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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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Basic data given: acceptable publication
Principles of method if other than guideline:
Method: other Fish, Acute Toxicity Test
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
not specified
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
24 h
Hardness:
12 °dH
Test temperature:
18 - 21 °C
Details on test conditions:
The fry were held in glass aquaria with aeration.
Sufficient oxygen content of the test water was ensured.
Tap water from the city of Hamburg was used in all experiments as test water.
3 animals were used per test concentration.
A second test series was run in parallel.
The fish were observed for at least 24 hours, in some cases even 48 hours and lethalities were recorded.
Duration:
24 h
Dose descriptor:
LC100
Effect conc.:
860 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
The authors noted that the disodium salt of EDTA showed significantly less toxicity towards aquatic organisms compared to the corresponding acid EDTA which was presumably caused by a pH effect.
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:
other: Acceptable, well documented publication
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
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
The test fish were obtained from the National Fish Hatchery at Hebron, Ohio, USA.
The fish had an average weight of 0.74 g (range 0.62-1.28 g) and an average total length of 34 mm (range 28-38 mm).
The fish were kept in a 16-h light/8-h dark cycle. Feed was stopped three days prior to the bioassay to empty the digestive tract. Fish were placed into the aquaria 24 h before adding the test material.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Three different water types were used:
very soft water: 10-13 mg/L CaCO3, reconstituted from destilled water
medium hard water: 103 mg/L as CaCO3, dilution water from Lake Huron
very hard water: 280-320 mg/L CaCO3, reconstituted from destilled water
Test temperature:
22 +/- 1°C
pH:
Three different water types were used:
very soft water reconstituted from destilled water, start: pH 6.4 - 6.8, end: unknown but low
medium hard water dilution water from Lake Huron: start pH 7.6, end pH 4
very hard water reconstituted from destilled water: start pH 8.0 - 8.4, end pH 4
Dissolved oxygen:
start of test: 8.2 mg/L
end of test: 4.2 mg/L
Salinity:
no data available
Nominal and measured concentrations:
no data available
Details on test conditions:
Water used came from Whitestone Point on the west side of Saginaw Bay, Lake Huron.
The water was sent through an activated charcoal bed to remove any free chlorine.
This water exhibits chemical characteristics in the following ranges:
total alkalinity: 85 mg/L as CaC03
specific conductivity: 1770 umhos/cm
resistivity: ca. 570 ohms/cm
This water passes the general requirements for dilution water as stated in COMMITTEE ON METHODS FOR TOXICITY WITH AQUATIC ORGANISMS (1975). In addition, VERSENE acid, VERSENE 100 were also tested for effect in very soft and hard waters. These waters were reconstituted distilled waters.

None of the products were neutralized prior to testing.

Bioassays were conducted by placing eight liters water in each vessel, a round glass aquarium measuring 22 cm deep with a 24.5 cm diameter, adding the fish, then aerating with plant air. If no deaths occurred in 24 h, the aerators were removed, and the test solution added with two liters of water for mixing, making a total of ten liters. Any vessel containing dead fish was cleaned, reset, and observed for another 24 h prior to compound addition. Ten fish were exposed to each concentration of the material. Between five and ten concentrations were set per product. Observations were made and recorded daily.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
other: NAEL
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: very soft water
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 34 - 62 mg/L, very soft water
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: very soft water
Duration:
96 h
Dose descriptor:
other: NAEL
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: medium hard water
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
159 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 136-204 mg/L, medium hard water
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
240 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: medium hard water
Duration:
96 h
Dose descriptor:
other: NAEL
Effect conc.:
420 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: very hard water
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
532 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 473-598 mg/L, very hard water
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
750 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: very hard water
Details on results:
Instead of the NOEC, in this study the NAEL (= no (observable) adverse effect level) is quoted.
Results with reference substance (positive control):
no data
Sublethal observations / clinical signs:

Two products illustrate the effect of pH in medium hard water: VERSENE acid and VERSENE powder. 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, VERSENE powder, 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, VERSENE 100, causes 100 % mortality at 1120 mg/L at a pH of 9.6. In hard water, total kill with the VERSENE acid occurs at 750 mg/L and pH 3.5 (again, a pH related kill), whereas VERSENE 100 killed all bluegill at 2400 mg/L and pH 9.8. A pH level of 9.5-10.0 may cause some mortality; values >10.0 are not tolerated by bluegill (COMMITTEE ON WATER QUALITY CRITERIA, 1972).

Validity criteria fulfilled:
not specified
Conclusions:
The test results of H4EDTA show that there is a relation between water hardness and ecotoxicity. Tests performed in very soft (10-13 mg/l CaCO3) and medium hard water (103 mg/I CaCO3) showed a higher toxicity than those performed in very hard water. As the hardness increased, more of the chelator was converted to the Ca-complex, thus more chelating agent was required to exert a toxic effect-less uncomplexed EDTA is available. This was proofed by a reported LC50 of 2340 mg/L for Versene Ca (where EDTA is already complexed with Ca) in the same publication. Thus, it should be noted, that under test conditions (buffer capacity) pH 4 alone leads to toxicity in 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:
other: Acceptable, well documented publication
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
Analytical monitoring:
not specified
Details on sampling:
no data available
Vehicle:
not specified
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
The test fish were obtained from the National Fish Hatchery at Hebron, Ohio, USA.
The fish had an average weight of 0.74 g (range 0.62-1.28 g) and an average total length of 34 mm (range 28-38 mm).
The fish were kept in a 16 -h light/8-h dark cycle. Feed was stopped three days prior to the bioassay to empty the digestive tract. Fish were placed into the aquaria 24 h before adding the test material.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
no data available
Hardness:
Three different water types were used:
very soft water: 10-13 mg/L CaCO3, reconstituted from destilled water
medium hard water: 103 mg/L as CaCO3, dilution water from Lake Huron
very hard water: 280-320 mg/L CaCO3, reconstituted from destilled water
Test temperature:
22 +/- 1 °C
pH:
Three different water types were used:
very soft water reconstituted from destilled water, start: pH 6.4 - 6.8, end: 7.5
medium hard water dilution water from Lake Huron: start pH 7.6, end pH 9.5
very hard water reconstituted from destilled water: start pH 8.0 - 8.4, end pH 9.5
Dissolved oxygen:
start of test: 8.2 mg/L
end of test: 4.2 mg/L
Salinity:
no data
Nominal and measured concentrations:
no data
Details on test conditions:
TEST WATER
Water used came from Whitestone Point on the west side of Saginaw Bay, Lake Huron.
The water was sent through an activated charcoal bed to remove any free chlorine.
This water exhibits chemical characteristics in the following ranges:
total alkalinity: 85 mg/L as CaCO3;
specific conductivity: 1770 µmhos/cm; and resistivity: ca. 570 ohms/cm (AMERICAN PUBLIC HEALTH ASSOCIATION 1975).
This water passes the general requirements for dilution water as stated in COMMITTEE ON METHODS FOR TOXICITY WITH AQUATIC ORGANISMS (1975).
None of the products were neutralized prior to testing.
Bioassays were conducted by placing eight liters water in each vessel, a round glass aquarium measuring 22 cm deep with a 24.5 cm diameter, adding the fish, then aerating with plant air. If no deaths occurred in 24 h, the aerators were removed, and the test solution added with two liters of water for mixing, making a total of ten liters. Any vessel containing dead fish was cleaned, reset, and observed for another 24 h prior to compound addition. Ten fish were exposed to each concentration of the material. Between five and ten concentrations were set per product. Observations were made and recorded daily.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
other: NAEL
Effect conc.:
88 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: very soft water
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
121 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: 113-130 mg/L, very soft water
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
138 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: very soft water
Duration:
96 h
Dose descriptor:
other: NAEL
Effect conc.:
669 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: medium hard water
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
792 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: 754-831 mg/L, medium hard water
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
861 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: medium hard water
Duration:
96 h
Dose descriptor:
other: NAEL
Effect conc.:
1 380 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: hard water
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1 592 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: 1493-1678 mg/L, hard water
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1 846 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: hard water
Details on results:
Instead of the NOEC, in this study the NAEL (= no (observable) adverse effect level) is quoted.
Results with reference substance (positive control):
no data available
Validity criteria fulfilled:
not specified
Conclusions:
The test results of Na4EDTA show that there is a relation between water hardness and ecotoxicity. Tests performed in very soft water (10-13 mg/L CaCO3) showed a higher toxicity than those performed in a medium hard water (103 mg/L CaCO3) or very hard water (280-320 mg CaCO3). The great variation in mortality resulted in LC50 values in a range of 121 mg/L (soft water) to 1592 mg/L (very hard water). The LC50 of 121 mg/L derived in very soft water which reflects spring water from lime-free geological zones is not considered as environmentally relevant as EDTA is not released to waters of such low hardness.
Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance.
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to the Analogue Approach Justification document provided in Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: very soft water; source, RA-A, CAS 60-00-4, Batchelder & al., 1980, L. macrochirus, 96 h, RL2
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
159 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: medium hard water; source, RA-A, CAS 60-00-4, Batchelder & al., 1980, L. macrochirus, 96 h, RL2
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
532 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: very hard water, source, RA-A, CAS 60-00-4, Batchelder & al., 1980, L. macrochirus, 96 h, RL2
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
121 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: very soft water; source, RA-A, CAS 64-02-8, Batchelder & al., 1980, L. macrochirus, 96 h, RL2
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
792 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: medium hard water; source, RA-A, CAS 64-02-8, Batchelder & al., 1980, L. macrochirus, 96 h, RL2
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1 592 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na4EDTA
Basis for effect:
mortality (fish)
Remarks on result:
other: hard water; source, RA-A, CAS 64-02-8, Batchelder & al., 1980, L. macrochirus, 96 h, RL2

Description of key information

LC50 (96 h) > 100 mg/L; read-across (Lepomis macrochirus)

Key value for chemical safety assessment

Additional information

Since no studies investigating the short-term toxicity trisodium hydrogen EDTA (CAS 150-38-9) to fish are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to structurally related EDTA species was conducted. This read-across is justified in detail in the overall summary (IUCLID Section 6.1) and within the analogue justification in IUCLID Section 13.
Several studies on different species of edetic acid (EDTA) are available. All studies were combined in a weight-of-evidence approach. The toxicity of EDTA on fish highly depends on water hardness, pH and metal speciation (EU Risk Assessment, 2004). Batchelder et al. (1980) investigated the toxicity of EDTA complexes to bluegill sunfish (Lepomis macrochirus) by taking into account the water hardness and pH of the test medium. The results testing the acidic form of EDTA lead to a decrease of pH in test medium < 4 with a LC100 of 240 mg/L. However, a pH of 4 leads to toxicity in bluegill sunfish (Committee on water quality criteria, 1972). Thus, the results testing the acidic form are only considered to be related to the very low pH of the test medium causing mortality.


The revealed LC50-values after 96 h resulting from test media with different hardness are in a range of 41 mg/L to 1592 mg/L (see table below).

EDTA Species

LC50 [mg/L]

Test conditions

H4-EDTA

41

Very soft water

 

159

Medium hard water

 

532

Very hard water

Na4-EDTA

121

Very soft water

 

792

Medium hard water

 

1592

Very hard water

Two consequences can be drawn from the results of Batchelder et al. First, the tests performed with the acid form lead to very low pH values under test conditions causing mortality. The low LC50-value of 41 mg/L in very soft water can be explained by a surplus of un-complexed EDTA which was present in the test media since species to be complexed are limited in very soft water. In medium hard and hard water the predominant species of EDTA is the complexed form. This effect is not expected to occur in the environment since EDTA is not released to waters of such low hardness (e.g. spring water from lime-free geological zones). Therefore, the results derived by soft water hardness and/or very low pH <4 are not relevant for the assessment.
One supporting study with disodium dihydrogen EDTA (CAS 139-33-3) supports this conclusion with a LC100 (24 h) of 860 mg/L. 

Consequently, the LC50 of EDTA is estimated to be higher 100 mg/L, due to the fact that this predicted value represents the lowest LC50 at acceptable pH which has been performed in natural non-synthetic water.