdf&Expires=1614295206&Signature=JrQUS8VIJAvpFPILOxV~23z4XNL1NGD~fMtpi1DLBY71GUft5kBcxzqC~k-43U49FZyll1ws90FYs4ZKKGCtbY1HfvyBgowGUiQ5l-ePHlXncmNMKLJfjecKUdnWkWz8k0MFArC1-oOzHGea25bnW1niFrMbrckseJNV-Rcuz7xyiYCpJQXxNz2S26helFXr729TXnyUnyM1TjJpMMnk0UnlLslvE~77KAcwdtTvsPzlc3DW1an64Uk02-mWoPaOssnPEYqAAK5iUqfVAZv4n86hcVbCc3heszU0qb-Jn3fnk8XvSPnW9lpH2qwPyVcWWNbvnDbcszk1NNrAQbCyhQ__&Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA).