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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19-29 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Physical state: white powder
- Lot/batch No.: A0102232 (01)
- Purity: > 99% CAS 139-33-3 as parent material.

The Fe-complex was prepared in situ: A stock solution/suspension of the test substance was prepared as follows: an accurately measured amount of 0.6526 g of parent substance (CAS 139-33-3) 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.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: assumed to be stable at storage conditions
- Storage condition of test material: at room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
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 (<-10 degree C) until analyses.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An accurately measured amount of 0.6526 g of parent substance was dissolved in deionized water. Exactly 4.0 mL of a 118.5 g/L FeCI3x6H20 was added with a pipette to reach an equimolar concentration of EDTA and Fe(lll). 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.
The test solutions were prepared by addition of the required amounts of stock Solution to the test vessels to obtain the following final nominal test concentrations: 60, 80 and 100 mg/L.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: P.subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, The Windermere Laboratory, Cumbria, Ambleside, United Kingdom.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 ± 2 °C
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations of 60.2, 79.4 and 99.9 mg/L (based on H4EDTA)
Measured (arith. mean): 38.1, 48.5 and 60.6 mg/L (based on H4EDTA)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer test flasks
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 100 mL, closed with cotton-wool stoppers

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity and quality: continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using 30 W fluorescent lamps


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: photometrically with a UVA/IS Spectrophotometer

Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 60 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: H4EDTA
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
48.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: H4EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
79.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Conclusions:
In conclusion the 72h-EC50 value for Fe(III)EDTA with P.subcapitata was > 100 mg/L based on nominal concentrations and > 60 mg/L based on the mean measured concentrations. The NOEC was 79.4 mg/L based on nominal concentrations and 48.4 mg/L based on the mean measured concentrations.
Executive summary:

The toxicity of Fe(lll)EDTA to exponentially growing Pseudokirchneriella subcapitata 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 (lll) EDTA (concentrations represented as H4EDTA). Due to photodegradation, the Fe(lll)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. In conclusion the 72 hour EC50 value for Fe(III)EDTA with P.subcapitata was greater than 100 mg/L based on nominal concentrations and greater than 60 mg/L based on the mean measured concentrations. The NOEC was 79.4 mg/L based on nominal concentrations and 48.4 mg/L based on the mean measured concentrations.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-07-22 to 2009-07-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
No analysis were performed. The screening study concerned was inaccordance with OECD guidelines but was not GLP audited. Control validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Study not audited
GLP compliance:
no
Remarks:
GLP-laboratory, but study not audited
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch/Lot. No. 428S5005
- Appearance/Colour Light Brown Powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Stability Expected to be stable during test duration
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The EDTA based test substance was found to be soluble in water, therefore a stock solution of 1000 mg/L was prepared in test medium as follows: To an accurately weighed amount of 0.1 g of the test substance freshly prepared OECD medium was added up to the mark in a 100 mL volumetric flask. The stock solution was then agitated with a magnetic stirrer until a homogeneous solution was achieved. This took approximately 1 hour. The pH of the stock solution was measured and adjustment was not necessary as the measurement fell within a half pH unit of the control values. The appropriate volume of the stock solution was then added to the test vessels to achieve the desired test concentration in 40 mL of test media of test substance as described above.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Nominal and measured concentrations:
Nominal: 1, 10, 100 and 1000 mg/L
Details on test conditions:
For the test an adequate amount of OECD test medium was prepared in an appropriately sized volumetric vessel. This medium was sterilized by filter sterilization (0.45 μm filter).
Adequate amounts of stock solution were added to the sterilized test vessels (100 mL Erlenmeyers) to achieve the desired test concentrations in 40 ml total volume. The test vessels were filled with medium up to 40 mL using a sterilized dispenser. The inoculum was added from an exponentially growing culture with a pipette. In addition, 6 replicates of the control without test solution were included. The extinction in each Erlenmeyer was measured after 0 and 72 hours. Algal medium was used as a blank in the spectrophotometer to correct for media absorbance.
No chemical analysis for quantification of the test substance concentration was performed during the study.
The pH of the highest and lowest concentrations and controls were measured at the beginning (t=0 h) and at the end (t=72 h) of the test and ranged from 8.1 to 9.2. The temperature in the culturing apparatus was continuously measured and read out at the end of the test. Temperature was set at 23 ºC and varied less than ± 2 ºC throughout the test period. The light intensity was measured at the beginning of the test and was measured at 100.0 μmol.m-2.s-1.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
307.63 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

This study was conducted as a series of preliminary tests for EDTA for metal complexes.

Concentration  Time (hours) Area Inhibition Specific Inhibition
(mg/L) 0 72 Percentage Growth Rate Percentage
0 0.007 0.501 17.784 0.059315222
0 0.007 0.492 17.46 0.059063452
0 0.006 0.35 12.384 0.056474635
0 0.008 0.285 9.972 0.049625662
0 0.007 0.464 16.452 0.058249644
0 0.008 0.265 9.252 0.048615115
Mean 0.007167 0.3928333 13.884 0.055610344 0
1 0.006 0.377 13.356 0.055081866
1 0.006 0.45 15.984 0.05753328
0 0
- -
Mean 0.006 0.4135 14.67 56.611.927 0.056361588 13.509.065
10 0.008 0.453 16.02 0.057564031
10 0.008 0.374 13.176 0.054897007
0
Mean 0.008 0.4135 14.598 5.1426102 0.056294438 1.2301551
100 0.025 0.365 12.24 0.052977253
100 0.024 0.563 19.404 0.061179004
0
Mean 0.0245 0.464 15.822 13.958513 0.057394055 -3.207516
1000 0 0.032 1.152 0.028630870
1000 0.005 0.116 3.996 0.035060394
0 0
Mean 0.0025 0.074 2.574 81.4606742 0.033274707 40.1645378
Validity criteria fulfilled:
yes
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no GLP
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
modified test system without iron limitation (medium differing to OECD)
GLP compliance:
no
Specific details on test material used for the study:
- Purity: 100%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Stock solutions:
249.75 mg/L (Na4-EDTA)
177.55 mg/L (FeCl3 x 6 H20)
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test species: Scenedesmus subspicatus
Strain: CHODAT SAG 86.81
Source: Algae collection Goettingen, Germany
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 ± 2 °C
pH:
ca. 8
Nominal and measured concentrations:
Nominal: 1.03, 2.06, 4.11, 8.22, 16.44, 32.88, 65.76, 131.52 and 263.04 µmol/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 mL test tubes containing 10 mL test solution
- Aeration: no
- Initial cells density: 10000 cells/mL (initial concentration in the test)
- Control end cells density: 36.7 x initial cell density
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium prepared using deionized water
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at the start and end of the test in one control and one treatment test tube. Temperature was recorded continuously.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: counting chamber (Neubauer)
- Chlorophyll measurement: fluorimeter (at 685 nm)
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC90
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Effect on Biomass:
------------------
EbC 10 = > 263.04 µmol/L (> 100 mg/L Na4-EDTA)
EbC 50 = > 263.04 µmol/L (> 100 mg/L Na4-EDTA)
EbC 90 = > 263.04 µmol/L (> 100 mg/L Na4-EDTA)

Effect on Growth rate:
----------------------
ErC 10 = > 263.04 µmol/L (> 100 mg/L Na4-EDTA)
ErC 50 = > 263.04 µmol/L (> 100 mg/L Na4-EDTA)
ErC 90 = > 263.04 mmol/L (> 100 mg/L Na4-EDTA)

NOEC (72 h) = 263.04 µmol/L (100 mg/L Na4 -EDTA)

LOEC (72 h) = > 263.04 µmol/L (> 100 mg/L Na4-EDTA)


Observations:
-------------
Over the whole range of test concentrations, a stimulation of the cell proliferation was observed (max. 30.4 % based on biomass).
Validity criteria fulfilled:
yes
Remarks:
Factor of the cell proliferation (cell count) after 72 h: 36.7
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Category justification document is attached in chapter 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 60.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: H4EDTA
Basis for effect:
growth rate
Remarks on result:
other: based on EDTA-FeNa
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
48.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: H4EDTA
Basis for effect:
growth rate
Remarks on result:
other: based on EDTA-FeNa
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on EDTA-Na4
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on EDTA-Na4

Description of key information

EC50 (72h) > 100 mg/L for growth rate of freshwater algae (WoE, read across)

EC10 (72h) = 48.4 mg/L for growth rate of freshwater algae (WoE, read across)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
48.4 mg/L

Additional information

Since no studies are available on algal toxicity of Disodium dihydrogen ethylenediaminetetraacetate (CAS No. 139-33-3), the assessment was based on studies conducted with other subcategory 1a member Tetrasodium ethylenediaminetetraacetate (CAS No. 64-02-8) and subcategory 1b member Sodium feredetate (CAS No. 15708-41-5) as part of a Weight-of-evidence read across approach. This is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Grouping of substance and read across approach. Further justification for suitability of read across between subcategory 1 members is given within the category justification attached to chapter 13.


The study with Tetrasodium ethylenediaminetetraacetate (CAS No. 64-02-8) was conducted according to EU method C.3 using medium without Fe limitation and Scenedesmus subspicatus as test organism (BASF 1995). Test concentrations between 1.03 and 263.04 µmol/L (0.39 - 100 mg/L) were tested. The 72h-EC50 and EC10 for the growth rate were determined to be nominal > 100 mg/L, as no analytical dose verification was performed.


In addition, two studies are available investigating the algal toxicity of Sodium feredetate (CAS No. 15708-41-5). A screening test was conducted according to OECD guideline 201 using Pseudokirchneriella subcapitata as test organism (Akzo 2009). Concentrations of 1, 10, 100 and 1000 mg/L were tested. No chemical analysis for quantification of the test substance concentration was performed during the study.


The pH of the highest and lowest concentrations and controls were measured at the beginning (0 h) and at the end (72 h) of the test and ranged from 8.1 to 9.2. The 72h-EC50 value is determined to be > 1000 mg/L. The 72h-EC10 value is determined to be 307.63 mg/L based on nominal concentrations. A second GLP study was also conducted according to OECD guideline 201 using Pseudokirchneriella subcapitata as test organism (Akzo 2001). The EDTA-FeNa-complex was prepared in situ: An accurately measured amount of 0.6526 g of EDTA-Na2H2 (CAS 139-33-3) 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. Concentrations (based on H4EDTA) of 60, 80 and 100 mg/L were tested. Due to photodegradation, the Fe(III)EDTA concentration declined during the test. The chemical analysis performed showed that the concentration of the test compound decreased to about 50% compared to the test initiation throughout the test. Thus, the effect values are related to the measured (arithm. mean) concentration. The test was conducted in a mineral salt medium in a climatized illuminated orbital incubator. The maximum variation in pH in the medium was 1.4 pH units. The 72h-EC50 is determined to be > 60 mg/L (> 100 mg/L nominal). The 72h-NOEC value is determined to be 48.4 mg/L based on the measured concentration.


Based on the outcome of the available studies it can be concluded that the 72h-EC50 for Disodium dihydrogen ethylenediaminetetraacetate and other members of subcategory 1 is > 100 mg/L and thus the substances in this subcategory are with a high probability acutely not harmful to freshwater algae. The 72h-EC10/NOEC value is determined to be 48.4 mg/L. However, since this is not the lowest long-term effect value it is not taken into account for the PNEC derivation.