Reaction mass of disodium [2,2'-(imino-κN)dibutanedioato-κ2O1,O4(4-)]calcium(2-), sodium chloride and aspartic acid disodium salt

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:

2013-09-20 - 2013-10-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Justification for type of information:

A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent as in a target substance (CaNa2IDHA). The only difference between the target and the source substance is presence of calcium (Ca) cation instead Mn2+ cations. As calcium is an essential macro element required by all forms of life, is considered not to influence the toxicological activity.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bureau for Chemical Substances, 30/34 Dowborczykow Street, 90-019 Lodz, Poland

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

yes

Details on sampling:

A sample (i.e. a control sample, a treated sample, or a sample fortified with the standard solution) in a volume between 1-5 mL was diluted with an acetonitrile : phosphate buffer mixture (50:50, v/v). Samples collected at the exposure initiation and at the exposure termination (72-hour-old samples) were analyzed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A solution at the concentration of 1.0 mg/mL was prepared by weighing the test item (240 mg) and mixing it with the (not modified / modified) AAP medium (240 mL). The solution was diluted by adding 10 mL of it to 90 mL of the (not modified / modified) AAP medium. As a result, a solution at the concentration of 0.1 mg/mL was obtained. The solutions at the concentrations of 1.0 and 0.1 mg/mL were diluted in order to prepare the remaining test item concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Source (laboratory, culture collection): Culture Collection of algae was received from Göttingen University, Germany. Test organisms were obtained from a standard laboratory culture cultivated at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology according to the recommendations specified in the OECD Guideline for the Testing of Chemicals No. 201.
- Age of inoculum (at test initiation): three days
- Method of cultivation: The algae were transferred from agar bevels to a fresh liquid medium (for Pseudokirchneriella subcapitata, the AAP medium is recommended), contained in Erlenmeyer flasks with a capacity of 250 mL, and incubated at temperature between 21 – 24 °C under constant illumination (a pre-culture). The algal culture was renewed (transferred to a fresh medium) twice a week under sterile conditions. The pre-culture whose density was 1 x 10 E4 cells/mL was renewed three days before the definitive test. The pre-culture was used for inoculation of the test item concentrations and the control. The sensitivity of the algal culture is monitored every six months using a reference substance, 3,5-dichlorophenol.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No information was provided.

Hardness:

not reported

Test temperature:

21.9 - 22.3 °C (first preliminary test)
22.1 - 22.7 °C (second and third preliminary test)

pH:

5.81 - 9.04 (definitive test)

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

0 (control), 0.1, 1.0, 10.0, and 100.0 mg/L (first preliminary test, nominal concentrations)
0 (control) and 100.0 mg/L (second and third preliminary test, nominal concentrations)
0 (control), 10, 32, 100, 320 and 1000 mg/L (definitive test, nominal concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (250 mL)
- Type (delete if not applicable): closed (plugged with air permeable stoppers)
- Material, size, headspace, fill volume: glass, 100 mL
- Aeration: no
- Initial cells density: initial algal density of 1 x 10 E4 cells/mL
- Control end cells density: 1.592 x 10 E6 cells/mL after 72 h (definitive test)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: It was prepared on the basis of deionised water by adding stock solutions of reagent grade chemicals. The
stock solutions were renewed, sterilised by autoclaving (except for sodium hydrogen carbonate which was a buffering ingredient), and stored in a refrigerator.
- Culture medium different from test medium: no
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not reported
- Photoperiod: constant illumination
- Light intensity and quality: mean light intensity (6804 – 7678 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometric measurements were made 24, 48, and 72 hours after the beginning of the exposure. The recorded absorbance values were converted into the number of cells using a nomogram.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: 3 preliminary studies were performed
- Test concentrations: 0 (control), 0.1, 1.0, 10.0, and 100.0 mg/L (first preliminary test, nominal concentrations); 0 (control) and 100.0 mg/L (second and third preliminary test, nominal concentrations)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

597.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 512.82 – 710.93

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

140.86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 108.87 – 172.34

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

66.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 45.17 – 88.10

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

108.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 89.16 – 133.21

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

42.27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 29.0 – 54.64

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

25.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 15.38 – 35.94

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- EC50: ErC50 (72h) = 2.61 mg/L; EyC50 (72h) = 1.74

Reported statistics and error estimates:

The statistical tests based on the growth rate data and the yield data were the Shapiro-Wilk’s Test on Normal Distribution which confirmed normal distribution of the data (respectively), the Levene’s Test on Variance Homogeneity which showed that the variances were homogeneous, and the Williams Multiple Sequential t-test Procedure which showed a significant difference between all concentrations and the control, respectively.

Reference test

Table 2. Reference test. Table A. 3,5-dichlorophenol - reference test results

Nominal concentration of the reference substance [mg/L]	Algal cell density [10 E6 cells/mL]			Growth Rate [10 E6cells/mL]				Yield [10 E6 cells/mL]
	24h	48h	72h	0-24h	24-48h	48-72h	0-72h	0-72h
Control	0.098	0.693	2.727	2.283	1.956	1.369	1.869	2.717
	0.091	0.602	2.344	2.212	1.886	1.359	1.819	2.334
	0.085	0.507	2.612	2.135	1.792	1.638	1.855	2.602
	0.085	0.721	2.963	2.135	2.142	1.414	1.897	2.953
	0.081	0.565	2.601	2.094	1.940	1.527	1.854	2.591
	0.085	0.788	3.160	2.135	2.232	1.388	1.919	3.150
mean	0.087	0.S4S	2.735	2.1SS	1.991	1.449	1.8S9	2.725
SD	0.006	0.106	0.289	0.069	0.165	0.111	0.035	0.289
0.56	0.054	0.521	2.524	1.689	2.264	1.578	1.844	2.514
	0.064	0.585	2.527	1.861	2.209	1.463	1.844	2.517
	0.061	0.565	2.206	1.807	2.228	1.362	1.799	2.196
mean	0.0S0	0.557	2.419	1.785	2.234	1.4S8	1.829	2.409
SD	0.005	0.033	0.185	0.088	0.028	0.108	0.026	0.185
1.0	0.064	0.423	2.124	1.861	1.884	1.614	1.786	2.114
	0.051	0.460	2.121	1.624	2.205	1.528	1.786	2.111
	0.054	0.450	2.199	1.689	2.118	1.587	1.798	2.189
mean	0.05S	0.444	2.148	1.725	2.0S9	1.57S	1.790	2.138
SD	0.007	0.019	0.044	0.122	0.166	0.044	0.007	0.044
1.8	0.068	0.206	1.580	1.912	1.115	2.035	1.687	1.570
	0.081	0.352	1.641	2.094	1.466	1.540	1.700	1.631
	0.081	0.369	1.597	2.094	1.513	1.466	1.691	1.587
mean	0.077	0.309	1.S0S	2.033	1.3S5	1.S80	1.S93	1.59S
SD	0.008	0.089	0.031	0.105	0.218	0.310	0.007	0.031
3.2	0.014	0.051	0.041	0.302	1.322	n.d.	0.467	0.031
	0.014	0.041	0.037	0.302	1.099	n.d.	0.438	0.027
	0.014	0.037	0.034	0.302	1.012	n.d.	0.406	0.024
mean	0.014	0.043	0.037	0.302	1.144	n.d.	0.437	0.027
SD	0.000	0.007	0.003	0.000	0.160	n.d.	0.030	0.003
5.6	0.014	0.020	0.020	0.302	0.405	0.000	0.236	0.232
	0.014	0.017	0.017	0.302	0.223	0.000	0.175	0.007
	0.007	0.010	0.007	n.d.	0.405	n.d.	n.d.	n.d.
mean	0.011	0.01S	0.015	n.d.	0.345	n.d.	n.d.	n.d.
SD	0.004	0.005	0.007	n.d.	0.105	n.d.	n.d.	n.d.
10.0	0.007	0.017	0.020	n.d.	0.916	0.182	0.236	0.010
	0.020	0.014	0.020	0.708	n.d.	0.405	0.236	0.010
	0.003	0.017	0.017	n.d.	1.609	0.000	0.175	0.007
mean	0.010	0.01S	0.019	n.d.	n.d.	0.19S	0.21S	0.009
SD	0.009	0.002	0.002	n.d.	n.d.	0.203	0.035	0.002

The coefficient of variation of the mean specific growth rates during the whole test period (0-72 hours) in the replicate control culture was 1.9%. The mean coefficient of variation for the section-by-section growth rate was 20.4%.

n.d. - not determined due to mathematical reasons

SD - standard deviation

Table 3. 3,5-dichlorophenol - growth rate and yield endpoints based on the nominal concentrations of the reference substance

Endpoint [mg/L]	Time of exposure
	24 h	48 h	72 h
ErC50	2.47 (1.93-3.16)	2.72 (2.47-2.99)	2.61 (2.42-2.78)
ErC20	1.55 (0.93- 1.97)	1.69 (1.41 - 1.92)	2.05 (1.79-2.23)
ErC10	1.21 (0.60-1.62)	1.32 (1.03- 1.55)	1.80 (1.52-2.01)
LOEC (growth rate)	n.d.	1.00	n.d.
NOEC(growth rate)	n.d.	0.56	n.d.
EyC50	1.58 (0.88-2.60)	1.46 (1.29-1.64)	1.74 (1.54-1.97)
EyC20	0.61 (0.11 - 1.04)	0.80 (0.63-9.33)	1.12 (0.88- 1.30)
EyC10	0.38 (0.03-0.73)	0.58 (0.42-0.71)	0.89 (0.63- 1.07)
LOEC(yield)	<0.56	<0.56	<0.56
NOEC(yield)	<0.56	<0.56	<0.56

(-) - 95% confidence limits n.d. - not determined due to mathematical reasons

Validity criteria fulfilled:

yes

Remarks:

The validity criteria were fulfilled.

Conclusions:

The ErC50 (72h) was determined as 597.57 mg/L while the NOEC (72h) amounts to < 10 mg/L.

Executive summary:

The impact of MnIDHA on the green algal growth was investigated during a 72-hour static toxicity study according to OECD Guideline 201 in compliance with GLP (Konfederak, 2013). The study was conducted in glass flasks with a capacity of 250 mL. Each replicate contained 100 mL. The initial density of the algae was 1 x 10 E4 cells/mL. The concentrations of the test item, MnIDHA of 10; 32; 100; 320 and 1000 mg/L and a control (0.0 mg/L) were used. Chemical analyses were performed using a validated method. Samples collected at the exposure initiation (t0) and at the exposure termination (t72) were analyzed. The average concentration of MnIDHA in samples collected at the exposure initiation (t0) was between 95.2 – 100.3% of the nominal concentration. The average concentration of MnIDHA in samples collected at the exposure termination (t72) was between 92.2 – 100.4% of the initial concentration. After 72 hours of the exposure, the algal cells were observed for changes in morphology. The cells in all test item concentrations did not differ from the control ones. Since the concentration of MnIDHA was in the range of 80 - 120% of the initial concentration at the exposure termination, the ECx values were determined on the basis of the nominal concentrations of the test item.

The concentration causing 50% inhibition of the growth rate of Pseudokirchneriella subcapitata, i.e. the ErC50 (72h) value is 597.57 mg/L. The ErC20 (72h) value is 140.86 mg/L, whereas the ErC10 (72h) value is 66.18 mg/L. For the growth rate, the LOEC (72h) is lower than or equal to 10 mg/L, whereas the NOEC (72h) is lower than 10 mg/L.

The mean concentration causing 50% inhibition of yield of Pseudokirchneriella subcapitata, i.e. the EyC50 (72h) value is 108.95 mg/L. The EyC20 (72h) value is 42.27 mg/L, whereas the EyC10 (72h) value is 25.76 mg/L. For yield, the LOEC (72h) is lower than or equal to 10 mg/L, whereas the NOEC (72h) is lower than 10 mg/L.

Description of key information

The ErC50 (72h) was determined as 597.57 mg/L while the NOEC (72h) amounts to < 10 mg/L.

Taking this information into account it can be concluded that results obtained for acute toxicity of Mn(2Na)IDHA are also applicable for Ca(2Na)IDHA and presence of Ca cation in Ca(2Na)IDHA molecule instead Mn²⁺could not influence of it acute toxicity to aquatic invertebrates.

Key value for chemical safety assessment

EC50 for freshwater algae:

597.57 mg/L

EC10 or NOEC for freshwater algae:

66.18 mg/L

Additional information

There are no available ecotoxicity studies for Ca-IDHA chelate as such, but it is well known that the toxicity of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body or environment. Ca-IDHA chelate in water environment, depending on pH, temperature and other physical conditions, could decompose on Ca²⁺ cations and IDHA chelating agent. There are studies for chelating agent IDHA- sodium salt, other IDHA-chelates like Mg-IDHA, Cu-IDHA, Fe-IDHA, Zn-IDHA and also for inorganic calcium salts (for table please see to attached additional justyfication for OECD part dossie,r C 45, key study for MnNa2IDHA).

Calcium as such is considered as low toxic, is common in environment and its inorganic salts like calcium chloride CaCl₂ and calcium sulphate CaSO₄ are approved as food additive. Also results of ecotoxicity studies with CaCl₂ confirm this, LC₅₀ and EC₅₀ values for fish, invertebrates and as well for algae are very high, average values of several thousand mg/L. The lowest values are for Daphnia manga:

LC50 (48 hr) = 2770 mg/L

EC50 (48 hr) = 1062 mg/L (immobilization)

LC50 (48 hr) = 1285 mg/L

(EPA/600/4-90/027, EPA/600/6-91/003) (INCHEM, 2002)

 

Taking into account nature of chelates toxicity (depends mainly on metal ion) and available data for calcium confirmed its low toxicity, we stand that results of ecotoxicity studies for IDHA chelating agents are the best way to evaluate the possible effect of Ca-IDHA on water organisms and environment. However there are available studies of acute ecotoxicity for other IDHA chelates, because metal ion decides about toxicity of chelate,  their results are treated as supporting information confirmed this rule.