Iron(3+) ion dipotassium 2-({2-[bis(carboxylatomethyl)amino]ethyl} (carboxylatomethyl)amino)acetate; hydrate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

19-29 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study done under GLP, with chemical analyses and certificate of analyses.

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The results as observed/predicted for EDTA-FeNa are read across to EDTA-Fe(OH)K2 as in diluted form in the aquatic environment both EDTA complexes will have a similar structure and related fate.

2. ANALOGUE APPROACH JUSTIFICATION
It is therefore considered justified to use data/predictions as observed for EDTA-FeNa also for EDTA-Fe(OH)K2.

3. ANALOGUE APPROACH JUSTIFICATION
It is considered justified to use the results as observed/predicted for EDTA-FeNa for read across to EDTA-Fe(OH)K2 because in diluted form in the aquatic environment both EDTA complexes will have a similar structure and related fate.

4. DATA MATRIX
See chapter 13 for Read across document

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The NaHCO3 concentrations was increased in order to maintain a more constant pH during the test.

Principles of method if other than guideline:

The NaHCO3 concentrations was increased in order to maintain a more constant pH during the test.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Chemical analyses were performed according to the analytical procedure described in Annex 3.
Samples of about 3 ml were taken from 3 control vessels and from each test vessels containing test substance at 0 24, 48 and 72 hours.
The samples were stored in the freezer (<-10ºC) until analyses.

Details on test solutions:

A stock solution/suspension of the test substance was prepared as follows: an accurately measured amount of 0.6526 g of test substance was
dissolved in deionized water. Exactly 4.0 ml of a 118.5 g/l FeCl3*6H2O was added with a pipette to reach an equimolar concentration of EDTA and
Fe(III). After stirring the pH of the solution was adjusted to pH 5.2 using a 1 M NaOH solution. The final concentration of 5.1237 g/l based on
H4EDTA was reached by adding deionized water to a final volume of 100 ml. The stock solution was stored in the refrigerator in the dark.

Test organisms (species):

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

Details on test organisms:

The test was carried out with the freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and
Protozoa, Institute of Freshwater Ecology, The Windermere Laboratory,Cumbria, Ambleside, United Kingdom. After purchasing this strain was
cultured and maintained according to Standard Operation Procedure E 3 (9.10). Cultures on sloped agar tubes were stored at 4°C until required.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

no data

Test temperature:

22.6-23.9 ºC

pH:

7.5-9.4

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

nominal concentrations: 0 - 60.2 - 79.4 - 99.9 mg/L
measured concentrations: 0 - 38.1 - 48.5 - 60.6 mg/L

Details on test conditions:

For the test an adequate amount of test medium was prepared in a 3-liter vessel. This medium was sterilized by filter sterilization (0.2 µm). Adequate amounts of stock solution were added to the test vessels (100 ml erlenmeyers). The test vessels were filled with medium up to a total volume of
40 ml using a sterilized dispenser. The inoculum was added from an exponentially growing culture with a pipette. In addition six control replicates
were included. The extinction in each erlenmeyer was measured after 0, 24, 48 and 72 hours. Algal medium was used as a blank in the
spectrophotometer.

Reference substance (positive control):

yes

Remarks:

potassium chromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

60.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

ErC50 & EbC50 are higher than 60.6 mg/L

Results with reference substance (positive control):

The sensitivity of the algae is checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year, when
the cultures are grafted from the sloped agar tubes. The sensitivity was tested for compliance with the Guidelines, which means that the EC50 values are between 0.25 and 2.0 mg/l.

Mean Extinction measured at 436 nm

Concentration mg/L (measured)	Time (hours)
	0	24	48	72
Control	0.001	0.042	0.201	0.798
38.1	0.024	0.191	0.350	0.847
48.5	0.032	0.234	0.395	0.854
60.6	0.042	0.299	0.432	0.797

Validity criteria fulfilled:

yes

Conclusions:

Good quality study under GLP, with analyses and certificate of analyses.
Therefore conclusion of report; effect concentration above 60.6 mg/L (mean measured concentration) is correct.

Executive summary:

The algal toxicity was determined in the Algal Growth Inhibition test in accordance with OECD, EEC and ISO test guidelines and with the OECD Principles of Good Laboratory Practice. The guidelines were slightly modified to ensure good growth and pH control of the cultures.

The toxicity of Fe(III)EDTA to exponentially growing Pseudokirchneriella subcapitata (formerly known asSelenastrum capricornutum) was determined over an exposure period of 72 hours. A test was carried out at nominal concentrations of 60, 80 and 100 mg/l Fe (III) EDTA (concentrations represented as H₄EDTA).

Due to photodegradation, the Fe(III)EDTA concentration declines during the test. The chemical analyses carried out 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 test was conducted in a mineral salt medium in a climatized illuminated orbital incubator. The maximum variation in pH in the test media was 1.4 pH unit.

The E_bC₅₀and E_rC₅₀ are both higher than 100 mg/l based on nominal concentrations and higher than 60 mg/l based on the mean measured concentrations.

The NOEC and LOEC based on nominal concentrations are 79.4 mg/l and 99.9 mg/l. Based on the mean measured concentrations the NOEC and LOEC are 48.4 and 60.6 mg/l.

The definitive test is valid as shown by the increase of the extinction of the control over 72 h by a factor of 489.

Description of key information

The ecotoxicity of EDTA-FeK is considered to be similar to the ecotoxicity of EDTA-Fe(OH)K2 as both substances will be tested in very diluted form in buffered matrices and under these conditions no significant effects of the additional OH are expected. The ecotoxicity test results as observed for EDTA-FeK will therefore be read across to EDTA-Fe(OH)K2. As a worst-case no molecular weight correction is applied (14% difference).

For EDTA-FeK the toxicity to algae Pseudokirchneriella subcapitata was observed to be ErC50 > 60.6 mg/L and NOEC = 48.4 mg/L. All concentrations in this study are presented as EDTA-H4 concentrations (72h. Geurts, 2001, Key study). Recalculated to EDTA-FeK this would result in a NOEC of 63.5 mg/L. These results are confirmed by the study from BASF (1995) where no toxicity was observed upto 100 mg/L of EDTA-H4.

Key value for chemical safety assessment

EC50 for freshwater algae:

79.4 mg/L

EC10 or NOEC for freshwater algae:

63.5 mg/L

Additional information

Two studies are summarised on the algal toxicity of the test material.

1. A study was carried out with Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) according to OECD, EEC and ISO test guidelines and under GLP (Geurts and Van Wijk 2001, Rel. 1, Key study). The guidelines were slightly modified to ensure good growth and pH control of the cultures.

The test was carried out on equimolar amounts of NaHEDTA.2H20 and FeCl3.6H20 and at nominal concentrations of 60, 80 and 100 mg/L (concentrations represented as EDTA-H4).

Due to photodegradation, the test substance concentration declines during the test. The chemical analyses carried out showed that the concentration at the end of the test had decreased to about half 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 test was conducted in a mineral salt medium in a climatized illuminated orbital incubator. The maximum variation in pH in the test media was 1.4 pH unit. The definitive test is valid as shown by the increase of the extinction of the control over 72 h by a factor of 489.

The EbC50and ErC50 are both higher than 100 mg/L based on nominal concentrations and higher than 60 mg/L based on the mean measured concentrations of EDTA-H4. Recalculated to the test substance FeK EDTA the 72h-EC50s are > 78.6 mg/L.

The NOEC and LOEC based on nominal concentrations are 79.4 mg/L and 99.9 mg/L (EDTA-H4). Based on the mean measured concentrations the NOEC and LOEC are 48.4 and 60.6 mg/L (EDTA-H4). Recalculated to the test substance FeK EDTA, the 72h-NOEC = 63.5 mg/L.

 

2. The European Union Risk Assessment Report Edetic Acid (EDTA) of 2004 reviews a study on the acute toxicity of equimolar amounts of EDTA-Na4 and Fe(III) towards Scenedesmus subspicatus. No inhibition was found up to a nominal concentration of 100 mg Na4EDTA. This implies that the NOEC expressed as the test substance is > 101 mg/L.