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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:
2001-03-19 to 2001-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD Guidelines and under GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
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
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. Exactiy 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 degree C
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations of 60, 80 and 100 mg/L
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: jes
- Light intensity and quality: continuous uniform illumination was provided in the spectral ränge 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
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:
EC50
Effect conc.:
> 60 mg/L
Nominal / measured:
meas. (geom. mean)
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
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
48.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
99.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
60.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Conclusions:
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.
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:
key study
Study period:
22-7-2009 - 25-7-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted to the OECD guidelines with a minor deviation (see below). No analysis were performed. The screening study concerned was in accordance 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
Principles of method if other than guideline:
The NaHCO3 concentration of the test medium was 150 mg/l instead of 50 mg/l, in order to maintain a more constant pH during the test.
GLP compliance:
no
Remarks:
GLP accredited laboratory not conducted to GLP
Analytical monitoring:
no
Vehicle:
no
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
Remarks on exposure duration:
This study was conducted as a series of preliminary tests for EDTA for metal complexes.
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:
EC50
Effect conc.:
> 1 000 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.:
1 000 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
Conclusions:
This study can be concluded reliable with restrictions.
Executive summary:

The study concerned was in accordance with OECD guidelines in a GLP accredited laboratory, but was not GLP audited. No chemical analysis was performed. Control growth was satisfactory and current reference test with potassium dichromate had been conducted on the algae species used for testing. This study can be considered reliable with the restrictions mentioned. And is therefore reliable as supporting evidence.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in compliance with GLP regulations
Qualifier:
according to guideline
Guideline:
other: EEC guideline 79/831/EEC, Annexe V, part C: Methods for the determination of ecotoxicity, algae: Test of growth inhibition, May 1988
GLP compliance:
yes
Analytical monitoring:
yes
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, SAG 86.81
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 +/- 2 °C
pH:
7.65 - 8.80
Details on test conditions:
Aeration: none
No. of replicates:
3 inoculated replicates
1 uninoculated replicate for measuring the pH-value of the uninoculated test concentrations after an incubation time of 72 h.
Controls: negative control

TEST MEDIUM / WATER PARAMETERS
Dilution water: according to EEC guideline 79/831/EEC, Annexe V, part C:
Methods for the determination of ecotoxicity, algae: Test of growth inhibition, May 1988 and the SOP of the laboratory
Deviation from the SOP: Each test concentration was mixed according to the following regimen:
1 part of inoculum (1E+05 cells/ml in test medium)
1 part of test medium (10 times concentrated)
8 parts of test substance (in appropriate water dilutions)

TEST CONDITIONS
Illumination: 120 uEinstein/m*m*s (permanent Illumination, Color of light was universal white, L 25 (Osram))
Inoculation density: 10000 cells/mL (initial concentration in the test)

TEST CONCENTRATIONS
Test concentrations: 100 mg/l - 0.39 mg/L
Stock solution: 125 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.39 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.78 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.7 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.77 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC90
Effect conc.:
>= 100 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Effect on the development of biomass [mg/L]:
EbC 10 = 0.48 mg/L
EbC 50 = 1.01 mg/L
EbC 90 = 100 mg/L

Effect on growth rate [mg/L]:
ErC 10 = 0.70 mg/L
ErC 50 = 2.77 mg/L
ErC 90 => 100.0 mg/L

No observed effect concentration (95 % significance level):
NOEC (72 h) = 0.39 mg/L

Lowest observed effect concentration:
LOEC (72 h) = 0.78 mg/L

VALIDTY CRITERIA:
The validity criteria were maintained.
Factor of cell division (cell count) after 72 hours: 46

SUMMARY OF THE CONCENTRATION CONTROL ANALYSIS:
The concentrations 100 and 12.5 mg/L, the untreated control and the stock solution (125 mg/L) were analysed. Test conditions and results are listed in the GLP-reports 94LOO650 and 94LOO663, dated 22 Nov 1994, from Dr. Scholtissek, BASF AG, ZAX/OF.

Time scan:
begin of test (0 h) - uninoculated replicate
end of test (72 h) - uninoculated replicate
end of test (72 h) - inoculated replicate after removal of algal cells through centrifugation

Recovery in %:
control / test / stock solution /
-----------------------------------------------------------
t= 0 h uninoculated <DL / 100-104 / 99.2 /
t= 72 h uninoculated <DL / 96-99 / 98.4 /
t= 72 h inoculated <DL / 99-104 / - /
-----------------------------------------------------------
DL= detection limit (1 mg/L)

Description of key information

Based on read-across from the related substance, ethylenediaminetetraacetic acid disodium salt dihydrate, an aquatic alga EC50 (growth rate) was determined to be >100 mg/L (nominal) / >60 mg/L (measured) for tripotassium hydrogen EDTA.

The NOEC was 79.4 mg/L based on nominal concentrations and 48.4 mg/L based on the mean measured concentrations. Sodium and potassium are both members of the 1a group of metals in the periodic table. Due to their low electronegativity they form ionic bonds and highly water soluble salts with inorganic and organic acids. The solution of these salts is therefore dominated by the property of the acid anion. This justifies reading across from the sodium to the potassium salt.

Key value for chemical safety assessment

EC50 for freshwater algae:
60 mg/L
EC10 or NOEC for freshwater algae:
48.4 mg/L

Additional information

Based on read-across from the related substance, ethylenediaminetetraacetic acid disodium salt dihydrate, an acute aqutic invertebrate EC50 of >100 mg/L (nominal) / >60 mg/L (measured) was determined for tripotassium hydrogen EDTA.

The test material was combined with FeCl3x6H20 to ensure that EDTA was in its complexed state, as it would be found in normal environmetnal conditions.

Sodium and potassium are both members of the 1a group of metals in the periodic table. Due to their low electronegativity they form ionic bonds and highly water soluble salts with inorganic and organic acids. The solution of these salts is therefore dominated by the property of the acid anion. This justifies reading across from the sodium to the potassium salt.