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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007.16.08-2007.08.13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L and control
- Sampling method: at time 0h from fresh test solution and after 24, 48 and 96 h from aged test solution
Vehicle:
no
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Source: Forellenzucht Lohmühle, Germany
- Length at study initiation (length definition, mean, range and SD): 4-6 cm
- Weight at study initiation (mean and range, SD):
- Method of breeding: 300 L containers with continuous renewal of water (5-10% per day) and permanent aeration of water.
- Feeding during test
- Food type: none
- Amount: none
- Frequency: none


ACCLIMATION
- Acclimation period: > 12 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: granular rearing food (size 0) with approximately 2% of their body weight. Food provided by the fish supplier
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no mortality above 5% was observed through the acclimatisation period
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
11 ºdH (178.5 mg/L as CaCO3)
Test temperature:
15.1-16.1 ºC
pH:
8.3-8.5
Dissolved oxygen:
> 94% of air saturation (approx. 9.4 mg O2/L)
Nominal and measured concentrations:
Nominal concentrations: 0, 1, 10 and 100 mg/L
The analytical verification of test item concentrations in test medium was done by analysing the content of Iron in the samples over the whole test period in intervals of 24 h. Samples were taken after initiation of the test (0 hours), after 24 hours, after 48 hours and at the end of the test after 96 hours at the concentration level of 100 mg/L and control.
The average content of the test item was determined to be 98.8% of nominal concentration at the concentration level of 100 mg/L from filtrated samples and 99.5% from unfiltrated samples.
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 L stainless steel tanks fillled with 24 L test solution
- Aeration: continuous aeration with a membrane pump using a Pasteur pipette
- No. of organisms per vessel: 10 fish per concentration
- No. of vessels per concentration (replicates): no replicates
- No. of vessels per control (replicates): no replicates
- Biomass loading rate: max. loading of 1 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Conductivity: 453 μS/cm
- Temperature, pH-values and dissolved oxygen concentration were measured n 24 h intervals from fresh and aged test solutions. Water hardness of untreated control was measured at the beginning of the test.

OTHER TEST CONDITIONS
- Photoperiod: 12-16 h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Behavioural abnormalities; observations on body length and weight; mortality
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 0, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no effects on mortality at 100 mg/L; therefore, no main study was required.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Measured concentrations not significantly different from nominal
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Measured concentrations not significantly different from nominal
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Measured concentrations not significantly different from nominal
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Measured concentrations not significantly different from nominal
Details on results:
- Behavioural abnormalities: no effects
- Observations on body length and weight: the average weight at termination of test was 1.37 ± 0.3 g; the average length was 53 ± 4 mm.
- Mortality of control: none

As the measured concentrations did not deviate significantly from the nominal concentrations, the endpoint is based on the nominal concentrations.
Reported statistics and error estimates:
Since no mortality was detected at the highest test concentration of 100 mg/L, the evaluation of toxicological data did not require the application of statistical methods.
Validity criteria fulfilled:
yes
Remarks:
The mortality in the control did not exceed 10% at the end of the test. The dissolved oxygen concentration was at least 60 % of the air saturation value throughout the test
Conclusions:
The LC50 (96h) of the test item was determined to be > 100 mg/L (nominal). The LC0 (96h) was observed at 100 mg/L (nominal). The NOEC was determined to be 100 mg/L (nominal). Measured concentrations were not significantly different from nominal.
Executive summary:

The assessment of the 96 h LC50 of FeNaEDTA in fish was done in an acute test according to the OECD Guideline 203, EC method C.1 (92/69/EEEC) and US EPA OPPTS 850.1075. The test item was evaluated in a range finding test with a static test at concentrations of 0, 1, 10 and 100 mg/L.

No mortalities of fish occurred up to a concentration of 100 mg/L within a period of 96 h. Therefore, no main test was performed.

The mean measured concentration of the test item was between 80 and 120% of the nominal through the entire test period and the toxicological endpoints were evaluated using nominal concentration of the test item.

The LC0 (96h) was determined to be 100 mg/L and the LC100 (96 h) was determined to be > 100 mg/L. The LC50 (96h) was determined to be > 100 mg/L with a probability of 99.9%. The NOEC (96 h) was determined to be 100 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Non-GLP study according to international guideline (EPA-600/3 -75 -009 and ASTM method E729 -80). Limited reporting on test species. No chemical analyses of test concentrations. No data available to determine whether validity criteria are met.
Qualifier:
according to guideline
Guideline:
other: EPA-600/3-75-009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ASTM Standard E 729-80
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
The chemicals used in the tests were reagent grade.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was added directly to the diluent water in each aquarium in the appropriate amounts to give nominal concentrations of 100, 10, 1 and 0.1 mg/L.
Test organisms (species):
Pimephales promelas
Details on test organisms:
All test organisms were acclimated to the control diluent water in the breeding/rearing tanks. Food was withheld for the 24 h preceding start of the test. Juveniles of each species, as uniform in size as possible, were collected from the colonies. Stratified randomization was accomplished by proportioning out no more than 20% of any one species into an aquarium at any one time. Biological loading was kept below 0.5 gram wet weight per liter of test solution. Approximate size is 0.2 - 0.5 g
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
130 mg/L as CaCO3
Test temperature:
20 ± 1 °C
pH:
6.5 - 8.5
Dissolved oxygen:
If the dissolved oxygen concentration in a test chamber fell below 40% of the starting level in a test, the test was repeated with 0.05 L/min glass-sparger aeration.
Conductivity:
260 µmhos/cm
Nominal and measured concentrations:
Nominal: 0.1, 1, 10 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: seamless glass, 30.5-cm cuboidal, Pyrex chromatography jars containing 20 L of test solution
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Activated carbon-filtered, dechlorinated and tempered industrial service water fromwas used in all tests.
- Total organic carbon: 1.8 mg/L
- Alkalinity: 93 mg/L as CaCO3
- Ca/mg ratio:

- Culture medium different from test medium: no
- Intervals of water quality measurement: Once the test solutions were prepared, the starting temperature, dissolved oxygen and pH values were determined for each exposure concentration and the control.

OTHER TEST CONDITIONS
- Adjustment of pH: When the starting pH of the test solution fell outside the extremes of 6.5 to 8.5, the pH was adjusted to 7.0 by the addition of 10% (v/v) NaOH or 10% (v/v) H2SO4.
- Photoperiod: 16h light: 8h dark
- Light intensity: pproximately 50 ft-c of cool-white fluorescent light

EFFECT PARAMETERS MEASURED : Survival, condition and behavorial information were recorded.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Validity criteria fulfilled:
not specified
Conclusions:
The acute toxicity (96h-LC50) of sodium ferric EDTA towards Pimephales promelas is >100 mg/l.
Executive summary:

The acute toxicity (96h-LC50) of sodium ferric EDTA towards Pimephales promelas was investigated according to EPA-600/3 -75 -009 and ASTM method E729 -80. The toxicity value was found to be >100 mg/l.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The static water acute toxicity tests followed the methods described in the EPA publication, "Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians" (COMMITTEE ON METHODS FOR TOXICITY WITH AQUATIC ORGANISMS, 1975).
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity: 40.1% active ingredient
Analytical monitoring:
not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Respective amounts of test substance was added to 2 L and then mixed with the 8 L of reconstituted water in the test vessel to achievet the final test concentration.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: bluegill
- Source: The test fish were obtained from the National Fish Hatchery at Hebron, Ohio, USA.
- Length at study initiation (length definition, mean, range and SD): average total length of 34 mm (range 28-38 mm)
- Weight at study initiation (mean and range, SD): average weight of 0.74 g (range 0.62-1.28 g)

ACCLIMATION
- Acclimation period:
10d prior to the test
- Acclimation conditions (same as test or not):
22 °C under laboratory conditions
- Type and amount of food during acclimation:
pulverized dry Master Mix fish pellets ad libidum (until 3 d prior to test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
103 mg/L as CaCO3
Test temperature:
22 +/- 1 °C
pH:
test start: pH 7.6
test end: pH 4.8
Dissolved oxygen:
start of test: 8.2 mg/L
end of test: 4.2 mg/L
Conductivity:
1770 µmhos/cm
Nominal and measured concentrations:
5 - 10 concentrations (not specified)
Details on test conditions:
TEST SYSTEM
- Test vessel: round glass aquarium measuring 22 cm deep with a 24.5 cm diameter containing 10 L test solution
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated Lake Huron water
- Alkalinity: 85 mg/L as CaC03
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was recorded constantly. DO and pH were measured at the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 -h light/8-h dark cycle

EFFECT PARAMETERS MEASURED: Mortality was recorded after 96h test duration.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
938 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
2 246 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
no data
Validity criteria fulfilled:
not specified
Conclusions:
Based on the results the LC50 of EDTA-(NH4)2H2 is > 100 mg/L and thus the substance is not acutely harmful to fish.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The static water acute toxicity tests followed the methods described in the EPA publication, "Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians" (COMMITTEE ON METHODS FOR TOXICITY WITH AQUATIC ORGANISMS, 1975).
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TA 04174022
- Purity: 39% active ingredient
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Respective amounts of test substance was added to 2 L and then mixed with the 8 L of restonstitued water in the test vessel to get the final test concentration.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: bluegill
- Source: The test fish were obtained from the National Fish Hatchery at Hebron, Ohio, USA.
- Length at study initiation (length definition, mean, range and SD): average total length of 34 mm (range 28-38 mm)
- Weight at study initiation (mean and range, SD): average weight of 0.74 g (range 0.62-1.28 g)

ACCLIMATION
- Acclimation period:
10d prior to the test
- Acclimation conditions (same as test or not):
22 °C under laboratory conditions
- Type and amount of food during acclimation:
pulverized dry Master Mix fish pellets ad libidum (until 3 d prior to test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
103 mg/L as CaCO3
Test temperature:
22 +/- 1 °C
pH:
test start: pH 7.6
test end: pH 8.1
Dissolved oxygen:
start of test: 8.2 mg/L
end of test: 4.2 mg/L
Conductivity:
1770 µmhos/cm
Nominal and measured concentrations:
5 - 10 concentrations (not specified)
Details on test conditions:
TEST SYSTEM
- Test vessel: round glass aquarium measuring 22 cm deep with a 24.5 cm diameter containing 10 L test solution
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated Lake Huron water
- Alkalinity: 85 mg/L as CaC03
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was recorded constantly. DO and pH were measured at the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 -h light/8-h dark cycle

EFFECT PARAMETERS MEASURED: Mortality was recorded after 96h test duration.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
275 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
390 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
no data
Validity criteria fulfilled:
not specified
Conclusions:
Based on the results the LC50 of EDTA-(NH4)4 is > 100 mg/L and thus the substance is not harmful to fish.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: #1072-69-1
- Purity : 42% (in water)

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under storage conditions:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Preparation of a nanomaterial dispersion (incl. dilution):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:
Analytical monitoring:
yes
Details on sampling:
Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, prior to and after renewal at 48 hours, and at test termination.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions were prepared in the aquaria for each replicate of each treatment group by dissolving a calculated amount of iron complexed EDTA in 15 L of Wildlife International, Ltd well water. The test solutions were stirred with a stainless steel whisk. New test solutions were prepared for renewal at approximately 48 hours. During the test, the 16 mgIL test solution appeared clear and colorless. The 26, 43, 72 and 120 mgIL test solutions appeared clear with a slight yellow color that increased in intensity with increasing concentration.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: obtained from Thomas Fish Company, Anderson, California.
- Length at study initiation (length definition, mean, range and SD): The average standard length of 10 negative control fish measured at the end of the test was 2.8 cm, with a range of 2.6 to 3.1 cm.
- Weight at study initiation (mean and range, SD): The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.35 grams, with a range of 0.26 to 0.50 grams.

ACCLIMATION
- Acclimation period: The fish were held for at least 14 days prior to the test.
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: During the holding period, the fish were fed daily a commercially-prepared diet supplied by Zeigler Brothers, Inc., Gardners, Pennsylvania. The fish were not fed for at least two days prior to the test or during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
106 mg/L as CaCO3
Test temperature:
11.4 - 12.2 °C
pH:
8.2 - 8.6
Dissolved oxygen:
9.6 - 10.8 mg/L
Conductivity:
270 µmhos/cm
Nominal and measured concentrations:
Nominal: 16, 26, 43, 72 and 120 mg a.i./L
Measured: 14, 24, 42, 68 and 116 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 38 L glass aquaria containing 15 L of test solution
- Renewal rate of test solution (frequency/flow rate): after 48h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water. The well water was passed through a sand filter to remove particles greater than approximately 25 J.Un, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 /.lm to remove microorganisms and particles.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was measured in each test chamber at the beginning and end of the test and at approximately 24-hour intervals during the test using a liquid-in-glass thermometer. Temperature
also was measured continuously during the test in one negative control test chamber using a Fulscope ERIC Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer. The target test temperature during the study was 12 ± 2°C. Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24-hour intervals during the test. Hardness, alkalinity and specific conductance were measured in the dilution water at the
beginning of the test. Measurements of pH were made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 51B dissolved oxygen meter.
Specific conductance was measured using a Yellow Springs Instrument Model 33 SalinityConductivity- Temperature meter. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods/or the Examination o/Water and Wastewater.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light: 8h dark
- Light intensity: 594 lux

EFFECT PARAMETERS MEASURED: Observations of mortality and other signs of toxicity were made approximately 4, 24, 48, 72 and 96 hours after test initiation.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 116 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Validity criteria fulfilled:
yes
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The static water acute toxicity tests followed the methods described in the EPA publication, "Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians" (COMMITTEE ON METHODS FOR TOXICITY WITH AQUATIC ORGANISMS, 1975).
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity: > 97% active ingredient
Analytical monitoring:
not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Respective amounts of test substance was added to 2 L and then mixed with the 8 L of reconstituted water in the test vessel to achievet the final test concentration.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: bluegill
- Source: The test fish were obtained from the National Fish Hatchery at Hebron, Ohio, USA.
- Length at study initiation (length definition, mean, range and SD): average total length of 34 mm (range 28-38 mm)
- Weight at study initiation (mean and range, SD): average weight of 0.74 g (range 0.62-1.28 g)

ACCLIMATION
- Acclimation period:
10d prior to the test
- Acclimation conditions (same as test or not):
22 °C under laboratory conditions
- Type and amount of food during acclimation:
pulverized dry Master Mix fish pellets ad libidum (until 3 d prior to test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
103 mg/L as CaCO3
Test temperature:
22 +/- 1 °C
pH:
test start: pH 7.6
test end: pH 7.4
Dissolved oxygen:
start of test: 8.2 mg/L
end of test: 4.2 mg/L
Conductivity:
1770 µmhos/cm
Nominal and measured concentrations:
5 - 10 concentrations (not specified)
Details on test conditions:
TEST SYSTEM
- Test vessel: round glass aquarium measuring 22 cm deep with a 24.5 cm diameter containing 10 L test solution
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated Lake Huron water
- Alkalinity: 85 mg/L as CaC03
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was recorded constantly. DO and pH were measured at the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 -h light/8-h dark cycle

EFFECT PARAMETERS MEASURED: Mortality was recorded after 96h test duration.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
2 340 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
4 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
no data
Validity criteria fulfilled:
not specified
Conclusions:
Based on the results the LC50 of EDTA-CaNa2 is > 100 mg/L and thus the substance is not acutely harmful to fish.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The static water acute toxicity tests followed the methods described in the EPA publication, "Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians" (COMMITTEE ON METHODS FOR TOXICITY WITH AQUATIC ORGANISMS, 1975).
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity: 39% a.i. (in water)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Respective amounts of test substance was added to 2 L and then mixed with the 8 L of reconstituted water in the test vessel to achieve the final test concentration.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: bluegill
- Source: The test fish were obtained from the National Fish Hatchery at Hebron, Ohio, USA.
- Length at study initiation (length definition, mean, range and SD): average total length of 34 mm (range 28-38 mm)
- Weight at study initiation (mean and range, SD): average weight of 0.74 g (range 0.62-1.28 g)

ACCLIMATION
- Acclimation period: 10d prior to the test
- Acclimation conditions (same as test or not): 22 °C under laboratory conditions
- Type and amount of food during acclimation: pulverized dry Master Mix fish pellets ad libidum (until 3 d prior to test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
103 mg/L as CaCO3
Test temperature:
22 +/- 1 °C
pH:
test start: pH 7.6
test end: pH 9.6
Dissolved oxygen:
start of test: 8.2 mg/L
end of test: 4.2 mg/L
Conductivity:
1770 µmhos/cm
Nominal and measured concentrations:
5 - 10 concentrations (not specified)
Details on test conditions:
TEST SYSTEM
- Test vessel: round glass aquarium measuring 22 cm deep with a 24.5 cm diameter containing 10 L test solution
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated Lake Huron water
- Alkalinity: 85 mg/L as CaC03
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was recorded constantly. DO and pH were measured at the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 -h light/8-h dark cycle

EFFECT PARAMETERS MEASURED: Mortality was recorded after 96h test duration.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
401.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
436 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
no data
Validity criteria fulfilled:
not specified
Conclusions:
Based on the results the LC50 of edetic acid is > 100 mg/L and thus the substance is not acutely harmful to fish. Effects observed (LC50 = 401.7 mg/L) are considered to be influenced by increasing pH values exceeding the recommended test conditions for bluegill.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The static water acute toxicity tests followed the methods described in the EPA publication, "Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians" (COMMITTEE ON METHODS FOR TOXICITY WITH AQUATIC ORGANISMS, 1975).
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity: 99,33% active ingredient
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Respective amounts of test substance was added to 2 L and then mixed with the 8 L of reconstituted water in the test vessel to achievet the final test concentration.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: bluegill
- Source: The test fish were obtained from the National Fish Hatchery at Hebron, Ohio, USA.
- Length at study initiation (length definition, mean, range and SD): average total length of 34 mm (range 28-38 mm)
- Weight at study initiation (mean and range, SD): average weight of 0.74 g (range 0.62-1.28 g)

ACCLIMATION
- Acclimation period: 10d prior to the test
- Acclimation conditions (same as test or not): 22 °C under laboratory conditions
- Type and amount of food during acclimation: pulverized dry Master Mix fish pellets ad libidum (until 3 d prior to test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
103 mg/L as CaCO3
Test temperature:
22 +/- 1 °C
pH:
start pH 7.6
end pH 4 (3.7 at 240 mg/L)
Dissolved oxygen:
start of test: 8.2 mg/L
end of test: 4.2 mg/L
Conductivity:
1770 µmhos/cm
Nominal and measured concentrations:
5 - 10 concentrations (not specified)
Details on test conditions:
TEST SYSTEM
- Test vessel: round glass aquarium measuring 22 cm deep with a 24.5 cm diameter containing 10 L test solution
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated Lake Huron water
- Alkalinity: 85 mg/L as CaC03
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was recorded constantly. DO and pH were measured at the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 -h light/8-h dark cycle

EFFECT PARAMETERS MEASURED: Mortality was recorded after 96h test duration.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
159 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
240 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
no data
Sublethal observations / clinical signs:

Effects are related to low pH (Edetic acid and Edetic acid sodium salt).

The acid kills 100 % of the bluegill at 240 mg/l in water at a pH of 3.7. Such acidity alone is lethal to bluegill (COMMITTEE ON WATER QUALITY CRITERIA, 1972). If the acid is made into the sodium salt, a 100 % mortality occurs at 521 mg/l. The resultant pH of 8.9 is not itself toxic to bluegill. A 50 % aqueous solution of this sodium salt, causes 100 % mortality at 1120 mg/l at a pH of 9.6.

Validity criteria fulfilled:
not specified
Conclusions:
Based on the results the LC50 of EDTA is > 100 mg/L and thus the substance is not acutely harmful to fish. Effects observed (LC50 = 159 mg/L) are considered to be influenced by strongly decreasing pH values over time, which clearly exceeds the test conditions recommended for bluegill..
Endpoint:
short-term toxicity to fish
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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
Reason / purpose for cross-reference:
read-across source
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:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Remarks on result:
other: based on EDTA, -CaNa2, -(NH4)2H2, -FeNa, -(NH4)4, - FeK

Description of key information

LC50 (96h) > 100 mg/L for mortality of freshwater fish (WoE, read across)

Key value for chemical safety assessment

Additional information

One study is available investigating the short-term toxicity of Tetrasodium ethylenediaminetetraacetate to freshwater fish. In addition, several studies with other subcategory 1a members edetic acid (CAS No. 60-00-4), Sodium calcium edetate (CAS No. 62-33-9), Tetraammonium ethylenediaminetetraacetate (CAS No. 22473-78-5), Diammonium dihydrogen ethylenediaminetetraacetate (CAS No. 20824-56-0) as well as Subcategory 1b members Sodium feredetate (CAS No. 15708-41-5) and Ethylenediaminetetraacetic acid iron(III) potassium salt (CAS No. 54959-35-2) were considered in 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 static study with edetic acid itself followed the methods described in the EPA publication Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians (Committee on methods for toxicity with aquatic organisms 1975) and used Lepomis macrochirus as test organism (Batchelder et al. 1980). Varius test concentrations up to 521 mg/L were tested. The 96h-LC50 is determined to be 401.7 mg/L and the 96h-LC100 is 436 mg/L, which clearly exceeds the test conditions recommended for bluegill. Such acidity alone is lethal to bluegill. A 96h-LC50 for pH neutralised treatments is not given. Thus, based on the results (not even excluding the pH effect) the 96h-LC50 of EDTA is > 100 mg/L and thus the substance can be considered not acutely harmful to fish.


Within the study of Batchelder et al. (1980) several other EDTA metal complexes of Subcategory 1 were tested using the same methodology and test organism: The 96h-LC50 values were determined to be 159 mg/L for edetic acid (CAS No. 60-00-4, pH effects observed), 2340 mg/L for Sodium calcium edetate (CAS No. 62-33-9), 275 mg/L for Tetraammonium ethylenediaminetetraacetate (CAS No. 22473-78-5) and 938 mg/L for Diammonium dihydrogen ethylenediaminetetraacetate (CAS No. 20824-56-0). In general, the EDTA-metal-complexes showed lower toxicity, since pH values did not decrease or to a lower extent. In addition, two studies are available with Sodium feredetate (CAS No. 15708-41-5). One study was performed according to OECD guideline 203 under GLP using Oncorhynchus mykiss as test organism (Borrmann 2007). Concentrations of 1, 10 and 100 mg/L were tested. Nominal concentrations were verified analytically. No mortality was observed in any treatment and the control throughout the test. Thus, the 96h-LC50 is determined to be > 100 mg/L. The second study was performed according to EPA guideline 600/3-75-009 using Pimephales promelas as test organism (Ewell et al. 1986). Concentrations between 0.1 and 100 mg/L were tested. The 96h-LC50 value is determined to be > 100 mg/L based on nominal concentrations. One additional study is available that investigated the short-term toxicity of Ethylenediaminetetraacetic acid iron(III) potassium salt (CAS No. 54959-35-2) to Oncorhynchus mykiss. The study was performed according to OECD guideline 203 under GLP (Palmer at al. 2002). Concentrations between 16 and 120 mg/l were tested and analytically verified. The test was conducted under semi-static conditions. The 96h-LC50 is determined to be > 116 mg/L based on mean measured concentrations.


Based on the outcome of the various studies available it can be concluded that the 96h-LC50 for edetic acid 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 fish.