The previous regulatory acceptable level (RAL) applied in the 2,4-D aquatic risk assessment was 30 µg/L. This was based on the EC50 of 2,4-D applied as the DEA salt in a Lemna gibba system, recognising that there were similar results between the different 2,4-D acid, salts and esters available at the time.
Since then, additional test data has been supplied to the APVMA, and a large body of work has become available indicating that submerged and rooted aquatic vascular plants may be more sensitive than Lemna gibba. In particular, an international ring test has been performed using a sediment free test system with the test plant Myriophyllum spicatum. The most sensitive result from this study was a geometric mean (n = 6) EC50 11 µg/L for root length. This end-point has been adopted by international regulators (EFSA, German Federal Environment Agency) with a level of concern of 10, resulting in a regulatory acceptable level of 1.1 µg/L.
There are some concerns about the validity of the root length value. For example, in the sediment free test system for Myriophyllum spicatum, the presence of sucrose and light (exposure of roots to light during the test) may have an influence on auxin (plant growth hormone) transport carriers, and that some chemicals may have an auxin type mode of action, the inclusion of root endpoints is questionable. However, in this case there is supporting evidence from the literature with a second Myriophyllum species that root growth is highly sensitive to 2,4-D acid with a second EC50 13 µg/L in a test system that contained rooting substrate. Given this additional line of evidence and similar results from different test systems (with and without rooting substrate/sediment), the very sensitive result for Myriphyllum spicatum root length obtained in the ring test is considered valid and appropriate for application in the end-point setting.
A number of species sensitivity distributions (SSDs) have been developed for informing the choice of end-point. These SSDs cannot be applied on their own as they contain a mix of end-points (biomass, yield, growth rate), and test systems (14 day, 28 day, sediment, no sediment etc). However, they do help demonstrate that the ring test Myriophyllum spicatum test result remains the most sensitive currently known and allow some confidence in applying a more relaxed level of concern.
Therefore, the aquatic RAL for 2,4-D was amended based on the root length geometric mean EC50 11 µg/L with an assessment factor of 3, resulting in a RAL of 3.7 µg/L.