Potassium; 2-[bis[2-[bis(carboxylatomethyl)amino]ethyl]amino]acetate; iron(3+)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

22-01-2013 - 25-1-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study according to most guideline requirements and incl CoA & GLP and clear reporting could justify reliability of 1. However, the coloration of test media due to the test substance and photo-instability of test substance raise some questions regarding effects expressed based on actual measured test concentrations. Conclusions based on measured concentrations must therefore be considered carefully.

Justification for type of information:

A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent (DTPA) as in a target substance (DTPA-FeK). The only difference between the target and the source substance is presence of potassium (K) cation instead Na+ cations. As potassium and sodium are the essential macro elements required by all forms of life, is considered not to influence the toxicological activity.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: There was no analytical confirmation of test substance and no replicate vessels used per test concentration, however the study was well documented and meets gernerally accepted scientific principles

Justification for type of information:

A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. Both EDTA and DTPA are chelates with similar structure and physico-chemical properties tham their toxicity are also similar. Please see RA statement in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

The action of EDTA in batch cultures of Scenedesmus quadricauda was investigated over a period of days. Concentrations of micro- and macro-nutrients were also manipulated in select test vessels dosed with increasing concentrations of EDTA.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100, 200, 400% EDTA as in the basic nutrient solution, which was 100 mg/L Na2EDTA * 2H2O
- Sampling method: Samples of the algal culture were taken every 24 hours for EDTA concentrations.

Vehicle:

no

Details on test solutions:

no data

Test organisms (species):

Scenedesmus quadricauda

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus quadricauda
- Strain: Greifswald/15
- Source (laboratory, culture collection): Collection of Autotrophic organisms, Institute of Botany, Czechoslovak Academy of Sceinces, Trebon.
- Method of cultivation:The algal were precultivated in a medium corresponding to 1/10 of the concentrations used in the test. The precultivated culture was filtered through filter paper, the cells were washed twice by repeated resuspension in freshly distilled water and filtration, and finally resuspended again in a small volume of distilled water to give a thick suspension. An adequate quantity of this suspension was then used for the inoculation of each test flask to the initial dry mass concentration of approximately 0.2 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

23 d

Post exposure observation period:

none

Hardness:

not reported

Test temperature:

25 plus or minus 1 degree C.

pH:

The pH of all media was adjusted to 6.8 with NaOH.

Dissolved oxygen:

The suspensions were bubbled with a mixture of air and 1.5% CO2; dissolved oxygen levels were not reported.

Salinity:

not applicable

Nominal and measured concentrations:

Nominal test concentrations of EDTA in test media: 100 (control nutrient media), 200, 400 mg/L
Measured concentrations of EDTA: In solutions containing 400 mg/L EDTA and 400% of the micronutrients supplied in the control nutrient media, concentrations of EDTA dropped as low as to one half of the initial concentration by the end of the experiments; all other concentrations (100 mg/L and 200 mg/L EDTA were relatively stable and close to nominal over the exposure period of 23 days)

Details on test conditions:

TEST SYSTEM
- Test vessel: 2L Roux flasks
- Aeration: The flasks were bubbled with a mixture of air and 1.5% CO2
- Initial cells density: 0.2 g/L (dry mass concentration)
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):


GROWTH MEDIUM

- Detailed composition if non-standard medium was used:
Macronutrients:
858 mg/L (NH2)2CO
1237 mg/L MgSO4 * 7H2O
953 mg/L KH2PO4
Micronutrients
16.2 mg/L FeCl3
6.2 mg/L H3BO3
21.9 mg/L CaCl2 * 6H2O
2.23 mg/L MnSO4 * 4H2O
2.50 mg/L CuSO4 * 5H2O
2.88 mg/L ZnSO4 * 7H2O
2.42 mg/L Na2MoO4 * 2H2O
2.81 mg/L CoSO4 * 7H2O
1.22 mg/L NaVO3
Chelating agent
100 mg/L Na2EDTA * 2H2O




TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: freshly distilled water

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: pH adjusted to 6.8 with NaOH
- Photoperiod: continuous
- Light intensity and quality: TESLA 40W natural white fluorescent tubes or high pressure sodium discharge lamps SHC 400 Tesla. The irradiance of the algal culture was 57 W/m2 PhAR (photosynthetically active radiation) in the case of the fluorescent tubes and 330 W/m2 in the case of the sodium discharge lamps.



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell number (using a Burker couting chamber macroscopically), and dry mass
- Chlorophyll measurement:Chlorophyll a and b were determined spectrophotometrically after cell disintigration and extraction with 80% acetone as described by Lukavsky et al (1979).

Reference substance (positive control):

no

Duration:

23 d

Dose descriptor:

NOEC

Effect conc.:

400 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

When the nutrient media was supplemented with micro and macronutrients in proportion with the percent increase of EDTA in the media, there were no observable effects on dry mass, cell number, or chlorophyll a and b content at the highest concentration of Na2EDTA test, which was 400 mg/L.

Results with reference substance (positive control):

not reported

Reported statistics and error estimates:

no data

Validity criteria fulfilled:

not applicable

Conclusions:

The authors state that the experiments conducted in this study show that it is not the absolute concentration of the chelating agent (for example 400 mg/L Na2EDTA) that is responsible for the inhibition of algal growth, but the excess over the concentrations of cations.

Executive summary:

The action of EDTA in batch cultures of Scenedesmus quadricauda grown under continuous illumination with either fluorescent tubes or high-pressure sodium discharge lamps was investigated. The effects of various concentrations and ratios of macroelements, microelements and EDTA in the nutrient solution on the growth characteristics of the algal suspensions were evaluated.

Higher concentrations of EDTA (Na2EDTA * 2H2O; 400 mg/L), when in surplus over trace elements in the nutrient solution, inhibited cell division, chlorophyll synthesis and the production of algal biomass, especially in the earlier phase of algal growth. No negative influence of the higher concentration of EDTA was observed when the concentration of trace elements in the nutrient solution was increased correspondingly to the increased EDTA concentrations. EDTA depletion from the solutions was observed in the experiments where a surplus of EDTA was accompanied by a corresponding surplus of trace elements. The highest decrease in EDTA concentration corresponded to the highest rate of cell division. The influence of light sources on the decomposition of EDTA in the algal nutrient solution was also studied, but no decomposition of EDTA by the light sources was proved.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

- NaCO3 concentration in test medium was increased to maintain a more constant pH.
- addition of nutrients other than Fe to 150% of guideline recommendations
- parallel test vessels without algae to try to mitigate effects of coloration and photo instability of the test substance

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

EC Number:

235-627-0

EC Name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

Cas Number:

12389-75-2

Molecular formula:

C14-H18-Fe-N3-O10.H.Na

IUPAC Name:

Iron(3+) ion sodium 5-[bis(carboxylatomethyl)amino]-3-{[bis(carboxylatomethyl)amino]methoxy}pentanoate

Constituent 2

Reference substance name:

Diethylenetriaminepentaacetic acid, ferric sodium complex

IUPAC Name:

Diethylenetriaminepentaacetic acid, ferric sodium complex

Constituent 3

Reference substance name:

Dissolvine D-Fe-11

IUPAC Name:

Dissolvine D-Fe-11

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Product Name Dissolvine : De-FE 11
Chemical Name: Diethylenetriaminepentaacetic acid, ferric sodium complex
Purity: 97.2
Appearance: Yellow / Green Crystals
Cas Number: 12389-75-2

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For the study samples of 30 mL were taken at the start and end of the test at all concentrations (including parallels), in the stock solution and in the control. All samples were shaken for approximately 10 minutes at 100 -150 RPM before being further diluted as required and then filtered through 0.45 µm acrodisk filters (as required) before chemical analysis.

Included were also storage stability samples at 1 mg/L and 100 mg/L.

Test solutions

Vehicle:

no

Details on test solutions:

General test principles and procedures
Culture medium was prepared by diluting the OECD stock mineral salts in an appropriate vessel with natural surface water. The Erlenmeyer flasks were then filled with test medium up to the appropriate volume using a calibrated dispenser pump. Adequate amounts of test substance were then added from the stock solution (see below) to achieve the desired test concentrations in a total volume of 40mL. Then the inoculum was added to the vessel from an exponentially growing culture. All test concentrations were tested in triplicate. In addition, six replicates of the control and a parallel replicate of every concentration without algae were included. The absorbance in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Test medium was used as a blank in the spectrophotometer.

Preparation of solutions
A stock solution of 525 mg/L of the test substance was prepared in the first and second studies respectively by weighing 0.0525 g of the test substance, on an analytical balance and making the volume accurately up to 100 ml with test medium in a volumetric flask. The test substance dissolved easily generating homogeneous bright yellow solution without visible precipitate. The stock solution was used to generate the test concentrations by dilution with test medium. The pH was adjusted from 5.8 to 8.1 with NaOH.

Test concentrations
A series of range finding tests were conducted using test solutions prepared as described above. Based on these results the following definitive nominal concentrations were prepared by adding the appropriate amounts of stock solution to test media so as to achieve the desired test concentration in a total volume of 40 mL: 1.0, 3.2, 10.24, 32.7 and 104.8 mg/L. Six replicates without the test substance were also prepared as a control to allow correction for the absorbance caused by the slight yellow colour of the test solutions and to investigate test substance stability without the presence of algae.


The following was added to demineralized water to generate the test medium:
Nutrient Concentration Stock solution (mg/L)
Macro-nutrients
NH4Cl 22.5 150 % of normal concentration
KH2PO4 2.5
CaCl2(H2O)2 18
MgSO4(H2O)7 27
MgCl2(H2O)6 18
Fe-EDTA
FeCl3 (H2O)6 0.064 Standard
Na2EDTA(H2O)2 0.1
Trace elements
H3BO3 0.185 150 % of normal concentration
ZnCl2 0.003
MnCl2(H2O)4 0.415
CoCl2(H2O)6 0.0015
CuCl2(H2O)2 1x10-5
Na2MoO4(H2O)2 0.007
NaHCO3
NaHCO3 150 Modified

Test flasks
The test was performed in sterilized 100 mL Erlenmeyer flasks containing 40 mL of mineral salts in spring water. The test flasks were closed with cotton wool stoppers.

Test organisms

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, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK. After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase. For the evaluation of the quality of the algae and the experimental conditions, the reference substance potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions and algae sensitivity.

Preparation of the inoculum
The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing. The absorbance of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1104 cells/mL in each of the test vessels.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

The temperature varied from 22.0 to 22.3 °C during the test

pH:

The pH measurements show a maximum increase of 1.4 pH units in a control replicate.
pH at the start 8.0 in all vessels, after 72 h range 8.2-9.6

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10.2, 32.7 and 104.8 mg/L
Geometric measured mean (0-72h): 0.85, 1.05, 2.91, 4.3 and 13.72 mg/L

Details on test conditions:

Culturing cabinet and test conditions
The test was carried out in a temperature-controlled illuminated orbital incubator in which the temperature was maintained at 23 ± 2°C. Uniform Illumination was provided in the spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about 0.36  0.02 m from the algal cultures. The test vessels were agitated continuously at a speed sufficient to prevent sedimentation of the algae (100 rpm approx)
The de-ionized water used contained not more than 0.01 mg/L of copper, had a TOC-content of not more than 2.0 mg/L and a conductivity of less than 5 S/cm. This water was produced from tap water in a water purification system according to the relevant Standard Operation Procedure.
The light intensity was 80.4 and 80.7 µE•m-2•s-1 at the beginning and end of the test respectively.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

70.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The ErC10 (72h) was calculated as 1.75 mg/L (Growth Rate) and the ErC50 (72h) was calculated as 9.39 mg/L (Growth rate) [Geometric measure mean values]. The NOEC was calculated as 1.05 mg/L and the LOEC as 2.91 mg/L. (Growth rate) [Geometric measure mean values].


Analytical quantification demonstrated a good initial level of the test substance in the test replicates indicating correct dosing of the test material. As would be expected with photosensitive test substances the recovery of the test material at the end of the study is low. Geometric means of the two concentrations were therefore used for endpoint expression.

The parallel measurements (without algae) actually were all determined as lower than the corresponding in test measurement with algae. This was an unexpected result. This is possibly explained by the fact that the algae themselves absorb light perhaps in this case mitigating the photo degradation of the test substance.

Any other information on results incl. tables

Expressed as nominal values, results would compare to an ErC50 of 70.2 mg/L and the ErC10 would be 6.6 mg/L, however due to the photo-instability and the coloration of the test solutions due to the test substance, it is difficult to conclude on actual concentrations.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

cell density, coefficient of variations, EC50 reference compound

Conclusions:

Study is well performed, well described and attempts were made to overcome any potential issues wrt chelating potential and coloration. However as actual measurements of test solutions and parallel samples showed, it is difficult to conclude on actual concentrations due to the photo- instability and the coloration of the test solutions due to the test substance.

Executive summary:

In order to predict the effects of diethylenetriaminepentaacetic acid, ferric sodium complex in an aquatic environment, an Algal Growth Inhibition test was conducted in accordance with OECD test guidelines 201 and with the OECD Principles of Good Laboratory Practice. Some minor modifications to the guideline were applied due to the chelating characteristics of the test substance.

 

The toxicity ofthe test chemical to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours.

Nominal concentrations of1.0, 3.2, 10.24, 32.7 and 104.8 mg/L were tested.

The E_rC₁₀(72h) was calculated as 1.75 mg/L (Growth Rate) and the ErC₅₀(72h) was calculated as 9.39 mg/L (Growth rate), based on geometric measure mean values. The NOEC was calculated as 1.05 mg/L and the LOEC as 2.91 mg/L. (Growth rate) [Geometric measure mean values].

Expressed as nominal values, this would compare to an ErC50 of 70.2 mg/L and the ErC10 would be 6.6 mg/L, however due to the photo- instability and the coloration of the test solutions due to the test substance, it is difficult to conclude on actual concentrations.