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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 21 February 2012 Experimental Completion Date: 14 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results
Justification for type of information:
Data from structural analogue sodium heptogluconate (CAS no.: 31138-65-5) is used for read across to (Gluconate)3 -FeNa3 (CAS no.: 2101772-89-6)
Currently an acute daphnia test with (Gluconate)3 -FeNa3 is running.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please see Chapter 13 for Read across document
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis. Samples were stored at approximately -20 deg C prior to analysis.

Duplicate samples were taken and stored at approximately -20 deg C for further analysis if necessary.

Samples at the No Observed Effect Concentration and above only were analyzed.
Vehicle:
no
Details on test solutions:
For both the range finding and definitive tests, reconstituted water was used. The theoretical hardness of reconstituted water is 250 mg/L as CaCO3. Therefore, in order to comply with the water characteristics of the OPPTS guideline, the prepared water was diluted in a ratio of 50:50 with deionized reverse osmosis water prior to use to reduce the total water hardness. At the start of the definitive test the diluent had a total water hardness of 148 mg/L as CaCO3 and an alkalinity of 26 mg/L as CaCO3.

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 deg C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
48 hours
Hardness:
The theoretical hardness of reconstituted water is 250 mg/L as CaCO3. Therefore, in order to comply with the water characteristics of the OPPTS guideline, the prepared water was diluted in a ratio of 50:50 with deionized reverse osmosis water prior to use to reduce the total water hardness. At the start of the definitive test the diluent had a total water hardness of 148 mg/L as CaCO3 and an alkalinity of 26 mg/L as CaCO3.
Test temperature:
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Temperature was maintained at approximately 20 °C to 21 °C throughout the test.
pH:
The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter.
Dissolved oxygen:
The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter.
Salinity:
Not applicable as fresh water study was conducted
Nominal and measured concentrations:
The following nominal concentrations were used: 100, 180, 320, 560 and 1000 mg active ingredient (ai)/L.

Measured concentrations: Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 80% to 83% of nominal value and so the results are based on nominal test concentrations only.
Details on test conditions:
Experimental preparation:
For the purpose of the definitive test the test item was dissolved directly in reconstituted water.

An amount of test item (10100 mg) was dissolved in reconstituted water and the volume adjusted to 5 liters to give the 1000 mg ai/L test concentration. Aliquots (100, 180, 320 and 560 mL) of the 1000 mg ai/L test concentration were added to a final volume of 1 liter to give the remaining test concentrations of 100, 180, 320 and 560 mg ai/L.

Each stock prepared concentration was inverted several times to ensure adequate mixing and homogeneity and the pH of each was adjusted to pH 7.

TEST SYSTEM
In the definitive test 250 mL glass jars containing 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20 °C to 21 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods with a light intensity ranging from 645 to 682 lux. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST MEDIUM / WATER PARAMETERS
- stock solutions: The following stock solutions were used to form the reconstituted water
a) CaCl2.2H2O 11.76 g/L
b) MgSO4.7H2O 4.93 g/L
c) NaHCO3 2.59 g/L
d) KCl 0.23 g/L

Analysis of the reconstituted water - Elendt M7 Medium
Solution Concentration of
stock solution (mg/L)
H3BO3 57190
MnCl2.4H2O 7210
LiCl 6120
RbCl 1420
SrCl2.6H2O 3040
NaBr 320
Na2MoO4.2H2O 1260
CuCl2.2H2O 335
ZnCl2 260
CoCl2.6H2O 200
KI 65
Na2SeO3 43.8
NH4VO3 11.5
Na2EDTA.2H2O 5000
FeSO4.7H2O 1991

- Source/preparation of dilution water: An aliquot (25 mL) of each of solutions a-d was added to each liter (final volume) of deionized water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
General observations on the test and control solutions e.g. appearance
Physical chemical properties of the test and control solutions were also investigated.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
Range finding study:
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10, 100 and 1000 mg ai/L. The test item was dissolved directly in reconstituted water.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg ai/L. All test sample concentrations were corrected for a water content of 50.5%.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
Range-finding Test:
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg ai/L. However, immobilization was observed at 1000 mg ai/L, as can be seen in table 1 (please see the section (any other information on results).

Chemical analysis of the test preparations at 0 and 48 hours showed that measured concentrations between 83% and 94% of nominal were obtained indicating that the test item was stable under test conditions. Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg ai/L were selected for the definitive test.

Definitive test:
There was no immobilization in 20 daphnids exposed to a range of test concentrations of 100, 180, 320, 560 and 1000 mg ai/L for a period of 48 hours. Please see table 2 in the any other information on results section for further details. The No Observed Effect Concentration after 24 and 48 hours exposure was 1000 mg ai/L.

Observations:
Throughout the duration of the test, the test preparations were observed to range from an extremely pale amber colored solution in the 100 mg ai/L test group to a dark amber solution in the 1000 mg ai/L test group.
Results with reference substance (positive control):
not applicable

Table 1 - Results for the range finding study:

Nominal
Concentration
(mg ai/L)

Cumulative Immobilized Daphnia (Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.10

0

0

1.0

0

1

10

0

0

100

0

0

1000

5

10

Table 2     Cumulative Immobilization Data in the Definitive Test

Nominal
Concentration
(mg/L)

Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)

24 Hours

48 Hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

100

0

0

0

0

0

0

0

0

0

0

0

0

180

0

0

0

0

0

0

0

0

0

0

0

0

320

0

0

0

0

0

0

0

0

0

0

0

0

560

0

0

0

0

0

0

0

0

0

0

0

0

1000

0

0

0

0

0

0

0

0

0

0

0

0


R1– R4= Replicates 1 to 4

Post study experiment:

Cumulative immobilization data from the exposure of Daphnia Magna to the test item during the post-study experiment are given in Table 3. Please see below:

Table 3     Cumulative Immobilization Data in the Post-Study Experiment

Nominal
Concentration
(mg ai/L)

Cumulative Immobilized Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.10

0

0

1.0

0

0

10

0

0

100

0

0

1000

0

0

No immobilization was observed at the test concentrations of 0.10, 1.0, 10, 100 and 1000 mg ai/L.

 

Chemical analysis of the 1000 mg ai/L test preparation at 0 and 48 hours (see Appendix 4) showed near nominal concentrations were obtained indicating that the test system was correctly dosed. It was therefore considered that the immobilization observed in the range finding test was due to contamination rather than a true toxic effect.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 1000 mg ai/L. The No Observed Effect Concentration was 1000 mg ai/L.
Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1010 (April 1996) and the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

 

Methods:

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 100, 180, 320, 560 and 1000 mg active ingredient (ai)/L for 48 hours at a temperature of 20°C to 21°C under static test conditions. The number of immobilized Daphnia were recorded after 24 and 48 hours.

 

 

Results

The 48-Hour EC50for the test item to Daphnia magna based on nominal test concentrations was greater than 1000 mg ai/L. The No Observed Effect Concentration was 1000 mg ai/L.

 

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 80% to 83% of nominal value and so the results are based on nominal test concentrations only.

Description of key information

No acute daphnia test results are available for the Reaction mass of (Gluconate)3Fe-Na3 and NaCl. For this reason the available study from the structurally similar substance Sodium glucoheptonate (CAS no 31138 -65 -5) has been read across to the Reaction mass of (Gluconate)3Fe-Na3 and NaCl. An acute daphnia study with the reaction mass of (Gluconate)3Fe-Na3 and NaCl3 is currently running.

The study with Sodium glucoheptonate (CAS no 31138 -65 -5) was conducted according to GLP and according to OECD guideline 202.  The test item was exposed to Daphnia magna for 48 hours under static test conditions. The EC50 value after 48 hours was 1000 mg a.i./L and the NOEC was 1000 mg a.i./L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1 000 mg/L

Additional information

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1010 (April 1996) and the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

Methods:

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 100, 180, 320, 560 and 1000 mg active ingredient (a.i.)/L for 48 hours at a temperature of 20°C to 21°C under static test conditions. The number of immobilized Daphnia were recorded after 24 and 48 hours.

 

Results:

The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 1000 mg a.i./L. The No Observed Effect Concentration was 1000 mg a.i./L.

 

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 80% to 83% of nominal value and so the results are based on nominal test concentrations only.