Ethylenediamine-N,N'-di(acetic acid)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-14 to 2018-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via LC-MS/MS after 0 (start of exposure) and 72 hours (end of the exposure) with algae.

Vehicle:

no

Details on test solutions:

6 concentrations were tested, with five concentrations in a geometric series with a factor of 4 and one additional higher concentration with the final concentration range of 0.391 - 1.56 - 6.25 - 25.0 - 100 - 1000 mg/L. The concentration levels are based on the results of a preliminary range finding test (non-GLP).
All test concentrations were weighed separately and added to dilution water. The test item solutions were treated with ultrasound for at least 5 minutes until the solutions were visually clear.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test organism: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81

Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)
Reason for the selection: Pseudokirchneriella subcapitata is a suitable green alga species of the test organism according to the guideline.
Origin: Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen
Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 hours per day.
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Standard OECD test medium

Test temperature:

Mean: 22.5 (21.0 - 23.5)

pH:

Nominal test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours

1000 6.80 7.01
100 7.28 7.77
25.0 7.57 8.31
6.25 7.63 8.65
1.25 7.69 8.89
0.391 7.74 9.46
Control 7.91 9.37

Nominal and measured concentrations:

Nominal concentration range of 0.391 - 1.56 - 6.25 - 25.0 - 100 - 1000 mg/L.
Measured: see any other information on materials and methods

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

161 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

corrected for A.I. 99.2%

Basis for effect:

growth rate

Remarks on result:

other: (95% C.I. 92.9 - 247 mg a.i./L)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.54 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Corrected for A.I. of 99.2%

Basis for effect:

growth rate

Remarks on result:

other: (95% C.I. <0.39 - 0.77 mg a.i./L)

Details on results:

The measured concentrations in the test solutions at the start of the exposure were in a range of 85 to 119% of the nominal concentrations. At the end of exposure after 72 hours, the measured concentrations were in the range of 82 to 109% of the nominal concentrations. All effect values given are therefore based on the nominal test item test item concentrations of Ethylenediaminediacetic acid (ED2A-H2).

Results with reference substance (positive control):

See any other information on material and methods. The results are in agreement with the required range.

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially, ≥ 16-fold corresponding to a specific growth rate of 0.92 day^-1	371-fold (specific growth rate 1.97 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤ 35%	11.0%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	1.24%

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled.

Conclusions:

In this study, the effect values of Ethylenediaminediacetic acid (ED2A-H2) (CAS 5657-17-0) on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follows (based on nominal test item concentrations):
The EC50-value for inhibition of growth rate (ErC50) after 72 hours was 161 (93 - 248) mg a.i./L. The EC10 for growth rate was 0.54 mg a.i./L.
Standard OECD test media was used and the wide dose response observed is likely caused by essential metal depletion.

Executive summary:

The toxicity of Ethylenediaminediaceticacid (ED2A-H2) to Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and EC Method C.3. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5523 cells/mL.Six concentrations were tested in relation to the flat dose response curve, with five concentrations in a geometrical series with factor of 4 and one additional higher concentration with the final concentration range of 0.391 - 1.56 - 6.25 - 25.0 - 100 - 1000 mg/L. All test item concentrations were weighed separately and added to dilution water. The test item solutions were treated with ultrasound for 5 minutes at room temperature until the solution were visually clear.

Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The measured concentrations in the test solutions at the start of the exposure were in a range of 85 to 119% of the nominal concentrations. At the end of exposure after 72 hours, the measured concentrations were in the range of 82 to 109% of the nominal concentrations. All effect values given are therefore based on the nominal test item test item concentrations of Ethylenediaminediacetic acid (ED2A-H2).

The EC50-value for inhibition of growth rate (ErC50) after 72 hours was 161 (93 - 248) mg a.i./L. The EC10 for growth rate was 0.54 mg a.i./L.

Description of key information

The final test was performed in standard OECD algae medium and a typical wide dose response was observed which was also observed in algae tests for other chelating agents.

The algae test with H2 -ED2A gave an EC50 for growth of 161 mg a.i./L. The EC10 for growth was 0.54 mg/L. The difference between EC10 and EC50 is normally about a factor 3 - 5.

Key value for chemical safety assessment

EC50 for freshwater algae:

161 mg/L

EC10 or NOEC for freshwater algae:

0.54 mg/L

Additional information

The algae test with H2 -ED2A gave an EC50 for growth of 161 mg a.i./L. Similar to H4 - EDTA an effect of the chelating properties of H2 -ED2A was anticipated but only minimal differences were observed with the 3*AAP (additional salts added) compared to the standard algae test medium in the range finding test. The final test was therefore performed in standard OECD algae medium and in contrast to the range finder a typical chelating agent dose response was observed (very wide dose response, big difference between EC50 based on growth and yield). The EC10 for growth was 0.54 mg/L where the difference between EC10 and EC50 is normally a factor 3 - 5. The ED2A algae test will therefore be repeated using filtered natural river water (with low DOC) supplemented with the salts of the minimal medium to evaluate the intrinsic algae toxicity of H2 -ED2A under representative environmental conditions.

The results observed in the Algae test with H2 -ED2A are line with what was observed for EDTA complexes which supports the read across from Na4-EDTA, FeNa -EDTA and Fe(III)EDTA to H2 -ED2A for ecotox datagaps.

According to the study performed by BASF with Na4 -EDTA an inhibitory effect of the test substance was shown, which was caused by complexing essential micronutrients from the minimal culture medium. Chelates can reduce the bioavailability of essential micronutrients in the culture medium to a level where growth is limited. This indirect nutrient deficiency effect is an artefact and should not be used for the effect assessment because it is unlikely to occur in the environment (see Guidance on Hazard to the Aquatic Environment Globally Harmonized System GHS Annex 9 A9.3.3.4, 2007).

To compensate for this secondary effect, Geurts and van Wijk performed an algae study with Pseudokirchneriela subcapitata where the stock solution of Na2H2 -EDTA was complexed with an equimolar amount of FeCl3 under acidic conditions. This stock solution was diluted in standard OECD test medium to obtain nominal test concentrations of 60, 80 and 100 mg/L (concentrations represented as H4-EDTA). Chemical analysis showed that due to photodegradation, the Fe(III)EDTA concentration declines during the test. Chemical analyses showed that the concentration of the test compound at the end of the test had decreased to about 50% of the concentration at the beginning of the test. The mean concentrations during the 72 hours of testing for the nominal concentrations of 60, 80 and 100 mg/L as measured by chemical analyses were 38.1, 48.4 and 60.6 mg/L, respectively. The E_bC₅₀ and E_rC₅₀ calculated are both higher than 60 mg/L based on the mean measured concentrations. The NOEC and LOEC based on the mean measured concentrations the NOEC and LOEC are 48.4 and 60.6 mg/L.

Finally the toxicity of FeNa-EDTA was tested in standard OECD medium. The calculated EC50 of nominal > 1000 mg/L FeNa-EDTA was based on range finding test with a broad range (Kean, 2009).

The results of these three studies show that the E_rC₁₀ and E_rC₅₀ for H4 EDTA and its salts are in any case higher than resp. 48 and 60 mg/L and indicate that the E_rC₅₀ could be higher than 300 mg/L which is in good agreement with the EU risk assessment (2004)

https://echa.europa.eu/documents/10162/65615721-ab6d-4f28-b48f-73cf9d8cc529