There is an urgent need to replace aqueous film-forming foam (AFFF) containing per- and polyfluoroalkyl substances (PFAS) with PFAS-free alternatives. Currently, little is known about the ecotoxicity and environmental impacts of existing alternatives. This proof-of-concept project aimed to comparatively assess ecotoxicity of a short-chain, PFAS-containing AFFF and six PFAS-free alternatives in terrestrial plants and soil invertebrates. Specifically, the objectives of this project were to:

• investigate chronic toxicity of the test products in a terrestrial plant species, the field mustard Brassica rapa;
• investigate lethal and sublethal toxicity of the test products in two soil invertebrates, the nematode Caenorhabditis elegans and the collembola Folsomia candida; and
• investigate the occurrence of the test products and their transformation products in plants.

The technology approach of this project was as follows:

1. The project team conducted a greenhouse experiment using product-amended soil to assess chronic toxicity of the test products in the terrestrial plant B. rapa. Toxicity endpoints included germination rate, survival rate, biomass, plant height, the first day of flowering, number of seeds, and development (i.e., number and length) of siliques.
2. The project team conducted toxicity tests with soil invertebrates using product-amended soil under laboratory conditions. They developed a high-content imaging method for rapidly counting and measuring C. elegans in soil and applied the method to assess sublethal toxicity (i.e., growth and reproduction) of the test formulations in C. elegans. They also assessed lethal toxicity of the test products in F. candida after 14-day incubation.
3. The project team developed methods of sample extraction and non-targeted analysis using high-resolution mass spectrometry and applied the methods to investigate the chemical composition of the test products and plant extracts.

The results of this project include:

1. Five of six PFAS-free products were more toxic than the short-chain, PFAS-containing reference product in B. rapa. Only one PFAS-free product was comparable to the short-chain, PFAS-containing AFFF, both of which showed very low chronic toxicity in B. rapa.
2. Similar to phytotoxicity test results, five of six PFAS-free products were more toxic than the short-chain, PFAS-containing AFFF to the reproduction of the nematode C. elegans.
3. Two of six PFAS-free products showed higher lethal toxicity than the short-chain, PFAS-containing AFFF in the collembola F. candida after a 14-day exposure period. The other four products had comparable or lower lethal toxicity to/than the reference product.
4. A total of 149 chemical ingredients were detected in the test products via nontargeted analysis. None of these ingredients were detected in the above-ground plant tissues. The toxic PFAS-free products were found to generate more transformation products with higher peak intensities in plant tissues than the weakly toxic products. The most abundant transformation products detected in plant extracts were suspect transformation products derived from diethylene glycol monobutyl ether.
5. Five PFAS-free products showed significant impacts on the growth and reproduction of B. rapa and C. elegans at concentrations lower than the practical application concentration, suggesting that replacement products may cause adverse effects on terrestrial plants and soil invertebrates.

This project provides ecotoxicological data that, combined with data from all related ongoing research, could be used in decision-making regarding recommendations for manufacturing and use of candidate PFAS-free AFFFs. The demonstrated toxicity of PFAS-free AFFFs to plant and invertebrate species suggests a need for further testing prior to field use. (Project Completion - 2023)