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Diss Factsheets

Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

Additional information

Abbreviations used: L-ASP for L-aspartic acid; SA for succinic acid.

A weight of evidence approach is applied for each of the acute aquatic toxicity endpoints fish, Daphnia, alga. This approach is based on:

1. A read across to data for the analogous substance succinic acid, which only lack a NH2-group compared to L-aspartic acid and which has comparable physical-chemical properties, is justified and was performed. More details on the read across approach are found in the attachments to the individual endpoints, where a read across was applied. Comparative results for L-ASP and SA are:
L-ASP, non-adjusted pH of 4.3, EC50algae = 8.5 mg/L
L-ASP, non-adjusted pH of 6.5, EC50Daphnia = 165 mg/L

SA, non-adjusted pH of 4.2, EC50algae = 46.8 mg/L
SA, adjusted pH to ca. neutral, EC50algae >100 mg/L
SA, non-adjusted pH of 4.7, EC50daphnia = 63 mg/L
SA, adjusted pH to ca. neutral, EC50daphnia >100 mg/L
SA, non-adjusted pH 6.2, LC50fish > 100 mg/L

2. It is assumed and is confirmed by the results presented above that the pH (or the H+ ion concentration) is the relevant parameter for describing toxicity and not the effects to specific targets of the organisms. The EC50/LC50 are lower in tests with initially low pH compared to the tests with pH adjusted to ca. neutral.
A correlation between pH and EC/LC50 exists for the tests with non-adjusted pH.

3. QSAR estimations predicted a low toxicity for each of the 3 aquatic organisms. This also confirms the assumption that no relevant toxicity is expected for L-ASP at neutral pH, because QSAR calculations generally refer to the undissociated molecule.
L-ASP, EC50algae = 2237 mg/L
L-ASP, EC50Daphnia = 607; 2187; >>1000 mg/L, for the 3 models used.
L-ASP, LC50fish = >100; 872, >>1000 mg/L, for the 3 models used.

4. The test media have a different buffer capacity so that the initial pH in the media with a concentration of ca. 100 mg test substance/L varies for the tests with the different organisms:
L-ASP, 120 mg/L, algae: pH = 4.3
L-ASP, 150 mg/L, Daphnia: pH = 6.5

SA, 100 mg/L, algae: pH = 4.2
SA, 100 mg/L, Daphnia: pH = 4.7
SA, 100 mg/L, fish: pH = 6.2
Especially for SA, for which each of the tests were performed in the same laboratory at 100 mg/L, it can be derived that the buffer capacity of the medium used increases from algae to Daphnia and being highest for fish.

5. From the last point, i.e. the highest pH of the medium in the fish study, it is further derived that in a fish test the highest EC50/LC50 will be obtained compared to tests with the other organisms. This is confirmed when looking at the results, as summarised in point 1, above.

6. It is therefore justified to waive the acute fish toxicity test because no new data, respectively a higher LC50 (compared to alga and Daphnia), are expected.
Instead of, the LC50 is transcribed from the LC50 >100 mg SA/L, by accounting for the slightly different molecular weight of SA and L-ASP, to give LC50fish > 113 mg L-ASP/L.

7. The performance of the test with non-adjusted pH is not in conformance with agreed procedures and therefore the results from these tests should not be used for classification or risk assessment. The remarkable lowering of the pH at the higher concentrations of L-ASP (and also SA) explains the inhibition of the growth rate of the algae or the lower EC50 for Daphnia (for SA), which is not due to the chemical structure of the substance but to the H+ concentration.
Although the OECD 202 method requires to perform first the test without adjusting the pH, it also describes to perform a second run with adjusting the pH: "If the pH does not remain in the range 6-9, then a second test could be carried out, adjusting the pH of the stock solution to that of the dilution water before addition of the test substance" and "The pH should normally not vary by more than 1.5 units in any one test."
No requirement is given in the OECD 201 method to adjust the pH in the case of ionised substances, but there are two relevant documents requiring it:
- The ECHA Guidance Document on Info Requirement and CSA, R.7b (Version November 2012, Table R. 7.8-3: "Summary of difficult substance testing issues", page 77 for pH effects) states: "Where a substance causes a change in pH of the test medium (e.g. strong acids and bases), the pH should be adjusted to lie within the specified range for the test using a suitable technique. ...".
- Also the OECD Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD Series on testing and assessment, No. 23 of 2000, states in Section 3.1: "Where the substance itself causes a change to the pH of the test medium, the pH should be adjusted to lie within the specified range for the test using acid, alkali or other suitable buffer. ...".
The studies without adjustment of the pH to ca. neutral are therefore considered to be of only restricted adequacy for REACH. It is foreseeable and has been shown for succinic acid (and also for other acids such as HCl) that a test with an adjusted pH to ca. neutral will cause no toxicity to the aquatic organisms.
It would be more reasonable to state that the aquatic toxicity is dependent on the initial pH and that a concentration of any test substance that lowers the pH below ca. 7 in the specific water medium used has a toxic effect to the aquatic organisms. The buffering capacity will be dependent on the actual aqueous environment, therefore relating the EC50 to a concentration is not relevant for the actual situation, only the resulting pH will be.

8. Test results at low initial pH are not relevant, also because a pH of between 6 and 9 has to be maintained anyway if the Directive 2006/44/EC of the European Parliament and of the Council of 6 September 2006 on "the quality of fresh waters needing protection or improvement in order to support fish life" is observed.

9. The test result of the algae test with L-ASP is therefore not used for classification and risk assessment. Instead of the EC50 is transcribed from the EC50 >100 mg SA/L, by accounting for the slightly different molecular weight of SA and L-ASP, to give EC50algae > 113 mg L-ASP/L.

10. The same procedure could be applied for the Daphnia result, but as a worst case it was accepted to use the EC50 = 165 mg/L, obtained from the study with L-ASP.

11. The inhibition of respiration of microorganisms could be waived, based on the readily biodegradability of L-ASP, which indicates a low toxicity to microorganisms. To obtain a more concrete data a read across was performed to SA.

12. The amino acid L-aspartic acid is probably a nutrient for most of the organisms.


Long-term tests are waived because no classification and no risk assessment has to be performed. Also, the long-term test results will be dependent on the resulting pH of the solutions of L-ASP and the result is therefore foreseeable, i.e. no toxicity will be observed at pH >6, endorsed by the EU Directive 2006/44/EC, see above.