Hexamethylenediamine

Toxicity to microorganisms

Description of key information

In an activated sludge respiration inhibition test according to OECD 209 (2010) nitrification  proved to be the most sensitive endpoint (highest test item concentration: 1000 mg/L):
EC50 (3h; total respiration): 1558 mg/L;
EC50 (3h; heteretrophic respiration): >> 1000 mg/L;
EC50 (3h; nitrification): 291 mg/L;

Key value for chemical safety assessment

EC50 for microorganisms:

291 mg/L

Additional information

In the reliable key study (RL1; GLP) the effect of the test item Hexamethylenediamine on the respiration of heterotrophic and nitrifying bacteria in activated sludge after 3 hours exposure was investigated in a static test system according to OECD 209 (2010). Five test item concentrations were applied (1000, 313, 97.7, 30.5, and 9.54 mg/L).

A concentration-response relationship was observed for inhibition of total and heterotrophic respiration. With heterotrophic respiration, however, inhibition was less than 50% of the control, so an EC50 was not calculated.

Also, for nitrification, inhibition followed a concentration-response relationship. At the lowest concentration level tested, however, there was considerable stimulation of nitrification (50.6% stimulation relative to the control), rather than inhibition. Non-GLP pre-tests performed with sludge from the same source, showed similar results (25% and 47% stimulation at 1.6 and 8 mg/L). The results of a further range-finding test performed with a sludge from a different source were equivocal in the low dose range (34%, -8% and -25% inhibition at 1.6, 8 and 40 mg/L; negative inhibition = stimulation). In conclusion, these data may indicate a stimulating effect of the test item in the low dose range, in any case considerable variability is observed at the lowest concentration levels. Therefore, the EC10 for nitrification is associated with considerable uncertainty and is therefore not reported here. All validity criteria specified by the guideline were fulfilled.

Nitrification proved to be the most sensitive endpoint:

EC50 (3h; total respiration): 1558 mg/L;

EC50 (3h; heteretrophic respiration): >> 1000 mg/L;

EC50 (3h; nitrification): 291 mg/L.

In a supporting study (scientific publication, RL3), an indicative IC50 for hexamethylenediamine of 85 mg/L was reported. In this work, several compounds were tested for nitrification inhibition using a stock culture of autotrophic nitrifiying bacteria as inoculum. By the authors, this degree of inhibitory action was considered to be "not likely to cause problems in waste water treatment plants" compared with other, more potent inhibitors. While these data seem scientifically valid, pure cultures (i.e. culture of autotrophic nitrifying bacteria instead of activated sludge) were used and actual relevance for STPs is questionable. Further, no raw data are presented and the rate constants were determined from only 3 time points.

A further study report is available. Colonies of Pseudomonas putida were exposed to increasing concentrations of test substance for 20 hours with and without pH neutralization. The bacterial density was measured by optical density at different time intervals in four replicates and EC10 was then determined. At the end of the test, EC10 of pH-neutralized test solutions was calculated as 12500 mg/L.

Concluding, for risk assessment purposes (derivation of the PNEC_STP-microorganisms) nitrification inhibition is the most relevant and sensitive endpoint and the EC50 (3h, nitrification) of 291 mg/L as determined in the reliable key study will be used.