Reaction mass of Trisodium tris(D-gluconato(2-)-κO1,κO2)ferrate(3-) and Sodium chloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Testing was conducted between 14th January 2013 and 14th 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:

Toxicity to aquatic algae used from an available structural analogue.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See chapter 13 for read across document

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 32, 100, 320 and 1000 mg active ingredient (ai)/L
- Sampling method:
Preparation of Calibration Standards
The test item (nominal 100 mg ai) was dissolved in diluent* (100 mL) to prepare a stock solution with a concentration of 1000 mg ai/L. This stock solution was further diluted with diluent to produce a solution of 100 mg ai/L. Defined volumes of this solution were then diluted with diluent to obtain calibration standards in the range of 0.10 to 1.25 mg ai/L. A second series of calibration standards was similarly prepared in the range of 0.25 to 1.5 mg ai/L. These solutions were used to determine the recovery and test sample concentrations.

Preparation of Linearity Standards
The test item (nominal 100 mg ai) was dissolved in diluent* (100 mL) to prepare a stock solution with a concentration of 1000 mg ai/L. This stock solution was further diluted with diluent* to produce a solution of 100 mg ai/L. Defined volumes of this solution were then diluted with diluent* to obtain calibration standards in the range of 0.10 to 1.25 mg ai/L. A second series of calibration standards was similarly prepared in the range of 0.25 to 1.5 mg ai/L. These standards were used to evaluate the linearity of the analytical system.

Preparation of Spiked Recovery Samples
To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and its recovery was assessed. The test item (nominal 100 mg ai) was initially dissolved in diluent* to prepare a stock solution with a concentration of 1000 mg ai/L. This stock solution was further diluted with diluent* to produce a solution of 10 mg ai/L. Defined volumes of this stock solution were diluted with test medium to obtain spiked recovery samples at concentrations of 1.0 mg ai/L. Five replicates at each concentration level were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

Preparation of Test Samples
The test samples were thawed with the aid of sonication. They were further diluted into the calibration range with solvent/mixture and were analysed using HPLC, please see the details below.

- Sample storage conditions before analysis: - 20 deg C

Vehicle:

no

Details on test solutions:

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The culture medium used in the definitive test had a hardness of 18 mg/L as CaCO3 and a Total Organic Carbon (TOC) content of less than 1.0 mg C/L.

For the purpose of the definitive test, the test item was dissolved directly in culture medium. All test concentrations were corrected for a test item water content of 50.5%.

Amounts of test item (2020 and 646 mg) were each separately dissolved in culture medium and the volume adjusted to 1 liter to give 1000 and 320 mg ai/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 100, 32 and 10 mg ai/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (13.5 mL) to give the required test concentrations of 10, 32, 100, 320 and 1000 mg ai/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 deg C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

96 hours

Hardness:

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The culture medium used in the definitive test had a hardness of 18 mg/L as CaCO3 and a Total Organic Carbon (TOC) content of less than 1.0 mg C/L.

Test temperature:

The temperature within the incubator was recorded hourly using the incubators’ internal pt-100 temperature probe and Multicom Software Package. The test vessels were kept at a temperature of 24 +/- 1 deg C, throughout the study.

pH:

At the request of the Sponsor, at the start of the test the pH of the control and test preparations was adjusted to pH 7. The pH of the control and each test preparation was determined at initiation of the test and after both 48 and 96 hours exposure. The pH was measured using a WTW pH 320 pH meter.

The pH value of the control cultures was observed to increase from pH 7.0 at 0 hours to pH 8.1 at 48 hours and pH 8.2 at 96 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 96 hours was in excess of 1 pH unit after 96 hours. This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Salinity:

Not applicable as freshwater study

Nominal and measured concentrations:

Nominal concentration: The following nominal concentrations were used:
10, 32, 100, 320 and 1000 mg active ingredient (ai)/L (there were three replicate flasks for each concentration).

Measured concentrations: Analysis of the test preparations at 0 and 96 hours showed measured test concentrations to range from 93% to 105% of the nominal concentrations, and therefore the results are based on nominal test concentrations only.

Details on test conditions:

Experimental Preparation:
Amounts of test item (2020 and 646 mg) were each separately dissolved in culture medium and the volume adjusted to 1 liter to give 1000 and 320 mg ai/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 100, 32 and 10 mg ai/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (13.5 mL) to give the required test concentrations of 10, 32, 100, 320 and 1000 mg ai/L.

TEST SYSTEM
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

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

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.68 x 105 cells per mL. Inoculation of 900 mL of test medium with 13.5 mL of this algal suspension gave an initial nominal cell density of 1 x 104 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 96 hours.

Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

GROWTH MEDIUM:
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Detailed composition of culture medium:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: At the request of the Sponsor, at the start of the test the pH of the control and test preparations was adjusted to pH 7. The pH of the control and each test preparation was determined at initiation of the test and after both 48 and 96 hours exposure.
- Photoperiod: The flasks were plugged and kept under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- A re-growth Test: A re-growth test was performed after 96 hours to determine the algicidal or algistatic effect of the test item. Aliquots were removed from each replicate control and test culture (0.25 mL and 0.50 mL respectively) and the replicates pooled. Fresh sterile culture medium (100 mL) was added to each to ensure that the test concentration was reduced to below the inhibiting level.

The flasks were plugged with polyurethane foam bungs are incubated (INFORS Multitron® Version 2 Incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 120 hours.


TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: not applicable
Range finding study
- Test concentrations: The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 1.0, 10, 100 and 1000 mg ai/L for a period of 96 hours.
- 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: 10, 32, 100, 320 and 1000 mg ai/L.

Reference substance (positive control):

yes

Remarks:

zinc chloride

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

790 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: * It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of confidence limits.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

220 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: 180 - 260

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% CL = 140 - 210

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1 (please see any other information on results section). The results showed no effect on growth at the test concentrations of 1.0 and 10 mg ai/L. However, growth was observed to be reduced at 100 and 1000 mg ai/L. Based on this information test concentrations of 10, 32, 100, 320 and 1000 mg ai/L were selected for the definitive test.

Definitive test:
Cell density values determined at each sampling time are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate, yield and biomass integral values for the control and test cultures after 96 hours and percentage inhibition values are given in Table 4 (for all the tables please see any other information in results section.

Results for growth:
Accordingly the following results were determined from the data:
ErC50 (0 - 96 h) : 790 mg ai/L*

Inhibition of yield
EyC50 (0 - 96 h) : 220 mg ai/L; (95% confidence limits 180 - 260 mg ai/L)

Statistical analysis of the yield data:
After 72 hours there were no statistically significant differences between the control, 10 and 32 mg ai/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 32 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100 mg ai/L. After 96 hours there were no statistically significant differences between the control and 10 mg ai/L test concentration (P0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 10 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 32 mg ai/L.

Inhibition of biomass integral:
EbC50 (0 - 96 h) : 180 mg ai/L; 95% confidence limits 140 - 210 mg ai/L

Statistical analysis of the biomass integral data:
There were no statistically significant differences between the control, 10 and 32 mg ai/L test concentrations (≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 32 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 100 mg ai/L.

Observations on cultures:
All test and control cultures were inspected microscopically at 96 hours. After 96 hours there were no abnormalities detected in the control or test cultures at 10, 32, 100 and 320 mg ai/L, however cell debris was observed to be present in the test cultures at 1000 mg ai/L.

Observations on test item solubility:
At the start of the test all control cultures were observed to be clear colorless solutions. The test cultures were observed to range from clear colorless solutions at 10 mg ai/L through to amber colored solutions at 1000 mg ai/L. After the 96-Hour test period all control, 10, 32, 100 and 320 mg ai/L test cultures were observed to be green dispersions whilst the 1000 mg ai/L test cultures were observed to be amber/green dispersions.

Re-growth test:
Re-growth was observed to have occurred in the control, 10, 32, 100 and 320 mg ai/L test cultures after 48 hours and in the 1000 mg ai/L test culture after 120 hours. These results indicate that the test item was algistatic in effect.

Results with reference substance (positive control):

A positive control used zinc chloride as the reference item at concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

Time Point
(Hours) Response Variable EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC) (mg/L) (LOEC) (mg/L)
72 Growth Rate 0.31 0.28 - 0.35 0.10 0.32
Yield 0.26 * 0.10 0.32
Biomass 0.27 * 0.10 0.32
96 Growth Rate 0.35 0.30 - 0.40 0.10 0.32
Yield 0.27 * 0.10 0.32
Biomass 0.26 * 0.10 0.32

The results from the positive control with zinc chloride were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). After 72 hours there were no statistically significant differences between the control, 10 and 32 mg ai/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 32 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100 mg ai/L. After 96 hours there were no statistically significant differences between the control, 10, 32 and 100 mg ai/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 320 mg ai/L.

Tables for the range finding study:

Table1     Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg ai/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	96 Hours	Growth Rate	Yield/Biomass Integral
Control	R1	1.14E+04	2.22E+06	-	-
	R2	1.16E+04	2.25E+06
	Mean	1.15E+04	2.23E+06
1.0	R1	1.07E+04	2.27E+06	0	3
	R2	1.04E+04	2.04E+06
	Mean	1.05E+04	2.16E+06
10	R1	1.03E+04	2.09E+06	0	12
	R2	1.00E+04	1.84E+06
	Mean	1.02E+04	1.97E+06
100	R1	1.04E+04	1.67E+06	4	22
	R2	1.07E+04	1.81E+06
	Mean	1.06E+04	1.74E+06
1000	R1	1.06E+04	1.59E+05	49	93
	R2	1.26E+04	1.78E+05
	Mean	1.16E+04	1.68E+05

Table 2     Cell Densities in the Definitive Test

Nominal Concentration (mg ai/L)		Cell Densities*(cells per mL)
		0 h	24 h	48 h	72 h	96 h
Control	R1	1.09E+04	5.43E+04	2.80E+05	1.41E+06	2.44E+06
	R2	1.00E+04	5.09E+04	3.58E+05	1.77E+06	2.45E+06
	R3	1.00E+04	6.18E+04	3.75E+05	1.85E+06	2.47E+06
	R4	1.09E+04	6.35E+04	3.59E+05	1.62E+06	2.55E+06
	R5	1.06E+04	6.10E+04	3.60E+05	1.50E+06	2.61E+06
	R6	1.06E+04	6.16E+04	4.14E+05	1.90E+06	2.50E+06
	Mean	1.05E+04	5.88E+04	3.58E+05	1.68E+06	2.51E+06
10	R1	1.13E+04	4.86E+04	3.13E+05	1.37E+06	2.74E+06
	R2	1.03E+04	6.35E+04	3.81E+05	1.79E+06	2.80E+06
	R3	1.31E+04	5.41E+04	2.89E+05	1.57E+06	2.57E+06
	Mean	1.16E+04	5.54E+04	3.28E+05	1.58E+06	2.70E+06
32	R1	1.27E+04	5.80E+04	2.59E+05	1.43E+06	2.24E+06
	R2	1.03E+04	5.36E+04	3.14E+05	1.74E+06	2.19E+06
	R3	1.15E+04	5.28E+04	3.57E+05	1.50E+06	2.14E+06
	Mean	1.15E+04	5.48E+04	3.10E+05	1.55E+06	2.19E+06
100	R1	1.05E+04	4.87E+04	2.60E+05	1.24E+06	2.06E+06
	R2	1.10E+04	4.37E+04	2.93E+05	1.16E+06	2.14E+06
	R3	1.15E+04	4.88E+04	3.00E+05	1.16E+06	1.70E+06
	Mean	1.10E+04	4.71E+04	2.84E+05	1.19E+06	1.97E+06
320	R1	1.15E+04	3.20E+04	5.82E+04	3.13E+05	9.14E+05
	R2	1.07E+04	3.59E+04	7.49E+04	2.90E+05	8.75E+05
	R3	1.04E+04	3.55E+04	7.26E+04	3.39E+05	1.01E+06
	Mean	1.09E+04	3.45E+04	6.86E+04	3.14E+05	9.31E+05
1000	R1	1.07E+04	1.91E+04	2.02E+04	4.49E+04	1.29E+05
	R2	1.09E+04	2.03E+04	2.11E+04	2.60E+04	8.90E+04
	R3	1.13E+04	2.21E+04	2.06E+04	4.34E+04	7.08E+04
	Mean	1.10E+04	2.05E+04	2.06E+04	3.81E+04	9.64E+04

Table 3     Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3	Day 3 - 4
Control	R1	0.070	0.068	0.067	0.023
	R2	0.068	0.081	0.067	0.013
	R3	0.076	0.075	0.066	0.012
	R4	0.077	0.072	0.063	0.019
	R5	0.075	0.074	0.060	0.023
	R6	0.076	0.079	0.064	0.011
	Mean	0.074	0.075	0.065	0.017

 

Table 4     Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test (0 – 96 Hour)

Nominal Concentration (mg ai/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)		Biomass Integral (cells/mL/hour)
		0 – 96 h	% Inhibition	0 – 96 h	% Inhibition*	0 – 96 h	% Inhibition
Control	R1	0.057		2.43E+06		7.04E+07
	R2	0.057		2.44E+06		8.09E+07
	R3	0.057		2.46E+06		8.36E+07
	R4	0.058	-	2.54E+06	-	7.87E+07	-
	R5	0.058		2.60E+06		7.67E+07
	R6	0.058		2.49E+06		8.63E+07
	Mean	0.058		2.49E+06		7.95E+07
	SD	0.001		6.65E+04		5.61E+06
10	R1	0.058	0	2.73E+06		7.36E+07	7
	R2	0.059	[2]	2.79E+06		8.65E+07	[9]
	R3	0.058	0	2.55E+06		7.59E+07	4
	Mean	0.058	[1]	2.69E+06	[8]	7.87E+07	1
	SD	0.001		1.26E+05		6.89E+06
32	R1	0.056	3	2.23E+06		6.79E+07	15
	R2	0.056	3	2.18E+06		7.59E+07	4
	R3	0.056	3	2.13E+06		7.06E+07	11
	Mean	0.056	3	2.18E+06	13	7.15E+07	10
	SD	0.000		5.13E+04		4.09E+06
100	R1	0.056	3	2.05E+06		6.12E+07	23
	R2	0.056	3	2.13E+06		6.08E+07	24
	R3	0.054	7	1.69E+06		5.58E+07	30
	Mean	0.055	4	1.96E+06	22	5.93E+07	26
	SD	0.001		2.34E+05		2.97E+06
320	R1	0.047	19	9.02E+05		1.98E+07	75
	R2	0.047	19	8.64E+05		1.93E+07	76
	R3	0.048	17	9.95E+05		2.20E+07	72
	Mean	0.047	18	9.20E+05	63	2.03E+07	74
	SD	0.001		6.75E+04		1.41E+06
1000	R1	0.027	53	1.19E+05		2.73E+06	97
	R2	0.023	60	7.81E+04		1.85E+06	98
	R3	0.020	66	5.95E+04		2.07E+06	97
	Mean	0.023	60	8.54E+04	97	2.22E+06	97
	SD	0.004		3.02E+04		4.60E+05

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Validation Criteria:

The following data show that the cell concentration of the control cultures increased by a factor of 160 after 72 hours and by a factor of 239 after 96 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours and the EPA/OPPTS Guidelines that states the enhancement must be at least by a factor of 100 after 96 hours.

 

Mean cell density of control at 0 hours         :          1.05 x 10⁴cells per mL

Mean cell density of control at 72 hours       :          1.68 x 10⁶cells per mL

Mean cell density of control at 96 hours       :         2.51 x 10⁶cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures over the test period (0-72 hours) was 9% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

The coefficient of variation of the cell density values in replicate control cultures was 12% after 72 hours and 3% after 96 hours and hence satisfied the validation criterion which states that this must not exceed 20%.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 96-Hour period and gave the following results:

Results for growth:
Accordingly the following results were determined from the data:
ErC50 (0 - 96 h) : 790 mg ai/L (95% confidence limits could not be determined)

Inhibition of yield
EyC50 (0 - 96 h) : 220 mg ai/L; (95% confidence limits 180 - 260 mg ai/L)

Inhibition of biomass integral:
EbC50 (0 - 96 h) : 180 mg ai/L; 95% confidence limits 140 - 210 mg ai/L

Executive summary:

Introduction:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400.

 

Methods:

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 10, 32, 100, 320 and 1000 mg active ingredient (ai)/L (three replicate flasks per concentration) for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results:

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

 

TiPoint (Hours)	Response Variable	EC50(mg ai/L)	95% Confidence Limits (mg ai/L)			No Observed Effect Concentration (NOEC) (mg ai/L)	Lowest Observed Effect Concentration (LOEC) (mg ai/L)
72	Growth Rate	510		*		32	100
	Yield	150	120	-	190	32	100
	Biomass	160	140	-	190	32	100
96	Growth Rate	790		*		100	320
	Yield	220	180	-	260	10	32
	Biomass	180	140	-	210	32	100

Analysis of the test preparations at 0 and 96 hours showed measured test concentrations to range from 93% to 105% of nominal and so the results are based on nominal test concentrations only.

A re-growth test was performed which showed the test item to be algistatic in effect.

Description of key information

No algae 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 algal growth study was conducted on the test material, this study was conducted according to OECD guideline 201 and EU Method C3. The results of this study showed Sodium Glucoheptonate to have an EC50 value of 790 mg a.i./L and a NOEC of 100 mg a.i./L.

Key value for chemical safety assessment

EC50 for freshwater algae:

790 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Introduction:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400.

 

Methods:

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 10, 32, 100, 320 and 1000 mg active ingredient (a.i.)/L (three replicate flasks per concentration) for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results:

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

 

Time Point (Hours)	Response Variable	EC50(mg a.i./L)	95% Confidence Limits (mg a.i./L)			No Observed Effect Concentration (NOEC) (mg a.i./L)	Lowest Observed Effect Concentration (LOEC) (mg a.i./L)
72	Growth Rate	510		*		32	100
	Yield	150	120	-	190	32	100
	Biomass	160	140	-	190	32	100
96	Growth Rate	790		*		100	320
	Yield	220	180	-	260	10	32
	Biomass	180	140	-	210	32	100

Analysis of the test preparations at 0 and 96 hours showed measured test concentrations to range from 93% to 105% of nominal and therefore the results are based on nominal test concentrations only, as this coincides within the acceptable range of between 80 and 120%.

A re-growth test was performed which showed the test item to be algistatic in effect.